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CCAAT/enhancer binding protein alpha (C/EBPα) is a transcription factor that influences immune cell fate and differentiation. However, the effect of C/EBPα on mast cells is not fully understood. In this study, we showed that C/EBPα suppressed granule formation in mast cells and increased macrophage inflammatory protein (MIP)‐2 production from mast cells upon bacterial stimulation. These results indicate that C/EBPα regulates the balance between the allergic response and the innate immune response of mast cells. Furthermore, we showed that stimulation of mast cells with the Lactobacillus casei JCM1134T strain during late differentiation up‐regulated C/EBPα expression in differentiated mast cells. This suggests that intestinal commensal bacteria modulate C/EBPα expression and thereby regulate mast cell function.C/EBPα mRNA expression is increasingly suppressed during mast cell differentiation. C/EBPα suppresses granule formation in mast cells. C/EBPα increases MIP‐2 production from mast cells in response to bacterial stimulation. C/EBPα expression in mast cells is upregulated by specific bacterial components. [ABSTRACT FROM AUTHOR]

Background: Basophils and mast cells are important initiator/effector cells capable of rapidly responding to IgE-mediated stimulation, but the precise mechanisms regulating their functions in vivo have not been fully identified. In this study, we assessed whether low levels of antigen can modulate activation of basophils and mast cells. Methods: Human basophils and cultured mast cells were pretreated with low concentrations of anti-FcεRI α-chain mAb (CRA-1 mAb), and their cell functions were assessed. Results: Basophils preincubated with CRA-1 mAb at as low as 1 ng/ml for 1 h showed significantly enhanced degranulation in response to various secretagogues such as MCP-1, FMLP, leukotriene B4 and Ca ionophore A23187. FMLP-induced leukotriene C4 production by basophils was also enhanced by CRA-1 mAb pretreatment. Degranulation was further enhanced when CRA-1 mAb-pretreated basophils were additionally treated with IL-3, IL-33 or leptin before stimulation with MCP-1. Priming by subthreshold CRA-1 mAb was a slow process, since 1 h of pretreatment was needed for maximal enhancement. Basophil priming also resulted from preincubation with subthreshold doses of an allergen, Der f 2. In parallel mAb experiments, CRA-1 mAb showed weak priming effects on human umbilical cord blood-derived cultured mast cells; a higher dose, 100 ng/ml, was necessary for this priming. Conclusion: These results indicate that subthreshold doses of CRA-1 mAb or allergens can prime basophils and induce exaggerated responses to various IgE-independent stimuli. This may be a potentially important mechanism that explains environmental allergen-induced exacerbation of IgE-mediated allergic diseases such as asthma. Copyright © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Oxysterols activating liver X receptors (LXRs) repress expression of pro-inflammatory genes and have anti-inflammatory effects. Here, we show for the first time that bone marrow-derived murine mast cells (BMMCs) predominantly express LXRβ. 25-hydroxycholesterol, a representative LXR activating oxysterol, suppressed IL-6 production and degranulation response in BMMCs following engagement of high-affinity IgE receptor (FcεRI). Interestingly, 25-hydroxycholesterol reduced cell-surface FcεRI expression by inhibiting assembly of FcεRIα and FcεRIβ. We demonstrate that LXR activation was involved in the suppression of IL-6 production in BMMCs, but that reduced FcεRI expression and degranulation response was mediated in an LXR-independent manner. [Copyright &y& Elsevier]

Phosphatidylinositol 3-kinase (PI3K) has been recognized as an important downstream effector of high-affinity receptor for IgE (FcεRI) signaling in mast cells, but little is known about the isoform specificity of PI3Ks on the FcεRI-mediated migration toward the antigen (Ag). In the present study, we explored the role of PI3Kγ on mast cell migration. The treatment of bone marrow-derived mast cells (BMMCs) with a PI3Kγ inhibitor, AS605240, significantly repressed FcεRI-induced degranulation and migration. The culture supernatants of wild-type mast cells stimulated with IgE and Ag attracted FcεRIβ–/– mast cells which did not express FcεRI on their cell surface, indicating that the migration appears to be dependent on an autocrine/paracrine secretion of soluble factors from the mast cells. Adenosine, which is produced by mast cells, showed a strong activity to attract mast cells. Pertussis toxin (PTX) significantly inhibited the migration toward both the supernatant and adenosine. The supernatant from mast cells pretreated with wortmannin (Wort) and stimulated with IgE and Ag still exhibited the activity as chemoattractant, while the BMMCs pretreated with Wort did not migrate toward the supernatant. Although PTX significantly reduced the activation of AKT/PKB and migration, PTX had no effects on degranulation. These results suggest that PI3Kγ activation through PTX-sensitive G-protein-coupled receptor as a secondary response of FcεRI cross-linking regulates FcεRI-mediated mast cell migration toward the Ag, while simultaneously activated PI3Kγ through a PTX-insensitive pathway might have an effect on degranulation. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

MIST (mast cell immunoreceptor signal transducer; also termed Clnk) is an adaptor protein structurally related to SLP-76-family hematopoietic cell-specific adaptor proteins. We demonstrate here that two major MIST-associated phosphoproteins expressed in mast cell lines are SLAP-130 and SKAP55, adaptors known to interact with the Src-homology (SH) 2 domain of Src-family protein tyrosine kinases (PTKs). MIST directly associated with SLAP-130 via its SH2 domain, and collaboration of SLAP-130 with SKAP55 was required for the recruitment of MIST to Lyn. Furthermore, MIST was preferentially recruited to Fyn rather than Lyn, which is regulated by higher affinity binding of SLAP-130 and SKAP55 with the Fyn-SH2 domain than the Lyn-SH2 domain. Our results suggest that the MIST–SLAP-130–SKAP55 adaptor complex functions downstream of high-affinity IgE receptor-associated Src-PTKs in mast cells. [Copyright &y& Elsevier]

The morbidity and mortality of elderly onset asthma have recently been on the increase. To evaluate the allergic status of the elderly onset asthmatics, we performed skin tests for allergens and estimated serum total IgE levels of the patients. The proportion of the skin-test-positive patients among the elderly onset asthmatics was significantly larger than that in the younger onset group, whereas no significant difference was observed in the frequency of patients with serum total IgE levels higher than the normal range (287.2 IU/ml). On the other hand, in the experiments with aged mice, isolated peritoneal mast cells (PMC) showed a considerable decrease in the FcγRIIB/III expression and higher degranulation triggered by 2.4G2, as compared to the PMC derived from young mice. Since FcγRIIB has been shown to act as a negative regulator, we speculate that in the aged mice Fc receptor-mediated mast cell function may be upregulated by a release from the negative regulation as a result of decreased FcγRIIB expression. Our results obtained from a mouse model system may help to understand pathophysiological mechanisms underlying elderly onset asthma. [ABSTRACT FROM AUTHOR]

Atopic dermatitis (AD) is a common pruritic disease that occurs primarily in infancy and childhood. AD is characterized by itching and the patient having an individual or family history of atopic diseases. Although AD is also frequently associated with elevated serum IgE levels and with common environmental factors contributing to its pathogenesis, the etiology of AD is still unknown. We examined NC/Nga mice (NC mice) that showed AD-like skin lesions with aging as a possible mouse model for AD. NC mice were maintained under conventional (Conv) or specific pathogen-free (SPF) conditions. Clinical symptoms, serum IgE levels and histopathology of the skin were compared between these 2 groups, and we explored their application as a model of human AD. It was found that the skin lesions of inbred NC mice were clinically and histologically very similar to human AD when the mice were raised under Conv conditions, but not under SPF conditions, and we assumed that some kinds of environmental factors might trigger AD-like signs and symptoms in NC mice. To further investigate the pathophysiology and treatment of AD, a suitable animal model is absolutely required, and NC mice are very useful for this purpose. [ABSTRACT FROM AUTHOR]

Abstract: The green tea polyphenol (−)-epigallocatechin-3-O-gallate (EGCG) has various biological activities, including anti-inflammatory, anti-neoplastic, anti- and pro-apoptotic, and neuroprotective effects. Although these are often associated with increased intracellular reactive oxygen species (ROS) and Ca2+ levels, their involvement in biological effects is poorly understood. Here we report that EGCG induces cytokine production in mast cells via Ca2+ influx and ROS generation. EGCG at concentrations of ≥50μM induced interleukin-13 and tumor necrosis factor-α production in RBL-2H3 and bone marrow-derived mast cells. The effects were dependent on extracellular Ca2+, and EGCG induced Ca2+ release from intracellular stores and Ca2+ influx. Ca2+ influx was suppressed by 2-aminoethoxydiphenyl borate, an inhibitor of store-operated Ca2+ (SOC) channels, including Ca2+ release-activated Ca2+ channels and transient receptor potential canonical channels. EGCG failed to induce Ca2+ influx through SOC channels. EGCG-activated Ca2+ channels were genetically and pharmacologically distinct from Cav1.2 L-type Ca2+ channels, another route of Ca2+ influx into mast cells. EGCG evoked release of superoxide (O2 −) into the extracellular space. Exogenous superoxide dismutase, but not catalase, inhibited EGCG-evoked Ca2+ influx and cytokine production, indicating that extracellular O2 − regulates these events. EGCG can serve as a powerful tool for studying O2 −-regulated Ca2+ channels, which may be selectively involved in the regulation of cytokine production but have yet to be elucidated. [Copyright &y& Elsevier]

Abstract: 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 Ca2+ 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 Ca2+ influx and store-operated Ca2+ entry (SOCE), the principal mode of Ca2+ influx into mast cells. EGCG, but not (–)-epicatechin (EC), inhibited antigen-induced degranulation, leukotriene (LT) C4 secretion, and Ca2+ influx. EGCG also blocked SOCE without reducing Ca2+ store emptying whereas EC did not, although it did reduce Ca2+ store emptying. EGCG, but not EC, also evoked intracellular reactive oxygen species (ROS) production, mitochondrial membrane potential (∆Ψm) collapse, cardiolipin oxidation, and mitochondrial Ca2+ ([Ca2+]m) release. Furthermore, FCCP, a potent inducer of ∆Ψm collapse, induced ROS production and [Ca2+]m dysfunction and inhibited degranulation, LTC4 secretion, Ca2+ influx, and SOCE. These data suggest that ROS production and ∆Ψm collapse are important mechanisms underlying the antiallergic effects of EGCG. These events may lead to [Ca2+]m dysfunction and impair mitochondria-mediated facilitation of SOCE, thereby attenuating mast cell activation. [Copyright &y& Elsevier]

Abstract: 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 Cav1.2 LTCCs are expressed on mast cell surfaces, activated by the high-affinity IgE receptor (FcɛRI) engagement and protect mast cells against activation-induced cell death (AICD). We also demonstrated that FcɛRI 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 Cav1.2 LTCC activation. FcɛRI 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 Cav1.2 LTCC revealed that eNOS was essential for NOS-dependent NO generation and Cav1.2 LTCC activation but not CRAC channel activation. Similar to Cav1.2 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 FcɛRI engagement. [Copyright &y& Elsevier]

Abstract: Ca2+ 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 Ca2+ from the extracellular fluid is required for sustained elevation of the cytosolic Ca2+ concentration and full activation of Ca2+-dependent processes. It is widely accepted that Ca2+ release-activated Ca2+ channels are the major routes of Ca2+ influx in electrically non-excitable cells, including hematopoietic cells, whereas voltage-gated Ca2+ channels such as L-type Ca2+ channels (LTCCs) serve as the principal routes of Ca2+ 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 Ca2+ 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 &y& Elsevier]

Background: Programmed death-1 (PD-1) is a marker for human neoplastic T cells. Here, we evaluated whether or not PD-1 was also a marker for human mastocytosis, and explored the role of PD-1 in human mastocytosis cells. Methods: Immunohistochemical analysis was used to evaluate the expression of PD-1 in clinical samples of human cutaneous mastocytosis. The expression of PD-1 in human mastocytosis cell lines was checked by RT-PCR, western blotting and flow cytometry. We stimulated human mastocytosis cell lines (LAD2 and HMC1.2) with recombinant ligand for PD-, PD-L1 (rPD-L1), and tested the proliferative activity and the status of signal molecules by Cell Counting Kit-8 and ELISA, respectively. Results: Ten of 30 human cutaneous mastocytosis cases (33.3%) expressed PD-1 protein. We also found that a human mastocytosis line LAD2 cells expressed PD-1 protein on their surfaces. The administration with rPD-L1 suppressed the stem cell factor-dependent growth of the LAD2 cells. And, rPD-L1 activated SHP-1 and SHP-2 simultaneously, and decreased the phosphorylation of AKT, in LAD2 cells. In contrast, we could not detect the expression of PD-, and the significant effect of rPD-L1 on the mutated KIT-driven growth of HMC1.2 cells. Conclusions: PD-1 could be a marker for human cutaneous mastocytosis and regulate the growth of human PD-1-positive mastocytosis cells. [ABSTRACT FROM AUTHOR]

Abstract: Mast cell is one of the central effectors in inflammatory responses. Recent studies suggest that a promising therapeutic approach for various inflammatory immune diseases, including rheumatoid arthritis, multiple sclerosis, and type I allergies, is to inhibit mast cell growth and/or survival. Studies also indicate that a balanced lipid metabolism is crucial for regulating the life span of cells. Oxysterol is a well-known regulator of lipid metabolism and has diverse functions, such as inhibition of the mevalonate isoprenoid pathway, efflux of free cholesterols, and synthesis of cholesterol esters. Here, we show that 24(S),25-epoxycholesterol, a representative endogenous oxysterol, induces apoptosis in bone marrow-derived murine mast cells. Furthermore, we have revealed, for the first time, that the accumulation of neutral lipids catalyzed by acyl-CoA:cholesterol acyltransferase in the cells was involved in induction of mast cell apoptosis. Our present findings confer new insights into the roles of lipid metabolism during oxysterol-mediated mast cell apoptosis. [ABSTRACT FROM AUTHOR]

Abstract: Protein kinase D (PKD) is a serine–threonine kinase involved in the activation of a variety of cells. In mast cells, activation of PKD by cross-linking of high affinity receptor for IgE (FcεRI) has been reported, but little is known for its effects on cytokine production. We investigated the roles of PKD on FcεRI-induced activator protein-1 (AP-1) activation and proinflammatory cytokine productions in mast cells. Pharmacological inhibition of PKD strongly inhibited production of interleukin (IL)-13 and tumor necrosis factor (TNF)-α induced by FcεRI stimulation, and the overexpression of PKD significantly increased the IL-13 and TNF-α production. Reporter assay revealed that the overexpression of PKD enhanced FcεRI-induced IL-13 promoter activation, and that the 5′-flanking region of IL-13 gene from positions −110 to −52 was under the regulation of PKD. The overexpression of PKD enhanced the induction of AP-1 luciferase activity by FcεRI stimulation, while it had no effect on luciferase activities dependent upon NF-κB and NF-AT activated by FcεRI stimulation. In EMSA, c-Jun and c-Fos appear to be the major components of AP-1 complexes activated by FcεRI stimulation. Moreover the overexpression of PKD strongly enhanced the phosphorylation of both c-Jun and c-Fos following FcεRI stimulation. Although stress-activated protein kinase/c-Jun N-terminal kinase (JNK) is known to be an important regulator for c-Jun phosphorylation and AP-1 activation, overexpression and inhibition of PKD had no effects on JNK phosphorylation. These results suggest that PKD may play a pivotal role in FcεRI-induced cytokine production in mast cells through the activation of c-Jun, c-Fos, and AP-1. [Copyright &y& Elsevier]

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: Advanced glycation end products (AGEs) accumulate during aging and to higher extents under pathological conditions such as diabetes. Since we previously showed that mast cells expressed the AGE-binding protein, receptor for AGEs (RAGE) on their cell surface, we examined whether AGE affected mast cell survival. Herein, we demonstrate that mast cells undergo apoptosis in response to AGE. Glycated albumin (GA), an AGE, but not stimulation with the high-affinity IgE receptor (FcɛRI), can induce mast cell death, as measured by annexin V/propidium iodide double-staining. GA (≥0.1 mg/ml) exhibited this pro-apoptotic activity in a concentration-dependent manner. GA and FcɛRI stimulation increased the cytosolic Ca2+ levels to a similar extent, whereas GA, but not FcɛRI stimulation, caused mitochondrial Ca2+ overload and membrane potential collapse, resulting in mitochondrial integrity disruption, cytochrome c release and caspase-3/7 activation. In addition, GA, but not FcɛRI stimulation, induced extracellular release of superoxide from mitochondria, and this release played a key role in the disruption of Ca2+ homeostasis. Knockdown of RAGE expression using small interfering RNA abolished GA-induced apoptosis, mitochondrial Ca2+ overload, and superoxide release, demonstrating that RAGE mediates the GA-induced mitochondrial death pathway. AGE-induced mast cell apoptosis may contribute to the immunocompromised and inflammatory conditions. [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: Galectin-1 and galectin-3 are the most ubiquitously expressed members of the galectin family and more importantly, these two molecules are shown to have opposite effects on pro-inflammatory responses and/or apoptosis depending on the cell type. Herein, we demonstrate for the first time that galectin-3 induces mast cell apoptosis. Mast cells expressed substantial levels of galectin-3 and galectin-1 and to a lesser extent the receptor for advanced glycation end products (RAGE) on their surfaces. Treatment of cells with galectin-3 at concentrations of ≥100 nM for 18–44 h resulted in cell death by apoptosis. Galectin-3-induced apoptosis was completely prevented by lactose, neutralizing antibody to RAGE, and the caspase-3 inhibitor z-DEVD-fmk. Galectin-3-induced apoptosis was also completely abolished by dithiothreitol and superoxide dismutase, but not inhibited by catalase. Moreover, galectin-3 but not galectin-1 induced the release of superoxide, which was blocked by lactose, anti-RAGE, and dithiothreitol. Finally, galectin-3-induced apoptosis was blocked by bongkrekic acid, an antagonist of the mitochondrial permeability transition pore (PTP), while atractyloside, an agonist of the PTP, greatly facilitated galectin-1-induced apoptosis. These data suggest that galectin-3 induces oxidative stress, PTP opening, and the caspase-dependent death pathway by binding to putative surface receptors including RAGE via the carbohydrate recognition domain. [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]