mast cells & basophils i: development & function feb. 23, 2004 advanced immunology course...
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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 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)