Generation Of Cytokines And Arachidonic Acid In Mast Cells

肥大细胞中细胞因子和花生四烯酸的产生

• 批准号: 7734947
• 负责人: Michael A Beaven
• 金额: $ 68.36万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7734947
• 关键词: Accounting; Adaptor Signaling Protein; Address; Adenosine; Adult asthma; Adverse effects; Affect; Allergens; Allergic; Allergic Reaction; Anti-Inflammatory Agents; Anti-inflammatory; Antigens; Arachidonic Acids; Ascaridil; Autoimmune Diseases; Bacterial Infections; Binding; Biological Assay; Blood; Calcineurin; Calcium; Calcium Signaling; Cell Line; Cells; Chronic; Collaborations; Curcumin; Cytokine Gene; Cytokine Suppression; DNA; Dactinomycin; Development; Dexamethasone; Dimensions; Dinoprostone; Disease; Elements; Endogenous Factors; Endothelial Cells; Enzymes; Evaluation; Event; Exposure to; Family; GTP-Binding Proteins; Generations; Genes; Genetic Transcription; Glucocorticoid Receptor; Glucocorticoids; Goals; Healed; Homeopathy; Hour; IgE; Immune; Immune response; In Vitro; Incidental Findings; Individual; Inflammatory Response; Interleukin-1; Interleukin-3; Interleukins; Lead; Ligands; Link; Luciferases; MAP3K7 gene; Manuscripts; Mediating; Metabolic; Mineralocorticoids; Mitogen-Activated Protein Kinases; Mutate; NFAT Pathway; National Institute of Allergy and Infectious Disease; Natural Immunity; Orphan; Passive Cutaneous Anaphylaxis; Pathologic; Pathway interactions; Pharmaceutical Preparations; Phospholipase C; Phospholipase D; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Plants; Play; Population; Postdoctoral Fellow; Primary Neoplasm; Process; Production; Prostaglandin E Receptor; Protein Isoforms; Protein Kinase C; Protein Overexpression; Purinergic P1 Receptors; Ras/Raf; Recruitment Activity; Reporter; Reporting; Response Elements; Reverse Transcriptase Polymerase Chain Reaction; Rheumatoid Arthritis; Role; Series; Signal Pathway; Signal Transduction; Specificity; Stem Cell Factor; Steroids; TNF receptor-associated factor 6; TRAF6 gene; Therapeutic Agents; Thinking; Tissues; Transactivation; Up-Regulation; Viral; chromatin immunoprecipitation; clinically relevant; cytokine; expectation; flavanoid; healing; hypothemycin; in vivo; inhibitor/antagonist; interest; mast cell; member; novel; programs; prostaglandin EP3 receptor; receptor; release of sequestered calcium ion into cytoplasm; response; transcription factor

CURRENT OBJECTIVES: 1) To identify endogenous factors that modulate mast cell reactivity to antigen: this year we have completed studies on PGE2. Like adenosine, receptors for PGE2 are expressed on mast cells, PGE2 accumulates in inflamed tissues, and by itself minimally stimulates mast cells. We have also examined another potential co-stimulant, IL-33. IL-33 is a recently discovered member of the IL-1 super family that acts through an orphan receptor, ST2, to activate TLR-mediated signaling pathways. Of particular interest, IL-33 accumulates in inflamed tissues associated with allergic and autoimmune diseases. The question addressed is whether IL-33, like TLR ligands, acts in synergy with antigen and if so by what mechanism. 2) To continue studies of the mechanisms by which glucocorticoids suppress activation of mast cells. In the current period we have examined the mechanism of upregulation of glucocorticoid-inducible inhibitory regulators at a transcriptional level. 3) To continue studies of the inhibitory actions of glucocorticoids on the activation of mast cells by TLR ligands. Of specific interest is the upregulation of suppressor of cytokine signaling1 (Socs1) and IKKalpha by dexamethasone. RESULTS: 1) IDENTIFICATION OF FACTORS THAT MODULATE RESPONSES TO ANTIGEN: In collaboration with Gilfillan and Metcalfe (LAD, NIAID) we found that PGE2, acting through its G protein-coupled EP3 receptor, markedly potentiated FceRI-mediated activation of phospholipase (PL) C, calcium signal, calcium-dependent PKC isoforms, and degranulation. Of note, activation of both EP3-linked PLCbeta and FceRI-linked PLCgamma were amplified by co-stimulation to reveal unexpected cross talk between these two PLCs (ref. 1). These and our previous studies with adenosine, Kit ligand (SCF) and TLR ligands, illustrate the remarkable ability of co-stimulants to augment mast cells to antigen. In fact, simultaneous inhibition of SCF (an essential growth factor for mast cells) and antigen mediated signaling events with hypothemycin is a particularly effective means of suppressing mast cell activation (ref. 2). An incidental finding some years ago was that the plant flavanoid, curcumin, was a potent inhibitor of mast cell cytokine production in vitro. A former post doctoral fellow of the PI has since continued studies with this compound and found that it is equally potent in suppressing passive cutaneous anaphylaxis (PCA) in vivo and acts at the level of Syk, a key kinase in the FceRI signaling pathways (ref. 3). IL-33 was found to be the most potent agent examined to date with respect to its ability to augment responses to antigen. At almost homeopathic concentrations (10 pg/ml), IL-33 markedly potentiated antigen-induced cytokine production in primary and tumor mast cell lines. This phenomenon was observed with concentrations of antigen that minimally stimulated cytokine production and degranulation. In effect, IL-33 finely tuned the mast cell response towards cytokine production. IL-33 was ineffective in MyD88-deficient mast cells indicating that IL-33 acted through TLR signaling pathways for which MyD88 is a critical adaptor protein. Preliminary evaluation of signaling mechanisms indicated that IL-33 robustly stimulated the NFkappB pathway and antigen the Ca2+/calcineurin/NFAT pathway which together produced a duality in signaling that was not evident with the individual stimulants. Therefore, production of IL-33 in inflamed tissue may profoundly affect mast cell cytokine production in response to antigen and perhaps other stimulants. 2) UPREGULATION OF INHIBITORY REGULATORS BY GLUCOCORTOIDS, A NOVEL MECHANISM OF ACTION OF GLUCORTICOIDS: The participation of GR and GRE in some inhibitory actions of dexamethasone in mast cells was verified by overexpression of a mutated GR that cannot interact with GREs. In such cells the ability of dexamethasone to inhibit degranulation and release of arachidonic acid was reduced without affecting its ability to suppress of cytokine production. This finding is consistent with the notion that suppression of cytokine production is due to "transrepression" of cytokine gene transcription which would not involve GRE whereas the upregulation of inhibitory regulators might involve "transactivation" via GRE (see Goals and Objectives section). The upregulation of the inhibitory regulators Dok1, SLAP1, and DUSP1 in the RBL-2H3 mast cell line was inhibited by actinomycin D and was thus dependent on gene transcription. Examination of the gene sequences by the MULAN program revealed a half GRE and a full GRE as potential regulators of the Dok1 and SLAP1 genes, respectively. As indicated by luciferase reporter assays, the SLAP1 GRE regulated gene transcription in dexamethasone treated cells whereas the half GRE of Dok1 did not do so. Binding of GR to the SLAP1 GRE was confirmed by chromatin immunoprecipitation assay. Similar studies with various steroids and a GR antagonist verified the GR/SLAP1 GRE connection. Therefore, upregulation of SLAP1 by dexamethasone is regulated primarily through interaction of GR with GRE while other transcription mechanisms come into play for the upregulation of Dok1 and DUSP1. Our findings add a new dimension to our understanding of the inhibitory actions of glucocorticoids. These findings are relevant to the current development of "dissociated" steroids that lack the "transactivation" potential of clinically approved glucococorticoids, a potential that was previously thought to account for the undesirable side-effects of glucocortoids. However, glucocorticoids lacking this "transactivation" potential may also fail to induce inhibitory regulators such as those discovered in our studies (manuscript submitted). 3) SYNERGY BETWEEN ANTIGEN AND TLR LIGANDS IS SUPPRESSED BY GLUCOCORTICOIDS: The enhanced production of cytokines on co-stimulation of mast cells with antigen and TLR ligands was attributed to the engagement of a more effective repertoire of transcription factors for cytokine gene transcription (Blood 107:610, 2006 and see previous reports). Glucocorticoids potently inhibit production of cytokines in response TLR ligands alone or in combination with antigen. As with antigen stimulation, the inhibitory effects were apparent after a delay of several hours on exposure to low concentrations of dexamethasone and were mediated via GR. Again in common with antigen stimulation, activation of MAP kinases by TLR ligands is inhibited by dexamethasone possibly through the induction of DUSP1 and Dok1. In addition, the activation of upstream TAK1 by TRAF6 and downstream NFkappB are key targets for dexamethasone in TLR activated mast cells. Use of chip arrays and RT-PCR revealed several additional dexamethasone-inducible inhibitory regulators, SOCS1, its cognate CIS, and IKKalpha. SOCS1 but not CIS was found to associate with TRAF6 and enhance its degradation. Knockdown of SOCS1, partially reverses the inhibitory effects of dexamethasone on TRAF6 degradation and phosphorylation of TAK1 to suggest that dexamethasone may act at this level through upregulation of SOCS1. Levels of IKKalpha, a negative regulator of NFkappaB, are increased more than 10 fold in dexamethasone treated cells. For this reason, IKKalpha is being studied as a fifth candidate for mediating the inhibitory actions of glucocorticoids. The ability of glucocorticoids to suppress both antigen and TLR-mediated signaling events suggests that they may be especially effective when allergic disease is exacerbated by viral or bacterial infection (ref. 4)

当前目标： 1）确定调节肥大细胞对抗原反应性的内源性因素：今年我们已经完成了对PGE2的研究。像腺苷一样，PGE2的受体在肥大细胞上表达，PGE2在发炎的组织中积聚，并且本身微微刺激了肥大细胞。我们还研究了另一种潜在的共同刺激IL-33。 IL-33是最近发现的IL-1超级家族的成员，该家族通过孤儿受体ST2起作用，以激活TLR介导的信号传导途径。特别有趣的是，IL-33在与过敏和自身免疫性疾病有关的发炎组织中积累。提出的问题是，IL-33（例如TLR配体）是否与抗原协同作用，如果是哪种机制。 2）继续研究糖皮质激素抑制肥大细胞激活的机制。在当前时期，我们检查了转录水平上糖皮质激素诱导的调节剂上调的机理。 3）继续研究糖皮质激素对TLR配体激活肥大细胞的抑制作用。特别感兴趣的是地塞米松的细胞因子信号1（SOCS1）和ikkalpha的抑制剂的上调。 结果： 1）识别调节对抗原反应的因素：与Gilfillan和Metcalfe（LAD，NIAID）合作，我们发现PGE2通过其G蛋白偶联EP3受体作用，显着增强FCERI介导的FCERI介导的磷脂酶（PL）C的激活，磷脂信号，钙信号，钙，钙化pkc iSoforms和Degrymems和Degryms和Degryms和Degryms和Degryms和Degryms＆Degryms和Degryms和Degryms和Degryms＆Degrym＆Degryms＆Degryms＆Degrym and Degryms＆Degryms和Degrym and Degry。值得注意的是，通过共刺激将EP3链接的PLCBETA和FCERI连接的PLCGAMMA的激活放大，以揭示这两个PLC之间意外的交叉交谈（参考文献1）。这些以及我们先前对腺苷，试剂盒配体（SCF）和TLR配体的研究，说明了共刺激剂增强肥大细胞对抗原的显着能力。实际上，同时抑制SCF（肥大细胞的基本生长因子）和抗原介导的信号事件，是抑制肥大细胞激活的一种特别有效的方法（参考文献2）。几年前的一个偶然发现是，植物类黄酮姜黄素是肥大细胞细胞因子在体外产生的有效抑制剂。此后，PI的前博士研究生一直在研究这种化合物，并发现它在体内抑制被动性皮肤过敏症（PCA）同样有效，并在Syk的水平上起作用，Syk是FCERI信号传导途径中的关键激酶（参考文献3）。 发现IL-33是迄今为止研究的最有效的药物，就其增强对抗原反应的能力而言。在几乎顺势疗法浓度（10 pg/mL）下，IL-33显着增强了抗原诱导的原代和肿瘤肥大细胞系中的细胞因子的产生。使用微小刺激细胞因子产生和脱粒的抗原浓度观察到了这种现象。实际上，IL-33对肥大细胞对细胞因子产生的反应进行了细微调整。 IL-33在MyD88缺陷型肥大细胞中无效，表明IL-33通过TLR信号传导途径作用，MyD88是关键的适配器蛋白。信号传导机制的初步评估表明，IL-33鲁棒刺激NFKAPPB途径和抗原Ca2+/钙调蛋白/NFAT途径，该途径共同产生了信号的双重性，而单个刺激物则不明显。因此，发炎组织中IL-33的产生可能会对抗原和其他兴奋剂的响应肥大细胞因子产生产生深远影响。 2）通过糖皮质激素对抑制性调节剂的上调，糖皮质激素是一种新型的糖脂作用机理：GR和GRE参与地塞米松在肥大细胞中的某些抑制作用中，通过过表达无法与GRE相互作用的突变GR来验证。在这样的细胞中，地塞米松抑制脱粒并释放蛛网膜含量的能力降低了，而不会影响其抑制细胞因子产生的能力。这一发现与以下观点一致：细胞因子产生的抑制是由于细胞因子基因转录的“反抑制”所致，这不会涉及GRE，而抑制性调节剂的上调可能涉及通过GRE进行“反式激活”（请参阅​​目标和目标部分）。放线霉素D抑制RBL-2H3肥大细胞系中抑制性调节剂DOK1，SLAP1和DUSP1的上调，因此依赖基因转录。通过Mulan程序对基因序列的检查分别显示了半GRE和全GRE作为DOK1和SLAP1基因的潜在调节剂。如荧光素酶报道测定法所示，slap1 GRE调节的基因转录在地塞米松处理的细胞中，而DOK1的一半GRE则没有。通过染色质免疫沉淀测定法证实了GR与Slap1 GRE的结合。与各种类固醇和GR拮抗剂的类似研究验证了GR/SLAP1 GRE连接。因此，地塞米松对slap1的上调主要是通过GR与GR的相互作用来调节的，而其他转录机制则起着DOK1和DUSP1的上调作用。我们的发现为我们对糖皮质激素的抑制作用的理解增加了一个新的维度。这些发现与当前缺乏临床认可的糖皮质激素的“反式激活”潜力的“解离”类固醇的发展有关，该潜力以前被认为是糖皮质激素的不良副作用。但是，缺乏这种“反式激活”潜力的糖皮质激素也可能无法诱导抑制性调节剂，例如我们的研究中发现的抑制剂（手稿提交）。 3）抗原和TLR配体之间的协同糖皮质激素受到抑制：在与抗原和TLR配体共同刺激的肥大细胞中的细胞因子的产生增强归因于对细胞因子基因转录的转录因子的更有效曲目的参与（Flood 107：610：610，2006，2006，2006年）。糖皮质激素可有效抑制单独或与抗原结合的反应TLR配体中的细胞因子的产生。与抗原刺激一样，在暴露于低浓度地塞米松的几个小时后，抑制作用显而易见，并通过GR介导。再次与抗原刺激共同，TLR配体对MAP激酶的激活可能通过诱导DUSP1和DOK1的诱导抑制了TLR配体的激活。此外，Traf6和下游NFKAPPB激活上游TAK1是TLR激活肥大细胞中地塞米松的关键目标。芯片阵列和RT-PCR的使用显示出了其他几个可毒剂诱导的抑制性调节剂SOCS1，其同源CIS和IKKALPHA。 SOCS1但没有CIS与TRAF6相关并增强其降解。 SOCS1的敲低部分逆转了地塞米松对TRAF6降解和TAK1磷酸化的抑制作用，以表明地塞米松可以通过上调SOCS1在此级别上起作用。 ikkalpha的水平是NFKAPPAB的负调节剂，在地塞米松处理的细胞中增加了10倍以上。因此，正在研究Ikkalpha作为介导糖皮质激素抑制作用的第五个候选者。糖皮质激素抑制抗原和TLR介导的信号事件的能力表明，当通过病毒或细菌感染加剧过敏性疾病时，它们可能特别有效（参考文献4）