Mast cells are critical effector and regulatory cells involved in the pathogenesis of allergic inflammation. Allergic responses are generally initiated in the presence of an antigen by activation of the high affinity IgE receptor (Fc-epsilon-RI) expressed on the cell surface of mast cells. Binding of the antigen to IgE initiates intracellular signaling events culminating in the release of vasoactive and inflammatory mediators that cause immediate and delayed allergic reactions. Despite their importance, the signaling pathways linking Fc-epsilon-RI aggregation to human mast cell activation are incompletely understood. In addition, activation of other receptors on mast cells, such as KIT (the receptor for stem cell factor, SCF), cytokines and growth factor receptors, may modify antigen-induced signals altering not only the threshold for and extent of mast cell activation but also, qualitatively, the combination of pro-inflammatory versus anti-inflammatory compounds that mast cells can secrete. This may impact on the severity and type of allergic response. A deeper understanding of the intracellular signals leading to mast cell activation as well as their modulation by signals from other receptors can help us understand human allergic disease and give clues on treatment options. An example of how cytokine receptors may alter IgE-induced responses is the inflammatory cytokine IL-6. We found that IL-6, which is elevated in association with mastocytosis, asthma and urticaria, causes mast cells to become hyper-responsive to an antigen stimulus. IL-6 was also necessary for optimal expansion of human mast cells from progenitor cells by promoting mast cell proliferation and maturation, which may markedly impact the mast cell burden associated with sites of inflammation. The sustained effect of IL-6 on mast cell responses was mediated by silencing SOCS3 expression, which negatively regulates STAT transcription factors, thus allowing for increased activation of the STAT3 pathway. Silencing of SOCS3 expression was caused by IL-6-mediated hypermetylation of the SOCS3 promoter. The findings indicate that prolonged exposure to IL-6 promotes development of a more mature and more reactive mast cell phenotype and suggest that IL-6 blockade may have a positive effect on amelioration of mast cell related symptoms and pathology in mast cell-related diseases associated with elevated IL-6 levels including mastocytosis. Another example is the cytokine IL-33. IL-33 plays a central role in type 2 immune responses and allergic inflammation. Long-term exposure of human and mouse mast cells to IL-33 results in a substantial reduction of mast cell responses by attenuating signaling processes necessary for mast cell activation. In contrast to chronic exposure, acute stimulation of mast cells with IL-33 alone or in combination with SCF or antigen causes a dramatic increase in the generation of cytokines. Under these conditions, mast cells also produce and release, with slower kinetics, abundant amounts of a soluble form of ST2 receptor (sST2) that functions to neutralize IL-33 activity. The late release of sST2 suggests that mast cell- derived sST2 may act to control inflammation and thus joins other mast cell derived products that may function to limit the spread of inflammatory. These studies give clues into how the acute or chronic presence of inflammatory signals may affect the way mast cells respond, helping to promote or dampen allergic inflammation. The beta-subunit of Fc-epsilon-RI (MS4A2) contributes to IgE-dependent mast cell signaling by trafficking the Fc-epsilon-RI receptor complex to the cell surface and amplifying FcRI-induced signaling. Members of the MS4A family cluster within chromosome 11q12-q13 and are linked to asthma, inflammatory diseases and cancer. Thus we were interested in investigating potential functions of this gene family on mast cell responses. We previously discovered that a truncated variant of MS4A2 named t- Fc-epsilon-RI-beta, is critical for microtubule formation, degranulation and cytokine release in mast cells, making it a therapeutic target for the downregulation of allergic inflammation. In addition, we found that silencing of MS4A4, another member of the MS4A family, altered ligand-induced KIT endocytosis pathways and reduced receptor recycling to the cell surface, thus promoting KIT signaling in the endosomes (AKT phosphorylation) while reducing it in the plasma membrane (PLCgamma1 phosphorylation). As a result of the increase in AKT signaling, SCF-induced mast cell proliferation and migration were enhanced in mast cells. These findings indicate that MS4A4 regulates mast cell function by promoting surface expression of KIT and signaling and imply that MS4A4 dysfunction in mast cells may result in aberrant cellular responses. Mast cells generate sphingosine-1-phosphate (S1P) after engagement of Fc-epsilon-RI, KIT or IL-3 receptors via the activation of the two isoforms of sphingosine kinase (SphK1 and SphK2). Antigen-mediated generation of S1P is important for mast cell reactivity; however, the function of KIT-generated S1P on mast cell responses is not known. Because neoplastic mast cells generally have gain of function mutations in KIT and S1P is a well-recognized mediator of growth/survival and resistance to anticancer drugs in various neoplastic cell types and cancers, we explored the role of S1P in the proliferation/survival of transformed human mast cells. Our studies suggest that targeting S1P generation in these cells may be an effective strategy to prevent growth of neoplastic mast cells with KIT mutations. This is relevant because the exploration of signaling targets other than receptor tyrosine kinase (RTK) has been a focus in the search for treatments in mastocytosis since RTK inhibitors are ineffective in patients carrying the most common activating mutation (D816V) in KIT.

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2016
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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
Kulinski, Joseph M; Proia, Richard L; Larson, Elisabeth M et al. (2018) S1P? Regulates Passive Systemic Anaphylaxis in Mice but Is Dispensable for Canonical IgE-Mediated Responses in Mast Cells. Int J Mol Sci 19:
Olivera, Ana; Beaven, Michael A; Metcalfe, Dean D (2018) Mast cells signal their importance in health and disease. J Allergy Clin Immunol 142:381-393
Bandara, Geethani; Muñoz-Cano, Rosa; Tobío, Araceli et al. (2018) Targeting Sphingosine Kinase Isoforms Effectively Reduces Growth and Survival of Neoplastic Mast Cells With D816V-KIT. Front Immunol 9:631
Yin, Yuzhi; Bai, Yun; Olivera, Ana et al. (2017) An optimized protocol for the generation and functional analysis of human mast cells from CD34+ enriched cell populations. J Immunol Methods 448:105-111
Cruse, Glenn; Yin, Yuzhi; Fukuyama, Tomoki et al. (2016) Exon skipping of Fc?RI? eliminates expression of the high-affinity IgE receptor in mast cells with therapeutic potential for allergy. Proc Natl Acad Sci U S A 113:14115-14120
Muñoz-Cano, Rosa; Pascal, Mariona; Bartra, Joan et al. (2016) Distinct transcriptome profiles differentiate nonsteroidal anti-inflammatory drug-dependent from nonsteroidal anti-inflammatory drug-independent food-induced anaphylaxis. J Allergy Clin Immunol 137:137-146
Boyden, Steven E; Metcalfe, Dean D; Komarow, Hirsh D (2016) Vibratory Urticaria and ADGRE2. N Engl J Med 375:95
Taguchi, Yoshimitsu; Allende, Maria L; Mizukami, Hiroki et al. (2016) Sphingosine-1-phosphate Phosphatase 2 Regulates Pancreatic Islet ?-Cell Endoplasmic Reticulum Stress and Proliferation. J Biol Chem 291:12029-38
Kirshenbaum, Arnold S; Cruse, Glenn; Desai, Avanti et al. (2016) Immunophenotypic and Ultrastructural Analysis of Mast Cells in Hermansky-Pudlak Syndrome Type-1: A Possible Connection to Pulmonary Fibrosis. PLoS One 11:e0159177

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