Avanti Desai

Stem Cell Factor Programs the Mast Cell Activation Phenotype

Mast cells, activated by Ag via FcεRI, release an array of proinflammatory mediators that contribute to allergic disorders, such as asthma and anaphylaxis. The KIT ligand, stem cell factor (SCF), is critical for mast cell expansion, differentiation, and survival, and under acute conditions, it enhances mast cell activation. However, extended SCF exposure in vivo conversely protects against fatal Ag-mediated anaphylaxis. In investigating this dichotomy, we identified a novel mode of regulation of the mast cell activation phenotype through SCF-mediated programming. We found that mouse bone marrow-derived mast cells chronically exposed to SCF displayed a marked attenuation of FcεRI-mediated degranulation and cytokine production. The hyporesponsive phenotype was not a consequence of altered signals regulating calcium flux or protein kinase C, but of ineffective cytoskeletal reorganization with evidence implicating a downregulation of expression of the Src kinase Hck. Collectively, these findings demonstrate a major role for SCF in the homeostatic control of mast cell activation with potential relevance to mast cell-driven disease and the development of novel approaches for the treatment of allergic disorders.

Vibratory Urticaria Associated with a Missense Variant in ADGRE2.

Patients with autosomal dominant vibratory urticaria have localized hives and systemic manifestations in response to dermal vibration, with coincident degranulation of mast cells and increased histamine levels in serum. We identified a previously unknown missense substitution in ADGRE2 (also known as EMR2), which was predicted to result in the replacement of cysteine with tyrosine at amino acid position 492 (p.C492Y), as the only nonsynonymous variant cosegregating with vibratory urticaria in two large kindreds. The ADGRE2 receptor undergoes autocatalytic cleavage, producing an extracellular subunit that noncovalently binds a transmembrane subunit. We showed that the variant probably destabilizes an autoinhibitory subunit interaction, sensitizing mast cells to IgE-independent vibration-induced degranulation. (Funded by the National Institutes of Health.).

IL-33 induces a hyporesponsive phenotype in human and mouse mast cells.

IL-33 is elevated in afflicted tissues of patients with mast cell (MC)–dependent chronic allergic diseases. Based on its acute effects on mouse MCs, IL-33 is thought to play a role in the pathogenesis of allergic disease through MC activation. However, the manifestations of prolonged IL-33 exposure on human MC function, which best reflect the conditions associated with chronic allergic disease, are unknown. In this study, we found that long-term exposure of human and mouse MCs to IL-33 results in a substantial reduction of MC activation in response to Ag. This reduction required >72 h exposure to IL-33 for onset and 1–2 wk for reversion following IL-33 removal. This hyporesponsive phenotype was determined to be a consequence of MyD88-dependent attenuation of signaling processes necessary for MC activation, including Ag-mediated calcium mobilization and cytoskeletal reorganization, potentially as a consequence of downregulation of the expression of phospholipase Cγ1 and Hck. These findings suggest that IL-33 may play a protective, rather than a causative, role in MC activation under chronic conditions and, furthermore, reveal regulated plasticity in the MC activation phenotype. The ability to downregulate MC activation in this manner may provide alternative approaches for treatment of MC-driven disease.

Estrogen increases the severity of anaphylaxis in female mice through enhanced endothelial nitric oxide synthase expression and nitric oxide production

BACKGROUND Clinical observations suggest that anaphylaxis is more common in adult women compared with adult men, although the mechanistic basis for this sex bias is not well understood. OBJECTIVES We sought to document sex-dependent differences in a mouse model of anaphylaxis and explore the role of female sex hormones and the mechanisms responsible. METHODS Passive systemic anaphylaxis was induced in female and male mice by using histamine, as well as IgE or IgG receptor aggregation. Anaphylaxis was assessed by monitoring body temperature, release of mast cell mediators and/or hematocrit, and lung weight as a measure of vascular permeability. A combination of ovariectomy, estrogen receptor antagonism, and estrogen administration techniques were used to establish estrogen involvement. RESULTS Anaphylactic responses were more pronounced in female than male mice. The enhanced severity of anaphylaxis in female mice was eliminated after pretreatment with an estrogen receptor antagonist or ovariectomy but restored after administration of estradiol in ovariectomized mice, demonstrating that the sex-specific differences are due to the female steroid estradiol. Estrogen did not affect mast cell responsiveness or anaphylaxis onset. Instead, it increased tissue expression of endothelial nitric oxide synthase (eNOS). Blockage of NOS activity with the inhibitor L-NG-nitroarginine methyl ester or genetic eNOS deficiency abolished the sex-related differences. CONCLUSION Our study defines a contribution of estrogen through its regulation of eNOS expression and nitric oxide production to vascular hyperpermeability and intensified anaphylactic responses in female mice, providing additional mechanistic insights into risk factors and possible implications for clinical management in the further exploration of human anaphylaxis.

IL-6 promotes an increase in human mast cell numbers and reactivity through suppression of suppressor of cytokine signaling 3

BACKGROUND IL-6, levels of which are reported to be increased in association with mastocytosis, asthma, and urticaria, is used in conjunction with stem cell factor to generate CD34(+) cell-derived primary human mast cell (HuMC) cultures. Despite these associations, the effects on and mechanisms by which prolonged exposure to IL-6 alters HuMC numbers and function are not well understood. OBJECTIVES We sought to study the effect of IL-6 on HuMC function, the mechanisms by which IL-6 exerts its effects, and the relationship of these findings to mastocytosis. METHODS HuMCs were cultured in stem cell factor with or without IL-6. Responses to FcεRI aggregation and expression of proteases and receptors, including the soluble IL-6 receptor (sIL-6R), were then quantitated. Epigenetic changes in suppressor of cytokine signaling 3 (SOCS3) were determined by using methylation-specific PCR. Serum samples from healthy control subjects and patients with mastocytosis were assayed for IL-6, tryptase, and sIL-6R. RESULTS IL-6 enhanced mast cell (MC) proliferation, maturation, and reactivity after FcεRI aggregation. IL-6 reduced expression of SOCS3, which correlated with methylation of the SOCS3 promoter and increased expression and activation of signal transducer and activator of transcription 3. IL-6 also suppressed constitutive production of sIL-6R, and serum levels of sIL-6R were similarly reduced in patients with mastocytosis. CONCLUSION IL-6 increases MC proliferation and formation of a more reactive phenotype enabled by suppressing proteolytic cleavage of sIL-6R from IL-6R and downregulation of the SOCS3 autoinhibitory pathway. We suggest IL-6 blockade might ameliorate MC-related symptoms and pathology in patients with MC-related diseases associated with increased IL-6 levels, including mastocytosis.

Examination of the role of TRPM8 in human mast cell activation and its relevance to the etiology of cold-induced urticaria

Mast cells are considered the primary initiators of allergic diseases as a consequence of the release of multiple inflammatory mediators on activation. Although predominately activated through antigen-mediated aggregation of IgE-occupied-FcɛRI, they can also be induced to release mediators by other receptors and environmental stimuli. Based on studies conducted in the RBL 2H3 rodent mast cell line, the transient receptor potential melastatin 8 (TRPM8) cation channel has been implicated in the activation of mast cells in response to cold and, by inference, the development of urticaria. Here we investigated the expression and role of TRPM8 receptor, in both human and mouse non-transformed cells, with the aim of exploring the potential link between TRPM8 and the pathology of cold urticaria in humans. Although expressed in mouse mast cells, we found no evidence of TRPM8 expression in human mast cells or functional mutations in TRPM8 in cold urticaria patients. Furthermore, neither mouse nor human primary cultured mast cells degranulated in response to cold challenge or TRPM8 agonists and mast cell reactivity was unaffected in Trpm8(-/-) mice. From these data, we conclude that TRPM8 is unlikely to directly regulate mast cell activation in cold urticaria. Thus, alternative mechanisms likely exist for the pathogenesis of this disease.

Knockout of the Trpc1 gene reveals that TRPC1 can promote recovery from anaphylaxis by negatively regulating mast cell TNF-α production.

Antigen-mediated mast cell (MC) degranulation is the critical early event in the induction of allergic reactions. Transient receptor potential channels (TRPC), particularly TRPC1, are thought to contribute to such MC activation. To explore the contribution of TRPC1 in MC-driven allergic reactions, we examined antigen-mediated anaphylaxis in Trpc1⁻/⁻ and WT mice, and TRPC1 involvement in the activation of MCs derived from the bone marrow (BMMCs) of these mice. In vivo, we observed a similar induction of passive systemic anaphylaxis in the Trpc1⁻/⁻ mice compared to WT controls. Nevertheless, there was delayed recovery from this response in Trpc1⁻/⁻ mice. Furthermore, contrary to expectations, Trpc1⁻/⁻ BMMCs responded to antigen with enhanced calcium signaling but with little defect in degranulation or associated signaling. In contrast, antigen-mediated production of TNF-α, and other cytokines, was enhanced in the Trpc1⁻/⁻ BMMCs, as were calcium-dependent events required for these responses. Additionally, circulating levels of TNF-α in response to antigen were preferentially elevated in the Trpc1⁻/⁻ mice, and administration of an anti-TNF-α antibody blocked the delay in recovery from anaphylaxis in these mice. These data thus provide evidence that, in this model, TRPC1 promotes recovery from the anaphylactic response by repressing antigen-mediated TNF-α release from MCs.

Elevated IgE and atopy in patients treated for early onset ADA-SCID

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Mast Cell Dependent Vascular Changes Associated with an Acute Response to Cold Immersion in Primary Contact Urticaria

Background While a number of the consequences of mast cell degranulation within tissues have been documented including tissue-specific changes such as bronchospasm and the subsequent cellular infiltrate, there is little known about the immediate effects of mast cell degranulation on the associated vasculature, critical to understanding the evolution of mast cell dependent inflammation. Objective To characterize the microcirculatory events that follow mast cell degranulation. Methodology/Principal Findings Perturbations in dermal blood flow, temperature and skin color were analyzed using laser-speckle contrast imaging, infrared and polarized-light colorimetry following cold-hand immersion (CHI) challenge in patients with cold-induced urticaria compared to the response in healthy controls. Evidence for mast cell degranulation was established by documentation of serum histamine levels and the localized release of tryptase in post-challenge urticarial biopsies. Laser-speckle contrast imaging quantified the attenuated response to cold challenge in patients on cetirizine. We found that the histamine-associated vascular response accompanying mast cell degranulation is rapid and extensive. At the tissue level, it is characterized by a uniform pattern of increased blood flow, thermal warming, vasodilation, and recruitment of collateral circulation. These vascular responses are modified by the administration of an antihistamine. Conclusions/Significance Monitoring the hemodynamic responses within tissues that are associated with mast cell degranulation provides additional insight into the evolution of the acute inflammatory response and offers a unique approach to assess the effectiveness of treatment intervention.

Exon skipping of FcεRIβ eliminates expression of the high-affinity IgE receptor in mast cells with therapeutic potential for allergy

Significance We identified an innovative use for the technique of antisense oligonucleotide-mediated exon skipping to specifically target and down-regulate IgE receptor expression in mast cells. Exon skipping is typically used as part of personalized medicine, where a mutant exon is skipped after sequencing the patients’ affected genes. Our approach, however, targets a nonmutated gene and an exon that is critical for surface IgE receptor expression. It does not require a personalized approach with genetic sequencing or multiple iterations of oligonucleotides that would require clinical trials. Furthermore, the diseases to be treated with this technology are ideal for local delivery of the oligonucleotides by aerosols or topical cream formulations. Allergic diseases are driven by activation of mast cells and release of mediators in response to IgE-directed antigens. However, there are no drugs currently available that can specifically down-regulate mast cell function in vivo when chronically administered. Here, we describe an innovative approach for targeting mast cells in vitro and in vivo using antisense oligonucleotide-mediated exon skipping of the β-subunit of the high-affinity IgE receptor (FcεRIβ) to eliminate surface high-affinity IgE receptor (FcεRI) expression and function, rendering mast cells unresponsive to IgE-mediated activation. As FcεRIβ expression is restricted to mast cells and basophils, this approach would selectively target these cell types. Given the success of exon skipping in clinical trials to treat genetic diseases such as Duchenne muscular dystrophy, we propose that exon skipping of FcεRIβ is a potential approach for mast cell-specific treatment of allergic diseases.