Mast cells & basophils I: Development & function

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Mast cells & basophils I: Development & function

Feb. 23, 2004Advanced Immunology Course

Stephen J. Galli([email protected])

Janet Kalesnikoff([email protected])

• The setting: Parasite immunity and allergic diseases

• Mast cell and basophil development (and their phenotypic heterogeity)

• Basic aspects of function: Regulation of FcεRI expression; secreted products

• Positive and negative regulation of activation via the FcεRI (Janet Kalesnikoff)

• Other activation mechansims; proposed roles in health & disease

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Asthma

Affects approximately 7% of population in U.S.

Features• Reversible airway narrowing• Immunologically non-specific

airway hyperresponsiveness (AHR)• Chronic inflammation of the airways• Airway remodeling

Asthma• PREVALENCE INCREASED 75% FROM 1980 TO 1994

• > 1.8 MILLION EMERGENCY ROOM VISITS IN 1995

• > 5,300 DEATHS FROM ASTHMA ANNUALLY

• DIRECT COSTS > $12.6 BILLION ANNUALLY

• INDIRECT COSTS > $2.8 BILLION ANNUALLY

• > 10 MILLION SCHOOL DAYS MISSED ANNUALLY

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C.M.M. Williams & S.J. Galli. J. Allergy. Clin. Immunol. 105:847, 2000.

Potential roles of mast cell products in atopic asthma

Mast cell & basophildevelopment, heterogeneity &

function: Mast cells & basophilsare not the same

T. Kawakami & S.J. Galli. Nature Rev Immunol. 2: 773, 2002. Reprinted with permission from A. Dvorak et al. Hum. Pathol. 11:606, 1980.

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Highly simplified diagram of mast cell & basophildevelopment (in the mouse)

Mast cell

Hematopoiesis:the formation ofblood cells

Pluripotent Stem Cell

LymphoidStem Cell

MyeloidStem Cell

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Pluripotentstem cell

Lymphoidstem cell

Myeloidstem cell

NKprecursor

NK cell

SCFIL-2

flk-2/flt-3 ligandSCF



Pre-T cell

Pre-B cell

IL-7flk-2/flt-3 ligand

SCF

IL-7flk-2/flt-3 ligand

SCF

IL-2IL-7

T cellIL-8

B cell

SCFIL-3

SCFIL-3

Mast cellCFU-mast

Basophil

Eosinophil

CFU-Bas

IL-3

SCFIL-3SCF

IL-3

SCFIL-3

CFU-Eos

IL-3GM-CSF

Neutrophil

Monocyte

CFU-GM

SCFIL-3

flk-2/flt-3 ligand

IL-3GM-CSFG-CSF

IL-3GM-CSFM-CSF

GM-CSFM-CSF

Osteoclast

Macrophage

Megakaryocyte

PlateletsCFU-Meg

SCFIL-3

GM-CSFIL-11IL-6TPO

TPOSCFIL-3

SCFIL-3

BFU-E CFU-E Erythrocyte

SCFIL-3

GM-CSFEPO

IL-3GM-CSF

EPO

IkarosPU.1

GATA-3

PU.1C/EBP

PU.1Pax5E2A

PU.1 PU.1

PU.1C/EBP

GATA-1C/EBPEts-1

GATA-1/2

GATA-1 GATA-1NF-E2EKLFFOG

GATA-1FOG

NF-E2

GATA-1/2Elf-1

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PL

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MC

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NK

Mφ

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DC

CLP

MEP

LT-HSC

GMP

Pro-T

Pro-B

CMP

ST-HSCMPP

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Ching-Cheng Chen (C3), Devavani Chatterjea (DC), et al.Murine mast cell/basophil development

Define lineage relationships of mast cells (C3)/basophils (DC) to other hematopoietic cells.

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FcεεεεRI-mediated activation pathways in mast cells

T. Kawakami & S.J. Galli. Nature Rev Immunol. 2: 773, 2002.

Activated mast cells/basophils release 3 major classesof “pro-inflammatory” mediators*

2) Newly synthesized AA metabolites (e.g. LTC4 & PGD2)

LTC4PGD2

3) Diverse cytokines; chemokines (e.g. TNFα, IL-4, IL-6; MIP-1α, IL-8)

IL-4, IL-5, IL-6, IL-8, IL-13, TNFαααα, MIP-1αααα, etc.

1) Preformed, granule-associated proteases, proteoglycans (e.g., heparin)and bioactive amines (e.g. histamine); released by DEGRANULATION

* Note: Underlined products are secreted by mast cells & basophils; basophils secrete a much more restricted set of cytokines than do mast cells.

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Reviewed in: C.M.M. Williams & S.J. Galli. J. Allergy. Clin. Immunol. 105:847, 2000.

IgE-dependent enhancement of FcεεεεRI surface expression: Functional consequences

As surface expression of FcεεεεRI increases, mast cells:• Can be “sensitized” to more antigens (Ags)• Can release mediators at lower Ag concentrations• Can release larger amounts of products in response to Ag• May release additional products (e.g., IL-4 in mouse mast

cells) in response to Ag challenge

cDNA microarray analysis of human mast cells stimulated via

aggregation of FcεRI *

1 Generate human umbilical cord blood-derived mast cells of >98-99% purity in SCF and IL-6.

2 Sensitize mast cells with human myeloma IgE (5 µg/ml) and rhIL-4 (10 ng/ml) for 4 days [to increase FcεRI on cell surface].

3 Challenge mast cells with anti-human IgE(10 µg/ml).

4 Assess % histamine release at 1 h; recover mRNA for microarray studies at baseline (0 hr), 1 & 2 h; measure released cytokines at various time intervals.

* K. Sayama, M. Diehn, K. Matsuda, C. Lunderius, M. Tsai, S-Y. Tam, D. Botstein, P.O. Brown & S.J. Galli. BMCImmunol. 3:5, 2002.

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0 1h 2h 0 1h 2hDonor 1 Donor 2

Clustering of > 2,400 genes in two human mast cell populations (> 98% pure, cord

blood-derived) stimulated via FcεεεεRI

K. Sayama, M. Diehn, K. Matsuda, C. Lunderius, M. Tsai, S.-Y. Tam, D. Botstein, P.O. Brown & S.J. Galli. BMC Immunol. 3:5, 2002.

Histamine Release (%) at 1 h

Vehicle Anti-IgEDonor 1 2 ± 0.1 57 ± 4

Donor 2 3 ± 0.2 63 ± 2

• 2,478 genes of ~ 14,000 tested exhibited 2-200 fold changes in expression vs. baseline (time 0)

• ~ 50% increase (red)

• ~ 50% decrease(green)

• ~ 50% of these 2,478 genes represented ESTs of genes of unknown function

“Reference” mRNA from: T cells (Jurkat), B cells (Raji), mast cells (HMC-1)

[80% “resting”: 20% 2h with 1 µM ionomycin, 25 ng/ml PMA]


Eotaxin Gro2IL-8MCP-1HCC-4 *MIP-1ββββRANTESLD78ββββ *MIP-1ααααMCP-3 LymphotactinMIP-3αααα *MCP-4 *

IL-16 †IL-1ββββIL-14 *MIFTNF-αααα convertingIL-11 * enzyme *IL-9IL-6IL-5IL-4CSF-1IL-18IL-12 p40 †TNF-ααααIL-3Pre-B cell enhancing factor *GM-CSFIL-1ααααLIF †

CytokinesDonor 1 Donor 2

0 1h 2h 0 1h 2h

0 1h 2h 0 1h 2h

ChemokinesDonor 1 Donor 2

‡‡

‡

‡

* = “new” for any mast cells‡ = “new” for human mast cells† = “new” information about FcεεεεRI-induced change in expression

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Gene products that regulate cell-cell interactions between mast cells & other cell types

T Cell, B Cell and Dendritic CellInteraction Molecules

CD40 ligand †CD82 †SLAM *Lymphotoxin-β β β β *ICAM-1 †CD83 *4-1BB ligand *OX40 ligand *

0 1h 2h 0 1h 2h

Donor 1 Donor 2

Adhesion Molecules

P-selectin glycoproteinCD103αααα * ligand †CD49F †ELAM-1 *CD49B †CD29 †ICAM-1 †

Protocadherin 43 *

0 1h 2h 0 1h 2hDonor 1 Donor 2


αααα

ββββ γγγγ γγγγ

surface bound Ig

αααα ββββ αααα ββββ

FcεεεεRI BCR

Immunoreceptors/antigen receptors: multiple subunits

Crosslink with multivalent Ag

MHC I/IIpeptide

αααα ββββ

TCR

CD3

ζζζζ ζζζζεεεε δδδδ γγγγ εεεε

CD4/8

Fyn LynFyn

LckLyn Blk & Fyn

• Associated with Src family tyrosine kinases →→→→ phosphorylate ITAM motifs (green) →→→→ serve as docking sites for adaptors or other NRTKs

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• 5 Classes of immunoglobulins or antibodies - IgG, IgM, IgA, IgD and IgE

• IgE heavy chain: εεεε-chain

• Binds high affinity FcεεεεRI

IgE

FC

Fab

Antigen-binding sites

αααα

ββββ

γγγγ γγγγ

• Antigen receptor superfamily

• Tetrameric structure

• 3 Distinct protein species

αααα - binds to Fc portion of IgE

ββββ - 4 transmembrane domains

γγγγ - 1 transmembrane domain- disulfide linked homodimer

FcεεεεRI

signal transduction

CURRENT IgE/MAST CELL DOGMA

• IgE binds to mast cells with high affinity (via FcεεεεRI)

• IgE binding is a “passive sensitization” step

• Activation occurs only when bound IgE is cross-linked, e.g., by binding to a multivalent Ag

IgE αααα

ββββ

γγγγγγγγ

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PI-3,4,5-P3PI-4,5 -P2

FcεεεεRI

IgE

FcεεεεRI

IgE

HSA

DNP

DNP DNP

Lyn FynGab2 PI3KFynPLC

Arachidonic acidmetabolism (LTC4, PGD2)

DegranulationCytokine/chemokineproduction

DAG + IP3

[Ca2+]PKC

BtkSyk PDK1

Akt

IgE & Ag-induced mast cell activation

LAT LATGTP

Grb2SosRas

Raf-1

MEK

ERK

PLA2

Slp-76Vav

Rac

JNK p38

Negative regulation of IgE & Ag-induced mast cell activation

IRS (inhibitory receptor superfamily)

IgE

FcεεεεRI

IgE

HSA

DNP

DNP DNP

PI-4,5 -P2

LynPI3K

DAG + IP3

[Ca2+]PKC

Btk PDK1

Akt

PI-3,4,5-P3

FynGab2FynPLC

Syk

LAT

• Contain 1+ ITIM motif (red)• Coaggregation with FcεεεεRI inhibits signalling

FcγγγγRIIB

IgGCrosslink with Ag

PI-3,4-P2

SHIP

ShcSHIP

DokRasGAP

Ras

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IRS (inhibitory receptor superfamily)

FcγγγγRIIB

# ITIMs Recruits Ligand

1 SHIP IgG+Ag

gp49B1 2 SHP-1 αvβ3

PIR-B

H. R. Katz (2002). Curr Opin Immunol 14:698-704.

4 SHP-1 SHP-2SHIP

?

SIRPαααα 4 SHP-1SHP-2

CD47

MAFA SHIP1 ?

PI-4,5 -P2

FcεεεεRI

IgE

FcεεεεRI

IgE

HSA

DNP

DNP DNP

Lyn

DegranulationCytokine/chemokineproduction

PI3K

DAG + IP3

[Ca2+]PKC

Btk

PDK1

Akt

PI-3,4,5-P3

FynGab2FynPLC

Arachidonic acidmetabolism (LTC4, PGD2)

SykLAT LAT

GTP

Grb2SosRas

Raf-1

MEK

ERK

PLA2

Slp-76Vav

Rac

JNK p38

PI-3,4-P2

SHIPSHIP2

RasGAPsRin1

RabGEF1?Lyn

Negative regulation of IgE & Ag-induced mast cell activation

Intracellular/cytoplasmic inhibitors

SHP1

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RabGEF1 knockout mouse

RabGEF1 is a negative regulator of mast cell activation and skin inflammation

See-Ying Tam, Mindy Tsai, John N. Snouwaert, Didier Scherrer, Devavani Chatterjea, Donna M. Bouley & Stephen J. Galli. Nature Immunology (submitted).

IgE & Ag-induced Ca2+ flux is elevatedin RabGEF1-/- BMCMCs

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IgE & Ag-induced release of pre-formed mediatorsis elevated in RabGEF1-/- BMCMCs

- 1ng/mlDNP

100ng/mlDNP

10ng/mlDNP

1µµµµg/mlDNP A&P

% D

egra

nula

tion

+/++/--/-

IgE & Ag-induced LTC4, PGD2 and cytokine production are elevated in RabGEF1-/- BMCMCs

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Ras

Raf-1

MEK

ERK

GTP

PLCγγγγPI3KShc

Sos

Stimulus

PI-4,5-P2PI-3,4,5-P3

PKC

PKB IP3 Ca2+

RabGEF1

DAG

Btk

Cellsurvival

Mitogenesis

EarlyEndosome

Rab5

Endocytosis

?

Diverse potential mast cell activationmechanisms in host defense/pathology *

• Products of pathogens (LPS/CD14-TLR4, E. coliFimH/CD48, C. difficile toxin, H. pylori, etc., etc.)

• Products of complement activation (C3a/C3aR, C5a/C5aR, ? C3bR/CD35, etc.)

• IgE (FcεRI, CD23, galectin-3), IgG1 (FcγRIII, mouse),LC via antigens or superAgs (e.g., S. aureus Protein A, P. magnus Protein L, HIV gp120, protein Fv in HBV & HCV)

• T cell-derived products (?)• Other: neuropeptides (e.g., VIP, Neurotensin, Substance

P), ET-1, SCF & other cytokines, leukocyte products,defensins/LL-37, insect/reptile venom components, etc., etc.

* Responsiveness (and responses) of different mast cell (basophil) populations can vary: “Mast cell (basophil) heterogeneity”

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Proposed mast cell (basophil ?) roles (a very incomplete list-I)

• Effector (& immunoregulatory) cell in: IgE- &/or (in mice) IgG1-associated immune responses, e.g., anaphylaxis, “asthma” & parasite immunity; some mouse models of “autoimmunity” (e.g., MS, RA)

• Regulate “immunologically non-specific” acute and chronic inflammation & “natural immunity” (e.g., IBD)• Regulate wound healing, angiogenesis & tissue-remodeling• Regulate T cell-dependent, “Ig-independent” responses (e.g., CS)

Proposed mast cell (basophil?) roles (a very incomplete list-II)

• Promote &/or retard tumor development, progression or metastasis

• Bi-directional interactions with peripheral nerves & promotion of “neurogenicinflammation” & neurite growth

• Promote protective responses to diverse endogenous or exogenous noxious (non-microbial) agents

• Regulate epithelial development, proliferation & function (e.g., “barrier function”, hair growth)

Mast cells & basophils I: Development & function

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