RESULTS: 1) IDENTIFICATION OF FACTORS THAT MODULATE RESPONSES TO ANTIGEN: As noted in the INTRODUCTION our past studies with adenosine, PGE2, Kit ligand (SCF) and TLR ligands, illustrate the remarkable ability of co-stimulants to shift the response of mast cells towards either degranulation and production of eicosanoids or towards production of cytokines and chemokines (reviewed in Refs. 1 and 2). In our most recent study with IL-33, which is produced mainly by endothelial and synovial cells and is elevated in asthmatic lung and arthritic joint fluids, we find that IL-33 markedly potentiated antigen-induced cytokine production in primary and tumor mast cell lines from humans and rodents. This phenomenon was observed with minimally effective concentrations of antigen. Under these conditions, the mast cell response was shifted almost exclusively to production of cytokines which were produced in amounts far exceeding those produced by optimal concentrations of either antigen or IL-33 alone. Synergy was apparent at the level of transforming growth factor-beta-activated kinase-1 (TAK1) and then transmitted downstream through JNK, p38 MAP kinase, AP-1, and NF-kappaB. Unlike antigen, IL-33 failed to stimulate Ca2+ mobilization and, in turn, degranulation and the Ca2+/calcineurin/NFAT pathway. However, stimulation of the NFAT pathway with thapsigargin in IL-33-stimulated cells markedly augmented cytokine production. Luciferase reporter assays revealed cooperative interactions between NFAT and AP-1 and among other transcription factors. The amplification of cytokine production by low concentrations of IL-33 and antigen can thus be attributed to synergy at the levels of TAK1 and gene transcription and may contribute to the severity of disease(manuscript in revision). 2) IL-33, A NOVEL REGULATOR OF OSTEOCLAST DEVELOPMENT AND FUNCTION. As a result of our studies of IL-33 and its receptor ST2, a member of the TLR superfamily of receptors, in mast cells, Dr Choi raised the question of whether IL-33 acted on other cells in arthritic lesions. One report had indicated that osteoclast differentiation was associated with mast cell activation and that IL-33 in elevated in synovial fluid from arthritic patients. Studies by Dr. Choi with the PI at NHLBI and his subsequent studies in Korea found that IL-33 mimics the actions of, but is distinguishable from, the well characterized osteoclast differentiation factor, receptor activator of nuclear factor B ligand (RANKL). IL-33 stimulates formation of multinuclear osteoclasts from peripheral blood CD14+ monocytes in a concentration-dependent manner. Studies with anti-ST-2 antibody indicated that IL-33 acts through ST-2, but not by antibody against RANKL. However, IL-33 and RANKL exhibit additive effects when added in combination. IL-33 induced bone resorption in vitro, an action blocked by anti-ST-2 antibody, but not by anti-RANKL antibody. As in mast cells, IL-33 stimulated phosphorylation of signaling molecules in CD14+ monocytes that are also thought to be involved in osteoclast differentiation. These included Syk, mitogen-activated protein kinases, TAK1, and NF-kB. IL-33 also elicited over a period of days substantial increases in expression of other key signaling molecules and bone resorption factors. This is the first indication of an osteoclastogenic role for IL-33 that may provide new opportunities for therapeutic intervention in degenerative bone diseases (Ref.3). 3) THE ACTION OF COACTIVATORS IN MAST CELLS EXTENDS TO CHEMOTAXIS. Our previous studies (with Alasdair Gilfillan) had shown remarkable synergy between PGE2 and antigen in promoting degranulation by a mechanism that involved trans-synergistic activation of phospholipase (PL)Cbeta (via the G protein linked EP3 receptor) and PLCgamma (via FceRI). Our most recent work, with Dr Gilfillan as lead investigator, shows that chemotaxis of mouse bone-marrow-derived mast cells (BMMCs) in response to the chemoattractants stem-cell factor (SCF) and prostaglandin (PG)E(2), is substantially enhanced by stimulation with antigen via FceRI. These responses were associated with enhanced activation of phosphoinositide 3-kinase (PI3K), and downstream activation of the tyrosine protein kinase Btk, with subsequent enhanced PLCgamma-mediated Ca2+ mobilization, Rac activation and F-actin rearrangement. Antigen-induced chemotaxis, and the ability of antigen to amplify responses mediated by SCF, adenosine and PGE2 were suppressed following inhibition of PI3K, and were impaired in BMMCs derived from Btk(-/-) mice. There were corresponding decreases in the PLCgamma-mediated Ca2+ signal, Rac activation and F-actin rearrangement which accounts for the impaired migration of Btk-deficient cells. By regulating signaling pathways that control F-actin rearrangement, Btk is crucial for the ability of antigen to amplify mast-cell chemotactic responses (Ref. 4). In summary, we have now demonstrated that mast cell coactivating factors can markedly modulate the pattern of inflammatory factors and mediators released as well as mast cell chemotaxis which may enable homing of mast cells to affected tisues.
