CURRENT OBJECTIVES: 1) To identify endogenous factors that modulate mast cell reactivity to antigen: this year we have focused on IL-33. IL-33 is a novel member of the IL-1 super family that acts through an orphan receptor, ST2, to activate TLR-mediated signaling pathways. Of particular interest, IL-33 accumulates in afflicted tissues in patients allergic and autoimmune diseases. The question addressed is whether IL-33, like TLR ligands, acts in synergy with antigen and if so by what mechanism. 2) A novel finding by one of my collaborators (Wahn Soo Choi) is that IL-33 is also a potent stimulant of osteoclast formation (from CD14+ monocytes)and function (bone resorption). He is now examining, while on Sabbatical here, the signaling pathways that regulate both osteoclastogenesis and osteoclast function in the light of our studies on mast cells. The broader question is;what is the link between the mast cell and osteoclast in rheumatoid arthritis (as described by others) and the role of IL-33 in this linkage. 3) To continue studies of the mechanisms by which glucocorticoids suppress activation of mast cells. In the current period we have examined the mechanism of upregulation of glucocorticoid-inducible inhibitory regulators at a transcriptional level. 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 Beaven, Eur. J. Immunol. 39:11, 2009). We found that IL-33 was the most potent of all agents examined to date in its ability to modulate responses to antigen. At surprisingly low concentrations (10 pg/ml), 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. With respect to signaling mechanisms, 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 augmented production of the cytokine, tumor necrosis factor-alpha, to the same extent as antigen. Luciferase reporter assays indicated that IL-33 enhanced activation of NFAT and AP-1 by antigen to reveal additional co-operative interactions among 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 submitted). 2) IL-33, A NOVEL REGULATOR OF OSTEOCLAST DEVELOPMENT AND FUNCTION. MC and OC relationship as a prelude for investigation of RA. The intial studies by Dr. Choi revealed 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. This action was suppressed by anti-ST-2 antibody, indicating 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 actioon was blocked by anti-ST-2 antibody, but not by anti-RANKL antibody. We find that, 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. These included TNF receptor-associated factor 6 (TRAF6), nuclear factor of activated T cells cytoplasmic 1 (NFATc1), c-Src, and cathepsin K. In conclusion, this is the first indication of an osteoclastogenic role for IL-33 that may provide new opportunities for therapeutic intervention in degenerative bone diseases. 3) UPREGULATION OF INHIBITORY REGULATORS BY GLUCOCORTOIDS, A NOVEL MECHANISM OF ACTION OF GLUCORTICOIDS: Last year we reported that interaction of GR and GRE was responsible for some inhibitory actions of dexamethasone in mast cells as indicated from experiments with a mutated GR that cannot interact with GREs but which retains the ability to suppress of cytokine production. This finding is consistent with the notion of the dual action of glucocorticoids. That is the suppression of cytokine production is due to """"""""transrepression"""""""" of cytokine gene transcription which would not involve GRE whereas the upregulation of inhibitory regulators might involve """"""""transactivation"""""""" via GRE. The glucocorticiod-inducible inhibitory regulators described by us so far include Src-like adaptor protein1 (SLAP), downstream of tyrosiine kinase1 (Dok1), and dual specificity protein phospahatase1 (DUSP1) (see previous reports in this series). We find that, the upregulation of SLAP and Dok1, but not DUSP1, was inhibited by actinomycin D and was thus dependent on gene transcription. Examination of the gene sequences revealed a glucocorticoid response element (GRE) and a half GRE as potential regulators of the SLAP and Dok1, respectively. As indicated by luciferase reporter assays, SLAP GRE, but not the Dok1 half GRE, robustly activated gene transcription after treatment of cells with glucocorticoids. Binding of the glucocorticoid receptor to the SLAP GRE was verified by chromatin immunoprecipitation assay. These findings further support our hypothesis that the immunosuppressive actions of glucocorticoids are exerted in part through upregulation of inhibitory regulators by various mechanisms including activation of gene transcription through the interaction of GR with GRE (Bibliography, Ref. 2). These and our previous findings are relevant to the recent development of """"""""dissociated"""""""" steroids that lack the """"""""transactivation"""""""" potential of glucococorticoids in current use, a potential that was previously thought to account for the undesirable side-effects of glucocortoids. However, glucocorticoids lacking this """"""""transactivation"""""""" potential may also fail to induce inhibitory regulators such as those described in our studies. An unintended finding was that antigen itself also leads to increased levels of SLAP transcripts and protein over a period of several hours. The effects of pharmacologic inhibitors indicate that this upregulation of SLAP is dependent on multiple signaling pathways but independent of GRE. Knockdown of SLAP with anti-SLAP siRNA is associated with enhanced phosphorylation of Syk, the linker for activation of T cells (LAT), phospholipase C, MAP kinases, and various transcription factors. Production of IL-3 and MCP-1, but not degranulation, is also enhanced. The upregulation of SLAP may thus serve to limit the duration of cytokine production in Ag-stimulated cells (Bibliography, Ref. 3).
