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Inflammatory mediators released from mast cells (MCs) through engagement of the high-affinity receptor for IgE (FcɛRI) have pivotal roles in chemical allergen-induced contact hypersensitivity (CHS) reactions, which suggests that the blockade of MC activation through FcɛRI stimulation may attenuate allergic contact dermatitis (CD). To address this possibility, we employed the following two approaches: (i) modulation of FcɛRI-mediated MC activation by introducing mutations in tyrosine residues of the FcɛRI β-chain immunoreceptor tyrosine-based activation motif (ITAM) and (ii) blockade of FcɛRI-mediated MC activation employing a recombinant soluble ecto-domain of the human FcɛRIα-chain (rsFcɛRIα). In this study, we show that optimal MC activation through the FcɛRI β-chain ITAM has essential roles in the onset of CHS to oxazolone (Oxa), a well-characterized chemical allergen. In addition, we demonstrate that administration of the rsFcɛRIα after sensitization successfully prevents murine CHS to Oxa. In a chronic CD model elicited by multiple challenges with low-dose Oxa, application of the rsFcɛRIα during the course of the challenges showed suppressive effects on CHS to Oxa. Taken together, our data indicate that inhibition of FcɛRI-dependent MC activation can suppress allergic CD. [ABSTRACT FROM AUTHOR]

Abstract: Src homology region 2 domain-containing phosphatase-1 (SHP-1) is known to act as a negative signal modulator in mast cells but its roles in cell survival and cell death are poorly understood. We previously reported that SHP-1 also positively regulates mast cell activation signaling by acting as an adaptor protein. In the present study, we examined whether SHP-1 plays a role in antigen (Ag)-induced activation-induced mast cell death. Bone marrow-derived mast cells (BMMCs) from SHP-1-deficient motheaten (me) mice (me-BMMCs) were significantly less susceptible to store-operated Ca2+ channel (SOC) activation, Ag-induced cell death and DNA fragmentation than BMMCs from their wild-type littermates (WT-BMMCs). Subsequent experiments revealed that the differences in these cellular susceptibilities to SOC activation and cell death resulted from the extent of the mitochondrial permeability transition pore (mPTP) opening. Specifically, mPTP opening was sufficiently persistent in WT-BMMCs to evoke mitochondrial integrity disruption, while mPTP opening was too transient to cause the minimal mitochondrial integrity collapse in me-BMMCs. In addition, pro-survival signaling including activation of mitogen-activated protein kinases (MAPKs) such as the extracellular signal-regulated protein kinases, c-Jun NH2 terminal kinases and p38 and the expression of Bcl-xL were significantly prolonged in me-BMMCs compared with WT-BMMCs. Taken together, these data demonstrate that a lack of SHP-1 prevents the mPTP-mediated mitochondrial integrity collapse and augments anti-apoptotic signaling such as MAPKs and Bcl-xL. These findings suggest that SHP-1 positively regulates mitochondrial death pathways and negatively regulates pro-survival signaling pathways. [Copyright &y& Elsevier]

Abstract: In non-excitable cells, store-operated Ca2+ channels (SOCs) are the principal routes of Ca2+ entry. Recently, store-independent Ca2+ channels which are pharmacologically and/or immunologically similar to L-type Ca2+ channels (LTCCs) have been shown to exist in various hematopoietic cells, including T cells, B cells and neutrophils. We previously reported that mast cells express LTCCs which regulate mast cell effector responses in a distinct manner from SOCs. In the present study, we examined the possible role for LTCCs in mast cell survival. Both RBL-2H3 mast cells and bone marrow-derived mast cells underwent considerable apoptosis after treatment with thapsigargin (Tg) but not stimulation through the high-affinity IgE receptor (FcɛRI). The LTCC-selective antagonists such as nifedipine greatly augmented FcɛRI-mediated apoptosis, while the LTCC-selective agonist (S)-BayK8644 blocked Tg-induced apoptosis. The modulation of apoptosis was accompanied by altered mitochondrial integrity, as measured with the mitochondrial membrane potential, cytochrome c release and caspase-3/7 activation. FcɛRI stimulation induced mitochondrial Ca2+ ([Ca2+]m) entry through both SOCs and LTCCs, while Tg evoked [Ca2+]m entry through LTCCs but not SOCs. The LTCC-selective antagonists blocked [Ca2+]m entry, whereas (S)-BayK8644 augmented Tg-induced [Ca2+]m entry. Moreover, blockade of the expression of the α1C subunit of Cav1.2 LTCC using small-interfering RNA strongly augmented FcɛRI-mediated apoptosis, mitochondrial integrity, and mitochondrial Ca2+ collapse, and abolished the protective effects of (S)-BayK8644 against Tg-induced apoptosis. These findings suggest that Cav1.2 LTCC protects mast cells against activation-induced cell death by preventing mitochondrial integrity disruption. [Copyright &y& Elsevier]

Abstract: Mast cells and T cells produce reactive oxygen species (ROS) after stimulation with the high-affinity IgE receptor (FcɛRI) and T cell receptor. A growing body of evidence suggests the existence of ROS-regulated intracellular and/or plasma membrane Ca2+ channels in these cells but their molecular entities remain to be identified. Here, we report that store-operated Ca2+ channel (SOC) activity is regulated by superoxide (O2 •−) and hydrogen peroxide (H2O2) in mast cells. MnTBaP (Mn(III)tetrakis(4-benzoic acid)porphyrin) and ebselen (2-phenyl-1,2-benziso-selenazol-3(2H)-one) selectively blocked the generation of O2 •− and H2O2, respectively, in antigen-stimulated cells. The H2O2 generation was dependent on the Src family kinase (SFK) and phosphatidylinositol-3-kinase (PI3K) activities but independent of extracellular Ca2+, and the FcɛRI β-chain immunoreceptor tyrosine-based activation motif played an essential role. On the other hand, O2 •− generation was strictly dependent on extracellular Ca2+, but negatively regulated by the SFK and PI3K activities. Inhibition of O2 •− generation resulted in increased H2O2 generation and reduced SOC activity, although it had a minimal effect on endoplasmic reticulum Ca2+ store depletion. On the contrary, inhibition of H2O2 generation resulted in increased intracellular O2 •− generation and augmented SOC activity. The findings suggest that O2 •− and H2O2, which are generated by separate signaling pathways/sources, reciprocally regulate SOC activity in mast cells. Such generations of multiple oxidant species and their distinct roles in the regulation of SOC activity may facilitate the fine tuning of Ca2+ signaling in mast cells. [Copyright &y& Elsevier]

Abstract: Store-operated Ca2+ channels (SOCs) are considered to be the principal route of Ca2+ influx in non-excitable cells. We have previously shown that in mast cells IgE+antigen (Ag) induces a dihydropyridine (DHP)-sensitive Ca2+ influx independently of Ca2+ store depletion. Since the DHP receptor is the α subunit of L-type Ca2+ channels (LTCCs), we examined the possible role of LTCCs in mast cell activation. Mast cells exhibited substantial expression of the α1C (CaV1.2) subunit mRNA and protein on their cell surface. IgE+Ag-induced Ca2+ influx was substantially reduced by the LTCC inhibitor nifedipine, and enhanced by the LTCC activator (S)-BayK8644, whereas these agents had minimal effects on thapsigargin (TG)-induced Ca2+ influx. These LTCC-modulating agents regulated IgE+Ag-induced cell activation but not TG-induced cell activation. Inhibition of SOCs by 2-aminoethoxydiphenyl borate reduced both degranulation and production of cytokines, including interleukin-13 and tumor necrosis factor-α, whereas LTCC modulation reciprocally regulated degranulation and cytokine production. IgE+Ag, but not TG, induced substantial plasma membrane depolarization, which stimulated a DHP-sensitive Ca2+ response. Moreover, IgE+Ag-, but not TG-induced mitochondrial Ca2+ increase was regulated by LTCC modulators. Finally, gene silencing analyses using small interfering RNA revealed that the α1C (CaV1.2) LTCC mediated the pharmacological effects of the LTCC-modulating agents. These results demonstrate that mast cells express LTCCs, which becomes activated by membrane depolarization to regulate cytosolic and mitochondrial Ca2+, thereby controlling mast cell activation in a distinct manner from SOCs. [Copyright &y& Elsevier]

Abstract: Aspirin is a well-known nonsteroidal anti-inflammatory drug (NSAID) that may potentiate some acute allergies and causes adverse immunological reactions collectively referred to as aspirin intolerance. Aspirin intolerance is accompanied by increased leukotriene (LT) synthesis, and high levels of serum IgE are a risk factor for NSAID sensitivity. Here we demonstrate that aspirin modulates LTC4 secretion in mast cells. Therapeutic levels of aspirin and salicylates (≤0.3 mM, i.e., the concentrations observed in vivo in the use of antipyretic analgesic) increased IgE-mediated LTC4 secretion. Aspirin-induced stimulation was accompanied by increased Ser-505 phosphorylation of cytosolic phospholipase A2, which occurred independently of extracellular signal-regulated protein kinase-1/2 and p38 mitogen-activated protein kinase pathways. Aspirin also increased IgE-mediated Ca2+ influx, whereas aspirin at concentrations of ≥0.3 mM dose-dependently reduced Ca2+ store emptying and Ca2+ release-activated Ca2+ channel activation. Instead, aspirin facilitated a dihydropyridine receptor-mediated Ca2+ influx, resulting in increased LTC4 secretion. This novel action of aspirin may play roles in exacerbation of immediate allergy and aspirin intolerance. [Copyright &y& Elsevier]

Abstract: Pharmacological modulation of IgE-mediated mast cell activation is important to the development of anti-allergic reagents. In this study, we investigated the effects of parthenolide (PTL) on high-affinity IgE receptor (FcεRI)-induced degranulation in mast cells. PTL dose-dependently inhibited degranulation induced by IgE·antigen stimulation in RBL-2H3 cells and BMMCs. Although PTL is a potent NF-κB inhibitor by targeting IκB kinase complex, NF-κB inhibition by other IκB kinase inhibitors did not inhibit degranulation in mast cells. IgE·antigen-induced microtubule formation is well known to be critical for degranulation in mast cells. Immunocytochemical study with anti-α-tubulin antibody revealed that PTL significantly inhibited IgE·antigen-induced microtubule formation. However, PTL, as well as nocodazol, had no significant effects on degranulation in the fyn-deficient BMMCs, suggesting that inhibitory effects of PTL in the microtubule formation are fyn dependent. We further demonstrated that in vivo administration of PTL in mice strongly inhibited passive cutaneous anaphylaxis reaction. The present study provides a possibility to develop potent reagents against mast cell activation based on an inhibition of microtubule formation. [Copyright &y& Elsevier]

Abstract: A growing body of evidence suggests that mitochondria take up calcium upon receptor (agonist) stimulation and that this contributes to the dynamics of spatiotemporal calcium signaling. We have previously shown that engagement of the high-affinity receptor for immunoglobulin E (FcɛRI) stimulates mitochondrial calcium ([Ca2+]m) uptake in mast cells. The present study was undertaken to investigate the mechanisms and biological significance of FcɛRI regulation of [Ca2+]m. Antigen stimulated [Ca2+]m uptake in a dose-dependent manner with a minimal effective dose of 0.03–3ng/ml. This [Ca2+]m uptake took place immediately, reaching its peak within minutes and was inhibited by the src family kinase inhibitor PP1 and phosphatidylinositol-3-kinase inhibitor wortmannin. Analyses using mast cells expressing the wild-type or the mutated type of the FcɛRIβ immunoreceptor tyrosine-based activation motif (ITAM) in which all tyrosine residues were replaced by phenylalanine revealed that the FcɛRIβ ITAM is essential for a sustained [Ca2+]m uptake. The FcɛRIβ ITAM was essential for overall calcium response upon weak FcɛRI stimulation (at low antigen concentration), while upon strong stimulation (at high antigen concentration) it appeared necessary selectively to an immediate calcium response that was sensitive to the dihydropyridine receptor (DHPR) antagonist nifedipine and wortmannin but not to the store-operated calcium entry (SOCE) antagonists such as 2-aminoethoxyphenyl borate and SK&F96365. These data demonstrate that the FcɛRIβ regulates [Ca2+]m uptake in mast cells via the ITAM and suggest that this plays a key role in regulating calcium influx especially that induced via a DHPR-mediated calcium channel. [Copyright &y& Elsevier]

Abstract: Prolactin (PRL) was originally identified by its ability to stimulate mammary development and lactation, and its essential roles other than lactation have recently been implicated in female reproduction. However, little is known about PRL-mediated events in pregnancy. The tryptophan catabolism enzyme indoleamine 2,3-dioxygenase (IDO) is interferon-γ (IFN-γ)-inducible and has recently become a focus for maternal–fetal tolerance for successful pregnancy. Based on recognition that PRL is one of the up-regulated hormones in pregnancy, in a previous study we have shown that PRL induces IDO expression in monocytes in cooperation with a suboptimal concentration of IFN-γ. Here, we demonstrate that PRL sensitizes monocytes to induce IDO expression in response to low doses of IFN-γ without affecting the typical IFN-γ signaling events, such as STAT1 phosphorylation and IRF-1 induction. In addition, IDO induction in these cell cultures was observed only after 24h pre-exposure to PRL. These results indicate a priming effect of PRL on monocytes that occurs before IFN-γ signaling and increases their sensitivity to IFN-γ for IDO induction, rather than a synergistic effect of PRL and IFN-γ on IDO induction. These results offer new insights into the roles of PRL in female reproduction, as well as provide a better understanding as to how IDO expression is regulated and achieved in pregnancy. [Copyright &y& Elsevier]

Abstract: In genetically susceptible human and/or experimental animals, heavy metals such as mercury, gold, and silver have been shown to highly induce adverse immunological reactions such as allergy and autoimmunity, in which mast cell degranulation is implicated as playing a role. We previously reported that silver activates mast cells and induces Ca2+ influx without stimulating intracellular signaling events required for activation of store-operated Ca2+ channels (SOCs). The purpose of the present study was to elucidate the possible involvement of reactive oxygen species (ROS) in the biological effects of silver. Analysis using oxidant-sensitive fluorescent probes such as dichlorodihydrofluorescein and scopoletin, as well as MCLA-amplified chemiluminescence, showed that silver induced intracellular production and/or extracellular release of ROS. Silver induced mast cell degranulation in a Ca2+-dependent manner. Unlike IgE antigen, silver-induced Ca2+ influx was not affected by depletion of internal Ca2+ stores. Instead, the metal-induced Ca2+ influx was abolished and reversed by the cell-impermeant thiol-reducing agent dithiothreitol, indicating the regulation by oxidation of vicinal thiols on the cell surface. Consistent with this view, Ca2+ influx was blocked by the glutathione peroxidase mimetic ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) and the superoxide dismutase mimetic manganese(III) tetrakis 4-(benzoic acid)porphyrin, but not by exogenously added catalase or superoxide dismutase. These findings indicate that silver evokes the release of ROS and oxidation of thiols critical for the activation of a Ca2+ channel other than SOC. Such a novel ROS-dependent pathway might play a role in mast cell degranulation in metal-induced allergic and autoimmune reactions. [Copyright &y& Elsevier]

Abstract: Fungal secondary metabolites such as gliotoxin, an epipolythiodioxopiperazine toxin produced by pathogenic fungi like Candida and Aspergillus, possess immunosuppressive activities and have been thought to contribute to pathology of fungal infections in animals and humans. Since recent studies show that mast cell plays a crucial role in the front of host defense, we examined whether fungal secondary metabolites affected mast cell activation. We found that gliotoxin had suppressive effects on FcεRI-dependent or -independent mast cell activation, including degranulation, leukotriene C4 secretion, and TNF-α and IL-13 production. Gliotoxin also suppressed intracellular Ca2+ rise through store-operated Ca2+ channels with a minimal effect on depletion of internal Ca2+ stores. Finally, gliotoxin induced intracellular production of superoxide possibly through a thiol redox cycling, which appeared to mediate suppressive effects on mast cell activation. These findings suggest that suppression of mast cell activation might contribute to the establishment of infections with gliotoxin-producing fungi. [Copyright &y& Elsevier]

Abstract: Platelet activation and the formation of platelet microaggregates in coronary vessels play pivotal roles in myocardial ischemia and reperfusion injury. The Fc receptor γ-chain (FcR γ) is coexpressed with glycoprotein (GP) VI, forming a platelet collagen receptor, and the activation of platelets by collagen is closely coupled with tyrosine phosphorylation of the FcRγ. To examine the functional significance of platelet FcR γ/GPVI complex in the early phase of myocardial ischemia and reperfusion injury in mice, we performed coronary occlusion and reperfusion experiments using wild type mice and FcRγ-deficient (FcRγ–/–) mice that lack GPVI. The infarct size was significantly smaller in FcRγ–/– mice subjected to occlusion and reperfusion of the coronary artery than in control FcR γ+/+ mice. Twenty-four hours after the reperfusion, electron microscopy of the injured tissue showed substantially more platelet aggregation and occlusive platelet microthrombi in the capillaries of the damaged areas of the wild type mice than in those of the FcR γ–/– mice. Platelet Syk was scarcely activated in the FcR γ–/– mice after myocardial ischemia and reperfusion, but significantly activated in the FcR γ+/+ mice. CD11b expression on neutrophils was elevated after myocardial ischemia and reperfusion in both mouse groups, whereas myeloperoxidase activity in the injured areas was significantly lower in the FcRγ–/– mice than in the FcRγ+/+ mice. These results suggest that the collagen-induced activation of platelets through the FcR γ plays a pivotal role in the extension of myocardial ischemia-reperfusion injury. FcRγ and GPVI may be important therapeutic targets for myocardial ischemia-reperfusion injury. [Copyright &y& Elsevier]

Summary: Background:: Atopic dermatitis (AD) and psoriasis are common inflammatory skin diseases. Although many reports implicate Th2 cytokines in the pathophysiology of AD and Th1 cytokines in psoriasis, the precise etiology of these diseases remains elusive. Objective:: We investigated novel AD- or psoriasis-related genes to further understand the pathogenesis of these diseases. Methods:: We performed a comprehensive analysis of mRNA expression in skin biopsies from AD or psoriasis patients using DNA microarrays. Quantitative PCR was then used to monitor the expression of novel disease-related genes in human keratinocytes or pinnae from NC/Nga mice. Results:: Levels of mRNA for IDO (indoleamine 2,3-dioxygenase) and kynureninase, enzymes constituting the tryptophan degradation pathway, were found to be upregulated in the skin lesions as compared to the uninvolved skin of patients with AD or psoriasis. Expression of these two genes was induced in human epidermal keratinocytes stimulated with IFN-γ in vitro. Moreover, in NC/Nga mice, the expression of kynureninase mRNA in the ear skin was induced following development of AD-like skin lesions. Conclusion:: The tryptophan degradation pathway may play an important role in the pathophysiology of AD and psoriasis. [Copyright &y& Elsevier]

Examines the bone marrow-derived mast cells from toll-like receptor 2 (TLR2) or TLR4-gene-targeted mice to clarify the functional roles of TLRs 2 and 4 in mast cells. Measurement of cytokine concentrations; Pattern of cytokine production by mast cells upon activation via TLR2 or TLR4; TLR2 on mast cell-dependent skin inflammation upon application of peptidoglycan.

ABSTRACTAnti-IgE and anti-FcℇRI α chain auto-antibodies have been detected in the sera of atopic and non-atopic subjects; however, the biological activity of these auto-antibodies is still unclear. These two kinds of auto-antibodies were examined in atopic dermatitis (AD) patients. In results, 12 of 92 AD patients have the anti-IgE, eight have the anti-FcℇRI α auto-antibodies and two have both auto-antibodies. Furthermore, biological characterization of these auto-antibodies has been performed. In results, three of 12 samples with the anti-IgE auto-antibodies exhibited inhibitory activity for IgE-FcℇRI α binding. Two of the eight samples with the anti-FcℇRI α auto-antibodies and one of the two samples with both auto-antibodies had histamine releasing activity. It is documented here that both anti-IgE and anti-FcℇRI α auto-antibodies were detected in some AD patients, some anti-IgE auto-antibodies had inhibitory activity for IgE-FcℇRI binding and also histamine releasing activity was detected in the anti-FcℇRI α chain auto-antibody. [ABSTRACT FROM AUTHOR]

We evaluated high-affinity receptor for IgE (FcεRI)-binding serum IgE in patients with atopic kerato- conjunctivitis (AKC; n = 31) and with seasonal allergic conjunctivitis (SAC; n = 13) by enzyme-linked immunosorbent assay (ELISA) using a recombinant soluble form of the human FcεRIα ectodomain (soluble α). The quantities of FcεRI-binding IgE are compared with those of total IgE measured by a conventional sandwich ELISA. Both of the quantities of FcεRI-binding and total IgE in AKC were significantly larger than those in SAC (P < 0.001). In contrast, the proportion of FcεRI-binding IgE (FcεRI-binding IgE/total IgE; %) in SAC was significantly larger than that in AKC (P < 0.001), although significant reverse correlation was observed between the proportion of FcεRI-binding IgE and total IgE in both AKC and SAC. Significantly, a higher proportion of FcεRI-binding IgE in SAC than that in AKC may reflect the differences in pathologic states of AKC and SAC that are caused by a disparity in immune responses in these diseases. [ABSTRACT FROM AUTHOR]

Background Interleukin (IL)-17A plays a pivotal role in the pathogenesis of rheumatoid arthritis (RA). The expression of IL-17A in synovial mast cells (MCs) in RA and osteoarthritis (OA) has been reported, but the frequencies of IL-17A expression in synovial MCs have varied. The aim of this study was to investigate whether IL-17A expression is upregulated in human synovial MCs in RA and to elucidate the mechanism of IL-17A expression in synovial MCs. Methods Synovial tissues were obtained from patients with RA or OA undergoing joint replacement surgery, and synovial MCs were enzymatically dispersed. Synovium-derived cultured MCs were generated by culturing synovial cells with stem cell factor. IL-17A expression was investigated using immunofluorescence in synovial tissues. IL-17A mRNA expression and its production from MCs were examined using RT-PCR and ELISA, respectively. Results The number of IL-17A-positive (+) synovial MCs and the percentage of IL-17A + MCs among all the IL-17A + cells from RA patients were not significantly increased compared with those from OA subjects. The synovium-derived cultured MCs spontaneously released small amounts of IL-17A. Neither IgE- nor IgG-dependent stimulation increased IL-17A production from the MCs. IL-33, tumor necrosis factor-α, C5a, lipopolysaccharide or IL-23 plus IL-1β did not affect IL-17A production in MCs. Conclusions The synovial MCs are not a main source of IL-17A in RA. [ABSTRACT FROM AUTHOR]

Background The activation of liver X receptor (LXR) α or LXRβ negatively regulates the expression of pro-inflammatory genes in mammalian cells. We recently reported that 25-hydroxycholesterol, a representative LXR-activating oxysterol, suppresses IL-6 production in mouse mast cells (MCs) following its engagement of the high-affinity IgE receptor (FcεRI). This finding suggests that murine MCs express functional LXRs; however, the mechanisms underlying the LXR-dependent repression of the MC-mediated production of pro-inflammatory cytokines, including IL-6, are poorly understood. Therefore, we employed the synthetic LXR ligand GW3965 to examine the functions of LXRα and LXRβ in the production of pro-inflammatory cytokines by murine bone marrow-derived MCs (BMMCs). Methods We prepared BMMCs from wild-type (WT), LXRα −/− , and LXRα/β −/− mice. Each group of BMMCs was pretreated with GW3965 and then stimulated with IgE+antigen (Ag) or lipopolysaccharide (LPS). Cytokine production was then analyzed using specific ELISA kits. Results The activation of LXRs by GW3965 significantly attenuated the production of IL-1α and IL-1β, but not of IL-6, in the WT and LXRα −/− BMMCs stimulated with IgE+Ag. However, GW3965 treatment decreased the production of IL-1α, IL-1β, and IL-6 in WT and LXRα −/− BMMCs upon stimulation with LPS, while the GW3965-mediated suppression of cytokine production was nearly absent from the LXRα/β −/− BMMCs. Conclusions These findings demonstrate, for the first time, that the activation of LXRs by GW3965 attenuates the antigen- or LPS-induced production of pro-inflammatory cytokines, such as IL-1α and IL-1β, in murine MCs and that LXRβ plays an important role in the LXR-mediated repression of cytokine production. [ABSTRACT FROM AUTHOR]

The CCAAT/enhancer-binding protein α (C/EBPα) is the member of a family of related basic leucine zipper (bZIP) transcription factors and is critical for granulopoiesis. We previously demonstrated that C/EBPα interacts with the ETS domain of widely expressed GABPα, which leads to cooperative transcriptional activation of the myeloid-specific promoter for human FCAR encoding the Fc receptor for IgA (FcαR, CD89) in part by facilitating recruitment of C/EBPα to the promoter. The C/EBPα molecule contains transactivation domains (TADs) at its N-terminus and a DNA-binding and dimerization bZIP structure at its C-terminus. We demonstrate here that GABPα interacts with the last 18 residues of the C/EBPα C-terminus beyond the bZIP DNA-binding and dimerizing region. Deletion of this C-terminus resulted in loss of GABPα interaction but not affecting its DNA binding ability, indicating that it is not required for homodimer formation. Moreover, the C-terminus confers the ability to functionally synergize with GABP on a heterologous TAD when fused to the C-terminus of the VP16 TAD. We identified a three-amino acid stretch (amino acids 341-343) that is important for both functional and protein interactions with GABP. Ectopic expression in K562 cells of C/EBPα mutant incapable of interacting with GABPα does not induce expression of granulocytic differentiation markers including CD15, CD11b, GCSF-R and C/EBPε, and does not inhibit proliferation, whereas wild type does. These results demonstrate the functional importance of the C/EBPα C-terminus beyond the bZIP DNA-binding and dimerization region, which may mediate cooperative activation by C/EBPα and GABP of myeloid-specific genes involved in C/EBPα-dependent granulopoiesis., (© 2013.)

The development of animal models that mimic human allergic responses is crucial to study the pathophysiology of disease and to generate new therapeutic methodologies. Humanized mice reconstituted with human immune systems are essential to study human immune reactions in vivo and are expected to be useful for studying human allergies. However, application of this technology to the study of human allergies has been limited, largely because of the poor development of human myeloid cells, especially granulocytes and mast cells, which are responsible for mediating allergic diseases, in conventional humanized mice. In this study, we developed a novel transgenic (Tg) strain, NOD/Shi-scid-IL2rγ(null) (NOG), bearing human IL-3 and GM-CSF genes (NOG IL-3/GM-Tg). In this strain, a large number of human myeloid cells of various lineages developed after transplantation of human CD34⁺ hematopoietic stem cells. Notably, mature basophils and mast cells expressing FcεRI were markedly increased. These humanized NOG IL-3/GM-Tg mice developed passive cutaneous anaphylaxis reactions when administered anti-4-hydroxy-3-nitrophenylacetyl IgE Abs and 4-hydroxy-3-nitrophenylacetyl. More importantly, a combination of serum from Japanese cedar pollinosis patients and cedar pollen extract also elicited strong passive cutaneous anaphylaxis responses in mice. Thus, to our knowledge, our NOG IL-3/GM-Tg mice are the first humanized mouse model to enable the study of human allergic responses in vivo and are excellent tools for preclinical studies of allergic diseases.

Microenvironment-based alterations in phenotypes of mast cells influence the susceptibility to anaphylaxis, yet the mechanisms underlying proper maturation of mast cells toward an anaphylaxis-sensitive phenotype are incompletely understood. Here we report that PLA2G3, a mammalian homolog of anaphylactic bee venom phospholipase A2, regulates this process. PLA2G3 secreted from mast cells is coupled with fibroblastic lipocalin-type PGD2 synthase (L-PGDS) to provide PGD2, which facilitates mast-cell maturation via PGD2 receptor DP1. Mice lacking PLA2G3, L-PGDS or DP1, mast cell-deficient mice reconstituted with PLA2G3-null or DP1-null mast cells, or mast cells cultured with L-PGDS-ablated fibroblasts exhibited impaired maturation and anaphylaxis of mast cells. Thus, we describe a lipid-driven PLA2G3-L-PGDS-DP1 loop that drives mast cell maturation.

Intracellular reactive oxygen species (ROS) such as hydrogen peroxide (H(2)O2()) are thought to mediate apoptosis induced by death receptor ligands, including tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). However, the role of H(2)O(2) is controversial, since some evidence suggests that H(2)O(2) acts as an anti-apoptotic factor. Here, we show that exogenously applied H(2)O(2) (30-100 µM) induces cell death in TRAIL-resistant human melanoma cells via intracellular superoxide (O(2)-) generation. H(2)O(2) induced apoptotic or necrotic cell death, depending on the concentration of the oxidant applied; low concentrations of H(2)O(2) preferentially activated the caspase-dependent apoptotic pathway, while high concentrations of H(2)O(2) induced apoptotic and necrotic cell death in a caspase-independent manner. The H(2)O(2)-induced cell death was associated with increased mitochondrial membrane potential collapse and caspase-3/7 activation and ER stress responses including caspase-12 and X-box-binding protein-1 (XBP-1) activation. H(2)O(2) induced intracellular O2- generation even within the mitochondria, while TRAIL did not. The superoxide dismutase mimetic antioxidant MnTBaP [Mn (III) tetrakis (4-benzonic acid) porphyrin chloride] inhibited the H(2)O(2)-induced O(2)- generation, apoptosis and XBP-1 and caspase-12 activation at comparable concentrations. Importantly, H(2)O(2) treatment caused minimal O(2)- generation and apoptosis in normal primary melanocytes. These data show that H(2)O(2) induces endoplasmic reticulum-associated cell death via intracellular O(2)- generation and that malignant melanoma cells are more susceptible than normal cells to this oxidative cell death. The findings suggest that H(2)O(2) has therapeutic potential in the treatment of TRAIL-resistant melanoma.

Background: Chronic urticaria (CU) appears to be of autoimmune origin in about half of all patients, since several autoreactive immunoglobulin Gs (IgGs), such as anti-FcεRIα and anti-IgE, are detected in the sera of such patients. However, whether autoreactive IgE is associated with CU remains unclear. In this study, we attempted to identify autoreactive IgE antibodies in sera from patients with CU., Methods: Sera were collected from 67 normal subjects, 85 patients with CU and 28 patients with atopic dermatitis (AD). An autologous serum skin test (ASST) was performed on 27 of the CU patients. Autoreactive IgE and IgG levels against self-antigens were measured using enzyme-linked immunosorbent assays. The basophils were activated with dsDNA, and the CD63 expression level was examined using a fluorescence-activated cell sorter., Results: The anti-dsDNA IgE levels were significantly higher in patients with CU and AD than in normal subjects, but no differences in the anti-dsDNA IgG levels were seen. The levels of thioredoxin-, peroxiredoxin- and thyroglobulin-reactive IgE and IgG were not significantly higher in the CU patients than in the other 2 groups. There was no significant difference in the levels of anti-dsDNA IgE between ASST-positive and ASST-negative patients. The basophils from 2 out of 9 CU patients exhibited degranulation in response to dsDNA., Conclusions: Our data suggest that anti-dsDNA IgE is involved in the pathogenesis of some cases of CU., (Copyright © 2013 S. Karger AG, Basel.)

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is promising for cancer treatment because of its selective cytotoxicity toward tumor cells. However, some cancer cell types including malignant melanoma cells are resistant to TRAIL cytotoxicity. Here, we show that diallyl trisulfide (DATS), a garlic organosulfur compound, sensitizes melanoma cells to TRAIL-induced apoptosis while sparing normal cells. DATS also potentiates apoptosis induced by agonistic antibodies against death receptors (DR) 4 and DR5. The amplification of DR-mediated apoptosis was associated with increased mitochondrial membrane potential collapse and caspase-3/7 activation. However, these events were not sufficient for full sensitization. TRAIL also induced endoplasmic reticulum (ER) stress, as indicated by the activation of X-box-binding protein 1 and caspase-12 and DATS poten-tiated both events. Moreover, inhibition of caspase-12, but not caspase-4, abolished the amplification of apoptosis, indicating that ER stress plays a crucial role. On the other hand, DATS and/or TRAIL induced minimal apoptosis and caspase-12 activation in melanocytes despite their substantial expression of DR4 and DR5 on the cell surface. Our data suggest that DATS amplifies death ligand-induced melanoma cell death by disrupting their adaptation to ER-mediated death pathway. The present findings raise the possibility that DATS may be combined with death ligands to treat TRAIL-resistance melanoma cells without impairing its tumor selectivity.

Purpose: Monomeric IgE molecules, when bound to the high-affinity receptor, exhibit a vast heterogeneity in their ability to induce survival promotion and cytokine production in mast cells. At one end of this spectrum, highly cytokinergic (HC) IgEs can induce potent survival promotion, degranulation, cytokine production, migration, etc., whereas at the other end, poorly cytokinergic (PC) IgEs can do so inefficiently. In this study, we investigated whether IgEs recognize autoantigens and whether IgEs' binding of autoantigens correlates with difference s in HC versus PC properties., Methods: Enzyme-linked immunosorbent assays were performed to test whether IgEs bind antigens. Histamine-releasing factor in human sera was quantified by western blotting. Cultured mast cells derived from human cord blood were used to test the effects of human sera on cytokine production., Results: Most (7/8) of mouse monoclonal HC IgEs exhibited polyreactivity to double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), β-galactosidase, thyroglobulin and/or histamine-releasing factor. By contrast, mouse PC IgEs failed to react with these antigens. A human monoclonal HC IgE also showed polyreactivity to histamine-releasing factor, dsDNA and ssDNA. Interestingly, sera from atopic dermatitis patients showed increased reactivity to ssDNA and β-galactosidase and increased levels of histamine-releasing factor. Some atopic dermatitis patients, but not healthy individuals, had substantial serum levels of HRF-reactive IgE. Sera from atopic dermatitis patients with high titers of DNA-reactive IgE could induce several fold more IL-8 secretion in human mast cells than sera from healthy individuals., Conclusions: The results show that most HC, but not PC, IgEs exhibit polyreactivity to autoantigens, supporting the autoimmune mechanism in the pathogenesis of atopic dermatitis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is promising for cancer treatment owing to its selective cytotoxicity against malignant cells. However, some cancer cell types, including malignant melanoma cells, are resistant to TRAIL-induced apoptosis. Therefore, drugs that can amplify TRAIL cytotoxicity are urgently required. Depolarization of the plasma membrane potential is associated with apoptosis induced by a variety of death-inducing agents but its role in apoptosis remains a matter of debate. We found that TRAIL treatment resulted in robust depolarization in human melanoma cells with a considerable lag (2-4 h). Moreover, membrane-depolarizing agents, including K+ and ATP-sensitive K+ (KATP) channel inhibitors glibenclamide and U37883A enhanced TRAIL-induced apoptosis. On the contrary, inhibitors of calcium- and voltage-dependent K+ channels and mitochondrial KATP channels had no such effects. Melanocytes were insensitive to TRAIL-induced depolarization and apoptosis as well as to the sensitization by membrane-depolarizing agents despite their substantial surface expression of death receptors. TRAIL induced robust activation of X-box-binding protein-1 and caspase-12, both of which were enhanced by the K+ and KATP channel inhibitors, but not by other K+ channel inhibitors. Finally, caspase-12-selective inhibitor completely abolished the amplification of apoptosis. These findings suggest that depolarization promotes endoplasmic reticulum stress-mediated death pathway, thereby amplifying TRAIL cytotoxicity. Thus, membrane-depolarizing agents such as KATP channel inhibitors may have therapeutic potential in the treatment of TRAIL-resistant cancer cells without impairing tumor-selectivity.

Mast cells play a key role in allergic reaction and disorders through the high affinity receptor for IgE (Fc(ε)RI) which is primarily activated by IgE and antigen complex. In humans, mast cells express two types of Fc(ε)RI on the cell surface, tetrameric αβγ(2) and trimeric αγ(2), whereas in mice, the tetrameric αβγ(2) type is exclusively expressed. In human allergic inflammation lesions, mast cells increase in number and preferentially express the αβγ(2) type Fc(ε)RI. By contrast, in the lesion of non-allergic inflammation, mast cells mainly express the αγ(2)type. Since the β chain amplifies the expression and signaling of FcεRI, mast cell effector functions and allergic reaction in vivo are enhanced in the presence of the β chain. In contrast, a truncated β chain-isoform (βT) inhibits FcεRI surface expression. The human Fc(ε)RIβ gene contains seven exons and a repressor element located in the forth intron, through which Fc(ε)RIβ transcription is repressed in the presence of GM-CSF. Regarding the additional signal regulatory function of the β chain, the β chain ITAM has dual (positive and negative) functions in the regulation of the mast cell activation. Namely, the Fc(ε)RIβ chain ITAM enhances the mast cell activation signal triggered by a low-intensity (weak) stimulation whereas it suppresses the signal triggered by high-intensity (strong) stimulation. In an oxazolone-induced mouse CHS model, IgE-mediated mast cell activation is required and the β chain ITAM is crucially involved. Adenosine receptor, one of the GPCRs, triggers a synergistic degranulation response with FcεRI in mast cells, for which the β chain ITAM critically plays positive role, possibly reflecting the in vivo allergic response. These regulatory functions of the FcεRIβ ITAM finely tune FcεRI-induced mast cell activation depending on the stimulation strength, enabling the Fc(ε)RIβ chain to become a potential molecular target for the development of new strategies for therapeutic interventions for allergies.

Previous studies suggested that Protein L (PpL), the bacterial Ig-binding protein, activates mast cells. PpL presumably performs the activation by interacting with membrane-bound IgEκ, but the underlying mechanisms behind the process remain unclear. In the current study, we found that cell-surface FcεRI expression is a critical factor participant in PpL-mediated full activation of murine mast cells, which includes cytokine production, the degranulation response, and leukotriene C(4) (LTC(4)) release, and that engagement of the FcεRI with IgEκ and PpL is enough to induce tyrosine phosphorylation of ITAM in the FcRβ- and γ-signaling subunits. Introduction of mutations in two canonical tyrosine residues (Y47F/Y58F) of the FcRγ-ITAM completely abolished the above-mentioned mast cell functions, with the exception of LTC(4) release. Importantly, the FcRβ-ITAM acts as a signal transducer that is responsible for LTC(4) release independently of the FcRγ-ITAM. Taken together, our results suggest crucial and distinct functions for the FcRβ- and γ-ITAMs in the FcεRI-dependent full activation of mast cells induced by IgEκ and PpL.

Mast cells play central roles in adaptive and innate immunity. IgE-dependent stimulation of the high-affinity IgE receptor (FcεRI) results in rapid secretion of various proinflammatory chemical mediators and cytokines. All of the outputs depend to certain degrees on an increase in the intracellular Ca(2+) concentration, and influx of Ca(2+) from the extracellular space is often required for their full activation. There is strong evidence that FcεRI stimulation induces two different modes of Ca(2+) influx, store-operated Ca(2+) entry (SOCE) and non-SOCE, which are activated in response to endoplasmic reticulum Ca(2+) store depletion and independently of Ca(2+) store depletion, respectively, in mast cells. Although Ca(2+) release-activated Ca(2+) channels are the major route of SOCE, recent evidence indicates that they are not the only Ca(2+) channels activated by Ca(2+) store depletion. The recent data suggest that L-type Ca(2+) channels, which were thought to be a characteristic feature of excitable cells, exist in mast cells to mediate non-SOCE, which is critical for protecting mast cells against activation-induced mitochondrial cell death. In this chapter, we provide an overview of recent advances in our understanding of Ca(2+) signaling in mast cells with a special attention to the emerging role for the L-type Ca(2+) channels as a regulator of mast cell survival.

Fcγ receptors I and III are thought to be involved in the development of lupus nephritis. Expression of Fc receptor common gamma chain (FcRγ) is necessary for the stable expression of Fcγ receptors I and III. The aim of this study was to develop a novel agent for the treatment of immune complex related renal disease using a gene regulator, pyrrole(Py)-imidazole(Im) (PI) polyamide, targeting the mouse FcRγ gene promoter. Two PI polyamides targeting FcRγ promoters were designed and synthesized. The effect of the PI polyamides on FcRγ mRNA expression was evaluated in J774.A cells by real-time polymerase chain reaction (PCR), and CD16/32 protein expression was determined by immunocytochemical analysis and flow cytometry. The effects of these polyamides on FcRγ gene expression and CD16/32 protein expression were evaluated in mouse peripheral blood mononuclear cells (PBMCs). One milligram per kilogram body weight of PI polyamide was injected via the tail vein every 2 d for 1 week and PBMCs were collected and analyzed. PI polyamide showed a specific binding to the target DNA in a gel mobility shift assay. Treatment of J774.A cells with 1.0 µM PI polyamide 1 significantly reduced FcRγ mRNA expression and CD16/32 surface protein expression in J774.A cells. Similarly, PI polyamide significantly decreased expression of FcRγ mRNA and CD16/32 in the PBMCs of C57B6 mice. PI polyamide designed to bind the FcRγ promoter decreased FcRγ gene and CD16/32 protein expression. PI polyamide targeting the FcRγ gene may be a novel gene regulator for the prevention of lupus nephritis or other immune complex-related disease.

Heavy metals, including gold, induce severe contact hypersensitivity and autoimmune disorders, which develop through an initial Th2-independent process followed by a Th2-dependent process. It has been shown that mast cell activation plays a role in the Th2-independent process and that gold stimulates histamine release in vitro. However, the mechanisms of the gold-induced mast cell activation remain largely unclear. Here we report that gold directly activates mast cells in a Ca2+-dependent manner. HAuCl4 [Au(III)] at nontoxic concentrations (≤50 μM) induced substantial degranulation and leukotriene C4 secretion in an extracellular Ca2+-dependent manner. Au(III) induced a robust Ca2+ influx but not Ca2+ mobilization from internal stores. Au(III) also stimulated intracellular production of reactive oxygen species, including H2O2, and blockade of the production abolished the mediator release and Ca2+ influx. Au(III) induced Ca2+ influx through multiple store-independent Ca2+ channels, including Cav1.2 L-type Ca2+ channels (LTCCs) and 2-aminoethoxydiphenyl borate (2-APB)-sensitive Ca2+ channels. The 2-APB-sensitive channel seemed to mediate Au(III)-induced degranulation. Our results indicate that gold stimulates Ca2+ influx and mediator release in mast cells through multiple H2O2-sensitive Ca2+ channels including LTCCs and 2-APB-sensitive Ca2+ channels. These findings provide insight into the roles of these Ca2+ channels in the Th2-independent process of gold-induced immunological disorders., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Recent studies suggest that homeostasis of lipid metabolism is crucial for the function of various immune cells. Oxygenated derivatives of cholesterol (oxysterols) are well-known regulators of lipid metabolism and have diverse functions, such as inhibition of cholesterol synthesis, efflux of intracellular cholesterol, synthesis of cholesterol esters, and activation of liver X receptors (LXRs). In this review, we introduce novel roles of the oxysterol receptors LXRs in the immune system, including regulation of inflammatory responses, T cell expansion, immunoglobulin production, and antitumor responses. We also discuss lipid-mediated signaling as a potential target for treatment of immune diseases.

Ets family members share a conserved DNA-binding ETS domain, and serve a variety of roles in development, differentiation and oncogenesis. Besides DNA binding, the ETS domain also participates in protein-protein interactions with other structurally unrelated transcription factors. Although this mechanism appears to confer tissue- or development stage-specific functions on individual Ets proteins, the biological significance of many of these interactions remains to be evaluated, because their molecular basis has been elusive. We previously demonstrated a direct interaction between the ETS domain of the widely expressed GABPalpha (GA-binding protein alpha) and the granulocyte inducer C/EBPalpha (CCAAT/enhancer-binding protein alpha), and suggested its involvement in co-operative transcriptional activation of myeloid-specific genes, such as human FCAR encoding FcalphaR [Fc receptor for IgA (CD89)]. By deletion analysis, we identified helix alpha3 and the beta3/beta4 region as the C/EBPalpha-interacting region. Domain-swapping of individual sub-domains with those of other Ets proteins allowed us to highlight beta-strand 3 and the subsequent loop, which when exchanged by those of Elf-1 (E74-like factor 1) reduced the ability to recruit C/EBPalpha. Further analysis identified a four-amino acid swap mutation of this region (I387L/C388A/K393Q/F395L) that reduces both physical interaction and co-operative transcriptional activation with C/EBPalpha without affecting its transactivation capacity by itself. Moreover, re-ChIP (re-chromatin immunoprecipitation) analysis demonstrated that GABPalpha recruits C/EBPalpha to the FCAR promoter, depending on these residues. The identified amino acid residues could confer the specificity of the action on the Ets proteins in diverse biological processes through mediating the recruitment of its partner factor.

The green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG) has been shown to reduce allergic inflammatory responses in animal models, but the mechanisms involved are poorly understood. Despite the essential role for Ca(2+) influx in mediating proinflammatory mediator release from mast cells, little is known about the effects of EGCG on this response. In the present study we found that EGCG inhibited antigen-induced Ca(2+) influx and store-operated Ca(2+) entry (SOCE), the principal mode of Ca(2+) influx into mast cells. EGCG, but not (-)-epicatechin (EC), inhibited antigen-induced degranulation, leukotriene (LT) C(4) secretion, and Ca(2+) influx. EGCG also blocked SOCE without reducing Ca(2+) store emptying whereas EC did not, although it did reduce Ca(2+) store emptying. EGCG, but not EC, also evoked intracellular reactive oxygen species (ROS) production, mitochondrial membrane potential (Psi(m)) collapse, cardiolipin oxidation, and mitochondrial Ca(2+) ([Ca(2+)](m)) release. Furthermore, FCCP, a potent inducer of Psi(m) collapse, induced ROS production and [Ca(2+)](m) dysfunction and inhibited degranulation, LTC(4) secretion, Ca(2+) influx, and SOCE. These data suggest that ROS production and Psi(m) collapse are important mechanisms underlying the antiallergic effects of EGCG. These events may lead to [Ca(2+)](m) dysfunction and impair mitochondria-mediated facilitation of SOCE, thereby attenuating mast cell activation., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Simultaneous stimulation with antigen and adenosine in mast cells induces a synergistic degranulation response at a low antigen dose that is insufficient to cause secretion by itself. This kind of stimulation is thought to be relevant to the immediate asthmatic response upon bronchial challenge with low-dose allergen. In this context, FcepsilonRI- and adenosine receptor-mediated signalings cooperate to increase degranulation in mast cells. In the present study, we prepared mast cells that have mutations (Y219F/Y225F/Y229F) in three tyrosine residues of the FcepsilonRI beta-chain (FcRbeta)-ITAM in order to elucidate the molecular mechanisms of degranulation response synergistically elicited by costimulation with low-dose antigen and adenosine. Introduction of mutations in the FcRbeta-ITAM abolished the synergistic degranulation response. Upon costimulation with low-dose antigen and adenosine, tyrosine phosphorylation of Grb2-associated binder 2, which is located upstream of PI3K-signaling, was significantly increased, but severely diminished in FcRbeta-ITAM mutant cells. These findings indicate that FcRbeta acts as a critical element in mast cell synergistic degranulation response through FcepsilonRI and adenosine receptors, and that PI3K-signaling through FcRbeta-ITAM is a crucial participant in augmentation of FcepsilonRI-mediated degranulation by adenosine.

Recent pharmacological and molecular genetic approaches have revealed the existence of functional L-type Ca2+ channels (LTCCs) in a variety of hematopoietic cells. We previously reported that Ca(v)1.2 LTCCs are expressed on mast cell surfaces, activated by the high-affinity IgE receptor (FcvarepsilonRI) engagement and protect mast cells against activation-induced cell death (AICD). We also demonstrated that FcvarepsilonRI engagement evokes nitric oxide (NO) generation in a phosphatidylinositol-3-kinase- and NO synthase (NOS)-dependent manner, which is also required for mast cell survival. Here we demonstrate that this endogenous NO mediates Ca(v)1.2 LTCC activation. FcvarepsilonRI engagement but not thapsigargin, a potent Ca2+ release-activated Ca2+ (CRAC) channel agonist, induced Ca2+ influx via NOS-dependent NO generation. RT-PCR analyses revealed predominant expression of eNOS in mast cells. Subsequent experiments involving siRNA-mediated gene silencing of eNOS or Ca(v)1.2 LTCC revealed that eNOS was essential for NOS-dependent NO generation and Ca(v)1.2 LTCC activation but not CRAC channel activation. Similar to Ca(v)1.2 LTCCs, eNOS prevented the dissipation of the mitochondrial membrane potential and mitochondrial integrity collapse, thereby protecting mast cells against AICD. Taken together, the present findings demonstrate the key roles of the eNOS-NO-LTCC axis in mast cell survival after FcvarepsilonRI engagement., (Copyright 2009 Elsevier B.V. All rights reserved.)

Background: Mast cell-released chemical mediators such as histamine, leukotriene (LT) C(4) and prostaglandin (PG) D(2) lead to the onset of allergic disorders. ATP provided from glycolysis is essential for histamine release and LTC(4) secretion from mast cells upon Fc epsilon RI cross-linking, indicating that glucose is a primary environmental factor for mast cell activation. In this study, we investigated whether increases in concentrations of glucose in culture media affect the activation of bone marrow-derived mouse mast cells (BMMCs) upon Fc epsilon RI cross-linking., Methods: BMMCs were cultured in RPMI-1640 supplemented with varying concentrations (5.5, 11, 16.5, 22, 27.5 and 33 mM) of D-glucose for 3 h, or 1, 3 or 7 days. D-Mannitol was added to the medium containing 5.5 mMD-glucose for osmotic control. After culturing, these cells were sensitized with anti-TNP IgE and then stimulated with TNP-BSA., Results: We found that long-term culture (7 days) of BMMCs with 33 mMD-glucose increases the Fc epsilon RI-dependent release of beta-hexosaminidase and LTC(4) without affecting surface expression levels of Fc epsilon RI, intracellular ATP levels or calcium signaling. Biochemical analyses demonstrated that Fc epsilon RI-dependent phosphorylation of cytosolic phospholipase A(2) (cPLA(2)) at the Ser505 residue was significantly increased by culturing with 33 mM glucose., Conclusions: Taken together, our data suggest that glucose can augment Fc epsilon RI-mediated mast cell activation, particularly the degranulation response and LTC(4) secretion after prolonged culture of mast cells with high-glucose medium. Moreover, it is suggested that increased phosphorylation of cPLA(2) at the Ser505 residue contributes to the enhancement of LTC(4) secretion., ((c) 2010 S. Karger AG, Basel.)

Ca(2+) is a highly versatile intracellular second messenger in many cell types, and regulates many complicated cellular processes, including cell activation, proliferation and apoptosis. Influx of Ca(2+) from the extracellular fluid is required for sustained elevation of the cytosolic Ca(2+) concentration and full activation of Ca(2+)-dependent processes. It is widely accepted that Ca(2+) release-activated Ca(2+) channels are the major routes of Ca(2+) influx in electrically non-excitable cells, including hematopoietic cells, whereas voltage-gated Ca(2+) channels such as L-type Ca(2+) channels (LTCCs) serve as the principal routes of Ca(2+) entry into electrically excitable cells such as neurons and myocytes. However, recent pharmacological and molecular genetic studies have revealed the existence of functional LTCCs and/or LTCC-like channels in a variety of immune cells including mast cells. In this article, we review recent advances in our understanding of Ca(2+) signaling in immune cells with a special interest in mast cells. We highlight roles for LTCCs in antigen receptor-mediated mast cell activation and survival., (Copyright 2010 Elsevier Ltd. All rights reserved.)

NO is generated by NOS activity and known to act as a negative regulator of mast cell activation. We reported previously that Ag (I) directly evokes mast cell degranulation and LTC(4) release via Ca(2+) influx through thiol-sensitive, store-independent channels. Here, we report that NO generated independently of NOS activity mediates the store-independent Ca(2+) influx. Exposure of mast cells to Ag (I) resulted in increased intracellular NO levels and NO(2)(-)/NO(3)(-) contents in the extracellular fluid. The NO increase was blocked by NO scavenger Hb and DTT but not by NOS inhibitors such as amino-BH(4) and L-NAME. This NO production occurred independently of the Src family kinase and PI3K activities, both of which were necessary for antigen-induced, NOS-dependent NO production. Hb and DTT reduced Ag (I)-induced beta-hexosaminidase release and LTC(4) release, whereas the NO scavengers and NOS inhibitors augmented antigen-induced mediator release. Moreover, Hb and DTT, but not the NOS inhibitors, abolished the Ag (I)-induced Ca(2+) influx, and none of the drugs blocked CRAC channel activity. Finally, Ag (I)-induced Ca(2+) influx was distinct from LTCC activity in terms of its sensitivities to wortmannin and LTCC antagonists and the effects of Ca(v)1.2 LTCC gene silencing. These data show that NOS-independent NO regulates mast cell activation positively via a unique store-independent Ca(2+) influx pathway. The present findings suggest multiple sources and functions of NO in mast cell biology.

IgE, a unique subclass of antibodies, leads to the development of allergic inflammation by eliciting mast cell activation. The molecular process of allergen specific IgE production which works for class switch recombination in B cells can be a good target for the treatment of allergic disorders. Here, we review the mechanism of IgE class switch recombination in B cells, and then introduce current topics including our findings; (1) preferable usage of IgE VH families in subjects with allergic diseases; (2) allergic march and epitope spreading; and (3) regulation of IgE synthesis by cognate interaction between B cells and mast cells in the local inflammatory sites such as nasal mucosa and skin.

Mast cells and T cells produce reactive oxygen species (ROS) after stimulation with the high-affinity IgE receptor (Fc epsilon RI) and T cell receptor. A growing body of evidence suggests the existence of ROS-regulated intracellular and/or plasma membrane Ca(2+) channels in these cells but their molecular entities remain to be identified. Here, we report that store-operated Ca(2+) channel (SOC) activity is regulated by superoxide (O(2)(*-)) and hydrogen peroxide (H(2)O(2)) in mast cells. MnTBaP (Mn(III)tetrakis(4-benzoic acid)porphyrin) and ebselen (2-phenyl-1,2-benziso-selenazol-3(2H)-one) selectively blocked the generation of O(2)(*-) and H(2)O(2), respectively, in antigen-stimulated cells. The H(2)O(2) generation was dependent on the Src family kinase (SFK) and phosphatidylinositol-3-kinase (PI3K) activities but independent of extracellular Ca(2+), and the Fc epsilon RI beta-chain immunoreceptor tyrosine-based activation motif played an essential role. On the other hand, O(2)(*-) generation was strictly dependent on extracellular Ca(2+), but negatively regulated by the SFK and PI3K activities. Inhibition of O(2)(*-) generation resulted in increased H(2)O(2) generation and reduced SOC activity, although it had a minimal effect on endoplasmic reticulum Ca(2+) store depletion. On the contrary, inhibition of H(2)O(2) generation resulted in increased intracellular O(2)(*-) generation and augmented SOC activity. The findings suggest that O(2)(*-) and H(2)O(2), which are generated by separate signaling pathways/sources, reciprocally regulate SOC activity in mast cells. Such generations of multiple oxidant species and their distinct roles in the regulation of SOC activity may facilitate the fine tuning of Ca(2+) signaling in mast cells.

In non-excitable cells, store-operated Ca(2+) channels (SOCs) are the principal routes of Ca(2+) entry. Recently, store-independent Ca(2+) channels which are pharmacologically and/or immunologically similar to L-type Ca(2+) channels (LTCCs) have been shown to exist in various hematopoietic cells, including T cells, B cells and neutrophils. We previously reported that mast cells express LTCCs which regulate mast cell effector responses in a distinct manner from SOCs. In the present study, we examined the possible role for LTCCs in mast cell survival. Both RBL-2H3 mast cells and bone marrow-derived mast cells underwent considerable apoptosis after treatment with thapsigargin (Tg) but not stimulation through the high-affinity IgE receptor (Fc epsilon RI). The LTCC-selective antagonists such as nifedipine greatly augmented Fc epsilon RI-mediated apoptosis, while the LTCC-selective agonist (S)-BayK8644 blocked Tg-induced apoptosis. The modulation of apoptosis was accompanied by altered mitochondrial integrity, as measured with the mitochondrial membrane potential, cytochrome c release and caspase-3/7 activation. Fc epsilon RI stimulation induced mitochondrial Ca(2+) ([Ca(2+)](m)) entry through both SOCs and LTCCs, while Tg evoked [Ca(2+)](m) entry through LTCCs but not SOCs. The LTCC-selective antagonists blocked [Ca(2+)](m) entry, whereas (S)-BayK8644 augmented Tg-induced [Ca(2+)](m) entry. Moreover, blockade of the expression of the alpha(1C) subunit of Ca(v)1.2 LTCC using small-interfering RNA strongly augmented Fc epsilon RI-mediated apoptosis, mitochondrial integrity, and mitochondrial Ca(2+) collapse, and abolished the protective effects of (S)-BayK8644 against Tg-induced apoptosis. These findings suggest that Ca(v)1.2 LTCC protects mast cells against activation-induced cell death by preventing mitochondrial integrity disruption.