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The Role of Nerve Growth Factor (NGF) on Mast Cells
During Allergic Inflammation
Natalie SandovalBiol 520
March 9, 2009
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Background Information
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Neurotrophins(Neurotrophic Factors)
Family of polypeptide growth factors
Responsible for the survival, development and function of neurons in the peripheral and central nervous systems (CNS)
Deister et al 2006 www.science-autism.org
Neurotrophins in the brain
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Neurotrophin Family Members Nerve Growth FactorNerve Growth Factor Brain-derived neurotrophic factor (BDNF):
– Active in areas, such as the hippocampus– Activates the TrkB receptor
Neurotrophin 3 (NT-3):– Stimulates a number of neurons– Activate two of the receptors (TrkC and TrkB)
Neurotrophin 4 (NT-4):– Recently discovered and least understood– Activates the TrkB receptor
Reichardt, et al 2006 Marinova et al 2007,
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Nerve Growth Factor (NGF) Discovered in 1950 by Rita Levi-
Montalcini Activates TrkA receptor 118 aminoacid polypeptide:
– α,β, and γ Mutation in gene encoding TrkA leads
to a role in pain production
Thacker et al. 2007
www.als.lbl.gov/als/science/sci_archive/89receptor.html
Marinova et al 2007,
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Neurotrophin Receptors
Kondyli et al 2005, Nockher et al. 2006
Tropomyosin-related kinase (Trk) receptor:– Ligand specific– High affinity receptor– TrkA, TrkB, TrkC
P75 neurotrophin receptor (p75NTR):– Tumour necrosis receptor
superfamily– All neurotrophins bind with
similar affinity– Low affinity recepter
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After Reichardt 2006,
NF-kB
Kondyli et al 2005,
Neurotrophin Signaling Pathway
Trk A
IKK
NF-kBIkB
Prosurvival genes
p75NTR
TRAF-6
NIK
IkB
Sh2Grb2SOS
RasRaf
MEK
MAPK
Prodifferentation genes
Cytoplasm
Nucleus
NGF
MAP kinase Pathway
NF-kB pathway
Molecular Cell Biology, 5 edition
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NFG Interacts with Immune cells NFG has a role outside the nervous
system.
Current data,– Show that cells of the immune
system express NGF – NGF can influence the developing
immune response
Mast cells were identified as the first non-neuronal cell type influenced by NGF.
Micera et al 2003
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Where do Mast Cells Come From?
Immunobiology (7edition)
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Mast Cells (MC) & Activation
First responders to infection Key effector cells of allergic inflammation
– Contains many granules rich in histamine and heparin
– Stimulated to degranulate by: Cross-linking of Immunoglobulin E
(IgE) receptors NT-3? NGF??
Releases its granules and various hormonal mediators:– Histamine– Tryptase
Bischoff et al. 2007
www.jralphb.co.uk/allerg.htm
Mast cell degranulation
Mast cell intact
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Bidirectional Communication between NGF and MCs
Allergies: are caused by an immune response to a normally harmless substance– Immune response is damaging rather than helpful to the host– Immediate responses
NGF plasma levels are increased in times of stress (allergic reactions)
Mast cells numbers are also increased Mast cells depend on NGF for homing, survival and
differentiation Could NGF possibly act as a chemoattractant for
these immune cells?
Vega et al. 2003
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Histamine and Tryptase
Histamine– Important protein involved
in many allergic reactions– The release of histamine
cause allergic symptoms Itching, burning
sensation, sneezing and wheezing
Tryptase – Secretory granule-derived
serine protease contained in mast cells
– Marker for mast cell activation
www.bio.davidson.edu/courses/Immunology/
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Allergic Diseases Atopic Dermatitis: chronic inflammatory skin disease
Associated with elevated IgE levels, eosinophils, and mast cell hyperplasia and NGF?
Any age (early infancy)
Affects both males and females
Not contagious
Extremely itchy skin
www.derm-student.com/Atopic_dermatitis.htmlPfaar et al. 2009
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Allergic Inflammation
After Nockher et al. 2006
ItchItch!
Fc receptor
IgE antibody
Histamines
TrK AMast Cell
Allergen
Epidermal keratinocytes
NGF ?
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Hypothesis
Nerve growth factor (NGF) activates mast cells during allergic inflammation.
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Overview Support Hypothesis:
– Clinical Relationship between NGF and allergic diseases in mice
– Molecular level of mast cell activation by NGF in mouse line
Refute Hypothesis:– No clinical relationship of NGF with allergic diseases– Mast cell activation in vivo studies of another neurotrophin
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Experimental Results:Support Hypothesis
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Mouse Model for Atopic Dermatitis
Yoshioka et al. 2005
Parent Mice ♂ DS-Nh (Nh/Nh) X ♀ DS (+/+)
F1 Generation ♂ ♀ Mice DS-Nh (Nh/+)
Raised under conventional conditions and after 1-2 weeks develop AD-like dermatitis symptoms, erythema, dry skin, etc.
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NGF, IgE and histamine levels increased in DS-Nh mice
Yoshioka et al. 2005
Conventional:Conventional: exposed to S. aureus
SPF:SPF: free from S. aureus
Levels increased only in DS-Nh mice conventionally housed.
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NGF increased in Human Patients with Atopic Dermatitis
Toyoda et al .2003
ControlControl: 31 subjects (no history allergies)18males, 13 females, mean age of 27
AD:AD: 42 subjects with (AD)26 males, 16 females, mean age of 24.9
(B):B): NGF levels
Significant increase NGF in patients with AD in comparison to the control subjects with no history of allergies (p<0.005).
No Y-axis
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NGF Activates Mast Cells Directly
Groneberg et al. 2005
((A)A) Tryptase Activity with different concentrations of NGF
(B)(B) Tryptase Activity w/ or w/o NGF(C)(C) Histamine Activity w/ or w/o
NGF
Black bars= with NGFWhite bars= without NGF
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NGF induces TrkA receptor mRNA expression in Human Mast Cell line-1
Groneberg et al. 2005
Lane 1:Lane 1: No cDNALane 2:Lane 2: 1 day with NGFLane 3:Lane 3: 2 days with NGFLane 4:Lane 4: 4 days with NGFLane 5:Lane 5: 10 days with NGF
Tryptase and TrkA mRNA levels increased in cells cultured in NGF for 1 to 10 days.
HD= Histidine decarboxylase
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p75NTR Expression Increased in Lesional MCs in Atopic Dermatitis Skin
Fischer et al. 2008
N: Normal skinAD: Atopic Dermatitis
(A) The neurotrophin p75 expression in found in skin cells of patients with AD.(B) p75 expression was significantly increased in MC of patients with AD.
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Experimental Results:Refute Hypothesis
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SCORAD SCORing Atopic Dermatitis: A clinical tool for assessing the severity of atopic
dermatitis.– Takes into account two main criteria:
The Extent The Intensity of the following symptoms:
– Erythema, edema/papulation, oozing/crustoozing/crust,, excoriation, lichenification and dryness
Subjective symptoms:– Pruritus and sleep loss
Schulte-Herbruggen et al. 2007adserver.sante.univ-nantes.fr/Scorad.html
STAGE 1
STAGE 2
STAGE 3
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No Correlation Between NGF levels and Atopic Dermatitis
Schulte-Herbruggen et al. 2007
NGF serum concentrations in AD patients do not differ from the control.
No correlation between NGF levels and severity of AD
AD: 57 patients(31 males, 26 females, mean age 33 yrs)
Healthy controls: 50 volunteers(26 males, 24 females, mean age 48 yrs)
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NGF Does Not Promote Growth of Human Intestinal Mast Cells
Lorentz et al. 2007
(A)SCF is needed to promote MC recovery.(B)Synergistic effect of SCF with NT-3.(C)No significant increase when NGF is added.
SCF=Stem Cell Factor
NT-3=Neurotrophin-3
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NT-3 Activates Mast Cells in vivo in Mice Skin
Metz et al. 2004
(A) NT-3 induces the degranulation of skin MCs
In Vivo Study
Biopsies of back skin of C57BL/6 mice
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Conclusion
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Summary
Support Hypothesis
Refute Hypothesis
Yoshioka et al. 2005
Fischer et al. 2005
Toyoda et al. 2003
Groneberg et al. 2005
Metz et al. 2004
Schulte-Herbruggen et al. 2007
Lorentz et al. 2007
NGF, histamine and IgE levels are increased in DS-Nh mice NGF levels are Increased human patients with AD
NGF activates MC directly (TrkA recptor)
NGF p75NTR receptor expression is
increased in lesional MC
NGF levels are not increased in human
patients with ADNGF does not promote growth of human intestional MCs
NT-3 activates MCs in vivo
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Take Home Message In allergic diseases (atopic dermatitis) NGF levels
are increased.
The NGF receptors, TrkA and p75NTR are expressed on MCs.
NGF activates mast cells to release mediators during allergic inflammation.
NGF
Histamine
TryptaseTrkAMCs
Fc
Allergies
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Conclusion Based on my finding my hypothesis is supported:
– Nerve growth factor activates mast cells during an allergic inflammation.
Mast cells are activated directly by NGF in HMC-1 (Groneberg et al 2005).
Mast cells are activated by SCF and NT-3 the paper was poorly written and they measure MC recovery using trypan blue (measures dead cells) (Lorentz et al 2007).
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Future Experiments
Yoshioka et al 2005
Block nerve growth factor (NFG) in DS-Nh mice
1 2 3 4
Mar
ker
PBS
Unt
reat
ed D
S-N
hTr
eate
d D
S-N
h
Less expression ofNGF
Remove tissue
Inject Anti-NGF No Anti-NGF
Make antibody against NGF
Remove tissue
IsolateMC
IsolateMC
RT-PCR
DS-Nh mice DS-Nh mice
SCORADSCORAD
RT-PCR
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References Fischer, T.C., Lauensteinw, H.D., Serowkaz, F., Pilznerw, C., Groneberg, D.A. and Welker, P. (2008) Pan-neurotrophin receptor
p75NTR expression is strongly induced in lesional atopic mast cells. Clinical and Experimental Allergy 38, 1168–1173. Groneberg, D.A., Serowka, F., Peckenschneider, Artuc, M., Gruzkau, A., Fischer, A., Henz, B.M., and Welker, P. (2005) Gene
expressionand regulation of nerve growth factor in atopic dermatitis mast cells and the human mast cell line-1. Journal of Neuroimmunology 161, 87- 92.
Kondyli, M., Varakis, J., and Assimakopoulou, M. (2005) Expression of p75NTR and Trk neurotrophin receptors in the enteric nervous system of human adults. Anatomical Science International 80, 223-228.
Lorentz, A., Hoppe, J., Worthmann, H., Gebhardt, T., Hesse, U., Bienenstock, J., and Bischoff. S.C. (2007) Neurotrophin-3, but not nervegrowth factor, promotes survival of human intestinal mast cells. Neurogastroenterol Motil 19, 301-308.
Marinova, T., Philipov, S. and Aloe, L. (2006) Nerve Growth Factor Immunoreactivity of Mast Cells in Acute Involuted Human Thymus. Inflammation 30, 38-42.
Metz, M., Botchkarev, V.A., Botchkareva, N.V., Welker, P., Tobin, D.J., Knop, J., Maurer, M. and Paus, R. (2004) Neurotrophin-3 regulates mast cell functions in neonatal mouse skin. Experimental Dermatology 13, 273-281.
Pfaar, O., Raap, U., Holz, M., Hormann, K., and Klimek, L. (2009) Pathophysiology of itching and sneezing in allergic rhinitis. Swiss Med Weekly 139, 35-40.
Schulte-Herbruggen, O., Folster-Holst, R., Von Elstermann, M., Augustin, M., and Hellweg, R. (2007) Clinical Relevance of Nerve Growth Factor Serum Levels in Patients with Atopic Dermatitis and Psoriasis. Int Arch Allergy Immunology 144, 211-216.
Toyoda, M., Nakamura, M., Makino, T., and Morohashi. (2003) Localization and content of nerve growth factor in peripheral blood eosinophils of atopic dermatitis patients. Clinical and Experimental Allergy 33, 950-955.
Yoshioka, T., Hikita, I., Asakawa, M., Hirasawa, T., Deguchi, M., Matsutani, T., Oku. H., Horikawa, T., and Arimura, A. (2006) Spontaneous scratching behavior in DS-Nh mice as a possible model for pruritus in atopic dermatitis.
Immunology 118, 293-301.
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References Deister, C. and Schmidt, C.E. (2006) Optimizing neurotrophic factor combinations for neurite
outgrowth. J. Neural Eng. 3, 172-179 Reichardt, L.F. (2006) Neurotrophin-regulated signaling pathways. Philosophical Transactions of the
Royal Society 361, 1545-1564. Thacker et al. (2007) Pathophysiology of Peripheral Neuropathic Pain: Immune Cells and Molecules. Anesthesia and analgesia 105, 839-847. Nockher, W.N., Renz, H., (2006) Neurotrophins in allergic diseases: From neuronal growth factors to
intercellular signaling molecules. Journal of Clinical Immunology 117, 583-588. Lodish, et al., Molecular Cell Biology, 5 edition Micera, A. et al. (2003) Nerve growth factor and tissue repair remodeling: trkA and p75NTR, two
receptors one fate. Cytokine & Growth Factor Reviews 18, 245–256. Janeway, et al., 7 edition 2008, Garland Publishing. Bischoff, S.C. (2007) Role of mast cells in allergic and non-allergic immune response: comparison of
human and murine data. Nature Reviews 7, 93-103. Vega, J.A., Garcia-Suarez, O., Hannestad, J., Perez-Perez, M., and Germana, A. (2003) Neurotrophins
and the Immune system. J. Anat. 203, 1-19. www.bio.davidson.edu/courses/Immunology/ www.jralphb.co.uk/allerg.htm www.als.lbl.gov/als/science/sci_archive/89receptor.html www.science-autism.org www.derm-student.com/Atopic_dermatitis.html
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Questions?