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). 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. The following observations were made since the last report: i. Under acute conditions, SCF enhances mast cell activation while chronic exposure to SCF appears to protect against fatal antigen-mediated anaphylaxis. We investigated this dichotomy by examining the manifestations of extended exposure to SCF as occurs in vivo. Mouse bone marrow-derived mast cells exposed to SCF for more than 72 hours displayed a marked attenuation of FceRI-mediated degranulation and cytokine production. The hypo-responsive phenotype was associated with a marked down-regulation of expression of the SRC kinase Hck and defective cytoskeletal reorganization. As such, these findings have important ramifications for the understanding of the homeostatic control of mast cell activation. ii.Similarly, 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 as 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 possibly a consequence of down-regulation of the expression of PLCg1 and Hck. iii. Migration of mast cells to sites of inflammation is regulated by chemotactic factors such as stem cell factor (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, then examined chemotaxis and mediator release in human mast cells induced by antigen or SCF. We found that disruption minimally affected early signaling pathways, but attenuated SCF-induced human mast cell chemotaxis. Unexpectedly, however, in the absence of other stimuli, SCF induced substantial degranulation in a concentration-dependent manner following actin disassembly. We interpret this data as consistent with the conclusion that processes regulating cell migration limit mast cell degranulation as a consequence of cytoskeletal reorganization. iv. Linkage analyses have implicated the MS4A2-containing gene locus (encoding for FceRI) as a candidate for allergy susceptibility. We have identified a truncation of FceRI (t-FceRI) in humans which contains a putative calmodulin binding domain and thus, we sought to identify the role of this variant in mast cell function. We determined that t-FceRI forms a complex with Fyn kinase, Gab2, p85 PI3K and -tubulin. Calmodulin bound to t-FceRI in the presence of Ca2+ initiating phosphorylation, which was critical for t-FceRI function. Confocal microscopy demonstrated localization of the t-FceRI complex to the Golgi surrounding the centrosome after IgE-dependent and IgE-independent activation. Knockdown of t-FceRI attenuated microtubule formation, degranulation and IL-8 production downstream of Ca2+ signals. These observations are consistent with the conclusion that t-FceRI mediates Ca2+-dependent microtubule formation, which promotes degranulation and cytokine release. v. We have derived a novel mouse MC line which 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 (KIT ligand). This cell line thus presents a novel system to delineate how mast cell function is modulated by native and mutated KIT and for the identification of inhibitors of these processes.

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Cruse, Glenn; Metcalfe, Dean D; Olivera, Ana (2014) Functional deregulation of KIT: link to mast cell proliferative diseases and other neoplasms. Immunol Allergy Clin North Am 34:219-37
Smrz, Daniel; Bandara, Geethani; Beaven, Michael A et al. (2013) Prevention of F-actin assembly switches the response to SCF from chemotaxis to degranulation in human mast cells. Eur J Immunol 43:1873-82
Smrz, Daniel; Bandara, Geethani; Zhang, Shuling et al. (2013) A novel KIT-deficient mouse mast cell model for the examination of human KIT-mediated activation responses. J Immunol Methods 390:52-62
Cruse, Glenn; Beaven, Michael A; Ashmole, Ian et al. (2013) A truncated splice-variant of the FcýýRI* receptor subunit is critical for microtubule formation and degranulation in mast cells. Immunity 38:906-17
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Gilfillan, Alasdair M; Peavy, Richard D; Metcalfe, Dean D (2009) Amplification mechanisms for the enhancement of antigen-mediated mast cell activation. Immunol Res 43:15-24
Bansal, Geetanjali; DiVietro, Jeffrey A; Kuehn, Hye Sun et al. (2008) RGS13 controls g protein-coupled receptor-evoked responses of human mast cells. J Immunol 181:7882-90
Kim, Mi-Sun; Radinger, Madeleine; Gilfillan, Alasdair M (2008) The multiple roles of phosphoinositide 3-kinase in mast cell biology. Trends Immunol 29:493-501

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