effects of lead on human microglia and neuronal … · microglia interaction, as well as during cns...
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Effects of Lead on Human Microglia
and Neuronal Precursor Cells
In Vitro
Samar Etemad, B.Sc., M.Sc.
This thesis is presented for the degree of
Doctor of Philosophy
The University of Western Australia
School of Anatomy, Physiology and Human Biology
2013
To my parents, Ladan and Reza who taught me
to work hard and dream big with their continous
love and support.
To my fiancé, Houman, whose endless love, support,
understanding and encouragement provided me the
strength and motivation I needed to achieve my goals
during the PhD
[ii]
Abstract
Lead is an environmental persistent pollutant with potent neurotoxic effects. In vitro and
in vivo studies have reported that lead induces neuronal death, astrocyte activation,
cognitive deficits, as well as learning and memory impairments. Involvement of the
immune system and inflammation has also been reported in lead toxicity. In humans, lead
concentrations are difficult to measure and assess, especially for the brain. However, 10
M of lead is expected to be the highest concentration that is reached in the central
nervous system (CNS). Australia is one of the main lead mining and processing countries
with reported lead toxicity in cities close to the mining sites and ports.
Microglia are the resident innate immune cells of the brain. As the macrophages of the
CNS, microglia share common characteristics with the monocyte-macrophage lineage
such as expression of particular surface markers and chemokine/cytokine receptors,
antigen presentation and phagocytic activity. Microglia activation in response to lead
exposure has been reported in animal studies. This activation is usually followed by
secretion of pro-inflammatory cytokines and chemokines as mediators of the innate
immunity. In the CNS, chemokines and their receptors are important in the neuron-
microglia interaction, as well as during CNS development by mediating neurogenesis,
migration and differentiation of neuronal progenitor cells.
Despite being the innate immune effectors of the CNS, the role of human microglia in
lead-induced toxicity and inflammation is still unknown. Furthermore, at the
commencement of this study, there was no standardized simple model available to
investigate the biology of human microglia.
To investigate the effects of lead on human microglia, first, a new human in vitro
microglia model (M-MG) using blood-derived monocyte was developed and established
(Samar Etemad et al., Journal of Neuroscience Methods 2012, 30, 79-89). In the next
step, the effects of lead exposure on human microglia, were studied to examine in
different aspects such as morphology, phenotype and function. In addition, the human
microglia cell line HMC3 and human neuronal precursors cell line HCN2 were used as
established human microglia and neuronal precursor cell lines in this study.
The human M-MG was clearly different in morphology, phenotype and function from
monocytes and macrophages, as well as from monocyte derived dendritic cells (MDC),
but shared many properties with the established HMC3 microglia cells. The human M-
MG acquired a ramified morphology with primary and secondary processes, comparable
[iii]
to HMC3. They expressed very low levels of CD45, CD14 and HLA-DR, CD11b and
CD11c; but a distinct pattern of chemokine receptors, including CCR1, CCR2, CCR3,
CCR4, CCR5, CXCR1, CXCR3, CX3CR1. Similar to HMC3, under non-activated
conditions, M-MG secreted IL-8 and IL-6. In comparison with MDC, M-MG displayed
lower T-lymphocyte stimulatory capacity, as well as lower phagocytosis activity.
To investigate the effects of lead on M-MG, cells were exposed to lead acetate at
different concentrations (1 to 100 µM, 24 hours to 5 days). Then, cells were
characterized morphologically, phenotypically (chemokine receptors expression) and
functionally using flow cytometry. Interestingly, lead exposure at lower concentrations
(<25 M) was not cytotoxic and had no effect on morphology or viability of the cells.
Most of the chemokine receptors were expressed by M-MG and HMC3 microglia under
non-stimulatory conditions. General down-regulation in most of the pro-inflammatory
chemokine receptors (CCR1, CCR2, CCR3, CCR5, CXCR1, CXCR5) was observed in
both cell types. On the other hand, IL-8 was the only cytokine that was up-regulated by
lead. In that respect, it is important to understand that IL-8 plays a relevant role in neuro-
development, neuro-regeneration and neuronal turn-over.
The effects of lead exposure on expression of CX3CR1 and TLR4 were also investigated
in M-MG and HMC3 cells due to their key role in inflammation and development.
CX3CR1 expression was dimorphic in microglia derived from different individuals,
having either CX3CR1low
or CX3CR1intermediate
expressing cells, supporting the presence
of functionally relevant genetic polymorphisms in humans. The human HMC3 microglia
cell line belongs to the CX3CRlow
expressing population. Lead had a differential effect on
changes of CX3CR1 and TLR4 expression. Lead significantly increased CX3CR1
expression in CX3CRlow
cells, whereas it significantly decreased CX3CR1 expression in
CX3CR1intermediate
cells. TLR4 was expressed at low levels in all microglia populations,
but TLR4 expression was increased by lead in the CX3CR1intermediate
microglia cells.
Given the important role of the chemokine-chemokine receptor axis in neuron-microglia
interactions, the effects of lead were studied on the human neuronal precursor cell line
HCN2 under optimal conditions (10 µM, 24 hours). Interestingly, lead was not cytotoxic
and had no effects on the morphology or viability of the human neuronal cells. However,
functional changes were observed, including down regulation of the pro-inflammatory
chemokine receptors. Expression of CX3CR1 and CXCR1 significantly increased in
HCN2, as well as their reciprocal chemokines, fractalkine and IL-8. Finally, IL-6
secretion decreased in lead-treated HCN2.
[iv]
In conclusion, the presented data show that human monocyte-derived microglia represent
an appropriate and feasible in vitro model to investigate the role of microglia in
neurotoxicity. Furthermore, the research presented in this thesis suggests that lead has
functional effects on human microglia, but with no significant direct cytotoxic effects.
Lead alters the expression patterns of chemokines and chemokine receptors in both,
human microglia and neuronal precursor cells. Therefore, lead exposed microglia may
interfere with neuronal turnover, recruitment and differentiation, enhancing direct lead
effects on neuronal precursors.
Keywords: human microglia, lead, in vitro model, monocyte-derived microglia, IL-8,
chemokine receptor, neuronal precursor cells, CX3CR1
[v]
Table of contents
Abstract..................................................................................................................... ii
Table of contents ....................................................................................................... v
List of Figures ............................................................................................................ x
List of Tables ............................................................................................................. xi
Statement of Candidate Contribution ....................................................................... xii
Publications and Proceedings ................................................................................... xiii
Conference presentations ......................................................................................... xiv
Acknowledgements................................................................................................... xv
Abbreviations ......................................................................................................... xvii
Chapter 1
1. Literature Reviews ............................................................................................... 1
1.1. Lead .................................................................................................................. 1
1.1.1. Lead neurotoxicity ................................................................................................................. 7
1.1.2. Lead entry into the brain ........................................................................................................ 8
1.1.3. Mechanisms involved in lead-induced neurotoxicity: role of neuroinflammation ................. 8
1.1.4. Lead toxicity in animal models ............................................................................................ 10
1.1.5. Lead and the immune system ............................................................................................... 12
1.2. Microglia ........................................................................................................ 13
1.2.1. Origin ................................................................................................................................... 13
1.2.2. Turnover ............................................................................................................................... 14
1.2.3. Distribution .......................................................................................................................... 14
1.2.4. Morphology .......................................................................................................................... 14
1.2.5. Microglia function: immune effectors of the CNS .............................................................. 16
1.2.6. Role of microglia: neuroprotection versus neurodegeneration ............................................. 18
1.2.7. Cytokines and neuroinflammation ....................................................................................... 18
1.3. Lead and microglia ......................................................................................... 19
1.4. Role of chemokines and chemokine receptors in CNS development ............. 20
1.5. Role of cytokines, chemokines and chemokine receptors in neuro
inflammation ..................................................................................................................... 22
Chapter 2
2. Research aims and outlines ................................................................................ 26
2.1. Aims ............................................................................................................... 26
2.2. Research outline and methodology ................................................................ 28
[vi]
Chapter 3
3. Effect of lead on human cortical neuronal precursor cells .................................... 31
3.1. About the paper .............................................................................................. 31
3.2. Abstracts ......................................................................................................... 32
3.3. Introduction .................................................................................................... 33
3.4. Material and methods ..................................................................................... 38
3.4.1. Characterization of HCN2 .................................................................................................... 38
3.4.2. Viability assay ...................................................................................................................... 39
3.4.3. Surface marker expression ................................................................................................... 39
3.4.4. Cytokine production measurement ....................................................................................... 40
3.4.5. Statistics ............................................................................................................................... 40
3.5. Results ............................................................................................................ 41
3.5.1. Characterization of human neuronal cells HCN2 ................................................................. 41
3.5.2. Lead effects on HCN2 morphology and viability ................................................................. 43
3.5.3. Effect of lead exposure on expression of chemokine receptors in HCN2 ............................ 44
3.5.4. Effect of lead on cytokine secretion in HCN2 cells .............................................................. 47
3.6. Discussion ...................................................................................................... 48
3.7. Conclusion ...................................................................................................... 53
Chapter 4
4. A novel in vitro human microglia model: characterization of human monocyte-
derived microglia .......................................................................................................... 55
4.1. Introduction to chapter ................................................................................... 55
4.2. Abstract .......................................................................................................... 57
4.3. Introduction .................................................................................................... 57
4.4. Materials and methods .................................................................................... 61
4.4.1. Cell isolation ........................................................................................................................ 61
4.4.2. Cell culture ........................................................................................................................... 62
4.4.3. Morphological studies .......................................................................................................... 62
4.4.4. Flow cytometry .................................................................................................................... 63
4.4.5. Phagocytosis assay ............................................................................................................... 63
4.4.6. Mixed leukocyte reaction (MLR) ......................................................................................... 64
4.4.7. Statistics ............................................................................................................................... 64
4.5. Results ............................................................................................................ 64
4.5.1. Morphological and phenotypic changes of monocyte-derived microglia in culture ............. 64
4.5.2. Expression pattern of specific surface markers .................................................................... 68
4.5.3. Phagocytic capacity .............................................................................................................. 71
4.5.4. T-lymphocyte stimulatory capacity of MGM ....................................................................... 73
4.6. Discussion ...................................................................................................... 74
4.7. Conclusion ...................................................................................................... 76
[vii]
Chapter 5
5. Lead modulates chemokine receptors pattern and induce interleukin-8 in human
microglia ...................................................................................................................... 78
5.1. Introduction to chapter ................................................................................... 78
5.2. Abstract .......................................................................................................... 79
5.3. Introduction .................................................................................................... 80
5.4. Material and methods ..................................................................................... 82
5.4.1. Cell culture ........................................................................................................................... 82
5.4.2. Lead exposure ...................................................................................................................... 83
5.4.3. Morphological studies .......................................................................................................... 83
5.4.4. Viability assays .................................................................................................................... 84
5.4.5. Detection of up-take and intracellular accumulation of lead ............................................... 84
5.4.6. Reactive oxygen species ....................................................................................................... 85
5.4.7. Expression of surface markers.............................................................................................. 85
5.4.8. Cytokine production ............................................................................................................. 86
5.4.9. Statistics ............................................................................................................................... 86
5.5. Results ............................................................................................................ 86
5.5.1. Detection of lead uptake and intracellular accumulation by human microglia ..................... 86
5.5.2. Effect of various lead concentration on human microglia cell morphology and activation .. 88
5.5.3. Effect of various concentration of lead on human microglia viability .................................. 90
5.5.4. Effects of lead exposure on reactive oxygen species production and lipid peroxidation in
human microglia ................................................................................................................... 91
5.5.5. Effect of lead exposure on expression of chemokine receptors and surface molecules in
human microglia ................................................................................................................... 91
5.5.6. Effect of lead on cytokine secretion patterns in human microglia ........................................ 95
5.6. Discussion ...................................................................................................... 97
5.7. Conclusion .................................................................................................... 102
Chapter 6
6. Lead alters the chemokine receptors expression pattern of the human microglia
cell line (HMC3) .......................................................................................................... 104
6.1. Introduction to the chapter .......................................................................... 104
6.2. Abstract ........................................................................................................ 105
6.3. Introduction .................................................................................................. 106
6.4. Material and methods ................................................................................... 109
6.4.1. Cell culture ......................................................................................................................... 109
6.4.2. Lead exposure .................................................................................................................... 109
6.4.3. Morphological studies ........................................................................................................ 109
6.4.4. Detection of up-take and intracellular accumulation of lead .............................................. 110
6.4.5. Viability assays .................................................................................................................. 110
[viii]
6.4.6. Expression of surface markers............................................................................................ 111
6.4.7. Mixed leukocyte reaction (MLR) ....................................................................................... 111
6.4.8. Phagocytosis assay ............................................................................................................. 112
6.4.9. Cytokine measurement ....................................................................................................... 112
6.4.10. Statistics ............................................................................................................................. 112
6.5. Results .......................................................................................................... 113
6.5.1. Intracellular detection of lead uptake in human microglia cell line HMC3 ........................ 113
6.5.2. Effect of low level lead exposure on the morphology and viability of HMC3 ................... 114
6.5.3. Effect of of lead exposure on expression of chemokine receptors and surface molecules on
HMC3 ....................................................................................................................... ..... 116
6.5.4. Effects of lead on phagocytic activity of HMC .................................................................. 120
6.5.5. Influence of lead on microglia T-cell stimulatory capacity ................................................ 121
6.5.6. Effect of lead on cytokine secretion in HMC3 ................................................................... 121
6.6. Discussion .................................................................................................... 124
6.7. Conclusion .................................................................................................... 128
Chapter 7
7. Differential effects of lead on CX3CR1 and TLR4 expression in human microglia in
vitro 130
7.1. Introduction to chapter ................................................................................. 130
7.2. Abstract ........................................................................................................ 131
7.3. Introduction .................................................................................................. 132
7.4. Material and Methods ................................................................................... 135
7.4.1. Cell culture and experimental setting ................................................................................. 135
7.4.2. Flow cytometry .................................................................................................................. 136
7.4.3. Statistical analysis .............................................................................................................. 136
7.5. Results .......................................................................................................... 137
7.5.1. Differential expression of CX3CR1 in human microglia in vitro ....................................... 137
7.5.2. Differential effects of lead on CX3CR1 expression in human microglia ........................... 138
7.5.3. Effect of immune modulators on CX3CR1 expression in HMC3 cells .............................. 140
7.5.4. Effect of lead on TLR4 expression human microglia ......................................................... 140
7.6. Discussion .................................................................................................... 142
7.7. .Conclusion ................................................................................................... 144
Chapter 8
8. General discussion and future directions .......................................................... 146
8.1. Summary of key findings ............................................................................. 146
8.2. Neuronal remodeling and lead toxicity ........................................................ 151
8.3. Why microglia are important in lead-neurotoxicity? ................................... 152
8.4. Microglia-neuron interaction: A possible target of lead toxicity ................. 153
8.5. Role of IL-8 / CXCR1 signaling in microglia-neurons interaction: ............. 154
[ix]
8.6. Importance of fractalkine/CX3CR1 in microglia-neuron interaction ........... 155
8.7. Immune modulation by lead and IL-6 in the CNS: between neuroprotection
and neuroinflammation ................................................................................................... 157
8.8. Research implication and future direction .................................................... 158
8.9. General conclusion ....................................................................................... 159
Chapter 9
Bibliography .......................................................................................................... 161
Chapter 10
Appendix ............................................................................................................... 186
[x]
List of Figures
Figure 1.1: Lead mining and processing centers in Australia. .......................................... 2
Figure 1.2: Effects of lead at different concentration on children vs. adults. ................... 3
Figure 1.3: The main sources of lead poisoning. .............................................................. 5
Figure 1.4: Lead toxicity targets in the human body......................................................... 6
Figure 1.5: Lead accumulation, storage and release in astrocytes. ................................... 9
Figure 1.6: Lead interacts with different sites of actions in neurons. ............................. 10
Figure 1.7: Microglia is the immune effectors cells in the brain. ................................... 15
Figure 1.8: Chemo taxis or phagocytosis-involved receptors in microglia and
correlation of the inflammatory or anti-inflammatory response. ..................................... 17
Figure 1.9: Constitutive vs. inducible chemokine ........................................................... 20
Figure 2.1: Research outline ........................................................................................... 29
Figure 3.1: Expression of neuronal markers by HCN2. .................................................. 42
Figure 3.2: Effect of various concentrations of lead on viability of HCN2 using the
MTS assay. ....................................................................................................................... 43
Figure 3.3: Effect of lead exposure on HCN2 morphology ............................................ 44
Figure 3.4: Effect of lead exposure on expression pattern of neural marker. ................. 45
Figure 3.5: Effect of lead on expression patterns of chemokine receptor in HCN2. ...... 46
Figure 3.6: Effect of lead on cytokine secretion in HCN2. ............................................. 47
Figure 4.1: Phase contrast microscopy of monocyte-derived microglia (M-MG) and
human microglia cell line HMC3 ..................................................................................... 65
Figure 4.2: Expression of Iba-1 in HMC3 and M-MG ................................................... 66
Figure 4.3: Differential expression of CD68 in freshly isolated blood monocytes ,
HMC3 and M-MG............................................................................................................ 67
Figure 4.4: Expression of surface markers CD45 (a), CD14 (b), HLA-DR (c), CCR2
(d) and CCR4 (e) measured with flow cytometry. ........................................................... 69
Figure 4.5: Expression of chemokine receptors CCR1 (a), CCR5 (b), CXCR1(c),
CXCR3 (d) and CX3CR1 (e) measured with flow cytometry. ...................................... 70
Figure 4.6: Phagocytic capacity of M-DC (Black line), M-MG (Dotted line) and
HMC3 (dashed line) towards TM Rhodamine-labelled S. aureus measured with flow
cytometry. ........................................................................................................................ 71
Figure 4.7: Up-take of TM Rhodamine-labelled S. aureus by M-MG using confocal
microscopy. ...................................................................................................................... 72
Figure 4.8: A selected Z-stack image taken with confocal microscopy showing the
up-take of TM Rhodamine-labeled S. aureus by M-MG. ................................................ 72
[xi]
Figure 4.9: Mixed leukocyte reactions (MLR) ............................................................... 73
Figure 5.1: Lead uptake by human microglia. ................................................................ 87
Figure 5.2: Effect of lead exposure on morphology of microglia. .................................. 89
Figure 5.3: Viability of human microglia after lead exposure. ....................................... 90
Figure 5.4: Effect of lead on ROS production and membrane peroxidation in human
microglia. ......................................................................................................................... 92
Figure 5.5: Effect of lead exposure (10 µM, 24 hours) on chemokine receptors and
surface marker expression patterns in human microglia as measured with flow
cytometry. ........................................................................................................................ 93
Figure 5.6: Comparison between expression pattern of surface markers in the absence
or presence of lead in human microglia ........................................................................... 94
Figure 5.7: Effect of lead on cytokine secretion in human microglia. ............................ 96
Figure 6.1: Lead uptake by human microglia cell line HMC3......................................113
Figure 6.2: Effect of lead on morphology and Viability of HMC3. .............................. 115
Figure 6.3: Effect of lead exposure (10 µM, 24hrs) on chemokine receptor expression
patterns in human microglia HMC3. .............................................................................. 117
Figure 6.4: Lead alters the expression of surface markers in HMC3 ............................ 118
Figure 6.5: Lead showed distinct immunomodulatory effects on the surface molecule
expression in HMC3. ..................................................................................................... 119
Figure 6.6: Effects of lead on phagocytic activity of HMC3 ........................................ 120
Figure 6.7: Influence of lead on microglia T-cell stimulatory capacity. ....................... 122
Figure 6.8: Effect of lead on cytokine secretion in HMC3 ........................................... 123
Figure 7.1: Expression levels of CX3CR1 in human M-MG and HMC3 cells. ............ 138
Figure 7. 2: Effect of lead on expression of CX3CR1 in human M-MG and HMC3 .. 139
Figure 7. 3: Comparison of various stimuli on CX3CR1 expression in HMC3 cells. .. 140
Figure 7. 4: Effect of lead on expression of TLR4 in human M-MG and HMC3 cells. 141
Figure 8.1: Neuromodulatory effects of lead on neurounal precursors and microglia . 160
List of Tables
Table 1.1: Chemokine and chemokine receptors in CNS.............................................. 24
Table 2.1: Testing hypotheses of microglia involvement in lead-induced
neurotoxicity. ................................................................................................................. 27
Table 2.2: The main aims of this thesis. ........................................................................ 27
Table 2.3: Main methods applied in this study and the corresponding chapters. .......... 28
[xii]
Statement of Candidate Contribution
All the experiments detailed in this thesis have been conducted at the School of
Anatomy, Physiology and Human Biology; University of Western Australia under the
supervision of Prof. Luis Filgueira, Dr. Marc Ruitenberg and A/Prof. Silvana Gaudieri. I
hereby declare that all the works presented here is entirely my own, except where the
contribution of others have been acknowledged.
Samar Etemad
Dec. 2013
[xiii]
Publications and Proceedings
PUBLICATIONS
Etemad S., Ruitenberg M.J. and Filgueira L. (2013). Differential effects of lead (Pb)
on CX3CR1 and TLR4 expression in human microglia in vitro. Journal of
Neuroinflammation (under review).
Etemad S., Ruitenberg M.J. and Filgueira L. (2013). Lead (Pb) modulates chemokine
receptor patterns and induces interleukin-8 in human microglia. Journal of
Neurotoxicology (under review).
Etemad S., Zamin, R.M., Ruitenberg M.J. and Filgueira L. (2012). A novel in vitro
human microglia model:Characterozation of human monocyte-derived microglia.
Journal of Neuroscience Methods 209,79-89 (Appendix E).
Etemad S., Ruitenberg M.J. and Filgueira L.(2012), In vitro generated microglia:
from blood precursors. Journal of Immunology; 118:111.14 (Meeting Abstract).
[xiv]
Conference presentations
ORAL PRESENTATIONS
S. Etemad, M. Ruitenberg and L. Filgueira (2013), Lead (Pb) modulates human microglia
inflammatory responses. Australian Society of Neuroscience (ANS), Melbourne,
Australia, February 3-6.
S. Etemad, M. Ruitenberg and L. Filgueira (2012), Effect of low-level lead exposure on
chemokine receptor expression in human microglia in vitro .School of Anatomy,
Physiology and Human Biology (APHB) Student Expo, Perth, Australia, July 21.
S. Etemad, M. Ruitenberg and L. Filgueira (2011), Effect of Lead on human microglia in
vitro. APHB Student Expo, Perth, Australia, July 15
S. Etemad, M. Ruitenberg and L. Filgueira (2010) A new in vitro model for human
microglia. ASMR (Australian Society for Medical Researches) meeting, Curtin
University, June 8.
S. Etemad, M. Ruitenberg and L. Filgueira (2010), A New In vitro Model for Human
Microglia. School of Anatomy, Physiology and Human Biology Student Expo, Perth,
Australia, July 15
S. Etemad, M. Ruitenberg and L. Filgueira (2009). Role of human microglia in lead
neurotoxicity. School of Anatomy, Physiology and Human Biology Student Expo, Perth,
Australia, July 16.
POSTER PRESENTATION
S. Etemad, M. Ruitenberg and L. Filgueira (2012), Effect of low-level lead exposure on
chemokine receptor expression using a novel human in vitro microglia model. Combined
Biological Sciences Meeting (CBSM), Perth, Australia, August 24. (Appendix A)
S. Etemad, M. Ruitenberg and L. Filgueira (2012), In vitro generated microglia: from
blood precursors. Immunology 2012 (99th American Association of Immunology),
Boston, MA, USA, May 4-9 . (Appendix B)
S. Etemad, M. Ruitenberg and L. Filgueira (2012), In vitro generated microglia derived
from blood precursors. Experimental Biology (American Association of Anatomists),
SanDiego, CA, USA, April 21-25. (Appendix C)
S. Etemad, M. Ruitenberg and L. Filgueira (2010), A new in vitro human microglia
model. 41st Annual Scientific Meeting of the ASI (Australian Society of Immunology),
Perth, Australia, December 5-9. (Appendix D).
[xv]
Acknowledgements
I would like to express my sincere appreciation for my main supervisor; Prof. Luis
Filgueira. I specially like to thank him for giving me the opportunity to come to Australia
and experience new life here. Luis had provided me his enduring guidance and support
during each and every stage of this project. Luis, you have helped me to become a better
scientist and you have taught me to believe in my ideas. Thank you also for the
opportunity to meet many top researchers in the field of Anatomy, Human Biology and
Immunology.
I also wish to extend my deepest gratitude to my supervisor Dr. Silavana Gaudieri. It has
been a great privilege for me to work under her supervision in particular over the last
year of PhD in absence of Luis.
A very special thank you to my co-supervisor, Dr. Marc Ruitenberg for his invaluable
advice and feedback on my research and for always being so supportive of my work
I particularly want to say thanks to my dear friends and PhD colleagues of School: Su-
ann Koh, Helen Dooley, Belinda Burns, Fritha Milne, Rebecca Dawson and Linda
Blomster who all helped me in numerous ways during various stages of my PhD Thanks
for your continuous support and encouragement. You made memorable moments during
my PhD and I will never forget.
Special thanks to my colleagues, Praseetha Prabhakaran, Pooja Deshpande and the staff
and postgraduate students in School of Anatomy, Physiology, and Human Biology
(APHB) for their encouragement. I would like to extend my thanks to Rasheeda Mohd
Zamin and acknowledge her assistance in confocal imaging.
I would like acknowledge the facilities (confocal microscopy and flow cytometry),
scientific and technical assistance of the Australian Microscopy & Microanalysis
Research Facility at the Centre for Microscopy, Characterization & Analysis, the
University of Western Australia. In particular, my deep appreciation goes to Dr. Kathy
Heel and Mrs. Tracey Lee-Pullen for their support and help.
My thanks also go out to the support I received from Prof. Link Schmitz and all the
academics of AHAB for their support and encouragement throughout my PhD . . . I am
indebted to Prof. Alan Harvey and Margret Pollett who accepted me as one of their
team membersand Margaret provided me with her constant support and advice regarding
neuronal cell culture as well as enabling me to use neuronal cell line.
I am also very grateful to all those at the APHB office, especially Vicki, Heather, Denis,
Debbie and others who were always so helpful and provided me with their assistance
throughout my dissertation.
I should say thanks to Cell Central and in special Guy Ben- Ary, center for microscopy at
APHB for providing the microscopic facilities. Thanks Greg Cozen, Mary Lee, Leonie
[xvi]
Khoo, and Steve Bamfort for your always help, advice and patience in answering my
questions. I also appreciate the Graduate Research School, The University of Western
Australia for supporting this project and me all these years.
On a personal note, I like to express my heartiest appreciation to my Mum and Dad for
their support beyond measures during all stages of my life in particular during the PhD.
Their everyday encouragement, optimisms, and strength made to overcome the obstacle
and without them I would not have contemplated this road. I am very lucky to have
parents, who understand the difficulties of research and science.
Finally, my very special thank is to Houman for his endless love, understanding,
encouragement, support and optimisms during my PhD, I don’t know how should I say
thank you. You were with me, with positive opinion and hope in every parts of these PhD
during happy and sad times. I am so lucky to have you in my life, one who understands
the real meaning of research and PhD. It would have not been possible without you.
[xvii]
Abbreviations
AD
Alzheimer’s Disease
ALS
APCs
Amyotrophic Lateral Sclerosis
Antigen Presenting Cells
BBB Blood-Brain Barrier
BDNF
CCL
Brain-Derived Neurotrophic Factor
Chemokine (C-C Motif) Ligand
CCR C-C Motif Chemokine Receptor
CNS
CXCL
CXCLR
DG
Central Nervous System
Chemokine C-X-C motif Ligand
Chemokine C-X-C motif Receptor
Dentate Gyrus
FKT Fractalkine/CX3CL1
GM-CSF
GFAP
Granulocyte-Macrophage Colony Stimulating Factor
Glial Fibrillary Acidic Protein
HLA-DR
HMGB1
Human Leukocyte Antigen-DR
High Mobility Group Box 1 Protein
HMC3 Human Microglia Cell Line Clone 3
Iba-1 Ionized Calcium Binding Adaptor Molecule 1
IL Interleukin
iNOS
ISF
LIF
Inducible Nitric Oxide Synthase
Interstitial Fluids
Leukaemia Inhibiting Factor
LPS
MBP
Lipopolysaccharide
Myelin Basic Proteins
MCP-1
MIP-1
Monocyte Chemoattraactant Protein 1
Macrophage Inflammatory Protein 1
M-CSF Macrophage Colony Stimulating Factor
M-DC Monocyte Derived Dendritic Cells
M-MG Monocyte-Derived Microglia
MS Multiple Sclerosis
NGF Nerve Growth Factor
NGFR Nerve Growth Factor Receptor
NPCs Neuronal Precursor Cell
[xviii]
NSCs
NSE
Neural Stem Cells
Neuronal Specific Enolase
PBMC Peripheral Blood Mononuclear Cells
PBS Phosphate Buffer Saline
PC12 Pheochromocytoma
PD Parkinson’s Disease
PFA Paraformaldehyde
PHA Phytohemagglutinin
PMA Phorbol-12-Myristate-13-Acetate
PVR
RANTES
SDF-1
SVZ
Proliferative Viteorethinopathy
Regulated And Normal T Cell Expressed And Secreted
Stromal Derived Factor 1
Subventricular Zone
TGF-β
TECK
Transforming Growth Factor Beta
Thymus Expressed Chemokine
TLR4 Toll-Like Receptor 4
TNF-
TGF-β
Tumor Necrosis Factor Alpha
Transforming Growth Factor Beta
Trk A Tyrosine Kinase Receptor A
Chapter 1
Literature Reviews
[1]
1. Literature Reviews
1.1. Lead
Lead is a persistent, widely spread environmental pollutant with potent toxic effects in
humans. Lead is a xenobiotic metal with many still unknown effects on cell growth,
proliferation and differentiation. Despite continuous attempts to reduce lead exposure in
humans, it is still a major global health concern. Lead can be naturally found in the
environment. However, human activities over the past three decades have increased the
lead level more than 1000 fold (NHMRC, 2009). Based on sources from the ancient
history literature, lead was known to humans as early as 4,000 BC. It has been used by
humans for thousands of years due to its wide geographical availability, easy mining and
enrichment, as well as convenient processing and usage. Lead is often found in ore with
other metals such as silver, zinc and copper which are usually extracted together. The
ancient Romans regarded lead as the father of all metals. However, Romans were aware
that lead could cause serious health problems such as madness and death (Woolley,
1984). Thus, lead and its toxicity have affected humans for many years. More recently,
lead has been widely used in building constructions, batteries, army equipment including
bullets and weapons, ceramic glazes and colouring, house paint, piping, and petrol
(Hernberg, 2000, Woolley, 1984). Epidemic incidents of lead poisoning during the 19th
century up until the mid-twentieth century, due to industrializing the world and changes
in human lifestyles, led to increased need for understanding and detection of lead
exposure sources. Thus, legislation was established in many countries in order to reduce
the level of environmental exposure from lead. For example in the 1920s, Austria,
Czechoslovakia, Finland, Norway, Poland, Spain and Sweden, abandoned the use of lead
in indoor white painting due to frequent toxic incidents in children (Hernberg, 2000).
Australia is one of the principal countries in lead mining, smelting and processing. Lead
was first mined in Australia in 1841 near Adelaide, South Australia. Hence, people
living next to lead-mining, smelting, and refining centers such as those in Broken Hill,
Port Pirie, and Mount Isa have been exposed to lead for a long time (Figure 1.1). In
1904, for the first time, lead poisoning has been reported in children from lead-based
paint in Brisbane by the Australian physician Lockhart Gibson (Rosner et al., 2005).
Later, it was shocking to discover that more than 3000 children in the town of Port Pirie,
South Australia, were reported lead poisoned in 1980-1990. Due to ignorance or
misinformation, generations of families living in lead mining and processing regions
have been and continue to be exposed to high environmental lead concentrations, despite
[2]
knowing about its neurotoxicity. A survey in Mt Isa , Queensland Health has shown
that 11% of children had blood lead concentrations in excess of 10 μg/dl (Health, 2008).
Similar to humans, over the past decade, several reports have shown lead poisoning in
animals such as birds and dolphins living close to the mining area in Western and South
Australia (Lavery et al., 2008).It is widely known that children, at the age of 1-5 years,
are a very vulnerable group (Finkelstein et al., 1998). In young children, lead
intoxication causes dramatic problems, including deficits in learning and memory,
behavioural disturbance and decreased IQ scores, even at low exposure levels (Nava-
Ruiz et al., 2012, White et al., 2007).
Figure 1.1: Lead mining and processing centers in Australia.
This map illustrates the many lead mining centers scattered throughout Australia. Sever lead
poisoning (BLL more than 10 µg/dl) in children living in the marked places are indicated by
black stars. Source: http://www.lead.org.au/lanv12n3/lanv12n3-2.html
Based on recent research and reports, it is not possible to determine safe levels of lead
exposure. In 1960-1990, due to the extensive world-wide efforts to decrease
environmental lead levels, average blood lead levels (BLL) were significantly reduced
from 60 to 25 µg/dl in most human populations. However, in 1991, the Centres for
Disease Control and Prevention (CDC) recommended lowering the BLL in individuals
to 15 µg/dl and started a world-wide lead poisoning prevention campaign in areas where
children had BLL > 10 µg/dl. In 2005, the CDC considered blood lead concentrations
greater than or equal 10 µg/dl (0.5 µM) excessive for infants and children (CDC),
[3]
2005).The CDC has set the standard elevated blood lead level for adults to be 25 µg/dl in
whole blood. However, according to recent reports about the toxic effects of lead,
reduction of recommended upper BLL in children has been now set at 5 µg/dl. The
Advisory Committee on Childhood Lead Poisoning Prevention (ACCLPP) in the United
States of America reported deficits in cognitive functions, IQ and academic skills, as
well as impairment in cardiovascular, immunological and endocrine functions in
children with BLL lower than 10 µg/dl (Figure 1.2).
Figure 1.2: Effects of lead at different concentration on children vs. adults.
This figure provides a summary of the organ-specific effects associated with different
concentrations of lead as well as physiological disorders in children versus adults. Source:
Preventing Lead Poisoning in Young Children, CDC, 1991, Gurer and Ercal, 2000.
Thus, they proposed the new upper references value of maximally 5 µg/dl as the base
line for lead poisoning in children (Prevention, 2012). Based on evidences on the effect
of low- level lead exposure, the Australian National Health and Medical Research
Council (NHMRC) suggested in 2009 that all Australians should have BLL below 10
μg/dl (NHMRC, 2009).Environmental lead is taken up through inhalation, ingestion and
dermal contact. Lead absorption in humans happens mainly through the respiratory and
gastrointestinal systems (Papanikolaou et al., 2005). Inhaling lead dust, as a result of
mining and smelting of lead, is still common in many countries, including Australia.
However, using lead in petrol was stopped in most countries by 2007. Lead
50 100 150
<
>5 10 20 40
Blood Lead
Level
learning disabilities
cognitive deficits
reduced IQ
neurobehavioral
disorders
encephalopathy
medical lead removal
chilation therapy
medical lead removal
chilation therapy
colic
anaemia
nephropathy
encephalopathy
death
Fatigue abdominal pain
Abdominal pain anaemia reproductive
problem
Reduced poor appetite
Sleep deprivation memory problem
Aggressive behaviour nausea
Headache kidney problem
nerve conducttion
velocity
hemoglubulin synthase
Vitamin D methabolisms
[4]
contaminated soil or water, via lead pipes, are other common sources of exposure. Lead-
based paint has been a major sources of lead exposure in children (Figure 1.3). Lead
paint tastes sweet, and thus encourages children to lick the paint. It can also easily be
peeled off walls or toys , and enters the infants’ or children’s body hand-to-mouth
(Schwartz and Levin, 1991). Approximately 40% of the inhaled lead enters the
circulation system (Philip and Gerson, 1994).
Adults absorb 10-15% of lead through the gastrointestinal tract, while infants and
children absorb as much as 50% through their gastrointestinal tract (Markowitz, 2000,
Papanikolaou et al., 2005, Goldstein, 1990a). Depending on the target tissue, lead can
persist for many years. For example the half-life of lead in the brain is about two years,
while it remains in bone for about 30-40 years (Verstraeten et al., 2008). Lead in blood
has a half-life of 35 days (Xu et al., 2012). Generally, the rate of lead transferred from
blood to soft tissue is slow and takes about 4-6 weeks. In the human body, lead has
detrimental effects on several important systems, such as the renal, reproductive,
cardiovascular, immune and central nervous system, even at low levels of exposure
(Figure 1.4). In children, the adverse effects of lead can vary from changes in cognitive
function to fatal lead poising depending on the amount of exposure (Needleman, 2004a).
It is noteworthy to mention that lead excretion from the body happens very slowly,
mainly through urinary excretion. Therefore, chelating agents are the best agents to
enhance lead excretion, these are currently being used in the therapeutic approach for
lead poisoning treatment.
[5]
Figure 1.3: The main sources of lead poisoning.
This image illustrates the most common sources of lead exposure: soil, drinking water, lead based
paint and leaded petrol. In Australia, due to the mining industries, lead-contaminated soil is one of
the main sources of exposure. Lead-based paint is another common source of lead exposure
especially among preschoolers. Children may eat, chew paint chips, or even lick the old-painted
window slides. Drinking water is usually contaminated with lead through lead water pipes, found
in many homes built before 1930.
Australia is one of the main leading countries in mining, smelting and processing
the lead
[6]
Figure 1.4: Lead toxicity targets in the human body.
The detrimental effects of lead can affect several organs during development and adulthood. The
central nervous system and the immune system are key targets of lead poisoning in human. Source:
WHO, 2010.
[7]
1.1.1. Lead neurotoxicity
The central nervous system (CNS) is one of the primary targets of lead toxicity,
especially during development. Lead can cross the blood–brain barrier (BBB) and
concentrates in the gray matter of the brain (Goyer and Clarkson 2001; Gwalteney-Brant
2002). Lead exposure has devastating effects on the developing nervous system,
producing morphological, cognitive, and behavioural deficits. Headaches, poor attention
span, irritability, loss of memory and dullness are early symptoms of lead exposure in the
central nervous system. High level exposure to lead, which results at blood lead levels of
70-100 µg/dl in children, may cause serious clinical encephalopathy or severely affect
neurological functions. Persistent vomiting, ataxia, stomach pain and loss of
consciousness are symptoms of acute encephalopathy (Kanwal and Kumar, 2011).
Furtunately, occurrence of this kind of lead poisoning is now extremely rare. Acute adult
lead poisoning occurs primarily as the result of inhalation or ingestion of lead particles
due to working in lead mines or smelting sites.
Despite the well-documented neurotoxic effects of lead, the mechanisms involved are not
fully understood. As lead affects numerous biological activities at different levels, several
cellular, intracellular and molecular mechanisms have been shown to be involved in lead
neurotoxicity. The prefrontal cerebral cortex, hippocampus and cerebellum have been
reported as preferred target regions in the brain for lead-induced damages (Selvin-Testa
et al., 1994). Based on observations from animal and human in vitro and in vivo models,
a number of neurotoxic effects of lead on the brain, especially during development, have
been observed. These include impaired synaptic plasticity, inhibition of glutamate
receptor activation (Guilarte et al., 1994), apoptosis (Oberto et al., 1996), learning and
cognitive deficits, memory impairment (Xu et al., 2009), excitotoxicity, altered
neurotransmitter storage and release, mitochondrial dysfunction, loss of neuronal myelin
sheaths, changes in cellular energy metabolism and disturbance in neuronal growth
(Toscano and Guilarte, 2005). Several studies have demonstrated the neurotoxic effects
of different metals, including zinc, mercury, and aluminum, on cortical neurons survival
and function, as well as their involvement in pathology of neurological and psychiatric
disorders, including autism spectrum disorders, Alzheimer’s disease, Parkinson’s disease,
epilepsy, depression and mood disorders (Xu et al., 2012, Chen and Liao, 2003).
Although there are several studies showing the developmental effects of low-levels of
lead exposure on neurons, such as reduction of the capacity of neurogenesis in the adult
rat hippocampus, deficits in synaptic plasticity, and effects on migration of the human
neural crest cells, (Gilbert et al., 2005, Zimmer et al., 2012), the mechanisms involved in
[8]
the effect of low-level exposure of lead on the CNS are still not known. Recent studies
have reported that lead could enhance neuron degeneration in cultured rat
cerebrocortical neurons without inducing neuronal cell death (Fujimura and Usuki,
2012).
1.1.2. Lead entry into the brain
The blood-brain barrier (BBB) is a specialized structure in the CNS, which separates the
systemic blood circulation from the interstitial fluids (ISF) and therefore protects the
brain’s microenvironment from fluctuations in ion and metabolite concentrations in the
blood (Zheng et al., 2003). The BBB is made up of the endothelial cells surrounded by
astrocytes. Toxicity of lead play a key role in the disruption of the astrocytes-endothelial
cell communication (Goldstein, 1990). Various receptor- mediated transport systems are
inherent to the BBB vascular structure in order to control the transport of metals within
the brain. The BBB has long been known as one of the targets of lead toxicity. Poisoning
by lead and other metals has been demonstrated to induce vascular destruction and
cerebral haemorrhage (Zheng et al., 2003). Lead may interact with astrocytes to
indirectly affect the BBB function (Zheng et al, 2003). Astrocytes are also known as the
metal depot cells of the brain.
Several studies suggest that lead enters the brain as a free ion or as a complex with small
molecules. In plasma, around 40% of lead is bound to albumin which stops brain lead
uptake, as albumin does not preferably cross the BBB. Lead may however, cross the
BBB by a passive process as an ion, such as Pb-OH+. In vitro studies of brain capillary
endothelial cells also revealed the involvement of the Ca-ATPase pump in the transport
of lead into the brain (Bradbury and Deane, 1993).
1.1.3. Mechanisms involved in lead-induced neurotoxicity: role of
neuroinflammation
Numerous research studies support the hypothesis that lead has an effect on altering the
cellular second messenger systems in neurons and astrocytes (Figures 1.5 and 1.6). The
ability of lead to substitute for Ca2+
the most common second messenger, is one of the
main proposed lead neurotoxicity mechanisms. It has been shown that lead enters
astrocytes and neurons via voltage sensitive calcium channels. Increased depletion of
stored Ca2+
, followed by lead exposure in HEK293 cells or glial C6 cells has been
reported, and suggests the possible of substitution of calcium by lead in these cells
(Bressler et al., 1999b). Perturbation of Ca2+
homeostasis, dysfunction of mitochondria,
[9]
glutamatergic excitability, disintegration of β-tubulin III protein and reactive oxygen
species (ROS) have been reported as several possible mechanisms in metal-induced
neuronal death. However, a recent study showed that the lead or Aβ-induced neurotoxic
mechanism was different to neuronal apoptosis or neuritic degeneration induced by other
metals or neurotoxicants (Fujimura and Usuki, 2012).
Figure 1.5: Lead accumulation, storage and release in astrocytes.
This diagram shows the possible mechanism involved in entry of lead into the cells, binding
with intracellular organelles, and neurotoxicity. Source: Tiffany-Castiglioni and Qian, 2001.
It has been shown that neuritic degeneration induced by exposure to 10 µM lead was not
associated with neuronal cell death (Fujimura and Usuki, 2012b). Lead can also
negatively affect the survival of neurons through changes in expression of neuronal cell
adhesion molecules. It has been well-documented that in the brain, lead interacts with a
variety of molecules involved in cell signalling pathways. Lead exposure has been shown
to stimulate the ERK1/2 and p38 MAPK
pathway in the hippocampus of the immature rats
in both in vivo and in vitro (Cordova et al., 2004). Induced apoptosis and alteration in
neurotrophic factors by lead has also been reported in the developing rat brain (Chao et
al., 2007). The ability of lead to substitute for zinc may also contribute to
neurodevelopmental toxicity. Lead can accumulate in the cell nuclei and can interfere
with the functioning of gene regulatory proteins (Hanas et al., 1999). Free zinc
concentration in microglia decreased after being challenged with lead 24 hours earlier,
which correlates with the previously mentioned mechanisms (personal communication).
In astrocytes, induced cytotoxicity and cell activation with enhanced cell detachment and
[10]
nuclear inclusion has been reported after exposure to lead. In addition, lead could
enhance the expression of astrocyte activation markers, glial fibrilary acidic protein
(GFAP) and S-100β protein, and induces astrocyte activation (Struzynska et al., 2007b).
Some studies suggested that lead may induce TNF-α expression, similar to
lipopolysaccharide (LPS) through activation of the PKC pathways (Cheng et al., 2004).
There is a possibility that lead suppresses the immune response through inhibiting the
signalling pathways involved in microglia activation. However, the exact molecular
mechanisms involved in lead–induced neurotoxicity/neuroinflammation are not
fully understood.
Figure 1.6: Lead interacts with different sites of actions in neurons.
This diagram demonstrates that lead can enter into the neuron through voltage channels, alter the
intracellular Ca2+
balance, affect cell signaling and interfere with the nucleus and damage DNA
and RNA. Source: Finkelstein et al. 1998.
1.1.4. Lead toxicity in animal models
Despite several studies on toxic effects of lead, understanding the underlying
mechanisms have generated much interest over past decades. The differential toxic
effects of lead exposure in young children and adults, as well as, inconsistency between
in vitro and in vivo models, imply the involvement of multiple mechanisms in lead-
induced toxicity. Due to the versatile effects of lead poisoning on human health,
[11]
particularly on nervous system functions, to study lead neurotoxicity using experimental
animal models is a convenient approach. Animal models are the beneficial systems in the
interpretation of behavioural and neurobiological mechanisms involved in observed
deficits in lead-exposed humans (Bellinger, 2004). The role of lead toxicity in the
pathogenesis of several neurodegenerative diseases, including Alzheimer’s disease and
Parkinson’s disease, has also been shown using animal models (Charlet et al., 2012). In
addition, involvement of microglia activation has been reported in inflammation-
mediated neurodegenerative diseases (Liu et al., 2012). Based on recent studies, lead
could induce the glial cell reactivity as a hallmark of brain inflammation. Enhanced
expression of beta amyloid precursor protein by lead has also been suggested as one
possible mechanism underlying the pathogenesis of Alzheimer’s disease (AD) (Monnet-
Tschudi et al., 2006). In vitro and in vivo animal experiments have revealed the dramatic
effects of lead on growth and development of the CNS. Studies in rat and mice show that
absorption of lead in pups is greater than in adults. Reduction in the number of neurons,
glia cells, as well as synapses per neurons was reported in lead-exposed pups. Pro-
inflammatory cytokine gene expression, IL-6 and TGF-β1, increased in the whole brain
of mice exposed to lead (Kasten-Jolly et al., 2011). Increased gene expression of IL-6
and TGF-β1 during development may adversely affect the growth and development of
neurons. In the hippocampus of developing rats, lead caused an increase in apoptosis
through suppressing the expression of apoptotic inhibitor proteins (Liu et al., 2010). Lead
exposure was also found to reduce hippocampal long-term potentiating. Recent studies
showed the developmental effects of lead on behaviour and gene expression patterns in
BALB/c mice. Lead caused significant impairment of spatial memory tested using the
Morris water maze (Kuhlmann et al., 1997). Lead-exposed mice manifested abnormal
exploratory behavior compared to controls in the activity monitor. However, lead showed
no changes in motor function (Gilbert et al., 2005). Lead differentially affected the
behavior of male and female mice. Lead-exposed males showed more violent behavior
towards the same cage mates (Gilbert et al., 2005). At the gene expression level,
developmental lead-exposure showed perturbation in pathways associated with region
formation in the developing brain. Lead has a high impact on genes related to the
immune system; for instance, increased transcription of genes for MHC protein and
decreases expression level of IL-10 in the cortex of lead-exposed mice were reported
(Kasten-Jolly et al., 2012). Altered cytokine gene expression patterns, ROS generation
and lipid peroxidation have been reported in developmental lead toxicity at relatively
low, physiological doses (Kasten-Jolly et al., 2011).
[12]
In vitro studies, using the rat PC12 neuronal cells, showed that lead could induce
apoptosis and DNA damage through changes in the expression of apoptotic genes. Lead-
induced neurotoxicity has been reported in a dose- dependent manner in PC12 (Xu et al.,
2006). Furthermore, mitochondrial activity and synaptosomal neurotransmitter decreased
in the lead-exposed rat brain (Devi et al., 2005).
1.1.5. Lead and the immune system
Immune modulatory effects of heavy metals, such as lead, zinc and copper, have been
reported previously, although not much is still known about the underlying mechanisms.
Changes in immune responses following lead exposure have been shown using both
animal and human models. For example, in vitro exposure of murine spleenocytes to lead
(1-100 µM) resulted in the enhancement of both B and T-cell activities (Lawrence and
McCabe Jr, 2002, McCabe and Lawrence, 1990). Interestingly, in this model, higher
levels of lead suppressed immune activity and increased bacterial susceptibility in vivo.
Thus, effects of lead on the immune system may differ depending on lead dosage and
exposure time, as well as the sub-population of responsive immune cells (Lawrence,
1981). Based on in vivo and in vitro studies, lead preferentially enhances Th2 cell
development and affects the development of B cells (Lawrence and McCabe Jr, 2002,
McCabe and Lawrence, 1990). Moreover, in vivo lead acetate treatment inhibits cell
adhesion properties and alters cell morphology in the splenic murine macrophages
(Sengupta and Bishayi, 2002). Gao and colleagues (2007) showed that lead can modulate
the development and function of the murine bone marrow derived dendritic cells. In
addition, lead-exposed dendritic cells skewed the immune response from Th1 to Th2
responses (Gao et al., 2007). It has been suggested that systemic immunosuppression,
which is characterized by a shift towards a T-helper cell type 2 (Th2) cytokine pattern, is
beneficial in the context of CNS lesions, as it results in reduced cell mediated and
humoral immune responses (Hendrix and Nitsch, 2007). Animal studies also showed that
lead may affect the immune-nervous system interaction through altering the
immunogenicity of two neuronal proteins, glial fibrilary acidic protein (GFAP) and
myelin basic proteins (MBP). Secretion of auto-antibodies against these proteins which
could enhance the progression of lead-induced neurotoxicity through auto-immunity
responses. Recent studies on human peripheral blood monocytes (PBMC) showed that
lead can activate IL-8 secretion, as well as induce secretion of pro-inflammatory
cytokines, such as IL-6 and TNF-α, though the mitogen-dependent activated pathway
(MAPK) (Gillis et al., 2012).
[13]
1.2. Microglia
Microglia are the resident macrophages of the brain and are highly responsive with key
functions in the host’s defense, as well as in neuroprotection and regeneration. Microglia
were first identified in the late 19th century. In 1899, the psychiatrist Franz Nissl came
across rod shaped cells in the CNS and described them as reactive glial elements
(Buchholz, 1899). Then, Santiago Ramón y Cajal in 1913 determined that glial cells were
the third major element of the CNS, in addition to neurons and astrocytes (Cajal, 1913).
However, Pio del Rio-Hortega, a Spanish neuroanatomist, was the first person to
determine that there were two types of glial cells, oligodendroglia and microglia, based
on morphological differences using silver- staining methods in 1932. He described
several fundamental characteristics of microglia that are still accepted and cited in
current microglia research (Del Rio-Hortega, 1932).
1.2.1. Origin
The origin of microglia has been debated for more than a century. Most researchers agree
that microglia have a myloid origin. Recently, Ginhoux et al (2010) showed that during
the embryonic and perinatal development of mice, two hematopoietic waves of microglia
are recruited to the CNS, where they differentiate. Ginhoux et al (2010) also reported that
myeloid cells expressing the hematopoietic marker CD45 and the adult
macrophage/microglia markers CD11b, F4/80, and CX3CR1 were detectable in the
developing brain starting from E9.5.It has also been reported that the primitive myeloid
progenitors have limited potential to give rise to adult blood leukocytes. Current evidence
suggests that in mammals, microglia are derived from primitive myeloid progenitors that
arise before embryonic day 8 in mice (age E8.0) (Ginhoux et al., 2010).
Throughout life, microglia are replenished by limitedproliferation of resident microglial
cells. Replenishment by bone marrow-derived progenitor cells is still underdebate.
Numerous reports have shown that bone-marrow-derived cells have the ability to
populate the CNS and differentiate into functional parenchymal microglia as well as
perivascular microglia (Soulet and Rivest, 2008, Simard and Rivest, 2004). Although
bone-marrow-derived cells can enter the brain parenchyma and throughout the CNS in
normal mice, studies using a panel of bone-marrow chimeric and transfer experiments
have shown that Ly-6Chigh
CCR2+ monocyte were preferentially recruited to the lesioned
adult brain and differentiated into microglia (Mildner et al., 2009). It has also been
reported that microglia and astrocytes chemokines could regulate monocyte recruitment
[14]
through the BBB in human immunodeficiency virus-1 encephalitis (Persidsky et al.,
1999).
1.2.2. Turnover
The turnover rate of microglia has been the subject of multiple studies since their
identification. Several studies have reported an amplified number of microglia during
pathological conditions. This may be due to the accelerated turnover in order to
compensate for the need of new cells. Investigators believe that microglia are a long-
lived population of macrophages, but how these cells are maintained during different
conditions is still unknown (Ransohoff et al., 2007). Two subsets of phagocytes have
been found in the adult brain: resident microglia in parenchymal microglia and
perivascular macrophages. Studies have shown that under healthy conditions, the
microglial replacement rate is low. However, during injury or inflammation, the turnover
rate of microglia is increased. Supplementation of resident populations of microglia with
bone-marrow derived cells has been seen in several CNS injury models. It has been
suggested that this phenomenon may also occur in the normal CNS, but at a very low rate
(Simard et al., 2006, Ajami et al., 2007).
1.2.3. Distribution
Microglia make up approximately 5-20% of the cells in the CNS (Lawson et al., 1990,
Benveniste, 1997, Tambuyzer et al., 2009). Based on animal and human studies, there
seems to be a differential distribution of microglia in separate regions of the brain. The
percentage of microglia in the healthy human brain show significant regional differences
ranging from 0.5% to 16.6% in the brain parenchyma with significantly
more microglia in white than in gray matter (Mittelbronn et al., 2001). In adult mice,
microglia are predominant in grey matter, with the highest concentration found in the
hippocampus, olfactory telencephalon, basal ganglia and substantia nigra (Lawson et al.,
1990).
1.2.4. Morphology
In the healthy brain, resting microglia manifest a typical ramified morphology, consisting
of a small cell body and long slender processes with secondary branching. In response to
stimuli, such as injury, toxins or inflammation, the processes are retracted and the cells
become more amoeboid-like, and transform into an activated phenotype (Figure 1.7)
(Xiang et al., 2006) . Del Rio-Hortega has also described the amoeboid cells showing
phagocytosis and migration similar to macrophages (Del Rio-Hortega, 1932). Resting
[15]
microglia with ramified morphology act as the gatekeepers of the brain and they restrict
changes or insults through constant surveying of the brain’s microenvironment. Recent
studies, using in vivo two-photon imaging in the neocortex, have reported that resting
microglia exhibit very dynamic processes that project and retract extensively to survey
and control the local environment (Kofler and Wiley, 2011, Nimmerjahn et al., 2005).
There is evidence showing that microglia activation occurs in response to recognition of
foreign pathogens, serum components, intercellular structures, stress, protein aggregates,
or receptor signaling pathways (Prinz et al., 1999). In the context of receptor signaling,
neuron-microglia interaction are involved in modulating the microglia phenotype and in
maintaining the ramified morphology. Disturbance in expression of some neuronal
ligands, such as fractalkine, CD200 and CD47, and reduced interaction with their
microglia receptors are associated with activated morphology (Lyons et al., 2009, Walker
et al., 2009).
Figure 1.7: Microglia is the immune effectors cells in the brain.
In the CNS, quick responses to stimuli results in activation of resting microglia, which is followed
by changes in function and morphology, from, ramified to amoeboid. Activated microglia increases
the expression of MHC molecules, enhances secretion of pro- and anti-inflammatory cytokines and
chemokines, and become potent APC as well as phagocytes. In the brain, activated microglia play
either a neuroprotective or neurotoxic role depending on the immune stimulation and secretions.
Source: Block et al., 2007, Trudler et al., 2010.
[16]
1.2.5. Microglia function: Immune effectors of the CNS
A quick response to any insult that disturbs the CNS homeostasis leads to microglia
activation, which results in changes in microglia morphology (from ramified to
amoeboid) and function. Based on recent published evidence, several surface molecules
expressed by microglia are involved in recognition of antigens, non-self or stressed-self
structures, phagocytosis, and migration, as well as pro- and anti-inflammatory responses,
which then stimulate the immune system (Figure 1.8). Like macrophages, microglia have
a wide range of receptors that are present on their surface which are responsible for the
detection of damage, associated with molecular pattern molecules (DAMP), which are
released by injured neurons or degenerating cells. Heat shock proteins, stress, oxidized
lipids, DNA, ATP and high mobility group box-1 (HMGB1) proteins are the main known
DAMPs. The receptors that detect DAMPs are scavenger receptors, Toll-like receptors
(TLRs), and receptors for advanced glycation end products (RAGE). TLRs play an
important role in recognition of the pathogen-associated pattern molecules (PAMP). TLR
signaling plays an important role not only in the innate immune system response, but also
in inflammation and demyelinating disorders. Ten TLRs have been identified in humans,
which can be expressed by microglia in the brain (Olson and Miller, 2004). Each member
of the TLR family recognizes different pathogens. TLR4 is the most studied member of
the Toll-like family, which is responsible for the recognition of LPS and the outer
membrane component of gram negative bacteria. The LPS-TLR4 interaction induces
activation of several intracellular signaling pathways, which initiate immune responses
through the secretion of pro-inflammatory cytokines (Luna et al., 2012, Kawai and Akira,
2011). Microglia express many other surface receptors, including phosphatidyl serine
(PS) receptor, LPS receptor CD14, the scavenger receptor CD36 and the purine receptor
P2Y6, which interact directly with the target to initiate phagocytosis (Hirt and Leist,
2003, Jones et al., 2012, Liu et al., 2005, Koizumi et al., 2007).
Microglia are in close contact with their cellular neighbours: neurons, astrocytes and
oligodendrocytes (HANSSON and RÖNNBÄCK, 2003). These connections have
significant effects on the microglia phenotype and function through receptor-ligand
interaction. There are a growing number of known molecules expressed or secreted by
neurons that bind to receptors on microglia. Fractalkine is one of these molecules which
is secreted by neurons and can modulate microglial activation through its receptor,
CX3CR1.
[17]
Figure 1.8: Chemo taxis or phagocytosis-involved receptors in microglia and correlation of the inflammatory or anti-inflammatory response.
Various receptors expressed by microglia are correlated with migration, phagocytosis, pro- and
anti-inflammatory responses. Source: Noda and Suzumura, 2012.
Microglia as the resident immune cells of the CNS have ability to act as an antigen-
presenting cell (APC) to stimulate the immune system through processing and presenting
antigens to T-cells. Previous studies provided evidence showing that constitutive MHC
class II molecules (HLA-DR) are expressed by resting microglia (Gehrmann et al., 1993).
Several studies have shown that microglia activation in response to neuronal damage,
infection, or inflammation is usually followed by increased expression of MHC class II,
as well as adhesion/co stimulatory molecules ICAM-1, CD40, CD80, and CD86. In
response to interferon-gamma (IFN-γ) and/or LPS stimulation in vitro, microglia are able
to prime allo-reactive T-cell responses and stimulate T-cell lines to proliferate and
secrete cytokines (Becher and Antel, 1996, Aloisi et al., 1998, Williams et al., 1993).
Microglia can be regarded as more efficient APC of the CNS than astrocytes for the
stimulation of both Th1 and Th2 cells (Aloisi et al., 1998).
The phagocytic ability of microglia plays a key role during development, as well as
during injuries and inflammation. Depending on the engulfed particles and the receptors
involved, phagocytosis can occur together with secretion of pro- or anti-inflammatory
mediators. During bacterial invasion, the activation of receptors, such as TLRs, induces
the secretion of pro-inflammatory cytokines, as well as active phagocytosis. However,
during debris removal, microglia secretion moves towards the anti-inflammatory
molecules (Hanisch and Kettenmann, 2007, Hanisch et al., 2001, Chan et al., 2001,
Magnus et al., 2001).
[18]
1.2.6. Role of microglia: neuroprotection versus neurodegeneration
The role of microglia in neuropathology is dichotomised into neurodegeneration and
neuroprotection (Ransohoff et al., 2007). There is increasing evidence that microglia
activation can either enhance or decrease neurogenesis depending on the stimuli and
situation (Gemma et al., 2010, Morrens et al., 2012). Microglia are important in synapse
remodelling and plasticity during development. The central nervous system is remarkable
for the large amount of developmental apoptosis that occurs during embryogenesis.
Microglia have been shown to play an important role in inducing developmental neuronal
apoptosis. Recent studies have demonstrated the important contribution of microglia to
synaptic remodelling. Microglia also play a role in synaptic striping in the pathogenesis
of neurodegenerative disorders (Perry and O'Connor, 2010).
A considerable amount of evidence supports the occurrence of adult neurogenesis in the
mature brain. One piece of supporting evidence for this view is the existence of neural
stem cells (NSCs) in the mammalian central nervous system. Microglia can promote
adult neurogenesis through the induction of NSCs proliferation, differentiation and
migration (Ekdahl et al., 2009).
It has been shown that disturbances in neurogenesis are also involved with induced
neuronal apoptosis as a result of injuries or inflammation during neurodegenerative
disorders. Notably, recent collective data indicate that neurogenesis is controlled by
endogenous factors such as cytokines, chemokines, neurotransmitters and reactive
oxygen species (ROS) that are released by activated microglia, astrocytes and injured
neurons (Whitney et al., 2009). It has been demonstrated in animal and human models
that microglia activation plays a critical role in the progression of various
neurodegenerative disorders, such as Parkinson's disease (PD), Alzheimer's disease (AD)
and Huntington's disease (HD), as well as neuro-inflammatory disorders including AIDS,
encephalitis and multiple sclerosis (Sugama et al., 2009, Nelson et al., 2002, McGeer and
McGeer, 2002).
1.2.7. Cytokines and neuroinflammation
Increased secretion of pro-inflammatory cytokines is one of the typical characteristics of
activated microglia, which induces an inflammatory reaction in the brain. Indeed, it is
well established that inflammation and activated microglia could play a positive role in
neurogenesis during brain injuries, such as brain ischemia. Interestingly, several
cytokines can induce both pro- and anti-inflammatory responses. For example, IL-6 has
[19]
the ability to induce NSC differentiation. On the other hand, an elevated level of IL-6 has
a dramatic effect on attenuating neurogenesis (Tang et al., 2009, Vallieres et al., 2002).
Opposing dual effects on neurogenesis has also been reported for IFN- (Song et al.,
2005). TNF- is a potent pro-inflammatory cytokine, mainly secreted by activated
microglia, and to a lesser extent, by astrocytes or neurons. Increased levels of TNF-
have been reported for many neurodegenerative disorders. Both, stimulatory and
inhibitory effects of TNF- have been shown in proliferation, differentiation and
migration of neural precursor cells (Ben-Hur et al., 2003).
1.3. Lead and microglia
Despite the importance of microglia in the physiology and pathology of the brain, there is
not much known about the exact role of microglia in lead-induced neurotoxicity. Recent
studies have raised the idea that microglia and astrocyte activation may be involved in
lead- induced neurotoxicity (Liu et al., 2012, Struzynska et al., 2007). However, most
research data have been obtained from in vitro and in vivo animal models, which may not
be the ideal models, especially in the context of the cellular immunity. To my knowledge,
there are no published data about the contribution of human microglia in lead-induced
toxicity. It is noteworthy that most of the studies on human microglia have been done
using fetal brain samples, post-mortem material and two non-commercialized cell lines.
In addition, the dosage of lead used in several studies was much higher than the level, the
human body may be exposed to in vivo. Recent studies found that lead-exposure induced
microglia activation was followed by the release of TNF-, IL-1β and enhanced
expression of inducible nitiric oxide syntase (iNOS) in vitro (Liu et al., 2012). Lead also
enhanced neuronal injuries in co-cultures of microglia with hippocampal neurons (Liu et
al., 2012). Low dose lead exposure caused considerable reduction in long-term
potentiation (LTP). It has been suggested that lead could impair spatial learning and
memory through the impairment of LTP. Indeed, microglia activation also could affect
LTP. Thus, in this study microglia activation has been suggested as a possible
mechanism involved in lead -induced LTP impairment (Liu et al., 2012).
In vitro studies have revealed that cytotoxicity and the activation of astrocytes occur
following lead exposure. An increase in densities of lysosomes, glial fibrillary acidic
protein (GFAP) and S 100 protein, along with the enlargement of rough endoplasmic
reticulum (RER), has also been reported. Enhanced detachments of astrocytes in presence
of lead in culture has been reported as well (Tiffany-Castiglioni and Qian, 2001).
[20]
1.4. Role of chemokines and chemokine receptors in CNS development
Chemokines, which are chemoattractant cytokines, and their G protein-coupled receptors
are essential for the selective recruitment of leukocytes to inflammatory sites. Besides
chemotaxis, chemokines are also involved in neuronal development, synaptic
transmission, modulation of cell adhesion, phagocytosis and cytokine secretion (Cartier
et al., 2005, Biber et al., 2002). Due to their wide range of biological functions,
chemokine receptors can be grouped as either constitutive or inflammatory (Figure 1.9),
depending on their involvement in development or inflammatory responses (Bennett et
al., 2011). The role of chemokines as neuromodulators is important in the neuron-
microglia interactions, as well as in the coordination of communication between the
nervous system and the immune system.
Figure 1.9: Constitutive vs. inducible chemokine
Chemokine receptors and their ligands can be broadly categorized into two classes depending on
whether they are constitutively produced or are inducible. Constitutive chemokine-receptors play a
basal role in leukocyte trafficking during development. However, inducible chemokine-receptors
are mainly involved in inflammatory responses. Source: Proudfoot, 2002.
There is increasing evidence that cytokines and chemokines in the CNS are involved in
neuronal development by regulating proliferation, survival, migration and differentiation
of the neuronal stem cells under physiological and inflammatory condition (Table 1.1).
Studies have shown that chemokines released from astrocytes, CCL3 (MIP-1) and CCL5
(RANTES) enhance the rat embryonic neuronal CCR5–mediated differentiation (Park et
al., 2009). Embryonic neuronal differentiation is promoted by the treatment with
[21]
astrocyte-microglia conditioned medium (Park et al., 2009). Chemokines have been
shown to be important in modulating the adult neurogenesis through expression of
chemokine receptors (CCR2, CXCR4) by neural stem cells. During inflammation,
expression of reciprocal ligands, CCL2 and CXCL12 is up-regulated by activated
microglia, as well as injured neurons, and can attract progenitor cells to sites of injuries
for repair (Belmadani et al., 2006, Liu et al., 2007, Miller et al., 2008).
Similar to the haematopoietic factor Granulocyte-colony stimulating factor (G-CSF),
granulocyte -macrophage colony stimulating factor (GM-CSF) have been reported to
have a stimulatory effect on rodent neuronal stem cells during differentiation (Kruger et
al., 2007). The expression of CCR9 and its ligand CCL25 (TECK) has been reported in
PC12 rat cells, a useful model for neuronal differentiation. It has also been shown that
CCL25/CCR9 interaction can promote PC12 survival (Cao et al., 2012).
Recent in vivo and in vitro studies have reported that some chemokines and chemokine
receptors, such as CCL2/CCR2, FRACTALKINE/CX3CR1, CXCL8/CXCR1/CXCR2
and CXCL12/CXCR4, are constitutively expressed in the CNS with roles in the
modulation of neuro-protection and neurogenesis, as well as neuro-inflammation
(Semple et al., 2010). Several studies have shown that along with their involvement in
inflammatory responses, cytokines play an active role in the CNS developmental process
especially in the proliferation, migration and differentiation of neuronal stem cells
(NSCs). Interleukin-4 (IL-4), interleukin-6 (IL-6), leukaemia inhibitory factor (LIF),
transforming growth factor beta (TGF-β), MCP-1/CCL2, GM-CSF and G-CSF have been
shown to be involved mainly in NSCs differentiation. Tumor necrosis factor alpha (TNF-
), LIF and stromal cell -derived factor (SDF-1)/ CXCL12 are considered to be the key
regulators of proliferation. Meanwhile, MCP-1 and SDF-1 play an active role in cell
migration (Taga and Fukuda, 2005, Gonzalez-Perez et al., 2010, Cameron et al., 1998,
Buisson et al., 2003). Like microglia, astrocytes also promote the NSCs differentiation.
Studies showed that microglial IL-6 can enhance the differentiation of astrocytes. On the
other hand, microglia can modulate the NSC differentiation either directly or indirectly,
through the secretion of IL-6 (Nakanishi et al., 2007).
Glia cells, microglia and astrocytes are the main sources of IL-6 in the CNS. However,
recent studies have shown the neuronal expression of IL-6 has a role in physiology and
inflammation. It has been reported that secretion of chemokines, such as IL-8, in
microglia can be regulated by pro- or anti-inflammatory cytokines. Human fetal
microglia showed enhanced secretion of IL-8 in the presence of LPS or pro-inflammatory
cytokines such as IL-1 β. However; IL-8 expression was attenuated when microglia were
[22]
treated with anti-inflammatory cytokines such as Il-4, Il-10 and TGF- β (Ehrlich et al.,
1998). IL-8/CXCL8 is a chemokine which is in humans can bind to both CXCR1 and
CXCR2 receptors. Mouse and rat can not express IL-8 and its receptos due to the
deletion of the genes encoding IL-8 and its receptor CXCR1 in a common ancestor of
mouse and rat, and therefore IL-8 is regarded as unique chemokine in human studies.
Human. IL-8 enhances progenitor cell division, neurogenesis, ventral midbrain
development and increases the number of dopaminergic neurons in the rat brain (Edman
et al., 2008). CXCR1 exclusively binds IL-8 and CXCL6 with high affinity, whereas
CXCR2 can be activated by additional ligands such as CXCL1,CXCL3 and CXLCL5-7.
Activation of the two IL-8 receptors triggers the formation of distinct second messengers,
which activate several signaling pathways through protein kinases and phospholipases.
The main signal transduction pathways induce the transient mobilization of free Ca+2
from internal stores.
For instance, distinct expression of CCL2/CCR2 has been identified during brain
development (Rezaie et al., 2002). CCL2 is mainly secreted by microglia and astrocytes.
Neuronal CCL2 secretion has also been shown to occur following trauma. Enhanced
secretion of CCL2 occurs in response to several stimuli during neuro-inflammation. It
has been shown that CCL2 could induce neuronal precursor migration to the site of
inflammation, and may be involved in neurogenesis (Semple et al., 2010).
Increased neuronal death has been reported in CCL2/CCR2 knock-out animal models of
methyl mercury neurotoxicity which implies a possible protective role of CCL2 in brain
deficits caused by toxicity (Godefroy et al., 2012). In addition to proliferation,
CCL2/CCR2 is involved in differentiation, as well as cell migration. Recent studies have
shown that treatment of embryonic rat cells with CCL2 and CCL7 enhances cell
differentiation towards dopaminergic neurons (Edman et al., 2008). CCL2 may also act
as the modulator of the blood-brain barrier permeability through interaction with
endothelial cells expressing CCR2 that may facilitate leukocyte trafficking to the brain.
1.5. Role of cytokines, chemokines and chemokine receptors in
neuroinflammation
Neuro-inflammation, neuronal damage and cell death in the CNS is usually accompanied
by the activation of microglia and astrocytes following the secretion of pro-inflammatory
cytokines and chemokines. There is published evidence showing that neuronal damage in
the CNS, after long term exposure to low-level lead can be attributed to the over
expression of TNF-, which is primarily released by microglia and astrocytes (Viviani et
[23]
al., 1998). Recent studies on human glioma cell lines have shown that lead and
liposaccharid, both share common signaling pathways that result in secretion of TNF-
(Cheng et al., 2004). Based on recent published evidence, chemokines and chemokine
receptors are expressed in CNS at low levels under healthy conditions and mediate the
innate immune system. In response to neuro-inflammatory stimuli, their expression is
rapidly induced and this has been reported in various neurological disorders (e.g. trauma,
stroke and Alzheimer's disease), in tumour induction and in neuroimmune-related
diseases, such as multiple sclerosis or acquired immunodeficiency syndrome (AIDS)
(Mennicken et al., 1999). Therefore, they play a key role in the pathogenesis of several
neurodegenerative or neuro-inflammatory diseases. Increased secretion of pro-
inflammatory cytokines and chemokines, such as IL-6, is one of the marked
characteristics of the activated microglia which can result in the inflammatory response,
activating more microglia, as well as astrocytes. Reactive gliosis, demonstrated by an
increase in the number and size of glia cells, has been reported as one of the symptoms of
neurodegenerative diseases ((Rubio-Perez and Morillas-Ruiz, 2012, Sheng et al., 2001,
Mrak and Griffin, 2005, Lee et al., 2013). Over-expression of cytokines may also cause
neuronal cell death through changes to neurotransmitter receptors and neuronal activation
(Kaindl et al., 2012).
[24]
Table 1.1: Chemokine and chemokine receptors in CNS
Chemokine receptor Chemokine
Common name Specific name
CCR1
MIP-1
RANTES
MCP-3
CCL3
CCL5
CCL7
CCR2
MCP-1
MCP-2
MCP-4
MCP-3
CCL2
CCL8
CCL13
CCL7
CCR3
Eotaxin
MEC
RANTES
MCP-4
CCL CCL11
CCL28
CCL5
CCL13
CCR4 TARC
MDC CCL17
CCL22
CCR5
MIP-1β
MIP-1
RANTES
CCL4
CCL3
CCL5
CCR6 MIP-3 CCL20
CCR7 SLC CCL21
CCR9 MEC CCL28
CXCR1 IL-8
GCP-2
CXCL8
CXCL6
CXCR2
NAP-2
ENA78
GRO-
GRO-β
GRO-
IL-8
CXCL7
CXCL5
CXCL1
CXCL2
CXCL3
CXCL8
CXCR3
IP-10
MIG
I-TAC
CXCL10
CXCL9
CXCL11
CXCR4 SDF-1 CXCL12
CXCR5 BCA-1 CXCL13
CX3CR1 Fractalkine CX3CL1
[25]
Chapter 2
Research aims and outline
[26]
2. Research aims and outlines
2.1. Aims
The literature review highlighted several potential mechanisms of lead poisoning in
humans. It focused on the current understanding and knowledge about the toxic effects of
lead, mainly on the developing central nervous system via changes in proliferation,
migration and differentiation of precursor cells, as well as possible involvement of
immune cells and inflammatory responses in lead-induced toxicity.
The literature review also underlined the key roles of microglia as the resident immune
cells of the brain during health and disease. In the context of microglia, more attention
was on the contribution of cytokines and chemokines as the immune modulator of the
CNS in both, neurogenesis and neuroinflammatory responses. The chemokine-
chemokine receptor axis has been shown to play an active role in the microglia- neuron
interaction.
Most of the data about the biology and function of microglia are gathered from in vitro
studies using the primary culture of embryonic or neonatal murine brain or established
cell lines. In humans, primary culture of microglia is not easily feasible due to ethical
reason and lack of sources. On the other hand, fetal microglia may behave differently in
some aspects from adult microglia. In addition, few established human cell lines have
been generated. Therefore, lack of a convenient and appropriate model for in vitro human
microglia studies is one of the current challenges for research into the human microglia.
It is well documented that lead exposure, even at low concentrations, can be associated
with deficits in neurogenesis and spatial learning abilities in adulthood through
inflammatory reactions.
Activation of microglia as the immune effectors of the brain has been reported in several
neurodegenerative diseases, including heavy metal toxicity. There is evidence showing
that a number of heavy metals can induce microglia activation followed by secretion of a
variety of immune modulators. In most studies, metal-induced microglia activation
enhanced the neuorinflammatory/neurodegenerative responses. Despite the reported
neurotoxic effects of lead, especially in children, little is known about the role of
microglia as the immune resident of the CNS in lead-induced
neurotoxicity/neuroinflammation. Therefore, in this thesis, I investigate the role of
[27]
microglia in lead-induced neuroinflammation using in vitro human models.The overall
aims of this thesis were to test the following hypotheses (Table 2.1).
Table 2.1: Testing hypotheses of microglia involvement in lead-induced neurotoxicity
In this thesis, several experimental studies were developed and designed to obtain
information to answer the hypotheses above. Main aims addressed in this thesis are
shown below (Table 2.2).
Table 2.2: The main aims of this thesis
Aim 1
Determine the effect of lead exposure on the in vitro human
neural precursor cells, with specific focus on immune-related
factors.
Aim 2
Establish and characterize the novel in vitro human microglia
model derived from human blood monocytes.
Aim 3 Study the effect of lead on the in vitro human microglia model
in the context of morphology, phenotype and function.
Aim 4
Investigate the effect of lead exposure on patterns of chemokine
and chemokine receptor expression in microglia in vitro
models.
Hypothesis 1
Lead has cytotoxic effects on survival and morphology of in
vitro microglia models.
Hypothesis 2
Lead activates microglia towards enhanced inflammatory
responses.
Hypothesis 3
Lead does not have a substantial effect on human neuronal
precursor cell in vitro.
[28]
2.2. Research outline and methodology
Several qualitative and quantitative tools and techniques, as well as support and
experience of experts were employed. Table 2.3 summarizes the main techniques applied
in this thesis. This study will provide new insights about the role of microglia in lead-
induced inflammation using the feasible and convenient in vitro human microglia model.
Knowledge on the effects of lead on the cytokine/chemokine receptor interactions in
microglia will provide new approaches to understand the role of lead as one of the known
environmental pollutants in modulating the immune responses in brain.
Table 2.3: Main methods applied in this study and the corresponding chapters.
Chapters Methods
Chapters 4, 5
and 6
Separation and isolation of the blood mononuclear cells
(PBMC) using human blood buffy coats
Chapters 3, 4,
5, 6 and 7
In vitro culture of the human blood monocytes, lymphocytes,
dendritic cells, microglia, human microglia and neuronal cell
lines
Chapters 3, 5
and 7
Viability test (MTS assay, SYTO-green and Propidium iodide
staining)
Chapters 3, 5
and 7 Proliferation test (BrdU Assay)
Chapters 3, 4,
5 and 7
Morphological studies ( phase-contrast and confocal
microscopy)
Chapters 3, 4,
5,6 and 7
Phenotype characterization using the flow cytometry and
immune-histochemistry staining
Chapter 4 Functional assay (phagocytosis, mixed leukocyte reaction -
MLR)
Chapters 5
and 7
Intracellular lead uptake using Leadmium green staining
Chapters 3, 5
and 7
Cytokine measurements
In order to investigate the proposed aims of this thesis, five separate studies were
undertaken. Each of these make-up their own chapter and each consists of their own
background information, methods, results, discussion, conclusion and references (Figure
2.1).
[29]
Figure 2.1: Research outline
To investigate the proposed aims, five separate studies were undertaken. The results of these
studies are presented in five papers found in this thesis.
[30]
Chapter 3
Effect of lead on human cortical neuronal
precursor cells
[31]
3. Effect of lead on human cortical neuronal precursor cells
3.1. About the paper
Lead poisoning remains a significant global health problem due to causing cognitive
deficits and neurological problem in children even at lower concentrations. In human,
brain formation begins in the third gestational week with the differentiation of neuronal
precursor cells (NPCs) and continues to develop in the first years of life. Therefore, brain
at the early time of development is sensitive to exposure of toxicants such as lead, which
can affect further developmental, and maturation processes. Despite the fact that lead
exposure can occur both during prenatal or postnatal exposure, it is still little known of
effect of lead exposure on neural precursor cells morphology and function, or to what
extent development and maturation of neuronal precursor cells are affected by lead.
There is evidence showing that lead exposure can differentially affect the proliferation
and differentiation of neuronal precursor cells originating from different region in brain.
Recent animal studies showed that, despite the region where neural precursor cells
originate from, the age of the animal play a key role in the degree of sensitivity to lead.
For example, hippocampal neuronal stem cells from newborn rats were more vulnerable
to the toxicity of lead than those from adult rats. Lead also could alter the differentiation
of neural stem cells towards oligodendrocytes or astrocytes. In summary, these data
implicate the importance of lead exposure prevention during brain development. One
suitable model to study the neurotoxic effects of metals such as lead, on neuronal cell
morphology and function is using a human neuronal precursor cell line. In this study, we
used the human cortical neuronal cell line. Undifferentiated HCN2 cells have been
reported to express several neuronal markers including neurofilament protein, neuron
specific enolase (NSE), β-tubulin III, vimentin, somatostatin (SST),etc. However, cells
have been reported to be negative for glial markers, such as glial fibrillary acidic protein
(GFAP) and myelin basis protein (MBP), which make them a suitable model for
investigating human neuronal precursor cell biology.
Neural precursor cells, due to their unique capacity for self renewal and multipotency,
can give rise to neurons, astrocytes and oligodendrocytes during development. Over the
past decades, it became evident that neurogenesis occurs in distinct areas of the adult
brain, where neural stem cells, progenitors and precursors reside. Alteration in
proliferation and differentiation of neuronal precursor cells (NPCs) has been reported in
impaired adult brain cognitive function, as well as in the pathology of neurodegenerative
[32]
diseases. In addition, it became apparent that the immune system plays a key role in
modulating the CNS function. Recent evidence shows that microglia have the capacity to
influence the differentiation of both, adult and embryonic neural precursor cells toward a
neuronal phenotype emphasizing the importance of the microglia- neuron interaction.
In order to answer the main question of this thesis, which is the role of microglia in lead
induced neurotoxicity, we investigated toxic effects of lead on the human neuronal cell
line HCN2 over 24 hours in vitro. The results of this study showed that lead at the
concentration of 10 µM had no detrimental effects on morphology or viability of these
cells. Reduction in the number of cells was observed in the presence of 100 µM lead
acetate. However, lead significantly altered the expression of a number of chemokine
receptors and cytokines. Given that the key role of the immune system in the
differentiation and migration of NPCs, these results indicate that immunomodulatory
effects of lead may have an impact on HCN2 maturation and function as well as their
interaction with other cells in the CNS.
Declaration of the work:
All the experiments were designed and conducted by SE involving cell culture,
proliferation assay, viability assay, morphology study, surface marker expression
(FACS), cytokine measurement and statistical analysis under supervision of Prof. Luis
Filgueira.
3.2. Abstracts
Lead is a developemental toxicant with no known physiological function. Lead toxicity
has been recognized for at least thousands of years with diverse harmful effects on
human body. It has been found that early childhood exposure to excessive amounts of
lead may produce lasting adverse effects upon brain function. Although, several studies
reported the negative effects of lead on adult neuronal cells, not many studies have been
investigated the effects of lead on human neuronal precursor cells.
In this study, the effects of lead on human neural precursor cells in vitro at
concentrations, which are expected to be found during lead intoxication in vivo were
investigated. For that purpose, the human cortical neuronal cell line HCN2 was used as a
model. First, we studied the expression of neuronal-restricted precursor marker such as
nestin, vimentin as well as β-tubulin III and NGF-R as developmental-dependent
maturation marker. After 10 days culture in standard medium, HCN2 were exposed to 1,
10 and 100 µM of lead acetate for 24 hours. Then, changes in morphology, viability,
[33]
chemokine receptor expression and cytokine secretion were investigated. Our result
showed that lead at lower concentration (1 and 10 µM) had no effect on morphology and
viability of HCN2 compared to negative and positive controls. HCN2 under non-
stimulatory control conditions expressed most of the chemokine receptors. Alteration in
expression pattern of chemokine receptors has been seen in response to lead exposure.
These changes included significant down regulation in pro-inflammatory chemokine
receptors CCR1, CCR2, CCR3 and CCR5. However, expression of CCR4, CXCR1 and
CXCR3 significantly increased in lead-treated HCN2. Interestingly, we have found
contrary effects of lead exposure on the expression pattern of neuronal fractalkine
(CX3CL1) and its receptor (CX3CR1). HCN2 at basal condition expressed fractalkine
and CX3CR1 at low and medium levels. After lead exposure, expression of CX3CL1
was enhanced and of CX3CR1 decreased. Beside the chemokine receptors, β-tubulin III,
neuron-specific marker showed significant reduction in lead-exposed HCN2, including a
reduction in differentiation potential of the cells. We have also investigated the effect of
lead on secretion of cytokines by HCN2. Of all cytokines tested, only IL-8 and IL-6 were
secreted by HCN2 at basal culture conditions. However, lead significantly reduced
secretion of IL-6, but had no effects on IL-8. In summary, lead (10 µM, 24 hrs) had no
cytotoxic effects on human neural precursor cells. However, lead changed functional
properties of the cells, indicating that lead may interfere with neuronal turnover,
recruitment and differentiation.
Keywords: human neuronal cell line, HCN2, lead, neuronal precursor, chemokine
receptor
3.3. Introduction
Lead is a xenobiotic metal with no apparent reported essential function in cell growth,
proliferation or differentiation. Lead exposure has devastating effects on the developing
nervous system, producing morphological, cognitive, and behavioural deficits.The
neurotoxicity of lead, even at blood concentrations as low as 10 µg/ dl (~ 0. 5 µM), has
been repported in children (Finkelstein et al., 1998). Despite the well-documented
neurotoxic effects of lead, related mechanism are not fully understood. Lead affects
numerous biological activities, including mechanisms at cellular, intracellular and
molecular level (Finkelstein et al., 1998, Zurich et al., 2002, Lidsky and Schneider,
2003). Lead crosses the blood-brain barrier and causes behavioral alterations, and may
even disrupt the main structural components of the BBB (Bressler et al., 1999b). The
prefrontal cerebral cortex, hippocampus and cerebellum have been shown to be the
preferred target region in the brain for lead-induced damages (Finkelstein et al., 1998). In
[34]
vitro and in vivo animal and human studies have shown neurotoxic effects of lead,
especially during development, including impaired synaptic plasticity, inhibition of
glutamate receptor activation (Guilarte et al., 1994), apoptosis (Oberto et al., 1996),
learning and cognitive deficits, memory impairment (Xu et al., 2009), exicitotoxicity,
altered neurotransmitter storage and release, mitochondrial dysfunction, loss of neuronal
myelin sheaths, changes in cellular energy metabolism and disturbance of neuronal
growth (Toscano and Guilarte, 2005). There is evidence showing reduced expression of
neuronal cell adhesion molecules (e.g. NCAM), delayed neurite outgrowth and reduced
survival rate of cultured hippocampal neurons after exposure to low level of lead (Hu et
al., 2011). Prospective studies in animal studies have also correlated the elevated blood
lead level with memory deficits associated with changes in neuronal adhesion molecules
in the hippocampus (Murphy and Regan, 1999). Neural cell adhesion molecule (NCAM)
plays an important role during neural development and in the adult brain (Boutin et al.,
2009). NCAM are also shown to be involved in synaptic plasticity and long term
memory formation in adults. Attenuation in expression of NCAM along with memory
deficits and changes in synaptic plasticity has been shown in animal models (Welzl and
Stork, 2003, Baydas et al., 2003). Previous studies also reported the morphological
changes in hippocampus, as well as long lasting decrease in density of cholinergic
neurons in rats exposed to lead at concentration less than 200 µM prenatally (Slomianka
et al., 1989).
In the context of sensitivity of neurons and glia cells to lead, different experimental
models have been used resulting in various ideas about lead toxicity. Holtzman et al.
(1987) showed that that lead induced neuronal necrosis in rat neuronal culture, while no
effects on astrocyte organelle morphology was seen. They reported that neurons seemed
to be more sensitive to lead toxicity than astrocytes. They also suggested that astrocytes
are able to sequester lead as protective mechanisms (Holtzman et al., 1987). On the other
hand, Zurich and colleagues showed that enriched culture of neurons store more lead
than astrocytes after 25 days exposure to 1µM lead. Moreover, they showed that glia are
more affected than neurons in glia-neuron culture (Zurich et al., 1998).
Although in mammals the process of neurogenesis is almost terminated after
development, neurogenesis occurs throughout the life in two regions in the hippocampus
(Zhao et al., 2008). Several studies showed a positive correlation between adult
hippocampal neurogenesis and memory function. Changes in hippocampal neurogenesis
have been reported in several neurodegenerative disease pathology and age-dependent
memory-learning deficits (Voloboueva and Giffard, 2011).The hippocampal region is
[35]
known to accumulate lead, which has been experimentally proven in animals or in
environmentally exposed humans (Sandhir et al., 1994). Recent studies reported that
perinatal exposure to even low level of lead with corresponding low blood lead
concentrations (below 10 µg/dl), decreased the number of hippocampal neurons with no
changes in apoptotic or necrotic numbers indicating that there may be reduced
neurogenesis (Baranowska-Bosiacka et al., 2012).
Several studies have reported the link between pro-inflammatory cytokines as immune
modulators and neurogenesis. There is evidence showing that inflammation could play a
positive role in neurogenesis during brain ischemia (Tang et al., 2009). Elevated levels of
pro-inflammatory cytokines with enhanced neurogenesis has also been reported in
Alzheimer disease as a chronic inflammatory condition (Yoneyama et al., 2011). Among
pro-inflammatory cytokines, IL-1 and IL-6 showed inhibitory effects on neural stem cells
(NSCs) proliferation or neurogenesis. Although, the ability of the cytokine to induce
NSC differentiation to astrocytes has also been shown (Vallieres et al., 2002, Wang et al.,
2007). Based on in vivo and also in in vitro studies, higher levels of pro-inflammatory
cytokine, such as TNF-α, IL-1β, IL-6, IL-8, monocyte chemoattractant protein-1,
macrophage inflammatory protein-1 and CXCL10, are involved in HIV-1-induced
neuropathogenesis (Guha et al., 2012, Kelder et al., 1998).
Growing evidences suggest that IL-8/CXCL8, pro-inflammatory cytokine/chemokine,
and its receptors, CXCR1/2 play a key role in modulating the neuronal function. The
neuroprotective role of IL-8 in the AD pathogenesis has recently been reported. IL-8
inhibits Aβ-induced neuronal apoptosis and up-regulates neuronal BDNF production
(Ashutosh et al., 2011).Up-regulation of IL-8 has been shown in AD brain tissue.
Increased neuronal secretion of IL-8 has also been reported in the presence of both Aβ
and pro-inflammatory cytokines such as IL-1β or TNF-α (Ashutosh et al., 2011). In
contrast, overexpression of IL-8 could induce the pro-apoptotic proteins and cell death in
AD models (Thirumangalakudi et al., 2007). IL-6 is another pro-inflammatory cytokine
shown to be involved in modulating neurogenesis. Both IL-6 and the IL-6 receptor are
expressed in the postnatal hippocampus (Vallieres and Rivest, 1997), overexpression of
IL-6 in the hippocampus suppresses hippocampal neurogenesis (Vallieres et al., 2002)
and complete lack of IL-6 attenuates the proliferation and survival of neuronal precursor
cells (NPCs) (Bowen et al., 2011). TNF- and IFN- are the pro-inflammatory cytokine
involved in neuronal precursor cells survival and differentiation during neurogenesis or
neuronal remodelling. TNF- is expressed predominantly by activated microglia and at
lesser extent by astrocytes and neurons. Several studies showed the role of TNF- in
[36]
proliferation and differentiation of newly generated cells in the CNS. TNF- showed
inhibitory effects on NSCs proliferation but stimulating its migration in the new born rat
(Keohane et al., 2010, Turbic et al., 2011). IFN-γ-induced neural differentiation and
neurite outgrowth has also been shown in murine neural precursor cells (Song et al.,
2005).
Chemokine and chemokine receptors play an important role in CNS health and pathology
(Bacon and Harrison, 2000, Tran and Miller, 2003). Studies showed that various
chemokine and chemokine receptors are expressed at different levels by neurons during
development, which implicate their role in development, differentiation and even
inflammation through recruitment of leukocyte to the site of injuries (Tissir et al., 2004).
Furthermore, the chemokine/receptor axis play a key role in the neuron-microglia
interaction in the brain in both, neurogenesis and neurotoxic pathway. Chemokines have
been shown to be involved in regulating the migration of NSCs and NPCs (Belmadani et
al., 2006). Expression of a number of chemokine and chemokine receptors have been
reported in rodent and human neurons during physiology and pathology of the brain
(Robin et al., 2006, Losy and Zaremba, 2001, Abrous et al., 2005). A recent study
demonstrated that the CXCR1 and CXCR2 transcripts are widely expressed in
glutamatergic, GABAergic, and cholinergic neurons in the rat brain where they may
modulate transmitter release or ion channel activities (Puma et al., 2001). CXCR4, alike
CX3CR1, is one of the chemokine receptors expressed constitutively in the brain
(Hesselgesser and Horuk, 1999). It has been shown that neurons and astrocytes
expressing CXCR4 in the cerebral cortex, hippocampus, and substantia nigra frequently
coexpress CCR2 (Coughlan et al., 2000, Van Der Meer et al., 2001) which has been
shown to play critical roles in the migration of the NPC during neurogenesis or after
injury.
Among the chemokine/receptors, the fractalkine/CX3CR1 axis was the focus of several
in vitro and in vivo studies in recent decades. Fractalkine is widely expressed in the CNS
with constitutive expression by neurons (Meucci et al., 2000). First, it has been thought
that CX3CR1, the only fractalkine receptor, is expressed by microglia and astrocytes,
considered as one of the main neuron-glia communication in the CNS. Later, studies
showed that CX3CR1 is expressed by neurons, as well. Several studies have shown the
neuroprotective and/or neurotrophic role of fractalkine/CX3CR1 activation in the CNS.
For instance, there are evidences showing fractalkine protects hippocampal neurons from
HIV induced neurotoxicity (Meucci et al., 2000). It has been noted that on one hand,
neuronal fractalkine could supress the migration of neurons by inducing the molecules
[37]
active in cell attachments, such as integrins. On the other hand, it could enhance
microglia migration by means of expressing the neuronal CX3CR1 and facilitate
microglia-neuron interactions (Lauro et al., 2006). Fractalkine is considered for its
therapeutic potential in limiting inflammation, microglial over-activation, and neuronal
injury in neurodegeneration models (Suzuki et al., 2011). CX3CR1 is mostly activated by
soluble fractalkine, which modulates the cell activation and survival through pro-survival
signalling pathway, such as the Akt pathways (Meucci et al., 2000). The neuronal
CX3CR1 receptor could mediate the neurotrophic effects of fractalkine, suggesting that
fractalkine and its receptor are involved in a complex network of both, paracrine and
autocrine interactions between neurons and glia. During the CNS development,
regulation of the neuronal survival and programmed cell death (apoptosis) has a key role
in formation of the neuronal network and different parts of the CNS. It has been shown
that cortex is one of the region which is greatly vulnerable to inflammation or infection
occurred during prenatal or early post natal stages. Published studies showed that LPS-
induced inflammation alters synchronized neuronal patterns and increases apoptosis
(Nimmervoll et al., 2012). Furthermore, decrease in neuronal survival and induced
apoptosis followed by LPS-induced inflammation in CNS has also been a reported effect
of fractalkine and its receptor (Yeh et al., 2008).
Quick and constant up-regulation of neuronal fractalkine along with TNF- as a
biomarker of inflammation, followed by induced neuronal death, has been reported in
patients with epilepsy (Xu et al., 2012). Neuronal expression of TLR2 and TLR4 has
also been reported. Up-regulation, activation of TLRs and down-stream signalling via
JNK has been reported in response to ischemic condition in neurons.
In this study, we used the human cortical neural cell line HCN2 as an appropriate model
for in vitro study of human neuronal precursor cells. HCN2 was derived from cortical
tissue removed from a 5 years old girl undergoing hemispherectomy.These cells are a
really slow growing cell line (doubling time greater than 120 hours).The growth rate of
HCN2 can be stimulated in the presence of phorbol esters (Ronnett et al., 1994). Induced
differentiation in the presence of nerve growth factor (NGF) has also been reported
(Ronnett et al., 1994). In addition, neurotrorphic effects of NGF on neuronal precursor
cell survival and differentiation has been reported (Ronnett et al., 1994). According to
reported studies, HCN2 is positive for several neuronal markers, such as β-tubulin III,
vimentin, somastatin (SST), glutamate, gamma aminobutyric acid (GABA),
cholecytokinin -8 (CCK-8). The cells are negative for glial fibillary acidic proteins
(GFAP) and myelin basis protein (MBP) (Ronnett et al., 1994).To our knowledge, there
[38]
is no study showing the effcts of lead exposure on human cortical neuronal cell line or
HCN2. As the CNS is one of the main targets of lead toxicity and several studies have
reported behavioural and cognitive deficits, as well as neural morphological
abnormalities on developing brain, we were interested in studying the effects of brief
exposure of lead (10 µM, 24 hrs) on HCN2, as an appropriate representitve of human
cortical neuronal precursor in vitro.
3.4. Material and methods
3.4.1. Characterization of HCN2
The human cortical neuronal cell line HCN2 (CRL-10742) was purchased from
American Type Culture Collection (ATCC, Manessa, VA) . Cells were thawed and
cultured in DMEM Glutamax (invitrogen, Carlsbad, CA) supplemented with 10% fetal
bovine serum (FBS, Serana, Bunbury, WA, Australia) and 1% antibiotic/antimycotic
mixed solution (10,000 units/mL of penicillin, 10,000 µg/mL of streptomycin, and 25
µg/mL of Fungizone, Gibco, Grand Island, NY) at 37oC, 5% CO2 . HCN2 is a slow
growing cell line (doubling time greater than 120 hours), therefore, they were maintained
for about 3 weeks after thawing. They did not reach the 100 % confluence (maximum
60%). NGF (10 ng/ml, PeproTech, Rocky Hill, NJ) was also added to culture medium to
induce differentiation and mature morphology in HCN2 (Ronnett et al., 1994). Under
other experimental conditions, we cultured the HCN2 in the presence of fractalkine (10
ng/ml, PeproTech) and LPS (10 ng/ml, E. coli serotype 0111:B4, Sigma -Aldrich, St.
Louis, MO).
Lead acetate [Pb(CH3CO2)2·3H2O] (Sigma-Aldrich) was prepared at the concentration of
1, 10 and 100 µM in medium. After 10 days in culture, HCN2 cells were exposed to
different concentration of lead for 24 hours at 37oC. Then, cells were used for further
characterization and functional studies.
To study the expression of neuronal markers, immunostaining was performed with
HCN2 cultured on cover slips, fixed with 3% paraformaldehyde in PBS, containing 2 %
sucrose for 10-15 min. After permeabilization with 0.1% TritonX100 in PBS for 10 mins,
cells were incubated overnight at 4°C with one of these primary antibodies: a mouse
anti- β-tubulin III (1:100, Stemgent, Cambridge, MA), mouse-anti nestin (1:50, Sigma-
Aldrich) and rabbit-anti ionized calcium-binding adapter molecule 1 (Iba-1) (1:100,Wako
Pure Chemical Industries, Osaka, Japan). Next day, cells were incubated for 1 hour with
Alexa Fluor 488-conjugated donkey anti-mouse and anti-rabbit antibodies (1:200,
[39]
Invitrogen) and DAPI (1:400, Roche, Manheim, Germany). Cells were also stained with
a mouse-anti NGFR (1:50, BD bioscience, San Jose, CA) and rabbit-anti fractalkine
antibody (1:50, AbD Serotec, Oxford, UK) to detect surface expression of tested
markers. Thus, fixed cells were directly incubated with primary antibody without being
perrmeabelized. In the next step, cells were incubated with Alexa Fluor 488-conjugated
donkey anti-mouse as secondary antibody, anti-rabbit Ig G antibodies as isotype control
(1:200, Invitrogen) and DAPI (1:400, Roche) for 1 hour. Stained cover slips were
mounted on slide using the fluorescent mounting medium (Dako, Glostrup, Denmark)
and slides were ready for analysis under fluorescent microscopy. It needs to be pointed
out that . excitation and emission wavelengths of Alexa Fluor-488 and DAPI were
respectively 495, 519 and 350,470.Morphology of HCN2 was studied after 10 days in
culture in the presence/absence of lead using an inverted Leica DM IL phase contrast
microscope. Images were taken with Photometrix Cool SNAP Fx digital camera and
processed with NIS-elements F3.0 software.
3.4.2. Viability assay
Cell viability was analyzed by the MTS assay. When cell reached the optimal confluency
(60%), they were challenged with 1, 10 and 100 µM lead for 24 hours at 37 °C. Then, 20
µl of MTS Aques solution working solution (1:100 from stock solution) was added
directly to each well and cells were incubated for 4 hours at 37°C. In addition, in three
experimental groups, HCN2 were treated with NGF (10 ng/ml, PeproTech), CX3CL1 (10
ng/ml, PeproTech) and LPS (10 ng/ml, Sigma-Aldrich) for 24 hours at 37°C to compare
the effect of these agents with lead treatment at different concentration on cell viability.
Samples were measured with a microplate reader (Multiskan) at 490 nm. Data were
analyzed and results are shown as percentages of cell survival (viability). Non-lead
treated cells viability was considered as 100 in this experiment. Same experimental
conditions (plated cell number, volumes of reagents) were applied in all tests.
Measurements were done in tetraplicate and repeated with three cellular experimental
repeats (n=3).
3.4.3. Surface marker expression
We studied the effects of lead exposure (10 µM) on the expression patterns of specific
surface molecules in HCN2 using flow cytometry. Cells were divided into two groups,
non-treated and lead-treated, which were exposed to lead for 24 hours. After detaching
and washing twice with PBS, HCN2 were stained with direct labelled antibodies, or
using primary unlabelled and corresponding labelled secondary antibodies. A panel of
[40]
directly labelled mouse anti-human antibodies, i.e. CCR1-AF647, CCR2-AF647, CCR3-
PE, CCR4-PE-Cy7,CCR5-FITC, CCR9-fitc, CXCR1-PE, CXCR3-AF488, CXCR4-PE
and CD 271-PE (NGFR p75) (all from BD Bioscience), were added at the supplier’s
recommended concentration. Corresponding labelled isotype control antibodies were
included in all experiments. A rabbit anti-human fractalkine (CX3CL1) and rabbit anti-
CX3CR1 polyclonal antibody was used at the final concentration of 1ug/ml (AbD
Serotec), followed by a secondary donkey anti-rabbit-AF647 used at the final
concentration of 1:200 (Invitrogen). A mouse anti-human β-tubulin III (Stemgent) was
also used at final a concentration of 1:200. In order to detect β-tubulin III intracellularly,
the cells were fixed with paraformaldehyde (PFA) 1% for 10-15 min. Then, they were
incubated with PBS Tween 0.5% as detergent for 10 min. After washing, they were
incubated with anti-β-tubulinIII, diluted in PBS Tween 0.5% for 1 hours, followed by
secondary donkey anti-mouse AF488 at final concentration of 1:200 for 30 min. After
washing twice with PBS-Tween 0.5%, cells were ready for measurement. Cells that were
not labelled with the primary antibody were used as negative control. Excitation and
emission wavelengths of the used fluorochrome were resspectively: FITC: 494, 519; PE:
496/546,578; PE-CY7:496/565,774; APC: 650, 660 and Alexa Fluor-647: 650, 668. The
stained samples were measured with a FACSCallibure (BD Biosciences). Data analysis
was performed using FlowJo software (Tree Star, San Carlos, CA). β
3.4.4. Cytokine production measurement
The cytometric bead array (CBA) human inflammatory kit (BD Biosciences) was used
for the quantitative measurement of the cytokine production and secretion by HCN2 cells
cultured under control and lead-exposed conditions. For that purpose, HCN2 cells were
incubated with lead 10 µM for 24 hours 37oC. The following day, after detaching cells
using cell scraper and washing twice with PBS, cells suspension were centrifuged to
obtain cell-free supernatant. Afterwards, 50-100 µl of the supernatants were collected
and stored at -20oC. The assay was performed according to the manufacturer’s instruction
using 50 µl of the human inflammatory cytokine standards and sample dilution. Cytokine
concentrations were measured using the FACSCanto flow cytometer (BD Bioscience).
FCAP Array version 3 software was used to analyse the data. Measurements were done
in duplicates and repeated with three cellular experimental repeats (n=3).
3.4.5. Statistics
Data were statistically analysed through the SPSS statistics 17.0 software. A one-way
ANOVA and uni-variant linear model were used to compare means. The LSD post-hoc
[41]
test was also used to determine the significant difference between groups. In all the
analysis P<0.05 were considered as statistically significant. Graphs with error bars
represent the standard deviation (SD).
3.5. Results
3.5.1. Characterization of human neuronal cells HCN2
The cortical neuronal cell line HCN2 has been established and reported as convenient
human neuronal precursor in vitro model (Zhang et al., 1994, Ronnett et al., 1994).
However, they have not been often used for research and only few reports have been
published. Therefore, these cells have not yet been characterized thoroughly.
Consequently, we examined the neuronal precursor properties of these cells. For this
purpose, we seeded HCN2 on glass cover slips under standard culture conditions for 7
days to cover 50-60% of the surface. It needs to be pointed out that HCN2 is very slow-
growing cell line and this range was the maximum could be reached over the 7 days.
Following fixation, the cells were stained with different markers specific for neuronal
precursor. Images were taken using the fluorescent microscopy. Nestin and β-tubulin III,
which are the significant markers of neural precursor cell, were highly expressed by the
immature HCN2 cells and decreased after induction of neuronal maturation upon nerve
growth factor (NGF) stimulation (Figure 3.1 A, B and C). We also tested expression of
nerve growth factor receptor p75 (P75 NGFR) and fractalkine. HCN2 expressed both
NGFR and CX3CL1, (Figure 3.1 D and E). No expression of Iba-1, a
microglia/macrophage marker was detectable in HCN2 (Figure 3.1 F). All these results
implicate that cultured HCN2 under basal (non-differentiated) condition show the
neuronal-restricted precursor properties, including expression of neural precursor
markers (nestin, vimentin, β-tubulin III), as well as specific morphology with elaborated
flat polygonal cell body with short neuritic processes. For further characterization, we
also studied the chemokine receptor expression patterns in HCN2 under non-lead treated
condition (Section 3.5.3).
[42]
Figure 3.1: Expression of neuronal markers by HCN2.
Fluorescent microscopy images represent the expression of β-tubulin III (A), nestin (B), vimentin
(C), NGFR (D), fractalkine (E), and Iba-1 (F) in HCN2. Green and blue colours represent the
expression of fluorescent markers and nucleus, respectively. Neural precursor markers (β-tubulin
III, vimentin and nestin) were expressed at higher levels, showing the precursor characteristics of
HCN2. Bar indicates 100 µM (n=3).
[43]
3.5.2. Lead effects on HCN2 morphology and viability
Several animal models have shown neuronal apoptosis and decreased number of neurons
after lead exposure in the developing brain (Tang et al., 2010).To verify whether lead
affects human neuronal cells in vitro, we studied the effect of lead on the viability of
HCN2 using the MTS assay (Figure 3.2). After 5 days in culture, HCN2 were treated
with various concentration of lead (1, 10 and 100 µM) for 24 hours. Viability of the non-
treated cells (control group) was considered as 100% (baseline) and the experimental
groups were correspondingly compared to the control. NGF and LPS were included as
negative and positive controls, respectively.
Interestingly, only high concentration of lead exposure at 100 µM reduced significantly
the viability of the HCN2 cells (P<0.05), whereas low lead concentrations (1 and 10 µM)
increased slightly, but not significantly the viability of the HCN2, similar to our positive
neurotrophic control agents, NGF and fractalkine (P>0.05). However, low lead
concentrations (≤10 µM) did not have a neurotoxic effect on HCN2 cells.
In the context of morphology, un-differentiated HCN2 showed the flat polygonal
morphology with few short-branched processes in the absence of lead. No significant
changes in morphology and number of cells were seen in presence of ≤10 µM
lead(Figure 3.3 A-B), although increases in length and number of branches as well as
typical differentiated morphology of neurons were observed in a number of cells in
culture in higher concentartion of lead (100 µM) (Figure 3.3 C-D).
Figure 3.2: Effect of various concentration of lead on viability of HCN2 using the MTS assay.
Bar charts represent the viability of HCN2
when challenged with 1, 10 and 100 µM
lead for 24 hours (black bars) when
compared to the non lead-treated cells as
negative control (white bar). HCN2 were
cultured in 96-well plate in standard
culture medium for 5 days in 37oC. Then,
cells were incubated with lead for 24
hours. Cells were also challenged with
LPS (10 ng/ml), fractalkine (10 ng/ml)
and NGF (10 ng/ml) for 24 hours as
positive controls. Viability of non-treated
cells considered as 100%. There were no
significant changes were seen in presence
of lower concentration of lead (1 and 10
µM).Only 100 µM lead reduced HCN2
viability at the significant level (n=3)
(P<0.05). Colorimetric measurements
were analyzed statically using the ANOVA and Dennett’s tests.
[44]
Figure 3.3: Effect of lead exposure on HCN2 morphology
HCN2 were cultured in DMEM supplemented with 10 % FCS for 10 days (A). Then, cells were
exposed to 10 µM lead (B) and 100 µM (C and D).for 24 hours, 37oC. Images were taken using the
phase contrast microscope (n=5). Bar indicates 100 µM (A-C) and 200 µM (D).
3.5.3. Effect of lead exposure on expression of chemokine receptors in
HCN2
As mentioned above, a variety of chemokine receptors are expressed by HCN2 cells.
Results showed that HCN2 expressed most of the chemokine receptors at moderate
levels. However, among the expressed receptors, high expression levels were found for
CCR1, CCR2, CCR3, CCR4 and CCR5 (MFI ± SD: 40.06 ± 2.09, 66.68 ±1.64, 114
±4.35, 25.4 ± 1.54 and 409.4 ± 37.9) (Figure 3.5 A-E). Both fractalkine and CX3CR1
(Figure 3.5 J and K) were expressed at high levels (101.2±10.23 and 175.8±17.07) In
accordance with immunohistochemistry data, high expression of β-tubulin III was also
shown by flow cytometry (201.62 ± 14.42) (Figure 3.4 A). As expected, low affinity
nerve growth factor (NGF) receptor (NGF-R p 75) was shown to be expressed by HCN2
at low levels (10.7 ± 1.27) (Figure 3.4 B). According to the important role of chemokine-
[45]
receptor axis in neuronal development, migration and even inflammation, we studied
possible changes in chemokine receptorexpression patterns after exposure to lead for 24
hours. Figure 3.5 summarizes the expression pattern of chemokine receptors in the
presence or absence of lead. Following 10 µM lead exposure, expression levels of CCR1,
CCR2, CCR3 and CCR5 drastically declined (MFI from 40.06 ± 2.09 to 23.86 ±1.96,
66.68 ± 1.64 to20.7 ± 2.54, 114 ± 4.35 to 48.08 ± 5.4, and 409.4 ± 37.9 to 67.64 ± 4.32)
(P=0.034, 0.005, 0.024, 0.020 respectively). Lead did not induce significant changes in
the expression of CCR9 nor CXCR4 (p>0.05). However, CCR4, CXCR1 and CXCR3
expression increased significantly in lead treated HCN2 (MFI from 25.4±1.54 to 60.54±
5.54, 51.22 ± 3.87 to84.52 ± 8.76 and 30.92 ± 1.17 to 42.16 ± 1.18) (P= 0.038, 0.023 and
0.037). Lead-exposed HCN2 showed significant down-regulation in β-tubulinIII
expression (from 201.62 ± 14.42 to 21.42 ± 2.3) (P=0.006). In terms of
fractalkine/CX3CR1 expression, lead exposure induced expression of membrane-bound
fractalkine while the expression of CX3CR1 decreased (MFI from 101.2 ± 10.23 to 239
± 14.2 and 175.8 ± 17.07 to71.32 ± 5.4) (P=0.017 and 0.024).
The effect of low level of lead exposure on the expression of β-tubulin III as young
neuronal marker and the p75 NGF receptor in HCN2 was also investigated. Result
showed that non-lead treated HCN2 expressed β-tubulin III at high level of intensity,
however, 10 µM lead exposure down-regulated significantly the expression of β-tubulin
III (P=0.006) (Figure 3.4. A). Lead had no significant effects on expression level of NGF
receptor p75 in HCN2, although a slight decrease was observed in lead treated cells
comparing with the control group (Figure 3.4. B).
Figure 3.4: Effect of lead exposure on expression pattern of neural marker.
Representative histograms of one
experiments showing the comparison
between the expression level of β-
tubulin III (A) and NGF-R (B) in
HCN2 in presence and absent of lead
(black and white histograms
respectively).Cells were incubated
with 10 µM lead for 24 hours at
37oC. Then, mean fluorescent
intensity of tested markers were
measured using the flow cytometry.
Data were analyzed using the FlowJo
software Analysis of the significant
differences between lead-treated cells
and non-treated cell was done by
Student’s t-test. (n=5).
[46]
Figure 3.5: Effect of lead on expression patterns of chemokine receptor in HCN2.
Flow cytometry data of one representative experiments is shown (n=5). Histograms represent the
expression level of CCR1-CCR5 (A-E), CCR9 (F), CXCR1 (G), CXCR3 (H), CXCR4 (I),
CX3CL1 (J) and CX3CR1 (K) in lead-treated HCN2 (black) comparing to control cells (white).
Cells were incubated with 10 µM lead for 24 hours at 37oC. Data were analyzed using the FlowJo
software Analysis of the significant differences between lead-treated cells and control ones was
done by Student’s t-test.
[47]
3.5.4. Effect of lead on cytokine secretion in HCN2 cells
Up-regulation of pro-inflammatory cytokines in the brain in response to lead exposure, as
well as in induced neuronal injuries and apoptosis has been reported in in vivo and in
vitro animal models (Struzynska et al., 2007). However, there is not much known about
possible changes of cytokine secretion patterns after lead exposure on human neuronal
precursors. Therefore, we studied whether lead exposure altered the expression of pro-
inflammatory cytokines. We measured the secretion of IL-1β, IL-6, IL-8, IL-10, IL-12,
TNF- and IFN-γ in lead-treated HCN2 compared to the control group. Results showed
that non-treated HCN2 secreted IL-8 and IL-6 (Figure 3.6). After exposure to 10 µM lead
for 24 hours, IL-8 expression remained unchanged, while IL-6 secretion decreased
significantly. HCN2 in both experimental condition, in the presence or absence of lead,
showed non-detectable secretion level of IL-10, IL-1, TNF- and IL-12 (n=4).
Figure 3.6. Effect of lead on cytokine secretion in HCN2.
HCN2 were incubated with 10 µM lead for 24 hours. Various secreted cytokines were measured in
collected culture media using the cytokine bead cytometric assay. Non-lead treated cells were
considered as control. Data was analyzed by FCAP software. Final concentration are shown in
pg/ml (n=4), * indicates P<0.05.Data analyzed using the Student’s t-test.
0
1000
2000
3000
4000
5000
6000
7000
IL-8 Il-6
Fin
al c
on
cen
trat
ion
(p
g/m
l)
control
pb
* p=0.000
[48]
3.6. Discussion
In this study, we investigated the effect of lead exposure on morphology, expression
pattern of chemokine receptors and cytokine secretion in the human cortical neural cell
line HCN2. To our knowledge, there are no published studies on HCN2 and lead. We
showed that at low concentrations (≤10 µM), lead had no effect on the morphology and
viability of HCN2. Lead altered the expression patterns of chemokine receptors and
cytokines in HCN2 with significant changes on expression of fractalkine/CX3CR1 axis,
as well as IL-6 secretion.
Lead is a well-known neurotoxicant with potent detrimental effects on the development
and function of the CNS. Lead exposure is still treated as a major global health problem
especially in children with long-lasting cognitive and behavioral impairment(Lidsky and
Schneider, 2003).
In humans, the CNS development begins in the third gestational week with proliferation,
differentiation and migration of neuronal precursor cells (NPCs) and continues in the
postnatal periods. The blood-brain barrier is not fully developed until the middle of the
first year of life (Stiles and Jernigan, 2010). The number of synaptic connections between
neurons reaches a peak around the age two and is then trimmed back to about half of it.
Similarly, there is great postnatal activity in the development of receptors and transmitter
systems, as well as in the production of myelin. All these facts support the sensitivity of
the developing brain to lead exposure (Rodier, 1995, Rodríguez-Martínez et al., 2012).
Recent studies demonstrated that low level of lead exposure produces blood lead levels,
near the accepted international safe limit, and triggers a wave of apoptotic
neurodegeneration in cortical and subcortical neurons in the neonatal mouse brain
(Dribben et al., 2011). There is also evidence from animal studies showing that short-
term exposure to moderate level of lead during neurogenesis can influence cell survival
and the process of development, by affecting neuronal precursor cells and by altering the
cortical arrangement (Davidovics and DiCicco-Bloom, 2005). Over the past decades,
several studies have reported lead-induced neurotoxicity in human and animals, using in
vivo and in vitro models. However, not many of them investigated the effect of lead on
human NPC.
Human cortical neurons are one of the principal cell types affected in neurotoxicity and
several neurodegenerative diseases. Due to their importance in healthy brain function, in
vitro and in vivo animal and human cortical neurons have been used commonly as
appropriate model, in neurotoxicity research (Schmuck et al., 2000). HCN2 are human
[49]
cortical neuronal non-transfected cells obtained from a 7 year old girl with Rasmussen's
encephalitis. Undifferentiated HCN2 showed characteristic of neural restricted precursor
cell including expression of neural precursor markers such as nestin, vimentin and β-
tubulin III, as well as polygonal flat soma with number of short neuronic branches
process resembling the immature/undifferentiated neuron morphology. These results are
in accordance with previous published data (Ronnett et al., 1994). HCN2 as immature
cortical neurons expressed the nerve growth factor receptor-gp75 and membrane-bound
fractalkine. HCN2 did not express Iba-1, a common microglia marker, which made it a
suitable in vitro neuronal precursor model for our study. Nerve growth factor (NGF) is a
well-characterized neurotrophic factor that plays a crucial role during development in
growth, differentiation and maintenance of brain neurons, as well as in the reparative
responses of the adult brain to damage. Expression of neurotrophin receptors by
neurosphere cultured from embryonic precursor cells has been reported (Lachyankar et
al., 1997, Maisonpierre et al., 1990). It has also been reported that NGF can induce the
differentiation of HCN2, which is in agreement with our data. Thus, based on our results,
undifferentiated HCN2 showed the properties of neuronal restricted precursor cells.
In the next step, we investigated the effect of lead exposure at different concentration (1,
10 and 100 µM) on the morphology and viability of the HCN2. HCN2 at the non-
stimulated condition showed the flat polygonal morphology with few short spin–like
processes. Piper et al. (2000) has reported that nestin positive human neuronal precursor
cells in an undifferentiated condition appeared morphologically flat with few processes,
and they formed tight clusters. When cells were replated into differentiation conditions,
cells altered their morphology and generated long processes (Piper et al., 2000,
Kerkovich et al., 1999). HCN2 showed no changes in morphology after exposure 0, 1 or
10 µM. However, in the presence of 100 µM lead, few cells showed the bipolar cell body
with long processes resembling the more differentiated cells. There is no evidence
showing the effect of lead in morphology of human neuronal precursor cells. However,
morphologic changes were observed in the rat hippocampus following low level (< 0.5
µM) exposure to lead such as a significant increase in the size and numerical density of
the dendrites and granulr cells. The opposite effects (i.e. decreased density of cell layers)
were observed at much higher concentration of lead, suggesting a bimodal effect of lead
on the developing hippocampus (Slomianka et al., 1989, Finkelstein et al., 1998). Lead
involvement has also been suggested to potentiate the PC12 morphological
differentiation that may be related to lead-induced alteration in binding transcription
factor to DNA (Crumpton et al., 2001).
[50]
As we did not observe any significant changes in morphology, we studied whether lead at
the low concentration could alter the HCN2 viability. Several in vitro and in vivo studies
have shown detrimental effects of lead on viability of CNS cells through different
mechanisms such as alteration of the cytoskeletal reorganization, increase in oxidative
stress, up-regulation in expression of apoptotic proteins and disturbance intracellular
Ca2+
(Choi et al., 2011, Penugonda et al., 2006). However, on the contrary, some studies
showed an inhibitory effect of lead on neurite outgrowth both, at high and low
concentration, with no effect at the intermediate levels (Kern et al., 1993, Schneider et
al., 2003). Thus, there is still debate on toxic effects of lead on neuronal cells depending
on the different concentration, exposure time, the roigin of the brain region cells and
developmental stage. Similar to previous studies in rat cortical neurons, no significant
toxic effect of lead (1 and 10 µM) on cell viability of HCN2 was observed in our study
(Fujimura and Usuki, 2012). In this study, we have also reported that lead could induce
cytotoxicity in HCN2 in vitro at the higher concentration (100 µM), over short period of
exposure (24 hours) a concentration which is not expected to be reached in vivo. A
similar cytotoxic effects of higher concentration of lead has been reported for rat cortical
neurons (Tang et al., 2010). It needs to be pointed out that MTS assay was used in this
study as a standardized cellular viability and cytotoxicity test. However, as MTS assay is
reflecting the cell death through indirect measure of the cell mitochondrial activity,
additional cytotoxic studies need to be conducted to investigate the exact cytotoxic
effects of lead on HCN2 viability. Functional chemokine receptors and chemokines are
expressed by glial cells within the CNSHowever, relatively little is known about the
patterns of chemokine receptor expression and function in neurons (Flynn et al., 2003,
Cartier et al., 2005). Recent studies showed that chemokines and their receptors play an
important role in regulating human neural stem cell (NSC) proliferation and survival. In
addition, there is not much known about the exact role of chemokine receptors in the
CNS development. It has been suggested that chemokines could have both stimulatory
and inhibitory effects on NPC proliferation (Coughlan et al., 2000, Krathwohl and
Kaiser, 2004). The expression of CCR1, CCR2, CCR5, CXCR3, CXCR4 and CX3CR1
has been reported in dividing adult mouse, rat and human NPC, which support our data
in HCN2 (Goczalik et al., 2008, Hesselgesser and Horuk, 1999, Ji et al., 2004, Wu et al.,
2012, Tran et al., 2007). We have observed the expression of all tested chemokine
receptors by HCN2 at a moderate to high level which suggests their involvement in
normal brain development, neurogenesis (i. e. regulating the migration of progenitor cells
in postnatal brain), as well as neuroinflammation. Several studies reported inhibitory
effects of chemokine receptors such as CCR3 and CXCR4 on neuronal precursor
proliferation and on neurogenesis (Krathwohl and Kaiser, 2004). In addition, activation
[51]
of pro-inflammaory chemokine receptors (CCR1, CCR5, CXCR4) have been reported to
play a role in neurodegenerative diseases, which implies their involvement in
inflammatory responses and induce neuronal death (Petito et al., 2001, Kaul et al., 2007).
In contrast, there is evidence showing a beneficial role of chemokine receptors on
neurogenesis during development. Several studies reported migration of neural
precursors to the site of inflammation in response to chemokine followed by injuries,
stoke, etc., to induce neurogeneration.
In this study, we have seen differential changes in expression patterns of chemokine
receptors following lead exposure. Most of the pro-inflammatory chemokine receptors,
such CCR1, CCR2, CCR3, CCR5 and CXCR4, showed down regulation, with one
exception, CXCR1, which significantly increased. Unlike other chemokine receptors,
there is little known about expression of CXCR1 in neurons. Expression of CXCR1
mRNA transcripts has been reported in glutamatergic, GABAergic, and cholinergic
neurons in the rat brain. Recently, Goczalik et al. (2008), reported for the first time the
prominent expression of CXCR1 in healthy human retina neuron. It has also been
suggested that CXCR1 and its ligand IL-8 play an important role as neuromdulator in
CNS development, neuronal survival, modulation of excitability, and neuroimmune
response (Danik et al., 2003). Increased expression of CXCR1 as well as IL-8 in human
retina has been reported in response to inflammation in proliferative viteoretinopathy
(PVR). Thus, during inflammation, elevated levels of IL-8 may activate neuronal
CXCR1/CXCR2 receptors to exert neuroprotective effects.
Among the neuronal chemokine/receptors, fractalkine and its unique receptor play a key
role in both, neuroprotection and neuroinflammation. The chemokine fractalkine, which
is the CX3CR1 ligand, is highly expressed in CNS neurons and is normally produced in a
membrane-bound form, from which the chemokine domain can be released (Harrison et
al., 1998). Over the past decades, it became clear that the interaction between neuronal
fractalkine and microglial CX3CR1 modulates microglia activation. Several studies have
shown the neuroprotective role of fractalkine in neurotoxicity models associated with
neurodegenerative diseases (Mizuno et al., 2003, Cardona et al., 2006). There is evidence
showing the correlation between decline in neurogenesis and reduced fractalkine in the
aged animal hippocampus (Bachstetter et al., 2011). Recent animal studies showed the
beneficial effects of increased fractalkine within hippocampus on NPC activation, which
in turn modulated microglia activation (Vukovic et al., 2012). Although expression of
CX3CR1 is thought to be restricted to microglia and astrocytes, recent findings showed
its expression by neurons which support our finding that fractalkine is expressed in
[52]
HCN2. The expression of CX3CR1 by hippocampal neurons indicates that these
receptors may also be a target for fractalkine produced by microglia and/or astroglia or
adjacent neurons. Despite the known paracrin interaction, recent studies suggested a
neuroprotective effect of fractalkine on neuronal survival and proliferation through
autocrine interaction (Meucci et al., 2000). Interestingly, we have seen a differential
effect of lead on fractalkine and CX3CR1 in HCN2. While lead suppressed the
expression of neuronal CX3CR1, it enhanced fractalkine expression. Thus, fractalkine
may interact with CX3CR1 expressed on neurons and other cells, including-like glia.
In this study, we also observed a significant reduction in expression of β-tubulin III.
In support of these results, it has been reported that cytoskeletal protein class III β-
tubulin isotype, a specific neuronal marker, is abundantly expressed in CNS during fetal
and postnatal development. Indeed, there is evidence indicating during brain maturation
alpha and beta tubulin expression is abundantly decreased (Bond and Farmer, 1983). In
addition, different expression patterns of β-tubulin subclasses during CNS development
has been noted. As exemplified, high expression level of class III β-tubulin was shown in
rat cortical neurons at the birth time which abundantly decreased during development and
maturation. While in dorsal root ganglia, β-tubulin III generally increased in postnatal
development (Jiang and Oblinger, 1992). In vitro and in vivo animal studies showed the
reduction in neurite outgrowth and axonal regeneration in knock-down β-tubulin III (Guo
et al., 2010). In this study, lead also reduced NGF receptor expression. Expression of
both low and high affinity receptors for NGF have been shown by NPC which implicates
their involvement in development and neurogenesis.
In this study,we reported that HCN2 under non-stimulatory condition secret IL-8.
However, no changes in secretion level of IL-8 in the supernatant of HCN2 was detected
following lead exposure. As mentioned previously, HCN2 expressed its receptor,
CXCR1 at non-stimulatory condition, which may implicate the role of this chemokine in
sustaining survival and growth of neuronal precursors, as well as during development.
Lead altered the receptor expression with no changes in secretion levels of IL-8.
We also observed the significant decrease in IL-6 secretion by HCN2 cells in the
presence of lead. It has been reported IL-6 has a neuropoitic effect on the NSC self
renewal and differentiation. On the other hand, in inflammatory conditions, enhanced
expression of IL-6 results in attenuation of NPC proliferation and differentiation. (Lan et
al., 2012). Microglia-derived IL-6 has also been shown to have suppressive effects on
neurosphere generation from adult human retina cells (Balasubramaniam et al., 2009).
Transgenic mice with over-expression of IL-6 showed alteration in hippocampal function
[53]
through changes in excitability of hippocampal neurons and synapses (Nelson et al.,
2012). Recent studies postulate that IL-6 does not have only influences on neurons
directly, but also is an important intercellular messenger in the neuronal-microglia
interaction.
3.7. Conclusion
To our knowledge, this is the first study reporting on the impact of lead exposure over a
short period of time on the human cortical neuronal cell line HCN2. In summary, lead
exposure (10 µM, 24 hous) had no cytotoxic effect on HCN2 cells. However, lead altered
the expresion patterns of chemokine receptors and IL-6 in HCN2. Lead caused changes
of these immune and neurogenesis related molecules and may affect the neuronal
communication with other cells, including neuronal precursors in areas important for
brain remodeling and development, which may finally be responsible for the neurotoxic
effects of lead.
Chapter 4
A novel in vitro human microglia model:
Characterization of human monocyte-
derived microglia
[55]
4. A novel in vitro human microglia model: characterization of human monocyte-derived microglia
4.1. Introduction to chapter
Microglia are the resident innate immune cells of the brain. As the brain macrophages,
microglia share common characteristics with the monocyte-macrophage lineage such as
expression of surface markers and chemokine/cytokine receptors, antigen presentation
and phagocytic activity. In response to stimuli such as pathogen, injuries, etc, resting
microglia with ramified morphology can become activated with amoeboid morphology.
This activation is usually followed by secretion of pro-inflammatory cytokine sand
chemokines as mediators of the innate immunity. Despite their known contribution to
pathological conditions, microglia also play a key role in CNS development and
neurogenesis through secretion of neurotrophic factors (Kettenmann et al., 2011,
Ransohoff and Perry, 2009, Kim and de Vellis, 2005, Chan et al., 2007).
To date, there is no standardized simple model available to investigate the biology of
human microglia. Consequently, the aim of this study was to establish a new in vitro
microglia model using blood-derived precursor cells. For that purpose, human peripheral
blood monocytes were cultured in serum free medium in the presence of a mixture of
cytokines and chemokines (M-CSF, GM-CSF, NGF and CCL2) to generate monocyte-
derived microglia (M-MG). Monocyte-derived dendritic cells (M-DC) were also
generated as a control population using GM-CSF and IL-4. The human microglia cell
line HMC3 was also used as control.
M-MG were clearly different in morphology, phenotype and function from M-DC, but
shared many properties with HMC3 cells. M-MG acquired a ramified morphology with
primary and secondary processes, comparable to HMC3. They expressed very low levels
of CD45, CD14 and HLA-DR, CD11b and CD11c; but a distinct pattern of chemokine
receptors, including CCR1, CCR2, CCR3, CCR4, CCR5, CXCR1, CXCR3, CX3CR1.
Similar to HMC3, under non-activated condition, the M-MG secreted of IL-8 and IL-6.
In comparison with M-DC, M-MG displayed lower T-lymphocyte stimulatory capacity,
as well as lower phagocytosis activity.
In summary, we have established a new protocol for the generation of human monocyte-
derived microglia, which is is feasible, well standardized and reliable, as it uses well
defined culture medium and recombinant cytokines, but no serum or conditioned
[56]
medium. This model will certainly be very helpful for future studies investigating the
biology and pathology of human microglia.
Declaration of work
All the experimental work were designed and conducted by SE including cell isolation,
cell culture, characterization, flow cytometry studies, functional assays, phagocytosis
tests and statistical analysis under supervision of Prof. Luis Filgueira. Confocal images
were taken with assistance of Mrs. Rasheeda Mohd Zamin. All the experimental work
described in this chapter has been published as manuscript in the Journal of Neuroscience
Methods (Appendix E).
[57]
4.2. Abstract
Microglia as the immune modulator cells of the CNS have the key functional roles both
in maintaining the healthy physiology of the brain and contributing in inflammation
responses especially in neuroinflammation–related pathogenesis. The aim of this study is
to generate the human microglia from blood monocyte .Microglia originate from distinct
sub-population of the monocytic lineage and share expressing various phenotype markers
of monocyte-macrophage lineage such as surface molecules, chemokine/cytokine
receptors, antigen presentation. To generate human microglia, PBMC were cultured in a
serum free condition using a novel mixture of four recombinant human cytokines, i.e. M-
CSF, GM-CSF, NGF and CCL2. These monocyte-derived microglia in presence of
mixture of cytokines acquired ramified morphology of with primary and secondary fine
processes. In context of surface marker expression, M-MG revealed different expression
in compare to monocyte. Microglia like cells showed down-regulation in expression level
of monocytic lineage markers (CD45, CD14, HLA-DR) and chemokine receptors, i.e.
CCR1, CCR2 and while up-regulation was detected in expression of CXCR3 and
CX3CR1. In light of these data, we developed a new in vitro protocol to generate human
microglia representing the specific morphological, phenotypic and functional properties
of the resting microglia. The described protocol is feasible, well standardized and
reliable, as it uses only well defined culture medium and recombinant cytokines, but no
serum or conditioned medium. This protocol will certainly be very helpful for future
studies investigating adult human microglia.
Keywords:Microglia, human peripheral blood monocytic cells, serum-free, characterization
4.3. Introduction
Microglia are resident innate immune cells of the central nervous system (CNS) and play
a crucial role in maintaining the healthy physiological homeostasis. Microglia also
contribute substantially to inflammation in response to injury, toxins, pathogens and
degenerative processes (Streit, 2001, 1996; Howell et al., 2010; Lee et al., 2010; Politis et
al., 2011). Microglia are characterized by a quick response to various stimuli, resulting in
activation with rapid changes in morphology, phenotype and function. Morphological
changes include shortening of cell processes and cellular hypertrophy. To date, there is
no specific microglia marker clearly separating this cell type from other cells of the
monocyte/macrophage lineage. Some studies suggested that primary microglia could be
distinguished from other tissue macrophages according to expression levels of markers
such as CD11b and CD45 (Aloisi et al., 2000; Ford et al., 1995). Under healthy
[58]
condition, resting microglia with typical ramified morphology show low expression
levels of these common myeloid lineage markers (Nimmerjahn et al., 2005). However,
microglia have been shown to share expression of surface markers common to other
immune cells of the macrophage family such as CD45, CD14, MHC-class II, CD68,
immunoglobulin Fc receptors and 2 integrins (Lambertsen et al., 2009; Wirenfeldt et al.,
2005). Expression levels of these surface markers may depend on the inflammatory status
of the CNS and on microglia activation. Similarly, functional inflammatory changes are
dominated by secretion of pro-inflammatory cytokines, chemokines, neurotrophic factors
and up-regulation of corresponding receptors, as well as production of nitric oxide and
reactive oxygen intermediates (Aloisi, 2001; Tambuyzer et al., 2009).
Microglia have recently been shown in a mouse model to originate from a myeloid yolk
sac population during embryogenesis (Ginhoux et al., 2010). In humans, the microglia
origin is still not known. Furthermore, little is known about the turnover rate of microglia
in the healthy CNS. It has been postulated that proliferation of the resident microglia
population is rather low and there may be immigration of bone marrow derived precursor
cells into the CNS. Invasion of bone-marrow derived microglia have been shown to
migrate into the CNS using chimeric irradiated mice (Simard et al., 2006; Zhang et al.,
2007). However, the proportion of bone-marrow progenitor cells in replenishment of
microglia is controversial.
A variety of cytokines have been shown to contribute to microglia development and
differentiation. Colony stimulating factor-1/M-CSF and its receptor CSF-1R play an
important role in the development of macrophage populations (Chitu and Stanley, 2006).
Recently, Ginhoux et al. (2010) reported the constant absence of microglia throughout
life in CSF-1R deficient mice, indicating that M-CSF is essential for microglia
development and differentiation. In addition, granulocyte-macrophage colony
stimulating factor (GM-CSF) also contributes to microglia development and
differentiation (Aloisi et al., 2000; Esen and Kielian, 2007). Both, M-CSF and GM-CSF
have crucial effects on proliferation and survival of primary human fetal and adult
microglia in culture with GM-CSF having a greater impact on proliferation (Esen and
Kielian, 2007; Lee et al., 1994). M-CSF plays a crucial role in the final maturation stage
of microglia. In op/op mice, a model of human osteopetrosis with a functional mutation
the M-CSF gene, the number of microglia are decreased and the cells are smaller and
have impaired activation ability in response to injury (Kalla et al., 2001;Wegiel et al.,
1998). Importantly, GM-CSF or IL-3 does not agile substitute for M-CSF (Blevins and
Fedoroff, 1995).
[59]
Nerve growth factor (NGF), a member of neurotrophins, has also been shown to act on
the proliferation and survival of microglia (Zhang et al., 2003). NGF binds and acts
through the P75 receptor, which has been shown to be expressed in microglia in multiple
sclerosis lesions (Valdo et al., 2002). It has also been shown that microglia express the
NGF receptor TrkA (Tonchev, 2011). NGF induces migration of the microglia cell
through activation of the TrkA (TrkA activation) (De Simone et al., 2007). NGF is
expressed by activated microglia, astrocytes and hippocampal neurons (Friedman, 2000;
Saez et al., 2006; Tonchev, 2011). However, inflammation certainly induces
neurotrophin secretion by human microglia (Heese et al., 1998; Nakajima et al., 2001).
Chemokines have also been shown to play an important role in microglia biology.
Monocyte chemoattractant protein-1 (MCP-1), or CCL2, is one of the prominent
chemokines in the regulation of the microglia migration to the site of inflammation in
experimental models (Leonard et al., 1991; Zhang et al., 2007). CCL2 acts through its
specific receptor CCR2 which is expressed by microglia and other cells of monocytic
lineage, such as macrophages and dendritic cells (Rebenko-Moll et al., 2006). CCL2 acts
also through CCR4, but little information is available about CCR4 expression in
microglia (Craig and Loberg, 2006; Zhang et al., 2006). Studies on normal rat CNS
showed that CCL2 is constitutively expressed by astrocytes and neurons and it can be
found in various brain regions, including the cerebral cortex, the hippocampus and the
hypothalamus (Banisadr et al., 2005). CCL2 is also secreted by astrocytes and neurons
under inflammatory condition (Banisadr et al., 2005; Farina et al., 2007; Tanuma et al.,
2006). It has been shown that CCL2 is secreted by both undamaged and damaged spinal
sensory neurons in rat, resulting in activation of spinal microglia and initiating
neuropathic-like pain (Thacker et al., 2009). Up-regulation of CCL2 expression during
Alzheimer’s disease and multiple sclerosis correlates with activation of microglia and
may contribute to pathogenesis of neurodegenerative diseases (Conductier et al., 2010;
Simpson et al., 2000). In that respect, CCL2 contributes to recruitment of mononuclear
cells into the inflamed CNS, followed by activation of the microglia. Zhang et al. (2007)
showed that CCL2 can induce spinal microglia activation in mice. They also reported in
chimeric mice that CCL2 recruits bone marrow-derived macrophages, which proliferate
and differentiate into microglia in the spinal cord and induce inflammation and
microgliosis after nerve injury. In experimental autoimmune encephalomyelitis (EAE), a
murine model of multiple sclerosis, CCR2 deficient mice do not develop the
mononuclear cell infiltration and inflammation. Indeed, no chemokines increase was
detected in the CNS in that model (Izikson et al., 2000). On the other hand, CCR2
deficiency in a mouse model of Alzheimer’s disease impairs significantly microglia
[60]
accumulation at sites of plaque formation, resulting in a decrease of amyloid-β clearance
and in accelerating early disease progression (El Khoury et al., 2007).
In vitro culture of mouse and human microglia has often been used as a model by various
researchers. Thereby, astrocyte-conditioned medium (ACM) has been used to keep
microglia in culture with a ramified resting morphology. ACM contains M-CSF, GM-
CSF and transforming growth factor (TGF-β), all known to be secreted by astrocytes.
Microglia lose their ramified morphology in the presence of antibodies against the
mentioned cytokines, indicating that M-CSF and GM-CSF are essential for microglia
culturing (Schilling et al., 2001). GM-CSF, even at low concentration in serum-free
medium, keeps microglia at a resting state with morphological ramifications (Fujita et al.,
1996). A recent study reported the proliferative effect of CCL2 on neonatal and
embryonic primary rat microglia in culture without inducing additional changes in
morphology, phenotype and cytokine expression (Hinze and Stolzing, 2011).
Most interestingly, murine bone marrow-derived precursor cells can be differentiated
towards microglia-like cells when cultured in the presence of astrocytes or in mixed glial
cultures. Thereby, M-CSF alone is not sufficient for microglia differentiation, which
indicates the importance of additional astrocyte-derived factors (Noto et al., 2010).
Differentiation of murine bone marrow stem cells toward microglia-like cells has
recently also been successful, using ACM supplemented with GM-CSF (Hinze and
Stolzing, 2011). Culturing fetal and adult human microglia in the presence of GM-CSF
induces functional maturation towards an antigen presenting cell type, especially in adult
microglia. However, GM-CSF does not induce maturation of microglia to acquire the
complete phenotype and function of mature dendritic cells (Lambert et al., 2008; Re et
al., 2002).
In vitro studies on microglia used mainly primary microglia culture from embryonic or
neonatal murine brains (Bassett et al., 2012; Hinojosa et al., 2011). In human, brain-
derived microglia is difficult to obtain for ethical reasons. In addition, only low numbers
of cells are collected. Also, fetal microglia seems to be quite different from adult
microglia. Only few human microglia cell lines have been generated, including HMO6
cells (Nagai et al., 2005) and HMC3 cells (Janabi et al., 1995). These cell lines cannot be
considered as an optimal model for microglia cells due the significant modification in
morphology and function as result of genetic manipulation and long-term culture.
Therefore, a more convenient and more appropriate model for in vitro microglia human
studies is still missing.
[61]
Most importantly, Leone et al. (2006) showed that human blood-derived monocyte,
cultured with astrocytes-conditioned medium (ACM), acquired the ramified morphology
of microglia and expressed substance P, calcium binding protein Iba-1 and dimly MHC
class II, three typical characteristics of microglia. There are also reports showing
successful differentiation of rat blood monocyte towards microglia using ACM
(Schmidtmayer et al., 1994; Sieverset al., 1994). Recently, Hinze and Stolzing (2011)
showed the differentiation of murine bone marrow stem cells towards microglia-like cells
in the presence of ACM with similar phenotypic and functional properties to primary
brain-derived microglia cultures. In the light of these data, we developed a new in vitro
human microglia model using human blood peripheral mononuclear cells, a serum-free
culture condition and a panel of factors, including M-CSF, GM-CSF, NGF and CCL2.
This new human microglia model has then been assessed for morphology, phenotype,
and function.
4.4. Materials and methods
4.4.1. Cell isolation
Human blood mononuclear cells (PBMC) were isolated from buffy coats (100 buffy
coats were used for this study) of healthy donors (Australian Red Cross Blood Service
(ARCBS), Perth, WA, Australia; ethics approval was granted by UWA and ARCBS)
using Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden) as published before
(Meagher et al., 2005). To obtain monocyte (adherent PBMC), the isolated blood cells
were cultured in T25 tissue culture flasks (Sarstedt, Numbrecht, Germany) (2 × 106 to 5 ×
106
cells/ml) using RPMI-1640 Glutamax medium (Invitrogen, Auckland, NZ)
supplemented with 1% antibiotics/antimycotic (10,000 units/ml penicillin G sodium,
10,000 µg/ml streptomycin sulfate and 25 µg/ml Amphotericin B, Invitrogen). It is
important to note that no serum was used in all microglia cultures. After overnight
incubation (humidified air, 37o
C, 5% CO), non-adherent cells (consisting mainly of T-
lymphocytes) were separated by thorough washing with PBS (Invitrogen) and used for
further experiments. The fresh adherent cells, which were mainly monocytes (>90%),
were used for the generation of microglia (M-MG). Dendritic cells (M-DC) and cultured
monocytes, as well as for comparison with the generated cell populations. All
experiments were done with cells from at least 6 different blood donors, if not otherwise
stated.
[62]
4.4.2. Cell culture
To induce the differentiation of M-MG, adherent PBMC (1×106
cells/ml) were cultured
in 6-well tissue culture plates (Sarstedt) using RPMI-1640 Glutamax supplemented with
1% antibiotic/antimycotic (serum-free condition) and a mixture of human recombinant
cytokines, including M-CSF (10 ng/ml; Pepro-Tech, Rocky Hill, NJ), GM-CSF (10
ng/ml; PeproTech), β-nerve growth factor (NGF-β; 10 ng/ml; PeproTech) and CCL2
(100 ng/ml; PeproTech) at standard humidified culture conditions (37oC, 5% CO) for up
to 14 days. Final yield of M-MG was 6-8×106 cells/60 ml of humanBuffy Coats.The cells
were used for experiments and characterization at different time points as indicated.
Monocytes were also cultured in RPMI-1640 Glutamax supplemented with 1%
antibiotic/antimycotic, but no serum, for up to 14 days as control cell population.
Furthermore, to obtain monocyte-derived dendritic cells (M-DC), adherent PBMC were
cultured in 6-well tissue culture plates (Sarstedt) using RPMI-1640 Glutamax, 1%
antibiotic/antimycotic supplemented with 10% fetal calf serum (FCS; Serana,
Bunbury,WA, Australia) in the presence of human recombinant GM-CSF(10 ng/ml;
PeproTech) and IL-4 (10 ng/ml; Pepro-Tech) for up to 14 days (Meagher et al., 2005).
The cells were used for experiments at different time points, as indicated.
Human microglia clone 3 (HMC3) cells, kindly provided by Prof. Karl-Heinz Krause
(University of Geneva, Switzerland) were cultured in T25 tissue culture flask (Sarstedt)
using DMEM/F12 Glutamax medium (Invitrogen) supplemented with 15% FCS and 1%
antibacterial/antimycotic. The cells were passaged and used for experiments when they
reached 70–80% confluence.
4.4.3. Morphological studies
Morphology and viability (using trypan blue dye exclusion: cell viability was >90% for
all experiments) of M-MG, M-DC and HMC3 during the 2 weeks in culture were studied
using an inverted Leica DM IL phase contrast microscope. Images were taken with
Photometrix CoolSNAP Fx digital camera and processed with NIS-elements F3.0
software. HMC3 and M-MG cells were also stained for expression of Iba-1, a well-
known characteristic marker for microglia cells. For that purpose, cells were grown on
glass cover-slips, fixed in 1% paraformaldehyde in PBS/sucrose and incubated with a
polyclonal rabbit anti-Iba-1 (Santa Cruz biotechnology, Santa Cruz, CA) and a donkey
anti-rabbit Alexa Fluor 488 (Invitrogen). Negative control stains were used by omitting
[63]
the primary antibody against Iba-1. Cell nuclei were stained with DAPI (1 µg/ml in PBS,
4,6-diamidine-2-phenylindole dihydrochloride; Roche Diagnostics, Mannheim,
Germany). Imaging was done using confocal microscopy (Leica TCS SP2 multiphoton
confocal microscope).
4.4.4. Flow cytometry
We studied the expression of specific surface molecules on freshly isolated and cultured
monocytes, M-MG, M-DC and HMC3 using flow cytometry. Cells (1×106/ml per
staining condition) were detached using a scraper (Sarstedt) and washed twice with PBS.
Staining was performed according to protocol recommended by BD. A panel of directly
labeled mouse anti-human antibodies, CD45-FITC, CD14-PE, CD40-PE, CD68-APC,
CD80-PE, CD11c-PE,HLA-DR–PERCP, CD11b-AF488, CD11c-PE, CCR1-AF647,
CCR2-AF647, CCR4-PE-Cy7, CCR5-FITC, CXCR1-PE, CXCR3-AF488
(BDPharmingen, San Jose, CA), CCR6-FITC, CCR7-APC (R&A systems, Minneapolis,
MN), NGFR-FITC (Santa Cruz Biotechnology, SantaCruz, CA) were added at the
supplier’s recommended concentration. Corresponding labeled isotype control antibodies
were included in all experiments. A rabbit anti-CX3CR1 polyclonal antibody was used at
the final concentration of 1 µg/ml (AbD Serotec, Oxford, UK), followed by a secondary
donkey anti-rabbit-AF 647 used at the final concentration of 1:200 (Invitrogen). The cells
were incubated with antibodies for 30 min to 1 h at 4o
C, washed with PBS, resuspended
and fixed with 1% formaldehyde in PBS containing 2% sucrose. PBS-2% sucrose
solution were used in combination with fixative to enhance the preservation of cells
morphology following by fixation (quality of fixation) as well as reduce the background
autofluorescence.The mixed samples were measured with a FACSCallibure (BD
Biosciences). Data analysis was performed using FlowJo software version 7.6.3 (Tree
Star, Ashland, OR).
4.4.5. Phagocytosis assay
Phagocytosis was assessed by measuring the uptake of tetra-methyl rhodamine (TMR)-
labeled Staphylococcus aureus (Invitrogen) as published before (Meagher et al., 2005).
Bacterial particles were added to the cells (107 bacteria/ml) at a ratio of 1:1000
(according to manufacturer’s protocol) and incubated for 4 h at 37oC. Cells were then
harvested and fixed with 1% paraformaldehyde in PBS/2% sucrose. Total ingested
fluorescent-labeled bacteria per cell was measured by flow cytometry. To verify that the
bacteria were located intra-cellularly, cells were cultured on glass coverslips, treated with
labeled bacteria, fixed and visualized using confocal microscopy (Leica TCS SP2
[64]
multiphoton confocal microscope). Dendritic cells and freshly isolated monocytes were
used as positive control populations. Untreated cells were used as negative controls.
4.4.6. Mixed leukocyte reaction (MLR)
The T-lymphocyte stimulatory capacity and corresponding antigen-presenting cell (APC)
properties of M-MG, in comparison with M-DC and HMC3, was assessed in MLRs
using a BrdU proliferation assay (Cadosch et al., 2010). Freshly processed non-adherent
PBMC containing mainly (>90%) T-lymphocytes (2 × 105 cells/well) were seeded in a
round bottom 96-well plate (Sarstedt). Allogeneic M-MG, M-DC and HMC3 were used
as antigen-presenting cells (APC) and added at various ratios (1:40 and 1:80) to the T-
lymphocyte (four wells for same condition). T-lymphocytes or APC only were used as
control conditions. To compare the effect of lead on activated T-lymphocytes, PHA was
added to each well at the concentration of 7 µg/well. PHA- activated T-cells with APC
was used as the positive controls. After 5 days in culture, BrdU proliferation assays
(Roche, Mannheim, Germany) were performed according to the supplier’s protocol and
the absorbance of the colorimetric reaction was measured by ELISA reader at 405 nm
(Labsystems Multiskan RC, Helsinki, Finland). Mean proliferation of the different
conditions was statistically analyzed and compared.
4.4.7. Statistics
Data were statistically analyzed through the SPSS statistics 17.0 software. A one-way
ANOVA and univariant linear model were used to compare means. The LSD post hoc
test was also used to determine the significant difference between groups. In all the
analysis p value < 0.05 were considered as statistically significant. Graphs with error bars
represent the standard deviation (SD).
4.5. Results
4.5.1. Morphological and phenotypic changes of monocyte-derived
microglia in culture
Adherent PBMC, representing mainly the monocytic population of blood leukocytes,
were cultured for up to 2 weeks in the presence M-CSF, GM-CSF, NGF-β and CCL2, in
RPMI 1640 medium without addition of serum to create optimally standardized
conditions. The cells were characterized for morphological changes over the period of 2
weeks in culture (Figure 4.1). The well established HMC3 human microglia cells line
was used as comparison. After 5 days in culture, the small (10–15 µm diameters) round-
[65]
shaped and semi-adhered monocytic cells expanded their cell body and developed few
unbranched processes (results not shown). During the second week, the cells reduced the
portion of the cell body and they increased the processes in length and number acquiring
ramified forms. M-MG obtained a ramified morphology after 10–14 days in culture,
including a polygonal or oval cell body and up to 7 primary processes and numerous
secondary process or spines. Over the 2 weeks in culture, the cells doubled in size and
increased in cell number indicating proliferation. After 2 weeks, M-MG displayed a very
similar morphology to the human microglia cell line HMC3 and to previously described
human brain-derived microglia in published studies (Leone et al., 2006).
Figure 4.1: Phase contrast microscopy of monocyte-derived microglia (M-MG) and human
microglia cell line HMC3
Monocyte derived microglia (M-MG) (A) and human microglia cell line HMC3 (B) Images were
taken from cells after 10 days in culture. Bar indicates 100 µM.
[66]
Basic known microglia markers, including expression of Iba-1 and CD68, were also
tested positive, supporting that the described protocol differentiates monocytes towards
M-MG (Figures 4.2 and 4.3).
Figure 4.2: Expression of Iba-1 in HMC3 and M-MG
Iba-1 is a characteristic microglia marker in HMC3(B) and M-MG (D). Corresponding negative
staining controls have been included (A and B). Bar indicates 30 µM.
[67]
Figure 4.3: Differential expression of CD68 in freshly isolated blood monocytes, HMC3 and M-MG.
Flow cytometry dot plots and histograms represent the differential expression of CD68 in freshly
isolated blood monocytes (A), HMC3 (B) and M-MG (C). The red dots/gray line indicates the
negative staining controls using a corresponding isotype control antibody. The black dots/line
indicates the specific test stain for CD68. Note that there is no CD68 expression in monocytes,
whereas two microglia populations clearly express CD68. Bar chart summarizes the mean
florescent intensity of studies groups (D). White bars are representing the isotope control, and
black representing the CD68 expression. M-MG and HMC3 showed significant differences in
expression of CD68 comparing to blood monocyte (P<0.05).
[68]
4.5.2. Expression pattern of specific surface markers
After culture in the presence or absence of the corresponding cytokines cocktails,
monocytes, M-MG, M-DC and the human microglia cell line (HMC3) were tested for
expression of surface markers relevant to human microglia. Figure 4.4 summarizes
expression of all relevant markers for all tested cell types, including CD45, CD14,
MHC class II, CCR2 and CCR4.
Firstly, the common leukocyte antigen CD45 was tested. CD45 is a characteristic
marker expressed by all leukocytes. As expected, CD45 was highly expressed in
monocytes, which did not change CD45 expression levels over the 2 weeks in culture in
serum-free medium without addition of cytokines. M-DC reduced only slightly CD45
expression over the 2 weeks in culture. However, M-MG reduced gradually CD45
expression levels over the 2 weeks in cul-ture reaching very low levels, as expected for
microglia and similar to HMC3 (Janabi et al., 1995; Li et al., 2009). CD14 is known to
be characteristic for monocytes, being expressed at highest level on these cells. As
predicted, M-MG reduced expression of CD14 to very low levels, similar to HMC3.
Activated microglia may process and present antigens on MHC class II. For that
purpose, activated microglia increase MHC classII expression, such as HLA-DR.
Usually, resting microglia express low levels of HLA-DR, whereas freshly isolated
monocytes express high levels of HLA-DR, which is also highly expressed in immature
M-DC. In our study, HLA-DR was clearly down-regulated in M-MG, reaching similar
levels detected in HMC3, indicating that M-MG may represent resting microglia cells.
Chemokines play a crucial role in recruitment and maturation of migratory, bone
marrow-derived microglia cells (Flynn et al., 2003). CCR1, CCR2, CCR5 expression
has been thus reported to be expressed at low levels in microglia (Bajetto et al., 2002;
Skuljecet al., 2011). In our study, monocytes expressed low levels of CCR2, but up-
regulation was seen when cultured in medium with-out cytokines. Interestingly, M-DC
decreased CCR2 expression to undetectable levels, whereas M-MG and HMC3
expressed clearly detectable CCR2 at rather low levels, as expected for microglia.
Expression of CCR4 and its ligand (CCL22 and CCL17) have been reported in the CNS
(Columba-Cabezas et al., 2002). Freshly isolated monocyte expressed CCR4 at very
low levels, while after one week in culture without cytokines, a sub-population (about
25%) expressed CCR4 at higher levels. However, M-DC and M-MG up-regulated
CCR4 expression to clearly detectable levels over time in culture to similar levels as
HMC3.
[69]
Figure 4.4: Expression of surface markers CD45 (a), CD14 (b), HLA-DR (c), CCR2 (d) and CCR4 (e) measured with flow cytometry.
Comparison of freshly isolated monocytes (A), cultured monocytes in the absence of cytokines
(B), monocyte derived dendritic cells (C), monocyte-derived microglia (D) and microglia cell line
HMC3 (E). The cells were cultured for 10 days.
Figure 4.5 summarizes the expression of various chemokine receptors on the different
cell populations that were investigated. Freshly isolated monocytes expressed
intermediate levels of CCR1. Differentiation towards M-MG decreased CCR1
expression to similar levels known for non-activated microglia and also seen in HMC3.
CCR5 expression level was detected at low levels in monocytes, but expressed at higher
levels in both M-MG and HMC3, as expected for microglia. CXCR1 was expressed at
very low levels in freshly isolated monocyte, but was clearly expressed at higher levels
[70]
in M-MG and HMC3. Freshly isolated monocyte did not express neither CXCR3 nor
CX3CR1. However, both these markers were clearly up-regulated in M-MG similar to
HMC3. CXCR3 expression in M-MG was at intermediate levels, while HMC3 showed
higher expression levels. Interestingly, almost similar expression of CX3CR1 was seen
in both M-MG and HMC3. In summary, M-MG and HMC3 expressed very similar
chemokine receptor patterns, supporting the microglia-like properties of M-MG.
Additional markers were also investigated (perosnal communiction), including CD 11c,
CD11b, CD40, CD80, CD83, CCR6 and CCR7. M-MG were negative for CCR6 and
CCR7. CD11c and CD11b were both expressed on M-MG and HMC3, but at much
lower levels than on M-DC. Immature M-DC expressed the dendritic cell markers
CD40 and CD83 at an intermediate intensity level. However, CD80 expression was
undetectable on M-DC. Most important, neither M-MG nor HMC3 expressed any of the
dendritic cell specific surface markers, such as CD83, CD80 or CD40, supporting that
M-MG are clearly different from dendritic cells.
Figure 4.5: Expression of chemokine receptors CCR1 (a), CCR5 (b), CXCR1(c), CXCR3 (d)
and CX3CR1 (e) measured with flow cytometry.
Expression levels in freshly isolated monocytes (A), monocyte-derived microglia (B) and the
microglia cell line HMC3 (C). The cells were cultured for 10 days.
[71]
4.5.3. Phagocytic capacity
Microglia play a major role in phagocytosis and clearance of dead cells and debris,
especially during inflammatory processes caused by injuries or neurodegenerative
diseases. To determine the phagocytic ability, we conducted phagocytosis experiments
(Meagher et al., 2005). M-MG were compared to immature M-DC, potent phagocytic
immune cells (Filgueira et al., 1996). HMC3 were used as microglia control cells.
Fluorescent-labeled S. aureus was used for measuring the phagocytic capacity of the
various cell populations.
Flow cytometry (Figure 4.6) and confocal microscopy (Figures 4.7 and 4.8) were used
for the detection and quantification of bacterial up-take. As expected, M-DC displayed
the highest phagocytosis, similar to HMC3, displayed even lower bacterial up-take.
However, both M-MG and HMC3 showed up-take of bacteria, with clear intracellular
location, as documented through confocal imaging. M-MG and HMC3 both showed
bimodal expression of labled bacteria (Figure 4.6) which may be related to the the sub-
population of cells with lower phagocytic activity.
Figure 4.6: Phagocytic capacity of M-DC (Black line), M-MG (Dotted line) and HMC3 (dashed line) towards TM Rhodamine-labelled S. aureus measured with flow cytometry.
Bacterial particles were added to culture medium at a ratio of 1:1000 (107
bacteria per ml) and
incubated for 4 hours at 37oC. Flow cytometry data represent the different levels of engulfment
fluorescent-labeled bacteria.
[72]
Figure 4.7: Up-take of TM Rhodamine-labeled S. aureus by M-MG using confocal microscopy.
M-MG were incubated for 4 hours with bacteria at a ration 1:1000. Note the intra-cellular
accumulation of red-labeled bacteria in the M-MG cells on the optical confocal section.
Figure 4.8: A selected Z-stack image taken with confocal microscopy showing the up-take of TM Rhodamine-labeled S. aureus by M-MG.
M-MG cells were incubated for 4 h with bacteria at a ration 1:1000. Note the intra-cellular
accumulation of red-labeled bacteria in the M-MG cells on the optical confocal section.
[73]
4.5.4. T-lymphocyte stimulatory capacity of MGM
As innate immune cells of the CNS, microglia are thought to be antigen presenting cells
(APC), able to process and present antigen to T-lymphocytes, activate them and induce
their proliferation. In this study, mixed leukocyte reactions (MLR) were used to
determine the T-lymphocytes stimulatory capacity of M-MG using proliferation assays.
Immature dendritic cells (M-DC) and the microglia cell line HMC3 were used as
comparison. A representative example was shown in Figure 4. 4. As expected, M-DC
induced the most pronounced and significant proliferation of allogeneic T-lymphocyte.
M-MG were also able to induce significant proliferation of allogeneic T-lymphocytes,
but to a much lower degree in comparison to M-DC. No significant T-lymphocyte
stimulatory effect was seen with HMC3, which is in accordance with previous studies
(Fischer and Reichmann, 2001, Janabi et al., 1995, Nagai et al., 2005, Zuiderwijk-Sick et
al., 2007).
Figure 4.9: Mixed leukocyte reactions (MLR)
Testing T-lymphocyte stimulatory capacity of HMC3 (black bars), M-MG (dotted bars) and M-DC
(hatched bars) at different cell ratios (antigen presenting cells: allogeneic T-lymphocytes). The
cells were cultured for 5 days before proliferation was measured using a BrdU incorporation assay.
Error bars represent the +/- 2 SD. Statistically significant differences between control and test
condition are indicated by *, P<0.05.
[74]
4.6. Discussion
Microglia play crucial roles in maintaining homeostasis and contributing to
neuroinflammation in response to any disturbances, such as injuries, toxins and pathogen
(Streit, 2001, Streit, 1996).To date, in vitro studies on microglia have used mainly
primary cells derived from embryonic or neonatal murine and human brains (Hinojosa et
al., 2011, Bassett et al., 2011). This study provides the first simple, standardized and
reliable protocol to generate human microglia-like cells from blood monocytes. The
protocol takes the advantage of a serum free culture condition, as well as a cocktail of 4
well defined recombinant human cytokines, i.e. M-CSF, GM-CSF, NGF and CCL-2. The
cells generated according to the described protocol display most known characteristics of
human microglia, including specific morphology, phenotype and functions.
In our model, the serum-free culture system is essential as it represents the physiological
condition of the extracellular fluid of the CNS, which is equal to the cerebrospinal fluid.
Normal cerebrospinal fluid (CSF) contains extremely low levels of proteins and bioactive
factors and is usually deficient in blood serum proteins (Reiber, 2003, Holman et al.,
2010). The blood-brain barrier prevents trafficking of blood serum proteins and other
factors into the extracellular space of the CNS, as they may damage glial and neuronal
cells through activation, resulting in apoptosis (Nadal et al., 1995, Abbott et al., 2010,
Hooper et al., 2009). For example, in vitro culture of rat microglia in presence of bovine
serum albumin, human or rat serum has been shown to enhance the production of
superoxide (O2−) as one of the mechanisms involved in pathogenesis of neuronal
damages (Elkabes et al., 1998). Knowing about the potential bio-active and even toxic
effects of blood serum, we established therefore this serum free protocol for the
generation of microglia-like cells derived from blood monocytes.
The cocktail of cytokines has been chosen according to the known effects of the
cytokines on microglia in published reports, as stated in the introduction section, and
extensive preliminary experiments. Thereby, the combination of M-CSF, GM-CSF, NGF
and CCL-2 has shown to differentiate M-MG with most similar characteristics to
microglia.
Under healthy condition, resting “surveying microglia” with ramified morphology is
responsible for maintaining the homeostatic condition of the CNS. Using the described
protocol, M-MG obtained a ramified morphology after 10-14 days in culture, including a
polygonal or oval cell body and up to seven primary processes and numerous fine
secondary process or fine spines. Our morphological findings for M-MG correspond to
[75]
the report by Leone et al. (2006), who used a less well-defined protocol. In addition, M-
MG displayed similar morphology to the human microglia cell line HMC3.
Most important was the phenotypic characterization of the cells using a broad panel of
antibodies and flow cytometry. Monocytes cultured over 2 weeks in the presence of the 4
cytokines changed significantly the expression of surface markers towards the known
pattern for microglia. CD45, CD14 and HLA-DR expression levels decreased
substantially in M-MG reaching levels expected for microglia. CD45 has been often used
for discriminating between tissue macrophages and microglia in CNS studies
(Lambertsen et al., 2009, Wirenfeldt et al., 2005). Penninger et al. (2001) states that in
the CNS, microglia express different levels of CD45 depending on their differentiation
state, with decreased expression levels in mature resting microglia. However, CD45
expression increases in activated microglia, e.g. in Alzheimer patients, as this surface
marker seems to play a role in degenerative and inflammatory CNS diseases (Masliah et
al., 1991). CD14 is also regarded as an important LPSreceptor (Triantafilou and
Triantafilou, 2002). Supporting our protocol, Lambert et al. (2008) reported down-
regulation of CD14 in monocyte-derived DC and microglia in presence of GM-CSF, M-
CSF and LPS.
The generation of T cell immunity requires the acquisition and presentation of Ag on
bone marrow-derived APCs. Both dendritic cells and macrophages with high expression
level of MHC class as well as co-stimulatory molecules, can stimulate naive CD8 T cells
in vivo to proliferate, develop effector function, and differentiate into memory cells
(Pozzi et al., 2005). Because of the low expression of MHC class II, B7 and CD40, the
essential molecules necessary for antigen presentation, microglia seem to be potential
APCs that can mount the immune response in the CNS. However, there is not much
known about antigen presenting ability of microglia as well as its role in physiological or
pathological immune responses in the CNS (Ulvestad et al., 1994). Compared with
resting microglia, microglia activation during CNS injury or infection could respond
more promptly to Th1-derived signals and behave as a more potent APC for T cell
restimulation (Aloisi et al., 2000, Streit et al., 1996). In line with previous data, our data
showed non-activated M-MG decreased HLA-DR expression to low levels, similar to
HMC3, but to much lower levels than freshly isolated monocytes or immature M-DC
(Lutz et al., 2000). Furthermore, it was important to discriminate between M-MG and M-
DC, as both may display similar morphological properties. In addition to the differences
in expression levels of MHC class II, CD11b and CD11c, M-MG did not express
characteristic M-DC markers, including CD40, CD80 and CD83. The differences
[76]
between M-MG and M-DC were further supported by the functional tests, such as MLR
and phagocytosis assays. As expected, M-MG displayed much lower T-lymphocyte
stimulatory capacity and bacterial up-take, in comparison with M-DC. In summary, our
data provide clear evidence that our M-MG protocol does not generate M-DC, but a
distinct microglia-like population.
Our study also revealed the expression level of various chemokine receptors, including
CCR1, CCR2, CCR4, CCR5, CXCR1, CXCR3 and CX3CR1 in M-MG and HMC3.
Freshly isolated monocytes showed intermediate expression level of CCR1, low
expression levels for CCR2, CXCR1 and CCR5, and undetectable expression levels of
CCR4, CXCR3 and CX3CR1, as reported previously by others (Geissmann et al., 2003).
M-MG changed expression levels of the various chemokine receptors substantially, in
comparison with freshly isolated monocytes. However, expression patterns of M-MG
were very similar to that of HMC3. M-MG showed down-regulation of CCR1 and CCR2
to still well detectable levels. On the other hand, M-MG displayed significant up-
regulation of CCR4 and CCR5. Interestingly, M-MG expressed CXCR3 and CX3CR1,
which were not detected in monocytes. Up-regulation of CXCR1 was also seen in M-
MG. Our findings are in accordance with the report by Bajetto et al. (2002), who
published that non-activated microglia express CCR1, CCR2 and CCR5 constitutively at
low levels. Several other researchers reported that activated microglia express higher
level of CCR2, CCR3, CCR4, CCR5, CX3CR1 (Simpson et al., 2000b, Eltayeb et al.,
2007, Szczuciński and Losy, 2007). However, little is known about chemokine receptor
expression in human microglia. Our protocol may help in future research to elucidate
regulation of these receptors and chemokine responses in human microglia.
4.7. Conclusion
We developed a new in vitro protocol for the generation of human microglia from blood
monocytes using a serum free culture condition and a novel mixture of four recombinant
human cytokines, i.e. M-CSF, GM-CSF, NGF and CCL2. Detailed characterization of
M-MG revealed a cell population representing resting microglia with their specific
morphological, phenotypic and functional properties. The described protocol is easy to
handle, well standardized and very reproducible, as it uses only well defined culture
medium and recombinant cytokines, but no serum or conditioned medium. This protocol
will certainly be very helpful for future studies investigating adult human microglia.
[77]
Chapter 5
Lead modulates chemokine receptors
expression pattern and induce
interleukin-8 in human microglia
[78]
5. Lead modulates chemokine receptors pattern and induce interleukin-8 in human microglia
5.1. Introduction to chapter
In the previous chapter, we showed differentiation and characterization of human
microglia derived from blood monocytic cells in the presence of a novel mixture of
cytokines. In this chapter, we investigated the effect of lead exposure on an in vitro
human monoycte-derived microglia model assessing morphology, phenotype and
function with the main focus on chemokine receptor expression pattern.
Lead is an environmental toxin with detrimental known effects on the functional and
development of CNS even at the low concentration. Despite the wealth of data on the
neurotoxic effect of lead in children, mechanism underlying its toxicity at the cellular and
molecular levels still need be studied. Microglia cells, the resident macrophages of the
brain, play important role in haemostasis of the CNS as neuroprotective cells (secretion
of anti-inflammatory cytokine, chemokine and their receptors, neurotrophic factors, etc).
Microglia activation which usually occurs in response to stimuli (toxin), has been shown
to be involved in many neuroinflammatory processes. Although recent animal studies
showed that lead may cause microglia activation and enhance hippocampal injuries
through changes in the secretion of pro-inflammatory cytokines, there is no evidence
showing the exact role of human microglia in lead induced toxicity or inflammation.
Therefore, we were interested to study whether exposure to low concentration of lead
over short period of time could affect the survival and function of the human microglia;
in particular, immune properties of these cells by alteration in chemokine receptors
expression pattern. Our results indicate that low level of lead exposure has no cytotoxic
effect on the morphology or viability of monocyte-derived microglia. General down
regulation in the expression of chemokine receptors expression was observed with
significant reduction in the expression of CCR1, CCR2, CCR3 and CXCR1, as well as
CD68. In addition, lead exposure induced increased secretion of interleukin-8 (IL-8), a
chemokine that is unique to humans and has recently been shown to contribute to brain
remodelling and memory loss. In summary, lead exposure can modulate
neuroinflammatory properties in human microglia. Declaration of work: All the
experiments were done by SE including cell culture, morphological studies, flow
cytometry, cytokine assay under supervision of Prof. Filgueira. A manuscript has been
prepared and submitted to Journal of Neurotoxicology (in revision at time of submission
of thesis).
[79]
5.2. Abstract
Lead (Pb) is a widely spread environmental pollutant with potentially detrimental effects
on major organ systems, including the central nervous system. Lead is therefore
considered a major health concern for humans. Several recent studies have confirmed the
neurotoxic effects of lead on development and function of the brain, even at low blood
concentrations (<10 µM). Thereby, lead has been shown to influence synaptogenesis and
neurogenesis, to negatively affect learning and memory, as well as to activate glia cells.
However, the molecular and cellular mechanisms involved in lead-induced neurotoxicity
in humans are still not well understood, especially for exposure to low lead
concentrations and for the contribution of microglia to lead toxicity. Microglia are the
resident immune cells of the CNS with a prominent role in physiology and pathology of
the brain. Over the past few years, environmental toxins, including lead, have been
reported to activate microglia in animal studies. Activated microglia have been shown to
contribute to neuroinflammation and neurodegenerative diseases. However, little is
known about the role of human microglia in lead induced neurotoxicity.
In this study, we investigated the effects of lead on human microglia in vitro at
concentrations, expected to represent in vivo concentrations found during lead
intoxication. For that purpose, a new human microglia model was applied. Human
microglia cells were exposed to 1-100 µM of lead acetate for 1 hour to 5 days.
Subsequently, changes in morphology, phenotype, cytokine secretion patterns and
function were investigated. Our results indicate that lead is taken up by microglia and
accumulates inside the cells, as visualized with specific fluorescent probes, using
fluorescence microscopy and flow cytometry. Lead exposure induced minor, but
significant morphological changes at higher concentrations (>50 µM), but had no
influence on morphology at lower concentrations (10 µM). Interestingly, lead was not
cytotoxic and did not influence microglia viability at lower concentrations. However,
lead had substantial effects on the expression patterns of surface markers that are
involved in inflammation, cell activation and migration, with significant reduction in
expression of CCR1, CCR2, CCR3 and CXCR1, as well as CD68. In addition, lead
exposure induced increased secretion of IL-8, a chemokine that is unique to humans and
that has recently been shown to contribute to brain remodeling and memory loss. In
summary, our results indicate that human microglia are significantly affected by lead
exposure, even at lower concentrations, resulting in discrete cell activation and
significant changes in chemokine receptor and surface marker expression, as well as
increased secretion of IL-8. We can therefore conclude that lead exposure, even at lower
[80]
concentrations, has a significant influence on microglia function, with possible
substantial consequences for homeostasis, tissue remodeling and immune responses in
the central nervous system.
Keywords: human microglia, in vitro model, M-MG, lead, IL-8, chemokine receptors
5.3. Introduction
Since humans started to process and use metals for their commodities, lead has widely
spread and become a persistent environmental pollutant. With that in mind, regulations of
how to deal with lead mining, processing and applications have reduced environmental
lead exposure in a number of countries, including Australia, Germany, Mexico, Sweden
and USA. Blood lead levels have declined on average below officially accepted toxic
concentrations in those populations (Canfield et al., 2003; Kasten-Jolly et al., 2011;
Koller et al., 2005; Sharma et al., 2011). However, lead exposure and corresponding
accumulation in people still happens at lower concentrations. Therefore, recent studies
have focused on mechanisms involved in low-level lead-induced toxicity (Bellinger,
2008; Dietert and Piepenbrink, 2006; Gidlow, 2004; Lanphear et al., 2000).
Lead is known to induce neurotoxicity even at low levels, resulting in irreversible,
detrimental effects on development, growth and cognitive functions (Finkelstein et al.,
1998). Lead enters the CNS by crossing the BBB. Foetuses and children are more
vulnerable than adults, as their BBB is not fully formed. In addition, their brain is more
susceptible to toxins, as it is still developing and growing. Lead seems though to interfere
with neuronal recruitment and differentiation (Baranowska-Bosiacka et al., 2013).
Several human and animal studies have reported the significant negative effects of lead,
even at lower blood levels (<2 µg/dl or <0.1 µM), on the cognitive function of
adolescents (Lucchini et al., 2012). Various published studies have reported that lead
exposure results in loss of hippocampal neurons, uncontrolled secretion of
neurotransmitters, alteration in neuronal growth rate, demyelination of nerve fibers,
proliferation and activation of astrocytes, as well as in breakdown of the BBB, causing a
deficit of learning and memory, and the decline of the IQ (Liu et al., 2012). It has been
shown that lead may substitute calcium and zinc resulting in its many toxic actions
(Marchetti, 2003). However, the molecular mechanisms in lead-induced neurotoxicity are
still not well understood.
Using animal models, lead exposure has been shown to potentially contribute to
pathogenesis of neurodegenerative diseases, such as Alzheimer’s disease, by inducing
glial cell activation and expression of the amyloid precursor protein (Monnet-Tschudi et
[81]
al., 2006). Lead has also been shown to modify cytokine gene expression patterns in the
murine CNS (Kasten-Jolly et al., 2011). In addition, lead-induced neurotoxicity has been
correlated with oxidative stress by generating reactive oxygen species (ROS), as well as
membrane lipid peroxidation (Adonaylo and Oteiza, 1999).
Interestingly, proliferation of T lymphocytes has also been observed in vitro after lead
exposure, indicating that lead has also an influence on immune responses (McCabe et al.,
2001). However, little is known about the effects of lead on human microglia.
Microglia are the innate resident immune cells of the CNS and play a crucial role in
maintaining the healthy and physiological homeostasis of the CNS. Resting microglia
with their branched processes form a dense cellular network, survey actively the nervous
parenchyma and respond rapidly to any disturbances, such as pathogens, toxins and
injuries, by retracting the processes and forming amoeboid activated microglia cells
(Block et al., 2007). Interestingly, the view of microglia function has been challenged
over the past decades. It has been recognized that activated microglia play an important
role in neuroinflammation and neurodegenerative diseases, even at early stages. Thereby,
microglia-derived soluble immune factors play a crucial role, including cytokines and
chemokines, as well as their receptors. Cytokines contribute to immune modulation in the
CNS, but they have also been reported to play an important role in neuronal development
and survival, by influencing neuronal activities, cell-cell interaction and neuronal-
endocrine communication (Boulanger, 2009; Deverman and Patterson, 2009). It has been
well documented that most CNS cells, including neurons, astrocytes and microglia, are
producer and targets of cytokines (Hopkins and Rothwell, 1995). In the healthy brain,
various cytokines, such as neurotrophins and pro- and anti- inflammatory factors, have
been shown to be constitutively expressed by various cell types (Elkabes et al., 1996).
Despite detection of cytokine mRNA under normal conditions, resting microglia have
been reported to secrete cytokine protein only at low or undetectable levels.
In turn, once microglia are activated, expression of various pro-inflammatory cytokines
are up-regulated, including Tumor Necrosis Factor (TNF)-α, Interleukin (IL)-1β, and IL-
6, contributing to neuroinflammation and neuronal cell death (Hanisch, 2002; Szelényi,
2001). In line with neurotoxic effects of activated microglia, several neurotrophic and
anti-inflammatory cytokines (IL-4, IL-10, TGF-β) have also been shown to be secreted
by those cells (Aloisi, 2001). Microglia, as well as astrocytes and neurons, also express a
large number of chemokines and chemokine receptors with potential effects on
development and homeostasis of the CNS, mainly through regulating cell migration (Cho
and Miller, 2002; Etemad et al., 2012; Flynn et al., 2003).
[82]
During microglia activation, up-regulation of chemokine receptors have been shown in
several neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease,
human immunodeficiency virus-associated dementia and multiple sclerosis (Wyss- Coray
and Mucke, 2002). Pro-inflammatory chemokines may also induce neuronal cell death
directly, or through microglia activation (Cartier et al., 2005).
To date, little is known about lead accumulation in microglia. However, astrocytes have
been shown to take up substantial amounts of lead, even more than neuronal cells, which
is enhanced in a neuron-glia co-culture models (Tiffany-Castiglioni, 1993; Tiffany-
Castiglioni and Qian, 2001). Recently, low–level, but long-term lead exposure has been
shown to activate rat microglia, resulting in memory deficiency (Liu et al., 2012). Lead is
well known for inducing deficits in spatial memory and learning (Carpenter et al., 1994).
However, the mechanisms for this phenomenon are still not well understood.
Interestingly, increased IL-8 secretion by microglia induces impairment of long-term
potentiation, responsible for memory deficiency (Xiong et al., 2003). Unfortunately, little
is known about IL-8 effects in the CNS, as this chemokine is unique to humans, but not
found in rodents (Edman et al., 2008). However, human peripheral blood mononuclear
cells (PBMC) have been shown to up-regulate expression of IL-8 when exposed to low-
level of lead (Gillis et al., 2012).
In this study, we investigated the effects of lead exposure on human microglia in vitro,
focusing on morphology, viability, chemokine receptors expression patterns and cytokine
secretion.
5.4. Material and methods
5.4.1. Cell culture
We used a novel human in vitro microglia model established by our group (Chapter 4).
Briefly, to generate human microglia, Human blood mononuclear cells (PBMC) were
isolated from buffy coats (100 buffy coats were used for this study) of healthy donors
(Australian Red Cross Blood Service (ARCBS), Perth, WA, Australia; ethics approval
was granted by UWA and ARCBS). The Monocytes (adherent PBMC) were seperated
from non-adherent cells (consisting mainly of T-lymphocytes) through plastic adherent
method (as mentioned in Chapter 4). Adherent PBMC (1×105
cells/ml) were cultured in
RPMI 1640 Glutamax medium and 1% antibacterial/antimycotic solution (Gibco),
supplemented with M-CSF (10 ng/ml; PeproTech, Rocky Hill, NJ), GM-CSF (10 ng/ml;
PeproTech), β-nerve growth factor (NGF-β; 10 ng/ml; PeproTech) and CCL2 (100
[83]
ng/ml; PeproTech), at standard humidified culture conditions (37oC, 5% CO2) for up to
14 days.
5.4.2. Lead exposure
Lead acetate [Pb(CH3CO2)2·3H2O] (Sigma-Aldrich, St. Louis, MO) was prepared at the
concentration of 100 mM as stock solution under sterile conditions. In order to study the
effect of lead on human microglia, lead was directly added at the concentration of 1, 5,
10, 25, 50 and 100 µM to the cells (depending on the experimental design) at the culture
days, 7-10 for different time period, including 1 hour, 24 hours, 3 and 5 days. To ensure
that lead effects were not caused by possible, even low endotoxin contamination, LPS(10
ng/ml, E. coli serotype 0111:B4, Sigma -Aldrich).were included in positive control
experiments, clearly showing completely different results.
5.4.3. Morphological studies
Microglia were cultured in the presence or absence of 10 µM lead for 24 hours.
Morphological appearance and changes of cell shape were documented by using an
inverted Leica DM IL microscope, equipped with phase contrast and various adequate
fluorescence filters. Images were taken with a Photometrix Cool SNAP Fx digital camera
and processed with the NIS- elements F3.0 software. Sytox green, a nucleic acid stain
(Invitrogen) was applied to exclude the dead cells in morphological quantification (Luo
et al., 2001). Cells were incubated with 1 µM Sytox Green for 30 min at 37 oC. Five
random optical fields were selected. Images were taken with fluorescent filter and bright
field microscopy. After overlaying the images, a total of 100 live cells were counted. In
repeated independent experiments, we also quantified the cell numbers with branching
changes by staining microglia with an antibody against Iba-1, a macrophage/microglia
marker, using a standard immunostaining protocol (Blomster et al., 2011).
Immunostaining was performed with microglia cultured on cover slips, fixed with 1%
paraformaldehyde in PBS, containing 2 % sucrose. After permeabilization with 0.01%
Triton X100 in PBS, cells were incubated overnight at 4°C with rabbit anti- Iba-
1(1:100,Wako Pure Chemical Industries), with subsequent incubation for 1 hour with
Alexa Fluor 488-conjugated donkey anti-rabbit antibody (1:200, Invitrogen) and DAPI (1
µg/ml, Roche, Manheim, Germany). Slides were analyzed using fluorescent microscopy
as described above and images were taken. According to previous studies, microglia with
less than two branches were scored as activated (Kauppinen et al., 2008).
[84]
5.4.4. Viability assays
To assess the effects of lead on viability of human microglia, MTS assays were carried
out (Cell Titer 96 AQueous Assay, Promega, Madison, WI). Briefly, cells were seeded in
96 well plates (5x105 cell/ml, 200 µl per well) and cultured to reach optimal confluence.
Then cells were incubated with various lead concentrations (0, 1, 10 and 50 µM,
quadruplets for same condition) for 24 hours. Thereafter, 20 µl of Cell Titer 96®
AQueous One Solution (MTS) was added directly to each well. Cells were incubated for
4 hours at 37oC and the absorbance of the colorimetric reaction was measured by a plate
reader (Labsystem Multiscan RC) at 490 nm. Viability of lead-treated microglia was
compared with the non-treated control and statistically analyzed using the Student’s t-test
and ANOVA (Vairano et al., 2004).
An additional method, namely the propidium iodide dead cell exclusion test, was also
used for testing microglia viability exposed to lead (Kauppinen et al., 2008). For that
purpose, cells were treated with different concentration of lead (as mentioned above) for
24 hours. Then, 2 µg/ml propidium iodide was added to the culture medium. Morphology
and viability of the cells were quantified using fluorescent microscopy. For
quantification, five randomly selected fields were chosen in each well and at least 100
cells were tabulated. Dead cells were labeled with propidium iodide and seen in orange
color under the fluorescent microscope. In addition, the samples were also assessed with
FACSCallibure (BD Biosciences, Mountain View, CA). Data analysis was performed
using FlowJo software.
5.4.5. Detection of up-take and intracellular accumulation of lead
We investigated the up-take and intracellular accumulation of lead by microglia with the
specific lead detection fluorescent probe Leadmium Green AM (Invitrogen) using flow
cytometry. In addition to Leadmium, we studied the expressdion level of DCF Newport
Green (Molecular Probes, Eugene, OR) as less specific lead detector. Human microglia
were incubated overnight with different concentrations of lead (5, 10, 25, 50 and 100
µM). Then, the cells were incubated with 1 µM Leadmium Green AM dye in saline for
30 minutes to 2 hours. To test the DCF Newport Green, in separate experiment, cell were
also cultured with 1 µM DCF Newport Green dye in saline for 30 minutes to 2 hours.
Cells were analyzed by flow cytometry using the 488nm excitation and measuring
fluorescent emission at 520nm. Non-lead treated cells were included in the experiment as
negative controls (Zeller et al., 2010). FlowJo software was used for further analysis of
data. Representative histograms for each experimental group were overlayed and mean
[85]
fluorescent intensity (MFI) were compared statistically using the Student’s t-test.
Experiments with cells from different donors were repeated five times. Another
fluorescent probe, which is less specific, Newport Green DCF (Invitrogen), was also used
to detect lead up-take by human microglia.
5.4.6. Reactive oxygen species
We studied the effect of lead on ROS production by human microglia using the
fluorescent dyes Dihydroethidium (DHE) to detect superoxide and BODIPY® 581⁄591
C11 to detect lipid peroxidation. Cells were incubated with different concentrations of
lead, including 0, 5, 10, 25 and 50 µM for 24 hours. Five separate experiments with cells
from different blood donors were performed. Subsequently, each sample was separately
incubated with DHE (1 µM) or BODIPY (2 µM) for 90 minutes. Cells also were cultured
with LPS (10 ng/ml, E.coli, Sigma-Aldrich) for 24 hours as positive control. Mean
fluorescence intensity of the samples was measured using the FACS Canto flow
cytometer (BD, Bioscience). After analyzing the flow cytometry data with FlowJo
software, they were statistically compared to controls using Student’s t-test, whereby
mean fluorescence intensity (MFI) of each group were calculated and compared for DHE
or Bodipy (Kauppinen et al., 2008; Yang et al., 2007).
5.4.7. Expression of surface markers
We studied the effect of lead (10 µM) on the expression patterns of specific surface
molecules in human microglia using flow cytometry. Cells were divided into two groups,
non-treated and lead-treated, which were exposed to lead for 24 hours. After detaching
and washing twice with PBS, the cells were stained with direct labeled antibodies, or
using primary unlabeled and corresponding labeled secondary antibodies.
A panel of directly labeled mouse anti-human antibodies was applied at the supplier’s
recommended concentration. The antibodies were directed against the following antigens
and containing corresponding label: CD45-FITC, CD14-PE, CD40-PE, CD80-PE, CD68-
AF647, HLADR–PERCP, CD11b-AF488, CD11c-PE, CCR1-AF647, CCR2-AF647,
CCR3-PE, CCR4-PE-Cy7, CCR5-FITC, CCR6-AF647, CCR7-PE, CCR9-fitc, CXCR1-
PE, CXCR3-AF488, CXCR4-PE and CXCR5-FITC (all from BD Bioscience).
Corresponding labeled isotype control antibodies were included in all experiments, as
well as non-treated cells. A rabbit anti-CX3CR1 polyclonal antibody was used at the
final concentration of 1 µg/ml (AbD Serotec, Oxford, UK), followed by a secondary
donkey anti-rabbit-AF 647 used at the final concentration of 1:200 (Invitrogen). Cells,
[86]
which were not labeled with the primary antibody, were used as negative control.
Corresponding excitation and emission wavelengths of the used fluorochrome were
FITC: 494, 519; PE: 496/546,578; PerCP: 482, 678; APC: 650, 660; Alexa Fluor -488:
495, 519; Alexa Fluor -647: 650, 668. The cells were incubated with antibodies for 30
minutes, washed with PBS, resuspended and fixed with 1% formaldehyde in PBS,
containing 2% sucrose. The fixed samples were measured with a FACSCallibure (BD
Biosciences). Data analysis was performed using FlowJo software.
5.4.8. Cytokine production
The cytometric bead array (CBA) human inflammatory kit (BD Biosciences) was used
for the quantitative measurement of cytokine production and secretion under control and
lead-exposed conditions. For that purpose, human microglia were incubated with and
without lead at various concentrations for 24 hours, as explained above. The following
day, 50-100 µl of the medium was collected and stored at -20oC. The assay was
performed according to the manufacturer’s instruction using 50 µl of the human
inflammatory cytokine standards and sample dilution. Cytokine concentrations were
measured using the FACSCanto flow cytometer (BD Bioscience). FCAP Array, version
3, software was used to analyze the data. Measurements were done in duplicates and
repeated once with 5 independent experimental repeats.
5.4.9. Statistics
Data were statistically analyzed with the SPSS statistical software (version 17.0). A one-
way ANOVA and univariant linear model were used to compare means. The
LSD/Dunnett post-hoc test also used to determine the significant difference between
groups. In all the analysis p value<0.05 were considered as statistically significant. Bars
in graphs represent the standard error (SED).
5.5. Results
5.5.1. Detection of lead uptake and intracellular accumulation by
human microglia
To study lead uptake and intracellular accumulation in human microglia, we used the
fluorescent probe Leadmium Green and flow cytometry. Leadmium Green has been
reported to be a very sensitive and specific probe for lead detection at nM levels. The
cells were incubated with lead at 5, 10, 25, 50 and 100 µM for 24 hours. Then, cells were
incubated with 1 µM Leadmium Greenand 1 µM DCF Newwport Green dyes for 30
[87]
minutes and mean fluorescence intensity (MFI) was calculated using flow cytometry.
Non- lead treated cells were included in each experiment as negative control. Lead
uptake was detectable as green fluorescence signal in the microglia cells from 5 µ
concentrations onwards in a concentration-dependentmanner (Figure 5.1 C). Mean of
fluorescence intensity ± SD of the Leadmium for the M-MG cultured with different
concentration of lead, 0 to 100 µM respectively were 3.2 ± 0.07,5.88 ± 0.19, 8.51 ± 0.32,
10.36 ± 0.05, 14.64 ± 2.3 and 30.38 ± 1.56. Similar results but at lesser extent were
obtained when using a different, less specific fluorescent probe, Newport Green DCF
(Figure 5.1 D). These data clearly indicate that human microglia are able to take up and
accumulate lead intracellularly.Mean of fluorescence intensity ± SD of the Newport
Green for the M-MG cultured with different concentration of lead, 0 to 100 µM
respectively were 4.26 ± 0.8, 4.68 ±0.45, 5.17 ±0.42, 7.34 ±1.4, 10.36 ±0.5 and 15.7
±0.87.
Figure 5.1: Lead uptake by human microglia.
These figures show uptake and accumulation of lead in human M-MG using the Leadmium and
DCF Newport Green using flow cytometry (n=5). Figures A and B are the representative FSC:SSC
dot plots of M-MG in absent/presence of 10 µM lead from one representative experiment out of
five with similar results. Histograms compare the relative expression of Leadmium (C) and DCF
Newport Green (D) to the corresponding amount of intracellular lead. the absence (a) or presence
of various concentration of lead, including 5 µM (b), 10 µM (c), 25 µM (d), 50 µM (e) and 100 µM
(f).
[88]
5.5.2. Effect of various lead concentration on human microglia cell
morphology and activation
Microglia react to toxic effects with quick morphological changes and activation,
resulting in a less branched and amoeboid appearance. Accordingly, we studied the effect
of lead on morphological changes in human microglia using live cell imaging. As
mentioned before, five random optical fields were selected. One hundred live cells from
each experiment were quantified and scored as activated, when displaying less than two
branches. In separate experiments, cells were stained with Iba-1 as microglia marker and
number of green fluorescent labeled branches was counted. Under control conditions (no
lead exposure) and at exposure of up to 10 µM lead, microglia showed a resting (non-
activated) morphology with usually more than four branched processes (Figure 5.2. A).
However, morphological changes were clearly seen from 50 µM lead concentration
onwards, causing the cells to retract their processes and to become more round shaped,
which suggests changes towards the activated form (Figure 5.2. B, C). Quantification
showed that more than 75% of the cells displayed ramified morphology in the control
group (0 µM lead). Only at lead concentrations above 25 M, cells showed significant
morphological changes compared to the control group (Figure 5.2. D). No significant
changes in morphology (size of the cell body, number of branches) were detected after
administration of the lower lead concentrations (<10 µM) for 24 hours. The
morphological changes documented with microscopy were confirmed and quantified
using the more sensitive method of fluorescent microscopy (data not shown; Kauppinen
et al., 2008; Minami et al., 2012).
[89]
Figure 5.2: Effect of lead exposure on morphology of microglia.
Morphological appearance of human microglia exposed to various concentrations of lead 0 µM
(A), 10 µM (B), 50 µM (C) for 24 hours as documented with phase contrast light microscopy
(representative of 5 experiments, bar indicates 100 µm). Quantification of branched and round
shaped morphology is shown in the bar chart in D as percentage of total live cells. Cells were
scored as activated/round shaped when displaying less than two branches. * indicates significance
at P<0.05, n=5. Error bars indicate SEM.
[90]
5.5.3. Effect of various concentration of lead on human microglia
viability
We investigated whether lead affected the viability of microglia cells. For that purpose,
human microglia were treated with various concentrations of lead (1, 10 and 50 µM) for
24 and 72 hours. Then, we assessed the viability of the cells using the MTS test (Figure
5.3. A). Viability of the non-treated cells (control group) was considered as 100%
(baseline) and the experimental groups were correspondingly compared to these controls.
Results showed that lead had no effect on viability at the concentration of 1 and 10 µM.
To assess microglia survival rate, we also calculated the ratio of dead to live microglia
using fluorescence microscopy, after exposure to 5, 10, 25, 50 µM lead for 24 hours.
Statistical analysis showed no significant differences between the lead-treated
experimental groups and the control cells at lower lead concentrations. Moderate increase
in number of dead cells was only seen at the concentration of 50 M, which was not
statistically significant. Overall, these data indicate that low lead concentrations (<10
µM) have no effect on the viability of human microglia.
Figure 5.3: Viability of human microglia after lead exposure.
A) Bar charts represent the percentages of live cells after exposure to various lead concentrations
(0, 1, 10 and 50 µM) for 24 hours (black bars) and 72 hours (white bars). Viability of the cells was
assessed by a MTS colorimetric test. Viability of negative control group (non-lead treated) was
used as baseline and normalized to 100%.Difference between two groups (lead-treated/non-treated
cell) were statistically analyzed using the Student’s t-test and Dunnett’s test (n=20, * indicates
significance with P<0.05). B) Stacked columns represent the fraction of dead (black) to live
(Green) microglia in the presence of various concentration of lead (0, 5, 10, 25 and 50 µM) for 24
hours. Dead cells were labeled with 2 µg/ml propidium iodide and counted using fluorescent
microscopy. Ratio of the dead to live cells is shown as percentage. Difference between two groups
(dead and live cells) were statistically analyzed using the Student’s t-test and Dunnett’s test (n=20,
P<0.05).
[91]
5.5.4. Effects of lead exposure on reactive oxygen species production
and lipid peroxidation in human microglia
Many studies have shown that activated microglia release reactive oxygen species (ROS)
in response to environmental stimuli, such as toxins. To study whether lead exposure
induced ROS production in human microglia, we used the fluorescent probes
Dihydroethidium (DHE) as superoxide detector and BODIPY® 581⁄591 C11 to measure
lipid peroxidation. Lead treated and untreated cells were stained with these fluorescent
probes. Propidium iodide and SYTO green labelling was included to determine live and
dead cells. Fluorescent intensity was measured using flow cytometry.Cells also cultured
overnight with LPS (10 ng/ml) as positive control. Only live cells were included in the
analysis. Figure 5.4. A. and B.shows the location of cells in FSC:SSC axis in absent and
presnt of lead treatmnet (10 μM lead). Figure 5.4. C is representative histograms from
one experiment out five experiments.. Overall, microglia treated with 5 µM lead showed
moderate increase of ROS production followed by a decrease of ROS towards 50 µM
lead exposure. Mean of fluorescence intensity ± SD of the DHE for the M-MG cultured
with different concentration of lead, 0 to 50 µM as well as LPS respectively were 151.22
±9.76, 176.6 ± 7.76, 159.44 ± 8.87, 144 ± 14.57, 151.2 ± 9.88 and 356.8 ± 52.2. The
mean ± SD of the isotype control was also 3.52 ± 0.67. Similarly, no effect of lead on
lipid peroxidation in the human microglia was seen at the tested lead concentrations
(Figure 5.4 D). One can conclude that low lead concentrations have no significant effect
on ROS production and lipid peroxidation in human microglia. Mean of fluorescence
intensity ± SD of the BODIPY for the M-MG cultured with different concentration of
lead, 0 to 50 µM as well as LPS respectively were 4.44 ± 0.29, 5.06 ± 1.06, 4.124 ± 0.85,
4.6 ± 0.77, 5.492 ± 0.87 and 163.6 ± 15.8. The mean ± SD of the isotype control was also
3.24 ± 0.82.
5.5.5. Effect of lead exposure on expression of chemokine receptors and
surface molecules in human microglia
Chemokines and chemokine receptors are considered to play a key role in the initiation
of inflammation and cell migration in the context of CNS development and remodelling.
As reported previously, most of the chemokine receptors are expressed by microglia, due
to their involvement in immune responses (Chapter 4; Flynn et al., 2003). We were
interested in investigating whether lead may affect the pattern of chemokine receptors
expression in human microglia. After 7-10 days in culture, monocyte-derived microglia
were exposed to 10 µM lead for 24 hours and compared with non-treated control cells.
[92]
Figure 5.5 shows representative histograms for the expression pattern of chemokine
receptors in lead treated cells as compared with control cells, including CCR1, CCR2,
CCR3, CCR4, CCR5, CCR9, CXCR1, CXCR3, CXCR4 and CXCR5. Figures 5.6. A and
5.6. B summarize the expression pattern of all relevant markers tested in all experiments
(n=30). Overall, the presence of lead changed expression of most chemokine receptors.
However, only CCR1, CCR2, CCR3 and CXCR1 expression was found to be
significantly decreased in comparison with the non-treated microglia. Down-regulation
was also found for CCR5, CCR6, CXCR4 and CXCR5, but which was not statistically
significant (P 0.86, 0.45, 0.45 and 0.67). However, CCR4, CCR9 and CXCR3 were up-
regulated, although this was not statistically significant. The cells expressed CCR7 at
very low level and no changes were seen after lead exposure.
Figure 5.4: Effect of lead on ROS production and membrane peroxidation in human microglia.
Cells were incubated for 24 with various lead concentration (0, 5, 10, 25 and 50 µM) before they
were incubated for 30 minutes with DHE (1 µM) for ROS detection, or BODIPY (2 µM) for
detection of membrane peroxidation. LPS (10 ng/ml) were used as positive control. Fluorescence
intensity was quantified with flow cytometry. A) Filled histograms represent DHE treated cells,
whereas the open curve to the left represents cells not labeled with DHE. B) Summary of changes
as mean fluorescent intensity of the DHE and BODIPY (n=5). Mean ± SD of the MFI were
calculated for all samples and statistically analyzed using the Student’s t-test.
.
[93]
Figure 5.5: Effect of lead exposure (10 µM, 24 hours) on chemokine receptors and surface marker expression patterns in human microglia as measured with flow cytometry.
Examples of one representative experiment. Black histograms for lead-untreated control cells and
white histograms for lead treaded cells. Blue histograms are the isotype controls. Cells were
cultured for 7-10 days (n=30). Shifting black comparing to white histogram to the right is
representing the increase in expression level of marker in presence of lead and vice versa. Double
picks in CCR5 and CXCR1 are showing sub-populations in M_MG express different levels of
these markers.
[94]
Figure 5.6: Comparison between expression pattern of surface markers in the absence and presence of lead in human microglia
Comparison between control cells (white bars) and lead-treated cells for the expression of various
surface markers. B) Comparison between control cells (white bars) and lead-treated cells 10 µM
lead, 24 hour, for expression of various chemokine receptors. Data represent the mean florescent
intensity. Analysis of the significant differences between two groups was done by ANOVA and
LSD/Dennett’s test. (* indicates, n=30).
A
B
A
[95]
5.5.6. Effect of lead on cytokine secretion patterns in human microglia
Microglia activation has always been associated with the expression of cytokines, which
contribute to neurotrophic and neuroinflammatory events. Cytokines may also contribute
to the modulation of microglia activation (Vila et al., 2001).We used a cytometric bead
array for human inflammatory cytokines to determine whether lead exposure induced
changes in inflammatory cytokine expression patterns in human microglia. Cells were
cultured in the presence of different concentration of lead (0, 10, 50, 100 µM) for 24
hours. Culture medium supernatants were collected and used for the measurement of a
panel of inflammatory cytokines, including IL-1β, IL-8, IL-6, IL-12p70, TNF- and IFN-
(Figure 5.7). Of the six evaluated cytokines, only IL-8 was found to increase two folds
which was statistically significant (P0.036), when human microglia were exposed to
10µM lead, namely from 4730 pg/ml (lead-untreated control cells) to 8986 pg/ml (10 µM
lead). Furthermore, highest lead exposure to 100 M not only induced significant
increase in IL-8 expression (37569 pg/ml, P=0.000) but also significant enhancement
was observed in expression of IL-6, TNF- and IL-1β respectively (406, 846 and 1099
pg/ml, P<0.05). Slight increase in expression of IL-6 was also seen following exposure to
10 µM lead, however this increase was not statistically significant.Expression of all other
cytokines was at low detectable levels and was not influenced by lead. It is noteworthy
that production of all tested cytokines was significantly stimulated by LPS (10 ng/ml),
which was used as a positive control.
[96]
Figure 5.7: Effect of lead on cytokine secretion in human microglia.
Cells were challenged with lead (0, 10, 50, 100 µM) or LPS 10 ng/ml for 24 hours. Negative
control, no lead or LPS treatment was also included in experiment. Various cytokines were
measured in the culture supernatants using a cytometric bead assay and flow cytometry. Data were
analyzed by FCAP software. Final concentration of cytokines are shown as pg/ml (n= 15, *
indicates P<0.05).
0.00
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Pb 50 µM
Pb 100 µM
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IL-1β
(p
g/m
l)
* p=0.000
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10
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* p=0.000
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* p=0.048
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*p=0.036
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* p=0.020
* p=0.000
[97]
5.6. Discussion
Lead has been used widely for many decades for the production of various commodities
(paint, gasoline, utensil, electronics, etc.) and it will continue to be used in the future for
many kinds of technological purposes, due to its unique properties (Luo et al., 2012).
Therefore, lead exposure and toxicity is still a major global health problem, especially in
lead mining and processing countries, where workers and corresponding local residents
are affected. The CNS is one of the main targets of lead toxicity. During development
and growth, the brain is most susceptible to toxic effects of lead, which correlates with
deficiency in neurogenesis and remodeling of the brain. These pathological changes in
the brain result in neurocognitive and functional deficits that are induced by lead even at
low blood concentration (< 10 µM) (Liu et al., 2012). Immune and inflammatory effects
of lead on the nervous system have also been reported (Waterman et al., 1994). In that
respect, one has to consider microglia as an important cell population, representing the
innate immune system in the CNS. Microglia are the resident macrophages of the CNS.
They act as the first line of defense in the brain and play a crucial role in health and
disease, including infections, trauma and neurodegenerative diseases. Activated
microglia can either induce or inhibit neurogenesis, depending on the condition and local
tissue response. Neurogenesis is of course important in the developing and growing CNS,
but adult neurogenesis contributes also to some important areas and functions in the
adult, such as the olfactory system, the forebrain and memory function in the
hippocampus (Lazarini et al., 2012; Morrens et al., 2012). Interestingly, using modern
imaging technologies, structural changes of the brain could be visualized in long-term
lead exposed workers, which could also be correlated to functional deficits (Bleecker et
al., 2007; Jiang et al., 2008). However, little is known about the molecular and cellular
mechanisms related to lead neurotoxicity.
In this study, we investigated the effects of low level lead exposure on human microglia,
using a novel human in vitro model (Chapter 4). We showed that at low concentration
(<10 µM), lead has only minor effect on morphology and viability of microglia.
However, functional and phenotypic tests indicate that lead induces specific changes in
chemokine receptors expression and secretion of IL-8.
Lead has a special and detrimental effect on the CNS during development and growth,
where cell proliferation, maturation and migration play a crucial role. Various studies
reported the inhibitory effect of lead on proliferation of the oligodendroglia and neuronal
[98]
precursors. Most interestingly, an in vitro study showed that low level exposure of lead
(0.01-10 µM) has no effects on rat neuronal stem cells viability, but results in a
concentration-dependentdecrease of proliferation of the cells. Reduction in neurons and
oligodendrocytes numbers, and a significant increase in astrocyte numbers has also been
reported following lead exposure (Huang and Schneider, 2004). However, there is no
clear evidence on apoptotic effects and decreased viability of astrocytes and microglia
after lead exposure. In contrast, astrocytes have been reported to be resistant to cell death
and morphologic changes following lead exposure (Opanashuk and Finkelstein, 1995).
Nevertheless, activation of astrocytes and microglia has been found (Garber and Heiman,
2002). These published reports are in concordance with our findings. Our results indicate
that lead, up to concentrations of 50µM, does not affect the survival of the human
microglia. In this context, it is worth mentioning that many lead neurotoxicity studies
have used very high lead concentrations in the range of 100µM and above, showing
substantial cytotoxicity. However, it is unlikely that such high lead concentrations are
reached in vivo. Therefore, we suggest that lead neurotoxicity is rather due to changes in
cell recruitment, turnover and function, and not due to increased cell death.
It has been well documented that resting microglia with ramified morphology are
activated by various stimuli, including toxins. Thereby, the cells change their
morphology, by shortening and losing branches, and by hypertrophy of the cell body,
resulting in a more mobile and migratory, “ameboid” microglia (Nakajima and Kohsaka,
2001; Liu et al., 2012). Loss of microglia ramification in the presence of LPS has been
published (Nakamura et al., 1999). Other factors and conditions have also been shown to
activate microglia, including exposure to interferon gamma (Kloss et al., 2001) and
astrocytes-conditioned medium (Bohatschek et al., 2001). Changes in morphology of the
macrophages in other organs have also been reported in response to lead intoxication
(Sengupta and Bishayi, 2002). However, our study indicate that low level lead exposure
(up to 10 µM) does not significantly change microglia morphology, and therefore does
not significantly activate the cells. Only exposure to higher lead concentrations (50 µM
and above) resulted in a significant morphological change, indicating substantial
microglia activation. The mechanisms of this transformation are still unknown. Elevation
of Ca2+
and cAMP, or inhibition of G-proteins and phosphatases have been postulated as
the possible mechanisms (Kalla et al., 2003). However, more research is required to
elaborate a better understanding of microglia activation in the context of lead
neurotoxicity.Oxidative stress has been postulated to play an important role in neuronal
cell death and pathogenesis of neurodegenerative diseases (Halliwell, 1992; Thomas et
al., 2007). Oxidative stress may induce cell death through increased production of ROS
[99]
and enhanced lipid peroxidation, resulting in substantial functional changes of cell
membranes and membranes of the organelles (Moreira et al., 2005). Activated microglia
has been shown to be an important source of ROS and have therefore been implicated in
the pathology of neurodegenerative diseases (Schilling and Eder, 2011). In addition,
mitochondrial alteration and ROS production has been correlated with microglia
activation (Verri et al., 2012). Disturbance in the pro-oxidative and anti-oxidative
balance in the brain has been considered as one of the possible mechanisms involved in
lead induced neurotoxicity. Previous studies have thus reported the enhanced regional
lipid peroxidation in the rat brain, especially the hippocampus, after low levels of lead
exposure (Villeda- Hernández et al., 2001). However, most studies have used rodent
models (Adonaylo and Oteiza, 1999, Qin et al., 2005, ), which are known to produce high
amounts of ROS, whereas humans produce much lower amounts, if at all. With this in
mind, the results of our study are reasonable, as they indicate that lead exposure does not
induce increased production of ROS and subsequent lipid peroxidation in human
microglia. The difference in ROS production between humans and rodents may thus be
responsible for discrepancies in the literature.
Cytokines and chemokines form an important part of an immune responses, regulating
the auto- and paracrine communications with other cells in various tissues, including the
CNS, where these factors support immune defense, repair and recovery processes. It has
been postulated that there is a constitutive base-level production of cytokines and
neurotrophic factors in the healthy CNS, supporting homeostasis and remodeling.
However, there has been more emphasis on the role of cytokines in causing damage in
the context of inflammation and degenerative diseases. Several studies have reported a
key role for pro-inflammatory cytokines, such as IL-1β, IL-6, tumor necrosis factor-
and transforming growth factor-β (TGF-β), in the pathogenesis of stroke, Parkinson's and
Alzheimer's disease (Mathias et al., 2004). Importantly, microglia have been shown to be
an important source of cytokines and chemokines in the CNS, as well as playing a role in
inflammation and degenerative diseases (Hanisch, 2002).Therefore, we investigated
whether lead exposure induced production of inflammatory cytokines by microglia.
However, no significant secretion of those cytokines was found, including IL-6, IL-10,
TNF-, IFN- and IL-12, independent of lead concentration. This indicates that lead-
exposed human microglia may not react with increased production of inflammatory
cytokines following lead exposure. However, lead induced increased secretion of IL-8,
an inflammatory chemokine. Chemokines are chemoattractant factors controlling
migration of cells of the immune systems. However, chemokines play also an important
role in the developing brain, controlling migration and differentiation of neural cells. In
[100]
addition, chemokines also contribute probably to cell replacement in the adult brain in
areas where tissue remodeling takes place. Additionally, chemokines contribute to cell
activation, control of metabolic functions and cell survival, as well as induction of
programmed cell death (Bacon and Harrison, 2000). In the CNS, various chemokines and
their receptors have been shown to play a role, including CCL2 (MCP-1)/CCR2, CCL3
(MIP-1α)/CCR1/CCR5, CCL4 (MIP-1β)/CCR5, CCL5 (RANTES)/ CCR1/CCR3/CCR5,
CXCL-1 (GRO-α)/CXCR2, CXCL8 (IL-8)/CXCR1, CXCL10 (IP-10)/CXCR3, CXCL12
(SDF-1)/CXCR4 and CX3CL1 (fractalkine)/CX3CR1 (Cartier et al., 2005; de Haas et al.,
2007). Among those chemokines, only CCL2, SDF-1 and fractalkine seem to be
constitutively expressed by neurons and astrocytes. However, expression of other
chemokines has also been reported in response to inflammation. IL-8 is one such
inflammatory chemokine, found only in humans, but not in rodents. IL-8 is produced by
astrocytes and microglia in the context of inflammation (Aloisi et al., 1992; Ehrlich et
al., 1998). The role of IL-8 and its receptors play a crucial role in the recruitment of
neutrophils (Horuk et al., 1997), bone marrow stromal cells (Wang et al., 2002), in cell
adhesion, neuronal protection and brain development (Goczalik et al., 2008). In humans,
IL-8 binds to two receptors, CXCR1 and CXCR2. CXCR1 has been shown to be
important in cell activation, while CXCR2 mediates the chemoattractant properties
(Semple et al., 2010). Interestingly, several in vitro studies have shown that IL-8 has also
a neurotrophic effect (Araujo and Cotman, 1993). However, little is known about the
role of Il-8 in the healthy brain and whether IL-8 is produced by resting microglia. On the
other hand, it is well known that activation of microglia induces IL-8 secretion (D'Aversa
et al., 2008). Our study clearly shows that human microglia produce detectable amounts
of Il-8 in our non-activated in vitro model. Furthermore, exposure to lead increased
significantly IL-8 production, both at low and high lead concentrations. This is consistent
with a recent study that showed increased IL-8 expression in peripheral blood monocytes,
when challenged with lead at the concentration of 10 µM and above (Gillis et al., 2012).
Interestingly, IL-8 has also been shown to be produced by microglia, astrocytes and
neurons in human Alzheimer disease, upon amyloid-β (Aβ) or pro-inflammatory
cytokine activation. Moreover, IL-8 has an inhibitory effect on Aβ-induced neuronal
apoptosis in vitro, along with an increase secretion of brain derived nerve growth factor
(Ashutosh et al., 2011). With this in mind, it is tempting to speculate that lead may affect
susceptible CNS regions, including the hippocampus, by inducing production of IL-8 in
microglia, which then interferes with cellular homeostasis and cell turnover, resulting in
the known effects of decreased neuronal density and corresponding functional
deficiencies.
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Chemokine receptors contribute to communication between neurons and glia cells,
including microglia. Most important, their action is not restricted to neuroinflammation,
but they play a role in cellular and molecular homeostasis (Bajetto et al., 2001). E.g. the
chemokine receptors CXCR3, CXCR4 and CCR3 play a role during brain development
and growth (Van Der Meer et al., 2001). Several studies have reported the contribution
of various chemokine receptors to the pathogenesis of neurodegenerative diseases, such
as Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, stroke and HIV-
associated dementia (Heneka et al., 2010; Mines et al., 2007). Interestingly, it has been
reported that knocking out CCR2 expression enhances microglia recruitment in a mouse
model of Alzheimer’s disease. Although in our study lead decreased expression of most
chemokine receptors, only CCR1, CCR2, CCR3 and CXCR1 were significantly reduced
in microglia. On the other hand, increased in expression level of CCR4, CCR9 and
CXCR3 was observed upon lead exposure. Interestingly, among the tested markers,
CCR5 and CXCR1 have shown to be expressed by M-MG at two different levels (two
picks in histograms) in both control and lead-treated cells. In accordance with our data, it
has been reported that that human monocytic consist of two principal subsets with
different expression level of CXCR1 and CCR5 (Geissmann et al., 2003). Therefore, our
results may suggest existence of two sub-populations of monocyte-derived microglia
with high and intermediate expression level of both markers. It is noteworthy to mention
that both sub-population showed decreased expression in response to lead exposure.
However, the role of these changes in expression levels and the molecular mechanisms
driving these changes are still not understood.
Accumulating evidence suggests that the diverse biological activity of macrophages is
mediated by functionally distinct subpopulations that are phenotypically polarized by
their microenvironment and by exposure to inflammatory mediators. M1 macrophages
exhibit potent microbicidal activity, and release IL-12, promoting strong Th1 immune
responses. M1 macrophages are activated by type I cytokines, IFNγ and TNFα, or after
recognition of pathogen associated molecular patterns or PAMPs (e.g., LPS, lipoproteins,
dsRNA, lipoteichoic acid) and endogenous “danger” signals (e.g., heat shock proteins,
HMGB1). M1 exert anti-proliferative and cytotoxic activities, which is due in part to the
release of reactive oxygen and nitrogen species and pro-inflammatory cytokines (e.g.,
TNFα, IL-1, IL-6). M1 population is thought to contribute to macrophage-mediated
tissue injury. In contrast, M2 macrophages support Th2-associated effector functions. M2
macrophages release IL-10 and exert selective immunosuppressive activity, and inhibit
T-cell proliferation. M2 macrophages also play a role in the resolution of inflammation
through phagocytosis of apoptotic neutrophils, reduced production of pro-inflammatory
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cytokines, and increased synthesis of mediators important in tissue remodeling,
angiogenesis, and wound repair (Benoit et al., 2008, Laskin, 2009). In the context of lead
poisoning, it seems that expression of certain chemokine receptors as well as quick
changes towards M1/M2 phenotype is affected in human microglia, which may
contribute to changes in cell migration, activation, turnover and function in the CNS and
finally be responsible for the neurotoxic effect of lead
5.7. Conclusion
In summary, low level lead exposure is not cytotoxic to human microglia, in the sense
that it does not affect viability, nor does it substantially activate the cells. However, lower
level lead exposure has functional and immune modulatory effects on the human
microglia, by increasing expression of IL-8 and changing significantly expression
patterns of various chemokine receptors. Those changes may be the relevant events in
acute and chronic lead toxicity by interfering with cell recruitment, cell replacement and
tissue remodeling in various areas of the CNS
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Chapter 6
Lead alters the chemokine receptors
expression pattern of the human
microglia cell line (HMC3)
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6. Lead alters the chemokine receptors expression pattern of the human microglia cell line (HMC3)
6.1. Introduction to the chapter
In the previous chapters, effect of lead exposure on human monocyte-derived mciroglia
was studied. Alteration in the expression pattern of chemokine IL-8 and pro-
inflammatory chemokine receptors was reported following by lead exposure (Chapters
4 & 5). Due to the important role of human microglia as the brain immune effectors
during inflammation, as well as neuroprotection against toxicity, the effect of lead
exposure was also investigated using the human microglia cell line HMC3 as an
additional estbalished human microglia model. HMC3 is an immortalized microlgia cell
line derived from human fetal brain (Janabi et al., 1995). There is not much known about
HMC3 characteristics and function. However, expression a number of chemokine
receptors has been reported by HMC3 (Flynn et al., 2003). Therefore, changes on
morphology, phenotype and function of HMC3 were investigated after exposure to
different concentration of lead acetate (1, 10 and 50 µM) for 24 hours. The main focus of
this chapter was to establish the underlying similarities in response to lead exposure
between the established human microglia cell line HMC3 with the human monocyte
derived microglia (M-MG) model.
Result showed that lead had no toxic effect on the morphology and viability of HMC3 at
concentrations below 10 µM. In addition, there was general down regulation pattern in
expression of chemokine receptors as well as surface molecules. Reduction in
stimulatory effect of HMC3 on activated PBMC was seen in presence of lead. Lead
exposure also correlated with changes in the secretion of IL-8 and IL-6 by HMC3.
In summary, the results showed in M-MG are valid, as they have been repeated and
shown to be similar, using the established HMC3 cells line. These findings also suggest
the immunomodulatory effect of lead on human microglia, through changes in the
chemokine/receptor expression patterns, which may affect CNS development,
remodeling and function.
Declaration of work:
All the experiments in this study were designed and conducted by SE under the
supervision of Prof. Luis Filgueira. The full draft of the manuscript has been prepared for
submission to an international peer reviewed journal.
[105]
6.2. Abstract
It has been well documented that lead even at low concentrations has neurotoxic effects
on the development and function of the brain. Its detrimental effects on the CNS can
vary from activation of glia cells, induced neuronal death, changes in synaptogenesis to
impaired learning and memory function. Microglia cells, the resident macrophages of the
brain, play an important role in homeostasis of the CNS as neuroprotective cells
(secretion of anti-inflammatory cytokine, chemokine and their receptors, neurotrophic
factors, etc). Microglia activation which usually occurs in response to stimuli (toxin), has
been shown to be involved in many neuroinflammatory processes. Involvement of glia
activation in lead-induced toxicity has been reported in animal studies. However, there is
no evidence showing the exact role of human microglia in lead induced toxicity of
inflammation.
Therefore, the aim of this study was to investigate the effects of low level of lead
exposure (10µM, 24 hrs) on a human microglia cell line at the cellular and functional
level. To obtain this aim, HMC3 were exposed to different concentration of lead 1-100
µM for 24 hours. Then, changes on its morphology, viability, chemokine and cytokine
expression pattern as well as its function as brain immune cells were studied.
Results showed that lead at lower concentration <10 µM had no toxic effects on the
viability and morphology of HMC3. Furthermor, non-activated HMC3 are able to uptake
lead and accumulate inside even at low level of lead exposure, as visualized with specific
fluorescent probe, using fluorescence microscopy and flow cytometry. In addition,
general down-regulation in the expression pattern of chemokine receptors and surface
molecules involved in microglia differentiation, activation and inflammation was
observed. Lead attenuated the stimulatory effects of HMC3 on activated T-cell
proliferation which implicating changes in antigen presenting ability in presence of lead..
Furthermore, lead at concentration of 1 and 10 µM significantly increased in the
secretion of IL-6 and IL-8, respectively. Several studies have reported the
neuromodulatory effect of IL-6 and IL-8 on CNS development and function.
In summary, our results indicate lead, even at low concentrations, has a significant
influence on the characteristics and function of the human microglia cell line HMC3.
These changes may be responsible for some of the effects seen in the context of lead
neurotoxicity.
Keywords: Human Microglia, Human microglia cell line HMC3, Chemokine receptor, lead
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6.3. Introduction
Lead is an potent neurotoxin, which causes long lasting neurobehavioral deficits and
cognitive impairments in children even at the very low doses (Zurich et al., 2002).The
developing nervous system is more vulnerable to toxic effects of lead (Bressler et al.,
1999b, Kasten-Jolly et al., 2012, Kasten-Jolly et al., 2011b, Struzynska et al., 2007b).
There is evidence showing that lead exposure alters the immune responses in the brain in
a way that resembles neuroinflammation. It has been known that lead accumulates more
in glia cells (astrocytes, microglia) than in neurons (Tiffany-Castiglioni and Qian, 2001).
Lead-induced astrocyte activation has been shown to increase the BBB permeability,
which makes lead entre to the brain easier (Dyatlov et al., 1998). It has been suggested
that lead potentiates chemokine and cytokine secretion, as well as glutamate induced
oxidative stress by astrocytes. However, there is not much known about the role of
microglia in lead induced neurotoxicity in human. Recent published animal studies
suggest that lead exposure can activate microglia and increase secretion of some
inflammatory cytokines, such as IL-1 β and TNF-, as well as hippocampal neuronal
injury. It has also been suggested that lead neurotoxicity may be mediated by microglia
activation (Liu et al., 2012). Recent studies showed a correlation between learning,
memory deficits and microglia activation (Tanaka et al., 2011). Several studies have
shown a correlation between increasing level of blood lead and the etiology of
neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease, and
amyotrophic lateral sclerosis (Liu et al., 2006, Kamel et al., 2002, Coon et al., 2006), Liu
et al., 2006). Several studies have reported the negative effects of lead on the nervous
system, especially motor neuron related disorders and cognitive reduction in
occupational lead exposed workers (Karimooy et al., 2010, Khalil et al., 2009, Zheng et
al., 2011). Recent studies suggest that long-term exposure lead may be responsible for
neurological deficiencies even at low concentrations (Baranowska-Bosiacka et. al.,
2012). Thus, there is growing interest in studying the effects of lead on the CNS, not
only in children but also in adults. According to the harmful effects of lead on memory,
learning and cognitive function, great attention has focused over the past decade on the
hippocampus as a target for lead toxicity, particularly in young children (Marchetti,
2003). However, underlying mechanisms of lead toxicity are still not well known.
Hence, understanding the mechanisms of lead neurotoxicity may provide a basis for
developing a new therapeutic strategy, aimed at preventing vital behaviour abnormalities
induced by lead poisoning.
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There is evidence of toxic effects of lead on the immune system (Fischbein et al., 1993).
Enhanced proliferation of T-cells has been reported previously, following lead exposure
at low concentrations (McCabe et al., 2001). In addition, it has been shown that exposure
to lead strongly enhances the susceptibility of rodents to endotoxin shock and parasitic
infections, by affecting the immune responses.
Microglia cells are the resident macrophages of the brain. As the immune cell of the
CNS, they play a key role in maintaining the homeostasis and function of the brain
through secretion of a variety of modulatory molecules, neurotrophic factors, and
through phagocytic ability and other immune-related functions during development and
adult life. In response to environmental stimuli, such as toxins or pathogens, “surveying”
microglia with ramified morphology rapidly turn into the amoeboid “activated” form.
Along with this morphological and functional alteration, microglia undergo several
changes at the cellular and molecular level, including changes in chemokine and
cytokine secretion, and surface molecules expression. Activated microglia act as the
effective phagocytes and antigen presenting cells in the CNS. It has been shown that
microglia phagocytosis play a critical role during neuronal development and
differentiation, as well. Furthermore, microglia express diverse membrane receptors in
order to recognize pathogen, toxins endogenous danger molecules and initiate immune
responses through presenting the antigen to immune cells (Block et al., 2007). Microglia
also can acquire the reactive or over activated form, which contributes to neurotoxicity
and neuroinflammation in the CNS. Microglia’s reactive phenotype displays a high
phagocytic and antigen presentation ability, as well as secretion of several pro-
inflammatory cytokine and reactive oxygen molecules (Tremblay et al., 2011,
Kettenmann et al., 2011, Ransohoff and Perry, 2009). There are still controversies about
turnover and proliferation capacity of the microglia during life span. Particular concern is
about the differentiation of the blood monocytes into the microglia in order to replenish
the resident population (Boche et al., 2013). There is published evidence that monocyte
migrate to the brain and become microglia during neurodegenerative diseases (Simard
and Rivest, 2004).
Most of the current data on microglia physiology and function has been obtained from
animal studies. Difficulties in obtaining human brain tissue samples, insufficient number
of cells in primary culture, as well as changes in its characteristics over time, are some of
the reasons restricting most of the studies to investigating microglia in animal models
instead of using human cells. Furthermore, there are only few established human
microglia cell lines that are commercially available. The human microglia cell line
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HMC3 is one of the few well established human microglia cell lines that are available for
research (Janabi et al., 1995). HMC3 cells show typical microglia characteristics,
including the constitutive expression of Iba-1, CD14, as well as IL-6, and IFN- induced
expression of CD11b, CD68 and MHC class II (Li et al., 2009).
Chemokines are chemoattractant cytokines, well known for their ability to recruit
inflammatory cells from the blood to the CNS and to enhance inflammatory responses.
However, recent studies have also shown their role in the development and migration of
neuronal precursor cells, as well as in mediating the microglia-neuronal interaction.
Chemokines and chemokine receptors have been detected under physiological and
pathological condition in brain tissues, as well as in neuronal and glia cultures (Bajetto et
al., 2002) (Xia and Hyman, 1999, Glabinski et al., 2002, Rezaie et al., 2002). Elevated
levels of chemokines and their reciprocal receptors have been found during
neuroinflammation and in neurodegenerative diseases (Simpson et al., 2000a, Cartier et
al., 2005). Despite the important role of these molecules and their receptors in mediating
brain inflammation, the expression patterns and functions of chemokines and their
receptors under physiological conditions in human microglia are still not fully
understood. In vitro studies showed that human microglia express several chemokine
receptors under non- stimulatory and under inflammatory conditions (Chapter 4, Flynn et
al., 2003).
In the context of lead and inflammation, recent animal studies showed the involvement
of glia activation in lead-induced neurotoxicity and impaired cognitive function. It has
been reported that astrocytes sequester lead ions, maintaining ionic balance in the
nervous system thus protecting neurons from lead toxicity (Selvin-Testa et al., 1991,
Raunio and Tahti, 2001)Activation of astrocytes with increased in their differentiation
have been also reported in rat model of lead-toxicity (Struzynska et al., 2007b).
Activation of astroglia may often lead to loss of the buffering function and contribute to
pathological processes. In addition, recent in vitro and in vivo studies in rat reported that
lead could cause microglia activation and hippocampal neuronal injuries through lead-
induced inflammation (Liu et al., 2012). All these studies implicate the significant
involvement of glia cells in both, protection and immune responses during lead toxicity.
However, there is no reported data by other researchers about the role of human
microglia in lead induced neurotoxicity/neuroinflammation. Therefore, we investigated
the effect of lead exposure on the human microglia cell line HMC3 to answer the main
question, whether human microglia are activated by lead and how these changes could
affect microglia functions as immune cells of the brain. In this study, HMC3 were
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exposed to different concentrations of lead over 24 hours. Changes in morphology,
phenotype and function were investigated. We also studied whether microglia as the
brain phagocytes are able to engulf and sequester the lead. Knowledge about the
influence of lead on human microglia cell focusing on immune modulators will help to
better understande the role of cell-cell communication in lead-induced neuronal
dysfunction and degeneration.
6.4. Material and methods
6.4.1. Cell culture
In this study, we used human microglia clone 3 (HMC3) cells, kindly provided by Prof.
Karl-Heinz Krause (University of Geneva, Switzerland). Cells were cultured in T25
tissue culture flask (Sarstedt) using DMEM/F12 Glutamax medium (Invitrogen)
supplemented with 5% FCS and 1% antibiotics/antimycotics (Invitrogen) at standard
humidified culture conditions (37oC, 5% CO2). The cells were passaged and used for
experiments when they reached 70–80% confluence.
6.4.2. Lead exposure
Lead acetate (Pb(CH3CO2)2 3H2Os (Sigma-Aldrich) was prepared at the concentration of
100 mM as stock solution under sterile conditions. In order to study the effect of lead on
HMC3, lead was directly added to the medium at the concentration of 5,10, 25, 50 and
100 µM (depending on the experimental design). Cells after 7 days in culture,were
challenged with lead for 24 hours before measurements were done.
6.4.3. Morphological studies
HMC3 cells were cultured in the presence or absence of 10 µM lead for 24 hours.
Morphological appearance and changes were documented by using an inverted Leica
DM IL phase contrast and fluorescent microscope. Images were taken with a
Photometrix Cool SNAP Fx digital camera and processed with the NIS- elements F3.0
software. Sytox green, nucleic acid stain (Invitrogen) was applied to exclude the dead
cells in morphological quantification (Luo et al., 2001). Cells were incubated with 1 µM
Sytox Green for 30 minutes at 37°C. Five random optical fields were selected. Images
were taken with fluorescent filter and at bright filed. After overlaying the images, a total
of 100 live cells were counted. In repeated independent experiments, we quantified the
numbers of branches in microglia stained with an antibody against Iba-1, a macrophage
and microglia marker, using a standard immunostaining protocol (Blomster et al., 2011).
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Immunostaining was performed with microglia cultured on cover slips, fixed with 1%
paraformaldehyde in PBS, containing 2% sucrose. After permeabilization with 0.01%
TritonX100 in PBS, cells were incubated overnight at 4°C with rabbit anti-Iba-1
(1:100,Wako Pure Chemical Industries), with subsequent incubation for 1 hour with
Alexa Fluor 488-conjugated donkey anti-rabbit antibody (1:200, Invitrogen) and DAPI
(1:400, Roche). Slides were analysed using fluorescent microscopy as described above
and images were taken. According to previous studies, microglia with less than two
branches were scored as activated (Kauppinen et al., 2008).
6.4.4. Detection of up-take and intracellular accumulation of lead
We investigated up-take and intracellular accumulation of lead by microglia with the
fluorescent probe Leadmium Green AM (Molecular Probes, Invitrogen, Carlsbad, CA)
using flow cytometry. HMC3 were incubated with different concentrations of lead (5, 10,
25, 50 and 100 µM) overnight. Then, M-MG were incubated with 1uM Leadmium Green
AM dye in saline for 30 minutes to 2 hours. Cells were analyzed by flow cytometry on
FACSCalibure flow cytometery (BD Biosciences, San Jose, CA) using the 488nm
excitation and measuring fluorescent emission at 520 nm. Non-lead treated cells were
included in the experiment as a negative control (Zeller et al., 2010). FlowJo software
was used for further analysis of the flow cytometry data. Representative histograms for
each experimental groups were overlayed and mean fluorescent intensities (MFI) were
compared using the Student’s t-test. The experiments were repeated for five times with
the same results (n=5). Another fluorescent probe, which is less specific, New port Green
DCF (Molecular Probes, Invitrogen, Carlsbad, CA), was also used to detect the lead
uptake.
6.4.5. Viability assays
To assess the effects of lead on the viability of HMC3, MTS assays were carried out
(Cell Titer 96 Aqueous assay, Promega). Briefly, cells were seeded in 96 well plates at
the concentration of 2x104 cells/200 µl/well and cultured to reach the 70-80%
confluence. After incubation of cells with 0, 1, 10, 50 µM of lead for 24 hours at 37oC,
20 µL of cell titer 96® AQueous One Solution (MTS) was added directly to each well.
After 4 hours incubation, absorbance of the colorimetric reaction was measured by a
plate reader (Labsystem Multiscan RC) at 490 nm. Viability of lead-treated cells was
compared the non-treated control cells and analysed using the Student’s t-test and
ANOVA (Vairano et al., 2004).
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6.4.6. Expression of surface markers
We studied the effect of lead (10 µM) on the expression pattern of chemokine receptors
in HMC3. Cells were incubated with 10 µM lead for 24 hours. After detaching and
washing twice with PBS, cells were stained with direct labelled antibodies, or using
primary unlabelled and corresponding secondary labelled antibodies. Non-lead treated
cells were included in all tests. In number of experiments, cells were exposed to IFN-γ
(10 ng/ml, Sigma-Aldrich) and dexamethasone (1 µM, Sigma -Aldrich) for 24 hours. A
panel of directly labelled mouse anti-human antibodies , CD45-FITC, CD14-PE, CD40-
PE, CD11c-PE, HLADR–PerCP, CD11b-AF488, CD11c-PE, CCR1-AF647, CCR2-
AF647, CCR4-PE-Cy7,CCR5-FITC, CXCR1-PE, CXCR3-AF488, CXCR5-FITC (BD
Pharmingen, San Jose, CA), CCR6-FITC and CCR7-APC (R&A systems, Minneapolis,
MN) were added at the supplier’s recommended concentration. Excitation and emission
wavelengths of the used fluorochrome were resspectively: FITC: 494, 519; PE:
496/546,578; PerCP: 482, 678; APC: 650, 660; Alexa Fluor-488: 495, 519; Alexa Fluor -
647: 650, 668. Corresponding labeled isotype control antibodies were included in all
experiments. The cells were incubated with antibodies for 30 minutes at 4oC, washed
with PBS and resuspended and fixed with 1% formaldehyde in PBS containing 2%
sucrose. The fixed samples were measured with a FACSCallibure (BD Biosciences).
Data analysis was performed using FlowJo software version 7.6.3 (TreeStar, Ashland,
OR).
6.4.7. Mixed leukocyte reaction (MLR)
The T-lymphocyte stimulatory capacity and corresponding antigen-presenting cell
(APC) properties of HMC3, in the presence and absence of lead (10 µM) compared to
phytohaemagglutinine (PHA) (7 µgr/ml, Sigma-Aldrich) activated T-lymphocytes, were
assesses in MLRs using a BrdU proliferation assay (Cadosch et al., 2010). Freshly
processed non-adherent PBMC containing mainly (>90%) T-lymphocytes (2x105/well)
were seeded in a round bottom 96–well plate (Sarstedt). Allogeneic HMC3 were used as
antigen-presenting cells (APC) and added at the ratio (1:80) to the T-lymphocyte (4 wells
for same condition). T-lymphocytes with APC only was used as the control condition. To
compare the effect of lead on activated T-lymphocytes, PHA was added to each well at
the concentration of 7 µg/well. PHA- activated T-cells with HMC3 was used as the
positive controls. After 5 days in culture, BrdU proliferation assays (Roche, Mannheim,
Germany) were performed according to the supplier’s protocol and the absorbance of the
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colorimetric reaction was measured by ELISA reader at 405nm (Labsystems Multiskan
RC). Mean proliferation of the different conditions was analyzed and compared.
6.4.8. Phagocytosis assay
Phagocytosis was assessed by measuring the uptake of tetramethylrhodamine (TMR)-
labelled Staphylococcus aureus (Invitrogen) as published before (Meagher et al., 2005).
To compare the effect of lead with IFN- and LPS on phagocytic activity of HMC3 and
its activation, cells were incubated overnight with 10 and 50 µM lead acetate, IFN- (10
ng/ml, Sigma-Aldrich)or LPS (10 ng/ml, E. coli serotype 0111:B4, Sigma -Aldrich).
. Next day, bacterial particles were added to the cells (107/ml) at a ratio of 1:1000
(according to manufacturer’s protocol) and incubated for 4 hours at 37oC. The cells were
then harvested and fixed with 1% paraformaldehyde in PBS containing 2% sucrose.
Total ingested fluorescent-labeled bacteria per cell were measured by flow cytometry.
6.4.9. Cytokine measurement
The cytometric bead array (CBA) human inflammatory kit (BD Biosciences) was used
for the quantitative measurement of cytokine production and secretion under control and
lead-exposed conditions. For that purpose, HMC3 were incubated with 1 and 10 µM
lead for 24 hours. To see whether HMC3 were responsive to IFN- and LPS activation,
cells were incubated with these activators at the concentration of 10 ng/ml for 24 hours.
The following day, 50-100 µl of the medium was collected and stored at -20oC. The
assay was performed according to the manufacturer’s instruction using 50ul of the
human cytokine standards and sample dilution. Cytokine concentrations were measured
using the FACSCanto flow cytometer (BD Bioscience). FCAP Array, version 3, software
was used to analyze the data. Measurements were done in duplicates and repeated once
with 3 independent experimental repeats.
6.4.10. Statistics
Data were analyzed with the SPSS statistical software (version 17.0). A one-way
ANOVA and univariant linear model were used to compare means. The LSD/Dunnett
post-hoc test also used to determine the significant difference between groups. In all the
analyses p value<0.05 was considered statistically significant. Bars in graphs represent
the standard error (SED).
[113]
6.5. Results
6.5.1. Intracellular detection of lead uptake in human microglia cell
line HMC3
Phagocytic ability of microglia play an important role in maintaining the brain
homoestasis in the healthy brain through clearance of debris and death cells. Low
phagocytic activity of HMC3 has been reported previously (Janabi et al., 1995).
Therefore, to detect the lead uptake by HMC3 and its intercellular accumulation, we used
the fluorescent probe Leadmium Green, which has been shown to be very sensitive and
specific probe for lead detection at nM levels. For this purpose, HMC3 were incubated
with different concentrations of lead (5, 10, 25, 50 and 100 µM) for 24 hours at 37oC.
Following incubation with 1µM Leadmium green for 30-60 minutes, the mean
fluorescent intensity (MFI) of the Leadmium was measured using flow cytometry and
compared to the non-lead treated cells as negative control (Figure 6.1). Increase in
intracellular amount of lead has been seen in a concentration-dependentmanner from 5
µM onwards. A major increase in green fluorescent intensity was seen from 10 µM to 25
µM. Mean of fluorescence intensity ± SD of the Leadmium Green for the HMC3
cultured with different concentration of lead, 0 to 100 µM respectively were 5.92 ± 0.96,
10.78 ± 0.95, 18.96 ± 0.58, 32.90 ± 1.84, 35.83 ± 2.24 and 40.02 ± 2.65.
Figure 6.1: Lead uptake by human microglia cell line HMC3
These figures show uptake
and intracellular accumulation
of lead in human microglia
using flow cytometry (n=5).
Figures A and B are the
representative FSC:SSC dot
plots of M-MG in
absent/presence of 10 µM lead
from one representative
experiment out of five with
similar results Histograms
compare the relative expression
(MFI) of fluorescent probes,
Leadmium (C) to the
corresponding amount of
intracellular lead. the absence
[114]
(a) or presence of various concentration of lead, including 5 µM (b), 10 µM (c), 25 µM (d), 50
µM (e) and 100 µM (f).
6.5.2. Effect of low level lead exposure on the morphology and
viability of HMC3
Quick changes in morphology from ramified to ameboid in response to stimuli is one of the
main characteristic of microglia. Under non-stimulatory condition (no lead) HMC3 showed
the typical resting morphology of microglia with long branches (Figure 6.2 A). Exposure to
10 µM for 24 hours lead did not have any significant effect on HMC3 morphology
regarding size of the cell and/or number and size of branches. However, exposure to higher
concentration of lead caused changes in morphology towards hypertrophy in cell body and
shortening of the branches. HMC3 treated with LPS, known microglia activation factor,
showed similar morphology to 50 µM lead which suggesting lead could induce activation in
HMC3 at higher concentrations of lead.
The effects of lead on the viability of the HMC3 at different concentrations and incubation
periods were studied using the MTS colorimetric assay. Cells were cultured with 1, 10 and
50 µM lead for 24 and 72 hours. Non-lead treated cells and IFN- stimulated HCM3 were
included in experiments as negative and positive controls, respectively. Cell viability were
determined by optical density (OD) measured at 490 nm. The measured optical densities
from different experimental condition are shown as percentages. The viability of non-lead
treated control cells was considered as 100%. The bar chart summarizes the effects of
different concentrations of lead 1, 10 and 50 µM over 24 hours and 72 hours (Figure 6.2. E).
at both exposure times, a slight viability increase was seen in at 1 µM, which was followed
by a slight decrease at 10 µM. In general, lead at lower concentrations (1 and 10 µM)
showed no significant effects on viability of HMC3 after 24 or 72 hours. However,
treatment with 50 µM lead acetate for 24 and 72 hours showed significant reduction in
viability (P<0.05) (n=20).
[115]
Figure 6.2: Effect of lead on morphology and Viability of HMC3.
Morphological changes of HMC3 exposed to different concentrations of lead 0 µM (A), 10
µM (B), 50 µM (C) for 24 hours. Cells were also cultured with LPS (10 ng/ml) for 24 hours to
obtain activated morphology (D). Images were taken with the phase contrast light microscopy
(n=5, bar indicates 100 µm). Arrows (red) show the morphological changes in HMC3 such as
hypertrophy in cell body as well as shortening of the branches E) Bar charts represent the
percentages of live cells after exposure to various lead concentrations (0, 1, 10 and 50 µM) for
24 hours (black bars) and 72 hours (white bars). Viability of the cells was assessed by a MTS
colorimetric test. IFN- was used as HMC3 activation marker (positive control).Viability of
negative control group (non-lead treated) was used as baseline and normalized to 100% (n=20,
* indicates significance with p < 0.05).
[116]
6.5.3. Effect of of lead exposure on expression of chemokine
receptors and surface molecules in HMC3
Chemokine and chemokine receptors play a key role in modulating the inflammatory
responses in the CNS. Previous studies have shown the microglial expression of different
chemokine receptors (Flynn et al., 2003). To study the influence of lead exposure on
chemokine and surface molecules expression patterns, cells were incubated with 10 µM
lead for 24 hours. Following staining, flourscent intenisty of tested markers were
measured using the flow cytometry. General down-regulation pattern were seen in the
expression level of studied chemokine receptors in HMC3 treated with 10 µM lead
(Figure 6.3). Among the tested chemokine receptors, CCR1, CCR2, CCR5, CXCR1 and
CXCR5 showed the siginificant down-regulation (P= 0.009,0.038, 0.042, 0.015 and
0.05) in lead tretaed HMC3 compating to the non-treated group (Figure 6.4). Although,
CCR9 expression level showed increase in lead-treated HMC3, this changes was not
statically significant.
In additon to chemokine receptors, we also compared the expressions pattern of surface
markerc in HMC3 before and after lead exposure. Similar to chemokine recetors, overall
down–regulation was observed among surface molecules following lead exposure
(Figure 6.4). Significant attenuation in the expression of CD45, HLA-DR and CD11b
and CD54 was observed (P=0.05,0.04, 0.028 and 0.042). Interstingly, lead showed no
significant effect on expression of CD68 as an activation marker, while CD11c
expression was up-regulated in lead treated HMC3 (P=0.022).
Studies showed that microglia during activation and inhibition expressed different level
of chemokine and chemokine receptor in distinct patterns. In order to identify whether
lead behaves as an activator or a suppressing agent on microglia, we compared the
chemokine receptor expression pattern in HMC3 exposed to lead with IFN-γ and
dexamethasone, well-known inflammation stimulator and inhibitors (Figure 6.5).
Therefore, cells were cultured with 10 µM lead, IFN-γ and dexamethsone for 24 hours.
The different expression level of chemokine receptors were compared with non-treated
control group. Most of the markers were responsive to IFN-γ and dexamethsone
stimulation , up- or down regulation of expression, respectively. Interstingly, lead did not
behave similar to IFN-γ nor dexamethasone in term of chemokine receptor expression
pattern.
[117]
Figure 6.3: Effect of lead exposure (10 µM, 24hrs) on chemokine receptor expression patterns in human microglia HMC3.
Examples of one representative experiment. Black histograms for lead-untreated control cells
and white histograms for lead treaded cells. Blue histograms are the isotype controls. Cells
were cultured for 7-10 days (n=20). Shifting black comparing to white histogram to the right is
representing the increase in expression level of marker in presence of lead and vice versa.
[118]
Figure 6.4: Lead alters the expression of surface markers in HMC3
A) Comparison between control cells (white bars) and lead-treated cells (black bars) for the
expression of various surface markers. B) Comparison between control cells (white bars) and
lead-treated cells 10 µM lead, 24 hour, for expression of various chemokine receptors. Data
represent the mean florescent intensity. Analysis of the significant differences between two
groups was done by ANOVA and LSD/Dennett’s test. (* indicates, n=20).
A
B
[119]
Figure 6.5: Lead showed distinct immunomodulatory effects on the surface molecule
expression in HMC3.
This diagram summarizes the comparison between stimulatory, inhibitory effects of IFN-
dexamethasone on the expression pattern of surface molecules in HMC3 and 10 µM lead-
treated HMC3. Non-lead treated HMC3 were included as a control. Data represents flow
cytometry measurements. Relative expression of marker were compared using the ANOVA
and LSD/Dennett’s test (n-=10).
CD11C CD68 CCR1 CCR2 CCR4 CCR5 CCR6 CCR7 CCR9 CXCR1 CXCR3 CXCR5
[120]
6.5.4. Effects of lead on phagocytic activity of HMC
Microglia are potent phagocytic cells of the brain and play a major role in the clearance
of dead cells and debris, especially during inflammatory processes caused by injuries or
neurodegenerative diseases. Phagocytic activity of microglia is also crucial in the
elimination of neurons during development. Phagocytic activity is usually enhanced
following microglia activation.and considered as one of the microglia activation marker.
Therfore, to determine the effect of lead on microlgia phagocytic ability and to find out
whether lead could enhance the microglia activation or not, we studied the different
concentration of lead (10 and 50 µM) on the phagocytic ability of HMC3 (Figure 6.6).
Changes on phagocytic ability of lead-treated HMC3 cells were also compared to IFN-
and LPS activated cells as positive controls. Non- treated HMC3 were considered as a
negative control. Fluorescent-labelled S. aureous was used for measuring the phagocytic
capacity of the various cell populations. Flow cytometry was used for the detection and
quantification of bacterial up-take. Our result showed that lead at the concentration of 10
µM did not change the phagocytic activity of HMC3. As we expected, lead at higher
concentration (50 µM) enhanced the bacterial engulfment similar to IFN- and LPS.
Figure 6.6: Effects of lead on phagocytic activity of HMC3.
Bar chart summarizes the phgocytic activity of HMC3 treated with different concentration of
lead (1, 10 and 50 µM) towards TM Rhodamin-lableled S. aureus measured with flow
cytometry. LPS and IFN- were used as the positive controls. Non-treated cells as negative
control were also included. Bacterial particles were added to culture medium at a ratio of
1:1000 (107 bacteria per ml) and incubated for 4 hours at 37
oC. Mean fluorescent intensity
represents the different levels of bacterial engulfment (P<0.05, n=7).
[121]
6.5.5. Influence of lead on microglia T-cell stimulatory capacity
Microglia as the resident immune cells of the brain have the unique capacity to process
and present antigens to T-lymphocytes which leads to activate and stimulation of T-cell
proliferation, as well as initiate the immune responses. To undestand whether lead could
enhance microglia antigen presentation ability through T-cell stimulation, we studied the
effect of lead exposure on T-lymphocyte stimulatory capacity of HMC3 using the mixed
leukocyte reactions (MLR) assay. In this study, non–activated and PHA- activated T-
cells were used as negative and positive controls, respectively. Co-culture of HMC3 and
T-cells were exposed to lead for 24 hours. To understand whether lead has more
stimulatory effect on activated T-cells, we included PHA and non-PHA treated T-cells in
one experimental condition. Optical density of culture was measured at 420 nm. The
higher number of cells represents the higher capacity in stimulation of T-cells. Optical
density of PHA-activated group was considered as 100%, which means the maximum
expected T-cell stimulation. A representative experiment is shown below (Figure 6.7).
Lead–treated HMC3 did not stimulate non-activated T-cells proliferation and showed
significant differences comparing to PHA-activated group (P=0.042). Regarding the
effect of lead on T-cell stimulation, result showed that lead–treated HMC3 had no
stimulatory effects on non-activated T-cells proliferation; however, lead-treated HMC3
significantly suppressed T-cell proliferation in PHA-activated experimental conditions
(P=0.036). In summary, low-level of lead exposure not only had no significant
stimulatory effect on T-cell proliferation by HMC3, it could attenuate antigen
presentation ability of microglia in response to activated T-cells.
6.5.6. Effect of lead on cytokine secretion in HMC3
Microglia activation has always been associated with the expression of cytokines, which
contribute to neurotrophic and neuroinflammatory events. Cytokines may also contribute
to the modulation of microglia activation (Vila et al., 2001).We used a flow cytometric
bead array for human inflammatory cytokines to determine whether lead exposure
induced changes in inflammatory cytokine expression patterns in human microglia. Cells
were cultured in the presence of different concentration of lead (1, 10µM) for 24 hours.
Cells were also incubated with HMC3 activators, IFN- and LPS (10 ng/ml) as a positive
control. Next day, culture medium supernatants were collected and used for the
measurement of a panel of inflammatory cytokines, including IL-1β, IL-8, IL-6, IL-
12p70, TNF- and IFN- (Figure 6.8). Of the six evaluated cytokines, only IL-8 and IL-6
showed changes in secretion level. It is worth to mention that HMC3 secreted cytokine at
the very low level. Interestingly, we found a significant increase in the secretion of IL-8
[122]
in a concentration-dependnet manner in the presence of lead (P=0.02, 0.04). When
HMC3 was treated with lead 10 µM, they showed only significnat decrease in IL-6
secretion (P=0.042). A slight increase was also showed in the presence of lead 1 µM,
however, it was not significant. HMC3 were responsive to stimulation with IFN- (10
ng/ml) followed by significant increase in secretion of both IL-6 and IL-8 (P=0.002,
0.000). LPS-treated HMC3(20 ng/ml) only showed increase in IL-8 expression
(P=0.026).
Figure 6.7: Influence of lead on microglia T-cell stimulatory capacity.
Comparing the T-lymphocyte stimulatory capacity of HMC3 in lead treated cells to PHA-
activated ones. Antigen presenting cells, allogenic T-cell were in cubated with HMC3 for 5
days at the ratio of 1:80 Cells. Non-treatd and PHA-activated cells were included as negative
and positive controls, respectively. Proliferation was measured using a BrdU incorporation
assay. Error bars represent the standard errors. No significant differences were seen between
control and test condition.
[123]
Figure 6.8: Effect of lead on cytokine secretion in HMC3
Cells were challenged with lead (0, 1 and 10µM), LPS 20 ng/ml and IFN- (10 ng/ml) for 24
hours. Negative control, no lead was also included in the experiment. Various cytokines were
measured in the culture supernatants using a cytometric bead assay and flow cytometry. Data
was analyzed by FCAP software. Final concentration of cytokines are shown as pg/ml (n= 10,
* indicates P<0.05).
[124]
6.6. Discussion
Despite the important role of microglia in the health and pathology of the CNS,
especially their involvement in inflammation-mediated neurotoxicity, there is not much
known about the role of human microglia in lead-exposed brain. Previously, we showed
the effects of lead exposure on human microglia using the novel in vitro model generated
from human blood monocytes (Chapter 4). In this study, we used the human microglia
cell line HMC3 as an additional human microglia model to investigate the effect of lead
on their morphology, phenotype and function, as well as to elucidate underlying
similarities between the HMC3 and M-MG models.
Lead is a widely spread persistent metal with potent toxic effects on human health.
Despite significant exposure reduction in humans, lead is still a major global health
concern. The developing central nervous system is one of the main targets of lead
toxicity. In children, neurotoxic effects of lead exposure can vary from changes in
neurocognitive function, in low-level exposures, to a potentially fatal encephalopathy in
acute lead poisoning, depending on the amount and length of exposure (Needleman,
2004b). In recent years, several studies have focused on the immunotoxic capacity of
lead. As a result, lead has been recognized as a new group of immunotoxicant with
potential capacity in shifting the immune function, even at the low to moderate levels of
exposure. It has been shown that lead could dramatically alter the function of the
immune system, even with moderate changes to immune cells (Dietert and Piepenbrink,
2006). There are evidences demonstrating a role of inflammation and inflammatory
mediators in pathogenesis of several CNS diseases (Lucas et al., 2006). In addition,
recent studies suggest the role of inflammatory responses in lead-induced neurotoxicity.
In that respect, microglia as the representative of the innate immune subsystem in the
CNS play a key role in inducing and modulating the immune responses in lead toxicity
(Struzynska et al., 2007b).
Microglia comprise 5-20 % of the glia population in CNS and their density varies in
different region of the brain (Denes et al., 2008). Under non-stimulatory condition,
microglia monitor surrounding environments and maintain homeostasis by removing
dead and injured cells, as well as by enhancing neuronal survival through secreting
neurotrophic and anti-inflammatory factors (Streit, 2002, Block et al., 2007). Microglia
activation, in response to injuries, trauma, toxin, etc., is followed by secretion of pro- and
anti-inflammatory cytokines, chemokines, expression of surface molecules, antigen
presentation, as well as phagocytosis in the CNS. It is becoming more accepted that,
although microglia activation is necessary and crucial for host defense and neuron
[125]
survival, over-activation of microglia could result in deleterious and neurotoxic
consequences.
During activation, microglia change their morphology from ramified to amoeboid
appearance, which usually is followed by an increase in the size of the cell body and by
shortening the branches. Microglia activation with changes in morphology under
inflammatory conditions in the presence of LPS in vitro has been reported (Sheng et al.,
2011). In addition, changes in morphology of microglia and astrocytes, following
activation, have also been reported in a number of lead-exposed animal models
(Struzynska et al., 2007b, Gillis et al., 2012). However, up to concentration of 50 µM,
lead had no toxic effects on the morphology or viability of the M-MG cells. Similarly,
we did not observe any changes in morphology or viability at lower concentration of lead
in HMC3. It seems that there is no clear evidence about negative effects of lead on
viability and proliferation of glia cells in CNS. A recent study has shown the broad
variability in size of microglia in low lead exposed animal (Sobin et al., 2013).
Interestingly, recent studies reported that treatment with LPS (100 ng/ml) and IFN- (10
ng/ml) could induce ramified morphology in a rat microglia cell line (Sheng et al., 2011).
However, in the presence of 50 µM or more lead, the size of HMC3 increased in a time-
dependent manner, similar to LPS treated cells, which is supported by published studies
(Espinosa-Oliva et al., 2009, Kim et al., 2000, Nakamura et al., 1999).
In the previous chapter, we showed that lead could alter the expression pattern of
chemokine receptors in M-MG model, general down regulation in expression of pro-
inflammatory chemokine receptors (Chapter 5). Chemokine receptors play an important
role in neuron-microglia communication, neuronal development as well as
neuroinflammation (Cartier et al., 2005, Mines et al., 2007). Therefore, novel therapeutic
strategies attempt to reduce or prevent the extent of chemokine/receptor expression by
microglia as well as other CNS cells to provoke neuroprotective processes. There is not
much known about the expression of chemokine receptors in healthy CNS in particular
non-activated microglia. Expression of a number of chemokine receptors by HMC3 has
been also reported (Flynn et al., 2003). Among the chemokine receptors, CCR1, CCR2,
CCR5, CXCR1 and CXCR5 showed higher levels of expression than others which may
implicate the involvement of these molecules in the natural function of microglia such
maintain homeostasis or cell turnover. Therefore, we were interested to investigate the
effect of lead exposure on chemokine receptor expression pattern in HMC3. We were
also wondering, if HMC3 would show similar responses to lead exposure. Our results
[126]
showed HMC3 expressed most of the chemokine receptors at low-medium level under
non-stimulatory condition similar to M-MG.
Following lead exposure, general down-regulation in expression patterns of chemokine
receptors were observed. Among the tested receptors, expression of CCR1, CCR2,
CCR5, CXCR1 and CXCR5 in lead-treated HMC3 showed the significant decrease
(P<0.05). Expression of CCR1, CCR2 and CXCR1 showed significant down-regulation
in lead-treated M-M. Recent evidence has shown involvement of CXCR1 and CXCR5 in
neurogenesis (Edman et al., 2008, Kizil et al., 2012). A distinct expression pattern of
CCL2/CCR2 has been documented during different stages of embryonic brain
development implicating a role for this chemokine network in development and
neurogenesis (Rezaie et al., 2002). On the other hand, enhanced expression of chemokine
receptors, CCR1, CCR2 and CCR5 have been demonstrated in neurodegenerative
diseases, such as Alzheimer’s disease, multiple sclerosis and Parkinson’s disease
(Eltayeb et al., 2007, Bagaeva et al., 2006). Therefore, it seems that lead alters the
expression of certain chemokine receptor in human microglia which may affect the cell
regeneration, migration and differentiation.
In this study, we also compared the effects of lead on chemokine receptor expression
pattern with dexamethasone and IFN- as well-known immune suppressant and
stimulants. We observed that lead did not behave similar to either of them, suggesting
lead has different effects on immune responses than dexamethasone and IFN-.
In the context of expression of surface molecules, in line with published data (Flynn et
al, 2003) and our M-MG model, HMC3 expressed CD45, CD11b, CD11c and CD68 ,at
low levels. Among the surface markers, CD54, adhesion molecules, showed the highest
expression level in HMC3 control cells. In the presence of lead (10 µM, 24 hours),
significant reduction in tested surface markers, including CD45, HLA-DR, CD11b,
CD11c and CD54, was seen (P<0.05). However, a similar decrease in expression of
surface markers was seen in lead-treated M-MG model.CD54, inter-cellular adhesion
molecules 1 (ICAM)-1, which has shown to be expressed by microglia in CNS, play
important role in trafficking leukocyte in the brain. Increased in expression of CD54
followed by microglia activation has been previously reported (Zuckerman et al, 1998,
Huber et al., 2006). In addition, it has been reported that CD54 plays an important role in
the antigen presentation ability of microglia together with MHC molecules and other co-
stimulatory molecules such as CD40 (Aloisi et al., 2000, Ma et al., 1999). Our results
showed that lead had no significant stimulatory effects on antigen presentation ability of
[127]
HMC3 and M-MG Decreased expression of HLA-DR, as well as co-stimulatory
molecules (CD40 and CD54) were observed.
Microglia are known as the phagocytes of the CNS (Nakajima and Kohsaka, 2001).
Increased phagocytic activity has been demonstrated by activated microglia which could
play both beneficial and detrimental during health and pathology (Rogers et al, 2001,
Fricker et al, 2012). HMC3 under basal condition showed low level of phagocytic
activity. Our result showed no influence of low level lead exposure on phagocytic
activity. However, at higher concentrations, significant increased phagocytosis was
observed, similar to that of LPS- and IFN--activated HMC3. In the context of effect of
lead on phagocytic activity of microglia, HMC3 and M-MG model showed a similar
responses (data not shown).
Using Leadmium Green as an specific lead detector probe, our result showed that HMC3
could take up lead intracellularly and accumulate inside the cell, similar to the M-MG
shown in the previous chapter. Otherwise, there is no other study showing lead uptake by
human microglia cells in vitro. However, lead uptake by astrocyte has been previously
reported. Similar to astrocytes, microglia may exert a neuroprotective mechanism against
toxic effects of lead on neuron especially during development by engulfing lead and
storing it intracellularly (Tiffany-Castiglioni and Qian, 2001).
We also compared the secretion of cytokines (IL-6, IL-8, TNF-, IFN-, IL-1β and IL-
12) in the absence and presence of lead in the HMC3 with M-MG models. HMC3 at the
non-stimulatory conditions secreted low detectable amount of IL-8 and IL-6. We have
observed both cytokines in supernants of non-tretaed M-MG. However, these cells
secreted IL-8 at the higher level than HMC3. In concordance with previous data, HMC3
exposed to different concentration of lead (1 and 10 µM) showed significant up-
regulation of IL-8 secretion, similar to M-MG. IL-8 is a chemokine secreted only in
human and shown to play an important factor in intercellular communication between
glia and neurons (Puma et al., 2001). IL-8 secretion by microglia has been previously
shown, which supports our findings in both M-MG and HMC3 (Ehrlich et al., 1998).
Interestingly, we also observed significant down-regulation in IL-6 secretion in HMC3
after lead exposure. IL-6 expression by non-activated human microglia has previously
reported (Janabi et al, 1995, Nagai et al, 2005) which supports our findings. Positive
effects of IL-6 has been reported on neurogenesis in the dentate gyrus of adult mice and
on memory performance of the animal (Baron et al., 2008).
[128]
6.7. Conclusion
In summary, findings of this study demonstrated that lead has similar effects on HMC3
and M-MG in the context of morphology, phenotype and function. These similarities
between M-MG and human microglia cells derived from the human fetal brain supports
M-MG as an novel, but valid in vitro model representing the characteristics of human
microglia. Like lead-treated M-MG, lead at low concentration (<10 µM) had no toxic
effects on the viability of HMC3. Lead-treated HMC3 showed no sign of activation
regarding the changes of its morphology or function. However, lead has a significant
influence on expression of chemokine receptors and markers important for microglia
proliferation, differentiation and activation. Similar to M-MG, HMC3 could take-up lead
under non-stimulatory conditions, accumulate lead intracellularly substantially, that may
cause the possible alteration in intracellular signaling and cellular functions. All these
results together suggest that despite not having direct toxic effects on human microglia,
lead exposure, even at low levels, may cause indirect negative effects on cell -cell
communication, which plays a key role on neuronal remodeling and synaptic activity.
In conclusion, one can postulate that both microglia models are equivalent and valid
to investigate lead toxicity in vitro in humans.
[129]
Chapter 7
Differential effects of lead on CX3CR1 and
TLR4 expression in human microglia
in vitro
[130]
7. Differential effects of lead on CX3CR1 and TLR4 expression in human microglia in vitro
7.1. Introduction to chapter
From the previous studies in chapters 5 and 6, lead had the functional and
immunomodulatory effects on human microglia. Lead exposure altered significantly the
expression patterns of various chemokine receptors in human in vitro microglia models.
In this chapter, we focus on the effects of lead on CX3CR1 and TLR4 expression pattern
in human microglia due to their important role in modulating inflammatory responses.
Over the past decade, several studies have shown the neurotoxic effects of lead exposure,
even at low concentration, on the impairment of learning and memory function
especially in children. Microglia as the immune effectors of the CNS are actively
involved in initiating the immune responses, secreting variety of cytokine and
chemokines as well as having normal role in the development and function of the brain.
The exact role of microglia in lead-induced neurotoxicity is still unknown. However, it
has been suggested that microglia activation may involve in lead-induced
neuroinflammation and neurotoxicity.
It has been known that chemokine and their receptors play a key role in modulating the
immune responses. There is evidence showing the interaction between fractalkine
(CX3CL1) and the fractalkine receptor (CX3CR1) regulates microglial activation in the
CNS. Numerous studies have revealed that the CX3CL1/CX3CR1 axis plays a direct role
in neurodegeneration/ or neuroprotection depending upon the CNS insult. CX3CR1
deficiency has been shown to be involved in impairment of hippocampal cognitive
function. Toll like receptor 4 (TLR4), the innate immune receptor, is expressed on
microglia and mediates the neuroinflammation. Numerous studies have shown TLR4-
dependent activation of microglia in neurodegenerative diseases. Developmental role of
TLR4 in learning and memory has also been demonstrated. In addition, a recent study
showed that lead could alter the susceptibility to different pathogens through TLR4
activation in murine macrophages.
In this study, first we investigated the differential expression of CX3CR1 and TLR4 in
non-treated M-MG and HMC3. Then, after 24 hours exposure to 10 µM lead acetate, the
changes on expression patterns of mentioned markers were measured using flow
cytometry. Results showed different expression level of CX3CR1, low and intermediate,
[131]
in M-MG derived from different individuals with dissimilar responses to lead exposure.
Previous studies have reported the polymorphisms in CX3CR1 gene. However, the
asscoaition between polymorphic variant of CX3CR1 and neuroinflammtion/
neurodegeeration remain incompletely defined. Human microglia cell line HMC3
expressed CX3CR1 at the low level. Lead has different effect on the expression pattern
of CX3CR1. Lead increased slightly the expression CX3CR1 in cells with low
expression level, however, CX3CR1 expression in the intermediate group down
regulated after lead exposure. These data may suggest the differential responses of
CX3CR1 to lead. We have observed the different responses to TLR4 as well which could
be possibly associated with different individual susceptibility to lead exposure. This
highlighted again that lead could altered the microglia function and inflammatory
responses through changing expression pattern of immune receptors.
Declaration of the work:
All the experiments were designed and conducted by SE involving cell culture, surface
marker expression (FACS) and statistical analysis under supervision Prof. Luis Filgueira
. SE was responsible for study design, experiments, data analysis and writing of the
manuscript. Dr. Marc J Ruitenberg contributed to study design, data analysis and editing
of the manuscript. Prof. Luis Filgueira contributed to study design, data analysis and
editing of the manuscript. A manuscript has been submitted to the Journal of
Neuroinflammation.
7.2. Abstract
Lead is a well-known environmental pollutant and neurotoxic heavy metal. Lead
neurotoxicity results in learning and memory impairment by probably altering
neurogenesis and neuronal turnover. Microglia cells have been proposed to contribute to
lead toxicity by promoting an inflammatory environment and interfering with neuronal
turnover. However, the exact mechanisms of how lead affects microglia phsyiology and
function at cellular and molecular levels is still not well understood. Due to the
importance of both receptors, CX3CR1 and TLR4, in modulation of neuroinflammation
towards neuroptotction or neurotoxicty; in this chapter, we hypothesized that lead may
alter inflammatory responses through changes on expression pattern of CX3CR1 and
TLR4 in human microglia. To conduct this aim, a newly established human monocyte-
derived microglia and the microglia cell line HMC3 were used as human in vitro
mcirolgia models. The cells were exposed to various lead concentrations (1µM to 100
µM). In addition,dexamethasone, as an immune suppressive agent, as well as interferon-
[132]
gamma, a known immune stimulatory agent were used to comapre the expression pattern
of CX3CR1 and TLR4 in presnce of lead. Expression levels of CX3CR1 and TLR4 was
measured using flow cytometry.
According to our results, CX3CR1 showed dimorphic expression in microglia cells
derived from different individuals, having either CX3CR1low
or CX3CR1intermediate
expressing cells. However, the human HMC3 microglia cell line belongs to the
CX3CRlow
expressing population. We showed that lead increased significantly CX3CR1
expression in CX3CR1low
cells, whereas it decreased significantly in CX3CR1intermediate
cells. TLR4 was expressed at low levels in all microglia populations. Intretsigly,
increased in TLR4 expression only was seen in the CX3CR1intermediate
microglia cells.
Dimorphic expression of CX3CR1 in human microglia from different individuals.is
supporting the presence of functionally relevant genetic polymorphisms in humans. In
addition, differential expression of CX3CR1 in the HMC3 cell line and the monocyte-
derived microglia may be possibly related to the different state of microglia
differentiation.
Furthermore, lead influences up-regulation of TLR4 expression in a CX3CR1
polymorphic dependent manner, indicating, that regulation of CX3CR1 and TLR4 must
be interconnected. Finally, our study indicates that lead interferes with regulation of
CX3CR1 and TLR4 expression. The detailed molecular mechanisms remain to be
investigated.
Keywords: human microglia, lead, CX3CR1, TLR4.
7.3. Introduction
Microglia are the resident immune cells of the central nervous system (CNS) and form a
dense cellular network throughout the nervous tissue. Thus, microglia contribute to the
first line of defence in the CNS. They play a key role in the homeostasis of the brain.
They are actively involved in removing cell debris and dead cells, including damaged
neurons. They also contribute to neurogenesis by producing neurotrophic factors (Aloisi,
2001, Ransohoff and Perry, 2009). They are activated in response to inflammatory
stimuli, following exposure to toxins or pathogens. Thereby, they change their dendritic
morphology towards round-shaped amoeboid cells, able to migrate, proliferate and
release inflammatory factors, as well as to increase the phagocytic activity. Several of
these functions are influenced or controlled by chemokines and corresponding receptors
[133]
expressed by microglia and the surrounding glia and neuronal cells (Bajetto et al.,
Kettenmann et al., 2011, Kim and de Vellis, 2005).
Chemokines are chemoattractant cytokines that play a vital role in leukocyte recruitment
and activation. The effect of chemokines is mediated by specific G-proteins-coupled
receptors. Interaction between the chemokine ligand and the receptor activates
downstream intracellular cascades, which act on regulation of gene expression,
modulation of cell cycle and changes in cell function. Chemokines and their receptors
have been shown to be expressed in all tissues and organs, including the CNS (Bacon
and Harrison, 2000). In the CNS, chemokines and their receptors contribute to attraction
and migration of neural stem and progenitor cells, to cell survival and differentiation,
cytokine production, myelination, neuro-vascularization and neuroprotection (Jaerve and
Müller, 2012). Fractalkine receptor (CX3CR1) is one such chemokine receptor. CX3CR1
is unique as it has fractalkine as its only, but very specific ligand. CX3CR1 has been
shown to control microglia activity and to contribute to migration and proliferation of the
cells. CX3CR1 deficiency results in microglia dysfunction, increased pathogen
susceptibility and increased neuronal cell death, as shown in CX3CR1 deficient animal
models (Lee et al., 2010). Importantly, CX3CR1 is constitutively expressed by microglia
in the CNS. On the other hand, fractalkine is expressed by neurons under healthy and
inflammatory conditions. Fractalkine has also been shown to be expressed by microglia
and astrocytes, especially following stimulation by inflammatory factors, including
tumor necrosis factor- or IL-1 β (Mizuno et al., 2003). Fractalkine can be found in two
forms, a membrane-bound and a soluble variant. The membrane form contributes to cell
adhesion, while the soluble form, produced from cleavage of the membrane bound form,
acts as a chemotactic agent and helps to recruit inflammatory cells (Chapman et al.,
2000). The membrane-bound form of fractalkine is cleaved by metalloproteases on
neurons upon cell activation (Hundhausen et al., 2003). Fractalkine has been reported to
be up-regulated and a neuroprotective factor in the context of neurodegenerative diseases
(Mizuno et al., 2003). Healthy neurons secrete fractalkine to modulate microglia
activation. On the other hand, interaction between neuronal fractalkine and its receptor
on microglia maintain microglia in their non-activated state (Gemma et al., 2010). The
neuroprotective properties of fractalkine are due to preventing neuronal cell death and
reducing the secretion of pro-inflammatory cytokines, including IL-1 β, IL-6 and tumour
necrosis factor-, as well as to enhance expression of iNOS by activated microglia
(Jones et al., 2010, Mizuno et al., 2003). Unfortunately, the exact role of CX3CR1
function in the brain remains controversial. In addition to the neuroprotective effects, the
fractalkine/CX3CR1 axis has also been shown to contribute to neurodegeneration.
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Disruption of this axis may activate the microglia and induce secretion of pro-
inflammatory cytokines. Lack of microglial expression of CX3CR1 has been shown to
be neurotoxic and to induce neuronal cell death in models of systemic inflammation,
Parkinson’s disease and amyotrophic lateral sclerosis (Cardona et al., 2006). On the other
hand, in models of ischemic stroke and Alzheimer’s disease, deficiency in CX3CR1
expression reduced inflammation and neuronal cell loss, implicating its role in the
various aspects of microglia functions (Denes et al., 2008, Fuhrmann et al., 2010).
However, the fractalkine/CX3CR1 axis seems to play a vital role in the crosstalk
between neurons and microglia.
Microglia express various other membrane receptor proteins for the interaction with
neurons and other cells of the CNS (Noda and Suzumura, 2012). Some of those receptors
belong to the toll-like receptor (TLR) family, which are essential in molecular pathogen
recognition and may also contribute to phagocytosis. TLR signaling is initiated by
specific ligands, mostly related to the recognition of pathogen associated molecular
patterns, such as bacterial LPS and other microbial and viral products. After binding of
the ligand to the receptor, the cells are activated by a down-stream cascade, which results
in production of pro-inflammatory cytokines and chemokines, as well as expression of
corresponding surface receptors. TLR4 is an important member of the TLR family. In
addition to binding LPS, TLR4 mediates strong activation of microglia, by enhancing
phagocytosis, including uptake of β amyloid, and production of pro-inflammatory
molecules. On the other hand, deficiency in TLR4 expression or in function results in
reduced microglia activity, as shown in TLR4 knock out animal models (Song et al.,
2011). TLR4 is therefore considered to be a crucial component of the innate immune
response in the brain (Jack et al., 2005). TLR4 expression has been shown to be
exclusively expressed by microglia (Tanga et al., 2005). Microglia has thus been shown
to be the most responsive cells to LPS in brain (Eklind et al., 2001). In addition to the
exogenous LPS, TLR4 is also able to bind endogenous ligands, known as molecules
representing the danger-associated molecular pattern (DAMP), such as heat shock
proteins, S100 protein and high mobility group box 1 protein (HMGB1), which are
released by injured and necrotic cells (Kakimura et al., 2002). TLR4 is unique, as its
activation induces production of interferon-β and CXCL-10. Moreover, TLR4 has been
shown to play a role in auto-regulatory apoptosis in activated microglia, through
interferon-regulatory factor 3 (IRF-3) activation and interferon-β production (Jung et al.,
2005).
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Despite the known roles of CX3CR1 and TLR4 in microglia activation, when broadly
investigated separately, no previous report has shown the direct or indirect interaction
between TLR4 and CX3CR1 under neuroinflammatory condition in human
microglia. However, there are evidences showing the activation via TLR4 can
regulate the CX3CL1/CX3CR1 expression in mouse microglia/macrophages
(Boddeke et al. 1999, Pachot et al., 2008). Furthermore, it has been reported that
activation of CX3CR1 can also potentiate the TLR4 activation through NF-kB
dependent signaling pathway in mouse macrophages (Donnelly et al., 2011, Ishida et
al., 2008).
Lead has been commonly accepted as a potent neurotoxin, especially in the developing
brain. However, the detailed mechanisms are still not well understood. A recent animal
study has reported the up-regulation of fractalkine in the lead exposed immature rat
brain, suggesting an inflammatory component in lead toxicity (Struzynska et al., 2007b).
Interestingly, CX3CR1 deficient mice have shown increased expression of IL-1 β in
response to LPS, with enhanced susceptibility to neurotoxins (Cardona et al., 2006,
Ransohoff et al., 2007). However, there is not much known about the effect of lead on
CX3CR1 expression in microglia. Other recent studies also suggest inflammatory effects
of lead by targeting TLR4 (Luna et al., 2012). However, the exact mechanisms are still
not known.
In this study, we investigated the effects of lead on expression of CX3CR1 and TLR4 in
human microglia using a novel human in vitro microglia model and the human microglia
cell line HMC3.
7.4. Material and Methods
7.4.1. Cell culture and experimental setting
In this study, we used a novel human in vitro microglia model, established by our group
(Etemad et al., 2012). Briefly, human peripheral blood monocytes were cultured in
RPMI 1640 with Glutamax and 1% antibiotics/antimycotic (all from Invitrogen,
Mulgrave, Victoria, Australia), supplemented with macrophage colony-stimulating factor
(M-CSF; 10 ng/ml; PeproTech, Rocky Hill, NJ), granulocyte-macrophage colony-
stimulating factor (GM-CSF; 10 ng/ml; PeproTech), β-nerve growth factor (NGF-β; 10
ng/ml; PeproTech) and CCL2 (100 ng/ml; PeproTech), at standard humidified culture
conditions (37oC, 5% CO2) for up to 14 days. Cell preparation of 30 blood donors were
used for this study
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We also used the human microglia cell line HMC3, derived from human fetal brain
tissue (kindly provided by Prof. Karl-Heinz Krause, University of Geneva, Switzerland).
HMC3 cells were cultured in T25 tissue culture flask (Sarstedt,) using DMEM/F12 with
Glutamax (Invitrogen) supplemented with 5% FCS and 1% antibiotics/antimycotic. The
cells were passaged and used for experiments when they reached 70–80% confluence.
In order to study the expression pattern of CX3CR1 and TLR4 under different
experimental conditions, the human microglia and HMC3 were treated with lead acetate
(1µM, 10µM, 50µM and 100µM, Sigma-Aldrich, St Louis, MO). The cells were also
exposed for 24 hours to immune stimulatory factors, such as LPS (10 ng/ml, Sigma-
Aldrich) and human recombinant interferon-gamma (10 ng/ml, PeproTech), as well as to
the immune-suppressant dexamethasone (1µM, Sigma-Aldrich).
7.4.2. Flow cytometry
Expression patterns of CX3CR1 and TLR4 in human microglia under various
experimental conditions was studied using flow cytometry. The cells were detached
using a scraper (Sarstedt) and washed twice with PBS. Cells (1x106 cells/ml) were
stained according to a protocol recommended by BD and used previously (Etemad et al.,
2012). Cells were incubated for 1 hour at 4oC with the primary antibody and blocking
agent (10% FCS), washed with PBS and incubated for 30 minutes with secondary
antibody. Following the final wash, cells were fixed with 1% formaldehyde in PBS
containing 2% sucrose. The fixed samples were measured with a FACSCalibure (BD
Biosciences). Data analysis was performed using the FlowJo software version 7.6.3
(TreeStar, Ashland, OR).
A rabbit anti-CX3CR1 polyclonal antibody (AbD Serotec, Oxford, UK; 1 /100) and
mouse monoclonal anti-TLR4 antibody (Santa Cruz Biotechnology, Santa Cruz, CA;
1:100) were used, followed by a secondary donkey anti-rabbit AF647 and anti-mouse
AF488 antibody at the final concentration of 1:200 (Invitrogen).
7.4.3. Statistical analysis
Data were statistically analyzed with the SPSS 17.0 software. Student t- test and one-way
ANOVA were used to compare means. The Dunnett post-hoc test was also used to
determine the significant difference between groups. In all the analysis P<0.05 was
considered as statistically significant. Graphs with error bars represent the standard error
(SED).
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7.5. Results
7.5.1. Differential expression of CX3CR1 in human microglia in vitro
We studied the expression of CX3CR1 in human microglia cells using two in vitro
models, monocyte-derived microglia cells (M-MG) and the microglia cell line HMC3.
Cells were cultured under standard protocols. It is noteworthy that M-MG were cultured
in serum free media, while HMC3 were culture in presence of 5% FCS. Once the cells
reached the optimal density, they were stained with anti-CX3CR1 antibody and the
fluorescence intensity was measured by flow cytometry. As expected, CX3CR1 was
expressed by non-lead treated, control M-MG. In addition, we observed that non-
activated, control M-MG from different human cell donors expressed CX3CR1 at
various levels, either low (n=18, MFI=19.87, CX3CR1low
) (Figure 7.1 A) or intermediate
expression levels (n=12, MFI=28.44, CX3CR1intermediate
) (Figure 7.1 B). The difference in
expression levels was statistically significant (P=0.027). However, the cells from the
different donors had the same morphological appearance (data not shown). We also
studied CX3CR1 expression in HMC3 cells, which was at low levels (n=20, MFI=17.15)
when the cells were non-activated (Figure 7.1 C), similarly to the CX3CR1low
M-MG and
statistically different from expression levels of CX3CR1intermediate
. CX3CR1 expression
levels of cells derived from low expressing (n=18) and intermediate (n=12) expressing
M-MG, as well as from HMC3 (n=20) were averaged and compared (Figure 7.1 D).
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Figure 7.1: Expression levels of CX3CR1 in human M-MG and HMC3 cells.
Depending on cell donor, CX3CR1 is expressed at low levels (A: CX3CR1 low) or
intermediate levels (B: CX3CR1 intermediate). Interestingly, CX3CR1 expression in the
microglia cell line HMC3 is at low levels (C), similar to the CX3CR1 low M-MG. In (D),
CX3CR1 expression levels of cells derived from low expressing (n=18) and intermediate
(n=12) expressing M-MG, as well as from HMC3 (n=20) were averaged and compared.
7.5.2. Differential effects of lead on CX3CR1 expression in human
microglia
To study the effect of lead on CX3CR1 expression, M-MG and HMC3 were cultured in
the presence of various concentrations of lead for 24 hours. Then, the cells were stained
and fluorescence intensity was measured using flow cytometry (Figure 7.2). We
observed two different responses in M-MG from the different cell donors after lead
exposure. M-MG with primarily lower expression showed up-regulation of CX3CR1
after exposure to lead (Figure 7.2. A). However, lead reduced expression of CX3CR1 in
M-MG with primarily intermediate expression levels (Figure 7.2. B). As we expected,
HMC3 with low expression level of CX3CR1 at non-stiumulatory condition showed
significant ehancement in CX3CR1 expression following exposure to lead, similar to
CX3CR1low
M-MG responses (n=20; from 17.154 to 38.942; P=0.009) (Figure 7.2. C).
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Figure 7.2. D summarizes the mean fluorescent intensity of lead- treated M-MG in
comparison with control in both responder populations and HMC3 cells. D. Lead
increased significantly expression of CX3CR1 in CX3CR1low M-MG (n=18; MFI: from
19.875 to 51.71; P=0.029), while CX3CR1intermediate
M-MG decreased CX3CR1
expression after lead exposure, although not significantly (n=12; MFI: from 28.44 to
18.9; P=0.05).
Figure 7. 2: Effect of lead on expression of CX3CR1 in human M-MG and HMC3 cells.
The cells were cultured for 24 hours in the presence of 10 µM lead. Histograms show
representative examples of CX3CR1 expression in M-MG (A and B) and HMC3 cells (C) after
lead exposure (white) in comparison with staining control (grey). Bar charts (D) compare lead
treated (black) with non-treated control (white) M-MG cells of basal low or intermediate
CX3CR1 expression, as well as HMC3 cells. Analysis of the significant differences between
two groups was done by ANOVA and LSD/Dunnett’s test. P<0.05.
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7.5.3. Effect of immune modulators on CX3CR1 expression in
HMC3 cells
To investigate whether the dichotomy in CX3CR1 expression and corresponding
response to lead exposure was unique or representing a basic pattern of response in
human microglia, we included treatment of HMC3 cells with interferon-gamma as
immune activator and dexamethasone as an immune suppressant. Interestingly,
dexamethasone did not influence CX3CR1 expression in HMC3 cells, whereas
interferon-gamma significantly increased CX3CR1 expression (P=0.000), similarly to
lead (Figure 7. 3).
Figure 7. 3: Comparison of various stimuli on CX3CR1 expression in HMC3 cells.
The cells were incubated for 24 hours in the presence of lead (Pb), dexamethasone (Dexa) or
interferon-gamma (IFN-). Interestingly, lead and interferon-gamma increased significantly
CX3CR1 expression in HMC3 cells.
7.5.4. Effect of lead on TLR4 expression human microglia
TLR4 as an important class of innate immune receptors, is expressed on microglia and
play key role in recognition of endogenous ligands as well as invaded pathogen. TLR4
medaites inflammtory respomses and participate both in development and in responses
associated with CNS injury.Therefore, expression of TLR4 was measured in M-MG and
HMC3 using flow cytometry. Under non-activated condition, both CX3CR1 low and
intermediate M-MG cell sub-pupulation as well as HMC3cell expressed TLR4 at low
levels (Figure 7.4 A-C. Following lead exposure, CX3CR1intermediate
M-MG cells showed
siginificant increse in expression of TLR4 (MFI: from 17.89 to 30.7775;
P=0.048),whereas no changes was seen in TLR4 expression level in CX3CR1low
M-MG(
MFI from 15.68 to 1.48, P=0.35) nor HMC3 cells was seen (MFI: from16.136 to
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13.821,P=0.45)(Figure 7.4 D-F). Expression levels of cells derived from low expressing
(n=18) and intermediate (n=12) expressing M-MG, as well as from HMC3 (n=20) were
averaged and compared (Figure 7.4 G).As expected, dexamethasone decreased
significantly TLR4 expression in HMC3 cells, whereas interferon-gamma increased
significantly TLR4 expression in HMC3 cells
Figure 7. 4: Effect of lead on expression of TLR4 in human M-MG and HMC3 cells.
The cells were cultured for 24 hours in the presence of 10 µM lead. Histograms (whites) show
representative examples of TLR4 expression in two M-MG sub-population and HMC3 cells in
absence (A-C) and presence of lead (D-F) and comparing the changes to iso type control
(gray).Bar charts (G) compare lead treated (black) with non-treated control (white) M-MG
cells of basal low or intermediate CX3CR1 expression, as well as HMC3 cells. Interestingly,
only CX3CR1intermediate
M-MG increased significantly TLR4 expression after lead exposure.
Analysis of the significant differences between two groups was done by ANOVA and
LSD/Dunnett’s test.
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7.6. Discussion
In this study, we investigated the effects of lead exposure on expression of CX3CR1 and
TLR4 in microglia using two human in vitro cellular models.
Fractalkine is expressed by neurons and astrocytes as a membrane-bound form for cell
adhesion and as a soluble form for chemotaxis. CX3CR1 is expressed mainly by
microglia in the CNS. The fractalkine-CX3CR1 axis mediates neuron-microglia
interactions, and contributes to the control of cell activation, proliferation, and migration
(Hatori et al., 2002, Zujovic et al., 2000). In microglia, fractalkine/CX3CR1 interaction
maintains the cells in a resting functional state (Rogers et al., 2011). Following neuronal
injury, decreased fractalkine expression levels is therefore associated with enhanced
microglia recruitment and activation (Rogers et al., 2011). However, little is known
about the regulation of CX3CR1 expression in human microglia. We have previously
shown that the human microglia cell line HMC3 express CX3CR1 at low levels (Etemad
et al., 2012). In the present study, we observed that human microglia expressed CX3CR1
at low and intermediate levels depending on the cell donor, which suggests heterogeneity
of CX3CR1 expression in human microglia depending on genetic polymorphism. In this
regard, previous studies have identified two single nucleotide polymorphisms (SNP) in
the CX3CR1 gene in humans (Chan et al., 2005). It is not known, whether these
polymorphisms influence expression levels of CX3CR1. However, CX3CR1
polymorphisms are associated with increased risk of macular degeneration (Raoul et al.,
2010), HIV-1related AIDS progression in children (Singh et al., 2005) and diverse
survival of patients suffering from glioblastoma (Rodero et al., 2008). It has also been
published that the two CX3CR1 variants may have differential effects on microglia
function, including cell migration and adherence (Donnelly et al., 2011b). Interestingly,
CX3CR1 expression levels in human macrophage populations also depends on
differentiation and functional status (Geissmann et al., 2003), indicating that CX3CR1
may be functionally regulated in human microglia, depending on activation. However,
our study confirms the existence of dimorphic CX3CR1 expression in humans. Now, it
was of interest to investigate, whether lead influences CX3CR1 expression levels in
CX3CR1low
and CX3CR1intermediate
microglia. Interestingly, lead somehow increased
significantly CX3CR1 expression levels in CX3CR1low
microglia, but decreased
CX3CR1 expression levels in CX3CR1intermediate
microglia. These results indicate that
lead has an influence on the regulation of CX3CR1 expression in human microglia cells,
which can be seen as an inflammatory effect of lead, similar to interferon-gamma, as
confirmed by our results in HMC3 ells. However, it remains to be further investigated of
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how and at what levels lead interferes with the regulation of CX3CR1 expression, and
whether the molecular interference of lead with CX3CR1 expression is different for the
two polymorphisms.
In this regard, recent studies showed the involvement of three polymorphic genes
related to lead toxicity (ALA-dehydratase, Vitamin D receptor gene, Hemochromatosis
gene), as well as sex differences in altering lead poisoning susceptibility in humans and
in animal models (Onalaja and Claudio, 2000). It has been suggested that these genetic
polymorphisms can potentially influence the accumulation of lead in the human body
and make some individuals more vulnerable to lead intoxication (Todd et al., 1996,
Sobin et al., 2009). Moreover, Schneider et al. (2011) reported that exposure to lead
results in significant changes in expression of various genes in the rat hippocampus and
various sex-dependent responses of the brain to lead exposure (Schneider et al., 2011).
These reports support our conclusion that lead interferes with the regulation of CX3CR1
expression and that further studies have to investigate the detailed mechanisms of this
interference. Considering the potential phenotypical and functional differences in
microglia between human individuals, one has to be careful in using only one given
microglia cell line, e.g. HMC3, which represents only one genetic make-up of only one
human individual. Consequently, we recommend to use our recently established new
human monocyte-derived microglia model for corresponding microglia studies to
consider genetic differences between human individuals with relevant functional impact.
When it comes to the discussion about the function of the fractalkine-CX3CR1 axis in
lead neurotoxicity, one has certainly to consider the controversial reports about the
function of this axis, as it includes neuroprotective and neuroinflammatory components.
However, all studies have been done in animal models and only little is known about this
axis in humans (Cardona et al., 2006). In the context of lead toxicity, knowledge about
the role of fractalkine-CX3CR1 axis remains also very scarce and further studies are
required (Struzynska et al., 2007b, Liu et al., 2012).In this study, we also investigated the
TLR4 expression pattern in human microglia models following lead exposure.
Interestingly, significant up-regulation in TLR4 expression was only seen in microglia
cells sub-population expressing the CX3CR1 at intermediate levels. These data indicate
that TLR4 expression is somehow connected to CX3CR1 expression in human microglia
as well as postulate the involvement of TLR4 in CX3CR1 dependent polymorphic
susceptibility against lead toxicity in a human population.
In that respect, the many different TLRs expressed on microglia and the TLR4 localized
on the surface of microglia is fisrt line of host defence against invading microorganisms
[144]
(Lehnardt et al., 2003). Microglia TLR4 is up-regulated upon brain inflammation and.
also mediates recognition of endogenous danger signals derived from stressed or necrotic
cells (Kakimura et al., 2002). Intriguingly, microglia TLRs tend to trigger a very
standard cytokine and chemokine response, irrespective of the type of TLR agonist they
meet. Which by microglia may be to recruit the assistance by other cells rather than to
immediately mount a destructive response towards invaders (van Noort and Bsibsi,
2009).
Although previous studies have reported that TLR4 activation, can regulate
CX3CL1/CX3CR1 expression and, vice versa, through activation of NF-kβ signaling
pathway (Boddeke et al., 1999, Donnelly et al., 2011, Ishida et al., 2008 and Pachot et
al., 2008), there is still not much known about interaction between TLR4 and CX3CR1
in the context of lead-induced neuroinflammation/neurotoxicity.
Since the shown reciprocal interaction has not been reflected in this study, our findings
may suggest the distinct effect of lead on expression pattern of TLR4 and CX3CR1 as
well as involvement of other signaling pathways in modulation of this interaction.
Therefore, more research is needed to identify the mechanisms behind the connection
between TLR4 and CX3CR1 regarding modulation of inflammatory responses in
microglia.
7.7. Conclusion
CX3CR1 is differentially expressed in human microglia, probably depending on genetic
polymorphism. Correspondingly, lead has differential effects on expression of CX3CR1
in microglia from different human individuals. Furthermore, lead influences up-
regulation of TLR4 expression in a CX3CR1 polymorphic dependent manner, indicating,
that regulation of CX3CR1 and TLR4 must be interconnected. Finally, our study
indicates that lead interferes with regulation of CX3CR1 and TLR4 expression. The
detailed molecular mechanisms remain to be investigated.
Chapter 8
General discussion
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8. General discussion and future directions
8.1. Summary of key findings
Lead has been known for a while as a potent neurotoxicant with severe detrimental
manifestations on the development and function of the CNS. Several human and animal
studies have reported significant effects of lead, even at lower blood lead levels (<2 µg
dl/0.1 µM), on learning ability and memory, as well as on cognitive functions of adults
(Olympio et al., 2009, Wang et al., 2012). Children and foetuses are two groups that are
highly susceptible to lead toxicity. It is evident that blood lead levels over 10
µg/dl/0.48µM (threshold limit) have negative effects on children’s brain development
and immune function (CDC, 2005, Finkelstein et al, Koller et al., 2004, Lanphear et al,
2000, Lidsky and Schneider, 2003).
CNS cells are the primary target of lead toxicity, especially during development. One of
the target cell populations are neurons, respectively neuronal precursor cells. They were
investigated in chapter 3.
In addition to neurons, involvement of glia cells in lead induced toxicity has been shown
in vitro and in vivo. Lead was shown to activate glia cells and to induce indirectly
neuronal cell death in animal models of lead toxicity. Astrocytes have been the major
glia population investigated in the past in the context of lead toxicity. They were shown
to take-up and store lead intra-cellularly, a phenomenon resembling the lead-
sequestering ability for mature astroglia in vivo (Tiffany-Castiglioni and Qian, 2001).
Microglia were investigated in chapters 4, 5, 6 and 7. Microglia as the immune cells in
the brain play an important role during health and pathology of the CNS (Kim and De
Vellis, 2005, Tremblay et al., 2011). As the brain macrophages, they share common
characteristics with other monocyte/macrophage lineages, including expression several
surface markers, expression of chemokine and chemokine receptors, secretion of
cytokines, antigen presentation and phagocytosis (Guillemin and Brew, 2004,
Kettenmann et al., 2011, Rock et al., 2004). Under healthy condition, microglia
constantly screen the microenvironment and modulate neuronal health and activity
through secretion of various neurotrophic factors (Nimmerjahn et al., 2005, Ransohoff
and Perry, 2009). Within the CNS, microglia are the first line of defense that respond
rapidly to any type of brain injury with changes in morphology and function towards
activation. Microglia activation is followed by secretion of several pro-inflammatory
cytokine and chemokine which can directly damage cells and lead to neuronal cell death
[147]
(Kreutzberg, 1995, Kettenmann et al., 2011, Ransohoff and Perry, 2009, Tambuyzer et
al, 2009). Thus, microglia are known to be the key cellular mediator of the
neuroinflammatory responses that can induce both, neuroprotection or neurotoxicity,
depending on the insult. Involvement of microglia activation has been recently reported
in animal model of lead-induced neurotoxicity, with enhanced secretion of pro-
inflammatory cytokines, as well as with induced neuronal injury (Liu et al., 2012).
However, there is not much known about the role of human microglia in lead induced
neurotoxicity/neuroinflammation. To investigate the role of human microglia in lead
toxicity in human, a new model was required, which was developed in the context of this
thesis (Chapter 4). Then the effect of low level of lead exposure (10 µM, 24 hours) was
investigated using the new human microglia model (Chapter 5). The results were then
confirmed using an established human microglia cell line (Chapter 6). Finally, two
important mechanisms were further investigated with these two microglia models,
namely fractalkine/CX3CR1 and TLR4 (Chapter 7).
The main aim of the first experimental chapter of this thesis (Chapter 3) was to
investigate the influence of lead on human neuronal precursor cells in vitro. The human
cortical neural cell line HCN2 was used as an in vitro human experimental model for this
study. Interestingly, lead was not really toxic to HCN2 cells, at least at lower
concentrations, which are probably those found in humans exposed to toxic lead levels.
With this in mind, it was of interest to investigate phenotypical and functional changes in
HCN2 cells. Since there were not many reported studies about the cellular characteristics
of HCN2 cells, further characterization was primarily needed. We showed that the non-
differentiated HCN2 cells represent the characteristics of neuronal precursor cells
(NPCs) in the context of morphology, as well as expressing neuronal precursor marker
(nestin, vimentin, β-tubulin III). Interestingly, expression of β-tubulin III was clearly
reduced in lead treated cells. Decrease in gene expression level of β-tubulin III in cortical
neurons with increasing postnatal development and differentiation has been reported
(Jiang & Oblinger, 1992). Lead driven differentiation was also supported by
corresponding morphological changes. This indicates that lead may drive differentiation
of HCN2 cells towards more mature neurons, something which may also happen in lead
exposed children and adults. In children, where brain development is still ongoing and
neuronal precursors differentiating in various brain regions contribute to it, lead may
have a more general detrimental effect on brain development by interfering with cell
differentiation. In adults, where neuronal precursors are mainly required in the
hippocampus, the memory function seems to be affected by lead toxicity. The
mechanisms of how lead influences directly HCN2 differentiation remains to be further
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investigated. Interestingly, lead suppressed the secretion of IL-6 in HCN2, which may be
a possible mechanism for blocking differentiation (Monje et al., 2003, Nakanishi et al,
2007). In vitro animal models have also reported the suppressive effects of IL-6 on
differentiation of neuronal stem cells into neurons, as well as diminished hippocampal
neurogenesis.
In the context of phenotype, HCN2 expressed a number of chemokine receptors, such as
CCR1, CCR2, CCR3, CCR5, CXCR4, CX3CR1, implicating the involvement of
chemokine signaling in neuronal development, as well. Previous studies also showed that
chemokines, such as CCL2, play a key role in migration of NPCs towards the site of
injury during neuroinflammation (Belmadani et al., 2006). Following lead exposure,
expression of the mentioned chemokine receptors were down-regulated in HCN2 cells.
However, a significant increase in expression of CXCR1 and fractalkine was observed.
Changes of these receptors and factors may play a major role in lead neurotoxicity
resulting in the well -known clinical effects. Summarizing, these findings suggest that
lead may affect the development and function of human NPCs through changes in
chemokine signaling pathways, cytokine secretion and metabolic differentiation
pathways.
For this study, we also needed a good model representing the characteristics of human
microglia in vitro, that would consider polymorphism and diversity usually seen in
humans. In the second experimental chapter (Chapter 4), we developed a novel protocol
to generate human blood monocyte-derived microglia model. Detailed characterization
of M-MG revealed a cell population representing resting microglia with their specific
morphological, phenotypic and functional properties. The described protocol is easy to
handle, well standardized and very reproducible, as it uses only well defined culture
medium and recombinant cytokines, but no serum or conditioned medium. This protocol
was very helpful for investigating the influence of lead on human microglia.
To date, a number of animal and human models of microglia and microglia-like cell
lines have been used to examine neuroinflammatory phenomena. These models include
primary microglia cultures, and immortalized microglia cell cultures, which were either
retrovirus transformed or non-retrovirus transformed. These culture models share
similarities, but show also crucial differences that must be considered when choosing an
appropriate model for neurodegenerative research. Most of the common microglia
models used in several studies were derived from rodents (mice and rats). Difficulties in
obtaining human brain tissue samples, limited number of cells in primary culture, as well
as changes in their characteristics after isolationhave been some of the reasons restricting
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microglia studies to animal models, instead of using human cells. In the context of
human immortalized microglia cell cultures, there are few established human cell lines
available for research. The human microglia cell line HMC3 is one of the few, well-
established human immortalized microglia cell lines (Janabi et al., 1995). HMC3 cells
were used in this study as an experimental model (Chapters 6 and 7). However, HMC3
are just representing one human, fetal individual, and results may be biased due to that
fact. Investigating the influence and effects of lead on human microglia has been the
main focus of this thesis (Chapters 5 and 6), with different aspects in mind, including
morphology, viability, surface marker expression, chemokine receptor expression
pattern, as well as cytokine secretion. For that purpose, monocyte-derived microglia and
the microglia cell line HMC3 have been used as human in vitro microglia models. In
both in vitro models, similar changes were observed in responses to lead exposure. Our
finding showed that lead had no cytotoxic effects on viability or morphology of M-MG
and HMC3 in culture. We also showed that lead at the low levels had no significant
impact on the phagocytic ability or the T- lymphocyte stimulatory capacity. Both
microglia cell types showed the ability to take-up lead in a concentration-dependent
manner. With these facts in mind, it is tempting to postulate that lead may not be really
cytotoxic to human microglia at lower concentrations, which are probably found in vivo
in exposed humans. However, there were substantial changes in other functions and
phenotype in microglia exposed to lead. In the context of phenotype, there was a general
down regulation of chemokine receptors. This indicates that lead may disrupt or change
migratory properties of microglia and the physiological interaction between microglia
and their neighbouring cells, including neuronal precursor cells. This interaction may be
further affected by lead-dependent changes in chemokine and cytokine secretion by
microglia and other cell types. In that context, one has to mention the increase in IL-8
secretion by M-MG and the enhanced secretion of IL-6.
Changes in these cytokines may well affect brain remodeling and recruitment of
neuronal precursor cells in the developing brain and in the adult hippocampus. In
summary, these studies provide a basis in understanding of the impacts of low-level of
lead exposure (<10µM, 24 hours) on human microglia in culture and changes in immune
properties of microglia as one of the possible involved mechanisms.
Finally, the last experimental chapter (Chapter 7) focused on the impacts of lead
exposure on expression of CX3CR1 and TL4 in human microglia. These two membrane
proteins pay a cruicial role in inflammatory responses, including inflammation in the
brain.
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TLR4 is one of toll-like receptors, which is expressed by microglia in CNS and plays a
key role in the recognition of danger molecules secreted by injured CNS upon exposure
to pathogens, toxins, mechanical damage, etc (Rock et al., 2004, Olson and Miller,
2004b). TLR4 is one of the LPS receptors in conjunction with CD14 and its activation
triggers the complex intracellular signaling cascades, which cause induction of
cytokines and other inflammatory mediators (Laflamme et al., 2003, Qin et al., 2005,
Triantafilou and Triantafilou, 2002). TLR4 has been reported to induce microglia
activation and cytokine production in several neurodegenerative diseases (Heneka et al.,
2010, Lehnardt et al., 2003). Recent studies suggest that lead could alter the immune
susceptibility of macrophages to pathogen probably through interfering with the
binding of LPS to TLR4 (Luna et al., 2012). The mechanisms of how lead influences
changes of TLR4 expression remains unknown and needs to be further investigated.
Our findings showed that lead has differential effects on expression of CX3CR1 in
microglia from different human individuals. Interestingly, polymorphism has been
shown in humans for CX3CR1 (Garin et al., 2003, Singh et al., 2005). This polymorphic
property may be responsible for the dichotomy in response to lead in different
individuals. However, a correlation between lead response and CX3CR1 polymorphism
has to be investigated through future experiments.
Furthermore, lead influences up-regulation of TLR4 expression in a CX3CR1
polymorphic dependent manner. On the other hand, activation of macrophages via
TLR4 can regulate fractalkine and CX3CR1 expression (Boddeke et al., 1999, Pachot
et al., 2008). In addition, it has been shown that CX3CR1 can also increase NFkB
dependent transcription signaling via TLR4-dependent inflammatory responses
implicating the prominent regulatory role of CX3CR1 and TLR4 signaling during
inflammation (Donnelly et al., 2011a, Ishida et al., 2008). In summary, these data
emphasizes the importance of CX3CR1 and TLR4 in lead toxicity.
As lead showed significant increase in expression of IL-8 and CX3CR1 in human
microglia as well as their reciprocal receptors/ligands, CXCR1 and fractalkine in human
NPCs, the following paragraphs will focus on the importance of IL-8/CXCR1 and
fractalkine/CX3CR1 signaling pathways in the microglia-neuron interaction. In addition,
possible outcomes of lead interference on neurogeneration or neuroinflammation will be
discussed.
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8.2. Neuronal remodeling and lead toxicity
There is evidence showing that lead exposure even at low concentration can cause
learning and memory impairments in animals and humans (Al-Saleh et al., 2009, Liu et
al., 2012). The hippocampus is a brain center for learning and memory (Bird and
Burgess, 2008). Long-term potentiation of the hippocampal excitatory synaptic
transmission is thought to be one of the underlying mechanisms for hippocampus-
dependent learning and memory (Bliss and Collingridge, 1993). Remodeling of brain
circuits, including the formation, modification and elimination of synaptic structures,
occurs throughout life as animals adapt to their environment. Until recently, known
mechanisms for experiment-dependent synaptic plasticity were focused on neuron-
astrocytes interaction (Stevens, 2008). However, microglia have been shown to be active
even under physiological condition and their processes actively in contact with dendritic
spines (Tremblay, 2011). Recent evidence shows that microglia may phagocytize
synaptic elements and modify synaptic plasticity during early postnatal development, as
well as after brain injury or during inflammation (Tremblay, 2011, Tremblay and
Majewska, 2011). Over the past decade, several studies suggested that lead interferes
with neuronal remodeling as one of the possible underlying mechanism in lead-induced
learning and memory deficits. It has been postulated that lead may specifically affect the
synaptic transmission; however, the molecular targets for lead are still unknown
(Bressler et al., 1999a, Goldstein, 1990b). In vitro studies suggested that exposure to lead
could cause changes in protein kinases activation in synapses and disrupted the normal
developmental sequence. Adhesion molecules also play key role in of synaptic plasticity.
Changes in adhesion molecules expression has been shown in lead induced neurotoxicity
studies (Davey and Breen, 1998, Hu et al., 2008, Hu et al., 2011). In general, the ability
of lead to substitute for other polyvalent cations such as calcium and zinc is regarded as
the main toxic mechanisms in CNS. These interactions allow lead to affect different
biologically significant processes, including metal transport, energy metabolism,
apoptosis, ionic conduction, cell adhesion, inter- and intracellular signaling, diverse
enzymatic processes, protein maturation, and genetic regulation. Membrane ionic
channels and signaling molecules seem to be the most relevant affected molecules
(Garza et al., 2006, Nemsadze et al., 2009, Rai et al., 2010, Xu et al., 2006b). Taken
together, lead could alter the neuronal remodeling process through changes in the
microenvironment of the brain. It also could affect signaling pathway, interfere with
communication of neuron with other cells (microglia, astrocytes) in the brain, and disrupt
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normal development without producing signs of cell death. In the future, it will be
important to provide better understanding of the role of microglia in neuronal synaptic
modification during lead poisoning, and examine whether microglia function, such as
phagocytosis or secretion of immune modulators proceeds differently in health and
disease.
8.3. Why microglia are important in lead-neurotoxicity?
The importance of microglia as the immune cells of the CNS in degenerative diseases
makes them an interesting target for therapeutic approaches. The involvement of
microglia in neurotoxicity induced by heavy metal such as cadmium (Yang et al., 2007),
manganese (Crittenden and Filipov, 2008), cobalt (Mou et al., 2012) has been previously
shown using the in vitro and in vivo animal models. Several studies reported that such
metals could trigger microglia activation followed by increased in number and size of
glia cells, enhanced secretion of pro inflammatory cytokine such as TNF and IL-6, more
expression of oxidative stress and nitiric oxide (NO) (Kauppinen et al., 2008, Chen et al.,
2006). Beside activation, microglia cell death also has been reported in rat methyl
mercury induced neurotoxicity(Nishioku et al., 2000). Nuclear factor kappa B (NF-κB)
and p38 mitogen-activated protein kinase (MAPK) signaling pathways has been
suggested as the main involved signaling pathway in metal -induced cytokine
production in activated microglia.
In the context of lead toxicity as a heavy metal, animal studies revealed induced
astrocyte activation as the CNS glia cells following lead exposure. Activation of
astrocytes may lead to loss of buffering function and induced neuronal cell death. It has
also been showed that astrocytes could accumulate lead ions intracellularly as possible
neuronal protection from lead toxicity (Struzynska et al., 2007). Astrocyte activation by
lead has been associated with an increased number of cells and activation markers such
as GFAP as well as increased secretion of pro-inflammatory cytokine (Selvin-Testa et
al., 1991). There is evidence showing the toxic effects of lead on both microglia and
astrocytes in immature culture of rat brain (Zurich et al., 2002). Interestingly, recent in
vitro and in vivo animal study showed that lead can cause microglia activation, which
can up-regulate the level of IL-1β, TNF-α and iNOS, these pro-inflammatory factors
may cause hippocampal neuronal injury as well as long-term potentiation (LTP) deficits.
However, to our knowledge no previous studies investigated the effect of lead exposure
on human microglia in vitro.
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On the other hand, learning and memory impairment as well as cognitive deficits are
one of the manifestations of lead toxicity in children. Over past decades, several studies
has focused on role of microglia in cognitive function of the brain (Ziv and Schwartz,
2008). Detrimental effects of microglia activation on learning and memory has been
reported (Cagnin et al., 2001). Inhibiting the activation can improve the cognitive
impairment in a murine model of human immunodeficiency virus (HIV) type 1
encephalitis (Keblesh et al., 2009). Therefore, better understanding of role of microglia
in lead-induced toxicity may be helpful to develop new pharmaceutical approaches in
treatment of clinical neurological disorders through inhibiting the microglia activation as
target mechanisms. As the main aim of this project, we were interested to investigate
whether low level of lead exposure could activate the human microglia through alteration
in its morphology, phenotype and function as well as neuronal precursor survival in
vitro.
8.4. Microglia-neuron interaction: A possible target of lead toxicity
Similar to previous reported data, in this thesis, we showed the expression of a variety of
chemokine receptors in both microglia and neuronal procures cells under non-
stimulatory conditions. Furthermore, secretion of several chemokines by neurons and
glia during CNS health and pathology has been reported, implicating the important role
of chemokine-receptor interaction in neuronal development and function (Bajetto et al.,
2001, Cartier et al., 2005, De Hass et al., 2007, Krathwohl et al., 2004, Sorensen et al.,
1999). It has been shown that microglia are actively involved in maintaining the brain
homeostasis and controlling the neuronal proliferation, migration as well as
differentiation during developmental period and adulthood (Bessis et al., 2007,
Cunningham et al, 2013, Morgan et al., 2004). As one of the remarkable finding of this
thesis, in the human neuronal precursor cells HCN2, down-regulation in expression level
of several chemokine receptors and surface markers was observed in the presence of
lead. Involvement of chemokine-receptors signaling in neuronal development
(proliferation, migration and differentiation), as well as neuroinflammation has been
previously reported. CCR2 is one of the chemokine receptors that play an important role
in recruiting monocytic cells to the site of injuries and initiate inflammatory responses
(Zhang et al., 2007, Wang et al., 2009). We showed down-regulation in CCR2
expression in both microglia and human neuronal precursor cells in the presence of lead.
Recruitments of the NPCs to the site of inflammation has been shown through CCR2
(Belmadani et al., 2006). Impaired microglia accumulation and early disease progression
has been reported in CCR2 deficient animal model of Alzheimer’s disease (El Khoury et
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al., 2007). These data implicate the importance of CCR2 for cell migration in health and
disease. Therefore, lead-dependent changes in chemokine signaling pathways could be
one of the possible explanations for detrimental effects of lead on neuronal development,
as well as microglia function. At the same time, neurons influence glia functions,
through direct cell-to-cell interactions, as well as the release of soluble mediators such as
neurotransmitters (Carnevale et al., 2007). Lead through its ability to substitute for
calcium can affect the intercellular signaling calcium pathway, release and storage, as
well as expression of receptors for neurotransmitter, secretion of growth factors,
adhesion molecules, etc. (Lidsky and Schneider, 2003).
All these facts together implicate the important role of microglia-neurons interaction in
modulating the neuronal turnover, differentiation and function. Therefore, this
interaction could be an important target in lead-induced neurotoxicity. More studies
about the impact of lead in the context of cell-cell contacts will be helpful toward finding
new approaches in attenuating lead-induced neuronal impairment in the future.
8.5. Role of IL-8 / CXCR1 signaling in microglia-neurons interaction:
One of the signaling pathways in this study suggested to be affected by lead exposure
was IL-8/CXCR1. Our finding show that low level of lead exposure induces secretion of
IL-8 in a concentration-dependentmanner in microglia (Chapter 5), while it enhances the
expression of its receptor, CXCR1 in human NPCs (Chapter 3). This observation has led
us to hypothesize that lead at the low level of exposure may have an effect on microglia-
neuronal precursor communication through the IL-8/CXCR1 signaling pathway, a
mechanism, which may be involved in neurogenesis/neuroregeneration. In this study,
much of interest has focused on the chemokine IL-8. IL-8 is secreted by human cells, but
not present in rodents. IL-8 is known to be synthesized by glia cells in the brain.
Increased in IL-8 secretion by human microglia and astrocytes in response to LPS or pro-
inflammatory stimuli (IL-1β, TNF-α), as well as in neurodegenerative disease models
has been reported. On the other hand, anti-inflammatory cytokines such as IL-4, IL-10
and TGF-β down-regulate the production of this IL-8 (Ehrlich et al., 1998). IL-8 has
traditionally been known to have an important role in neuro-inflammation. However,
increasing evidence suggests that IL-8 also plays an important roles in CNS
development, neuronal survival, modulation of excitability, and neuroimmune responses
(Danik et al., 2003). Neurotrophic effects of IL-8 on survival of cortical neurons in
culture has previously been shown (Araujo and Cotman, 1993). Furthermore, IL-8 has
shown inhibitory effects on amyloid-β induced neuronal cell death, as well as
stimulatory effects on BDNF production in vitro (Ashutosh et al., 2011). Expression of
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both IL-8 and its receptor CXCR1 by CNS cells postulate that the IL-8/CXCR1 axis may
exert neuroprotective effects and may regulate neuronal function by both, a paracrine
and an autocrine loop.
Interestingly, recent studies reported that human neuronal precursor cells express
receptors for IL-8 and CXCL13 (CXCR1 and CXCR5, respectively) and migrate across
brain endothelial cells in vitro, in response to these chemokines. We have observed the
expression of CXCR1 in human neuronal precursor cells (Chapter 3). Danik et al. (2003)
for the first time reported that CXCR1 mRNAs were expressed in different regions of rat
brain (septum, striatum, hippocampus, cerebellum, and cortex) at different levels and
appeared to be regulated independently from CXCR2 during development. They showed
expression of CXCR1 transcripts in different subtypes of neurons and astrocytes in the
rat brain (Danik et al., 2003). To our knowledge, there is no previous evidence showing
the lead toxicity induced IL-8 secretion in CNS (Chapter 5). However, enhanced
expression of IL-8 by human peripheral blood monocytes cultured in the presence of
lead, along with increased secretion of pro-inflammatory cytokines, has been recently
reported (Gillis et al., 2012). This indicates the involvement of IL-8 in lead-driven
inflammatory responses. Taken together, this information suggests the neuromodulatory
effects of the IL-8/CXCR1 axis in the CNS as part of lead neurotoxicity.
8.6. Importance of fractalkine/CX3CR1 in microglia-neuron interaction
Fractalkine (CX3CL1) is a unique chemokine because it is the only known member to
date of the CX3C class of chemokines (Bazan et al., 1997). Fractalkine is expressed as a
trans-membrane anchored protein that can be cleaved to yield the soluble forms. The
soluble form of fractalkine is thought to act more as chemoattractant molecules, while
the membrane-bound form functions as an adhesion molecule and provides cell-cell
interaction (Garton et al., 2001, Hundhausen et al., 2003, Tsou et al., 2001). In CNS,
fractalkine is mainly secreted by neurons. However, microglia and astrocytes have also
been shown fractalkine expression, but at a lower extent. Unlike other chemokines, there
is only one receptor for fractalkine, to date, i.e. CX3CR1 which make this
chemokine/receptor of interest to researchers. CX3CR1 is a G-protein-coupled receptor
that appears most highly expressed on all cells of monocytic lineage i.e.
monocytes/macrophages, dendritic cells and microglia (Imai et al., 1997, Harrison et al.,
1998, Ruitenberg et al., 2008). From extensive studies, it appears that the
fractalkine/CX3CR1 axis plays a direct role in neurodegeneration and/or neuroprotection
depending on the CNS insult. In this study, we o observed increased secretion of
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fractalkine by neuronal precursor cells, as well as increased CX3CR1 in microglia
following by lead exposure (Chapters 3 and 7). Similar to our results, enhanced
expression of fractalkine has been shown in the hippocampus and forebrain of lead
exposed rat brain, suggesting that increase in fractalkine may act as chemoattractant for
microglia/macrophage recruitment and may be the primary responses of neuronal cell to
inflammation and toxicity (Struzynska et al., 2007). It has been surmised that the role of
fractalkine may be either chemotactic or pro-inflammatory in nature, or a protective
reaction against pro-inflammatory cytokines. Numerous studies reported the activation of
CX3CR1 could have neuroprotective effects, mainly through inhibiting the production of
pro-inflammatory cytokines (Cardona et al., 2006, Blomster et al., 2011). It has been
reported that fractalkine could suppress the production of pro-inflammatory cytokine,
such as IL-6 and TNF-, as well as nitric oxide by activated microglia in vitro.
Furthermore, suppression of neuronal cell death induced by microglia activation was
observed in presence of fractalkine (Mizuno et al., 2003, Zujovic et al., 2000).
In addition to neuroprotective/chemotactic role of fractalkine, interactions between
fractalkine and CX3CR1 contribute to microglia ability to maintain a resting phenotype.
However, when neurons are injured, fractalkine levels decrease, which results in
microglia recruitment and activation. Interestingly, polymorphisms in CX3CR1
associated with neuroinflammatory disorders, such as human age-related macular
degeneration (Chan et al., 2005), have been identified. In this study, differential
expression of CX3CR1 was also observed in monocyte -derived microglia generated
from different individuals that may be linked to the different susceptibility of the person
to immune responses and inflammation, and thus to polymorphism.
Recent studies also showed the importance of fractalkine/CX3CR1 signaling in
hippocampal cognitive functions. Disruption in fractalkine signaling has lead to
impairment in cognitive function and synaptic plasticity via increased action of IL-1β
(Rogers et al., 2011). Since cognitive impairment is one of the manifestations of lead
poisoning in children, fractalkine/CX3CR1 may play an important role in lead-induced
cognitive deficits.
To our knowledge, there is not much known about the exact role of fractalkine/CX3CR1
in lead induced inflammation/toxicity. However, increased fractalkine and CX3CR1 in
both microglia and neuron could be related to the neuroprotective properties through
modulate microglia activation as well as enhance neurogenesis. Therefore, future
investigation should focus on the interaction of microglia and neuronal precursor cells
(NPCs) considering the fractalkine/CX3CR1 axis.
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8.7. Immune modulation by lead and IL-6 in the CNS: between
neuroprotection and neuroinflammation
Among the CNS cytokines, IL-6 is a cytokine that has demonstrated both
neurodegenerative and neuroprotective properties (Morales et al., 2010, Godbout and
Johnson, 2004, Peng et al., 2005). Emerging evidence suggest possible function of IL-6
as a physiological regulatory molecule and as a neuroprotective agent in brain pathology.
In this thesis, changes in cytokine and chemokine receptors expression pattern followed
by lead exposure has been shown in both human microglia and neuronal precursor
models in vitro, suggesting the possible immune modulatory effects of lead on CNS cell
development and function. Although IL-6 has been regarded a major inducer of immune
and inflammatory responses, accumulating evidence points to a crucial role of IL-6
within the CNS (Rose-John et al., 2006, Juttler et al., 2002). For instance, IL-6 promotes
the differentiation of cortical precursor cells into oligodendrocytes and astrocytes
(Nakanishi et al., 2007, Rajan and McKay, 1998), and also activates adult astrocytes
(Campbell et al., 1993). In addition, IL-6 may act as a developmental neurotrophic
factor (Marz et al., 1997, Schafer et al., 1999, Thier et al., 1999). It has been shown to
improve survival of neurons in culture (Akaneya et al., 1995, Hama et al., 1991),
protecting neurons from excitotoxic and ischemic insults (Loddick et al., 1998, Yamada
and Hatanaka, 1994, Matsuda et al., 1996), and promoting the growth of axons and
consequently the number of synapses in a region (Ihara et al., 1996, Gadient and Otten,
1997). Moreover, it has been shown that single and local blockade of IL-6 signaling
promotes neuronal differentiation from transplanted embryonic stem cell-derived neural
precursor cells (Gomi et al., 2011). On the other hand, impairment in synaptic plasticity,
changes in length and distribution of dendritic spine, reduced long term potentiation and
memory deficits has been shown following elevation in IL-6 in brain (Wei et al., 2012,
Bellinger et al., 1995). In response to prolonged exposure of lead, inflammation like glia
responses with increased secretion of pro-inflammatory cytokines such as IL-6, IL-1β
and TNF- and enhanced neuronal cell death has been reported in the immature rat brain
(Struzynska et al., 2007). On the other hand, recently Sobin et al. (2013) reported a
concentration-dependentreduction in IL-6 secretion in young mice with early chronic
lead exposure (Sobin et al., 2013). Consequently, rodent studies have to date provided
contradictory data.
In this thesis, using human in vitro models, low level of lead exposure decreased the
neuronal secretion of IL-6 (Chapter 3) with no effects on IL-6 secretion by microglia
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(Chapters 5 and 6). Interestingly, it has been reported that during neurodegeneration,
injured neurons are able to secret the soluble IL-6 receptors while IL-6 is secreted by
microglia and astrocyte. Via trans-signaling, IL-6 bound to the soluble receptor can act
on neuronal precursor cells and induce proliferation and differentiation (Islam et al.,
2009). Overall, these findings from previous studies implicate that constant level of IL-6
play key role in healthy neuronal development and function. However, changes in
regular IL-6 patterns can induce neurodegenerative effects. Taken together, findings
from this thesis suggest that lead may interfere with neuronal turnover, differentiation
and protection through changes in expression pattern of IL-6 in neuronal precursor cells.
Further studies are required. Studies investigating the effect of lead exposure on neurons
in IL-6 knocked-out animal models or the blocking of IL-6 signaling may provide new
information about involvement of this cytokine in lead induced
neuroinflammation/neurotoxicity.
8.8. Research implication and future direction
This research has used human monocyte derived microglia as a human model for
investigating the effects of lead, which will provide valuable information about human
microglia function as immune effectors of the brain. Due to the important role of
microglia as both, neuroprotective and neurotoxic cells, the novel method introduced in
this work, mixture of cytokines in serum free condition, could be used as a cornerstone
for future human cellular microglia models as a reliable and convenient model in
comparison with current established cell lines such as HMC3.
Cognitive deficits, as well as neurobehavioral impairment, especially in children, are the
prominent manifestation of inflammation-mediated neurotoxicity by lead even at low
concentration. Therefore, further studies are needed to elucidate the exact role of human
microglia in lead-induced inflammation as key players in learning and memory. To
better understand the impact of lead on microglia in the brain, as well as on neuronal
development, migration and differentiation, it would be of much interest to investigate
the effect of lead exposure on the neuron- microglia interaction in a human in vitro co-
culture system, which was beyond the scope of this project.
At this point, results from this thesis have shed some light into the role of microglia
biology in lead toxicity in human with the precise concern on involvement of chemokine
and chemokine receptors. As chemokine-receptors biology accounts for a yet not well
understood point in neuroscience, more research about chemokines and lead may
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contribute to the development of therapeutic strategies in mediating neuro-inflammatory
responses induced by lead.
8.9. General conclusion
In summary, this study, conducted as part of this thesis, provides new insights to the role
of microglia in lead neurotoxicity with a major focus on chemokine-receptor expression.
A novel human blood monocyte-derived microglia model has been developed. We
demonstrated that human microglia are able to uptake lead. Furthermore, we reported
that low-level of lead exposure has a significant influence on chemokine-receptor
expression patterns in both, human microglia and neuronal precursor cells in vitro.
Interestingly, our findings showed that lead could induce reciprocal enhancement of IL-
8/CXCR1 and fractalkine/CX3CR1 signaling in human microglia and neuronal precursor
cells, respectively. These results implicate that lead may have impacts on development
and function of CNS cells through altering the microglia-neuron interaction (Figure 8.1).
The data generated from this study present novel findings and indicate the potential
avenue for future research on human microglia and lead toxicity. These findings may
serve as a basis for a new approach to design future pharmacological therapies for the
treatment of lead neurotoxicity in children through the selective targeting of chemokine-
receptor function in the brain and modulate immune response to attenuate
neuroinflammation induced by lead poisoning in human.
Figure 8.1: Neuromodulatory effects of lead on neuronal precursors and microglia
This figure summarizes the findings of this thesis and showing how lead may possibly have modulatory effects on the microglia neuron interaction
Chapter 9
Bibliography
[162]
Abbott, N. J., Patabendige, A. A. K., Dolman, D. E. M., Yusof, S. R. & Begley, D. J. (2010) Structure
and function of the blood-brain barrier. Neurobiology of Disease, 37, 13-25. Abrous, D. N., Koehl, M. & Le Moal, M. (2005) Adult neurogenesis: from precursors to network
and physiology. Physiological Reviews, 85, 523-569. Adonaylo, V. & Oteiza, P. (1999) Lead intoxication: antioxidant defenses and oxidative damage
in rat brain. Toxicology, 135, 77 - 85. Adonaylo, V. & Oteiza, P. (1999) Pb2+ promotes lipid oxidation and alterations in membrane
physical properties. Toxicology, 132, 19 - 32. Ajami, B., Bennett, J. L., Krieger, C., Tetzlaff, W. & Rossi, F. M. (2007) Local self-renewal can
sustain CNS microglia maintenance and function throughout adult life. Nature Neuroscience, 10, 1538-43.
Akaneya, Y., Takahashi, M. & Hatanaka, H. (1995) Interleukin-1 beta enhances survival and interleukin-6 protects against MPP+ neurotoxicity in cultures of fetal rat dopaminergic neurons. Experimental Neurology, 136, 44-52.
Aloisi, F. (2001) Immune function of microglia. Glia, 36, 165-179. Aloisi, F., Carè, A., Borsellino, G., Gallo, P., Rosa, S., Bassani, A., Cabibbo, A., Testa, U., Levi, G. &
Peschle, C. (1992) Production of hemolymphopoietic cytokines (IL-6, IL-8, colony-stimulating factors) by normal human astrocytes in response to IL-1 beta and tumor necrosis factor-alpha. The Journal of Immunology, 149, 2358-66.
Aloisi, F., De Simone, R., Columba-Cabezas, S., Penna, G. & Adorini, L. (2000) Functional Maturation of Adult Mouse Resting Microglia into an APC Is Promoted by Granulocyte-Macrophage Colony-Stimulating Factor and Interaction with Th1 Cells. The Journal of Immunology, 164, 1705-1712.
Aloisi, F., Ria, F., Penna, G. & Adorini, L. (1998) Microglia Are More Efficient Than Astrocytes in Antigen Processing and in Th1 But Not Th2 Cell Activation. The Journal of Immunology, 160, 4671-4680.
Aloisi, F. (2001) Immune function of microglia. Glia, 36, 165-179. Al-Saleh, I., Nester, M., Mashhour, A., Moncari, L., Shinwari, N., Mohamed, G. E.-D. & Rabah, A.
(2009) Prenatal and postnatal lead exposure and early cognitive development: longitudinal study in Saudi Arabia. Journal of Environmental Pathology, Toxicology and Oncology, 28, 283-302.
Araujo, D. M. & Cotman, C. W. (1993) Trophic effects of interleukin-4, -7 and -8 on hippocampal neuronal cultures: potential involvement of glial-derived factors. Brain Research, 600, 49-55.
Ashutosh, Kou, W., Cotter, R., Borgmann, K., Wu, L., Persidsky, R., Sakhuja, N. & Ghorpade, A. (2011) CXCL8 protects human neurons from amyloid-beta-induced neurotoxicity: relevance to Alzheimer's disease. Biochemical and Biophysical Research Communications, 412, 565-71.
Bachstetter, A., Xing, B., de Almeida, L., Dimayuga, E., Watterson, D. & Van Eldik, L. (2011) Microglial p38alpha MAPK is a key regulator of proinflammatory cytokine up-regulation induced by toll-like receptor (TLR) ligands or beta-amyloid (Abeta). Journal of Neuroinflammation, 8, 79.
Bacon, K. B. & Harrison, J. K. (2000) Chemokines and their receptors in neurobiology: perspectives in physiology and homeostasis. Journal of Neuroimmunology, 104, 92-7.
Bagaeva, L. V., Rao, P., Powers, J. M. & Segal, B. M. (2006) CXC chemokine ligand 13 plays a role in experimental autoimmune encephalomyelitis. Journal of Immunology, 176, 7676-7685.
Bajetto, A., Bonavia, R., Barbero, S., Florio, T. & Schettini, G. (2001) Chemokines and their receptors in the central nervous system. Frontiers in Neuroendocrinology, 22, 147-184.
Bajetto, A., Bonavia, R., Barbero, S. & Schettini, G. (2002) Characterization of chemokines and their receptors in the central nervous system: physiopathological implications. Journal of Neurochemistry, 82, 1311-1329.
Balasubramaniam, B., Carter, D. A., Mayer, E. J. & Dick, A. D. (2009) Microglia derived IL-6 suppresses neurosphere generation from adult human retinal cell suspensions. Experimental Eye Research, 89, 757-766.
[163]
Banisadr, G., Gosselin, R.-D., Mechighel, P., Kitabgi, P., Rostène, W. & Parsadaniantz, S. M. (2005) Highly regionalized neuronal expression of monocyte chemoattractant protein-1 (MCP-1/CCL2) in rat brain: Evidence for its colocalization with neurotransmitters and neuropeptides. The Journal of Comparative Neurology, 489, 275-292.
Baranowska-Bosiacka, I., Gutowska, I., Rybicka, M., Nowacki, P. & Chlubek, D. (2012) Neurotoxicity of lead. Hypothetical molecular mechanisms of synaptic function disorders. Neurologia i Neurochirurgia Polska, 46, 569-578.
Baron, R., Nemirovsky, A., Harpaz, I., Cohen, H., Owens, T. & Monsonego, A. (2008) IFN-γ enhances neurogenesis in wild-type mice and in a mouse model of Alzheimer’s disease. The FASEB Journal, 22, 2843-2852.
Bassett, T., Bach, P. & Chan, L. H. (2011) Effects of methylmercury on the secretion of pro-inflammatory cytokines from primary microglial cells and astrocytes. Neurotoxicology, 33, 229-234.
Baydas, G., Nedzvetskii, V. S., Nerush, P. A., Kirichenko, S. V. & Yoldas, T. (2003) Altered expression of NCAM in hippocampus and cortex may underlie memory and learning deficits in rats with streptozotocin-induced diabetes mellitus. Life Sciences, 73, 1907-1916.
Bazan, J. F., Bacon, K. B., Hardiman, G., Wang, W., Soo, K., Rossi, D., Greaves, D. R., Zlotnik, A. & Schall, T. J. (1997) A new class of membrane-bound chemokine with a CX3C motif. Nature, 385, 640-4.
Becher, B. & Antel, J. P. (1996) Comparison of phenotypic and functional properties of immediately ex vivo and cultured human adult microglia. Glia, 18, 1-10.
Bellinger, D. C. (2004) Lead. Pediatrics, 113, 1016-1022. Bellinger, D. C. (2008) Very low lead exposures and children's neurodevelopment. Current
Opinion in Pediatrics, 20, 172-1777. Bellinger, F. P., Madamba, S. G., Campbell, I. L. & Siggins, G. R. (1995) Reduced long-term
potentiation in the dentate gyrus of transgenic mice with cerebral overexpression of interleukin-6. Neuroscience Letters, 198, 95-98.
Belmadani, A., Tran, P. B., Ren, D. & Miller, R. J. (2006) Chemokines Regulate the Migration of Neural Progenitors to Sites of Neuroinflammation. The Journal of Neuroscience, 26, 3182-3191.
Blevins, G. & Fedoroff, S. (1995) Microglia in colony-stimulating factor 1-deficient op/op mice. Journal of Neuroscience Research, 40, 535-544.
Ben-Hur, T., Ben-Menachem, O., Furer, V., Einstein, O., Mizrachi-Kol, R. & Grigoriadis, N. (2003) Effects of proinflammatory cytokines on the growth, fate, and motility of multipotential neural precursor cells. Molecular and Cellular Neurosciences, 24, 623-631.
Bennett, L. D., Fox, J. M. & Signoret, N. (2011) Mechanisms regulating chemokine receptor activity. Immunology, 134, 246-56.
Benveniste, E. N. (1997) Role of macrophages/microglia in multiple sclerosis and experimental allergic encephalomyelitis. Journal of Molecular Medicine (Berl), 75, 165-173.
Bessis, A., Béchade, C., Bernard, D. & Roumier, A. (2007) Microglial control of neuronal death and synaptic properties. Glia, 55, 233-238.
Biber, K., Dijkstra, I., Trebst, C., De Groot, C. J. A., Ransohoff, R. M. & Boddeke, H. W. G. M. (2002) Functional expression of CXCR3 in cultured mouse and human astrocytes and microglia. Neuroscience, 112, 487-497.
Bird, C. M. & Burgess, N. (2008) The hippocampus and memory: insights from spatial processing. Nature Reviews. Neuroscience, 9, 182-194.
Bleecker, M. L., Ford, D. P., Vaughan, C. G., Walsh, K. S. & Lindgren, K. N. (2007) The association of lead exposure and motor performance mediated by cerebral white matter change. Neurotoxicology, 28, 318-323.
Blevins, G. & Fedoroff, S. (19F95) Microglia in colony-stimulating factor 1-deficient op/op mice. Journal of Neuroscience Research, 40, 535-544.
Bliss, T. V. & Collingridge, G. L. (1993) A synaptic model of memory: long-term potentiation in the hippocampus. Nature, 361, 31-39.
Block, M. L., Zecca, L. & Hong, J. S. (2007) Microglia-mediated neurotoxicity: uncovering the molecular mechanisms. Nature Reviews. Neuroscience, 8, 57-69.
[164]
Blomster, L. V., Vukovic, J., Hendrickx, D. A., Jung, S., Harvey, A. R., Filgueira, L. & Ruitenberg, M. J. (2011) CX(3)CR1 deficiency exacerbates neuronal loss and impairs early regenerative responses in the target-ablated olfactory epithelium. Molecular and Cellular Neurosciences, 48, 236-245.
Boche, D., Perry, V. H. & Nicoll, J. A. R. (2013) Review: Activation patterns of microglia and their identification in the human brain. Neuropathology and Applied Neurobiology, 39, 3-18.
Boddeke, E. W., Meigel, I., Frentzel, S., Biber, K., Renn, L. Q. & Gebicke-Harter, P. (1999) Functional expression of the fractalkine (CX3C) receptor and its regulation by lipopolysaccharide in rat microglia. European Journal of Pharmacology, 374, 309-313.
Bohatschek, M., Kloss, C. U. A., Kalla, R. & Raivich, G. (2001) In vitro model of microglial
deramification: Ramified microglia transform into amoeboid phagocytes following addition of brain cell membranes to microglia-astrocyte cocultures. Journal of Neuroscience Research, 64, 508-522.
Bond, J. F. & Farmer, S. R. (1983) Regulation of tubulin and actin mRNA production in rat brain: expression of a new beta-tubulin mRNA with development. Molecular and Cellular Biology, 3, 1333-1342.
Boulanger, L. M. (2009) Immune proteins in brain development and synaptic plasticity. Neuron, 64, 93-109.
Boutin, C., Schmitz, B., Cremer, H. & Diestel, S. (2009) NCAM expression induces neurogenesis in vivo. European Journal of Neuroscience, 30, 1209-1218.
Bowen, K. K., Dempsey, R. J. & Vemuganti, R. (2011) Adult interleukin-6 knockout mice show compromised neurogenesis. Neuroreport, 22, 126-130.
Bradbury, M. W. & Deane, R. (1993) Permeability of the blood-brain barrier to lead. Neurotoxicology, 14, 131-136.
Brenneman, D. E., Schultzberg, M., Bartfai, T. & Gozes, I. (1992) Cytokine regulation of neuronal survival. Journal of Neurochemistry, 58, 454-460.
Bressler, J., Kim, K., Chakraborti, T. & Goldstein, G. (1999) Molecular mechanisms of lead neurotoxicity. Neurochemical Research, 24, 595 - 600.
Buchholz (1899) Ueber einen Fall syphilitischer Erkrankung des Centralnervensystems. Archiv fur Psychiatrie und Nervenkrankheiten, 32, 1-56.
Buisson, A., Lesne, S., Docagne, F., Ali, C., Nicole, O., MacKenzie, E. T. & Vivien, D. (2003) Transforming growth factor-beta and ischemic brain injury. Cellular and Molecular Neurobiology, 23, 539-550.
Butovsky, O., Ziv, Y., Schwartz, A., Landa, G., Talpalar, A. E., Pluchino, S., Martino, G. & Schwartz, M. (2006) Microglia activated by IL-4 or IFN-gamma differentially induce neurogenesis and oligodendrogenesis from adult stem/progenitor cells. Molecular and Cellular Neurosciences, 31, 149-160.
Cadosch, D., Sutanto, M., Chan, E., Mhawi, A., Gautschi, O. P., von Katterfeld, B., Simmen, H. P. & Filgueira, L. (2010) Titanium uptake, induction of RANK-L expression, and enhanced proliferation of human T-lymphocytes. Journal of Orthopaedic Research, 28, 341-347.
Cagnin, A., Brooks, D. J., Kennedy, A. M., Gunn, R. N., Myers, R., Turkheimer, F. E., Jones, T. & Banati, R. B. (2001) In-vivo measurement of activated microglia in dementia. The Lancet, 358, 461-467.
Cajal, S. R. (1913) Contribución al conocimiento de la neuroglia del cerebro humano Trabajos del Instituto Cajal de Investigaciones Biologicas, 11, 255-345
Cameron, H. A., Hazel, T. G. & McKay, R. D. (1998) Regulation of neurogenesis by growth factors and neurotransmitters. Journal of Neurobiology, 36, 287-306.
Campbell, I. L., Abraham, C. R., Masliah, E., Kemper, P., Inglis, J. D., Oldstone, M. B. & Mucke, L. (1993) Neurologic disease induced in transgenic mice by cerebral overexpression of interleukin 6. Proceedings of the National Academy of Sciences of the United States of America, 90, 10061-5.
Canfield, R. L., Henderson, C. R., Cory-Slechta, D. A., Cox, C., Jusko, T. A. & Lanphear, B. P. (2003) Intellectual Impairment in Children with Blood Lead Concentrations below 10 μg per Deciliter. New England Journal of Medicine, 348, 1517-1526.
[165]
Cao, X., Ma, J., Wu, G., Zhang, C., Wang, L., Dai, S. & Xu, W. (2012) Thymus-expressed chemokine promotes survival of PC12 cells via PI3K pathway. Neurochemistry International, 60, 163-169.
Cardona, A. E., Pioro, E. P., Sasse, M. E., Kostenko, V., Cardona, S. M., Dijkstra, I. M., Huang, D., Kidd, G., Dombrowski, S., Dutta, R., Lee, J.-C., Cook, D. N., Jung, S., Lira, S. A., Littman, D. R. & Ransohoff, R. M. (2006) Control of microglial neurotoxicity by the fractalkine receptor. Nature Neuroscience, 9, 917-924.
Carnevale, D., De Simone, R. & Minghetti, L. (2007) Microglia-neuron interaction in inflammatory and degenerative diseases: role of cholinergic and noradrenergic systems. CNS and Neurological Disorders Drug Targets, 6, 388-397.
Carpenter, D. O., Matthews, M. R., Parsons, P. J. & Hori, N. (1994) Long-term potentiation in the piriform cortex is blocked by lead. Cellular and Molecular Neurobiology, 14, 723-33.
Cartier, L., Hartley, O., Dubois-Dauphin, M. & Krause, K.-H. (2005) Chemokine receptors in the central nervous system: role in brain inflammation and neurodegenerative diseases. Brain Research Reviews, 48, 16-42.
CDC 1991. Preventing lead poisoning in young children-United States. Morbidity and Mortality Weekly Report. Centers for Disease Control and Prevention.
CDC 2005. Blood lead levels- United States, 1999–2002. Morbality and Mortality Weekly Report. Centers for Disease Control and Prevention.
Chalasani, S., Sabelko, K., Sunshine, M., Littman, D. & Raper, J. (2003) A chemokine, SDF-1, reduces the effectiveness of multiple axonal repellents and is required for normal axon
pathfinding.The Journal of Neuroscience, 23, 1360-1371. Chan, A., Magnus, T. & Gold, R. (2001) Phagocytosis of apoptotic inflammatory cells by
microglia and modulation by different cytokines: mechanism for removal of apoptotic cells in the inflamed nervous system. Glia, 33, 87-95.
Chan, C. C., Tuo, J., Bojanowski, C. M., Csaky, K. G. & Green, W. R. (2005) Detection of CX3CR1 single nucleotide polymorphism and expression on archived eyes with age-related macular degeneration. Histology and Histopathology, 20, 857-863.
Chan, W. Y., Kohsaka, S. & Rezaie, P. (2007) The origin and cell lineage of microglia—New concepts. Brain Research Reviews, 53, 344-354.
Chao, S. L., Moss, J. M. & Harry, G. J. (2007) Lead-induced alterations of apoptosis and neurotrophic factor mRNA in the developing rat cortex, hippocampus, and cerebellum. Journal of Biochemical and Molecular Toxicology, 21, 265-272.
Chapman, G. A., Moores, K., Harrison, D., Campbell, C. A., Stewart, B. R. & Strijbos, P. J. L. M. (2000) Fractalkine Cleavage from Neuronal Membranes Represents an Acute Event in the Inflammatory Response to Excitotoxic Brain Damage. The Journal of Neuroscience, 20, RC87.
Charlet, L., Chapron, Y., Faller, P., Kirsch, R., Stone, A. T. & Baveye, P. C. (2012) Neurodegenerative diseases and exposure to the environmental metals Mn, Pb, and Hg. Coordination Chemistry Reviews, 256, 2147-2163.
Chen, C. J. & Liao, S. L. (2003) Zinc toxicity on neonatal cortical neurons: involvement of glutathione chelation. Journal of Neurochemistry, 85, 443-453.
Chen, C. J., Ou, Y. C., Lin, S. Y., Liao, S. L., Chen, S. Y. & Chen, J. H. (2006) Manganese modulates pro-inflammatory gene expression in activated glia. Neurochemistry International, 49, 62-71.
Cheng, Y.-J., Liu, M.-Y., Wu, T.-P. & Yang, B.-C. (2004) Regulationof tumor necrosis factor-alpha in glioma cells by lead and lipposaccahride:involvement of common signaling pathway. Toxicology Letters, 152, 127-137
Chitu, V. & Stanley, E. R. (2006) Colony-stimulating factor-1 in immunity and inflammation. Current Opinion in Immunology, 18, 39-48.
Cho, C. & Miller, R. J. (2002) Chemokine receptors and neural function. Journal of Neurovirology, 8, 573-584.
Choi, W. S., Kim, S. J. & Kim, J. S. (2011) Inorganic lead (Pb)- and mercury (Hg)-induced neuronal cell death involves cytoskeletal reorganization. Laboratory Animal Research, 27, 219-225.
Columba-Cabezas, S., Serafini, B., Ambrosini, E., Sanchez, M., Penna, G., Adorini, L. & Aloisi, F. (2002) Induction of macrophage-derived chemokine/CCL22 expression in experimental
[166]
autoimmune encephalomyelitis and cultured microglia: implications for disease regulation. Journal of Neuroimmunology, 130, 10-21.
Conductier, G., Blondeau, N., Guyon, A., Nahon, J.-L. & Rovère, C. (2010) The role of monocyte
chemoattractant protein MCP1/CCL2 in neuroinflammatory diseases. Journal of Neuroimmunology, 224, 93-100.
Coon, S., Stark, A., Peterson, E., Gloi, A., Kortsha, G., Pounds, J., Chettle, D. & Gorell, J. (2006) Whole-body lifetime occupational lead exposure and risk of Parkinson's disease. Environmental Health Perspectives, 114, 1872-1876.
Cordova, F. M., Rodrigues, A. L., Giacomelli, M. B., Oliveira, C. S., Posser, T., Dunkley, P. R. & Leal, R. B. (2004) Lead stimulates ERK1/2 and p38MAPK phosphorylation in the hippocampus of immature rats. Brain Research, 998, 65-72.
Coughlan, C. M., McManus, C. M., Sharron, M., Gao, Z. Y., Murphy, D., Jaffer, S., Choe, W., Chen, W., Hesselgesser, J., Gaylord, H., Kalyuzhny, A., Lee, V. M. Y., Wolf, B., Doms, R. W. & Kolson, D. L. (2000) Expression of multiple functional chemokine receptors and monocyte chemoattractant protein-1 in human neurons. Neuroscience, 97, 591-600.
Craig, M. J. & Loberg, R. D. (2006) CCL2 (Monocyte Chemoattractant Protein-1) in cancer bone metastases. Cancer and Metastasis Reviews, 25, 611-619.
Crittenden, P. L. & Filipov, N. M. (2008) Manganese-induced potentiation of in vitro proinflammatory cytokine production by activated microglial cells is associated with persistent activation of p38 MAPK. Toxicology In vitro, 22, 18-27.
Crumpton, T., Atkins, D. S., Zawia, N. H. & Barone Jr, S. (2001) Lead Exposure in Pheochromocytoma (PC12) Cells Alters Neural Differentiation and Sp1 DNA-Binding. Neurotoxicology, 22, 49-62.
Cunningham, C. L., Martinez-Cerdeno, V. & Noctor, S. C. (2013) Microglia regulate the number of neural precursor cells in the developing cerebral cortex. Journal of Neuroscience, 33, 4216-4233.
Danik, M., Puma, C., Quirion, R. & Williams, S. (2003) Widely expressed transcripts for chemokine freceptor CXCR1 in identified glutamatergic, γ-aminobutyric acidergic, and cholinergic neurons and astrocytes of the rat brain: A single-cell reverse transcription-multiplex polymerase chain reaction study. Journal of Neuroscience Research, 74, 286-295.
D'Aversa, T. G., Eugenin, E. A. & Berman, J. W. (2008) CD40-CD40 ligand interactions in human microglia induce CXCL8 (interleukin-8) secretion by a mechanism dependent on activation of ERK1/2 and nuclear translocation of nuclear factor-κB (NFκB) and activator protein-1 (AP-1). Journal of Neuroscience Research, 86, 630-639.
Davey, F. D. & Breen, K. C. (1998) Stimulation of sialyltransferase by subchronic low-level lead exposure in the developing nervous system. A potential mechanism of teratogen action. Toxicology and Applied Pharmacology, 151, 16-21.
Davidovics, Z. & DiCicco-Bloom, E. (2005) Moderate lead exposure elicits neurotrophic effects in cerebral cortical precursor cells in culture. Journal of Neuroscience Research, 80, 817-825.
De Haas, A. H., van Weering, H. R., de Jong, E. K., Boddeke, H. W. & Biber, K. P. (2007) Neuronal chemokines: versatile messengers in central nervous system cell interaction. Molecular Neurobiology, 36, 137-151.
Denes, A., Ferenczi, S., Halasz, J., Kornyei, Z. & Kovacs, K. J. (2008) Role of CX3CR1 (fractalkine receptor) in brain damage and inflammation induced by focal cerebral ischemia in mouse. Journal of Cerebral Blood Flow and Metabolism, 28, 1707-1721.
De Simone, R., Ambrosini, E., Carnevale, D., Ajmone-Cat, M. A. & Minghetti, L. (2007) NGF promotes microglial migration through the activation of its high affinity receptor: Modulation by TGF-[beta]. Journal of Neuroimmunology, 190, 53-60.
Del Rio-Hortega, P. (1932) Microglia. Cytology and Cellular Pathology of the Nervous System, 2, 481-534.
Deverman, B. E. & Patterson, P. H. (2009) Cytokines and CNS Development. Neuron, 64, 61-78. Devi, C. B., Reddy, G. H., Prasanthi, R. P. J., Chetty, C. S. & Reddy, G. R. (2005) Developmental lead
exposure alters mitochondrial monoamine oxidase and synaptosomal catecholamine levels in rat brain. International Journal of Developmental Neuroscience, 23, 375-381.
[167]
Dheen, S. T., Kaur, C. & Ling, E. A. (2007) Microglial activation and its implications in the brain diseases. Current Medicinal Chemistry, 14, 1189-1197.
Dietert, R. R. & Piepenbrink, M. S. (2006) Lead and immune function. Critical Reviews in Toxicology, 36, 359-385.
Donnelly, D. J., Longbrake, E. E., Shawler, T. M., Kigerl, K. A., Lai, W., Tovar, C. A., Ransohoff, R. M. & Popovich, P. G. (2011) Deficient CX3CR1 signaling promotes recovery after mouse spinal cord injury by limiting the recruitment and activation of Ly6Clo/iNOS+ macrophages. Journal of Neuroscience, 31, 9910-9922.
Dribben, W. H., Creeley, C. E. & Farber, N. (2011) Low-level lead exposure triggers neuronal apoptosis in the developing mouse brain. Neurotoxicology and Teratology, 33, 473-480.
Dyatlov, V. A., Platoshin, A. V., Lawrence, D. A. & Carpenter, D. O. (1998) Lead potentiates cytokine- and glutamate-mediated increases in permeability of the blood-brain barrier. Neurotoxicology, 19, 283-291.
Edman, L. C., Mira, H. & Arenas, E. (2008) The beta-chemokines CCL2 and CCL7 are two novel differentiation factors for midbrain dopaminergic precursors and neurons. Experimental Cell Research, 314, 2123-2130.
Edman, L. C., Mira, H., Erices, A., Malmersjo, S., Andersson, E., Uhlen, P. & Arenas, E. (2008) Alpha-chemokines regulate proliferation, neurogenesis, and dopaminergic differentiation of ventral midbrain precursors and neurospheres. Stem Cells, 26, 1891-900.
Ehrlich, L. C., Hu, S., Sheng, W. S., Sutton, R. L., Rockswold, G. L., Peterson, P. K. & Chao, C. C. (1998) Cytokine Regulation of Human Microglial Cell IL-8 Production. The Journal of Immunology, 160, 1944-1948.
Ekdahl, C. T., Kokaia, Z. & Lindvall, O. (2009) Brain inflammation and adult neurogenesis: The dual role of microglia. Neuroscience, 158, 1021-1029.
Eklind, S., Mallard, C., Leverin, A. L., Gilland, E., Blomgren, K., Mattsby-Baltzer, I. & Hagberg, H. (2001) Bacterial endotoxin sensitizes the immature brain to hypoxic--ischaemic injury. European Journal of Neuroscience, 13, 1101-6.
Elkabes, S., DiCiccoBloom, E. M. & Black, I. B. (1996) Brain microglia macrophages express neurotrophins that selectively regulate microglial proliferation and function. Journal of Neuroscience, 16, 2508-2521.
Elkabes, S., Peng, L. & Black, I. B. (1998) Lipopolysaccharide differentially regulates microglial trk receptor and neurotrophin expression. Journal of Neuroscience, 54, 117-122.
El Khoury, J., Toft, M., Hickman, S. E., Means, T. K., Terada, K., Geula, C. & Luster, A. D. (2007) Ccr2 deficiency impairs microglial accumulation and accelerates progression of Alzheimer-like disease. Nature Medicine, 13, 432-438.
Eltayeb, S., Berg, A.-L., Lassmann, H., Wallstrom, E., Nilsson, M., Olsson, T., Ericsson-Dahlstrand, A. & Sunnemark, D. (2007) Temporal expression and cellular origin of CC chemokine receptors CCR1, CCR2 and CCR5 in the central nervous system: insight into mechanisms of MOG-induced EAE. Journal of Neuroinflammation, 4, 1- 14.
Esen, N. & Kielian, T. (2007) Effects of low dose GM-CSF on microglial inflammatory profiles to diverse pathogen-associated molecular patterns (PAMPs). Journal of Neuroinflammation, 4- 10.
Espinosa-Oliva, A. M., de Pablos, R. M., Villaran, R. F., Arguelles, S., Venero, J. L., Machado, A. & Cano, J. (2011) Stress is critical for LPS-induced activation of microglia and damage in the rat hippocampus. Neurobiology of Aging, 32, 85-102.
Etemad, S., Ruitenberg, M. & Filgueira, L. (2012) In vitro generated human microglia derived from blood precursors. The Journal of Immunology, 188, 111-114.
Fanarraga, M. L., Avila, J. & Zabala, J. C. (1999) Expression of unphosphorylated class III beta-tubulin isotype in neuroepithelial cells demonstrates neuroblast commitment and differentiation. European Journal of Neuroscience, 11, 517-527.
Farina, C., Aloisi, F. & Meinl, E. (2007) Astrocytes are active players in cerebral innate immunity. Trends in Immunology, 28, 138-145.
Finkelstein, Y., Markowitz, M. E. & Rosen, J. F. (1998) Low-level lead-induced neurotoxicity in children: an update on central nervous system effects. Brain Research Reviews, 27, 168-176.
[168]
Fischbein, A., Tsang, P., Luo, J. C. & Bekesi, J. G. (1993) The immune system as target for subclinical lead related toxicity. British Journal of Industrial Medicine, 50, 185-186.
Fischer, H.-G. & Reichmann, G. (2001) Brain Dendritic Cells and Macrophages/Microglia in Central Nervous System Inflammation. Journal of Immunology, 166, 2717-2726.
Flynn, G., Maru, S., Loughlin, J., Romero, I. A. & Male, D. (2003) Regulation of chemokine receptor expression in human microglia and astrocytes. Journal of Neuroimmunology, 136, 84-93.
Ford, A., Goodsall, A., Hickey, W. & Sedgwick, J. (1995) Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein- reactive CD4+ T cells compared. The Journal of Immunology, 154, 4309-4321.
Fricker, M., Oliva-Martin, M. & Brown, G. (2012) Primary phagocytosis of viable neurons by microglia activated with LPS or Abeta is dependent on calreticulin/LRP phagocytic signalling. Journal of Neuroinflammation, 9, 196.
Friedman, W. J. (2000) Neurotrophins induce death of hippocampal neurons via the p75 receptor. Journal of Neuroscience, 20, 6340-6346.
Fuhrmann, M., Bittner, T., Jung, C. K., Burgold, S., Page, R. M., Mitteregger, G., Haass, C., LaFerla, F. M., Kretzschmar, H. & Herms, J. (2010) Microglial Cx3cr1 knockout prevents neuron loss in a mouse model of Alzheimer's disease. Nature Neuroscience, 13, 411-413.
Fujimura, M. & Usuki, F. (2012) Differing effects of toxicants (methylmercury, inorganic mercury, lead, amyloid β and rotenone) on cultured rat cerebrocortical neurons: Differential expression of Rho proteins associated with neurotoxicity. Toxicological Sciences, 126, 506-514.
Fujita, H., Tanaka, J., Toku, K., Tateishi, N., Suzuki, Y., Matsuda, S., Sakanaka, M. & Maeda, N. (1996) Effects of GM-CSF and ordinary supplements on the ramification of microglia in culture: A morphometrical study. Glia, 18, 269-281.
Gadient, R. A. & Otten, U. H. (1997) Interleukin-6 (IL-6)—A molecule with both beneficial and destructive potentials. Progress in Neurobiology, 52, 379-390.
Gao, D., Mondal, T. K. & Lawrence, D. A. (2007) Lead effects on development and function of bone marrow-derived dendritic cells promote Th2 immune responses. Toxicology and Applied Pharmacology, 222, 69-79.
Garber, M. M. & Heiman, A. S. (2002) The in vitro effects of Pb acetate on NO production by C6 glial cells. Toxicology In vitro, 16, 499-508.
Garin, A., Tarantino, N., Faure, S., Daoudi, M., Lécureuil, C., Bourdais, A., Debré, P., Deterre, P. & Combadiere, C. (2003) Two Novel Fully Functional Isoforms of CX3CR1 Are Potent HIV Coreceptors. The Journal of Immunology, 171, 5305-5312.
Garton, K. J., Gough, P. J., Blobel, C. P., Murphy, G., Greaves, D. R., Dempsey, P. J. & Raines, E. W. (2001) Tumor necrosis factor-alpha-converting enzyme (ADAM17) mediates the cleavage and shedding of fractalkine (CX3CL1). Journal of Biological Chemistry, 276, 37993-8001.
Garza, A., Vega, R. & Soto, E. (2006) Cellular mechanisms of lead neurotoxicity. Medical Science Monitor, 12, 57-65.
Gehrmann, J., Banati, R. B. & Kreutzberg, G. W. (1993) Microglia in the immune surveillance of the brain: human microglia constitutively express HLA-DR molecules. Journal of Neuroimmunology, 48, 189-198.
Geissmann, F., Jung, S. & Littman, D. R. (2003) Blood Monocytes Consist of Two Principal Subsets with Distinct Migratory Properties. Immunity, 19, 71-82.
Gemma, C., Bachstetter, A. D. & Bickford, P. C. (2010) Neuron-Microglia Dialogue and Hippocampal Neurogenesis in the Aged Brain. Aging and Diesease, 1, 232-244.
Gidlow, D. A. (2004) Lead toxicity. Occupational Medicine, 54, 76-81. Gilbert, M. E., Kelly, M. E., Samsam, T. E. & Goodman, J. H. (2005) Chronic develometal lead
exposure reduces neurogenesis in adult rat hippocampus but doesn't impair spatial learning. Toxicological Sciences, 86, 365-374.
Gillis, B., Arbieva, Z. & Gavin, I. (2012) Analysis of lead toxicity in human cells. BMC Genomics, 13, 344.
[169]
Ginhoux, F., Greter, M., Leboeuf, M., Nandi, S., See, P., Gokhan, S., Mehler, M. F., Conway, S. J., Ng, L. G., Stanley, E. R., Samokhvalov, I. M. & Merad, M. (2010) Fate Mapping Analysis Reveals That Adult Microglia Derive from Primitive Macrophages. Science, 330, 841-845.
Giulian, D. & Baker, T. J. (1986) Characterization of ameboid microglia isolated from developing mammalian brain. Journal of Neuroscience, 6, 2163-2178.
Glabinski, A. R., Bielecki, B., O'Bryant, S., Selmaj, K. & Ransohoff, R. M. (2002) Experimental autoimmune encephalomyelitis: CC chemokine receptor expression by trafficking cells. Journal of Autoimmunity, 19, 175-181.
Goczalik, I., Ulbricht, E., Hollborn, M., Raap, M., Uhlmann, S., Weick, M., Pannicke, T., Wiedemann, P., Bringmann, A., Reichenbach, A. & Francke, M. (2008) Expression of CXCL8, CXCR1, and CXCR2 in neurons and glial cells of the human and rabbit retina. Investigative Ophthalmology and Visual Science, 49, 4578-4589.
Godbout, J. P. & Johnson, R. W. (2004) Interleukin-6 in the aging brain. Journal of Neuroimmunology, 147, 141-144.
Godefroy, D., Gosselin, R. D., Yasutake, A., Fujimura, M., Combadiere, C., Maury-Brachet, R., Laclau, M., Rakwal, R., Melik-Parsadaniantz, S., Bourdineaud, J. P. & Rostene, W. (2012) The chemokine CCL2 protects against methylmercury neurotoxicity. Toxicological Sciences, 125, 209-218.
Goldstein, G. W. (1990) Lead poisoning and brain cell function. Environmental Health Perspectives, 89, 91-94.
Gomi, M., Aoki, T., Takagi, Y., Nishimura, M., Ohsugi, Y., Mihara, M., Nozaki, K., Hashimoto, N., Miyamoto, S. & Takahashi, J. (2011) Single and local blockade of interleukin-6 signaling promotes neuronal differentiation from transplanted embryonic stem cell-derived neural precursor cells. Journal of Neuroscience Research, 89, 1388-1399.
Gonzalez-Perez, O., Quinones-Hinojosa, A. & Garcia-Verdugo, J. M. (2010) Immunological control of adult neural stem cells. Journal of Stem Cells, 5, 23-31.
Goyer, R. A.& Clarsksom, W. T. (2001). Toxic effects of metals. In: C.D. Klaassen (Ed.), Casarett and Doull’s Toxicology. The Basic Science of Poisons, McGraw-Hill, NY. Guha, D., Nagilla, P., Redinger, C., Srinivasan, A., Schatten, G. & Ayyavoo, V. (2012) Neuronal
apoptosis by HIV-1 Vpr: contribution of proinflammatory molecular networks from infected target cells. Journal of Neuroinflammation, 9, 138.
Guilarte, T. R., Miceli, R. C. & Jett, D. A. (1994) Neurochemical aspects of hippocampal and cortical Pb2+ neurotoxicity. Neurotoxicology, 15, 459-466.
Guillemin, G. J. & Brew, B. J. (2004) Microglia, macrophages, perivascular macrophages, and pericytes: a review of function and identification. Journal of Leukocyte Biology, 75, 388-397.
Guo, J., Walss-Bass, C. & Luduena, R. F. (2010) The beta isotypes of tubulin in neuronal differentiation. Cytoskeleton (Hoboken), 67, 431-441.
Gurer, H. & Ercal, N. (2000) Can antioxidants be beneficial in the treatment of lead poisoning? Free Radical Biology and Medicine, 29, 927 - 945.
Gwaltney-Brant, S. M. & Rumbeiha, W. K. (2002) Newer antidotal therapies. Veterinary Clinics of North America. Small Animal Practice, 32, 323-339.
Halliwell, B. (1992) Reactive Oxygen Species and the Central Nervous System. Journal of Neurochemistry, 59, 1609-1623.
Hama, T., Kushima, Y., Miyamoto, M., Kubota, M., Takei, N. & Hatanaka, H. (1991) Interleukin-6 improves the survival of mesencephalic catecholaminergic and septal cholinergic neurons from postnatal, two-week-old rats in cultures. Neuroscience, 40, 445-452.
Hanas, J. S., Rodgers, J. S., Bantle, J. A. & Cheng, Y. G. (1999) Lead inhibition of DNA-binding mechanism of Cys(2)His(2) zinc finger proteins. Molecular Pharmacology, 56, 982-988.
Hanisch, U. K. & Kettenmann, H. (2007) Microglia: active sensor and versatile effector cells in the normal and pathologic brain. Nature Neuroscience, 10, 1387-1394.
Hanisch, U. K., Prinz, M., Angstwurm, K., Hausler, K. G., Kann, O., Kettenmann, H. & Weber, J. R. (2001) The protein tyrosine kinase inhibitor AG126 prevents the massive microglial cytokine induction by pneumococcal cell walls. European Journal of Immunology, 31, 2104-2115.
Hanisch, U.-K. (2002) Microglia as a source and target of cytokines. Glia, 40, 140-155.
[170]
Hansson, E. & Rönnbäck, L. (2003) Glial neuronal signaling in the central nervous system. The FASEB Journal, 17, 341-348.
Hatori, K., Nagai, A., Heisel, R., Ryu, J. K. & Kim, S. U. (2002) Fractalkine and fractalkine receptors in human neurons and glial cells. Journal of Neuroscience Research, 69, 418-426.
Harrison, J. K., Jiang, Y., Chen, S., Xia, Y., Maciejewski, D., McNamara, R. K., Streit, W. J., Salafranca, M. N., Adhikari, S., Thompson, D. A., Botti, P., Bacon, K. B. & Feng, L. (1998) Role for neuronally derived fractalkine in mediating interactions between neurons and CX3CR1-expressing microglia. Proceedings of the National Academy of Sciences of the United States of America, 95, 10896-10901.
Health, Q. 2008. A report into the results of blood-lead screening program of 1- 4-year-oldchildren in Mt Isa, Queensland.
Heese, K., Hock, C. & Otten, U. (1998) Inflammatory Signals Induce Neurotrophin Expression in Human Microglial Cells. Journal of Neurochemistry, 70, 699-707.
Hendrix, S. & Nitsch, R. (2007) The role of T helper cells in neuroprotection and regeneration. Journal of Neuroimmunology, 184, 100-112.
Heneka, M., O’Banion, M. K., Terwel, D. & Kummer, M. (2010) Neuroinflammatory processes in Alzheimer’s disease. Journal of Neural Transmission, 117, 919-947.
Hernberg, S. (2000) Lead poisoning in a historical perspective. American Journal of Industrial Medicine, 38, 244-254.
Hesselgesser, J. & Horuk, R. (1999) Chemokine and chemokine receptor expression in the central nervous system. Journal of Neurovirology, 5, 13-26.
Hinojosa, A. E., Garcia-Bueno, B., Leza, J. C. & Madrigal, J. L. (2011) CCL2/MCP-1 modulation of microglial activation and proliferation. Journal of Neuroinflammation, 8, 77-78.
Hirt, U. A. & Leist, M. (2003) Rapid, noninflammatory and PS-dependent phagocytic clearance of necrotic cells. Cell Death and Differentiation, 10, 1156-1164.
Holtzman, D., Olson, J. E., DeVries, C. & Bensch, K. (1987) Lead toxicity in primary cultured cerebral astrocytes and cerebellar granular neurons. Toxicology and Applied Pharmacology, 89, 211-225.
Hooper, C., Pinteaux-Jones, F., Fry, V. A. H., Sevastou, I. G., Baker, D., Heales, S. J. & Pocock, J. M.
(2009) Differential effects of albumin on microglia and macrophages; implications for neurodegeneration following blood–brain barrier damage. Journal of Neurochemistry, 109, 694-705.
Hopkins, S. J. & Rothwell, N. J. (1995) Cytokines and the nervous system. I: Expression and recognition. Trends in Neurosciences, 18, 83-88.
Howell, O. W., Rundle, J. L., Garg, A., Komada, M., Brophy, P. J. & Reynolds, R. (2010) Activated Microglia Mediate Axoglial Disruption That Contributes to Axonal Injury in Multiple Sclerosis. Journal of Neuropathology and Experimental Neurology, 69, 1017-1033.
Horuk, R., Martin, A. W., Wang, Z., Schweitzer, L., Gerassimides, A., Guo, H., Lu, Z., Hesselgesser, J., Perez, H. D., Kim, J., Parker, J., Hadley, T. J. & Peiper, S. C. (1997) Expression of chemokine receptors by subsets of neurons in the central nervous system. The Journal of Immunology, 158, 2882-2890.
Hu, Q., Fu, H., Ren, T., Wang, S., Zhou, W., Song, H., Han, Y. & Dong, S. (2008) Maternal low-level lead exposure reduces the expression of PSA-NCAM and the activity of sialyltransferase in the hippocampi of neonatal rat pups. Neurotoxicology, 29, 675-681.
Hu, Q., Fu, H., Song, H., Ren, T., Li, L., Ye, L., Liu, T. & Dong, S. (2011) Low-level lead exposure attenuates the expression of three major isoforms of neural cell adhesion molecule. Neurotoxicology, 32, 255-260.
Huang, F. & Schneider, J. S. (2004) Effects of lead exposure on proliferation and differentiation of neural stem cells derived from different regions of embryonic rat brain. Neurotoxicology, 25, 1001-1012.
Huber, J. D., Campos, C. R., Mark, K. S. & Davis, T. P. (2006) Alterations in blood-brain barrier ICAM-1 expression and brain microglial activation after λ-carrageenan-induced inflammatory pain. American Journal of Physiology - Heart and Circulatory Physiology, 290, H732-H740.
[171]
Hundhausen, C., Misztela, D., Berkhout, T. A., Broadway, N., Saftig, P., Reiss, K., Hartmann, D., Fahrenholz, F., Postina, R., Matthews, V., Kallen, K. J., Rose-John, S. & Ludwig, A. (2003) The disintegrin-like metalloproteinase ADAM10 is involved in constitutive cleavage of CX3CL1 (fractalkine) and regulates CX3CL1-mediated cell-cell adhesion. Blood, 102, 1186-1195.
Ihara, S., Iwamatsu, A., Fujiyoshi, T., Komi, A., Yamori, T. & Fukui, Y. (1996) Identification of interleukin-6 as a factor that induces neurite outgrowth by PC12 cells primed with NGF. Journal of Biochemistry, 120, 865-868.
Imai, T., Hieshima, K., Haskell, C., Baba, M., Nagira, M., Nishimura, M., Kakizaki, M., Takagi, S., Nomiyama, H., Schall, T. J. & Yoshie, O. (1997) Identification and molecular characterization of fractalkine receptor CX3CR1, which mediates both leukocyte migration and adhesion. Cell, 91, 521-30.
Ishida, Y., Hayashi, T., Goto, T., Kimura, A., Akimoto, S., Mukaida, N. & Kondo, T. (2008) Essential involvement of CX3CR1-mediated signals in the bactericidal host defense during septic peritonitis. Journal of Immunology, 181, 4208-4218.
Islam, O., Gong, X., Rose-John, S. & Heese, K. (2009) Interleukin-6 and neural stem cells: more than gliogenesis. Molecular Biology of the Cell, 20, 188-199.
Izikson, L., Klein, R. S., Charo, I. F., Weiner, H. L. & Luster, A. D. (2000) Resistance to Experimental Autoimmune Encephalomyelitis in Mice Lacking the Cc Chemokine Receptor (Ccr2). The Journal of Experimental Medicine, 192, 1075-1080.
Jack, C. S., Arbour, N., Manusow, J., Montgrain, V., Blain, M., McCrea, E., Shapiro, A. & Antel, J. P. (2005) TLR Signaling Tailors Innate Immune Responses in Human Microglia and Astrocytes. The Journal of Immunology, 175, 4320-4330.
Jaerve, A. & Müller, H. (2012) Chemokines in CNS injury and repair. Cell and Tissue Research, 349, 229-248.
Janabi, N., Peudenier, S., Héron, B., Ng, K. H. & Tardieu, M. (1995) Establishment of human microglial cell lines after transfection of primary cultures of embryonic microglial cells with the SV40 large T antigen. Neuroscience Letters, 195, 105-108.
Ji, J. F., He, B. P., Dheen, S. T. & Tay, S. S. W. (2004) Expression of chemokine receptors CXCR4, CCR2, CCR5 and CX3CR1 in neural progenitor cells isolated from the subventricular zone of the adult rat brain. Neuroscience Letters, 355, 236-240.
Jiang, Y. M., Long, L. L., Zhu, X. Y., Zheng, H., Fu, X., Ou, S. Y., Wei, D. L., Zhou, H. L. & Zheng, W. (2008) Evidence for altered hippocampal volume and brain metabolites in workers occupationally exposed to lead: a study by magnetic resonance imaging and (1)H magnetic resonance spectroscopy. Toxicology Letters, 181, 118-125.
Jiang, Y. Q. & Oblinger, M. M. (1992) Differential regulation of beta III and other tubulin genes during peripheral and central neuron development. Journal of Cell Science, 103, 643-651.
Jones, B. A., Beamer, M. & Ahmed, S. (2010) Fractalkine/CX3CL1: a potential new target for inflammatory diseases. Molecular Interventions, 10, 263-270.
Jones, R. S., Minogue, A. M., Connor, T. J. & Lynch, M. A. (2012) Amyloid-beta-Induced Astrocytic Phagocytosis is Mediated by CD36, CD47 and RAGE. Journal Neuroimmune Pharmacology, 8, 301-311.
Jung, D. Y., Lee, H., Jung, B.-Y., Ock, J., Lee, M.-S., Lee, W.-H. & Suk, K. (2005) TLR4, but Not TLR2, Signals Autoregulatory Apoptosis of Cultured Microglia: A Critical Role of IFN-β as a Decision Maker. The Journal of Immunology, 174, 6467-6476.
Juttler, E., Tarabin, V. & Schwaninger, M. (2002) Interleukin-6 (IL-6): A Possible Neuromodulator Induced by Neuronal Activity. The Neuroscientist, 8, 268-275.
Kaindl, A. M., Degos, V., Peineau, S., Gouadon, E., Chhor, V., Loron, G., Le Charpentier, T., Josserand, J., Ali, C., Vivien, D., Collingridge, G. L., Lombet, A., Issa, L., Rene, F., Loeffler, J. P., Kavelaars, A., Verney, C., Mantz, J. & Gressens, P. (2012) Activation of microglial N-methyl-D-aspartate receptors triggers inflammation and neuronal cell death in the developing and mature brain. Annals of Neurology, 72, 536-549.
Kakimura, J., Kitamura, Y., Takata, K., Umeki, M., Suzuki, S., Shibagaki, K., Taniguchi, T., Nomura, Y., Gebicke-Haerter, P. J., Smith, M. A., Perry, G. & Shimohama, S. (2002) Microglial activation and amyloid-beta clearance induced by exogenous heat-shock proteins. FASEB Journal, 16, 601-603.
[172]
Kalla, R., Bohatschek, M., Kloss, C. U. A., Krol, J., Von Maltzan, X. & Raivich, G. (2003) Loss of microglial ramification in microglia-astrocyte cocultures: Involvement of adenylate cyclase, calcium, phosphatase, and Gi-protein systems. Glia, 41, 50-63.
Kalla, R., Liu, Z., Xu, S., Koppius, A., Imai, Y., Kloss, C. U. A., Kohsaka, S., Gschwendtner, A., Möller, J. C., Werner, A. & Raivich, G. (2001) Microglia and the early phase of immune surveillance in the axotomized facial motor nucleus: Impaired microglial activation and lymphocyte recruitment but no effect on neuronal survival or axonal regeneration in macrophage-colony stimulating factor-deficient mice. The Journal of Comparative Neurology, 436, 182-201.
Kamel, F., Umbach, D. M., Munsat, T. L., Shefner, J. M., Hu, H. & Sandler, D. P. (2002) Lead Exposure and Amyotrophic Lateral Sclerosis. Epidemiology, 13, 311-319.
Kanwal, S. K. & Kumar, V. (2011) High prenatal and postnatal lead exposure associated lead encephalopathy in an infant. Indian Journal of Pediatrics, 78, 1420-1423.
Kauppinen, T. M., Higashi, Y., Suh, S. W., Escartin, C., Nagasawa, K. & Swanson, R. A. (2008) Zinc triggers microglial activation. Journal of Neuroscience, 28, 5827-5835.
Karimooy, H. N., Mood, M. B., Hosseini, M. & Shadmanfar, S. (2010) Effects of occupational lead exposure on renal and nervous system of workers of traditional tile factories in Mashhad (northeast of Iran). Toxicology and Industrial Health, 26, 633-638.
Kasten-Jolly, J., Heo, Y. & Lawrence, D. A. (2011) Central nervous system cytokine gene expression: modulation by lead. Journal of Biochemical and Molecular Toxicology, 25, 41-54.
Kasten-Jolly, J., Pabello, N., Bolivar, V. J. & Lawrence, D. A. (2012) Developmental lead effects on behavior and brain gene expression in male and female BALB/cAnNTac mice. Neurotoxicology, 33, 1005-1020.
Kaul, M., Ma, Q., Medders, K. E., Desai, M. K. & Lipton, S. A. (2007) HIV-1 coreceptors CCR5 and CXCR4 both mediate neuronal cell death but CCR5 paradoxically can also contribute to protection. Cell Death and Differentiation, 14, 296-305.
Kawai, T. & Akira, S. (2011) Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity, 34, 637-650.
Keblesh, J. P., Dou, H., Gendelman, H. E. & Xiong, H. (2009) 4-Aminopyridine improves spatial memory in a murine model of HIV-1 encephalitis. Journal of Neuroimmune Pharmacology, 4, 317-327.
Kelder, W., McArthur, J., Nance-Sproson, T., McClernon, D. & Griffin, D. (1998) Beta-chemokines MCP-1 and RANTES are selectively increased in cerebrospinal fluid of patients with human immunodeficiency virus-associated dementia. Annals of Neurology, 44, 831 - 835.
Keohane, A., Ryan, S., Maloney, E., Sullivan, A. M. & Nolan, Y. M. (2010) Tumour necrosis factor-alpha impairs neuronal differentiation but not proliferation of hippocampal neural precursor cells: Role of Hes1. Molecular and Cellular Neurosciences, 43, 127-135.
Kerkovich, D. M., Sapp, D., Weidenheim, K., Brosnan, C. F., Pfeiffer, S. E., Yeh, H. H. & Busciglio, J. (1999) Fetal human cortical neurons grown in culture: morphological differentiation, biochemical correlates and development of electrical activity. International Journal of Developmental Neuroscience, 17, 347-356.
Kern, M., Audesirk, T. & Audesirk, G. (1993) Effects of inorganic lead on the differentiation and growth of cortical neurons in culture. Neurotoxicology, 14, 319-327.
Kettenmann, H., Hanisch, U., Noda, M. & Verkhratsky, A. (2011) Physiology of microglia. Physiological Reviews, 91, 461 - 553.
Khalil, N., Morrow, L. A., Needleman, H., Talbott, E. O., Wilson, J. W. & Cauley, J. A. (2009) Association of cumulative lead and neurocognitive function in an occupational cohort. Neuropsychology, 23, 10-19.
Kim, S. U. & de Vellis, J. (2005) Microglia in health and disease. Journal of Neuroscience Research, 81, 302-313.
Kim, W.-G., Mohney, R. P., Wilson, B., Jeohn, G.-H., Liu, B. & Hong, J.-S. (2000) Regional Difference in Susceptibility to Lipopolysaccharide-Induced Neurotoxicity in the Rat Brain: Role of Microglia. The Journal of Neuroscience, 20, 6309-6316.
Kizil, C., Dudczig, S., Kyritsis, N., Machate, A., Blaesche, J., Kroehne, V. & Brand, M. (2012) The chemokine receptor cxcr5 regulates the regenerative neurogenesis response in the adult zebrafish brain. Neural Devevlopment, 7, 27-40.
[173]
Kloss, C. U. A., Bohatschek, M., Kreutzberg, G. W. & Raivich, G. (2001) Effect of Lipopolysaccharide on the Morphology and Integrin Immunoreactivity of Ramified Microglia in the Mouse Brain and in Cell Culture. Experimental Neurology, 168, 32-46.
Kofler, J. & Wiley, C. A. (2011) Microglia: key innate immune cells of the brain. Toxicologic Pathology, 39, 103-114.
Koizumi, S., Shigemoto-Mogami, Y., Nasu-Tada, K., Shinozaki, Y., Ohsawa, K., Tsuda, M., Joshi, B. V., Jacobson, K. A., Kohsaka, S. & Inoue, K. (2007) UDP acting at P2Y6 receptors is a mediator of microglial phagocytosis. Nature, 446, 1091-1095.
Koller, K., Brown, T., Spurgeon, A. & Levy, L. (2004) Recent developments in low-level lead exposure and intellectual impairment in children. Environmental Health Perspectives, 112, 987-994.
Koller, k., Levy, L. & Brown, T. (2005) Low-Level Lead Exposure and Intellectual Impairment in Children: Koller et al. Respond. Environmental Health Perspectives, 113, A16-A17.
Krathwohl, M. D. & Kaiser, J. L. (2004) Chemokines Promote Quiescence and Survival of Human Neural Progenitor Cells. Stem Cells, 22, 109-118.
Kruger, C., Laage, R., Pitzer, C., Schabitz, W.-R. & Schneider, A. (2007) The hematopoietic factor GM-CSF (Granulocyte-macrophage colony-stimulating factor) promotes neuronal differentiation of adult neural stem cells in vitro. BMC Neuroscience, 8, 88-95.
Kreutzberg, G. W. (1995) Microglia, the first line of defence in brain pathologies. Arzneimittel-Forschung, 45, 357-360.
Kuhlmann, A. C., McGlothan, J. L. & Guilarte, T. R. (1997) Developmental lead exposure causes spatial learning deficits in adult rats. Neuroscience Letters, 233, 101-114.
Lachyankar, M. B., Condon, P. J., Quesenberry, P. J., Litofsky, N. S., Recht, L. D. & Ross, A. H. (1997) Embryonic precursor cells that express Trk receptors: induction of different cell fates by NGF, BDNF, NT-3, and CNTF. Experimental Neurology, 144, 350-360.
Laflamme, N., Echchannaoui, H., Landmann, R. & Rivest, S. (2003) Cooperation between toll-like receptor 2 and 4 in the brain of mice challenged with cell wall components derived from gram-negative and gram-positive bacteria. European Journal of Immunology, 33, 1127-1138.
Lambert, C., Desbarats, J., Arbour, N., Hall, J. A., Olivier, A., Bar-Or, A. & Antel, J. P. (2008) Dendritic Cell Differentiation Signals Induce Anti-Inflammatory Properties in Human Adult Microglia. The Journal of Immunology, 181, 8288-8297.
Lambertsen, K. L., Clausen, B. H., Babcock, A. A., Gregersen, R., Fenger, C., Nielsen, H. H., Haugaard, L. S., Wirenfeldt, M., Nielsen, M., Dagnaes-Hansen, F., Bluethmann, H., Færgeman, N. J., Meldgaard, M., Deierborg, T. & Finsen, B. (2009) Microglia Protect Neurons against Ischemia by Synthesis of Tumor Necrosis Factor. The Journal of Neuroscience, 29, 1319-1330.
Lan, X., Chen, Q., Wang, Y., Jia, B., Sun, L., Zheng, J. & Peng, H. (2012) TNF-alpha Affects Human Cortical Neural Progenitor Cell Differentiation through the Autocrine Secretion of Leukemia Inhibitory Factor. PLoS ONE, 7(12): e50783. doi:10.1371/journal.pone.0050783
Lanphear, B. P., Dietrich, K., Auinger, P. & Cox, C. (2000) Cognitive deficits associated with blood lead concentrations <10 microg/dL in US children and adolescents. Public Health Reports, 115, 521-9.
Lauro, C., Catalano, M., Trettel, F., Mainiero, F., Ciotti, M. T., Eusebi, F. & Limatola, C. (2006) The Chemokine CX3CL1 Reduces Migration and Increases Adhesion of Neurons with Mechanisms Dependent on the β1 Integrin Subunit. The Journal of Immunology, 177, 7599-7606.
Lavery, T. J., Butterfield, N., Kemper, C. M., Reid, R. J. & Sanderson, K. (2008) Metals and selenium in the liver and bone of three dolphin species from South Australia, 1988-2004. Science of the Total Environment, 390, 77-85.
Lawrence, D. A. (1981) In vivo and in vitro effects of lead on humoral and cell-mediated immunity. Infection and Immunity, 31, 136-143.
Lawrence, D. A. & McCabe Jr, M. J. (2002) Immunomodulation by metals. International Immunopharmacology, 2, 293-302.
Lawson, L. J., Perry, V. H., Dri, P. & Gordon, S. (1990) Heterogeneity in the distribution and morphology of microglia in the normal adult mouse brain. Neuroscience, 39, 151-170.
[174]
Lazarini, F., Gabellec, M.-M., Torquet, N. & Lledo, P.-M. (2012) Early Activation of Microglia Triggers Long-Lasting Impairment of Adult Neurogenesis in the Olfactory Bulb. The Journal of Neuroscience, 32, 3652-3664.
Lee, M., McGeer, E. & McGeer, P. L. (2013) Neurotoxins released from interferon-gamma-stimulated human astrocytes. Neuroscience, 229, 164-175.
Lee, S., Varvel, N. H., Konerth, M. E., Xu, G., Cardona, A. E., Ransohoff, R. M. & Lamb, B. T. (2010) CX3CR1 Deficiency Alters Microglial Activation and Reduces Beta-Amyloid Deposition in Two Alzheimer's Disease Mouse Models. The American journal of pathology, 177, 2549-2562.
Leonard, E. J., Skeel, A. & Yoshimura, T. (1991) Biological aspects of monocyte chemoattractant protein-1 (MCP-1). Advances in Experimental Medicine and Biology, 305, 57-64.
Leone, C., Le Pavec, G., Même, W., Porcheray, F., Samah, B., Dormont, D. & Gras, G. (2006) Characterization of human monocyte-derived microglia-like cells. Glia, 54, 183-192.
Li, B., Bedard, K., Sorce, S., Hinz, B., Dubois-Dauphin, M. & Krause, K. H. (2009) NOX4 Expression in Human Microglia Leads to Constitutive Generation of Reactive Oxygen Species and to Constitutive IL-6 Expression. Journal of Innate Immunity, 1, 570-581.
Lidsky, T. I. & Schneider, J. S. (2003) Lead neurotoxicity in children: basic mechanisms and clinical correlates. Brain, 126, 5-19.
Liu, G., Huang, W., Moir, R. D., Vanderburg, C. R., Lai, B., Peng, Z., Tanzi, R. E., Rogers, J. T. & Huang, X. (2006) Metal exposure and Alzheimer’s pathogenesis. Journal of Structural Biology, 155, 45-51.
Liu, J., Han, D., Li, Y., Zheng, L., Gu, C., Piao, Z., Au, W. W., Xu, X. & Huo, X. (2010) Lead affects apoptosis and related gene XIAP and Smac expression in the hippocampus of developing rats. Neurochemical Research, 35, 473-9.
Liu, M.-C., Liu, X.-Q., Wang, W., Shen, X.-F., Che, H.-L., Guo, Y.-Y., Zhao, M.-G., Chen, J.-Y. & Luo, W.-J. (2012) Involvement of Microglia Activation in the Lead Induced Long-Term Potentiation Impairment. PLoS ONE, 7, e43924.
Liu, X. S., Zhang, Z. G., Zhang, R. L., Gregg, S. R., Wang, L., Yier, T. & Chopp, M. (2007) Chemokine ligand 2 (CCL2) induces migration and differentiation of subventricular zone cells after stroke. Journal of Neuroscience Research, 85, 2120-2125.
Liu, Y., Walter, S., Stagi, M., Cherny, D., Letiembre, M., Schulz-Schaeffer, W., Heine, H., Penke, B., Neumann, H. & Fassbender, K. (2005) LPS receptor (CD14): a receptor for phagocytosis of Alzheimer's amyloid peptide. Brain, 128, 1778-89.
Loddick, S. A., Turnbull, A. V. & Rothwell, N. J. (1998) Cerebral interleukin-6 is neuroprotective during permanent focal cerebral ischemia in the rat. Journal of Cerebral Blood Flow and Metabolism, 18, 176-9.
Losy, J. & Zaremba, J. (2001) Monocyte chemoattractant protein-1 is increased in the cerebrospinal fluid of patients with ischemic stroke. Stroke, 32, 2695-6.
Lucas, S. M., Rothwell, N. J. & Gibson, R. M. (2006) The role of inflammation in CNS injury and disease. British Journal of Pharmacology, 147, S232-240.
Lucchini, R. G., Zoni, S., Guazzetti, S., Bontempi, E., Micheletti, S., Broberg, K., Parrinello, G. & Smith, D. R. (2012) Inverse association of intellectual function with very low blood lead but not with manganese exposure in Italian adolescents. Environmental Research, 118, 65-71.
Luna, A. L., Acosta-Saavedra, L. C., Martínez, M., Torres-Avilés, N., Gómez, R. & Calderón-Aranda, E. S. (2012) TLR4 is a target of environmentally relevant concentration of lead. Toxicology Letters, 214, 301-306.
Luo, W., Ruan, D., Yan, C., Yin, S. & Chen, J. (2012) Effects of chronic lead exposure on functions of nervous system in Chinese children and developmental rats. Neurotoxicology, 33, 862-71.
Lutz, M. B., Suri, R. M., Niimi, M., Ogilvie, A. L., Kukutsch, N. A., Rossner, S., Schuler, G. & Austyn, J. M. (2000) Immature dendritic cells generated with low doses of GM-CSF in the absence of IL-4 are maturation resistant and prolong allograft survival in vivo. European Journal of Immunology, 30, 1813-22.
Lyons, A., Lynch, A. M., Downer, E. J., Hanley, R., O'Sullivan, J. B., Smith, A. & Lynch, M. A. (2009) Fractalkine-induced activation of the phosphatidylinositol-3 kinase pathway attentuates microglial activation in vivo and in vitro. Journal of Neurochemistry, 110, 1547-1556.
[175]
Ma, N & Streilein, J.W. (1999) T cell immunity induced by allogeneic microglia in relation to neuronal retina transplantation. Journal of Immunology, 162, 4482-4489.
Magnus, T., Chan, A., Grauer, O., Toyka, K. V. & Gold, R. (2001) Microglial phagocytosis of apoptotic inflammatory T cells leads to down-regulation of microglial immune activation. Journal of Immunology, 167, 5004-5010.
Maisonpierre, P. C., Belluscio, L., Friedman, B., Alderson, R. F., Wiegand, S. J., Furth, M. E., Lindsay, R. M. & Yancopoulos, G. D. (1990) NT-3, BDNF, and NGF in the developing rat nervous system: Parallel as well as reciprocal patterns of expression. Neuron, 5, 501-509.
Marchetti, C. (2003) Molecular targets of lead in brain neurotoxicity. Neurotoxicity Research, 5, 221 - 236.
Markowitz, M. (2000) Lead poisoning. Pediatrics in Review, 21, 327-335. Marz, P., Herget, T., Lang, E., Otten, U. & Rose-John, S. (1997) Activation of gp130 by IL-
6/soluble IL-6 receptor induces neuronal differentiation. European Journal of Neuroscience, 9, 2765-2773.
Masliah, E., Mallory, M., Hansen, L., Alford, M., Albright, T., Terry, R., Shapiro, P., Sundsmo, M. & Saitoh, T. (1991) Immunoreactivity of CD45, a protein phosphotyrosine phosphatase, in Alzheimer's disease. Acta Neuropathology, 83, 12-20.
Mathias, C., Nathalie, L., Simon, C. & Denis, V. (2004) Cytokines in Neuroinflammation and Alzheimers Disease. Current Drug Targets, 5, 529-534.
Matsuda, S., Wen, T. C., Morita, F., Otsuka, H., Igase, K., Yoshimura, H. & Sakanaka, M. (1996) Interleukin-6 prevents ischemia-induced learning disability and neuronal and synaptic loss in gerbils. Neuroscience Letters, 204, 109-112.
McCabe, M. J., Jr. & Lawrence, D. A. (1990) The heavy metal lead exhibits B cell-stimulatory factor activity by enhancing B cell Ia expression and differentiation. Journal of Immunology, 145, 671-677.
McCabe, M. J., Jr., Singh, K. P. & Reiners, J. J., Jr. (2001) Low level lead exposure in vitro stimulates the proliferation and expansion of alloantigen-reactive CD4(high) T cells. Toxicology and Applied Pharmacology, 177, 219-231.
McGeer, P. L. & McGeer, E. G. (2002) Local Neuroinflammation and the Progression of Alzheimer's Disease. Journal of Neurovirology, 8, 529-538.
Meagher, J., Zellweger, R. & Filgueira, L. (2005) Functional Dissociation of the Basolateral Transcytotic Compartment from the Apical Phago-lysosomal Compartment in Human Osteoclasts. Journal of Histochemistry and Cytochemistry, 53, 665-670.
Mennicken, F., Maki, R., de Souza, E. B. & Quirion, R. (1999) Chemokines and chemokine receptors in the CNS: a possible role in neuroinflammation and patterning. Trends in Pharmacological Sciences, 20, 73-78.
Mestas, J. & Hughes, C. C. W. (2004) Of Mice and Not Men: Differences between Mouse and Human Immunology. The Journal of Immunology, 172, 2731-2738.
Meucci, O., Fatatis, A., Simen, A. A. & Miller, R. J. (2000) Expression of CX3CR1 chemokine receptors on neurons and their role in neuronal survival. Proceedings of the National Academy of Sciences, 97, 8075-8080.
Mildner, A., Mack, M., Schmidt, H., Brück, W., Djukic, M., Zabel, M. D., Hille, A., Priller, J. & Prinz, M. (2009) CCR2+Ly-6Chi monocytes are crucial for the effector phase of autoimmunity in the central nervous system. Brain, 132, 2487-2500.
Miller, R. J., Rostene, W., Apartis, E., Banisadr, G., Biber, K., Milligan, E. D., White, F. A. & Zhang, J. (2008) Chemokine action in the nervous system. Journal of Neuroscience, 28, 11792-11795.
Minami, S. S., Sun, B., Popat, K., Kauppinen, T., Pleiss, M., Zhou, Y., Ward, M. E., Floreancig, P., Mucke, L., Desai, T. & Gan, L. (2012) Selective targeting of microglia by quantum dots. Journal of Neuroinflammation, 9, 22.
Mines, M., Ding, Y. & Fan, G. H. (2007) The many roles of chemokine receptors in neurodegenerative disorders: emerging new therapeutical strategies. Current Medicinal Chemistry, 14, 2456-70.
Mittelbronn, M., Dietz, K., Schluesener, H. J. & Meyermann, R. (2001) Local distribution of microglia in the normal adult human central nervous system differs by up to one order of magnitude. Acta Neuropathology, 101, 249-255.
[176]
Mizuno, T., Kawanokuchi, J., Numata, K. & Suzumura, A. (2003) Production and neuroprotective functions of fractalkine in the central nervous system. Brain Research, 979, 65-70.
Modi, W. S. & Yoshimura, T. (1999) Isolation of novel GRO genes and a phylogenetic analysis of the CXC chemokine subfamily in mammals. Molecular Biology and Evolution, 16, 180-193.
Monnet-Tschudi, F., Zurich, M. G., Boschat, C., Corbaz, A. & Honegger, P. (2006) Involvement of environmental mercury and lead in the etiology of neurodegenerative diseases. Reviews on Environmental Health, 21, 105-117.
Monje, M. L., Toda, H. & Palmer, T. D. (2003) Inflammatory Blockade Restores Adult Hippocampal Neurogenesis. Science, 302, 1760-1765.
Morales, I., Farias, G. & Maccioni, R. B. (2010) Neuroimmunomodulation in the pathogenesis of Alzheimer's disease. Neuroimmunomodulation, 17, 202-204.
Moreira, P. I., Smith, M. A., Zhu, X., Nunomura, A., Castellani, R. J. & Perry, G. (2005) Oxidative Stress and Neurodegeneration. Annals of the New York Academy of Sciences, 1043, 545-552.
Morgan, S. C., Taylor, D. L. & Pocock, J. M. (2004) Microglia release activators of neuronal proliferation mediated by activation of mitogen-activated protein kinase, phosphatidylinositol-3-kinase/Akt and delta-Notch signalling cascades. Journal of Neurochemistry, 90, 89-101.
Morrens, J., Van Den Broeck, W. & Kempermann, G. (2012) Glial cells in adult neurogenesis. Glia, 60, 159-174.
Mou, Y. H., Yang, J. Y., Cui, N., Wang, J. M., Hou, Y., Song, S. & Wu, C. F. (2012) Effects of cobalt chloride on nitric oxide and cytokines/chemokines production in microglia. International Immunopharmacology, 13, 120-125.
Mrak, R. E. & Griffin, W. S. (2005) Glia and their cytokines in progression of neurodegeneration. Neurobiology of Aging, 26, 349-354.
Murphy, K. J. & Regan, C. M. (1999) Low-Level Lead Exposure in the Early Postnatal Period Results in Persisting Neuroplastic Deficits Associated with Memory Consolidation. Journal of Neurochemistry, 72, 2099-2104.
Nadal, A., Fuentes, E., Pastor, J. & McNaughton, P. A. (1995) Plasma albumin is a potent trigger of calcium signals and DNA synthesis in astrocytes. Proceedings of the National Academy of Sciences, 92, 1426-1430.
Nagai, A., Mishima, S., Ishida, Y., Ishikura, H., Harada, T., Kobayashi, S. & Kim, S. U. (2005) Immortalized human microglial cell line: Phenotypic expression. Journal of Neuroscience Research, 81, 342-348.
Nakajima, K., Honda, S., Tohyama, Y., Imai, Y., Kohsaka, S. & Kurihara, T. (2001) Neurotrophin secretion from cultured microglia. Journal of Neuroscience Research, 65, 322-331.
Nakamura, Y., Si, Q. S. & Kataoka, K. (1999) Lipopolysaccharide-induced microglial activation in culture: temporal profiles of morphological change and release of cytokines and nitric oxide. Neuroscience Research, 35, 95-100.
Nakanishi, M., Niidome, T., Matsuda, S., Akaike, A., Kihara, T. & Sugimoto, H. (2007) Microglia-derived interleukin-6 and leukaemia inhibitory factor promote astrocytic differentiation of neural stem/progenitor cells. European Journal of Neuroscience, 25, 649-658.
Nava-Ruiz, C., Méndez-Armenta, M. & Ríos, C. (2012) Lead neurotoxicity: effects on brain nitric oxide synthase. Journal of Molecular Histology, 1-11.
Needleman, H. (2004) Lead poisoning. Annual Review of Medicine, 55, 209 - 222. Nelson, P. T., Soma, L. A. & Lavi, E. (2002) Microglia in diseases of the central nervous system.
Annals of Medicine, 34, 491-500. Nelson, T. E., Olde Engberink, A., Hernandez, R., Puro, A., Huitron-Resendiz, S., Hao, C., De
Graan, P. N. & Gruol, D. L. (2012) Altered synaptic transmission in the hippocampus of transgenic mice with enhanced central nervous systems expression of interleukin-6. Brain, Behavior, and Immunity, 26, 959-971.
Nemsadze, K., Sanikidze, T., Ratiani, L., Gabunia, L. & Sharashenidze, T. (2009) Mechanisms of lead-induced poisoning. Georgian Med News, 92-96.
NHMRC. 2009. Blood Lead level for asutralian [Online]. Available: http://www.nhmrc.gov.au/_files_nhmrc/publications/attachments/gp3-lead-pub-stmnt.pdf.
[177]
Ni, M. & Aschner, M. (2010) Neonatal rat primary microglia: isolation, culturing, and selected applications. Current Protocols in Toxicology, Chapter 12, Unit 12 17.
Nimmerjahn, A., Kirchhoff, F. & Helmchen, F. (2005) Resting Microglial Cells Are Highly Dynamic Surveillants of Brain Parenchyma in vivo. Science, 308, 1314-1318.
Nimmervoll, B., White, R., Yang, J. W., An, S., Henn, C., Sun, J. J. & Luhmann, H. J. (2012) LPS-Induced Microglial Secretion of TNFalpha Increases Activity-Dependent Neuronal Apoptosis in the Neonatal Cerebral Cortex. Cerebral Cortex, 23, 1742-1755.
Nishioku, T., Takai, N., Miyamoto, K., Murao, K., Hara, C., Yamamoto, K. & Nakanishi, H. (2000) Involvement of caspase 3-like protease in methylmercury-induced apoptosis of primary cultured rat cerebral microglia. Brain Research, 871, 160-164.
Noda, M. & Suzumura, A. (2012) Sweepers in the CNS: Microglial Migration and Phagocytosis in the Alzheimer Disease Pathogenesis. International Journal of Alzheimer's Disease, 891087. doi: 10.1155/2012/891087. Epub 2012 May 14
Noto, D., Takahashi, K., Miyake, S. & Yamada, M. (2010) In vitro differentiation of lineage-negative bone marrow cells into microglia-like cells. European Journal of Neuroscience, 31, 1155-1163.
Oberto, A., Marks, N., Evans, H. L. & Guidotti, A. (1996) Lead (Pb+2) promotes apoptosis in newborn rat cerebellar neurons: pathological implications. Journal of Pharmacology and Experimental Therapeutics, 279, 435-442.
Olson, J. K. & Miller, S. D. (2004) Microglia initiate central nervous system innate and adaptive immune responses through multiple TLRs. Journal of Immunology, 173, 3916-3924.
Olympio, K. P., Goncalves, C., Gunther, W. M. & Bechara, E. J. (2009) Neurotoxicity and aggressiveness triggered by low-level lead in children: a review. Revista Panamericana de Salud Publica, 26, 266-275.
Onalaja, A. O. & Claudio, L. (2000) Genetic susceptibility to lead poisoning. Environmental Health Perspectives, 108 Suppl 1, 23-28.
Opanashuk, L. A. & Finkelstein, J. N. (1995) Relationship of lead-induced proteins to stress response proteins in astroglial cells. Journal of Neuroscience Research, 42, 623-632.
Pachot, A., Cazalis, M.-A., Venet, F., Turrel, F., Faudot, C., Voirin, N., Diasparra, J., Bourgoin, N., Poitevin, F., Mougin, B., Lepape, A. & Monneret, G. (2008) Decreased Expression of the Fractalkine Receptor CX3CR1 on Circulating Monocytes as New Feature of Sepsis-Induced Immunosuppression. The Journal of Immunology, 180, 6421-6429.
Papanikolaou, N. C., Hatzidaki, E. G., Belivanis, S., Tzanakakis, G. N. & Tsatsakis, A. M. (2005) Lead toxicity update. A brief review. Medical Science Monitor, 11, RA329-336.
Park, M. H., Lee, Y. K., Lee, Y. H., Kim, Y.-B., Yun, Y. W., Nam, S. Y., Hwang, S. J., Han, S. B., Kim, S. U. & Hong, J. T. (2009) Chemokines released from astrocytes promote chemokine receptor 5-mediated neuronal cell differentiation. Experimental Cell Research, 315, 2715-2726.
Peng, Y. P., Qiu, Y. H., Lu, J. H. & Wang, J. J. (2005) Interleukin-6 protects cultured cerebellar granule neurons against glutamate-induced neurotoxicity. Neuroscience Letters, 374, 192-196.
Penninger, J. M., Irie-Sasaki, J., Sasaki, T. & Oliveira-dos-Santos, A. J. (2001) CD45: new jobs for an old acquaintance. Nature Immunology, 2, 389-396.
Penugonda, S., Mare, S., Lutz, P., Banks, W. A. & Ercal, N. (2006) Potentiation of lead-induced cell death in PC12 cells by glutamate: Protection by N-acetylcysteine amide (NACA), a novel thiol antioxidant. Toxicology and Applied Pharmacology, 216, 197-205.
Perry, V. H. & O'Connor, V. (2010) The role of microglia in synaptic stripping and synaptic degeneration: a revised perspective. ASN Neuro, 2, e00047.
Persidsky, Y., Ghorpade, A., Rasmussen, J., Limoges, J., Liu, X. J., Stins, M., Fiala, M., Way, D., Kim, K. S., Witte, M. H., Weinand, M., Carhart, L. & Gendelman, H. E. (1999) Microglial and astrocyte chemokines regulate monocyte migration through the blood-brain barrier in human immunodeficiency virus-1 encephalitis. American Journal of Pathology, 155, 1599-611.
Petito, C. K., Roberts, B., Cantando, J. D., Rabinstein, A. & Duncan, R. (2001) Hippocampal injury and alterations in neuronal chemokine co-receptor expression in patients with AIDS. Journal of Neuropathology and Experimental Neurology, 60, 377-385.
[178]
Philip, A. & Gerson, B. (1994) Lead poisoning-Part I. Incidence, etiology, and toxicokinetics. Clinics in Laboratory Medicine, 14, 423 - 444.
Piper, D. R., Mujtaba, T., Rao, M. S. & Lucero, M. T. (2000) Immunocytochemical and Physiological Characterization of a Population of Cultured Human Neural Precursors. Journal of Neurophysiology, 84, 534-548.
Politis, M., Pavese, N., Tai, Y. F., Kiferle, L., Mason, S. L., Brooks, D. J., Tabrizi, S. J., Barker, R. A. & Piccini, P. (2011) Microglial activation in regions related to cognitive function predicts disease onset in Huntington's disease: A multimodal imaging study. Human Brain Mapping, 32, 258-270.
Prevention, Advisory Committee on Childhood Lead Poisoning Prevention. 2012. Low Level Lead Exposure Harms Children: A Renewed Call for Primary Prevention. Centers for Disease Control and Prevention.
Prinz, M., Kann, O., Draheim, H. J., Schumann, R. R., Kettenmann, H., Weber, J. R. & Hanisch, U.-K. (1999) Microglial Activation by Components of Gram-Positive and -Negative Bacteria: Distinct and Common Routes to the Induction of Ion Channels and Cytokines. Journal of Neuropathology and Experimental Neurology, 58, 1078-1089.
Proudfoot, A. E. (2002) Chemokine receptors: multifaceted therapeutic targets. Nature Reviews Immunology, 2, 106-115.
Puma, C., Danik, M., Quirion, R., Ramon, F. & Williams, S. (2001) The chemokine interleukin-8 acutely reduces Ca(2+) currents in identified cholinergic septal neurons expressing CXCR1 and CXCR2 receptor mRNAs. Journal of Neurochemistry, 78, 960-971.
Qin, L., Li, G., Qian, X., Liu, Y., Wu, X., Liu, B., Hong, J.-S. & Block, M. L. (2005) Interactive role of the toll-like receptor 4 and reactive oxygen species in LPS-induced microglia activation. Glia, 52, 78-84.
Rai, A., Maurya, S. K., Khare, P., Srivastava, A. & Bandyopadhyay, S. (2010) Characterization of developmental neurotoxicity of As, Cd, and Pb mixture: synergistic action of metal mixture in glial and neuronal functions. Toxicological Sciences, 118, 586-601.
Rajan, P. & McKay, R. D. (1998) Multiple routes to astrocytic differentiation in the CNS. Journal of Neuroscience, 18, 3620-3629.
Ransohoff, R. & Perry, V. (2009) Microglial physiology: unique stimuli, specialized responses. Annual Review of Immunology, 27, 119 - 145.
Ransohoff, R. M., Liu, L. & Cardona, A. E. (2007) Chemokines and chemokine receptors: multipurpose players in neuroinflammation. International Review of Neurobiology, 82, 187-204.
Raoul, W., Auvynet, C., Camelo, S., Guillonneau, X., Feumi, C., Combadiere, C. & Sennlaub, F. (2010) CCL2/CCR2 and CX3CL1/CX3CR1 chemokine axes and their possible involvement in age-related macular degeneration. Journal of Neuroinflammation, 7, 87.
Raunio, S. & Tahti, H. (2001) Glutamate and calcium uptake in astrocytes after acute lead exposure. Chemosphere, 44, 355-359.
Re, F., Belyanskaya, S. L., Riese, R. J., Cipriani, B., Fischer, F. R., Granucci, F., Ricciardi-Castagnoli, P., Brosnan, C., Stern, L. J., Strominger, J. L. & Santambrogio, L. (2002) Granulocyte-macrophage colony-stimulating factor induces an expression program in neonatal microglia that primes them for antigen presentation. Journal of Immunology, 169, 2264-2273.
Reale, M., Iarlori, C., Thomas, A., Gambi, D., Perfetti, B., Di Nicola, M. & Onofrj, M. (2009) Peripheral cytokines profile in Parkinson’s disease. Brain, Behavior, and Immunity, 23, 55-63.
Rebenko-Moll, N. M., Liu, L., Cardona, A. & Ransohoff, R. M. (2006) Chemokines, mononuclear cells and the nervous system: heaven (or hell) is in the details. Current Opinion in Immunology, 18, 683-689.
Reiber, H. (2003) Proteins in cerebrospinal fluid and blood: barriers, CSF flow rate and source-related dynamics. Restorative Neurology and Neuroscience, 21, 79-96.
Rezaie, P., Trillo-Pazos, G., Everall, I. P. & Male, D. K. (2002) Expression of β-chemokines and chemokine receptors in human fetal astrocyte and microglial co-cultures: Potential role of chemokines in the developing CNS. Glia, 37, 64-75.
Robin, A. M., Zhang, Z. G., Wang, L., Zhang, R. L., Katakowski, M., Zhang, L., Wang, Y., Zhang, C. & Chopp, M. (2006) Stromal cell-derived factor 1alpha mediates neural progenitor cell
[179]
motility after focal cerebral ischemia. Journal of Cerebral Blood Flow and Metabolism, 26, 125-134.
Rock, R. B., Gekker, G., Hu, S., Sheng, W. S., Cheeran, M., Lokensgard, J. R. & Peterson, P. K. (2004) Role of Microglia in Central Nervous System Infections. Clinical Microbiology Reviews, 17, 942-964.
Rodero, M., Marie, Y., Coudert, M., Blondet, E., Mokhtari, K., Rousseau, A., Raoul, W., Carpentier, C., Sennlaub, F., Deterre, P., Delattre, J.-Y., Debré, P., Sanson, M. & Combadière, C. (2008) Polymorphism in the Microglial Cell-Mobilizing CX3CR1 Gene Is Associated With Survival in Patients With Glioblastoma. Journal of Clinical Oncology, 26, 5957-5964.
Rodier, P. M. (1995) Developing brain as a target of toxicity. Environmental Health Perspectives, 103 Suppl 6, 73-76.
Rogers, J. & Lue, L.-F. (2001) Microglial chemotaxis, activation, and phagocytosis of amyloid [beta]-peptide as linked phenomena in Alzheimer's disease. Neurochemistry International, 39, 333-340.
Rogers, J. T., Morganti, J. M., Bachstetter, A. D., Hudson, C. E., Peters, M. M., Grimmig, B. A., Weeber, E. J., Bickford, P. C. & Gemma, C. (2011) CX3CR1 deficiency leads to impairment of hippocampal cognitive function and synaptic plasticity. Journal of Neuroscience, 31, 16241-50.
Ronnett, G. V., Hester, L. D., Nye, J. S. & Snyder, S. H. (1994) Human cerebral cortical cell lines from patients with unilateral megalencephaly and Rasmussen's encephalitis. Neuroscience, 63, 1081-1099.
Rose-John, S., Scheller, J., Elson, G. & Jones, S. A. (2006) Interleukin-6 biology is coordinated by membrane-bound and soluble receptors: role in inflammation and cancer. Journal of Leukocyte Biology, 80, 227-236.
Rosner, D., Markowitz, G. & Lanphear, B. (2005) J. Lockhart Gibson and the discovery of the impact of lead pigments on children's health: a review of a century of knowledge. Public Health Reports, 120, 296-300.
Rostene, W., Kitabgi, P. & Parsadaniantz, S. M. (2007) Chemokines: a new class of neuromodulator? Nature Review Neuroscience, 8, 895-903.
Rothwell, N. J. & Strijbos, P. J. (1995) Cytokines in neurodegeneration and repair. International Journal of Developmental Neuroscience, 13, 179-185.
Rubio-Perez, J. M. & Morillas-Ruiz, J. M. (2012) A review: inflammatory process in Alzheimer's disease, role of cytokines. Scientific World Journal, 2012, 756357.
Ruitenberg, M. J., Vukovic, J., Blomster, L., Hall, J. M., Jung, S., Filgueira, L., McMenamin, P. G. & Plant, G. W. (2008) CX3CL1/fractalkine regulates branching and migration of monocyte-derived cells in the mouse olfactory epithelium. Journal of Neuroimmunology, 205, 80-85.
Saez, E. T., Pehar, M., Vargas, M. R., Barbeito, L. & Maccioni, R. B. (2006) Production of nerve growth factor by beta-amyloid-stimulated astrocytes induces p75NTR-dependent tau hyperphosphorylation in cultured hippocampal neurons. Journal of Neuroscience Research, 84, 1098-1106.
Sandhir, R., Julka, D. & Dip Gill, K. (1994) Lipoperoxidative Damage on Lead Exposure in Rat Brain and its Implications on Membrane Bound Enzymes. Pharmacology and Toxicology, 74, 66-71.
Schafer, K. H., Mestres, P., Marz, P. & Rose-John, S. (1999) The IL-6/sIL-6R fusion protein hyper-IL-6 promotes neurite outgrowth and neuron survival in cultured enteric neurons. Journal of Interferon and Cytokine Research, 19, 527-532.
Schilling, M., Strecker, J.-K., Ringelstein, E. B., Schäbitz, W.-R. & Kiefer, R. (2009) The role of CC chemokine receptor 2 on microglia activation and blood-borne cell recruitment after transient focal cerebral ischemia in mice. Brain Research, 1289, 79-84.
Schilling, T. & Eder, C. (2011) Amyloid-beta-induced reactive oxygen species production and priming are differentially regulated by ion channels in microglia. Journal of Cellular Physiology, 226, 3295-3302.
Schilling, T., Nitsch, R., Heinemann, U., Haas, D. & Eder, C. (2001) Astrocyte-released cytokines induce ramification and outward K+ channel expression in microglia via distinct signalling pathways. European Journal of Neuroscience, 14, 463-473.
[180]
Schmidtmayer, J., Jacobsen, C., Miksch, G. & Sievers, J. (1994) Blood monocytes and spleen macrophages differentiate into microglia-like cells on monolayers of astrocytes: Membrane currents. Glia, 12, 259-267.
Schmuck, G., Ahr, H. J. & Schluter, G. (2000) Rat cortical neuron cultures: an in vitro model for differentiating mechanisms of chemically induced neurotoxicity. In vitro and Molecular Toxicology, 13, 37-50.
Schneider, J. S., Anderson, D. W., Sonnenahalli, H. & Vadigepalli, R. (2011) Sex-based differences in gene expression in hippocampus following postnatal lead exposure. Toxicology and Applied Pharmacology, 256, 179-190.
Schneider, J. S., Huang, F. N. & Vemuri, M. C. (2003) Effects of low-level lead exposure on cell survival and neurite length in primary mesencephalic cultures. Neurotoxicology and Teratology, 25, 555-559.
Schwartz, J. & Levin, R. (1991) The risk of lead toxicity in homes with lead paint hazard. Environmental Research, 54, 1-7.
Selvin-Testa, A., Loidl, C. F., Lopez-Costa, J. J., Lopez, E. M. & Pecci-Saavedra, J. (1994) Chronic lead exposure induces astrogliosis in hippocampus and cerebellum. Neurotoxicology, 15, 389-401.
Semple, B. D., Kossmann, T. & Morganti-Kossmann, M. C. (2010) Role of chemokines in CNS health and pathology: a focus on the CCL2/CCR2 and CXCL8/CXCR2 networks. Journal of Cerebral Blood Flow and Metabolism, 30, 459-473.
Sengupta, M. & Bishayi, B. (2002) Effect of lead and arsenic on murine macrophage response. Drug and Chemical Toxicology, 25, 459-472.
Sharma S, Sharma V, Paliwal R & Pracheta (2011) Lead toxicity, oxidative damage and health implications. A review. International Journal for Biotechnology and Molecular Biology Research, 2, 215-221.
Sheng, J. G., Jones, R. A., Zhou, X. Q., McGinness, J. M., Van Eldik, L. J., Mrak, R. E. & Griffin, W. S. (2001) Interleukin-1 promotion of MAPK-p38 overexpression in experimental animals and in Alzheimer's disease: potential significance for tau protein phosphorylation. Neurochemistry International, 39, 341-348.
Sheng, W., Zong, Y., Mohammad, A., Ajit, D., Cui, J., Han, D., Hamilton, J. L., Simonyi, A., Sun, A. Y., Gu, Z., Hong, J. S., Weisman, G. A. & Sun, G. Y. (2011) Pro-inflammatory cytokines and lipopolysaccharide induce changes in cell morphology, and upregulation of ERK1/2, iNOS and sPLA(2)-IIA expression in astrocytes and microglia. Journal of Neuroinflammation, 8, 121.
Sievers, J., Parwaresch, R. & Wottge, H.-U. (1994) Blood monocytes and spleen macrophages differentiate into microglia-like cells on monolayers of astrocytes: Morphology. Glia, 12, 245-258.
Simard, A. R., Soulet, D., Gowing, G., Julien, J.-P. & Rivest, S. (2006) Bone Marrow-Derived Microglia Play a Critical Role in Restricting Senile Plaque Formation in Alzheimer's Disease. Neuron, 49, 489-502.
Simpson, J., Rezaie, P., Newcombe, J., Cuzner, M. L., Male, D. & Woodroofe, M. N. (2000) Expression of the β-chemokine receptors CCR2, CCR3 and CCR5 in multiple sclerosis central nervous system tissue. Journal of Neuroimmunology, 108, 192-200.
Singh, K. K., Hughes, M. D., Chen, J. & Spector, S. A. (2005) Genetic polymorphisms in CX3CR1 predict HIV-1 disease progression in children independently of CD4+ lymphocyte count and HIV-1 RNA load. Journal of Infectious Diseases, 191, 1971-1980.
Škuljec, J., Sun, H., Pul, R., Bénardais, K., Ragancokova, D., Moharregh-Khiabani, D., Kotsiari, A., Trebst, C. & Stangel, M. (2011) CCL5 induces a pro-inflammatory profile in microglia in vitro. Cellular Immunology, 270, 164-171.
Slomianka, L., Rungby, J., West, M. J., Danscher, G. & Andersen, A. H. (1989) Dose-dependent bimodal effect of low-level lead exposure on the developing hippocampal region of the rat: a volumetric study. Neurotoxicology, 10, 177-190.
Smith, J. A., Das, A., Ray, S. K. & Banik, N. L. (2012) Role of pro-inflammatory cytokines released from microglia in neurodegenerative diseases. Brain Research Bulletin, 87, 10-20.
Sobin, C., Gutierrez, M. & Alterio, H. (2009) Polymorphisms of delta-aminolevulinic acid dehydratase (ALAD) and peptide transporter 2 (PEPT2) genes in children with low-level lead exposure. Neurotoxicology, 30, 881-887.
[181]
Soulet, D. & Rivest, S. (2008) Bone-marrow-derived microglia: myth or reality? Current Opinion in Pharmacology, 8, 508-518.
Song, J. H., Wang, C. X., Song, D. K., Wang, P., Shuaib, A. & Hao, C. (2005) Interferon gamma induces neurite outgrowth by up-regulation of p35 neuron-specific cyclin-dependent kinase 5 activator via activation of ERK1/2 pathway. Journal of Biological Chemistry, 280, 12896-12901.
Song, M., Jin, J., Lim, J.-E., Kou, J., Pattanayak, A., Rehman, J., Kim, H.-D., Tahara, K., Lalonde, R. & Fukuchi, K.-i. (2011) TLR4 mutation reduces microglial activation, increases Abeta deposits and exacerbates cognitive deficits in a mouse model of Alzheimer's disease. Journal of Neuroinflammation, 8, 92.
Sorensen, T. L., Tani, M., Jensen, J., Pierce, V., Lucchinetti, C., Folcik, V. A., Qin, S., Rottman, J., Sellebjerg, F., Strieter, R. M., Frederiksen, J. L. & Ransohoff, R. M. (1999) Expression of specific chemokines and chemokine receptors in the central nervous system of multiple sclerosis patients. Journal of Clinical Investigation, 103, 807-815.
Stevens, B. (2008) Neuron-Astrocyte Signaling in the Development and Plasticity of Neural Circuits. Neurosignals, 16, 278-288.
Streit, W. J. (1996) The role of microglia in brain injury. Neurotoxicology, 17, 671-678. Streit, W. J. (2001) Microglia and Macrophages in the Developing CNS. Neurotoxicology, 22,
619-624. Streit, W. J. (2002) Microglia as neuroprotective, immunocompetent cells of the CNS. Glia, 40,
133-139. Struzynska, L., Dabrowska-Bouta, B., Koza, K. & Sulkowski, G. (2007) Inflammation-like glial
response in lead-exposed immature rat brain. Toxicological Sciences, 95, 156 - 162. Sugama, S., Takenouchi, T., Cho, B. P., Joh, T. H., Hashimoto, M. & Kitani, H. (2009) Possible roles
of microglial cells for neurotoxicity in clinical neurodegenerative diseases and experimental animal models. Inflammation and Allergy- Drug Targets, 8, 277-284.
Suzuki, M., El-Hage, N., Zou, S., Hahn, Y. K., Sorrell, M. E., Sturgill, J. L., Conrad, D. H., Knapp, P. E. & Hauser, K. F. (2011) Fractalkine/CX3CL1 protects striatal neurons from synergistic morphine and HIV-1 Tat-induced dendritic losses and death. Molecular Neurodegeneration, 6, 78.
Szczuciński, A. & Losy, J. (2007) Chemokines and chemokine receptors in multiple sclerosis. Potential targets for new therapies. Acta Neurologica Scandinavica, 115, 137-146.
Szelényi, J. (2001) Cytokines and the central nervous system. Brain Research Bulletin, 54, 329-338.
Taga, T. & Fukuda, S. (2005) Role of IL-6 in the neural stem cell differentiation. Clinical Reviews in Allergy and Immunology, 28, 249-256.
Tambuyzer, B. R., Ponsaerts, P. & Nouwen, E. J. (2009) Microglia: gatekeepers of central nervous system immunology. Journal of Leukocyte Biology, 85, 352-370.
Tanaka, S., Kondo, H., Kanda, K., Ashino, T., Nakamachi, T., Sekikawa, K., Iwakura, Y., Shioda, S., Numazawa, S. & Yoshida, T. (2011) Involvement of interleukin-1 in lipopolysaccaride-induced microglial activation and learning and memory deficits. Journal of Neuroscience Research, 89, 506-14.
Tang, H., Wang, Y., Xie, L., Mao, X., Won, S. J., Galvan, V. & Jin, K. (2009) Effect of neural precursor proliferation level on neurogenesis in rat brain during aging and after focal ischemia. Neurobiology of Aging, 30, 299-308.
Tang, Y., Jia, Q., Ma, P., Li, F., Lv, T. & Ha, X. (2010) [The effects of lead exposure on primary cultured hippocampus neurons apoptosis and the expressions of Bcl-2, Bax, p53 and NOS1]. Wei Sheng Yan Jiu, 39, 235-8.
Tanga, F. Y., Nutile-McMenemy, N. & DeLeo, J. A. (2005) The CNS role of Toll-like receptor 4 in innate neuroimmunity and painful neuropathy. Proceedings of the National Academy of Sciences of the United States of America, 102, 5856-5861.
Tanuma, N., Sakuma, H., Sasaki, A. & Matsumoto, Y. (2006) Chemokine expression by astrocytes plays a role in microglia/macrophage activation and subsequent neurodegeneration in secondary progressive multiple sclerosis. Acta Neuropathologica, 112, 195-204.
Thacker, M. A., Clark, A. K., Bishop, T., Grist, J., Yip, P. K., Moon, L. D. F., Thompson, S. W. N., Marchand, F. & McMahon, S. B. (2009) CCL2 is a key mediator of microglia activation in neuropathic pain states. European Journal of Pain (London, England), 13, 263-272.
[182]
Thier, M., Marz, P., Otten, U., Weis, J. & Rose-John, S. (1999) Interleukin-6 (IL-6) and its soluble receptor support survival of sensory neurons. Journal of Neuroscience Research, 55, 411-22.
Thirumangalakudi, L., Yin, L., Rao, H. V. & Grammas, P. (2007) IL-8 Induces Expression of Matrix Metalloproteinases, Cell Cycle and Pro-Apoptotic Proteins, and Cell Death in Cultured Neurons. Journal of Alzheimer's Disease, 11, 305-311.
Thomas, M. P., Chartrand, K., Reynolds, A., Vitvitsky, V., Banerjee, R. & Gendelman, H. E. (2007) Ion channel blockade attenuates aggregated alpha synuclein induction of microglial reactive oxygen species: relevance for the pathogenesis of Parkinson's disease. Journal of Neurochemistry, 100, 503-519.
Tiffany-Castiglion, E. & Qian, Y. (2001) Astroglia as metal depots: molecular mechanisms for metal accumulation, storage and release. Neurotoxicology, 22, 577-592.
Tiffany-Castiglioni, E. (1993) Cell culture models for lead toxicity in neuronal and glial cells. Neurotoxicology, 14, 513-536.
Tiffany-Castiglioni, E. & Qian, Y. (2001) Astroglia as Metal Depots: Molecular Mechanisms for Metal Accumulation, Storage and Release. Neurotoxicology, 22, 577-592.
Tissir, F., Wang, C.-E. & Goffinet, A. M. (2004) Expression of the chemokine receptor Cxcr4 mRNA during mouse brain development. Developmental Brain Research, 149, 63-71.
Todd, A. C., Wetmur, J. G., Moline, J. M., Godbold, J. H., Levin, S. M. & Landrigan, P. J. (1996) Unraveling the chronic toxicity of lead: an essential priority for environmental health. Environmental Health Perspectives, 104 Suppl 1, 141-146.
Tonchev, A. B. (2011) The nerve growth factor in health and unhealth neurons. Archives Italiennes de Biologie, 149, 225-231.
Toscano, C. & Guilarte, T. (2005) Lead neurotoxicity: from exposure to molecular effects. Brain Research. Brain Research Reviews, 49, 529 - 554.
Tran, P. B., Banisadr, G., Ren, D., Chenn, A. & Miller, R. J. (2007) Chemokine receptor expression by neural progenitor cells in neurogenic regions of mouse brain. Journal of Comparative Neurology, 500, 1007-1033.
Tran, P. B. & Miller, R. J. (2003) Chemokine receptors: signposts to brain development and disease. Nature Review Neuroscience, 4, 444-455.
Tremblay, M. E. (2011) The role of microglia at synapses in the healthy CNS: novel insights from recent imaging studies. Neuron Glia Biology, 7, 67-76.
Tremblay, M. E. & Majewska, A. K. (2011) A role for microglia in synaptic plasticity? Communicative and Integrative Biology, 4, 220-222.
Tremblay, M. E., Stevens, B., Sierra, A., Wake, H., Bessis, A. & Nimmerjahn, A. (2011) The Role of Microglia in the Healthy Brain. Journal of Neuroscience, 31, 16064-16069.
Triantafilou, M. & Triantafilou, K. (2002) Lipopolysaccharide recognition: CD14, TLRs and the LPS-activation cluster. Trends in Immunology, 23, 301-304.
Trudler, D., Farfara, D. & Frenkel, D. (2010) Toll-Like Receptors Expression and Signaling in Glia Cells in Neuro-Amyloidogenic Diseases: Towards Future Therapeutic Application. Mediators of Inflammation, 2010.
Tsou, C. L., Haskell, C. A. & Charo, I. F. (2001) Tumor necrosis factor-alpha-converting enzyme mediates the inducible cleavage of fractalkine. Journal of Biological Chemistry, 276, 44622-6.
Turbic, A., Leong, S. Y. & Turnley, A. M. (2011) Chemokines and inflammatory mediators interact to regulate adult murine neural precursor cell proliferation, survival and differentiation. PLoS ONE, 6, e25406.
Vairano, M., Graziani, G., Tentori, L., Tringali, G., Navarra, P. & Russo, C. D. (2004) Primary cultures of microglial cells for testing toxicity of anticancer drugs. Toxicology Letters, 148, 91-94.
Valdo, P., Stegagno, C., Mazzucco, S., Zuliani, E., Zanusso, G., Moretto, G., Raine, C. S. & Bonetti, B. (2002) Enhanced expression of NGF receptors in multiple sclerosis lesions. Journal of Neuropathology and Experimental Neurology, 61, 91-98.
Vallieres, L., Campbell, I. L., Gage, F. H. & Sawchenko, P. E. (2002) Reduced hippocampal neurogenesis in adult transgenic mice with chronic astrocytic production of interleukin-6. Journal of Neuroscience, 22, 486-492.
[183]
Vallieres, L. & Rivest, S. (1997) Regulation of the genes encoding interleukin-6, its receptor, and gp130 in the rat brain in response to the immune activator lipopolysaccharide and the proinflammatory cytokine interleukin-1 beta. Journal of Neurochemistry, 69, 1668-1683.
Van Der Meer, P., Goldberg, S. H., Fung, K. M., Sharer, L. R., González-Scarano, F. & Lavi, E. (2001) Expression Pattern of CXCR3, CXCR4, and CCR3 Chemokine Receptors in the Developing Human Brain. Journal of Neuropathology and Experimental Neurology, 60, 25-32.
Verri, M., Pastoris, O., Dossena, M., Aquilani, R., Guerriero, F., Cuzzoni, G., Venturini, L., Ricevuti, G. & Bongiorno, A. I. (2012) Mitochondrial alterations, oxidative stress and neuroinflammation in Alzheimer’s disease. International Journal of Immunopathology and Pharmacology, 25, 345-353.
Verstraeten, S., Aimo, L. & Oteiza, P. (2008) Aluminium and lead: molecular mechanisms of brain toxicity. Archives of Toxicology, 82, 789 - 802.
Vila, M., Jackson-Lewis, V., Guegan, C., Wu, D. C., Teismann, P., Choi, D. K., Tieu, K. & Przedborski, S. (2001) The role of glial cells in Parkinson's disease. Current Opinion in Neurology, 14, 483-489.
illeda-Herna ndez, J., Barroso-Moguel, R., Me ndez-Armenta, M., Nava-Ru z, C., Huerta-Romero, R. & R os, C. (2001) Enhanced brain regional lipid peroxidation in developing rats exposed to low level lead acetate. Brain Research Bulletin, 55, 247-251.
Viviani, B., Corsini, E., Galli, C. L. & Marinovich, M. (1998) Glia Increase Degeneration of Hippocampal Neurons through Release of Tumor Necrosis Factor-α. Toxicology and Applied Pharmacology, 150, 271-276.
Voloboueva, L. A. & Giffard, R. G. (2011) Inflammation, mitochondria, and the inhibition of adult neurogenesis. Journal of Neuroscience Research, 89, 1989-1996.
Vukovic, J., Colditz, M. J., Blackmore, D. G., Ruitenberg, M. J. & Bartlett, P. F. (2012) Microglia modulate hippocampal neural precursor activity in response to exercise and aging. Journal of Neuroscience, 32, 6435-6443.
Walker, D. G., Dalsing-Hernandez, J. E., Campbell, N. A. & Lue, L. F. (2009) Decreased expression of CD200 and CD200 receptor in Alzheimer's disease: a potential mechanism leading to chronic inflammation. Experimental Neurology, 215, 5-19.
Wang, L., Li, Y., Chen, X., Chen, J., Gautam, S., Xu, Y. & Chopp, M. (2002) MCP-1, MIP-1, IL-8 and Ischemic Cerebral Tissue Enhance Human Bone Marrow Stromal Cell Migration in Interface Culture. Hematology, 7, 113-117.
Wang, X., Fu, S., Wang, Y., Yu, P., Hu, J., Gu, W., Xu, X. M. & Lu, P. (2007) Interleukin-1beta mediates proliferation and differentiation of multipotent neural precursor cells through the activation of SAPK/JNK pathway. Molecular and Cellular Neurosciences, 36, 343-354.
Wang, X. M., Liu, W. J., Zhang, R. & Zhou, Y. K. (2012) Effects of exposure to low-level lead on spatial learning and memory and the expression of mGluR1, NMDA receptor in different developmental stages of rats. Toxicology and Industrial Health, published on February 23, 2012, DOI: 10.1177/0748233712436641.Waterman, S. J., el-Fawal, H. A. & Snyder, C. A. (1994) Lead alters the immunogenicity of two neural proteins: a potential mechanism for the progression of lead-induced neurotoxicity. Environmental Health Perspectives, 102, 1052-1056.
Węgiel, J., Wiśniewski, H. M., Dziewiątkowski, J., Tarnawski, M., Kozielski, R., Trenkner, E. & Wiktor-Jędrzejczak, W. (1998) Reduced number and altered morphology of microglial cells in colony stimulating factor-1-deficient osteopetrotic op/op mice. Brain Research, 804, 135-139.
Wei, H., Chadman, K. K., McCloskey, D. P., Sheikh, A. M., Malik, M., Brown, W. T. & Li, X. (2012) Brain IL-6 elevation causes neuronal circuitry imbalances and mediates autism-like behaviors. Biochimica et Biophysica Acta, 1822, 831-42.
Welzl, H. & Stork, O. (2003) Cell Adhesion Molecules: Key Players in Memory Consolidation? Physiology, 18, 147-150.
White, L., Cory-Slechta, D., Gilbert, M., Tiffany-Castiglioni, E., Zawia, N., Virgolini, M., Rossi-George, A., Lasley, S., Qian, Y. & Basha, M. (2007) New and evolving concepts in the neurotoxicology of lead. Toxicology and Applied Pharmacology, 225, 1 - 27.
[184]
Whitney, N. P., Eidem, T. M., Peng, H., Huang, Y. & Zheng, J. C. (2009) Inflammation mediates varying effects in neurogenesis: relevance to the pathogenesis of brain injury and neurodegenerative disorders. Journal of Neurochemistry, 108, 1343-1359.
WHO. 2010. Chilhood Lead Poisioning [Online]. World Health Organization. Available: http://www.who.int/ceh/publications/leadguidance.pdf.
Williams, K., Jr., Ulvestad, E., Cragg, L., Blain, M. & Antel, J. P. (1993) Induction of primary T cell responses by human glial cells. Journal of Neuroscience Research, 36, 382-390.
Wirenfeldt, M., Ladeby, R., Dalmau, I., Banati, R. B. & Finsen, B. (2005) Microglia - Biology and relevance to disease. Mikroglia - Biologi og sygdomsmæssig relevans, 167, 3025-3030.
Woolley, D. E. (1984) A perspective of lead poisoning in antiquity and the present. Neurotoxicology, 5, 353-61.
Wu, Y., Chen, Q., Peng, H., Dou, H., Zhou, Y., Huang, Y. & Zheng, J. (2012) Directed migration of human neural progenitor cells to interleukin-1beta is promoted by chemokines stromal cell-derived factor-1 and monocyte chemotactic factor-1 in mouse brains. Translational Neurodegeneration, 1, 15.
Wyss-Coray, T. & Mucke, L. (2002) Inflammation in Neurodegenerative Disease—A Double-Edged Sword. Neuron, 35, 419-432.
Xia, M. & Hyman, B. T. (1999) Chemokines/chemokine receptors in the central nervous system and Alzheimer's disease. Journal of Neurovirology, 5, 32-41.
Xiang, Z., Chen, C. J., Ping, J., Dunn, P., Lv, J., Jiao, B. & Burnstock, G. (2006) Microglial Morphology and Its Transformation After Challenge by Extracellular ATP In vitro. Journal of Neuroscince Research, 83, 91-101.
Xiong, H., Boyle, J., Winkelbauer, M., Gorantla, S., Zheng, J., Ghorpade, A., Persidsky, Y., Carlson, K. A. & Gendelman, H. E. (2003) Inhibition of long-term potentiation by interleukin-8: Implications for human immunodeficiency virus-1-associated dementia. Journal of Neuroscience Research, 71, 600-607.
Xu, F., Farkas, S., Kortbeek, S., Zhang, F.-X., Chen, L., Zamponi, G. & Syed, N. (2012) Mercury-induced toxicity of rat cortical neurons is mediated through N-methyl-D-Aspartate receptors. Molecular Brain, 5, 30.
Xu, J., Ji, L.-D. & Xu, L.-H. (2006) Lead-induced apoptosis in PC 12 cells: Involvement of p53, Bcl-2 family and caspase-3. Toxicology Letters, 166, 160-167.
Xu, J., Yan, H. C., Yang, B., Tong, L. S., Zou, Y. X. & Tian, Y. (2009) Effects of lead exposure on hippocampal metabotropic glutamate receptor subtype 3 and 7 in developmental rats. Journal of Negative Results in Biomedicine, 8, 5.
Xu, S. Z., Shan, C. J., Bullock, L., Baker, L. & Rajanna, B. (2006) Pb2+ reduces PKCs and NF-kappaB in vitro. Cell Biology and Toxicology, 22, 189-198.
Xu, Y., Zeng, K., Han, Y., Wang, L., Chen, D., Xi, Z., Wang, H., Wang, X. & Chen, G. (2012) Altered expression of CX3CL1 in patients with epilepsy and in a rat model. American Journal of Pathology, 180, 1950-1962.
Yamada, M. & Hatanaka, H. (1994) Interleukin-6 protects cultured rat hippocampal neurons against glutamate-induced cell death. Brain Research, 643, 173-180.
Yang, C. S., Lee, H. M., Lee, J. Y., Kim, J. A., Lee, S. J., Shin, D. M., Lee, Y. H., Lee, D. S., El-Benna, J. & Jo, E. K. (2007) Reactive oxygen species and p47phox activation are essential for the Mycobacterium tuberculosis-induced pro-inflammatory response in murine microglia. Journal of Neuroinflammation, 4, 27.
Yang, Z., Yang, S., Qian, S. Y., Hong, J. S., Kadiiska, M. B., Tennant, R. W., Waalkes, M. P. & Liu, J. (2007) Cadmium-induced toxicity in rat primary mid-brain neuroglia cultures: role of oxidative stress from microglia. Toxicological Sciences, 98, 488-494.
Yeh, S.-H., Hung, J.-J., Gean, P.-W. & Chang, W.-C. (2008) Hypoxia-Inducible Factor-1α Protects Cultured Cortical Neurons from Lipopolysaccharide-Induced Cell Death via Regulation of NR1 Expression. The Journal of Neuroscience, 28, 14259-14270.
Yoneyama, M., Shiba, T., Hasebe, S. & Ogita, K. (2011) Adult neurogenesis is regulated by endogenous factors produced during neurodegeneration. Journal of Pharmacological Sciences, 115, 425-432.
Zeller, I., Knoflach, M., Seubert, A., Kreutmayer, S. B., Stelzmuller, M. E., Wallnoefer, E., Blunder, S., Frotschnig, S., Messner, B., Willeit, J., Debbage, P., Wick, G., Kiechl, S., Laufer, G. & Bernhard, D. (2010) Lead contributes to arterial intimal hyperplasia through nuclear
[185]
factor erythroid 2-related factor-mediated endothelial interleukin 8 synthesis and subsequent invasion of smooth muscle cells. Arteriosclerosis, Thrombosis, and Vascular Biology, 30, 1733-1740.
Zhang, J., Geula, C., Lu, C., Koziel, H., Hatcher, L. M. & Roisen, F. J. (2003) Neurotrophins regulate proliferation and survival of two microglial cell lines in vitro. Experimental Neurology, 183, 469-481.
Zhang, J., Shi, X. Q., Echeverry, S., Mogil, J. S., De Koninck, Y. & Rivest, S. (2007) Expression of CCR2 in Both Resident and Bone Marrow-Derived Microglia Plays a Critical Role in Neuropathic Pain. The Journal of Neuroscience, 27, 12396-12406.
Zhang, T., Somasundaram, R., Berencsi, K., Caputo, L., Gimotty, P., Rani, P., Guerry, D., Swoboda, R. & Herlyn, D. (2006) Migration of cytotoxic T lymphocytes toward melanoma cells in three-dimensional organotypic culture is dependent on CCL2 and CCR4. European Journal of Immunology, 36, 457-467.
Zhang, Z., Drzewiecki, G. J., Hom, J. T., May, P. C. & Hyslop, P. A. (1994) Human cortical neuronal (HCN) cell lines: a model for amyloid β neurotoxicity. Neuroscience Letters, 177, 162-164.
Zhao, C., Deng, W. & Gage, F. H. (2008) Mechanisms and functional implications of adult neurogenesis. Cell, 132, 645-660.
Zheng, G., Tian, L., Liang, Y., Broberg, K., Lei, L., Guo, W., Nilsson, J., Bergdahl, I. A., Skerfving, S. & Jin, T. (2011) delta-Aminolevulinic acid dehydratase genotype predicts toxic effects of lead on workers' peripheral nervous system. Neurotoxicology, 32, 374-382.
Zheng, W., Aschner, M. & Ghersi-Egea, J. F. (2003) Brain barrier systems: a new frontier in metal neurotoxicological research. Toxicology and Applied Pharmacology, 192, 1-11.
Zimmer, B., Lee, G., Stiegler, N. V., Meganathan, K., Sachinidis, A., Studer, L. & Leist, M. (2012) Evaluation of Developmental Toxicants and Signaling Pathways in a Functional Test Based on the Migration of Human Neural Crest Cells. Environmental Health Perspectives, 120, 1116-1122.
Ziv, Y. & Schwartz, M. (2008) Immune-based regulation of adult neurogenesis: Implications for learning and memory. Brain, Behavior, and Immunity, 22, 167-176.
Zuckerman, S. H., Gustin, J. & Evans, G. F. (1998) Expression of CD54 (intercellular adhesion molecule-1) and the beta 1 integrin CD29 is modulated by a cyclic AMP dependent pathway in activated primary rat microglial cell cultures. Inflammation, 22, 95-106.
Zuiderwijk-Sick, E. A., van der Putten, C., Bsibsi, M., Deuzing, I. P., de Boer, W., Persoon-Deen, C., Kondova, I., Boven, L. A., van Noort, J. M., t Hart, B. A., Amor, S. & Bajramovic, J. J. (2007) Differentiation of primary adult microglia alters their response to TLR8-mediated activation but not their capacity as APC. Glia, 55, 1589-1600.
Zujovic, V., Benavides, J., Vigé, X., Carter, C. & Taupin, V. (2000) Fractalkine modulates TNF-α secretion and neurotoxicity induced by microglial activation. Glia, 29, 305-315.
Zurich, M. G., Monnet-Tschudi, F., Bérode, M. & Honegger, P. (1998) Lead acetate toxicity in vitro: Dependence on the cell composition of the cultures. Toxicology in vitro, 12, 191-196.
Zurich, M.-G., Eskes, C., Honegger, P., Bérode, M. & Monnet-Tschudi, F. (2002) Maturation-dependent neurotoxicity of lead acetate in vitro: Implication of glial reactions. Journal of Neuroscience Research, 70, 108-116.
Chapter 10
Appendix
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Journal of Neuroscience Methods
jou rna l h om epa ge: www.elsev ier .com/ locate / jneumeth
asic Neuroscience
novel in vitro human microglia model: Characterization of humanonocyte-derived microglia
amar Etemada, Rasheeda Mohd Zamina,b, Marc J. Ruitenbergc, Luis Filgueiraa,∗
School of Anatomy, Physiology and Human Biology, University of Western Australia, WA 6009, AustraliaDepartment of Anatomy, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, MalaysiaSchool of Biomedical Sciences, University of Queensland, Brisbane, QLD 4072, Australia
i g h l i g h t s
New culture protocol for the gen-eration of human monocyte-derivedmicroglia in vitro.Morphological, phenotypic and func-tional characterization of humanmonocyte-derived microglia.Expression of surface markers byhuman microglia.Chemokine receptors expressionpattern in human microglia.Comparison of human microglia withmonocytes and dendritic cells.
g r a p h i c a l a b s t r a c t
r t i c l e i n f o
rticle history:eceived 21 December 2011eceived in revised form 20 April 2012ccepted 22 May 2012
eywords:icrogliauman peripheral blood monocyteserum-free mediumlow cytometryhemokine receptors-CSF
a b s t r a c t
Microglia are the innate immune cells of the central nervous system. They help maintaining physiologicalhomeostasis and contribute significantly to inflammatory responses in the course of infection, injuryand degenerative processes. To date, there is no standardized simple model available to investigate thebiology of human microglia. The aim of this study was to establish a new human microglia model. Forthat purpose, human peripheral blood monocytes were cultured in serum free medium in the presenceof M-CSF, GM-CSF, NGF and CCL2 to generate monocyte-derived microglia (M-MG). M-MG were clearlydifferent in morphology, phenotype and function from freshly isolated monocytes, cultured monocytesin the absence of the cytokines and monocyte-derived dendritic cells (M-DC) cultured in the presenceof GM-CSF and IL-4. M-MG acquired a ramified morphology with primary and secondary processes. M-MG displayed a comparable phenotype to the human microglia cell line HMC3, expressing very lowlevels of CD45, CD14 and HLA-DR, CD11b and CD11c; and undetectable levels of CD40, CD80 and CD83,
M-CSFCL2GFhagocytosis
and a distinct pattern of chemokine receptors (positive for CCR1, CCR2, CCR4, CCR5, CXCR1, CXCR3,CX3CR1; negative for CCR6 and CCR7). In comparison with M-DC, M-MG displayed lower T-lymphocytestimulatory capacity, as well as lower phagocytosis activity. The described protocol for the generationof human monocyte-derived microglia is feasible, well standardized and reliable, as it uses well definedculture medium and recombincertainly be very helpful for fu
Abbreviations: CNS, central nervous system; PBMC, peripheral mononuclear bloacrophage colony stimulating factor; NGF, nerve growth factor; CCL2, c-c motif ligand
CR1, c-c motif receptor 1; CCR2, c-c motif receptor 2; CXCR3, c-x-c motif receptor 3;
ulbecco’s Modification of Eagle’s Medium; FBS, fetal bovine serum.∗ Corresponding author at: School of Anatomy and Human Biology, University of Weste
Tel.: +61 8 64883907; fax: +61 8 64881051.E-mail address: [email protected] (L. Filgueira).
165-0270/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jneumeth.2012.05.025
ant cytokines, but no serum or conditioned medium. This protocol willture studies investigating the biology and pathology of human microglia.
© 2012 Elsevier B.V. All rights reserved.
od cells; M-CSF, macrophage-colony stimulating factor; GM-CSF, granulocyte- 2; M-MG, monocyte-derived microglia; M-DC, monocyte-derived dendritic cell;CX3CR1, cxc3 chemokine receptor 1; CSF-1, colony stimulating factor 1; DMEM,
rn Australia, 35 Stirling Highway, M309, Crawley, WA 6009, Australia.
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. Introduction
Microglia are resident innate immune cells of the central ner-ous system (CNS) and play a crucial role in maintaining theealthy physiological homeostasis. Microglia also contribute sub-tantially to inflammation in response to injury, toxins, pathogensnd degenerative processes (Streit, 2001, 1996; Howell et al., 2010;ee et al., 2010; Politis et al., 2011). Microglia are characterized by auick response to various stimuli, resulting in activation with rapidhanges in morphology, phenotype and function. Morphologicalhanges include shortening of cell processes and cellular hypertro-hy. To date, there is no specific microglia marker clearly separatinghis cell type from other cells of the monocyte/macrophage lineage.ome studies suggested that primary microglia could be distin-uished from other tissue macrophages according to expressionevels of markers such as CD11b and CD45 (Aloisi et al., 2000; Fordt al., 1995). Under healthy condition, resting microglia with typicalamified morphology show low expression levels of these com-on myeloid lineage markers (Nimmerjahn et al., 2005). However,icroglia have been shown to share expression of surface mark-
rs common to other immune cells of the macrophage family suchs CD45, CD14, MHC-class II, CD68, immunoglobulin Fc receptorsnd �2 integrins (Lambertsen et al., 2009; Wirenfeldt et al., 2005).xpression levels of these surface markers may depend on thenflammatory status of the CNS and on microglia activation. Simi-arly, functional inflammatory changes are dominated by secretionf pro-inflammatory cytokines, chemokines, neurotrophic factorsnd up-regulation of corresponding receptors, as well as produc-ion of nitric oxide and reactive oxygen intermediates (Aloisi, 2001;ambuyzer et al., 2009).
Microglia have recently been shown in a mouse model to orig-nate from a myeloid yolk sac population during embryogenesisGinhoux et al., 2010). In humans, the microglia origin is still notnown. Furthermore, little is known about the turnover rate oficroglia in the healthy CNS. It has been postulated that prolif-
ration of the resident microglia population is rather low and thereay be immigration of bone marrow derived precursor cells into
he CNS. Invasion of bone-marrow derived microglia have beenhown to migrate into the CNS using chimeric irradiated miceSimard et al., 2006; Zhang et al., 2007). However, the proportionf bone-marrow progenitor cells in replenishment of microglia isontroversial.
A variety of cytokines have been shown to contribute toicroglia development and differentiation. Colony stimulating
actor-1/M-CSF and its receptor CSF-1R play an important role inhe development of macrophage populations (Chitu and Stanley,006). Recently, Ginhoux et al. (2010) reported the constantbsence of microglia throughout life in CSF-1R deficient mice,ndicating that M-CSF is essential for microglia development andifferentiation. In addition, granulocyte-macrophage colony stim-lating factor (GM-CSF) also contributes to microglia developmentnd differentiation (Aloisi et al., 2000; Esen and Kielian, 2007).oth, M-CSF and GM-CSF have crucial effects on proliferation andurvival of primary human fetal and adult microglia in cultureith GM-CSF having a greater impact on proliferation (Esen andielian, 2007; Lee et al., 1994). M-CSF plays a crucial role in thenal maturation stage of microglia. In op/op mice, a model of humansteopetrosis with a functional mutation the M-CSF gene, the num-er of microglia are decreased and the cells are smaller and have
mpaired activation ability in response to injury (Kalla et al., 2001;egiel et al., 1998). Importantly, GM-CSF or interleukin-3 does not
ubstitute for M-CSF (Blevins and Fedoroff, 1995).
Nerve growth factor (NGF), a member of neurotrophins, has alsoeen shown to act on the proliferation and survival of microgliaZhang et al., 2003). NGF binds and acts through the P75 receptor,hich has been shown to be expressed in microglia in multiple
nce Methods 209 (2012) 79– 89
sclerosis lesions (Valdo et al., 2002). It has also been shown thatmicroglia express the nerve growth factor receptor TrkA (Tonchev,2011). NGF induces migration of the microglia cell through activa-tion of the TrkA (TrkA activation) (De Simone et al., 2007). NGF isexpressed by activated microglia, astrocytes and hippocampal neu-rons (Friedman, 2000; Saez et al., 2006; Tonchev, 2011). However,inflammation certainly induces neurotrophin secretion by humanmicroglia (Heese et al., 1998; Nakajima et al., 2001).
Chemokines have also been shown to play an important role inmicroglia biology. Monocyte chemoattractant protein-1 (MCP-1),or CCL2, is one of the prominent chemokines in the regulation ofthe microglia migration to the site of inflammation in experimentalmodels (Leonard et al., 1991; Zhang et al., 2007). CCL2 acts throughits specific receptor CCR2 which is expressed by microglia and othercells of monocytic lineage, such as macrophages and dendritic cells(Rebenko-Moll et al., 2006). CCL2 acts also through CCR4, but littleinformation is available about CCR4 expression in microglia (Craigand Loberg, 2006; Zhang et al., 2006). Studies on normal rat CNSshowed that CCL2 is constitutively expressed by astrocytes andneurons and it can be found in various brain regions, including thecerebral cortex, the hippocampus and the hypothalamus (Banisadret al., 2005). CCL2 is also secreted by astrocytes and neurons underinflammatory condition (Banisadr et al., 2005; Farina et al., 2007;Tanuma et al., 2006). It has been shown that CCL2 is secreted by bothundamaged and damaged spinal sensory neurons in rat, result-ing in activation of spinal microglia and initiating neuropathic-likepain (Thacker et al., 2009). Up-regulation of CCL2 expression dur-ing Alzheimer’s disease and multiple sclerosis correlates withactivation of microglia and may contribute to pathogenesis of neu-rodegenerative diseases (Conductier et al., 2010; Simpson et al.,2000a). In that respect, CCL2 contributes to recruitment of mononu-clear cells into the inflamed CNS, followed by activation of themicroglia. Zhang et al. (2007) showed that CCL2 can induce spinalmicroglia activation in mice. They also reported in chimeric micethat CCL2 recruits bone marrow-derived macrophages, which pro-liferate and differentiate into microglia in the spinal cord andinduce inflammation and microgliosis after nerve injury. In exper-imental autoimmune encephalomyelitis (EAE), a murine model ofmultiple sclerosis, CCR2 deficient mice do not develop the mononu-clear cell infiltration and inflammation. Indeed, no chemokinesincrease was detected in the CNS in that model (Izikson et al.,2000). On the other hand, CCR2 deficiency in a mouse model ofAlzheimer’s disease impairs significantly microglia accumulation atsites of plaque formation, resulting in a decrease of �-amyloid (A�)clearance and in accelerating early disease progression (El Khouryet al., 2007).
In vitro culture of mouse and human microglia has oftenbeen used as a model by various researchers. Thereby, astrocyte-conditioned medium (ACM) has been used to keep microglia inculture with a ramified resting morphology. ACM contains M-CSF,GM-CSF and transforming growth factor � (TGF �), all known to besecreted by astrocytes. Microglia lose their ramified morphology inthe presence of antibodies against the mentioned cytokines, indi-cating that M-CSF and GM-CSF are essential for microglia culturing(Schilling et al., 2001). GM-CSF, even at low concentration in serumfree medium, keeps microglia at a resting state with morphologi-cal ramifications (Fujita et al., 1996). A recent study reported theproliferative effect of CCL2 on neonatal and embryonic primary ratmicroglia in culture without inducing additional changes in mor-phology, phenotype and cytokine expression (Hinze and Stolzing,2011).
Most interestingly, murine bone marrow-derived precursor
cells can be differentiated towards microglia-like cells when cul-tured in the presence of astrocytes or in mixed glial cultures.Thereby, M-CSF alone is not sufficient for microglia differentiation,which indicates the importance of additional astrocyte-derived
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S. Etemad et al. / Journal of Neu
actors (Noto et al., 2010). Differentiation of murine bone mar-ow stem cells toward microglia-like cells has recently also beenuccessful, using ACM supplemented with GM-CSF (Hinze andtolzing, 2011). Culturing fetal and adult human microglia in theresence of GM-CSF induces functional maturation towards anntigen presenting cell type, especially in adult microglia. How-ver, GM-CSF does not induce maturation of microglia to acquirehe complete phenotype and function of mature dendritic cellsLambert et al., 2008; Re et al., 2002).
In vitro studies on microglia used mainly primary microglia cul-ure from embryonic or neonatal murine brains (Bassett et al., 2012;inojosa et al., 2011). In human, brain-derived microglia is diffi-ult to obtain for ethical reasons. In addition, only low numbersf cells are collected. Also, fetal microglia seems to be quite dif-erent from adult microglia. Only few human microglia cell linesave been generated, including HMO6 cells (Nagai et al., 2005)nd HMC3 cells (Janabi et al., 1995). These cell lines cannot beonsidered as an optimal model for microglia cells due the signif-cant modification in morphology and function as result of genetic
anipulation and long-term culture. Therefore, a more convenientnd more appropriate model for in vitro microglia human studiess still missing.
Most importantly, Leone et al. (2006) showed that humanlood-derived monocyte, cultured with astrocytes-conditionededium (ACM), acquired the ramified morphology of microglia
nd expressed substance P, calcium binding protein Iba1 and dimlyHCII, three typical characteristics of microglia. There are also
eports showing successful differentiation of rat blood monocytesowards microglia using ACM (Schmidtmayer et al., 1994; Sieverst al., 1994). Recently, Hinze and Stolzing (2011) showed the differ-ntiation of murine bone marrow stem cells towards microglia-likeells in the presence of ACM with similar phenotypic and functionalroperties to primary brain-derived microglia cultures. In the lightf these data, we developed a new in vitro human microglia modelsing human blood peripheral mononuclear cells, a serum-free cul-ure condition and a panel of factors, including M-CSF, GM-CSF, NGFnd CCL2. This new human microglia model has then been assessedor morphology, phenotype, and function.
. Materials and methods
.1. Cell Isolation
Human blood mononuclear cells (PBMC) were isolated fromuffy coats (50 buffy coats were used for this study) of healthyonors (Australian Red Cross Blood Service (ARCBS), Perth, WA,ustralia; ethics approval was granted by UWA and ARCBS)sing Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden) asublished before (Meagher et al., 2005). To obtain monocytesadherent PBMC), the isolated blood cells were cultured in T25issue culture flasks (Sarstedt, Numbrecht, Germany) (2 × 106 to
× 106 cells/ml) using RPMI-1640 Glutamax medium (Invitro-en, Auckland, NZ) supplemented with 1% antibiotics/antimycotic10,000 units/ml penicillin G sodium, 10,000 �g/ml streptomycinulfate and 25 �g/ml Amphotericin B, Invitrogen). It is importanto note that no serum was used in all microglia cultures. Aftervernight incubation (humidified air, 37 ◦C, 5% CO2), non-adherentells (consisting mainly of T-lymphocytes) were separated byhorough washing with PBS (Invitrogen) and used for furtherxperiments. The fresh adherent cells, which were mainly mono-ytes (>90%), were used for the generation of microglia (M-MG).
endritic cells (M-DC) and cultured monocytes, as well as for com-arison with the generated cell populations. All experiments wereone with cells from at least 6 different blood donors, if not other-ise stated.nce Methods 209 (2012) 79– 89 81
2.2. Cell culture
To induce the differentiation of M-MG, adherent PBMC(about 105/ml) were cultured in 6-well tissue culture plates(Sarstedt) using RPMI-1640 Glutamax supplemented with 1%antibiotic/antimycotic (serum-free condition) and a mixture ofhuman recombinant cytokines, including M-CSF (10 ng/ml; Pepro-Tech, Rocky Hill, NJ), GM-CSF (10 ng/ml; PeproTech), beta-nervegrowth factor (NGF-�; 10 ng/ml; PeproTech) and CCL2 (100 ng/ml;PeproTech) at standard humidified culture conditions (37 ◦C, 5%CO2) for up to 14 days. The cells were used for experiments andcharacterization at different time points as indicated.
Monocytes were also cultured in RPMI-1640 Glutamax supple-mented with 1% antibiotic/antimycotic, but no serum, for up to 14days as control cell population.
Furthermore, to obtain monocyte-derived dendritic cells (M-DC), adherent PBMC were cultured in 6-well tissue culture plates(Sarstedt) using RPMI-1640 Glutamax, 1% antibiotic/antimycoticsupplemented with 10% fetal calf serum (FCS; Serana, Bunbury,WA, Australia) in the presence of human recombinant GM-CSF(10 ng/ml; PeproTech) and interleukin-4 (IL-4; 10 ng/ml; Pepro-Tech) for up to 14 days (Meagher et al., 2005). The cells were usedfor experiments at different time points, as indicated.
Human microglia clone 3 (HMC3) cells, kindly provided byProf. Karl-Heinz Krause (University of Geneva, Switzerland) werecultured in T25 tissue culture flask (Sarstedt) using DMEM/F12Glutamax medium (Invitrogen) supplemented with 15% FCS and1% antibacterial/antimycotic. The cells were passaged and used forexperiments when they reached 70–80% confluence.
2.3. Morphological studies
Morphology and viability (using trypan blue dye exclusion: cellviability was >90% for all experiments) of M-MG, M-DC and HMC3during the 2 weeks in culture were studied using an invertedLeica DM IL phase contrast microscope. Images were taken withPhotometrix CoolSNAP Fx digital camera and processed with NIS-elements F3.0 software. HMC3 and M-MG cells were also stainedfor expression of Iba1, a well-known characteristic marker formicroglia cells. For that purpose cells were grown on glass cover-slips, fixed in 1% paraformaldehyde in PBS/sucrose and incubatedwith a polyclonal rabbit anti-Iba1 (Santa Cruz biotechnology, SantaCruz, CA) and a donkey anti-rabbit Alexa Fluor 488 (Invitrogen).Negative control stains were used by omitting the primary anti-body against Iba1. Cell nuclei were stained with DAPI (1ug/ml inPBS, 4′,6-diamidine-2′-phenylindole dihydrochloride; Roche Diag-nostics, Mannheim, Germany). Imaging was done using confocalmicroscopy (Leica TCS SP2 multiphoton confocal microscope).
2.4. Flow cytometry
We studied the expression of specific surface molecules onfreshly isolated and cultured monocytes, M-MG, M-DC and HMC3using flow cytometry. Cells (105/ml per staining condition) weredetached using a scraper (Sarstedt) and washed twice with PBS.Staining was performed according to protocol recommended byBD.
A panel of directly labeled mouse anti-human antibodies,CD45-FITC, CD14-PE, CD40-PE, CD68-APC, CD80-PE,CD11c-PE,HLA-DR–PERCP, CD11b-AF488, CD11c-PE, CCR1-AF647, CCR2-AF647, CCR4-PE-Cy7, CCR5-FITC, CXCR1-PE, CXCR3-AF488 (BDPharmingen, San Jose, CA), CCR6-FITC, CCR7-APC (R&A systems,
Minneapolis, MN), NGFR-FITC (Santa Cruz Biotechnology, SantaCruz, CA) were added at the supplier’s recommended concen-tration. Corresponding labeled isotype control antibodies wereincluded in all experiments. A rabbit anti-CX3CR1 polyclonal
82 S. Etemad et al. / Journal of Neuroscience Methods 209 (2012) 79– 89
d hum
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Fig. 1. Phase contrast microscopy of monocyte derived Microglia (M-MG) (A) an
ntibody was used at the final concentration of 1 �g/ml (AbDerotec, Oxford, UK), followed by a secondary donkey anti-rabbit-F 647 used at the final concentration of 1:200 (Invitrogen). Theells were incubated with antibodies for 30 min to 1 h at 4 ◦C,ashed with PBS, resuspended and fixed with 1% formaldehyde in
BS containing 2% sucrose. The fixed samples were measured with aacsCallibure (BD Biosciences). Data analysis was performed usingowjo software version 7.6.3 (Tree Star, Ashland, OR).
.5. Phagocytosis assay
Phagocytosis was assessed by measuring the uptake of tetram-thylrhodamine (TMR)-labeled Staphylococcus aureus (Invitrogen)s published before (Meagher et al., 2005). Bacterial particles weredded to the cells (107/ml) at a ratio of 1:1000 (according to man-facture’s protocol) and incubated for 4 h at 37 ◦C. Cells were thenarvested and fixed with 1% paraformaldehyde in PBS/2% sucrose.otal ingested fluorescent-labeled bacteria per cell was measuredy flow cytometry. To verify that the bacteria were located intra-ellularly, cells were cultured on glass coverslips, treated withabeled bacteria, fixed and visualized using confocal microscopyLeica TCS SP2 multiphoton confocal microscope). Dendritic cellsnd freshly isolated monocytes were used as positive control pop-lations. Untreated cells were used as negative controls.
.6. Mixed leukocyte reaction (MLR)
The T-lymphocyte stimulatory capacity and correspondingntigen-presenting cell (APC) properties of M-MG, in comparisonith M-DC and HMC3, was assessed in MLRs using a BrdU prolifer-
tion assay (Cadosch et al., 2010). Freshly processed non-adherentBMC containing mainly (>90%) T-lymphocytes (2 × 105/well) wereeeded in a round bottom 96-well plate (Sarstedt). Allogeneic-MG, M-DC and HMC3 were used as antigen-presenting cells
APC) and added at various ratios (1/20, 1/40 and 1/80) to the T-ymphocyte (4 wells for same condition). T-lymphocytes or APCnly were used as control conditions. After 5 days in culture, BrdUroliferation assays (Roche, Mannheim, Germany) were performedccording to the supplier’s protocol and the absorbance of theolorimetric reaction was measured by ELISA reader at 405 nmLabsystems Multiskan RC, Helsinki, Finland). Mean proliferation ofhe different conditions was statistically analyzed and compared.
.7. Statistics
Data were statistically analyzed through the SPSS statistics 17.0oftware. A one-way ANOVA and uni-variant linear model were
an microglia cell line HMC3 (B) after 10 days in culture. Bar indicates 100 �M.
used to compare means. The LSD post hoc test was also used todetermine the significant difference between groups. In all theanalysis p value < 0.05 were considered as statistically significant.Graphs with error bars represent the standard deviation (SD).
3. Results
3.1. Morphological and phenotypic changes of monocyte-derivedmicroglia in culture
Adherent PBMC, representing mainly the monocytic populationof blood leukocytes, were cultured for up to 2 weeks in the presenceM-CSF, GM-CSF, NGF-� and CCL2, in RPMI 1640 without addition ofserum to create optimally standardized conditions. The cells werecharacterized for morphological changes over the period of 2 weeksin culture (Fig. 1). The well established HMC3 human microgliacells line was used as comparison. After 5 days in culture, the small(10–15 �m diameter) round-shaped and semi-adhered monocyticcells expanded their cell body and developed few unbranched pro-cesses. During the second week, the cells reduced the portion of thecell body and they increased the processes in length and numberacquiring ramified forms. M-MG obtained a ramified morphologyafter 10–14 days in culture, including a polygonal or oval cell bodyand up to 7 primary processes and numerous fine secondary pro-cess or fine spines. Over the 2 weeks in culture, the cells doubledin size and increased in cell number indicating proliferation. After2 weeks, M-MG displayed a very similar morphology to the humanmicroglia cell line HMC3 and to previously described human brain-derived microglia in published studies (Leone et al., 2006).
Basic known microglia markers, including expression of Iba1and CD68, were also tested positive, supporting that the describedprotocol differentiates monocytes towards M-MG (Figs. 2 and 3).
3.2. Expression pattern of specific surface markers
After culture in the presence or absence of the correspond-ing cytokines cocktails, monocytes, M-MG, M-DC and the humanmicroglia cell line (HMC3) were tested for expression of surfacemarkers relevant to human microglia. Fig. 4 summarizes expres-sion of all relevant markers for all tested cell types, including CD45,CD14, MHC class II, CCR2 and CCR4.
Firstly, the common leukocyte antigen CD45 was tested. CD45is a characteristic marker expressed by all leukocytes. As expected,
CD45 was highly expressed in monocytes, which did not changeCD45 expression levels over the 2 weeks in culture in serum-freemedium without addition of cytokines. M-DC reduced only slightlyCD45 expression over the 2 weeks in culture. However, M-MG
S. Etemad et al. / Journal of Neuroscience Methods 209 (2012) 79– 89 83
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ig. 2. Expression of Iba1, a characteristic microglia marker in HMC3(B) and M-MG
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educed gradually CD45 expression levels over the 2 weeks in cul-ure reaching very low levels, as expected for microglia and similaro HMC3 (Janabi et al., 1995; Li et al., 2009).
CD14 is known to be characteristic for monocytes, beingxpressed at highest level on these cells. As predicted, M-MGeduced expression of CD14 to very low levels, similar to HMC3.
Activated microglia may process and present antigens on MHClass II. For that purpose, activated microglia increase MCH classI expression, such as HLA-DR. Usually, resting microglia expressow levels of HLA DR, whereas freshly isolated monocytes expressigh levels of HLA-DR, which is also highly expressed in immature-DC. In our study, HLA-DR was clearly down-regulated in M-MG,
eaching similar levels detected in HMC3, indicating that M-MGay represent resting microglia cells.Chemokines play a crucial role in recruitment and maturation
f migratory, bone marrow-derived microglia cells (Flynn et al.,003). CCR1, CCR2, CCR5 expression has been thus reported to bexpressed at low levels in microglia (Bajetto et al., 2002; Skulject al., 2011). In our study, monocytes expressed low levels ofCR2, but up-regulation was seen when cultured in medium with-ut cytokines. Interestingly, M-DC decreased CCR2 expression tondetectable levels, whereas M-MG and HMC3 expressed clearlyetectable CCR2 at rather low levels, as expected for microglia.
Expression of CCR4 and their ligand (CCL22 and CCL17) has beeneported in the CNS (Columba-Cabezas et al., 2002). Freshly iso-ated monocyte expressed CCR4 at very low levels, while after one
eek in culture without cytokines, a sub-population (about 25%)xpressed CCR4 at higher levels. However, M-DC and M-MG up
egulated CCR4 expression to clearly detectable levels over time inulture to similar levels as HMC3.Fig. 5 summarizes expression of various chemokine receptorsn the different cell populations that were investigated. Freshly
rresponding negative staining controls have been included (A and B). Bar indicates
isolated monocytes expressed intermediate levels of CCR1. Dif-ferentiation towards M-MG decreased CCR1 expression to similarlevels known for non-activated microglia and also seen in HMC3.CCR5 expression level was detected at low levels in monocytes, butexpressed at higher levels in both M-MG and HMC3, as expectedfor microglia. CXCR1 was expressed at very low levels in freshlyisolated monocyte, but was clearly expressed at higher levels inM-MG and HMC3.
Freshly isolated monocyte did not express neither CXCR3 norCX3CR1. However, both these markers were clearly up-regulatedin M-MG similar to HMC3. CXCR3 expression in M-MG was atintermediate levels, while HMC3 showed higher expression levels.Interestingly, almost similar expression of CX3CR1 was seen in bothM-MG and HMC3. In summary, M-MG and HMC3 expressed verysimilar chemokine receptor patterns, supporting the microglia-like properties of M-MG.Additional markers were also investigated(data not shown), including CD 11c, CD11b, CD40, CD80, CD83,CCR6 and CCR7. M-MG were negative for CCR6 and CCR7. CD11cand CD11b were both expressed on M-MG and HMC3, but at muchlower levels than on M-DC. Immature M-DC expressed the den-dritic cell markers CD40 and CD83 at an intermediate intensitylevel. However, CD80 expression was undetectable on M-DC. Mostimportant, neither M-MG nor HMC3 expressed any of the den-dritic cell specific surface markers, such as CD83, CD80 or CD40,supporting that M-MG are clearly different from dendritic cells.
3.3. Phagocytic capacity
Microglia play a major role in phagocytosis and clearance ofdead cells and debris, especially during inflammatory processescaused by injuries or neurodegenerative diseases. To determinethe phagocytic ability, we conducted phagocytosis experiments
84 S. Etemad et al. / Journal of Neuroscie
Fig. 3. Expression of CD68 in freshly isolated blood derived monocytes (A), HMC3(B) and M-MG (C). The grey line indicates the negative staining control using a corre-sponding isotype control antibody. The black line indicates the specific test stain forCD68. Note that there is no CD68 expression in monocytes, whereas the 2 microgliapopulations clearly express CD68.
nce Methods 209 (2012) 79– 89
(Meagher et al., 2005). M-MG were compared to immature M-DC,potent phagocytic immune cells (Filgueira et al., 1996). HMC3 wereused as microglia control cells. Fluorescent-labeled S. aureus wasused for measuring the phagocytic capacity of the various cell pop-ulations. Flow cytometry (Fig. 6) and confocal microscopy (Fig. 7)was used for the detection and quantification of bacterial up-take.As expected, M-DC displayed the highest phagocytosis capacity,whereas M-MG showed clearly decreased phagocytosis, similar toHMC3, which displayed even lower bacterial up-take. However,both M-MG and HMC3 showed up-take of bacteria, with clear intra-cellular location, as documented through confocal imaging.
3.4. T-lymphocyte stimulatory capacity of M-GM
As innate immune cells of the CNS, microglia are thought tobe antigen presenting cells (APC), able to process and presentantigen to T-lymphocyte, activate them and induce their prolifer-ation. In this study, mixed leukocyte reactions (MLR) were usedto determine the T-lymphocyte stimulatory capacity of M-MGusing proliferation assays. Immature dendritic cells (M-DC) andthe microglia cell line HMC3 were used as comparison. A repre-sentative example is shown in Fig. 8. As expected, M-DC inducedthe most pronounced and significant proliferation of allogeneicT-lymphocyte. M-MG were also able to induce significant prolif-eration of allogeneic T-lymphocytes, but to a much lower degreein comparison to M-DC. No significant T-lymphocyte stimulatoryeffect was seen with HMC3, which is in accordance with previousstudies (Fischer and Reichmann, 2001; Janabi et al., 1995; Nagaiet al., 2005; Zuiderwijk-Sick et al., 2007).
4. Discussion
Microglia play crucial roles in maintaining homeostasis and con-tributing to neuroinflammation in response to any disturbances,such as injuries, toxins and pathogen (Streit, 2001, 1996). To date,in vitro studies on microglia have used mainly primary cells derivedfrom embryonic or neonatal murine and human brains (Bassettet al., 2012; Hinojosa et al., 2011). This study provides the firstsimple, standardized and reliable protocol to generate humanmicroglia-like cells from blood monocytes. The protocol takes theadvantage of a serum-free culture condition, as well as a cocktailof 4 well defined recombinant human cytokines, i.e. M-CSF, GM-CSF, NGF and CCL-2. The cells generated according to the describedprotocol display most known characteristics of human microglia,including specific morphology, phenotype and functions.
In our model, the serum-free culture system is essential as itrepresents the physiological condition of the extracellular fluid ofthe CNS, which is equal to the cerebrospinal fluid. Normal cere-brospinal fluid (CSF) contains extremely low levels of proteins andbioactive factors and is usually deficient in blood serum proteins(Reiber, 2003). The blood–brain barrier prevents trafficking of bloodserum proteins and other factors into the extracellular space of theCNS, as they may damage glial and neuronal cells through activa-tion, resulting in apoptosis (Abbott et al., 2010; Hooper et al., 2009;Nadal et al., 1995). For example, in vitro culture of rat microgliain presence of bovine serum albumin, human or rat serum hasbeen shown to enhance the production of superoxide (O2
−) asone of the mechanisms involved in pathogenesis of neuronal dam-ages (Elkabes et al., 1998). Knowing about the potential bio-activeand even toxic effects of blood serum, we established thereforethis serum-free protocol for the generation of microglia-like cells
derived from blood monocytes.The cocktail of cytokines has beenchosen according to the known effects of the cytokines on microgliain published reports, as stated in the introduction, and exten-sive preliminary experiments. Thereby, the combination of M-CSF,
S. Etemad et al. / Journal of Neuroscience Methods 209 (2012) 79– 89 85
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ig. 4. Expression of surface markers CD45 (a), CD14 (b), HLA-DR (c), CCR2 (d) andultured monocytes in the absence of cytokines (B), monocyte derived dendritic ceultured for 10 days.
M-CSF, NGF and CCL-2 has shown to differentiate M-MG withost similar characteristics to microglia.Under healthy condition, resting “surveying microglia” with
amified morphology is responsible for maintaining the homeo-
tatic condition of the CNS. Using the described protocol, M-MGbtained a ramified morphology after 10–14 days in culture, includ-ng a polygonal or oval cell body and up to 7 primary processes andumerous fine secondary process or fine spines. Our morphological(e) measured with flow cytometry: comparison of freshly isolated monocytes (A),, monocyte derived microglia (D) and microglia cell line HMC3 (E). The cells were
findings for M-MG correspond to the report by Leone et al. (2006),who used a less well defined protocol. In addition, M-MG displayedsimilar morphology to the human microglia cell line HMC3. Inaddition, M-MG expressed Iba1 and CD68, well known microglia
markers.Most important was the phenotypic characterization of thecells using a broad panel of antibodies and flow cytometry. Mono-cytes cultured over 2 weeks in the presence of the 4 cytokines
86 S. Etemad et al. / Journal of Neuroscience Methods 209 (2012) 79– 89
Fig. 5. Expression of chemokine receptors CCR1 (a), CCR5 (b), CXCR1(c), CXCR3 (d) andmonocytes (A), monocyte derived microglia (B) and the microglia cell line HMC3 (C). The
Fig. 6. Phagocytic capacity of M-DC (black line), M-MG (dotted line) and HMC3(dashed line) towards TM Rhodamine-labeled S. aureus measured with flow cytom-etry. Bacterial particles were added to culture medium at a ratio of 1:1000 (107
bacteria per ml) and incubated for 4 h at 37 ◦C. Flow cytometry data represented thedifferent levels of engulfed fluorescent-labeled bacteria.
CX3CR1 (e) measured with flow cytometry: expression levels in freshly isolated cells were cultured for 10 days.
changed significantly expression of surface markers towards theknown pattern for microglia. CD45, CD14 and HLA-Dr expressionlevels decreased substantially in M-MG reaching levels expectedfor microglia. CD45 expression levels have been often used fordiscriminating between tissue macrophages and microglia in CNSstudies (Lambertsen et al., 2009; Wirenfeldt et al., 2005). Penningeret al. (2001) states that in the CNS, microglia express differ-ent levels of CD45 depending on their differentiation state, withdecreased expression levels in mature resting microglia. How-ever, CD45 expression increases in activated microglia, e.g. inAlzheimer patients, as this surface marker seems to play a rolein degenerative and inflammatory CNS diseases (Masliah et al.,1991). CD 14 is regarded as an important lipopolysaccharide (LPS)receptor (Triantafilou and Triantafilou, 2002). Supporting our pro-tocol, Lambert et al. (2008) reported down-regulation of CD14 inmonocyte-derived DC and microglia in presence of GM-CSF, M-CSFand LPS.
Activated microglia is considered as the antigen presenting cellsof the CNS, as they express MHC class II molecules. However, rest-ing microglia express low levels of MHC class II. M-MG decreasedHLA-DR expression to low levels, similar to HMC3, but to muchlower levels than freshly isolated monocytes or immature M-DC(Lutz et al., 2000). Furthermore, it was important to discriminate
between M-MG and M-DC, as both may display similar morpholog-ical properties. In addition to the differences in expression levels ofMHC class II, CD11b and CD11c, M-MG did not express characteris-tic M-DC markers, including CD40, CD80 and CD83. The differences
S. Etemad et al. / Journal of Neuroscience Methods 209 (2012) 79– 89 87
F -stacb eria in
btdtpM
c
Foccac
ig. 7. Up-take of TM Rhodamine-labeled S. aureus by M-MG using on a selected Zacteria at a ration 1:1000. Note the intra-cellular accumulation of red labeled bact
etween M-MG and M-DC were further supported by the functionalests, such as MLR and phagocytosis assays. As expected, M-MGisplayed much lower T-lymphocyte stimulatory capacity and bac-erial up-take, in comparison with M-DC. In summary, our datarovide clear evidence that our M-MG protocol does not generate
-DC, but a distinct microglia-like population.Our study also revealed the expression level of varioushemokine receptors, including CCR1, CCR2, CCR4, CCR5, CXCR1,
ig. 8. Mixed leukocyte reactions (MLR) testing T-lymphocyte stimulatory capacityf HMC3 (black bars), M-MG (dotted bars) and M-DC (hatched bars) at differentell ratios (antigen presenting cells: allogeneic T-lymphocytes). The cells were co-ultured for 5 days before proliferation was measured using a BrdU incorporationssay. Error bars represent the ±2 SD. Statistically significant differences betweenontrol and test condition are indicated by *p < 0.05.
k image taken with confocal microscopy. M-MG cells were incubated for 4 h with the M-MG cells on the optical confocal section.
CXCR3 and CX3CR1 in M-MG and HMC3. Freshly isolatedmonocytes showed intermediate expression level of CCR1, lowexpression levels for CCR2, CXCR1 and CCR5, and undetectableexpression levels of CCR4, CXCR3 and CX3CR1, as reported previ-ously by others (Geissmann et al., 2003). M-MG changed expressionlevels of the various chemokine receptors substantially, in compari-son with freshly isolated monocytes. However, expression patternsof M-MG were very similar to that of HMC3. M-MG showeddown-regulation of CCR1 to still well detectable levels. On theother hand, M-MG displayed significant up-regulation of CCR4 andCCR5. Interestingly, M-MG expressed CXCR3 and CX3CR1, whichwere not detected in monocytes. Up-regulation of CXCR1 was alsoseen in M-MG. Our findings are in accordance with the report byBajetto et al. (2002), who published that non-activated microgliaexpress CCR1, CCR2 and CCR5 constitutively at low levels. Sev-eral other researchers reported that activated microglia expresshigher level of CCR2, CCR3, CCR4, CCR5, CX3CR1 (Eltayeb et al.,2007; Simpson et al., 2000b; Szczucinski and Losy, 2007). However,little is known about chemokine receptor expression in humanmicroglia. Our protocol may help in future research to elucidateregulation of these receptors and chemokine responses in humanmicroglia.
5. Conclusion
We developed a new in vitro protocol for the generation ofhuman microglia from blood monocytes using a serum-free culturecondition and a novel mixture of 4 recombinant human cytokines,i.e. M-CSF, GM-CSF, NGF and CCL2. Detailed characterization of M-MG revealed a cell population representing resting microglia withtheir specific morphological, phenotypic and functional properties.The described protocol is easy to handle, well standardized and
very reproducible, as it uses only well defined culture medium andrecombinant cytokines, but no serum or conditioned medium. Thisprotocol will certainly be very helpful for future studies investigat-ing the biology and pathology of human microglia.
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onflict of interest statement
None of the authors have any conflict of interest to disclose.
cknowledgements
Microscopes were provided by Cell Central, School of Anatomy,hysiology and Human Biology. In that respect, we thank Guy Ben-ry and Steve Parkinson for their excellent support. The authorscknowledge the facilities (Confocal microscopy and flow cytome-ry), scientific and technical assistance of the Australian Microscopy
Microanalysis Research Facility at the Centre for Microscopy,haracterisation & Analysis, The University of Western Australia,
facility funded by the University, State and Commonwealth Gov-rnments. In that respect, we acknowledge the support by Kathyeel, Paul Rigby and Tracey Lee-Pullen. The project was supportedy a UWA Collaboration Award.
eferences
bbott NJ, Patabendige AAK, Dolman DEM, Yusof SR, Begley DJ. Structure and func-tion of the blood–brain barrier. Neurobiol Dis 2010;37:13–25.
loisi F. Immune function of microglia. Glia 2001;36:165–79.loisi F, De Simone R, Columba-Cabezas S, Penna G, Adorini L. Functional maturation
of adult mouse resting microglia into an APC is promoted by granulocyte-macrophage colony-stimulating factor and interaction with Th1 cells. J Immunol2000;164:1705–12.
ajetto A, Bonavia R, Barbero S, Schettini G. Characterization of chemokines andtheir receptors in the central nervous system: physiopathological implications.J Neurochem 2002;82:1311–29.
anisadr G, Gosselin R-D, Mechighel P, Kitabgi P, Rostène W, ParsadaniantzSM. Highly regionalized neuronal expression of monocyte chemoattractantprotein-1 (MCP-1/CCL2) in rat brain: evidence for its colocalization with neuro-transmitters and neuropeptides. J Comp Neurol 2005;489:275–92.
assett T, Bach P, Chan HM. Effects of methylmercury on the secretion ofpro-inflammatory cytokines from primary microglial cells and astrocytes. Neu-roToxicology 2012;33:229–34.
levins G, Fedoroff S. Microglia in colony-stimulating factor 1-deficient op/op mice.J Neurosci Res 1995;40:535–44.
adosch D, Sutanto M, Chan E, Mhawi A, Gautschi OP, von Katterfeld B, et al. Titaniumuptake, induction of RANK-L expression, and enhanced proliferation of humanT-lymphocytes. J Orthop Res 2010;28:341–7.
hitu V, Stanley ER. Colony-stimulating factor-1 in immunity and inflammation.Curr Opin Immunol 2006;18:39–48.
olumba-Cabezas S, Serafini B, Ambrosini E, Sanchez M, Penna G, Adorini L, et al.Induction of macrophage-derived chemokine/CCL22 expression in experimen-tal autoimmune encephalomyelitis and cultured microglia: implications fordisease regulation. J Neuroimmunol 2002;130:10–21.
onductier G, Blondeau N, Guyon A, Nahon J-L, Rovère C. The role of monocytechemoattractant protein MCP1/CCL2 in neuroinflammatory diseases. J Neuroim-munol 2010;224:93–100.
raig MJ, Loberg RD. CCL2 (Monocyte Chemoattractant Protein-1) in cancer bonemetastases. Cancer Metastasis Rev 2006;25:611–9.
e Simone R, Ambrosini E, Carnevale D, Ajmone-Cat MA, Minghetti L. NGF pro-motes microglial migration through the activation of its high affinity receptor:modulation by TGF-[beta]. J Neuroimmunol 2007;190:53–60.
l Khoury J, Toft M, Hickman SE, Means TK, Terada K, Geula C, et al. Ccr2 deficiencyimpairs microglial accumulation and accelerates progression of Alzheimer-likedisease. Nat Med 2007;13:432–8.
lkabes S, Peng L, Black IB. Lipopolysaccharide differentially regulates microglial trkreceptor and neurotrophin expression. J Neurosci Res 1998;54:117–22.
ltayeb S, Berg A-L, Lassmann H, Wallstrom E, Nilsson M, Olsson T, et al. Temporalexpression and cellular origin of CC chemokine receptors CCR1, CCR2 and CCR5in the central nervous system: insight into mechanisms of MOG-induced EAE. JNeuroinflamm 2007;4:14.
sen N, Kielian T. Effects of low dose GM-CSF on microglial inflammatory profilesto diverse pathogen-associated molecular patterns (PAMPs). J Neuroinflamm2007;4:10.
arina C, Aloisi F, Meinl E. Astrocytes are active players in cerebral innate immunity.Trends Immunol 2007;28:138–45.
ilgueira L, Nestle F, Rittig M, Joller H, Groscurth P. Human dendritic cells phagocy-tose and process Borrelia burgdorferi. J Immunol 1996;157:2998–3005.
ischer H-G, Reichmann G. Brain dendritic cells and macrophages/microglia in cen-tral nervous system inflammation. J Immunol 2001;166:2717–26.
lynn G, Maru S, Loughlin J, Romero IA, Male D. Regulation of chemokinereceptor expression in human microglia and astrocytes. J Neuroimmunol2003;136:84–93.
ord A, Goodsall A, Hickey W, Sedgwick J. Normal adult ramified microglia separatedfrom other central nervous system macrophages by flow cytometric sorting.
nce Methods 209 (2012) 79– 89
Phenotypic differences defined and direct ex vivo antigen presentation to myelinbasic protein-reactive CD4+ T cells compared. J Immunol 1995;154:4309–21.
Friedman WJ. Neurotrophins induce death of hippocampal neurons via the p75receptor. J Neurosci 2000;20:6340–6.
Fujita H, Tanaka J, Toku K, Tateishi N, Suzuki Y, Matsuda S, et al. Effects of GM-CSF and ordinary supplements on the ramification of microglia in culture: amorphometrical study. Glia 1996;18:269–81.
Geissmann F, Jung S, Littman DR. Blood monocytes consist of two principal subsetswith distinct migratory properties. Immunity 2003;19:71–82.
Ginhoux F, Greter M, Leboeuf M, Nandi S, See P, Gokhan S, et al. Fate mapping anal-ysis reveals that adult microglia derive from primitive macrophages. Science2010;330:841–5.
Heese K, Hock C, Otten U. Inflammatory signals induce neurotrophin expression inhuman microglial cells. J Neurochem 1998;70:699–707.
Hinojosa AE, Garcia-Bueno B, Leza JC, Madrigal JL. CCL2/MCP-1 modulation ofmicroglial activation and proliferation. J Neuroinflamm 2011;8:77.
Hinze A, Stolzing A. Differentiation of mouse bone marrow derived stem cells towardmicroglia-like cells. BMC Cell Biol 2011;12:35.
Hooper C, Pinteaux-Jones F, Fry VAH, Sevastou IG, Baker D, Heales SJ, et al. Differentialeffects of albumin on microglia and macrophages; implications for neurodegen-eration following blood–brain barrier damage. J Neurochem 2009;109:694–705.
Howell OW, Rundle JL, Garg A, Komada M, Brophy PJ, Reynolds R. Acti-vated microglia mediate axoglial disruption that contributes to axonalinjury in multiple sclerosis. J Neuropathol Exp Neurol 2010;69:1017–33,http://dx.doi.org/10.1097/NEN.0b013e3181f3a5b1.
Izikson L, Klein RS, Charo IF, Weiner HL, Luster AD. Resistance to experimentalautoimmune encephalomyelitis in mice lacking the cc chemokine receptor(Ccr2). J Exp Med 2000;192:1075–80.
Janabi N, Peudenier S, Héron B, Ng KH, Tardieu M. Establishment of human microglialcell lines after transfection of primary cultures of embryonic microglial cells withthe SV40 large T antigen. Neurosci Lett 1995;195:105–8.
Kalla R, Liu Z, Xu S, Koppius A, Imai Y, Kloss CUA, et al. Microglia and the earlyphase of immune surveillance in the axotomized facial motor nucleus: impairedmicroglial activation and lymphocyte recruitment but no effect on neuronal sur-vival or axonal regeneration in macrophage-colony stimulating factor-deficientmice. J Comp Neurol 2001;436:182–201.
Lambert C, Desbarats J, Arbour N, Hall JA, Olivier A, Bar-Or A, et al. Dendritic celldifferentiation signals induce anti-inflammatory properties in human adultmicroglia. J Immunol 2008;181:8288–97.
Lambertsen KL, Clausen BH, Babcock AA, Gregersen R, Fenger C, Nielsen HH, et al.Microglia protect neurons against ischemia by synthesis of tumor necrosis fac-tor. J Neurosci 2009;29:1319–30.
Lee S, Varvel NH, Konerth ME, Xu G, Cardona AE, Ransohoff RM, et al. CX3CR1 defi-ciency alters microglial activation and reduces beta-amyloid deposition in twoAlzheimer’s disease mouse models. Am J Pathol 2010;177:2549–62.
Lee SC, Liu W, Brosnan CF, Dickson DW. GM-CSF promotes proliferation of humanfetal and adult microglia in primary cultures. Glia 1994;12:309–18.
Leonard EJ, Skeel A, Yoshimura T. Biological aspects of monocyte chemoattractantprotein-1 (MCP-1). Adv Exp Med Biol 1991;305:57–64.
Leone C, Le Pavec G, Même W, Porcheray F, Samah B, Dormont D, et al. Characteri-zation of human monocyte-derived microglia-like cells. Glia 2006;54:183–92.
Li B, Bedard K, Sorce S, Hinz B, Dubois-Dauphin M, Krause KH. NOX4 expression inhuman microglia leads to constitutive generation of reactive oxygen species andto constitutive IL-6 expression. J Innate Immun 2009;1:570–81.
Lutz MB, Suri RM, Niimi M, Ogilvie AL, Kukutsch NA, Rossner S, et al. Immaturedendritic cells generated with low doses of GM-CSF in the absence of IL-4are maturation resistant and prolong allograft survival in vivo. Eur J Immunol2000;30:1813–22.
Masliah E, Mallory M, Hansen L, Alford M, Albright T, Terry R, et al. Immunoreactivityof CD45, a protein phosphotyrosine phosphatase, in Alzheimer’s disease. ActaNeuropathol 1991;83:12–20.
Meagher J, Zellweger R, Filgueira L. Functional dissociation of the basolateral tran-scytotic compartment from the apical phago-lysosomal compartment in humanosteoclasts. J Histochem Cytochem 2005;53:665–70.
Nadal A, Fuentes E, Pastor J, McNaughton PA. Plasma albumin is a potent trig-ger of calcium signals and DNA synthesis in astrocytes. Proc Natl Acad Sci1995;92:1426–30.
Nagai A, Mishima S, Ishida Y, Ishikura H, Harada T, Kobayashi S, et al. Immor-talized human microglial cell line: phenotypic expression. J Neurosci Res2005;81:342–8.
Nakajima K, Honda S, Tohyama Y, Imai Y, Kohsaka S, Kurihara T. Neurotrophinsecretion from cultured microglia. J Neurosci Res 2001;65:322–31.
Nimmerjahn A, Kirchhoff F, Helmchen F. Resting microglial cells are highly dynamicsurveillants of brain parenchyma in vivo. Science 2005;308:1314–8.
Noto D, Takahashi K, Miyake S, Yamada M. In vitro differentiation of lineage-negativebone marrow cells into microglia-like cells. Eur J Neurosci 2010;31:1155–63.
Penninger JM, Irie-Sasaki J, Sasaki T, Oliveira-dos-Santos AJ. CD45: new jobs for anold acquaintance. Nat Immunol 2001;2:389–96.
Politis M, Pavese N, Tai YF, Kiferle L, Mason SL, Brooks DJ, et al. Microglialactivation in regions related to cognitive function predicts disease onset inHuntington’s disease: a multimodal imaging study. Hum Brain Mapp 2011;32:
258–70.Re F, Belyanskaya SL, Riese RJ, Cipriani B, Fischer FR, Granucci F, et al. Granulocyte-macrophage colony-stimulating factor induces an expression program inneonatal microglia that primes them for antigen presentation. J Immunol2002;169:2264–73.
roscie
R
R
S
S
S
S
S
S
S
S
S
SS
S. Etemad et al. / Journal of Neu
ebenko-Moll NM, Liu L, Cardona A, Ransohoff RM. Chemokines, mononuclear cellsand the nervous system: heaven (or hell) is in the details. Curr Opin Immunol2006;18:683–9.
eiber H. Proteins in cerebrospinal fluid and blood: barriers, CSF flow rate andsource-related dynamics. Restor Neurol Neurosci 2003;21:79–96.
aez ET, Pehar M, Vargas MR, Barbeito L, Maccioni RB. Production of nerve growthfactor by beta-amyloid-stimulated astrocytes induces p75NTR-dependenttau hyperphosphorylation in cultured hippocampal neurons. J Neurosci Res2006;84:1098–106.
chilling T, Nitsch R, Heinemann U, Haas D, Eder C. Astrocyte-released cytokinesinduce ramification and outward K+ channel expression in microglia via distinctsignalling pathways. Eur J Neurosci 2001;14:463–73.
chmidtmayer J, Jacobsen C, Miksch G, Sievers J. Blood monocytes and spleenmacrophages differentiate into microglia-like cells on monolayers of astrocytes:membrane currents. Glia 1994;12:259–67.
ievers J, Parwaresch R, Wottge H-U. Blood monocytes and spleen macrophagesdifferentiate into microglia-like cells on monolayers of astrocytes: morphology.Glia 1994;12:245–58.
imard AR, Soulet D, Gowing G, Julien J-P, Rivest S. Bone marrow-derived microgliaplay a critical role in restricting senile plaque formation in Alzheimer’s disease.Neuron 2006;49:489–502.
impson J, Rezaie P, Newcombe J, Cuzner ML, Male D, Woodroofe MN. Expressionof the [beta]-chemokine receptors CCR2, CCR3 and CCR5 in multiple sclerosiscentral nervous system tissue. J Neuroimmunol 2000a;108:192–200.
impson J, Rezaie P, Newcombe J, Cuzner ML, Male D, Woodroofe MN. Expression ofthe �-chemokine receptors CCR2, CCR3 and CCR5 in multiple sclerosis centralnervous system tissue. J Neuroimmunol 2000b;108:192–200.
ˇkuljec J, Sun H, Pul R, Bénardais K, Ragancokova D, Moharregh-Khiabani D, et al.CCL5 induces a pro-inflammatory profile in microglia in vitro. Cell Immunol2011;270:164–71.
treit WJ. Microglia and macrophages in the developing CNS. Neurotoxicology
2001;22:619–24.treit WJ. The role of microglia in brain injury. Neurotoxicology 1996;17:671–8.zczucinski A, Losy J. Chemokines and chemokine receptors in multiple scle-
rosis. Potential targets for new therapies. Acta Neurol Scand 2007;115:137–46.
nce Methods 209 (2012) 79– 89 89
Tambuyzer BR, Ponsaerts P, Nouwen EJ. Microglia: gatekeepers of central nervoussystem immunology. J Leukoc Biol 2009;85:352–70.
Tanuma N, Sakuma H, Sasaki A, Matsumoto Y. Chemokine expression by astrocytesplays a role in microglia/macrophage activation and subsequent neurodegen-eration in secondary progressive multiple sclerosis. Acta Neuropathol (Berl)2006;112:195–204.
Thacker MA, Clark AK, Bishop T, Grist J, Yip PK, Moon LDF, et al. CCL2 is a key mediatorof microglia activation in neuropathic pain states. Eur J Pain (London, England)2009;13:263–72.
Tonchev AB. The nerve growth factor in health and unhealth neurons. Arch Ital Biol2011;149:225–31.
Triantafilou M, Triantafilou K. Lipopolysaccharide recognition: CD14, TLRs and theLPS-activation cluster. Trends Immunol 2002;23:301–4.
Valdo P, Stegagno C, Mazzucco S, Zuliani E, Zanusso G, Moretto G, et al. Enhancedexpression of NGF receptors in multiple sclerosis lesions. J Neuropathol ExpNeurol 2002;61:91–8.
Wegiel J, Wisniewski HM, Dziewiatkowski J, Tarnawski M, Kozielski R, Trenkner E,et al. Reduced number and altered morphology of microglial cells in colony stim-ulating factor-1-deficient osteopetrotic op/op mice. Brain Res 1998;804:135–9.
Wirenfeldt M, Ladeby R, Dalmau I, Banati RB, Finsen B. Microglia – Biologyand relevance to disease. Mikroglia – Biologi og sygdomsmæssig relevans2005;167:3025–30.
Zhang J, Geula C, Lu C, Koziel H, Hatcher LM, Roisen FJ. Neurotrophins regu-late proliferation and survival of two microglial cell lines in vitro. Exp Neurol2003;183:469–81.
Zhang J, Shi XQ, Echeverry S, Mogil JS, De Koninck Y, Rivest S. Expression of CCR2in both resident and bone marrow-derived microglia plays a critical role inneuropathic pain. J Neurosci 2007;27:12396–406.
Zhang T, Somasundaram R, Berencsi K, Caputo L, Gimotty P, Rani P, et al. Migra-tion of cytotoxic T lymphocytes toward melanoma cells in three-dimensionalorganotypic culture is dependent on CCL2 and CCR4. Eur J Immunol 2006;36:
457–67.Zuiderwijk-Sick EA, van der Putten C, Bsibsi M, Deuzing IP, de Boer W, Persoon-Deen C, et al. Differentiation of primary adult microglia alters their responseto TLR8-mediated activation but not their capacity as APC. Glia 2007;55:1589–600.