Regulation Of Mast Cell Activation Research Articles

Epigenetic Regulation via Altered Histone Acetylation Results in Suppression of Mast Cell Function and Mast Cell-Mediated Food Allergic Responses.

Mast cells are highly versatile cells that perform a variety of functions depending on the immune trigger, context of activation, and cytokine stimulus. Antigen-mediated mast cell responses are regulated by transcriptional processes that result in the induction of numerous genes contributing to mast cell function. Recently, we also showed that exposure to dietary agents with known epigenetic actions such as curcumin can suppress mast cell-mediated food allergy, suggesting that mast cell responses in vivo may be epigenetically regulated. To further assess the effects of epigenetic modifications on mast cell function, we examined the behavior of bone marrow-derived mast cells (BMMCs) in response to trichostatin A (TSA) treatment, a well-studied histone deacetylase inhibitor. IgE-mediated BMMC activation resulted in enhanced expression and secretion of IL-4, IL-6, TNF-α, and IL-13. In contrast, pretreatment with TSA resulted in altered cytokine secretion. This was accompanied by decreased expression of FcεRI and mast cell degranulation. Interestingly, exposure to non-IgE stimuli such as IL-33, was also affected by TSA treatment. Furthermore, continuous TSA exposure contributed to mast cell apoptosis and a decrease in survival. Further examination revealed an increase in I-κBα and a decrease in phospho-relA levels in TSA-treated BMMCs, suggesting that TSA alters transcriptional processes, resulting in enhancement of I-κBα transcription and decreased NF-κB activation. Lastly, treatment of wild-type mice with TSA in a model of ovalbumin-induced food allergy resulted in a significant attenuation in the development of food allergy symptoms including decreases in allergic diarrhea and mast cell activation. These data therefore suggest that the epigenetic regulation of mast cell activation during immune responses may occur via altered histone acetylation, and that exposure to dietary substances may induce epigenetic modifications that modulate mast cell function.

Epigenetic regulation of mast cell function via inhibition of histone acetylation results in suppression of mast cell responses

Abstract Mast cells are highly versatile cells that perform a variety of functions depending on the immune trigger, context of activation and cytokine stimulus. Antigen-mediated mast cell responses are regulated by transcriptional processes that result in the induction of numerous genes contributing to mast cell function. Recently, we also showed that exposure to dietary agents with known epigenetic actions such as curcumin can suppress mast cell-mediated food allergy, suggesting that mast cell responses in vivo may be epigenetically regulated. To further assess the effects of epigenetic modifications on mast cell function, we examined the behavior of bone marrow-derived mast cells (BMMCs) in response to trichostatin A (TSA) treatment, a well-studied histone deacetylase inhibitor. IgE-mediated BMMC activation resulted in enhanced expression and secretion of IL-4, IL-6, TNF-a and IL-13. In contrast, pretreatment with either curcumin or TSA resulted in altered cytokine secretion. This was accompanied by decreased expression of FceRI and mast cell degranulation. Interestingly, exposure to non-IgE stimuli such as IL-33, was also affected by TSA treatment. Furthermore, long-term TSA exposure contributed to mast cell apoptosis and a decrease in survival. Further examination revealed a decrease in phospho-relA levels in TSA-treated BMMCs, suggesting that TSA alters transcriptional processes, resulting in decreased NF-kB activation. These data therefore suggest that the epigenetic regulation of mast cell activation during immune responses may occur via regulation of histone acetylation, and that exposure to dietary substances such as curcumin may induce epigenetic modifications that modulate mast cell function.

Tanshinone IIA suppresses FcεRI-mediated mast cell signaling and anaphylaxis by activation of the Sirt1/LKB1/AMPK pathway

AMP-activated protein kinase (AMPK) and its upstream mediators liver kinase B1 (LKB1) and sirtuin 1 (Sirt1) are generally known as key regulators of metabolism. We have recently reported that the AMPK pathway negatively regulates mast cell activation and anaphylaxis. Tanshinone IIA (Tan IIA), an active component of Salvia miltiorrhiza extract that is currently used for the treatment of cardiovascular and cerebrovascular diseases, shows anti-diabetic activity and improves insulin resistance in db/db mice through activation of AMPK. The aim of this study was to evaluate the anti-allergic activity of Tan IIA in vivo and to investigate the underlying mechanism in vitro in the context of AMPK signaling. The anti-allergic effect of Tan IIA was evaluated using mouse bone marrow-derived mast cells (BMMCs) from AMPKα2−/− or Sirt1−/− mice, or BMMCs transfected with siRNAs specific for AMPKα2, LKB1, or Sirt1. AMPKα2−/− and Sirt1−/− mice were used to confirm the anti-allergic effect of Tan IIA in anaphylaxis in vivo. Tan IIA dose-dependently inhibited FcεRI-mediated degranulation and production of eicosanoids and cytokines in BMMCs. These inhibitory effects were diminished by siRNA-mediated knockdown or genetic deletion of AMPKα2 or Sirt1. Moreover, Tan IIA inhibited a mast cell-mediated local passive anaphylactic reaction in wild-type mice, but not in AMPKα2−/− or Sirt1−/− mice. In conclusion, Tan IIA suppresses FcεRI-mediated mast cell activation and anaphylaxis through activation of the inhibitory Sirt1-LKB1-AMPK pathway. Thus, Tan IIA may be useful as a new therapeutic agent for mast cell-mediated allergic diseases.

SHIP negatively regulates type II immune responses in mast cells and macrophages.

SHIP is a hematopoietic-specific lipid phosphatase that dephosphorylates PI3K-generated PI(3,4,5)-trisphosphate. SHIP removes this second messenger from the cell membrane blunting PI3K activity in immune cells. Thus, SHIP negatively regulates mast cell activation downstream of multiple receptors. SHIP has been referred to as the "gatekeeper" of mast cell degranulation as loss of SHIP dramatically increases degranulation or permits degranulation in response to normally inert stimuli. SHIP also negatively regulates Mϕ activation, including both pro-inflammatory cytokine production downstream of pattern recognition receptors, and alternative Mϕ activation by the type II cytokines, IL-4, and IL-13. In the SHIP-deficient (SHIP-/- ) mouse, increased mast cell and Mϕactivation leads to spontaneous inflammatory pathology at mucosal sites, which is characterized by high levels of type II inflammatory cytokines. SHIP-/- mast cells and Mϕs have both been implicated in driving inflammation in the SHIP-/- mouse lung. SHIP-/- Mϕs drive Crohn's disease-like intestinal inflammation and fibrosis, which is dependent on heightened responses to innate immune stimuli generating IL-1, and IL-4 inducing abundant arginase I. Both lung and gut pathology translate to human disease as low SHIP levels and activity have been associated with allergy and with Crohn's disease in people. In this review, we summarize seminal literature and recent advances that provide insight into SHIP's role in mast cells and Mϕs, the contribution of these cell types to pathology in the SHIP-/- mouse, and describe how these findings translate to human disease and potential therapies.

MicroRNA-4443 regulates mast cell activation by T cell–derived microvesicles

The mechanism by which mast cells (MCs) are activated in T cell-mediated inflammatory processes remains elusive. Recently, we have shown that microvesicles derived from activated T cells (mvT*s) can stimulate MCs to degranulate and release several cytokines. The aim of this study was to characterize the contribution of microRNAs (miRs) delivered by microvesicles to MC activation. miR profiling was performed with NanoString technology and validated by using real-time PCR. The biological role of mvT* miR was verified by overexpression of miRs in MCs using mimic or inhibitory molecules and analyzing the effect on their predicted targets. mvT*s were found to downregulate the expression of the tyrosine phosphatase protein tyrosine phosphatase receptor type J (PTPRJ), a known extracellular signal-regulated kinase inhibitor. Bioinformatics analysis predicted that miR-4443 regulates the PTPRJ gene expression. Indeed, miR-4443, which was present in mvT*s, was also found to be overexpressed in human MCs stimulated with these MVs. α-Amanitin insensitivity confirmed that overexpression of miR-4443 was not due to transcriptional activation. The luciferase reporter assay indicated that the 3' untranslated region of PTPRJ was targeted by this miR. Transfection of MCs with mimic or inhibitor of miR-4443 resulted in decreased or enhanced PTPRJ expression, respectively. Furthermore, miR-4443 regulated extracellular signal-regulated kinase phosphorylation and IL-8 release in MCs activated by mvT*s. These results support a scenario by which T cell-derived microvesicles act as intercellular carriers of functional miR-4443, which might exert heterotypic regulation of PTPRJ gene expression in MCs, leading to their activation in the context of T cell-mediated inflammatory processes.

The effect of Rho drugs on mast cell activation and degranulation.

Mast cells are tissue-resident immune cells that produce potent proinflammatory mediators, which are stored in cytoplasmic granules. Stimulation triggers degranulation, a process that mobilizes granules to dock and fuse to the plasma membrane, releasing mediators. Mast cell degranulation has an important role in immunity but can also intensify inflammation and contribute to allergic disorders. Hence, it is important to understand signaling pathways that regulate mast cell degranulation. Here, we examined the role of Rho proteins in regulating mast cell activation leading to degranulation. RBL-2H3 cells and bone marrow-derived mast cells (BMMCs) were stimulated through aggregation of FcεRI receptors. Stimulated cells showed a large increase in the levels of activated Rac and, to a lesser extent, RhoA. Drugs were used to acutely inhibit the function of specific Rho proteins. The Rac inhibitor EHT-1864 and the RhoA inhibitor rhosin inhibited degranulation. Microscopic characterization showed that, upon stimulation, RBL-2H3 cells formed surface ridges that grew into large protrusions reminiscent of circular dorsal ruffles, which flattened into large lamellipodia. LysoTracker-labeled cells showed granules stream into peripheral protrusions. EHT-1864 reduced granule motility, whereas rhosin increased motility; both drugs affected the formation of peripheral protrusions. These results showed that, in response to stimuli, Rho proteins control discrete cytoskeletal remodeling processes that are needed for granule exocytosis. Rac is required to stimulate the remodeling of mast cells, triggering actin-mediated flattening of the cell periphery to create an active degranulation zone, whereas RhoA controls the streaming of highly motile granules into the active zone.

Dnmt3a restrains mast cell inflammatory responses

DNA methylation and specifically the DNA methyltransferase enzyme DNMT3A are involved in the pathogenesis of a variety of hematological diseases and in regulating the function of immune cells. Although altered DNA methylation patterns and mutations in DNMT3A correlate with mast cell proliferative disorders in humans, the role of DNA methylation in mast cell biology is not understood. By using mast cells lacking Dnmt3a, we found that this enzyme is involved in restraining mast cell responses to acute and chronic stimuli, both in vitro and in vivo. The exacerbated mast cell responses observed in the absence of Dnmt3a were recapitulated or enhanced by treatment with the demethylating agent 5-aza-2'-deoxycytidine as well as by down-modulation of Dnmt1 expression, further supporting the role of DNA methylation in regulating mast cell activation. Mechanistically, these effects were in part mediated by the dysregulated expression of the scaffold protein IQGAP2, which is characterized by the ability to regulate a wide variety of biological processes. Altogether, our data demonstrate that DNMT3A and DNA methylation are key modulators of mast cell responsiveness to acute and chronic stimulation.

Mast Cell Regulatory Gene Variants Are Common in Mast Cell Activation Syndrome

Recently recognized but likely prevalent, mast cell (MC) activation syndrome (MCAS) presents as heterogeneous chronic multisystem polymorbidity of generally inflammatory ± allergic theme; severity can be disabling. MCAS features aberrant MC reactivity and constitutive MC activation with little MC accumulation [Afrin, Ann Med 48:190-201], distinct from mastocytosis (rare and notable for recurrent KIT codon 816 variants). Studies at Univ. of Bonn [Molderings, Scand J Gastroenterol 42:1045-53 and Immunogenetics 62:721-7] suggest most MCAS patients (pts) bear in peripheral blood (PB) MCs somatic, likely constitutively activating, non-codon-816 variants, and some splicing variants, in mRNA for KIT, the dominant MC regulatory gene (MCRG). Other studies show most mastocytosis, too, bears variants in many MCRGs beyond KIT [e.g., Soverini, Blood 126:4085], suggesting the same is likely in MCAS. Confirmation of MCRG variants in MCAS would spur new direction in research in MCAS and chronic inflammatory diseases (CIDs) possibly born of MCAS [Afrin, Transl Res 174:33-59]. We are studying (UMN IRB-approved protocol 2015NTLS023) mRNA for KIT and other MCRGs in PB MCs from MCAS pts and controls (ctls).Methods: We extracted (Miltenyi CD117 immunomagnetic bead method per Bonn) PB MCs from 20 pts aged 18-50 diagnosed at UMN Jul-Dec 2015 with MCAS per criteria [Molderings, J Hematol Oncol 4:10] which in our experience (>1000 pts) reflects MCAS behavior better than other criteria [Valent, Int Arch Allergy Immunol 157:215-25]. We plan the same for 20 demographically matched ctls meeting rigorous good health criteria to exclude illness possibly from undiagnosed MCAS (9 ctls accrued so far, sequencing pending). Immunohistochemical staining with anti-human-c-kit antibodies (Thermo Scientific RB-9038-R7) confirmed extracts were pure bright CD117/c-kit-positive cells. mRNA was isolated (Qiagen RNeasy) and sequenced (Agilent HiSeq). Variant analysis of reverse-transcribed sequences from 38 genes (selected [Molderings, Crit Rev Oncol Hematol 93:75-89 and other reports] as regulating MC activation (receptor activation, signal transduction, cytokine production/release)) was performed via well-established GATK-based bioinformatic pipelines [Onsongo, BMC Res Notes 7:314], plus kallisto [Bray, Biotechnology 34:525-7] for finding RNA splice variants.Results: Table 1 shows key pt characteristics (families similarly afflicted). Percentages of pts with elevations of various MC mediators tested in diagnosing MCAS were similar to prior report [Zenker, Blood 126:5174].Exonic RNAseq showed many variants across many MCRGs in all 20 pts (median 11,276 variants per pt across studied genes; median 3,277 per pt were novel) but no recurrent variant signatures. Table 2 shows high- and medium-impact variants (HMIVs, i.e., frameshifts and missenses) found with high confidence (≥20 reads aligned to the region of the variant in all 20 pts); 8 KIT variants found were low impact (3’ untranslated region). As ctls are not yet fully accrued, we show comparator allele frequencies from 3 reference datasets.Other HMIVs were found with less confidence (<20 reads aligned to variant region in ≥1 pt) in ASXL1, CBL, DNMT3B, ETV6, EZH2, HNMT, IDH1, IL13, JAK2, KIT, KMT2A, KRAS, MS4A2, NLRP3, RASGRP4, SETBP1, SF3B1, TBXA2R, TET2, and TP53 (analysis ongoing). No HMIVs were found in ADGRE2, DNMT3A, HRH4, IL4, IL33, KITLG, MRGPRX2, NRAS, PDGFRα/β, RUNX1, SRSF2, SWAP-70, TNF, U2AF1, or VEGFA. We confirmed far greater expression of potentially more activating del(510-513) KIT splice variant in all pts.Discussion: We did not find HMIVs in PB MC KIT mRNA in our pts (we soon will study another 20), but we found all bear many mRNA variants in many MCRGs, requiring further study for significance. Reference datasets may be inadequate comparators, skewed by unrecognized MCAS (suspected prevalence as high as 17% [Molderings, PLoS One 8(9):e76241]), so we are accruing rigorously healthy ctls for similar study. If our ctls are largely devoid of HMIVs in MCRG mRNA from PB MCs (as in [Immunogenetics, op. cit.]), a stronger case will be made that MCAS is primarily a clonal disease, if complicated, heterogeneous, and notwithstanding MC-targeted/activating autoimmunity. Further study of variants will include validation by direct DNA sequencing, characterization, and probing for recurrence in CID-specific MCAS subpopulations. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CD13/aminopeptidase N is a negative regulator of mast cell activation.

Antigen-induced mast cell (MC) activation via cross-linking of IgE-bound high-affinity receptors for IgE (FcεRI) underlies type I allergy and anaphylactic shock. Comprehensive knowledge of FcεRI regulation is thus required. We have identified a functional interaction between FcεRI and CD13 in murine MCs. Antigen-triggered activation of IgE-loaded FcεRI results in cocapping and cointernalization of CD13 and equivalent internalization rates of up to 40%. Cointernalization is not unspecific, because ligand-driven KIT internalization is not accompanied by CD13 internalization. Moreover, antibody-mediated cross-linking of CD13 causes IL-6 production in an FcεRI-dependent manner. These data are indicative of a functional interaction between FcεRI and CD13 on MCs. To determine the role of this interaction, CD13-deficient bone marrow-derived MCs (BMMCs) were analyzed. Intriguingly, antigen stimulation of CD13-deficient BMMCs results in significantly increased degranulation and proinflammatory cytokine production compared to wild-type cells. Furthermore, in a low-dose model of passive systemic anaphylaxis, antigen-dependent decrease in body temperature, reflecting the anaphylactic reaction, is substantially enhanced by the CD13 inhibitor bestatin (-5.9 ± 0.6°C) and by CD13 deficiency (-8.8 ± 0.6°C) in contrast to controls (-1.2 ± 1.97°C). Importantly, bestatin does not aggravate anaphylaxis in CD13-deficient mice. Thus, we have identified CD13 as a novel negative regulator of MC activation in vitro and in vivo-Zotz, J. S., Wölbing, F., Lassnig, C., Kauffmann, M., Schulte, U., Kolb, A., Whitelaw, B., Müller, M., Biedermann, T., Huber, M. CD13/aminopeptidase N is a negative regulator of mast cell activation.

Selective Cannabinoid Receptor-1 Agonists Regulate Mast Cell Activation in an Oxazolone-Induced Atopic Dermatitis Model.

BackgroundMany inflammatory mediators, including various cytokines (e.g. interleukins and tumor necrosis factor [TNF]), inflammatory proteases, and histamine are released following mast cell activation. However, the endogenous modulators for mast cell activation and the underlying mechanism have yet to be elucidated. Endogenous cannabinoids such as palmitoylethanolamide (PEA) and N-arachidonoylethanolamine (anandamide or AEA), were found in peripheral tissues and have been proposed to possess autacoid activity, implying that cannabinoids may downregulate mast cell activation and local inflammation.ObjectiveIn order to investigate the effect of cannabinoid receptor-1 (CB1R) agonists on mast cell activation, AEA-derived compounds were newly synthesized and evaluated for their effect on mast cell activation.MethodsThe effects of selected compounds on FcεRI-induced histamine and β-hexosaminidase release were evaluated in a rat basophilic leukemia cell line (RBL-2H3). To further investigate the inhibitory effects of CB1R agonist in vivo, an oxazolone-induced atopic dermatitis mouse model was exploited.ResultsWe found that CB1R inhibited the release of inflammatory mediators without causing cytotoxicity in RBL-2H3 cells and that CB1R agonists markedly and dose-dependently suppressed mast cell proliferation indicating that CB1R plays an important role in modulating antigen-dependent immunoglobulin E (IgE)-mediated mast cell activation. We also found that topical application of CB1R agonists suppressed the recruitment of mast cells into the skin and reduced the level of blood histamine.ConclusionOur results indicate that CB1R agonists down-regulate mast cell activation and may be used for relieving inflammatory symptoms mediated by mast cell activation, such as atopic dermatitis, psoriasis, and contact dermatitis.

Interleukin-33 as a Potential Therapeutic Target for Interstitial Cystitis: New Perspectives on Mast Cell Regulation.

There are many problems still unresolved by modern medicine. In the field of voiding disorders, interstitial cystitis (IC) remains a representative disease yet to be conquered. The exact pathophysiologic mechanism of IC is not understood. Although several hypotheses have been proposed, including leaky epithelium, mast cell activation, and neurogenic inflammation, no theory has been clearly established yet [1]. IC may have multiple causes and may represent a final common reaction of the bladder to multifactorial etiology. Most researchers believe that the mast cell plays a key role in the common pathophysiology of IC. Despite many efforts to clarify the etiology of IC, our knowledge is insufficient for useful insights into effective treatments [2]. The current therapeutic goal is the improvement of quality of life, and not curative treatment. It is believed that histamine, which is secreted from mast cells, causes specific symptoms of IC such as pain, hyperemia, and fibrosis. Therefore, antihistamine treatment to adjust mast cell activation is considered a scientifically robust and rational therapy for IC. However, results using antihistamines in clinical practice were disappointing. Sufficient evidence does not exist to conclusively state that antihistamines effectively regulate the activation of mast cells in the bladder [3]. In this issue of the International Neurourology Journal, we have published an article reviewing new perspectives on mast cell regulation [4]. It was confirmed in other organs that interleukin (IL) 33 can modulate allergic inflammation. IL-33 is closely related to mast cell activation, which is considered the core of IC pathogenesis. Therefore, it may be possible to regulate mast cell activation by inhibiting IL-33. Promising results have been reported in a number of studies. However, because the reaction of the anti–IL-33 could vary in different organs, we have many challenges to overcome before systemic administration of anti–IL-33 as a therapeutic agent can be implemented. Nevertheless, this is valuable work as it presents a potential therapeutic target for an incurable disease yet unconquered by modern medicine. The most important message of this review for urologists is included in the last sentence of conclusion: “co-operative research among allergologists and urologists could yield more meaningful conclusions about the pathophysiologic mechanism and treatment strategies for interstitial cystitis” [4].

Rictor negatively regulates high-affinity receptors for IgE-induced mast cell degranulation.

Rictor is a regulatory component of the mammalian target of rapamycin (mTOR) complex 2 (mTORC2). We have previously demonstrated that rictor expression is substantially downregulated in terminally differentiated mast cells as compared with their immature or transformed counterparts. However, it is not known whether rictor and mTORC2 regulate mast cell activation. In this article, we show that mast cell degranulation induced by aggregation of high-affinity receptors for IgE (FcεRI) is negatively regulated by rictor independently of mTOR. We found that inhibition of mTORC2 by the dual mTORC1/mTORC2 inhibitor Torin1 or by downregulation of mTOR by short hairpin RNA had no impact on FcεRI-induced degranulation, whereas downregulation of rictor itself resulted in an increased sensitivity (∼50-fold) of cells to FcεRI aggregation with enhancement of degranulation. This was linked to a similar enhancement in calcium mobilization and cytoskeletal rearrangement attributable to increased phosphorylation of LAT and PLCγ1. In contrast, degranulation and calcium responses elicited by the G protein-coupled receptor ligand, C3a, or by thapsigargin, which induces a receptor-independent calcium signal, was unaffected by rictor knockdown. Overexpression of rictor, in contrast with knockdown, suppressed FcεRI-mediated degranulation. Taken together, these data provide evidence that rictor is a multifunctional signaling regulator that can regulate FcεRI-mediated degranulation independently of mTORC2.

Spiraeoside inhibits mast cells activation and IgE-mediated allergic responses by suppressing phospholipase C-γ-mediated signaling.

Mast cells are responsible for IgE-mediated allergic responses through the secretion of various inflammatory cytokines and mediators. Therefore, the pharmacological regulation of mast cell activation is an important goal in the development of novel anti-allergic drugs. In this study, we found that spiraeoside (SP) inhibits mast cell activation and allergic responses in vivo. SP dose-dependently inhibited the degranulation induced by IgE-antigen (Ag) stimulation in RBL-2H3 mast cells without cytotoxic effects. At the molecular level, SP reduced the Ag-induced phosphorylation and subsequent activation of phospholipase C-γ2 (PLC-γ2). Moreover, SP inhibited the phosphorylation of spleen tyrosine kinase (Syk), linker for activation of T cells (LAT), and downstream MAPKs, such as ERK1/2, p38, and JNK, eventually attenuating expression of TNF-α and IL-4. Finally, we found that SP significantly inhibited IgE-mediated passive cutaneous anaphylaxis (PCA) in mice. Taken together, our results strongly suggest that SP suppresses IgE-mediated mast cell activation and allergic responses by inhibiting Lyn-induced PLC-γ2/MAPK signaling in mast cells.

Assay of mast cell mediators.

Mediator release from activated mast cells is a major initiator of the symptomology associated with allergic disorders such as anaphylaxis and asthma. Thus, methods to monitor the generation and release of such mediators have widespread applicability in studies designed to understand the processes regulating mast cell activation and for the identification of therapeutic approaches to block mast cell-driven disease. In this chapter, we discuss approaches used for the determination of mast cell degranulation, lipid-derived inflammatory mediator production, and cytokine/chemokine gene expression as well as cytokine release.

Protein tyrosine phosphatase-PEST (PTP-PEST) regulates mast cell-activating signals in PTP activity-dependent and -independent manners

Aggregation of the high-affinity IgE receptor (FcεRI) in mast cells leads to degranulation and production of numerous cytokines and lipid mediators that promote allergic inflammation. Tyrosine phosphorylation of proteins in response to FcεRI aggregation has been implicated in mast cell activation. Here, we determined the role of PTP-PEST (encoded by PTPN12) in the regulation of mast cell activation using the RBL-2H3 rat basophilic leukemia cell line as a model. PTP-PEST expression was significantly induced upon FcεRI-crosslinking, and aggregation of FcεRI induced the phosphorylation of PTP-PEST at Ser39, thus resulting in the suppression of PTP activity. By overexpressing a phosphatase-dead mutant (PTP-PEST CS) and a constitutively active mutant (PTP-PEST SA) in RBL-2H3 cells, we showed that PTP-PEST decreased degranulation and enhanced IL-4 and IL-13 transcription in FcεRI-crosslinked RBL-2H3 cells, but PTP activity of PTP-PEST was not necessary for this regulation. However, FcεRI-induced TNF-α transcription was increased by the overexpression of PTP-PEST SA and suppressed by the overexpression of PTP-PEST CS. Taken together, these results suggest that PTP-PEST is involved in the regulation of FcεRI-mediated mast cell activation through at least two different processes represented by PTP activity-dependent and -independent pathways.

アレルギーにおけるマスト細胞の活性化とその制御

マスト細胞は骨髄の多能性血液幹細胞由来の細胞で，末梢組織において成熟分化する．全身のほとんどあらゆる臓器に亘って分布し，その数はリンパ球の総数にも匹敵するとされており，特に皮膚や気道，消化管粘膜などの外界に直接する臓器の血管周囲に多数存在することが知られている．この位置取りは，マスト細胞が外界からの抗原（アレルゲン）に曝されアレルギーのエフェクター細胞として働くばかりではなく，感染防御や免疫系の基礎的な領域においても重要な役割を演じていることを示唆するものである．本記念講演では，最近明らかになってきたマスト細胞の多彩な役割の中で，IgE受容体(FcεRI) を中心に我々が解明してきたその活性化と制御の分子的な仕組みについて紹介し，アレルギーの新しい分子標的薬の開発に向けた取り組みについても言及する．

P66Shc regulates vesicle-mediated secretion in mast cells by affecting F-actin dynamics

The extracellular vesicular compartment has emerged as a novel system of intercellular communication; however, the mechanisms involved in membrane vesicle biogenesis and secretion are as yet unclear. Among immune cells releasing membrane vesicles-mast cells that reside near tissues exposed to the environment-are master modulators of immune responses. Here, we have addressed the role of p66Shc, a novel regulator of mast cell activation and homeostasis, in the dynamic reorganization of the actin cytoskeleton that is associated with morphological changes during secretion. We show that p66Shc is recruited as a complex with the lipid phosphatase SHIP1 to the F-actin skeleton and impairs antigen-dependent cortical F-actin disassembly and membrane ruffling through the inhibition of Vav and paxillin phosphorylation. We also show that in addition to acting as a negative regulator of antigen-dependent mast cell degranulation, p66Shc limits the basal release of granule contents by inhibiting microvesicle budding from the plasma membrane and piecemeal degranulation. These findings identify p66Shc as a critical regulator of actin dynamics in mast cells, providing a basis for understanding the molecular mechanisms involved in vesicle-mediated secretion in these cells.

Deciphering New Molecular Mechanisms of Mast Cell Activation

Deciphering New Molecular Mechanisms of Mast Cell Activation

DJ-1 regulates mast cell activation and IgE-mediated allergic responses

DJ-1 is an antioxidant protein known to reduce levels of reactive oxygen species (ROS), but its presence or function in mast cells and allergic diseases is unknown. We sought to determine the role and mechanism of DJ-1 in allergic responses in vitro and in vivo. ROS and DJ-1 levels in serum or culture medium were measured with ELISA kits. The role of DJ-1 was evaluated in mast cell cultures and passive cutaneous anaphylaxis in normal or DJ-1 knockout (KO) mice. The mechanism of DJ-1 action was examined by using immunoblotting, immunoprecipitation, RT-PCR, and other molecular biological approaches. Patients with atopic dermatitis had increased levels of ROS and diminished levels of DJ-1. DJ-1 KO mice exhibited enhanced passive cutaneous anaphylaxis and augmented ROS levels in sera and bone marrow-derived mast cells (BMMCs). Furthermore, antigen-induced degranulation and production of TNF-α and IL-4 were significantly amplified in DJ-1 KO and anti-DJ-1 small interfering RNA-transfected BMMCs compared with that seen in wild-type (WT) BMMCs. Studies with these cells and BMMCs transfected with small interfering RNAs against the phosphatases Src homology domain 2-containing protein tyrosine phosphatase (SHP) 1 and SHP-2 revealed that the DJ-1 KO phenotype could be attributed to suppression of SHP-1 activity and enhancement of SHP-2 activity, leading to strengthened signaling through linker for activation of T cells, phospholipase Cγ, and mitogen-activated protein kinases. A deficiency or constitutive activation of DJ-1 can have implications in mast cell-driven allergic diseases, such as asthma and anaphylaxis.

The Receptor LMIR3 Negatively Regulates Mast Cell Activation and Allergic Responses by Binding to Extracellular Ceramide

Mast cells (MCs) are key effector cells in allergic reactions. However, the inhibitory mechanism that prevents excessive activation of MCs remains elusive. Here we show that leukocyte mono-immunoglobulin-like receptor 3 (LMIR3; also called CD300f) is a negative regulator of MC activation in vivo. LMIR3 deficiency exacerbated MC-dependent allergic responses in mice, including anaphylaxis, airway inflammation, and dermatitis. Both physical binding and functional reporter assays via an extracellular domain of LMIR3 showed that several extracellular lipids (including ceramide) and lipoproteins were possible ligands for LMIR3. Importantly, MCs were frequently surrounded by extracellular ceramide in vivo. Upon engagement of high-affinity immunoglobulin E receptor, extracellular ceramide-LMIR3 binding inhibited MC activation via immunoreceptor tyrosine-based inhibitory and switch motifs of LMIR3. Moreover, pretreatment with LMIR3-Fc fusion protein or antibody against either ceramide or LMIR3 interfered with this binding in vivo, thereby exacerbating passive cutaneous anaphylaxis. Thus, the interaction between extracellular ceramide and LMIR3 suppressed MC-dependent allergic responses.