the mast cell: a cell for all seasons€¦ · allergen/ige c3a, c5a il-1, il-33 endothelin lps...
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4/6/2014 Copyright by Dr. TC Theoharides 1
Theoharis C. Theoharides, MS, PhD, MD, FAAAAI Professor of Pharmacology, Internal Medicine and
Biochemistry; Associate Professor of Psychiatry Director, Molecular Immunopharmacology
and Drug Discovery Tufts University School of Medicine-Tufts Medical Center
Boston, MA, USA drtheoharides@gmail.com www.mastcellmaster.com www.autismfreebrain.org
www.brain-gate.org
1 4/6/2014 Copyright by Dr. TC Theoharides
The Mast Cell: A Cell for All Seasons
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What Are Mast Cells and What is Mastocytotsis?
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Allergies Angioneurotic edema Atopy Atopic dermatitis Eczema Food allergy Food intolerance Idiopathic urticaria Idiopathic mast
activation disorder Mastocytosis Mast cell activation
syndrome Non-IgE food allergy
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Histamine and kinins are responsible for the cardinal signs of inflammation, the “Triple Response of Lewis”: Swelling Heat Pain (wheal owing to local edema, flare due to axon reflexes) as well as itching (pruritus)
Allergic hives (urticarial wheals)
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Typical Skin Mast Cell Degranulation Observed by Metachromasia as First Noted by Paul Ehrlich in 1887
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Mast Cell Activation Nomarski Optics
Allergen/IgEC3a, C5a
IL-1, IL-33Endothelin
LPSNeuropeptides
Thrombin
HistamineChymaseTryptase
LeukotrienesPAFProstaglandinsChemokines IL-1, IL-6, IL-8GM-CSF, TNF-αVEGF
Triggers Mediators
Activated mast cell
Normal mast cells
Mast cell degranulation leads to the release of mediators with potent
vasodilatory, nociceptive, and inflammatory properties 6 4/6/2014
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Typical Skin Mast Cell Degranulation
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Mast Cells Communicate with Many Pathogens and Other Immune Cells
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10 μm
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Mast Cell Degranulation Video
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Can mast cells secrete mediators selectively?
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Human mast cells have heterogeneous response to non-maximally activating
stimulus
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Single cell degranulation assay
• Utilizes fluorescently-tagged anti-CD63
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Multiple Measurements in Individual Human Mast Cells Using the Live Cell Array
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Degranulation ROS Calcium
Rel
ativ
e flu
ores
cenc
e
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IL-1 Stimulates Differential Release of IL-6 Without Degranulation
0
250
500
750
1000 spontstim *
A
IL-1 anti-IgE
Try
ptas
e (n
g/5x
105
MC
)
0
25
50
75
100
125**B
IL-1 anti-IgE
IL-6
(pg/
5x10
5M
C)
n=6, *p<0.05 paired t-test
8-16 wk hCBMCs + IL-1 6 hourscell free supernatants assay IL-6 (ELISA) or tryptase (fluoroimmunoenzyme assay).
Tryptase IL-6
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Ultrastructural Cryo-immunocytochemistry of IL-6 Released from a Mast Cell Stimulated with IL-1
140 nm
The secretory vesicle is 1/10th the diameter of that of secretory granules
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• Mast cells can be activated by bacterial or viral antigens, cytokines, growth factors, neuropeptides and stress hormones, leading to selective release of distinct mediators without degranulation. – Trigger Mediator Pathpophysiologic Implications CRH VEGF Disrupts BBB IL-1 IL-6 Th-17 cell maturation IL-33 IL-6, VEGF Disrupt BBB LPS TNF-a Th-17 cell maturation SCF TGF-beta Th-17 cell maturation TLR-9 IL-6 Th-17 cell maturation
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Mast Cell Function is Not Static
0
1
2
3
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5
6
7 * *
IL-33 (100 ng/ml) - + - + - +SP (0.1 µM) - - + + - -SP (0.2 µM) - - - - + +
VE
GF
mR
NA
exp
ress
ion
050
100150200250300350400450
*
IL-33 (100 ng/ml) - + - +
SP (1 µM) - - + +
VEG
F (p
g/10
6 cel
ls)
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Divergent Actions of Mast Cells
ANAPHYLACTIC DEGRANULATION DIFFERENTIAL RELEASE WITHOUT DEGRANULATION
Allergic and anaphylactic responses Inflammation
IgE + antigen Neuropeptides
Compound exocytosis Intragranular activation
CRH, SCF, IL-1, LPS
Do Mast Cells Secrete Autopathogens?
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Lynn Margulis (1938 –2011)
Margulis L. Symbiotic theory of the origin of eukaryotic organelles;criteria for proof Symp Soc Exp Biol. 1975;(29):21-38.
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J Allergy Clin Immunol 2011;127:1522-31
Control
SP (2 µM)
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Mitochondrial Fragmentation (fission) During Mast Cell Degranulation
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Mitochondria Undergo Fission and Move to the Cell Surface During MC Secretion
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Effects of Mitochondrial Components on Different Cell Types
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Do Mast Cells Have Any Useful Function?
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Wound Healing
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Are Mast Cells Associated with Other Diseases?
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Atherosclerosis
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Cancer
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Multiple Chemical
Sensitivity s.
Allergy & Asthma
Mast cell activation s.
Food allergy &
intolerance
Interstitial cystitis
Chronic fatigue s.
Fibromyalgia Autism
Mast Cell
Mast Cells-Related Immune Disorders
Edvard Munch, The Scream (Skrik, 1893) 4/6/2014 46
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Do Mast Cells Exist in the Brain and are Do They Get Stressed Out?
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Stress and Skin Mast Cells
Hypothalamus: CRH Pituitary: ACTH
SKIN
Blood vessel
HPA axis
Local effects
SCG
Sensory nerve
CRH, IL-1 NT, SP, viruses
LC
Limbic& Neurosensory
signals
Hypothesis Stress activates
mast cells leading to skin inflammation
PROINFLAMMATORY ↑ vascular permeability +
Mast cell
Triggering or exacerbating Dermatitis, Flushing
Histamine, IL-6, TNF, tryptase
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Stress-induced Brain Vascular Permeability is Dependent on Mast Cells
0
50
100
150
200
*
+/+ W/Wv
% c
hang
e fr
om c
ontr
ol
+/+ W/W v0
100
200
300
400
500
600 controlstress
*p<0.05
*p<0.05
cort
icos
tero
ne (n
g/m
l)
99-Technetium-gluceptate extravasation
HPA axis activation
Esposito P, TC. J Pharmacol Exp Ther. 2002 Dec;303(3):1061-6.
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Intradermal CRH Injection Induces Skin Vascular Permeability, but not in W/Wv Mast Cell Deficient Mice
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CRH, NT and SP Released from DRG Stimulate Skin Mast Cells and Induce Vascular Permeability
Donelan J et al. Proc Natl Acad Sci U S A. 2006 May 16;103(20):7759-64.
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Culture of human umbilical cord blood-derived mast cells (hCBMCs)
Culture with SCF (100 ng/ml) and IL-6 (50 ng/ml) for 8-16 wk yields 100% tryptase-positive mast cells
CD34+ cells
mast cells Kempuraj D., Saito H. et al. Blood 93: 3338-3346, 1999.
RBCs
MNCs
AC133 mAb
MACs
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Immunocytochemistry of CRH-R in human mast cells
F G
B
-
R1 no IL-4
D C A E
E F
B
R1 + IL-4
A B
R2 no IL-4 R2 + IL-4
D B
No R1 primary DAPI
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01020304050
control 1nM 10nM 100nM 1uM
CRH
VEG
F
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• A 38-year-old woman presented with a pronounced increase in symptoms and proliferation of urticaria pigmentosa (UP) after acute psychological stress, which was quantified using the Spielberger’s State–Trait Anxiety Inventory.
• Immunohistochemical examination of a skin biopsy from a new UP lesion showed
Mnay CRH-R1-postive cells. • There were many activated mast cells expressing corticotropin-releasing hormone
receptor-1 (CRH-R1). • There was high serum CRH.
• This is the first documented report to our knowledge of UP worsening associated
with acute stress, possibly through activation of skin mast-cell CRH-R1.
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Lesional UP Skin Showing Positive CRH-R1 Immunocytochemistry
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Do Allergic Symptoms Affect Behavior ?
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There was significant association with food intolerance (P = 0.001).
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Diencephalon (coordinates sensory input and emotions)
Mast Cell Activation
Affected Behavior
The Genesis of Brain Immunity Storms
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Is there Anything Linking Genes,
Environment and Mast cells?
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Mast Cell Triggers
PI3K
AKT
mTORC1
PTEN Mast cell number
mast cell activation
Mast cell number
Rapamycin
Luteolin
Calcium influx via receptor
Luteolin inhibits mTOR activation
P-mTOR (Ser 2448)
P-4EBP1 (Thr37/46)
mTOR
β-Actin
289
289
45
15-20
kDa 1 2 3
1-Control 2-Rapamycin 3-Luteolin
MAST CELL SECRETION
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Are there Any Effective Mast Cell Blockers?
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Are there Any Mast Cell Blockers ?
81
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Flavonoids are potent: 1. Anti-oxidant
2. Anti-inflammatory
3. Mast cell inhibitors
4. Metal chelators
5. Neuroprotective
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Allergic stimulation of human mast cells results in secretion of histamine, the proteolytic enzyme tryptase, and the pro-inflammatory cytokines IL-6, IL-8 and TNF-α, all of which are inhibited by quercetin.
01020
3040506070
8090
100
Histamine Tryptase IL-6 IL-8 TNF-alpha
Per
cen
t in
hib
itio
n
*
** *
*
Inhibition of inflammatory molecules by quercetin from human mast cells
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Mast Cell Inhibition Video
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Disclosure Dr. Theoharides is Medical Director
and holds a major interest in Algonot, LLC www.algonot.com
NeuroProtek® US Trademark
3,225,924
Portion of any profits will be donated to autism research. NeuroProtek®
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Effect of Luteolin and Methoxyluteolin on MC Secretion
Beta
-Hex
rele
ase
(%)
Control Mµ
SP 2 Mµ10 Mµ
50 Mµ100
0
10
20
30
40
50LutMethlut
**
*
TNF
(pg/
mL/
106 c
ells
)
Control Mµ
SP 2 Mµ
10 Mµ
50 Mµ
100
0
100
200
300
400LutMethlut
**
Hist
amin
e (p
g/m
L/10
6 cel
ls)
Control Mµ
SP 2 Mµ
10 Mµ
50 Mµ
100
0
500
1000
1500
2000LutMethlut
*
A B
C
PGD
2 (p
g/m
L/10
6 cel
ls)
Control
IgE only
IgE/Anti-I
gE Mµ10
Mµ50
Mµ10
0
0
1000
2000
3000
4000
5000LutMethlut
**
D
Flavonoids Trigger 30 min 30 min
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Intracellular Calcium Elevation in MC
Cell viability > 95%
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Research Funding: • DK62861 • AR47652 • NS071361 • NS55681 • AR60951 • NS66205 • Autism Research
Collaborative • National Autism
Association • Safe Minds • Autism Research
Institute • Jane B Johnson Fnd.
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drtheoharides@gmail.com www.algonot.com
www.mastcellmaster.com www.autismfreebrain.org
www.brain-gate.org
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