Monomer hapten and hapten‐specific IgG inhibit mast cell activation evoked by multivalent hapten with different mechanisms

Aggregation of IgE bound to high affinity IgE receptor (FcεRI) by multivalent antigen induces mast cell activation. Reportedly, disaggregation of aggregated FcεRI immediately terminated degranulation, and formation of co‐ligated FcεRI and low affinity IgG receptor FcγRIIB blocked degranulation by inhibitory signal via SH2‐containing inositol 5’‐phosphatase 1 (SHIP1) phosphorylation. However, their molecular mechanisms to inhibit mast cell activation have been unclear in detail. Herein, we found that addition of excess monomeric hapten (TNP‐alanine) to multivalent antigen (TNP‐OVA)‐activated rat basophilic leukemia cells and mouse bone marrow‐derived mast cells induced immediate and transient Syk dephosphorylation, which was previously phosphorylated by TNP‐OVA addition. Syk dephosphorylation correlated to rapidly decreased intracellular Ca²⁺ concentrations ([Ca²⁺]_i), terminated degranulation, and suppressed cytokine production through inhibition of Akt and ERK phosphorylation. Addition of hapten‐specific IgG monoclonal antibody (anti‐TNP IgG1) to activated mast cells induced translocation of SHIP1 to the plasma membrane and its phosphorylation, indicating that co‐ligation of FcεRI and FcγRIIB after FcεRI aggregation can lead to SHIP1 activation. SHIP1 phosphorylation led to gradually decreased [Ca²⁺]_i, weak inhibition of degranulation, and strong inhibition of cytokine production. Our findings clearly show the inhibitory mechanism of cell function in activated mast cells by operating Fc receptor crosslinking.     

Direct interaction of Syk and Lyn protein tyrosine kinases in rat basophilic leukemia cells activated via type I Fcε receptors

Activation of rat mast cells through the receptor with high affinity for IgE (FcεRI) requires a complex set of interactions involving transmembrane subunits of the FcεRI and two classes of nonreceptor protein tyrosine kinase (PTK), the Src family PTK p53/p56^lyn (Lyn) and the Syk/ZAP-family PTK p72^syk (Syk). Early activation events involve increased activity of Lyn and Syk kinases and their translocation into membrane domains containing aggregated FcεRI, but the molecular mechanisms responsible for these changes have remained largely unclear. To determine the role of FcεRI subunits in this process, we have analyzed Syk- and Lyn-associated proteins in activated rat basophilic leukemia (RBL) cells and their variants deficient in the expression of FcεRI β or γ subunits. Sepharose 4B gel chromatography of postnuclear supernatants from Nonidet-P40-solubilized antigen (Ag)- or pervanadate-activated RBL cells revealed extensive changes in the size of complexes formed by Lyn and Syk kinases and other cellular components. A fusion protein containing Src homology 2 (SH2) and SH3 domains of Lyn bound Syk from lysates of nonactivated RBL cells; an increased binding was observed when lysates from Ag- or pervanadate-activated cells were used. A similar amount of Syk was bound when lysates from pervanadate-activated variant cells deficient in the expression of FcεRI β or γ subunits were used, suggesting that FcεRI does not function as the only intermediate in the formation of the Syk-Lyn complexes. Further experiments have indicated that Syk-Lyn interactions occur in Ag-activated RBL cells under in vivo conditions and that these interactions could involve direct binding of the Lyn SH2 domain with phosphorylated tyrosine of Syk. The physical association of Lyn and Syk during mast-like cell activation supports the recently proposed functional cooperation of these two tyrosine kinases in FcεRI signaling.     

The PH domain adaptor protein Bam32/DAPP1 functions in mast cells to restrain FcɛRI-induced calcium flux and granule release

Mast cell activation triggered by IgE binding to its high affinity receptor Fc?RI is highly dependent on signaling via phosphoinositde 3-kinases (PI3K). The phosphoinositide phosphatase SHIP controls mast cell activation by regulating accumulation of D3 phosphoinositide second messengers generated by PI3K. The PH domain adaptor protein Bam32/DAPP1 binds specifically to the D3 phosphoinositides PI(3,4,5)P3 and PI(3,4)P2 (the substrate and product of SHIP respectively). In B cells, Bam32 is phosphorylated by Src family kinases including Lyn, and is required for antigen receptor-induced activation; however the function of Bam32 in mast cells is unknown. Here we report that Bam32 is expressed in mast cells, is recruited to the plasma membrane upon stimulation and functions in Fc?RI signaling. Examination of bone marrow-derived mast cells (BMMC) isolated from Bam32-deficient mice revealed enhanced Fc?RI-induced degranulation and IL-6 production, indicating that Bam32 may function to restrain signaling via Fc?RI. These enhanced degranulation responses were PI3K-dependent, as indicated by blockade with PI3K inhibitors wortmannin or IC87114. While Bam32-deficient BMMC showed reduced Fc?RI-induced activation of mitogen-activated protein kinases ERK and JNK, Fc?RI-induced calcium flux and phosphorylation of PLCγ1 and Akt were increased. Bam32-deficient BMMC showed significantly reduced phosphorylation of Lyn and SHIP, indicating reduced activity of inhibitory signaling pathways. Together our results identify Bam32 as a novel regulator of mast cell activation, potentially functioning in membrane-proximal integration of positive and negative signaling pathways.     

Microfilaments make mast cells migrate (rather than degranulate)

Expression of the high‐affinity receptor for IgE (FcεRI) provides mast cells with the ability to react in a proinflammatory manner to antigens (Ags). In particular, the immediate secretion of preformed mediators from secretory lysosomes (degranulation) is typical for FcεRI‐mediated mast cell activation. In addition to the FcεRI, the stem cell factor receptor, KIT, is expressed at high levels on the surface of mast cells. KIT activation controls mast cell differentiation and survival in vivo and potently stimulates the chemotaxis of these cells. Although FcεRI and KIT initiate many of the same early signaling events in mast cells, FcεRI activation results in potent degranulation and a poor chemotactic response while KIT activation triggers very little degranulation and a strong chemotactic response. Novel data published in this issue of the European Journal of Immunology [Smr? et al. Eur. J. Immunol. 2013. 43: 1873‐1882] demonstrate that actin de‐ and repolymerization, involved in both degranulation and chemotaxis, make all the difference: Pharmacological suppression of F‐actin formation converts activated KIT into a strong degranulator. The possible implications for mast cell physiology and pathophysiology are discussed in this Commentary.     

Downstream signals initiated in mast cells by FcεRI and other receptors

The significant contributions this past year to our understanding of IgE receptor (FcεRI) signaling in mast cells include studies with truncated Syk in a vaccinia expression system and Syk-negative variants of rat basophilic (RBL-2H3) cells. These studies demonstrate an essential role for Syk in initiating signals for secretion and release of arachidonic acid via phospholipase A₂ and mitogen-activated protein kinase. A newly recognized addition to the repertoire of FcεRI-mediated signaling systems is the activation of sphingosine kinase, which contributes to calcium mobilization in mast cells. Advances have been made in our understanding of other receptors that regulate proliferation and differentiation of mast cells, and in our understanding of the ability of mast cells to mount acquired and acute responses to antigenic and bacterial challenge.     

Adenine suppresses IgE-mediated mast cell activation

Nucleobase adenine is produced by dividing human lymphoblasts mainly from polyamine synthesis and inhibits immunological functions of lymphocytes. We investigated the anti-allergic effect of adenine on IgE-mediated mast cell activation in vitro and passive cutaneous anaphylaxis (PCA) in mice. Intraperitoneal injection of adenine to IgE-sensitized mice attenuated IgE-mediated PCA reaction in a dose dependent manner, resulting in a median effective concentration of 4.21 mg/kg. In mast cell cultures, only adenine among cytosine, adenine, adenosine, ADP and ATP dose-dependently suppressed FcɛRI (a high affinity receptor for IgE)-mediated degranulation with a median inhibitory concentration of 1.6 mM. It also blocked the production of LTB₄, an inflammatory lipid mediator, and inflammatory cytokines TNF-α and IL-4. In addition, adenine blocked thapsigargin-induced degranulation which is FcɛRI-independent but shares FcɛRI-dependent signaling events. Adenine inhibited the phosphorylation of signaling molecules important to FcɛRI-mediated allergic reactions such as Syk, PLCγ2, Gab2, Akt, and mitogen activated protein kinases ERK and JNK. From this result, we report for the first time that adenine inhibits PCA in mice and allergic reaction by inhibiting FcɛRI-mediated signaling events in mast cells. Therefore, adenine may be useful for the treatment of mast cell-mediated allergic diseases. Also, the upregulation of adenine production may provide another mechanism for suppressing mast cell activity especially at inflammatory sites.     

An involvement of neurokinin-1 receptor in FcεRΙ-mediated RBL-2H3 mast cell activation

Objective and design

To determine whether the neurokinin-1 receptor (NK1R) plays a role in the activation of RBL-2H3 mast cells after FcεRΙ aggregation.

Materials and methods

NK1R expression in RBL-2H3 cells was inhibited by small hairpin RNA (shRNA) against NK1R, and determined by western blotting. For activation, both NK1R knockdown and control RBL-2H3 cells were sensitized by dinitrophenol (DNP)-specific IgE and stimulated with the antigen DNP-bovine serum albumin (BSA). Following the activation of RBL-2H3 cells, monocyte chemoattractant protein (MCP-1) production and intracellular calcium flux were monitored by ELISA and confocal microscopy assay, respectively. For investigation of the signaling mechanism, phosphorylation of mitogen-activated protein kinases (MAPKs) after RBL-2H3 cell activation was assessed by western blotting.

Results

shRNA-NK1R mediated an effective inhibition of NK1R expression in RBL-2H3 cells. Protein production of MCP-1 was reduced by more than 55?% in NK1R knockdown RBL-2H3 cells compared with control RBL-2H3 cells. In addition, both calcium mobilization and phosphorylation levels of MAPKs (Erk1/2, JNK, and p38) after DNP-BSA stimulation (via FcεRΙ) were decreased due to the inhibition of NK1R expression.

Conclusion

NK1R is required for the activation of RBL-2H3 cells following FcεRΙ engagement and involved in the regulation of MAPK signaling pathways.     

Decrease of cerebral mast cell degranulation after systemic administration of lipopolysaccharide

Introduction

The mast cell is an immunocyte, but its functions in the brain remain unclear.

Materials and methods

In a rat model of weak inflammation, we analyzed the effect of a gram-negative bacterial lipopolysaccharide injection (100?μg/kg) on thalamic mast cell (MC) population.

Conclusion

We demonstrate a significant decrease of their degranulation, which suggests the implication of MC in preventing sepsis on the brain.     

芝麻素通过PKCα/ NF-κB 信号途径抑制肥大细胞活化

目的:观察芝麻素对肥大细胞活化和炎症介质释放的影响并探讨其可能的作用机制。方法:体外培养HMC-1细胞,用10 μg/ ml 的anti-DNP IgE 处理细胞6 h 后,再用100 ng/ ml 的DNP-HAS 孵育10 min 诱导HMC-1 细胞活化,在DNP-HAS 处理前给予最终浓度25、50 和100 μg/ L 芝麻素预处理30 min 后光镜下观察芝麻素对肥大细胞脱颗粒的影响,ELISA 法检测芝麻素对肥大细胞组胺、TNF-α、IL-6、IL-1β、IL-8 释放的影响,Western blot 方法观察芝麻素对Fc RI 受体下游信号Lyn 和 Syk,PKC琢激活,IκB琢磷酸化和NF-κB 活化的影响。结果:DNP-HAS 能明显诱导HMC-1 细胞脱颗粒,促进组胺和TNF-α、IL-6、IL-1β和IL-8 等细胞因子的释放,激活Fc着RI 受体下游信号分子Lyn、Syk 和PKCα,磷酸化IκBα,促进NF-κB 活化(P<0.05)。芝麻素高(100 μg/ L)、中(50 μg/ L)浓度能明显抑制HMC-1 细胞脱颗粒和炎症介质的释放,阻断Fc着RI 受体下游信号激活,抑制NF-κB 活化(P<0.05)。结论:芝麻素通过PKCα/ NF-κB 途径抑制肥大细胞活化和炎症介质释放。     

Spleen tyrosine kinase: An Src family of non-receptor kinase has multiple functions and represents a valuable therapeutic target in the treatment of autoimmune and inflammatory diseases

Spleen tyrosine kinase (Syk) is involved in the development and function of B and T cells, the Fc receptor-mediated degranulation of basophils and mast cells. Recent work has assigned important roles for Syk in the aberrant function of T cells in patients with systemic lupus erythematosus (SLE), osteoclasts, and urate crystal-induced neutrophil stimulation. Preclinical and early clinical studies have urged Syk inhibition for the treatment of patients with rheumatoid arthritis, whereas ex vivo experiments and preclinical studies point to a therapeutic potential of Syk inhibition in patients with SLE and crystal-induced arthritides.     

Src family-selective tyrosine kinase inhibitor,PP1, inhibits both FcεRI- and Thy-1-mediated activation of rat basophilic leukemia cells

Cross-linking of the surface receptor with high affinity for IgE (FcεRI) by multivalent antigen/immunoglobulin E complexes, as well as aggregation of Thy-1 glycoprotein by monoclonal antibodies lead in rat basophilic leukemia cells, clone RBL-2H3, to tyrosine phosphorylation of several cellular proteins, followed by a release of secretory components. To investigate the molecular mechanisms of FcεRI- and Thy-1-mediated transmembrane signaling and to map a step at which they converge into a common secretory pathway, we used a novel Src family-selective tyrosine kinase inhibitory, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1), and analyzed its inhibitory activity on cell activation. Here we show that in RBL-2H3 cells PP1 demonstrates substrate specificity for a Src family kinase Lyn. In immunocomplex kinase assays in vitro, PP1 inhibited the Lyn kinase activity at nanomolar levels without any effect on Syk kinase activity. However, in RBL cells activated via aggregation of FcεRI, phosphorylation of both Syk and Lyn kinases was inhibited. FcεRI- and Thy-1-mediated early (protein-tyrosine phosphorylation) and late (release of β-hexosaminidase) activation events were similarly affected by PP1. The inhibition was specific for membrane receptor-mediated signaling and was not observed in cells activated by an exposure to pervanadate. The combined data suggest that activation of Lyn is the early activation step at which the FcεRI- and Thy-1-mediated activation pathways of mast cells and basophils may converge.     

Allergic FcεRI‐ and pseudo‐allergic MRGPRX2‐triggered mast cell activation routes are independent and inversely regulated by SCF

While allergic mast cell (MC) degranulation occurs by FcεRI aggregation and varies in strength among subjects, the analogous pseudo‐allergic route was recently uncovered to proceed via MRGPRX2. Here, we examine interindividual variability in skin MC responses to FcεRI triggering vs those evoked by MRGPRX2. While population‐based variability is comparable between the routes, FcεRI‐ and MRGPRX2‐stimulated pathways are completely independent from each other, and responsiveness to one has therefore no predictive value for the other. Conversely, degranulation triggered by compound 48/80 is highly correlated to the process elicited by substance P. MRGPRX2 mRNA shows pronounced population‐based variability (coefficient of variation 102.9%). Surprisingly, stem cell factor (SCF) as the MC‐supportive mediator par excellence potently inhibits pseudo‐allergic degranulation, while it simultaneously promotes allergic stimulation via FcεRI . We conclude that SCF can have selective MC‐dampening functions. Clinically, the data imply that subjects highly reactive in one pathway are not automatically hyper‐responsive in terms of the alternative route.     

Disparity in FcεRI-induced degranulation of primary human lung and skin mast cells exposed to adenosine

Inhaled and intravenously administered adenosine induces mast cell-mediated (histamine-dependent) bronchospasm in asthmatics without causing urticaria. A differential response to adenosine by human lung and skin mast cells is shown: low concentrations potentiate FcεRI-induced degranulation of human lung mast cells but not that of skin mast cells. Human lung mast cells were found to express ∼3-fold more A3AR messenger RNA (mRNA) than skin mast cells, suggesting the involvement of the G_i-linked A3AR. Indeed, the adenosine-induced potentiation was sensitive to inhibition by pertussis toxin and, furthermore, could be induced with an A3AR-specific agonist. This study reveals a previously unrecognized disparity in the response to adenosine by primary human mast cells from lung and skin that might explain why adenosine induces a pulmonary but not dermatologic allergy-like response in vivo. In addition, we identify the A3AR as a potentiating receptor of FcεRI-induced degranulation, thereby implicating it in the in vivo bronchoconstrictive response to adenosine in asthmatics.     

Changing the threshold—Signals and mechanisms of mast cell priming

Mast cells play a key role in allergy and other inflammatory diseases involving engagement of multivalent antigen with IgE bound to high-affinity IgE receptors (FcεRIs). Aggregation of FcεRIs on mast cells initiates a cascade of signaling events that eventually lead to degranulation, secretion of leukotrienes and prostaglandins, and cytokine and chemokine production contributing to the inflammatory response. Exposure to pro-inflammatory cytokines, chemokines, bacterial and viral products, as well as some other biological products and drugs, induces mast cell transition from the basal state into a primed one, which leads to enhanced response to IgE-antigen complexes. Mast cell priming changes the threshold for antigen-mediated activation by various mechanisms, depending on the priming agent used, which alone usually do not induce mast cell degranulation. In this review, we describe the priming processes induced in mast cells by various cytokines (stem cell factor, interleukins-4, -6 and -33), chemokines, other agents acting through G protein-coupled receptors (adenosine, prostaglandin E₂, sphingosine-1-phosphate, and β-2-adrenergic receptor agonists), toll-like receptors, and various drugs affecting the cytoskeleton. We will review the current knowledge about the molecular mechanisms behind priming of mast cells leading to degranulation and cytokine production and discuss the biological effects of mast cell priming induced by several cytokines.     

Adenosine closes the K+ channel KCa3.1 in human lung mast cells and inhibits their migration via the adenosine A2A receptor

Human lung mast cells (HLMC) express the Ca2+-activated K+ channel KCa3.1, which opens following IgE-dependent activation. This hyperpolarises the cell membrane and potentiates both Ca2+ influx and degranulation. In addition, blockade of KCa3.1 profoundly inhibits HLMC migration to a variety of diverse chemotactic stimuli. KCa3.1 activation is attenuated by the beta2adrenoceptor through a Galphas-coupled mechanism independent of cyclic AMP. Adenosine is an important mediator that both attenuates and enhances HLMC mediator release through the Galphas-coupled A2A and A2B adenosine receptors, respectively. We show that at concentrations that inhibit HLMC degranulation (10(-5)-10(-3) M), adenosine closes KCa3.1 both dose-dependently and reversibly. KCa3.1 suppression by adenosine was reversed partially by the selective adenosine A2A receptor antagonist ZM241385 but not by the A2B receptor antagonist MRS1754, and the effects of adenosine were mimicked by the selective A2A receptor agonist CGS21680. Adenosine also opened a depolarising current carried by non-selective cations. As predicted from the role of KCa3.1 in HLMC migration, adenosine abolished HLMC chemotaxis to asthmatic airway smooth muscle-conditioned medium. In summary, the Galphas-coupled adenosine A2A receptor closes KCa3.1, providing a clearly defined mechanism by which adenosine inhibits HLMC migration and degranulation. A2A receptor agonists with channel-modulating function may be useful for the treatment of mast cell-mediated disease.     

Leptin stimulates tissue rat mast cell pro-inflammatory activity and migratory response

Objective

The aim of this study was to determine whether leptin, a member of the adipocytokines involved in immune and inflammatory response regulation, may influence some aspects of mast cell biology.

Materials and methods

Experiments were done in vitro on fully mature tissue rat mast cells isolated from the peritoneal cavity, and leptin was used at concentrations 0.001–100 ng/ml. The effect of leptin on mast cell degranulation (histamine release assay), intracellular Ca²⁺ level (fluorimetry), pro-inflammatory mediator release (ELISA technique), surface receptor expression (flow cytometry and confocal microscopy), and migration (Boyden microchamber assay) was estimated.

Results

Leptin was found to stimulate mast cells to degranulation and histamine release. It induced the intracellular Ca²⁺ increase, as well. In response to leptin stimulation, mast cells generated and released cysLTs and chemokine CCL3. Leptin-induced upregulation of CYSLTR1 and CYSLTR2 surface expression was observed. Moreover, this adipocytokine stimulated mast cells to migratory response, even in the absence of extracellular matrix (ECM) proteins.

Conclusions

Our observations clearly documented that leptin promotes the pro-inflammatory activity of mast cells, and it thereby engages these cells in the inflammatory processes.

     

Autoreactive IgE in Chronic Spontaneous/Idiopathic Urticaria and Basophil/Mastocyte Priming Phenomenon,as a Feature of Autoimmune Nature of the Syndrome

Recent years of research have shed a new light on the role of IgE in immune reactions. It seems to be more than just a contribution to immediate type of allergic response. It appears that monomeric IgE may enhance mast cell activity without cross-linking of FcεRI by IgE specific allergen or autoreactive IgG anti-IgE antibodies. Monomeric IgE molecules are heterogeneous concerning their ability to induce survival and activation of mast cells only by binding the IgE to FcεRI, but not affecting degranulation of cells. It also turned out that IgE may react to autoantigens occurring in the blood not only in chronic spontaneous urticaria (CSU) but also in other autoimmune diseases. The aforementioned phenomena may promote the activity of mast cells/basophils in CSU that easily degranulate when influenced by various inner (autoreactive IgG against IgE and FcεRI, autoreactive IgE for self-antigens) and outer factors (cold, heat, pressure) or allergens. These findings forced the new approach to the role of autoimmunity, self-antigens and IgE autoantibodies in the pathology of CSU. CSU put in the scheme of autoreactive IgG and autoreactive IgE seems to be either a kind of an autoimmune disease or a clinical manifestation of some other defined autoimmune diseases or both.

     

Post-translational suppression of the high affinity IgE receptor expression on mast cells by an intestinal bacterium

Mast cells, which express the high-affinity IgE receptor (FcεRI) on their surface, play a crucial role in inducing allergic inflammation. Since mast cells are activated by crosslinking of FcεRI with IgE and allergens, the cell surface expression level of FcεRI is an important factor in determining the sensitivity to allergens. Recently, the involvement of gut microbiota in the prevalence and regulation of allergy has attracted attention but the precise underlying mechanisms are not fully understood. In this study, the effect of intestinal bacteria on cell surface expression of FcεRI was examined. Bacteroides acidifaciens type A 43 specifically suppressed cell surface expression of FcεRI on mouse bone marrow-derived mast cells (BMMCs) without reduction in FcεRI α and β-chain mRNA and total protein expression. The suppressive effect required sustained exposure to this bacterium, with a corresponding reduction in Erk activation. Inhibition of Erk decreased cell surface distribution of FcεRI in BMMCs, at least in part, through facilitated endocytosis of FcεRI. These results indicate that B. acidifaciens type A 43 suppresses cell surface expression of FcεRI on mast cells in a post-translational manner via inhibition of Erk. The suppression of FcεRI expression on mast cells by specific bacteria might be the underlying mechanism involved in the regulation of allergy by gut microbiota.     

Interleukin-33 amplifies IgE synthesis and triggers mast cell degranulation via interleukin-4 in naïve mice

Background

The regulation and function of IgE in healthy individuals and in antigen‐naïve animals is not well understood. IL‐33 administration increases serum IgE in mice with unknown mechanism. We tested the hypothesis that IL‐33 provides an antigen‐independent stimulus for IgE production and mast cell degranulation.

Methods

IL‐33 was administered to naïve wild‐type (WT), nude and ST2^?/?, IL‐4^?/?, IL4Rα^?/? and T‐or B‐cell‐specific IL‐4Rα^?/? mice. IgEand cytokines were quantified by ELISA. T‐ and B‐lymphocyte numbers and CD40L expression were determined by flow cytometry. Anaphylaxis was measured by temperature, mast cell degranulation and histamine release.

Results

IL‐33 enhanced IgE production in naïve WT, T‐IL‐4Rα^?/? but not in ST2^?/?, IL‐4^?/?, IL‐4Rα^?/? or B‐cell‐specific IL‐4Rα^?/? mice, demonstrating IL‐33 specificity and IL‐4 dependency. Moreover, IL‐4 was required for IL‐33‐induced B‐cell proliferation and T‐cell CD40L expression, which promotes IgE production. IL‐33‐induced IL‐4 production was mainly from innate cells including mast cells and eosinophils. IL‐33 increased mast cell surface IgE and triggered degranulation and systemic anaphylaxis in allergen‐naïve WT but not in IL‐4Rα^?/? mice.

Conclusion

IL‐33 amplifies IgE synthesis and triggers anaphylaxis in naïve mice via IL‐4, independent of allergen. IL‐33 may play an important role in nonatopic allergy and idiopathic anaphylaxis.