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FcεRI is the primary receptor in mast cells that mediates allergic reactions by inducing rapid release of mediators, an adaptive immune response that might have evolved as a host defense against parasites and venoms. Yet it is apparent that mast cells are also activated through non-IgE receptors, the significance of which is just beginning to be understood. This includes the Mas-related G protein-coupled receptor X2, which might contribute to reactions to diverse antimicrobials and polybasic compounds, and the adhesion G protein-coupled receptor E2, variants of which are associated with familial vibratory urticaria and are activated by mechanical vibration. Similarly, mast cells have long been recognized as the main repository for histamine, heparin, and proteases. Recent evidence also points to new functions, modes of delivery, and mechanisms of action of mast cell proteases that add new dimensions to the roles of mast cells in human biology. In addition, exposure of mast cells to environmental cues can quantitatively and qualitatively modulate their responses and thus their effect on allergic inflammation. Illustrating this paradigm, we summarize a number of recent studies implicating the injury/tissue damage cytokine IL-33 as a modulator of allergen-induced mast cell responses. We also discuss the discovery of markers associated with transformed mast cells and new potential directions in suppressing mast cell activity., (Published by Elsevier Inc.)

Background: DJ-1 is a redox-sensitive protein with multiple roles in cell homeostasis, levels of which are altered in patients with mast cell (MC)-related disorders. However, whether DJ-1 can regulate human MC function is unknown., Objective: We sought to investigate the potential role of DJ-1 in the responses of human MCs to antigen stimulation., Methods: DJ-1 was silenced in human CD34 + -derived MCs and in the LAD2 MC line by using lentiviral short hairpin RNA constructs. Release of β-hexosaminidase, prostaglandin D 2 , and GM-CSF and changes in reactive oxygen species levels were measured after FcεRI engagement. Enzymatic assays, sucrose density gradient centrifugation, immunoprecipitation, dot and Western blotting, and confocal imaging were performed for signaling, cellular localization, and coassociation studies., Results: DJ-1 knockdown substantially reduced mediator release, as well as Lyn kinase and spleen tyrosine kinase activation and signaling through mechanisms that appeared largely unrelated to DJ-1 antioxidant activity. Following FcεRI activation, nonoxidized rather than oxidized DJ-1 translocated to lipid rafts, where it associated with Lyn, an interaction that appeared critical for maximal Lyn activation and initiation of signaling. Using purified recombinant proteins, we demonstrated that DJ-1 directly bound to Lyn but not to other Src kinases, and this interaction was specific for human but not mouse proteins. In addition, DJ-1 reduced Src homology 2 domain-containing phosphatase 2 phosphatase activity by scavenging reactive oxygen species, thus preventing spleen tyrosine kinase dephosphorylation and perpetuating MC signaling., Conclusion: We demonstrate a novel role for DJ-1 in the early activation of Lyn by FcεRI, which is essential for human MC responses and provides the basis for an alternative target in allergic disease therapy., (Published by Elsevier Inc.)

Receptor activator of NF-kB ligand (RANKL) generates intracellular reactive oxygen species (ROS), which increase RANKL-mediated signaling in osteoclast (OC) precursor bone marrow macrophages (BMMs). Here we show that a ROS scavenging protein DJ-1 negatively regulates RANKL-driven OC differentiation, also called osteoclastogenesis. DJ-1 ablation in mice leads to a decreased bone volume and an increase in OC numbers. In vitro, the activation of RANK-dependent signals is enhanced in DJ-1-deficient BMMs as compared to wild-type BMMs. DJ-1 suppresses the activation of both RANK-TRAF6 and RANK-FcRγ/Syk signaling pathways because of activation of Src homology region 2 domain-containing phosphatase-1, which is inhibited by ROS. Ablation of DJ-1 in mouse models of arthritis and RANKL-induced bone disease leads to an increase in the number of OCs, and exacerbation of bone damage. Overall, our results suggest that DJ-1 plays a role in bone homeostasis in normal physiology and in bone-associated pathology by negatively regulating osteoclastogenesis.

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., Competing Interests: Carrying out this study resulted in the filing of US Provisional Patent Application No. 62/289,447 by G.C. and D.D.M.

Neoplastic accumulation of mast cells in systemic mastocytosis (SM) associates with activating mutations in the receptor tyrosine kinase KIT. Constitutive activation of tyrosine kinase oncogenes has been linked to imbalances in oxidant/antioxidant mechanisms in other myeloproliferative disorders. However, the impact of KIT mutations on the redox status in SM and the potential therapeutic implications are not well understood. Here, we examined the regulation of reactive oxygen species (ROS) and of the antioxidant protein DJ-1 (PARK-7), which increases with cancer progression and acts to lessen oxidative damage to malignant cells, in relationship with SM severity. ROS levels were increased in both indolent (ISM) and aggressive variants of the disease (ASM). However, while DJ-1 levels were reduced in ISM with lower mast cell burden, they rose in ISM with higher mast cell burden and were significantly elevated in patients with ASM. Studies on mast cell lines revealed that activating KIT mutations induced constant ROS production and consequent DJ-1 oxidation and degradation that could explain the reduced levels of DJ-1 in the ISM population, while IL-6, a cytokine that increases with disease severity, caused a counteracting transcriptional induction of DJ-1 which would protect malignant mast cells from oxidative damage. A mouse model of mastocytosis recapitulated the biphasic changes in DJ-1 and the escalating IL-6, ROS and DJ-1 levels as mast cells accumulate, findings which were reversed with anti-IL-6 receptor blocking antibody. Our findings provide evidence of increased ROS and a biphasic regulation of the antioxidant DJ-1 in variants of SM and implicate IL-6 in DJ-1 induction and expansion of mast cells with KIT mutations. We propose consideration of IL-6 blockade as a potential adjunctive therapy in the treatment of patients with advanced mastocytosis, as it would reduce DJ-1 levels making mutation-positive mast cells vulnerable to oxidative damage., Competing Interests: The authors have declared that no competing interests exist.

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., (Published by Elsevier Inc.)

Food allergy is a hypersensitive immune reaction to food proteins. We have previously demonstrated the presence of IL-10-producing CD5(+) B cells and suggested their potential role in regulating cow's milk casein allergy in humans and IgE-mediated anaphylaxis in mice. In this study, we determined whether IL-10-producing CD5(+) regulatory B cells control casein-induced food allergic responses in mice and, if so, the underlying mechanisms. The induction of oral tolerance (OT) by casein suppressed casein-induced allergic responses including the decrease of body temperature, symptom score, diarrhea, recruitment of mast cells and eosinophils into jejunum, and other biological parameters in mice. Notably, the population of IL-10-producing CD5(+) B cells was increased in mesenteric lymph node (MLN), but not in spleen or peritoneal cavity (PeC) in OT mice. The adoptive transfer of CD5(+) B cells from MLN, but not those from spleen and PeC, suppressed the casein-induced allergic responses in an allergen-specific and IL-10-dependent manner. The inhibitory effect of IL-10-producing CD5(+) B cells on casein-induced allergic response was dependent on Foxp3(+) regulatory T cells. Taken together, mesenteric IL-10-producing regulatory B cells control food allergy via Foxp3(+) regulatory T cells and could potentially act as a therapeutic regulator for food allergy.

IL-33 released from damaged cells plays a central role in allergic inflammation by acting through its membrane-bound receptor, ST2 receptor (ST2L). IL-33 activity can be neutralized by the soluble spliced variant of ST2 (sST2) that has been associated with allergic inflammation but its source is not well defined. We investigated whether mast cells (MCs) are a significant source of sST2 following activation through FcεRI or ST2. We find that antigen and IL-33 induce substantial production and release of sST2 from human and mouse MCs in culture and do so synergistically when added together or in combination with stem cell factor. Moreover, increases in circulating sST2 during anaphylaxis in mice were dependent on the presence of MCs. Human MCs activated via FcεRI failed to generate IL-33 and IL-33 produced by mouse bone marrow-derived MCs was retained within the cells. Therefore, FcεRI-mediated sST2 production is independent of MC-derived IL-33 acting in an autocrine manner. These results are consistent with the conclusion that both mouse and human MCs when activated are a significant inducible source of sST2 but not IL-33 and thus have the ability to modulate the biologic impact of IL-33 produced locally by other cell types during allergic inflammation., (Published 2015. This article is a U.S. Government work and is in the public domain in the USA.)

MS4A family members differentially regulate the cell cycle, and aberrant, or loss of, expression of MS4A family proteins has been observed in colon and lung cancer. However, the precise functions of MS4A family proteins and their mechanistic interactions remain unsolved. Here we report that MS4A4 facilitates trafficking of the receptor tyrosine kinase KIT through endocytic recycling rather than degradation pathways by a mechanism that involves recruitment of KIT to caveolin-1-enriched microdomains. Silencing of MS4A4 in human mast cells altered ligand-induced KIT endocytosis pathways and reduced receptor recycling to the cell surface, thus promoting KIT signaling in the endosomes while reducing that in the plasma membrane, as exemplified by Akt and PLCγ1 phosphorylation, respectively. The altered endocytic trafficking of KIT also resulted in an increase in SCF-induced mast cell proliferation and migration, which may reflect altered signaling in these cells. Our data reveal a novel function for MS4A family proteins in regulating trafficking and signaling, which could have implications in both proliferative and immunological diseases., (© 2015 Cruse et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Subsets of B cells inhibit various immune responses through their production of the cytokine interleukin-10 (IL-10). We found that IL-10-producing CD5(+) B cells suppressed the immunoglobulin E (IgE)- and antigen-mediated activation of mast cells in vitro as well as allergic responses in mice in an IL-10-dependent manner. Furthermore, the suppressive effect of these B cells on mast cells in vitro and in vivo depended on direct cell-to-cell contact through the costimulatory receptor CD40 on CD5(+) B cells and the CD40 ligand on mast cells. This contact enhanced the production of IL-10 by the CD5(+) B cells. Through activation of the Janus-activated kinase-signal transducer and activator of transcription 3 pathway, IL-10 decreased the abundance of the kinases Fyn and Fgr and inhibited the activation of the downstream kinase Syk in mast cells. Together, these findings suggest that an important function of IL-10-producing CD5(+) B cells is inhibiting mast cells and IgE-mediated allergic responses., (Copyright © 2015, American Association for the Advancement of Science.)

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.

The protein prohibitin (PHB) is implicated in diverse cellular processes, including cell signaling, transcriptional control, and mitochondrial function. We found that PHB was abundant in the intracellular granules of mast cells, which are critical for allergic responses to antigens. Thus, we investigated whether PHB played a role in signaling mediated by the high-affinity receptor for antigen-bound immunoglobulin E (IgE), FcεRI. PHB-specific small interfering RNAs (siRNAs) inhibited antigen-mediated signaling, degranulation, and cytokine secretion by mast cells in vitro. Knockdown of PHB inhibited the antigen-dependent association of the tyrosine kinase Syk with FcεRI and inhibited the activation of Syk. Fractionation studies revealed that PHB translocated from intracellular granules to plasma membrane lipid rafts in response to antigen, and knockdown of PHB suppressed the movement of FcεRIγ and Syk into lipid rafts. Tyrosine phosphorylation of PHB by Lyn was observed early after exposure to antigen, and point mutations in PHB indicated that Tyr(114) and Tyr(259) were required for the recruitment of Syk to FcεRIγ and mast cell activation. In mice, PHB-specific siRNAs inhibited antigen-initiated mast cell degranulation, passive cutaneous anaphylaxis, and passive systemic anaphylaxis. Together, these results suggest that PHB is essential for FcεRI-mediated mast cell activation and allergic responses in vivo, raising the possibility that PHB might serve as a therapeutic target for the treatment of allergic diseases.

Following antigen/IgE-mediated aggregation of high affinity IgE-receptors (FcεRI), mast cells (MCs) degranulate and release inflammatory mediators leading to the induction of allergic reactions including anaphylaxis. Migration of MCs to resident tissues and sites of inflammation is regulated by tissue chemotactic factors such as stem cell factor (SCF (KIT ligand)). 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 MC chemotaxis are rate limiting for MC mediator release. To investigate this issue, we disrupted actin polymerization, a requirement for MC chemotaxis, with latrunculin B and cytochalasin B, then examined chemotaxis and mediator release in human (hu)MCs induced by antigen or SCF. As expected, such disruption minimally affected early signaling pathways, but attenuated SCF-induced human mast cell chemotaxis. In contrast, SCF, in the absence of other stimuli, induced substantial degranulation in a concentration-dependent manner following actin disassembly. It also moderately enhanced antigen-mediated human mast cell degranulation which was further enhanced in the presence of SCF. These observations suggest that processes regulating cell migration limit MC degranulation as a consequence of cytoskeletal reorganization., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: 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., Objectives: We sought to determine the role and mechanism of DJ-1 in allergic responses in vitro and in vivo., Methods: 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., Results: 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., Conclusions: A deficiency or constitutive activation of DJ-1 can have implications in mast cell-driven allergic diseases, such as asthma and anaphylaxis., (Published by Mosby, Inc.)

Human linkage analyses have implicated the MS4A2-containing gene locus (encoding FcεRIβ) as a candidate for allergy susceptibility. We have identified a truncation of FcεRIβ (t-FcεRIβ) in humans that 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-FcεRIβ is critical for microtubule formation and degranulation and that it may perform this function by trafficking adaptor molecules and kinases to the pericentrosomal and Golgi region in response to Ca2+ signals. Mutagenesis studies suggest that calmodulin binding to t-FcεRIβ in the presence of Ca2+ could be critical for t-FcεRIβ function. In addition, gene targeting of t-FcεRIβ attenuated microtubule formation, degranulation, and IL-8 production downstream of Ca2+ signals. Therefore, t-FcεRIβ mediates Ca2+ -dependent microtubule formation, which promotes degranulation and cytokine release. Because t-FcεRIβ has this critical function, it represents a therapeutic target for the downregulation of allergic inflammation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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., (Published by Elsevier India Pvt Ltd.)

Activation of KIT, by its ligand, stem cell factor (SCF), results in the initiation of signal transduction pathways that influence mast cell survival and proliferation. Activating mutations in KIT have thus been linked to clonal MC proliferation associated with systemic mastocytosis. SCF also modulates MC function by inducing MC chemotaxis and by potentiating antigen (Ag)/IgE-mediated MC degranulation. Thus, mutations in KIT also have the potential to affect these processes in allergic and other mast cell-related diseases. Studies to determine how native and mutated KIT may modulate MC chemotaxis and activation have, however, been limited due to the lack of availability of a suitable functional MC line lacking native KIT which would allow transduction of KIT constructs. Here we describe a novel mouse MC line which allows the study of normal and mutated KIT constructs. These cells originated from a bone marrow-derived mouse MC culture out of which a rapidly dividing mast cell sub-population spontaneously arose. Over time, these cells lost KIT expression while continuing to express functional high affinity receptors for IgE (FcεRI). As a consequence, these cells degranulated in response to Ag/IgE but did not migrate nor show any evidence of potentiation of Ag/IgE degranulation in response to SCF. Retroviral transduction of the cells with a human (hu)KIT construct resulted in surface expression of huKIT which responded to huSCF by potentiation of Ag/IgE-induced degranulation and chemotaxis. This cell line thus presents a novel system to delineate how MC function is modulated by native and mutated KIT and for the identification of novel inhibitors of these processes., (Published by Elsevier B.V.)

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 Cγ(1) 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.

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.

Resveratrol, a polyphenol in red wine, has been reported as a calorie restriction mimetic with potential antiaging and antidiabetogenic properties. It is widely consumed as a nutritional supplement, but its mechanism of action remains a mystery. Here, we report that the metabolic effects of resveratrol result from competitive inhibition of cAMP-degrading phosphodiesterases, leading to elevated cAMP levels. The resulting activation of Epac1, a cAMP effector protein, increases intracellular Ca(2+) levels and activates the CamKKβ-AMPK pathway via phospholipase C and the ryanodine receptor Ca(2+)-release channel. As a consequence, resveratrol increases NAD(+) and the activity of Sirt1. Inhibiting PDE4 with rolipram reproduces all of the metabolic benefits of resveratrol, including prevention of diet-induced obesity and an increase in mitochondrial function, physical stamina, and glucose tolerance in mice. Therefore, administration of PDE4 inhibitors may also protect against and ameliorate the symptoms of metabolic diseases associated with aging., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Unlabelled: Reports indicate that prostaglandin (PG)E(2) markedly enhances antigen-mediated degranulation in mouse bone marrow-derived mast cells (BMMCs) but not in human mast cells (HuMCs). We have examined the underlying mechanism(s) for this disparity in HuMCs derived from the peripheral blood of multiple donors in addition to mouse BMMCs. HuMCs from half of these donors failed to respond to PGE(2) and the PGE(2) EP3 receptor agonist, sulprostone. However, HuMCs from the remaining donors and the LAD2 human MC line responded to PGE(2) and sulprostone with marked enhancement of antigen-mediated degranulation and IL-8 production in a similar manner to that observed in mouse BMMCs. The EP2 agonist, butaprost, failed to modulate antigen-mediated responses in any type of MCs. These distinct phenotypes could not be explained by differences in EP2 or EP3 expression nor by differences in the ability of PGE(2) to elevate levels of cAMP, a signal recognized to down-regulate mast cell activation. Moreover, both responder and non-responder HuMC populations exhibited similar activation of phosphatidylinositol 3-kinase, and MAP kinases. However, translocation of PLCγ(1) to the cell membrane and the associated calcium signal were enhanced only in the responder HuMC population indicating that the link between EP3 and PLCγ is impaired in the non-responder HuMCs., Conclusions: These data provide a cautionary note for the translating of observations in the mouse to human mast cell-dependent disorders, but may also provide a basis for examining the effects of co-activating receptors in patients susceptible to allergic conditions., (Published by Elsevier B.V.)

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., (Published by Elsevier India Pvt Ltd.)

Mast cells are critical for various allergic disorders. Mast cells express Src family kinases, which relay positive and negative regulatory signals by Ag. Lyn, for example, initiates activating signaling events, but it also induces inhibitory signals. Fyn and Hck are reported to be positive regulators, but little is known about the roles of other Src kinases, including Fgr, in mast cells. In this study, we define the role of Fgr. Endogenous Fgr associates with FcεRI and promotes phosphorylation of Syk, Syk substrates, which include linkers for activation of T cells, SLP76, and Gab2, and downstream targets such as Akt and the MAPKs in Ag-stimulated mast cells. As a consequence, Fgr positively regulates degranulation, production of eicosanoids, and cytokines. Fgr and Fyn appeared to act in concert, as phosphorylation of Syk and degranulation are enhanced by overexpression of Fgr and further augmented by overexpression of Fyn but are suppressed by overexpression of Lyn. Moreover, knockdown of Fgr by small interfering RNAs (siRNAs) further suppressed degranulation in Fyn-deficient bone marrow-derived mast cells. Overexpression of Fyn or Fgr restored phosphorylation of Syk and partially restored degranulation in Fyn-deficient cells. Additionally, knockdown of Fgr by siRNAs inhibited association of Syk with FcεRIγ as well as the tyrosine phosphorylation of FcεRIγ. Of note, the injection of Fgr siRNAs diminished the protein level of Fgr in mice and simultaneously inhibited IgE-mediated anaphylaxis. In conclusion, Fgr positively regulates mast cell through activation of Syk. These findings help clarify the interplay among Src family kinases and identify Fgr as a potential therapeutic target for allergic diseases.

IL-33 is associated with atopic and autoimmune diseases and, as reported here, it interacts synergistically with Ag to markedly enhance production of inflammatory cytokines in rodent mast cells even in the absence of degranulation. Investigation of the underlying mechanisms revealed that synergy in signaling occurred at the level of TGF-β-activated kinase 1, which was then transmitted downstream through JNK, p38 MAP kinase, and AP-1. Stimulation of the Ca(2+) /calcineurin/NFAT pathway by Ag, which IL-33 did not, was critical for the synergy between Ag and IL-33. For example, selective stimulation of the NFAT pathway by thapsigargin also markedly enhanced responses to IL-33 in a calcineurin-dependent manner. As indicated by luciferase-reporter assays, IL-33 failed to stimulate the transcriptional activities of NFAT and AP-1 but augmented the activation of these transcription factors by Ag or thapsigargin. Robust stimulation of NF-κB transcriptional activity by IL-33 was also essential for the synergy. These and pharmacologic data suggested that the enhanced production of cytokines resulted in part from amplification of the activation of AP-1 and NFAT as well as co-operative interactions among transcription factors. IL-33 may retune mast cell responses to Ag toward enhanced cytokine production and thus determine the symptoms and severity of Ag-dependent allergic and autoimmune diseases., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Prostaglandin (PG) E(2), a potent mediator produced in inflamed tissues, can substantially influence mast cell responses including adhesion to basement membrane proteins, chemotaxis, and chemokine production. However, the signaling pathways by which PGE(2) induces mast cell chemotaxis and chemokine production remains undefined. In this study, we identified the downstream target of phosphatidylinositol 3-kinase, mammalian target of rapamycin (mTOR), as a key regulator of these responses. In mouse bone marrow-derived mast cells, PGE(2) was found to induce activation of mTORC1 (mTOR complexed to raptor) as indicated by increased p70S6K and 4E-BP1 phosphorylation, and activation of mTORC2 (mTOR complexed to rictor), as indicated by increased phosphorylation of AKT at position Ser(473). Selective inhibition of the mTORC1 cascade by rapamycin or by the use of raptor-targeted shRNA failed to decrease PGE(2)-mediated chemotaxis or chemokine generation. However, inhibition of the mTORC2 cascade through the dual mTORC1/mTORC2 inhibitor Torin, or through rictor-targeted shRNA, resulted in a significant attenuation in PGE(2)-mediated chemotaxis, which was associated with a comparable decrease in actin polymerization. Furthermore, mTORC2 down-regulation decreased PGE(2)-induced production of the chemokine monocyte chemoattractant protein-1 (CCL2), which was linked to a significant reduction in ROS production. These findings are consistent with the conclusion that activation of mTORC2, downstream of PI3K, represents a critical signaling locus for chemotaxis and chemokine release from PGE(2)-activated mast cells.

Mast cells are multifunctional cells that initiate not only IgE-dependent allergic diseases but also play a fundamental role in innate and adaptive immune responses to microbial infection. They are also thought to play a role in angiogenesis, tissue remodeling, wound healing, and tumor repression or growth. The broad scope of these physiologic and pathologic roles illustrates the flexible nature of mast cells, which is enabled in part by their phenotypic adaptability to different tissue microenvironments and their ability to generate and release a diverse array of bioactive mediators in response to multiple types of cell-surface and cytosolic receptors. There is increasing evidence from studies in cell cultures that release of these mediators can be selectively modulated depending on the types or groups of receptors activated. The intent of this review is to foster interest in the interplay among mast cell receptors to help understand the underlying mechanisms for each of the immunological and non-immunological functions attributed to mast cells. The second intent of this review is to assess the pathophysiologic roles of mast cells and their products in health and disease. Although mast cells have a sufficient repertoire of bioactive mediators to mount effective innate and adaptive defense mechanisms against invading microorganisms, these same mediators can adversely affect surrounding tissues in the host, resulting in autoimmune disease as well as allergic disorders.

A calcium signal is essential for degranulation, generation of eicosanoids and optimal production of cytokines in mast cells in response to antigen and other stimulants. The signal is initiated by phospholipase C-mediated production of inositol1,4,5-trisphosphate resulting in release of stored Ca(2+) from the endoplasmic reticulum (ER) and Golgi. Depletion of these stores activates influx of extracellular Ca(2+), usually referred to as store-operated calcium entry (SOCE), through the interaction of the Ca(2+)-sensor, stromal interacting molecule-1 (STIM1 ), in ER with Orai1(CRACM1) and transient receptor potential canonical (TRPC) channel proteins in the plasma membrane (PM). This interaction is enabled by microtubular-directed reorganization of ER to form ER/PM contact points or "punctae" in which STIM1 and channel proteins colocalize. The ensuing influx of Ca(2+) replenishes Ca(2+) stores and sustains elevated levels of cytosolic Ca(2+) ions-the obligatory signal for mast-cell activation. In addition, the signal can acquire spatial and dynamic characteristics (e.g., calcium puffs, waves, oscillations) that encode signals for specific functional outputs. This is achieved by coordinated regulation of Ca(2+) fluxes through ATP-dependent Ca(2+)-pumps and ion exchangers in mitochondria, ER and PM. As discussed in this chapter, studies in mast cells revealed much about the mechanisms described above but little about allergic and autoimmune diseases although studies in other types of cells have exposed genetic defects that lead to aberrant calcium signaling in immune diseases. Pharmacologic agents that inhibit or activate the regulatory components of calcium signaling in mast cells are also discussed along with the prospects for development of novel SOCE inhibitors that may prove beneficial in the treatment inflammatory mast-cell related diseases.

Interleukin (IL)-33 is a recently described pro-inflammatory cytokine. Here we demonstrate IL-33 as a regulator of functional osteoclasts (OCs) from human CD14(+) monocytes. IL-33 stimulates formation of tartrate-resistant acid phosphatase (TRAP)(+) multinuclear OCs from monocytes. This action was suppressed by anti-ST2 antibody, suggesting that IL-33 acts through its receptor ST2, but not by the receptor activator of NF-κB ligand (RANKL) decoy, osteoprotegerin, or anti-RANKL antibody. IL-33 stimulated activating phosphorylations of signaling molecules in monocytes that are critical for OC development. These included Syk, phospholipase Cγ2, Gab2, MAP kinases, TAK-1, and NF-κB. IL-33 also enhanced expression of OC differentiation factors including TNF-α receptor-associated factor 6 (TRAF6), nuclear factor of activated T cells cytoplasmic 1, c-Fos, c-Src, cathepsin K, and calcitonin receptor. IL-33 eventually induced bone resorption. This study suggests that the osteoclastogenic property of IL-33 is mediated through TRAF6 as well as the immunoreceptor tyrosine-based activation motif-dependent Syk/PLCγ pathway in human CD14(+) monocytes.

Mast cells infiltrate the sites of inflammation associated with chronic atopic disease and during helminth and bacterial infection. This process requires receptor-mediated cell chemotaxis across a concentration gradient of their chemotactic ligands. In vivo, mast cells are likely to be exposed to several such agents, which can cooperate in a synergistic manner to regulate mast cell homing. Here, we report that chemotaxis of mouse bone-marrow-derived mast cells (BMMCs) in response to the chemoattractants stem-cell factor (SCF) and prostaglandin (PG)E(2), is substantially enhanced following antigen-dependent ligation of the high-affinity receptor for IgE (FcεRI). These responses were associated with enhanced activation of phosphoinositide 3-kinase (PI3K), and downstream activation of the tyrosine protein kinase Btk, with subsequent enhanced phospholipase (PL)Cγ-mediated Ca(2+) mobilization, Rac activation and F-actin rearrangement. Antigen-induced chemotaxis, and the ability of antigen to amplify responses mediated by SCF, adenosine and PGE(2) were suppressed following inhibition of PI3K, and were impaired in BMMCs derived from Btk(-/-) mice. There were corresponding decreases in the PLCγ-mediated Ca(2+) signal, Rac activation and F-actin rearrangement, which, as they are essential for BMMC chemotaxis, accounts for the impaired migration of Btk-deficient cells. Taken together, these data demonstrate that, by regulating signaling pathways that control F-actin rearrangement, Btk is crucial for the ability of antigen to amplify mast-cell chemotactic responses.

Initial IgE-dependent signaling events are associated with detergent-resistant membrane microdomains. Following Ag stimulation, the IgE-receptor (Fc(epsilon)RI ) accumulates within these domains. This facilitates the phosphorylation of Fc(epsilon)RI subunits by the Src kinase, Lyn, and the interaction with adaptor proteins, such as the linker for activation of T cells. Among the phospholipases (PL) subsequently activated, PLD is of interest because of its presence in lipid microdomains and the possibility that its product, phosphatidic acid, may regulate signal transduction and membrane trafficking. We find that in Ag-stimulated RBL-2H3 mast cells, the association of Fc(epsilon)RI with detergent-resistant membrane fractions is inhibited by 1-butanol, which subverts production of phosphatidic acid to the biologically inert phosphatidylbutanol. Furthermore, the knockdown of PLD2, and to a lesser extent PLD1 with small inhibitory RNAs, also suppressed the accumulation of Fc(epsilon)RI and Lyn in these fractions as well as the phosphorylation of Src kinases, Fc(epsilon)RI , linker for activation of T cells, and degranulation. These effects were accompanied by changes in distribution of the lipid microdomain component, ganglioside 1, in the plasma membrane as determined by binding of fluorescent-tagged cholera toxin B subunit and confocal microscopy in live cells. Collectively, these findings suggest that PLD activity plays an important role in promoting IgE-dependent signaling events within lipid microdomains in mast cells.

Our studies in the RBL-2H3 mast cell line suggest that responses to antigen (Ag) are negatively modulated through upregulation of Src-like adaptor protein (SLAP). Ag stimulation of RBL-2H3 cells leads to increased levels of SLAP (but not SLAP2) transcripts and protein over a period of several hours. The effects of pharmacologic inhibitors indicate that the upregulation of SLAP is dependent on multiple signaling pathways. Knockdown of SLAP with anti-SLAP siRNA is associated with enhanced phosphorylation of Syk, the linker for activation of T cells (LAT), phospholipase C gamma, MAP kinases, and various transcription factors. Production of IL-3 and MCP-1, but not degranulation, is also enhanced. The upregulation of SLAP may thus serve to limit the duration of cytokine production in Ag-stimulated cells.

Calcium signals mediate diverse cellular functions in immunological cells. Early studies with mast cells, then a preeminent model for studying Ca2+-dependent exocytosis, revealed several basic features of calcium signaling in non-electrically excitable cells. Subsequent studies in these and other cells further defined the basic processes such as inositol 1,4,5-trisphosphate-mediated release of Ca2+ from Ca2+ stores in the endoplasmic reticulum (ER); coupling of ER store depletion to influx of external Ca2+ through a calcium-release activated calcium (CRAC) channel now attributed to the interaction of the ER Ca2+ sensor, stromal interacting molecule-1 (STIM1), with a unique Ca2+-channel protein, Orai1/CRACM1, and subsequent uptake of excess Ca2+ into ER and mitochondria through ATP-dependent Ca2+ pumps. In addition, transient receptor potential channels and ion exchangers also contribute to the generation of calcium signals that may be global or have dynamic (e.g., waves and oscillations) and spatial resolution for specific functional readouts. This review discusses past and recent developments in this field of research, the pharmacologic agents that have assisted in these endeavors, and the mast cell as an exemplar for sorting out how calcium signals may regulate multiple outputs in a single cell.

Glucocorticoids suppress mast cell activation by inhibiting signaling events as well as transcription of cytokine genes. The inhibition of signaling events has been attributed to upregulation of inhibitory regulators such as Src-like adaptor protein1 (SLAP), downstream of tyrosine kinase1 (Dok1), and dual specificity protein phospahatase1 (DUSP1). As reported here, the upregulation of SLAP and Dok1, but not DUSP1, in the RBL-2H3 mast cell line was inhibited by actinomycin D and was thus dependent on gene transcription. Examination of the gene sequences revealed a glucocorticoid response element (GRE) and a half GRE as potential regulators of the SLAP and Dok1, respectively. As indicated by luciferase reporter assays, SLAP GRE, but not the Dok1 half GRE, robustly activated gene transcription after treatment of cells with glucocorticoids. Binding of the glucocorticoid receptor to the SLAP GRE was verified by chromatin immunoprecipitation assay. These findings further support the notion that the immunosuppressive actions of glucocorticoids are exerted in part through upregulation of inhibitory regulators by various mechanisms. In the case of SLAP specifically, this requires activation of gene transcription through the interaction of the glucocorticoid receptor with GRE.

Just over a century ago Paul Ehrlich received the Nobel Prize for his studies of immunity. This review describes one of his legacies, the histochemical description of the mast cell, and the research that has ensued since then. After a long period of largely descriptive studies, which revealed little about the biological role of the mast cell, the field was galvanized in the 1950s by the recognition that the mast cell was the main repository of histamine and a key participant in anaphylactic reactions. Although the mast cell was long-viewed in these terms, recent research has now shown that the mast cell also plays a key role in innate and adaptive immune responses, autoimmune disease, and possibly tissue homeostasis by virtue of its expression of a diverse array of receptors and biologically active products. In addition, the responsiveness of mast cells to immunological and pathological stimulants is highly modulated by the tissue cytokine environment and by synergistic, or inhibitory, interactions among the various mast cell receptor systems. This once enigmatic cell of Paul Ehrlich has proved to be both adaptable and multifunctional.

Activated mast cells are a major source of the eicosanoids PGD(2) and leukotriene C(4) (LTC(4)), which contribute to allergic responses. These eicosanoids are produced following the ERK1/2-dependent activation of cytosolic phospholipase A(2), thus liberating arachidonic acid, which is subsequently metabolized by the actions of 5-lipoxygenase and cyclooxygenase to form LTC(4) and PGD(2), respectively. These pathways also generate reactive oxygen species (ROS), which have been proposed to contribute to FcepsilonRI-mediated signaling in mast cells. In this study, we demonstrate that, in addition to ERK1/2-dependent pathways, ERK1/2-independent pathways also regulate FcepsilonRI-mediated eicosanoid and ROS production in mast cells. A role for the Tec kinase Btk in the ERK1/2-independent regulatory pathway was revealed by the significantly attenuated FcepsilonRI-dependent PGD(2), LTC(4), and ROS production in bone marrow-derived mast cells of Btk(-/-) mice. The FcepsilonRI-dependent activation of Btk and eicosanoid and ROS generation in bone marrow-derived mast cells and human mast cells were similarly blocked by the PI3K inhibitors, Wortmannin and LY294002, indicating that Btk-regulated eicosanoid and ROS production occurs downstream of PI3K. In contrast to ERK1/2, the PI3K/Btk pathway does not regulate cytosolic phospholipase A(2) phosphorylation but rather appears to regulate the generation of ROS, LTC(4), and PGD(2) by contributing to the necessary Ca(2+) signal for the production of these molecules. These data demonstrate that strategies to decrease mast cell production of ROS and eicosanoids would have to target both ERK1/2- and PI3K/Btk-dependent pathways.

Background: Activation of mast cells through the high-affinity receptor for IgE (FcepsilonRI) underlies atopic allergic reactions. Curcumin can block this activation, but the mechanism and the effects of curcumin on IgE-mediated allergic reactions are unknown., Objectives: We sought to determine the antiallergic activity of curcumin in vivo and its mechanism of action in mast cells., Methods: The antiallergic activity of curcumin was evaluated in mast cell cultures and the passive cutaneous anaphylaxis model. The effects of curcumin on mast cell signaling events were examined by using immunoblotting, immunoprecipitation, RT-PCR, and other molecular biologic approaches., Results: Curcumin inhibited antigen-mediated activation of mast cells and passive cutaneous anaphylaxis in mice. Suppression of degranulation and secretion of TNF-alpha and IL-4 was apparent at concentrations as low as 3 micromol/L curcumin in activated mast cells. Similar concentrations of curcumin suppressed Syk-dependent phosphorylations of the adaptor proteins linker of activated T cells and Grb2-associated binder 2, which are critical for mast cell activation. Although curcumin did not inhibit the phosphorylation of Syk itself, it directly inhibited Syk kinase activity in vitro. Further downstream, activating phosphorylations of Akt and the mitogen-activated protein kinases p38, p44/42 (extracellular signal-regulated kinase 1/2), and c-Jun N-terminal kinase, which are critical for the production of inflammatory cytokines, were also inhibited., Conclusions: Curcumin inhibits Syk kinase-dependent signaling events in mast cells and might thus contribute to its antiallergic activity. Therefore curcumin might be useful for the treatment of mast cell-related immediate and delayed allergic diseases.

Antigen/IgE-mediated mast cell activation via FcvarepsilonRI can be markedly enhanced by the activation of other receptors expressed on mast cells and these receptors may thus contribute to the allergic response in vivo. One such receptor family is the G protein-coupled receptors (GPCRs). Although the signaling cascade linking FcvarepsilonRI aggregation to mast cell activation has been extensively investigated, the mechanisms by which GPCRs amplify this response are relatively unknown. To investigate this, we utilized prostaglandin (PG)E2 based on initial studies demonstrating its greater ability to augment antigen-mediated degranulation in mouse mast cells than other GPCR agonists examined. This enhancement, and the ability of PGE2 to amplify antigen-induced calcium mobilization, was independent of phosphoinositide 3-kinase but was linked to a pertussis toxin-sensitive synergistic translocation to the membrane of phospholipase (PL)Cgamma and PLCbeta and to an enhancement of PLCgamma phosphorylation. This "trans-synergistic" activation of PLCbeta and gamma, in turn, enhanced production of inositol 1,4,5-trisphosphate, store-operated calcium entry, and activation of protein kinase C (PKC) (alpha and beta). These responses were critical for the promotion of degranulation. This is the first report of synergistic activation between PLCgamma and PLCbeta that permits reinforcement of signals for degranulation in mast cells.

Degranulation of mast cells in response to Ag or the calcium mobilizing agent, thapsigargin, is dependent on emptying of intracellular stores of Ca(2+) and the ensuing influx of external Ca(2+), also referred to as store-operated calcium entry. However, it is unlikely that the calcium release-activated calcium channel is the sole mechanism for the entry of Ca(2+) because Sr(2+) and other divalent cations also permeate and support degranulation in stimulated mast cells. In this study we show that influx of Ca(2+) and Sr(2+) as well as degranulation are dependent on the presence of the canonical transient receptor potential (TRPC) channel protein TRPC5, in addition to STIM1 and Orai1, as demonstrated by knock down of each of these proteins by inhibitory RNAs in a rat mast cell (RBL-2H3) line. Overexpression of STIM1 and Orai1, which are known to be essential components of calcium release-activated calcium channel, allows entry of Ca(2+) but not Sr(2+), whereas overexpression of STIM1 and TRPC5 allows entry of both Ca(2+) and Sr(2+). These and other observations suggest that the Sr(2+)-permeable TRPC5 associates with STIM1 and Orai1 in a stoichiometric manner to enhance entry of Ca(2+) to generate a signal for degranulation.

Although primarily required for the growth, differentiation, and survival of mast cells, Kit ligand (stem cell factor) is also required for optimal antigen-mediated mast cell activation. Therefore, concurrent inhibition of Kit- and FcepsilonRI-mediated signaling would be an attractive approach for targeting mast cell-driven allergic reactions. To explore this concept, we examined the effects of hypothemycin, a molecule that we identified as having such properties, in human and mouse mast cells. Hypothemycin blocked Kit activation and Kit-mediated mast cell adhesion in a similar manner to the well characterized Kit inhibitor imatinib mesylate (imatinib). In contrast to imatinib, however, hypothemycin also effectively inhibited FcepsilonRI-mediated degranulation and cytokine production in addition to the potentiation of these responses via Kit. The effect of hypothemycin on Kit-mediated responses could be explained by its inhibition of Kit kinase activity, whereas the inhibitory effects on FcepsilonRI-dependent signaling were at the level of Btk activation. Because hypothemycin also significantly reduced the mouse passive cutaneous anaphylaxis response in vivo, these data provide proof of principle for a coordinated approach for the suppression of mast cell activation and provide a rationale for the development of compounds with a similar therapeutic profile.

Generation of sphingosine-1-phosphate by sphingosine kinases (SphKs) promotes allergic inflammatory diseases, but the roles of the individual SphK isoforms are unclear. Olivera et al. (2007) show that SphK2 regulates mast cell activation whereas SphK1 enhances susceptibility to antigen challenge in vivo.

Mast cells play a pivotal role in inflammatory and immediate-type allergic reactions by secreting a variety of potent inflammatory mediators, including sphingosine-1-phosphate (S1P). However, it is not known how S1P is released from cells. Here, we report that S1P is exported from mast cells independently of their degranulation and demonstrate that it is mediated by ATP binding cassette (ABC) transporters. Constitutive and antigen-stimulated S1P release was inhibited by MK571, an inhibitor of ABCC1 (MRP1), but not by inhibitors of ABCB1 (MDR-1, P-glycoprotein). Moreover, down-regulation of ABCC1 with small interfering RNA, which decreased its cell surface expression, markedly reduced S1P export from both rat RBL-2H3 and human LAD2 mast cells. Transport of S1P by ABCC1 influenced migration of mast cells toward antigen but not degranulation. These findings have important implications for S1P functions in mast cell-mediated immune responses.

We have identified Src-like adaptor protein (SLAP) as one of several dexamethasone-inducible inhibitory regulators in mast cells. SLAP is a known inhibitor of T cell signaling and interacts with the tyrosine kinase, Zap70. Exposure of RBL-2H3 mast cells to dexamethasone markedly increased expression of SLAP. Cells so exposed or made to overexpress SLAP exhibited reduced Ag-stimulated phosphorylation of Syk (a cognate of Zap70), linker for activation of T cells, phospholipase Cgamma, and ERK. Ca(2+) mobilization, Ca(2+)-dependent degranulation, and ERK-dependent release of arachidonic acid were suppressed as well. Small interfering RNA directed against SLAP blocked the induction of SLAP and reversed the inhibitory effects of dexamethasone on phosphorylation of Syk, linker for activation of T cells, and phospholipase Cgamma, but not downstream events, which are likely suppressed by up-regulation of downstream of tyrosine kinase-1 and MAPK phosphatase-1. The induction of these inhibitory regulators may contribute to the immunosuppressive activity of dexamethasone in mast cells.

Mast cells are responsible for IgE-mediated allergic reactions. Phospholipase D1 (PLD1) and PLD2 regulate mast cell activation, but the mechanisms remain unclear. Here we show that PLD2 associates with and promotes activation of Syk, a key enzyme in mast cell activation. Antigen stimulation resulted in increased association and colocalization of Syk with PLD2 on the plasma membrane as indicated by coimmunoprecipitation and confocal microscopy. This association was dependent on tyrosine phosphorylation of Syk but not on PLD2 activity. In vitro, PLD2 interacted via its Phox homology (PX) domain with recombinant Syk to induce phosphorylation and activation of Syk. Furthermore, overexpression of PLD2 or catalytically inactive PLD2K758R enhanced antigen-induced phosphorylations of Syk and its downstream targets, the adaptor proteins LAT and SLP-76, while expression of a PLD2 siRNA blocked these phosphorylations. Apparently, the interaction of PLD2 with Syk is an early critical event in the activation of mast cells.

Engagement of the FcepsilonRI expressed on mast cells induces the production of phosphatidylinositol 3, 4, 5-trisphosphate by PI3K, which is essential for the functions of the cells. PTEN (phosphatase and tensin homologue deleted on chromosome ten) directly opposes PI3K by dephosphorylating phosphatidylinositol 3, 4, 5-trisphosphate at the 3' position. In this work we used a lentivirus-mediated short hairpin RNA gene knockdown method to study the role of PTEN in CD34(+) peripheral blood-derived human mast cells. Loss of PTEN caused constitutive phosphorylation of Akt, p38 MAPK, and JNK, as well as cytokine production and enhancement in cell survival, but not degranulation. FcepsilonRI engagement of PTEN-deficient cells augmented signaling downstream of Src kinases and increased calcium flux, degranulation, and further enhanced cytokine production. PTEN-deficient cells, but not control cells, were resistant to inhibition of cytokine production by wortmannin, a PI3K inhibitor. The findings demonstrate that PTEN functions as a key regulator of mast cell homeostasis and FcepsilonRI-responsiveness.

Mast cells mediate both IgE-dependent allergic reactions and protective responses against acute infections, possibly through the activation of Toll-like receptors (TLRs). We find that antigen interacts synergistically with TLR2 and TLR4 ligands to markedly enhance production of cytokines in murine mast cell lines. However, the TLR ligands neither stimulated degranulation and release of arachidonic acid nor influenced such responses to antigen, probably because these ligands failed to generate a necessary calcium signal. The enhanced cytokine production could be attributed to synergistic activation of mitogen-activated protein kinases in addition to the engagement of a more effective repertoire of transcription factors for cytokine gene transcription. The synergistic interactions of TLR ligands and antigen might have relevance to the exacerbation of IgE-mediated allergic diseases by infectious agents.

Stem cell factor (SCF) acts in synergy with antigen to enhance the calcium signal, degranulation, activation of transcription factors, and cytokine production in human mast cells. However, the underlying mechanisms for this synergy remain unclear. Here we show, utilizing bone marrow-derived mast cells (BMMCs) from Btk and Lyn knock-out mice, that activation of Btk via Lyn plays a key role in promoting synergy. As in human mast cells, SCF enhanced degranulation and cytokine production in BMMCs. In Btk-/- BMMCs, in which there was a partial reduction in the capacity to degranulate in response to antigen, SCF was unable to enhance the residual antigen-mediated degranulation. Furthermore, as with antigen, the ability of SCF to promote cytokine production was abrogated in the Btk-/- BMMCs. The impairment of responses in Btk-/- cells correlated with an inability of SCF to augment phospholipase Cgamma1 activation and calcium mobilization, and to phosphorylate NFkappaB and NFAT for cytokine gene transcription in these cells. Similar studies with Lyn-/- and Btk-/-/Lyn-/- BMMCs indicated that Lyn was a regulator of Btk for these responses. These data demonstrate, for the first time, that Btk is a key regulator of a Kit-mediated amplification pathway that augments Fc epsilonRI-mediated mast cell activation.

Activation of phospholipase D (PLD) and protein kinase C (PKC) as well as calcium mobilization are essential signals for degranulation of mast cells. However, the exact role of PLD in degranulation remains undefined. In this study we have tested the hypothesis that the PLD product, phosphatidic acid, and diacylglycerides generated therefrom might promote activation of PKC. Studies were conducted in two rodent mast cell lines that were stimulated with Ag via FcepsilonRI and a pharmacologic agent, thapsigargin. Diversion of production of phosphatidic acid to phosphatidylbutanol (the transphosphatidylation reaction) by addition of l-butanol suppressed both the translocation of diacylglyceride-dependent isoforms of PKC to the membrane and degranulation. Tertiary-butanol, which is not a substrate for the transphosphatidylation, had a minimal effect on PKC translocation and degranulation, and 1-butanol itself had no effect on PKC translocation when PKC was stimulated directly with phorbol ester, 12-O-tetradecanoylphorbol-13-acetate. Also, in cells transfected with small inhibitory RNAs directed against PLD1 and PLD2, activation of PLD, generation of diacylglycerides, translocation of PKC, and degranulation were all suppressed. Phorbol ester, which did not stimulate degranulation by itself, restored degranulation when used in combination with thapsigargin whether PLD function was disrupted with 1-butanol or the small inhibitory RNAs. However, degranulation was not restored when cells were costimulated with Ag and phorbol ester. These results suggested that the production of phosphatidic acid by PLD facilitates activation of PKC and, in turn, degranulation, although additional PLD-dependent processes appear to be critical for Ag-mediated degranulation.

The glucocorticoid dexamethasone suppresses antigen-induced degranulation, cytokine production, and intermediate signaling events in RBL-2H3 mast cells, although the exact mechanisms are uncertain. By microarray analysis, we discovered that expression of the inhibitory adaptor protein, downstream of tyrosine kinase (Dok)-1, was up-regulated 4-fold in dexamethasone-treated RBL-2H3 cells. The up-regulation was apparent with as little as 1 to 10 nM dexamethasone. Treatment with dexamethasone also enhanced tyrosine phosphorylation of Dok-1, augmented recruitment of Ras GTPase-activating protein (RasGAP) by Dok-1, and inhibited activation of the mitogen-activated protein (MAP) kinase pathway in antigen-stimulated cells. The same effects were obtained by transient overexpression of Dok-1 but not by overexpression of Dok-1 that was mutated in RasGAP-binding domain. The negative regulatory role of Dok-1 was further validated by the expression of small interfering RNA directed against Dok-1, which enhanced activation of MAP kinase and subsequent release of arachidonic acid and tumor necrosis factor-alpha. These findings identify Dok-1 as mediator of the antiallergic actions of dexamethasone and as a negative regulator of the MAP kinase pathway and downstream release of inflammatory mediators.