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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15

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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

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 210

2

4

6

8

10

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14

16

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 210.5

0.7

0.9

1.1

1.3

1.5

1.7

1.9

2.1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

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

4

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