Mast cells play a pivotal role in the pathogenesis of allergic inflammation. These reactions are generally initiated by antigen-dependent aggregation of the high affinity IgE receptor (Fc-epsilon-RI) expressed on the cell surface and subsequent release of pro-inflammatory mediators. Ligands for other receptors such as KIT and various GPCRs may serve to prime mast cells for, or act as co-activators of, antigen-mediated mast cell activation. The signaling pathways linking Fc-epsilon-RI aggregation to human mast cell activation and function and how other receptors modify these Fc-mediated signaling events are not well understood. Thus the primary focus of the research is the elucidation of signaling mechanisms associated with the activation of mast cells via the Fc-epsilon-RI and especially how the signaling pathways initiated by other receptors may integrate with those initiated by the Fc-epsilon-RI for synergistic mast cell activation and/or inhibition. The ability of mast cells to impact disease states in vivo also depends on their growth and differentiation from their progenitor cells, migration of the mast cells to their resident tissues, and their survival at these sites. Therefore, the integrated receptor-mediated signaling events regulating these processes are also being examined. Recent key observations have been made relating to the effect of IL-33 on human mast cell function, cytoskeletal rearrangement, and the MS4A2-containing gene locus. These studies in part also resulted in identification of a novel mouse mast cell line which now has been reported and allows the study of normal and mutated KIT constructs. These cells originated from a bone marrow-derived mouse mast cell culture as a rapidly dividing mast cell sub-population. Over time, these cells lost KIT expression while continuing to express functional high affinity receptors for IgE. Retroviral transduction of the cells with a human KIT construct resulted in surface expression of human KIT which responded to human stem cell factor (SCF;KIT ligand). In examining IL-33 on mast cell function, we discovered that long-term exposure of human and mouse mast cells to IL-33 results in a substantial reduction of mast cell activation in response to antigen. This appears to be a consequence of MyD88-dependent attenuation of signaling processes necessary for mast cell activation including antigen-mediated calcium mobilization and cytoskeletal reorganization. These changes were related to down-regulation of the expression of PLCg1 and Hck. Linkage analyses have implicated the MS4A2-containing gene locus (encoding for Fc-epsilon-RI) as a candidate for allergy susceptibility. We have identified a truncation of Fc-epsilon-RI (t-Fc-epsilon-RI) in humans which contains a putative calmodulin binding domain. We thus sought to identify the role of this variant in mast cell function. We determined that t-Fc-epsilon-RI forms a complex with Fyn kinase, Gab2, p85 PI3K and -tubulin. Calmodulin bound to t-Fc-epsilon-RI in the presence of Ca2+ initiating phosphorylation, which was critical for t-Fc-epsilon-RI function. Confocal microscopy demonstrated localization of the t-Fc-epsilon-RI complex to the Golgi surrounding the centrosome after IgE-dependent and IgE-independent activation. Knockdown of t-Fc-epsilon-RI attenuated microtubule formation, degranulation and IL-8 production downstream of Ca2+ signals. These observations are consistent with the conclusion that t-Fc-epsilon-RI mediates Ca2+-dependent microtubule formation, which promotes degranulation and cytokine release. Migration of mast cells to sites of inflammation is known to be regulated by chemotactic factors such as SCF. Despite inducing similar early signaling events to antigen, chemotactic factors (including SCF) produce minimal degranulation in the absence of other stimuli. We therefore investigated whether processes regulating mast cell chemotaxis are rate limiting for mast cell mediator release. In these experiments, we disrupted actin polymerization, a requirement for mast cell chemotaxis. We then examined chemotaxis and mediator release in human mast cells induced by antigen or SCF. We found and reported that disruption of actin polymerization minimally affected early signaling pathways, but attenuated SCF-induced human mast cell chemotaxis. Unexpectedly, in the absence of other stimuli, SCF induced substantial degranulation in a concentration-dependent manner following actin disassembly. We interpreted this data as consistent with the conclusion that processes regulating cell migration limit mast cell degranulation as a consequence of cytoskeletal reorganization.
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