Distinct roles of Ca2+ mobilization and G protein usage on regulation of Toll-like receptor function in human and murine mast cells

Toll-like receptors (TLRs) expressed in mast cells play important roles in orchestrating host defence against bacterial pathogens. Previous studies demonstrated that TLR2 agonist tripalmitoyl-S-glycero-Cys-(Lys)4 (Pam3Cys) stimulates both degranulation and cytokine production in human mast cells but only induces cytokine production in murine mast cells. To determine the molecular basis for this difference, we utilized a human mast cell line LAD 2, murine lung and bone marrow-derived mast cells (MLMC and BMMC). We found that Pam3Cys caused a sustained Ca2+ mobilization and degranulation in LAD 2 mast cells but not in MLMC or BMMC. Despite these differences, Pam3Cys stimulated equivalent chemokine CCL2 generation in all mast cell types tested. Cyclosporin A (CsA), an inhibitor of Ca2+/calcineurin-mediated nuclear factor of activated T cells (NFAT) activation, blocked chemokine production in LAD 2 but not in MLMC or BMMC. In contrast, inhibitors of nuclear factor kappa B (NF-kappaB) completely blocked CCL2 production in MLMC and BMMC but not in LAD 2 mast cells. Pertussis toxin and U0126, which, respectively, inhibit Galphai, extracellular signal-regulated kinase (ERK) phosphorylation substantially inhibited Pam3Cys-induced CCL2 generation in LAD 2 mast cells but had little or no effect on chemokine generation in MLMC and BMMC. These findings suggest that TLR2 activation in human LAD 2 mast cells and MLMC/BMMC promotes the release of different classes of mediators via distinct signalling pathways that depend on Ca2+ mobilization and G protein usage.     

Catestatin, a neuroendocrine antimicrobial peptide, induces human mast cell migration, degranulation and production of cytokines and chemokines

Catestatin, a neuroendocrine peptide with effects on human autonomic function, has recently been found to be a cutaneous antimicrobial peptide. Human catestatin exhibits three single nucleotide polymorphisms: Gly364Ser, Pro370Leu and Arg374Gln. Given reports indicating that antimicrobial peptides and neuropeptides induce mast cell activation, we postulated that catestatin might stimulate numerous functions of human mast cells, thereby participating in the regulation of skin inflammatory responses. Catestatin and its naturally occurring variants caused the human mast cell line LAD2 and peripheral blood-derived mast cells to migrate, degranulate and release leukotriene C(4) and prostaglandins D(2) and E(2). Moreover, catestatins increased intracellular Ca(2+) mobilization in mast cells, and induced the production of pro-inflammatory cytokines/chemokines such as granulocyte-macrophage colony-stimulating factor, monocyte chemotactic protein-1/CCL2, macrophage inflammatory protein-1α/CCL3 and macrophage inflammatory protein-1β/CCL4. Our evaluation of possible cellular mechanisms suggested that G-proteins, phospholipase C and the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) are involved in catestatin-induced mast cell activation as evidenced by the inhibitory effects of pertussis toxin (G-protein inhibitor), U-73122 (phospholipase C inhibitor) and U0126 (ERK inhibitor), respectively. We also found that human mast cells express the α7 subunit of the nicotinic acetylcholine receptor at both the mRNA and protein levels. Given that silencing the α7 receptor mRNA and an α7-specific inhibitor did not affect catestatin-mediated activation of mast cells, however, we concluded that this receptor is not likely to be functional in human mast cell stimulation by catestatins. Our finding that the neuroendocrine antimicrobial peptide catestatin activates human mast cells suggests that this peptide might have immunomodulatory functions, and provides a new link between neuroendocrine and cutaneous immune systems.     

CRAC channels and Ca2+ signaling in mast cells

Summary:  Mast cells are integral members of the immune system. Upon activation by a rise in cytoplasmic Ca²⁺, they release a battery of paracrine signals, chemokines, and cytokines, which help sculpt the subsequent immune response. Ca²⁺ entry through store-operated Ca²⁺ release-activated Ca²⁺ (CRAC) channels in the plasma membrane is central for driving most of these responses. The molecular basis of the CRAC channel has been identified, with Orai1 forming the channel pore. Recent work has revealed that a range of mast cell responses are activated by spatially restricted Ca²⁺ signals just below the plasma membrane. These Ca²⁺ microdomains can activate cytosolic enzymes, leading to the generation of intracellular messengers as well as proinflammatory molecules like LTC₄. In this review, we describe key features of CRAC channels in mast cells, how they generate local Ca²⁺ signals, and how the cell can decode these restricted signals to generate a raft of responses.     

The role of mast cells and basophils in inflammation

Mast cells are positioned in the asthmatic airways so that they are able to respond to the inhaled environment. During active disease, the cells are primed to secrete an array of preformed and newly generated inflammatory mediators including histamine, neutral proteases and heparin sulphate, prostaglandins and cysteinyl leukotrienes as well as an array of cytokines and chemokines that are involved in leucocyte recruitment and activation. These cells are a potent source of mediators in both allergen- and exercise-induced asthma and possibly in asthma provoked by other stimuli such as adenosine and inhaled air pollutants. The important role played by mast cells in maintaining airway dysfunction in asthma is underpinned by the efficacy of mediator inhibitors, such as those interfering with the release or action of the leukotrienes, agents that inhibit mast cell activation such as sodium cromoglycate and the recently studied E-20 humanized monoclonal antibody that binds to and removes IgE. The recent discovery of novel inhibitory pathways involving inhibitory motifs (ITIMS) on critical cell surface signalling molecules has opened up new possibilities for preventing mast cell activation. Future research will focus on more effective ways for inhibiting the mast cell's contribution to asthma and understanding what role this unique cell has in the pathogenesis of airway wall remodelling.     

Endogenous protein and enzyme fragments induce immunoglobulin E‐independent activation of mast cells via a G protein‐coupled receptor,MRGPRX2

Mast cells play a central role in inflammatory and allergic reactions by releasing inflammatory mediators through 2 main pathways, immunoglobulin E‐dependent and E‐independent activation. In the latter pathway, mast cells are activated by a diverse range of basic molecules (collectively known as basic secretagogues) through Mas‐related G protein‐coupled receptors (MRGPR s). In addition to the known basic secretagogues, here, we discovered several endogenous protein and enzyme fragments (such as chaperonin‐10 fragment) that act as bioactive peptides and induce immunoglobulin E‐independent mast cell activation via MRGPRX 2 (previously known as MrgX2), leading to the degranulation of mast cells. We discuss the possibility that MRGPRX 2 responds various as‐yet‐unidentified endogenous ligands that have specific characteristics, and propose that MRGPRX 2 plays an important role in regulating inflammatory responses to endogenous harmful stimuli, such as protein breakdown products released from damaged or dying cells.     

Mechanical stretch activates type 2 cyclooxygenase via activator protein-1 transcription factor in human myometrial cells

The uterus is subject to stretch throughout pregnancy, which, in the presence of progesterone, is a potent stimulus for uterine growth. However, in the absence of progesterone or when stretch is excessive, as in multiple pregnancy, it may provoke the onset of labour. We have investigated the effect of stretch on prostaglandin synthesis in primary human uterine myocytes [non-pregnant (NP), pregnant not in labour (NL) and pregnant in labour (L)]. The cells were grown on flexible bottom culture plates and subjected to 1 or 6 h static stretch. Expression of type 2 cyclooxygenase (COX-2) mRNA was similar in samples obtained from NP and L groups and both were significantly greater than those found in the NL group. Stretch of cells from all groups resulted in increased COX-2 mRNA expression. In further studies carried out on cells taken from the NL group, 6 h of stretch resulted in increased COX-2 protein levels and, in the media, increases in prostaglandin (PG) I(2) metabolite and PGE(2) concentrations and a reduction in the concentration of PGF(2)alpha metabolites. After stretch, EMSA studies showed increased activator protein-1 (AP-1) nuclear protein DNA binding activity but not of nuclear factor kappaB. These data demonstrate that stretch of human myocytes results in increased COX-2 activity and suggest that this may occur through activation of the AP-1 system.     

Midostaurin (PKC412) inhibits immunoglobulin E-dependent activation and mediator release in human blood basophils and mast cells

Background KIT tyrosine kinase inhibitors (TKI), such as nilotinib or midostaurin (PKC412), are increasingly used in clinical trials to counteract neoplastic cell growth in patients with aggressive mast cell (MC) disorders. However, these patients suffer not only from MC infiltration and consecutive organ damage but also from MC mediator-related symptoms.
Methods We examined the effects of three KIT TKI, imatinib, nilotinib, and midostaurin, on IgE-dependent mediator release in normal human blood basophils and cultured cord blood cell-derived MC, and on spontaneous histamine secretion in the MC leukaemia cell line HMC-1 and the basophil cell line KU812.
Results The multi-kinase inhibitor midostaurin that interacts with KIT and protein kinase C was found to counteract anti-IgE-induced mediator release in blood basophils and cultured cord blood cell-derived MC in all samples examined. By contrast, no effects of imatinib or nilotinib on histamine secretion in basophils or MC were found. The effects of midostaurin on histamine release were dose-dependent and occurred at pharmacologic concentrations (IC₅₀ 10-100 n m ). Midostaurin was also found to inhibit the IgE-dependent up-regulation of CD63 on cultured cord blood cell-derived human MC, but did not inhibit IgE-dependent up-regulation of CD63 or CD203c in human blood basophils.
Conclusion Midostaurin may be a beneficial drug in aggressive systemic mastocytosis not only because of its growth-inhibitory effects but also because of its additional effects on activation and mediator release in MC and basophils.     

Role of β-chemokines in mast cell activation and type I hypersensitivity reactions in the conjunctiva: in vivo and in vitro studies

Summary:  Chemokines have a clearly defined role in mobilizing the recruitment of leukocytes to both healthy and inflamed tissues. This review details work from our and other laboratories, indicating that β-chemokines may play important roles (i) in driving the terminal differentiation of mast cell precursors in mucosal tissues and (ii) in providing priming or costimulatory signals required for mast cell activation, leading to an antigen-driven inflammatory response. These data stem from in vivo , ex vivo , and in vitro studies. Data are also presented that suggest that FcɛRI:chemokine receptor cross talk may involve spatiotemporal dynamics that may control the strength and nature of the complex activating signals controlling mast cell effector function.     

Detection of novel leukocyte differentiation antigens on basophils and mast cells by HLDA8 antibodies

BACKGROUND: Basophils (BA) and mast cells (MC) are important effector cells in allergic reactions. Development, growth and effector cell functions are regulated by a network of cytokines, other ligands, and respective cell surface antigens. METHODS: We examined the expression of novel CD antigens on human BA, lung MC, the BA cell line KU-812, and the MC line HMC-1. Expression of surface antigens was analyzed by monoclonal antibodies (mAb) of the HLDA8 workshop and flow cytometry. RESULTS: Basophils were found to stain positive for CXCR1 (CD181), CCR1 (CD191), CCR2 (CD192), CCR7 (CD197), IL-18Ralpha (CDw218a), IL-18Rbeta (CDw218b), TRAIL-R1 (CD261), TRAIL-R2 (CD262), TACI (CD267), TLR-4 (CD284), LAIR1 (CD305), EMR-2 (CD312), JAM1 (CD321), and JAM2 (CD322). Lung MC were found to react with mAb against EMR-2 (CD312) and JAM1 (CD321). KU-812 cells were found to stain positive for CXCR1 (CD181), TRAIL-R2 (CD262), B7H2 (CD275), TLR-4 (CD284), JAM1 (CD321), and E-Cadherin (CD324). HMC-1 cells were recognized by mAb against TRAIL-R2 (CD262), B7H2 (CD275), LAIR1 (CD305), EMR-2 (CD312), JAM1 (CD321), and Siglec-6 (CDw327). CONCLUSIONS: Extensive phenotyping with antibodies against novel CD antigens provides further evidence that BA and MC represent two separate hematopoietic cell lineages with unique phenotypic properties observed in mature cells as well as malignant immature cells. Further studies are required to define the functional role of these CD antigens expressed in BA and MC.     

Differential modulation of mediator release from human basophils and mast cells by mizolastine

BACKGROUND: Basophils and mast cells play a major role in the pathogenesis of allergic disorders by releasing several proinflammatory mediators. Some histamine H1 receptor antagonists exert anti-inflammatory activities by modulating mediator release from basophils and mast cells. OBJECTIVE: To study the in vitro effects of mizolastine, an H1 receptor antagonist, on the release of eicosanoids, histamine and IL-4 from human basophils and lung mast cells. METHODS AND RESULTS: Mizolastine (10(-7)-10(-5) M) concentration-dependently inhibited the release of cysteinyl leukotriene C4 from anti-IgE-stimulated basophils (IC(50): 3.85+/-0.28 microM) and mast cells (IC(50): 3.92+/-0.41 microM). The same concentrations of mizolastine did not affect anti-IgE-induced prostaglandin D2 release from lung mast cells. In contrast, mizolastine enhanced up to 80% IgE-mediated histamine release (EC(50): 4.63+/-0.14 microM) from basophils, but not from mast cells and it significantly potentiated IL-4 release from basophils induced by anti-IgE. Mizolastine did not affect histamine release from basophils induced by formyl peptide, whereas it inhibited cysteinyl leukotriene C4 release (IC(50): 1.86+/-0.24 microM). Blockade of cytosolic phospholipase A2 and arachidonic acid mobilization by pyrrolidine-1 did not alter the effect of mizolastine on histamine release from basophils, thereby excluding accumulation of arachidonic acid metabolic intermediates as the cause of this effect. Mizolastine did not influence anti-IgE-induced activation of extracellular signal-regulated kinase-1 and -2 (ERK-1 and -2) in human basophils. CONCLUSIONS: Mizolastine efficiently inhibits LTC4 synthesis in human basophils and mast cells presumably by interfering with 5-lipoxygenase. In contrast, it enhances histamine and IL-4 release only from anti-IgE-stimulated basophils. Therefore, mizolastine differentially regulates the production of mediators from basophils and mast cells in a cell- and stimulus-specific fashion.     

TRAIL mediated signaling in human mast cells: the influence of IgE-dependent activation

Background:  Mast cells activation through FcɛRI cross-linking has a pivotal role in the initiation of allergic reactions. The influence of this activation on programmed cell death of human mast cells has not yet been clarified. This study evaluates the influence of IgE-dependent activation alone and in synergy with TRAIL on the expression of molecules involved in the apoptotic signal transduction.
Methods:  Human cord blood derived mast cells (CBMC) were cultured with myeloma IgE followed by activation with anti-human IgE. The expression of proteins involved in apoptotic signal transduction was assessed by immunoblot analysis. To test the effect of activation on a pro-apoptotic stimulus, activated, IgE-treated and resting CBMC were incubated with TRAIL, or in a medium with suboptimal concentrations of stem cell factor (SCF).
Results:  In accordance with a previous study of ours, it was found that IgE-dependent activation increased TRAIL-induced caspase-8 and caspase-3 cleavage. However, it did not have a significant influence on CBMC death induced by SCF withdrawal. IgE-dependent activation increased the expression of FLIP and myeloid cell leukemia 1 (MCL-1) anti-apoptotic molecules as well as the pro-apoptotic one, BIM. In addition, a decrease in BID expression was observed. TRAIL could reverse the increase in FLIP but did not influence the upregulation of MCL-1 and of BIM.
Conclusions:  These findings suggest that IgE-dependent activation of human mast cells induces an increase in both pro-survival and pro-apoptotic molecules. We therefore hypothesized that IgE-dependent activation may regulate human mast cell apoptosis by fine-tuning anti-apoptotic and pro-apoptotic factors.     

Assessment of histamine-releasing activity of sera from patients with chronic urticaria showing positive autologous skin test on human basophils and mast cells

BACKGROUND: All previous studies agree that only a proportion of sera from patients with chronic urticaria (CU) positive on the autologous serum skin test (ASST) are able to induce histamine release in vitro. A non-specific release of bradykinins during clotting of blood samples has been suggested; however, ASST seems rather specific and some data point to the existence of a mast cell-specific histamine-releasing factor. OBJECTIVE: To assess whether, and to what extent, the use of both human basophils and mast cells increases the sensitivity of in vitro histamine release assays (HRAs) in ASST-positive patients with CU. METHODS: The histamine-releasing activity of sera from 93 patients with CU selected on the basis of strong skin reactivity on ASST was assessed in vitro on basophils from 1 (n=86), 2 (n=31), or 3 (n=20) normal donors, and on mast cells from 1 (n=3), 2 (n=3), or 3 (n=87) normal donors. RESULTS: Sera from 88/93 (95%) patients induced significant histamine release from mast cells or basophils on at least one HRA. 76/93 (82%), 45/90 (50%), 22/80 (28%), and 6/12 (50%) sera were able to induce significant histamine release from cells of 2/5, 3/5, 4/5 and 5/5 donors, respectively. CONCLUSION: Sera from nearly all ASST-positive patients with CU are able to induce histamine release in vitro. However, the serum from each single patient seems to show its maximal activity on autologous mast cells in vivo, and functional in vitro tests show much variability and seem less sensitive than ASST in the detection of patients with histamine-releasing factors in their blood.     

哮喘大鼠模型信号转导子和转录激活子6在气道上皮细胞表达增强

目的探讨哮喘大鼠肺组织中信号转导子和转录激活子6(STAT6)的活化规律及其意义。方法将SD大鼠随机分为对照组和哮喘组,后者根据末次激发后处死时相不同分成0、3、8、24、72、120和144 h组。采用透射电镜、细胞计数、免疫组化和图像分析等,分别观察肺组织超微结构、支气管肺泡灌洗液(BALF)中嗜酸性粒细胞(EOS)数量及STAT6蛋白表达。结果①哮喘组肺组织EOS炎性浸润较对照组明显增加;②哮喘组STAT6蛋白主要在气道上皮细胞表达;③3 h时STAT6即开始明显升高,24 h达到峰值,其后有所下降;④STAT6蛋白表达的变化与BALF中EOS绝对值及EOS%的动态变化均显著正相关(P<0.01)。结论哮喘大鼠气道上皮细胞存在STAT6的持续活化及过度表达,并与EOS的生成密切相关,提示STAT6的活化在哮喘气道炎症调控中起重要作用。