Mast cells play a pivotal role in the pathogenesis of asthma and other allergic diseases. 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 (e.g. histamine, prostanoids, proteases and cytokines). However, 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 is unclear. 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. The following observations were made since the last report: 1. Prostaglandin E2- and SCF-mediated mast cell chemotaxis is synergistically enhanced by antigen: Critical role of the PI3K-Btk axis in the necessary Rac-dependent cystoskeletal reorganization. We, and others, have shown that that PGE2, which is generated in inflamed tissues, and the KIT ligand SCF, which is expressed by fibroblasts, markedly enhance mast cell mediator release. As mast cells home to sites of inflammation, we examined whether the process of mast cell chemotaxis could be similarly enhanced. Both PGE2 and SCF alone induced robust mast cell chemotaxis. However, antigen alone only produced a small, but significant, response. Nevertheless antigen, in combination with either PGE2 or SCF, produced a dramatic enhancement of the chemotaxis response. Such synergy was determined to be due to PI3K/Btk-mediated, Rac-dependent synergistic cytoskeletal reorganization. These observations may have particular relevance to understanding how mast cells, and indeed other inflammatory cells, migrate to sites of inflammation. ii. mTORC2 is a central locus for prostaglandin (PG)E2-induced mast cell chemotaxis and mediator release. In our studies described above, we determined that the mechanism by which PGE2 alone induced mast cell chemotaxis differed from that required by SCF. We hence investigated the signaling pathways regulating this response. These studies reveled that PGE2 activated mTOR-regulated signaling cascades in mast cells and that, not only did the mTORC2 complex (mTOR complexed to rictor and other regulatory molecules) contribute to PGE2 mediated chemotaxis, but also to the generation of reactive oxygen species leading to cytokine generation. These obsevations provided further support for our conclusions that mTOR complexes, by regulating multiple processes in mast cells, represents a central signaling locus for mast cell function as regulated by multiple receptors. iii. TLR-mediated signaling pathways circumvent the requirement for DAP12 in mast cells for the induction of inflammatory mediator release. Toll-like receptor (TLRs) function in multiple cell types, including mast cells, to induce cytokine generation. In various cell types, such processes are modulated by DAP12 a membrane associate molecule that, once phosphorylated, recruits the critical tyrosine kinase Syk. As Syk is a critical signaling molecule in mast cell degranulation and cytokine generation, we hypothesized that DAP12 may play a role in TLR-mediated mast cell activation via Syk. We however observed that, whereas DAP12 mediated responses by other receptors expressed on mast cells, TLRs circumvented the requirement for DAP12, likely as a conesquence of utilization of the adaptor molecule MyD88. These studies thus demonstrated that TLRs have differential requirement for DAP12 depending on the cell types in which they are expressed.

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Kim, Do-Kyun; Beaven, Michael A; Metcalfe, Dean D et al. (2018) Interaction of DJ-1 with Lyn is essential for IgE-mediated stimulation of human mast cells. J Allergy Clin Immunol 142:195-206.e8
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