Mast cells & basophils I: Development & function

Feb. 23, 2004Advanced Immunology Course

Stephen J. Galli (sgalli@stanford.edu)

Janet Kalesnikoff(jkalesni@stanford.edu)

• The setting: Parasite immunity and allergic diseases

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

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

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

• Other activation mechansims; proposed roles in health & disease

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

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 & basophil development, heterogeneity &

function: Mast cells & basophils are 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.

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

Mast cell

Hematopoiesis:the formation ofblood cells

Pluripotent Stem Cell

LymphoidStem Cell

MyeloidStem Cell

Pluripotentstem cell

Lymphoidstem cell

Myeloidstem cell

NKprecursor

NK cell

SCFIL-2

flk-2/flt-3 ligandSCF

flk-2/flt-3 ligandSCF

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

Figure 1: A tentative scheme ofhematopoiesis and its growth factors. The pluripotent stem cell divides infrequently to generate either more stem cells (self-renewal;as indicated by ) or committed progenitor cells which can produce only one or a few types of bloodcells. Progenitor cells are stimulated to proliferate anddifferentiate by specific growth factors (indicated on the arrows) but progressively lose their capacity for division and develop into terminally differentiated blood cells, which live for only a few days or weeks. Only mature T cells, B cells, mast cells and macrophages are known to carry proliferative potential (as indicated by ). Dotted arrows represent uncertain pathways. Transcription factors involved in the differentiation of specfic pathwas are indicated in italics under the arrows. SCF = stem cell factor; IL = interleukin; GM-CSF = granulocyte monocyte colony stimulating factor; G-CSF = granulocyte colony stimulating factor; M-CSF = monocyte colony stimulating factor; TPO = thrombopoietin; EPO = erythropoietin. CFU = colony forming unit. BFU = burst forming unit. Adapted from Alberts et al.4

PL

E

MC

T

NK

M

GR

B

DC

CLP

MEP

LT-HSC

GMP

Pro-T

Pro-B

CMP

ST-HSCMPP

?

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.

FcRI-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)

LTC4

PGD2

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

IL-4, IL-5, IL-6, IL-8, IL-13, TNF, MIP-1, 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.

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

IgE-dependent enhancement of FcRI surface expression: Functional consequences

As surface expression of FcRI 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 FcRI *

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 g/ml) and rhIL-4 (10 ng/ml) for 4 days [to increase FcRI on cell surface].

3 Challenge mast cells with anti-human IgE (10 g/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. BMC Immunol. 3:5, 2002.

0 1h 2h 0 1h 2h

Donor 1 Donor 2

Clustering of > 2,400 genes in two human mast cell populations (> 98% pure, cord blood-derived) stimulated via FcRI

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 M ionomycin, 25 ng/ml PMA]

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.

Eotaxin Gro2IL-8MCP-1HCC-4 *MIP-1RANTESLD78*MIP-1MCP-3 LymphotactinMIP-3*MCP-4 *

IL-16 †IL-1IL-14 *MIFTNF- convertingIL-11 * enzyme *

IL-9IL-6IL-5IL-4CSF-1IL-18

IL-12 p40 †TNF-IL-3Pre-B cell enhancing factor *GM-CSFIL-1LIF †

Cytokines

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

0 1h 2h 0 1h 2h

Chemokines

Donor 1 Donor 2

‡‡

‡

‡

* = “new” for any mast cells

‡ = “new” for human mast cells

† = “new” information about FcRI-induced change in expression

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-*

ICAM-1 †

CD83 *

4-1BB ligand *

OX40 ligand *

0 1h 2h 0 1h 2h

Donor 1 Donor 2

Adhesion Molecules

P-selectin glycoprotein

CD103 *

ligand †

CD49F †ELAM-1 *

CD49B †

CD29 †

ICAM-1 †

Protocadherin 43 *

0 1h 2h 0 1h 2h

Donor 1 Donor 2

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.

surface bound Ig

FcRI BCR

Immunoreceptors/antigen receptors: multiple subunits

Crosslink with multivalent Ag

MHC I/IIpeptide

TCR

CD3

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

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

• IgE heavy chain: -chain

• Binds high affinity FcRI

IgE

FC

Fab

Antigen-binding sites

• Antigen receptor superfamily

• Tetrameric structure

• 3 Distinct protein species

- binds to Fc portion of IgE

- 4 transmembrane domains

- 1 transmembrane domain - disulfide linked homodimer

FcRI

signal transduction

CURRENT IgE/MAST CELL DOGMA

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

• 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

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

FcRI

IgE

FcRI

IgE

HSA

DNP

DNP DNP

LynFyn

Gab2 PI3KFyn

PLC

Arachidonic acidmetabolism (LTC4, PGD2)

DegranulationCytokine/chemokineproduction

DAG + IP3

[Ca2+]PKC

BtkSyk PDK1

Akt

IgE & Ag-induced mast cell activation

LAT LATGTP

Grb2Sos

Ras

Raf-1

MEK

ERK

PLA2

Slp-76Vav

Rac

JNK p38

Negative regulation of IgE & Ag-induced mast cell activation

IRS (inhibitory receptor superfamily)

IgE

FcRI

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 FcRI inhibits signalling

FcRIIB

IgG

Crosslink with Ag

PI-3,4-P2

SHIP

ShcSHIP

DokRasGAP

Ras

IRS (inhibitory receptor superfamily)

FcRIIB

# ITIMs Recruits Ligand

1 SHIP IgG+Ag

gp49B1 2 SHP-1 v3

PIR-B

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

4 SHP-1 SHP-2SHIP

?

SIRP 4 SHP-1SHP-2

CD47

MAFA SHIP1 ?

PI-4,5 -P2

FcRI

IgE

FcRI

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)

Syk

LAT LATGTP

Grb2Sos

Ras

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

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

2

4

6

8

10

12

14

16

0 60 120 180 240 300

5M3+/+5M5-/-

20 ng/mlDNP

Flu

o-4/

Sna

rf-1

0

10

20

30

40

50

60

70

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

-1ng/ml

DNP100ng/ml

DNP10ng/ml

DNP1g/mlDNP

A&P

% D

egra

nu

lati

on

+/++/--/-

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

Ras

Raf-1

MEK

ERK

GTP

PLCPI3KShc

Sos

Stimulus

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

PKC

PKB IP3Ca2+

RabGEF1

DAG

Btk

Cellsurvival

Mitogenesis

EarlyEndosome

Rab5

Endocytosis

?

Diverse potential mast cell activation mechanisms in host defense/pathology *

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

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

• IgE (FcRI, CD23, galectin-3), IgG1 (FcRIII, 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”

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 “neurogenic inflammation” &

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)