Neutrophil-epithelial interaction mediated by adenosine

腺苷介导的中性粒细胞-上皮相互作用

• 批准号: 6930349
• 负责人: SHANTHI Vasudevan SITARAMAN
• 金额: $ 26.93万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6930349
• 关键词: adenosine; basolateral membrane; binding sites; biological signal transduction; cell adhesion molecules; cell cell interaction; clinical research; gastrointestinal epithelium; gene deletion mutation; human subject; immunofluorescence technique; interleukin 6; metalloendopeptidases; neutrophil; nuclear factor kappa beta; point mutation; receptor expression; site directed mutagenesis

DESCRIPTION (provided by applicant): Acute flares of inflammatory diseases of the intestine are characterized by the migration of neutrophils across the intestinal epithelium into the lumen to form 'crypt abscess'. Adenosine is generated during active intestinal inflammation by the conversion of neutrophil-derived 5'AMP into adenosine by the epithelial 5'ectonucleotidase. Adenosine, acting via the A2b receptor, not only mediates electrogenic chloride secretion but also induces an apically polarized secretion of the pro-inflammatory cytokine, interleukin-6 (IL-6). IL-6, in turn, induces a calcium flux in neutrophils, thus providing a paracrine signal to neutrophils. In this R01 application, we propose to investigate the hypothesis that the A2b receptors play a central role in orchestrating both the diarrheal and inflammatory components of the intestinal inflammatory response. If so, pharmacological manipulation of the A2b receptor may be therapeutic for intestinal inflammation. The overall goals of this proposal are to characterize the intestinal A2b receptor (specific aim 1), to examine the regulation of adenosine-induced IL-6 secretion (specific aim 2) and to determine the effect of IL-6 on neutrophil function (specific aim 3). Specific aim 1: We will use model intestinal cell line, T84, expressing native A2b receptor and Caco2-BBE (which do not express A2b receptor) overexpressing GFP-A2b to study: i) the recruitment of the A2b receptor to apical vs. basolateral membrane using domain-specific biotinylation and immunoflourescence ii) the molecular mechanism of interaction of A2b receptor with E3KARP will be studied using deletion constructs and site-directed mutagenesis iii) the role of A2b receptor recruitment in receptor signaling and function and desensitization. Specific aim 2: We will investigate: i) the mechanism of adenosine-mediated IL-6 induction using IL-6 promoter constructs with deletion and point mutations of the various nuclear factor binding sites, ii) the effect of IL-6 on intestinal epithelia by examining STAT-3 and NF-?B activation and expression of ICAM-1 and metalloproteinases, proteins regulated by STAT-3 and/or NF-?B and known to play key role in the pathogenesis of intestinal inflammation. Specific aim 3: We will study: i) the effect of IL-6 on degranulation of neutrophils and ii) the role of IL-6 in neutrophil adhesion and transmigration.

描述（由申请方提供）：肠道炎性疾病急性发作的特征是中性粒细胞穿过肠上皮迁移到管腔中，形成“隐窝扩张”。腺苷是在活动性肠道炎症期间通过上皮细胞5 '外核苷酸酶将嗜酸性粒细胞衍生的5' AMP转化为腺苷而产生的。腺苷通过A2 b受体起作用，不仅介导产电氯化物分泌，而且诱导促炎细胞因子白细胞介素-6（IL-6）的顶端极化分泌。IL-6又诱导嗜中性粒细胞中的钙流，从而向嗜中性粒细胞提供旁分泌信号。在R 01申请中，我们提出研究A2 b受体在协调肠道炎症反应的肠道和炎症组分中发挥核心作用的假设。如果是这样的话，A2 b受体的药理学操作可能是治疗肠道炎症。该提案的总体目标是表征肠道A2 b受体（具体目标1），检查腺苷诱导的IL-6分泌的调节（具体目标2），并确定IL-6对中性粒细胞功能的影响（具体目标3）。具体目标1：我们将使用表达天然A2 b受体和Caco 2-BBE的模型肠细胞系T84，（其不表达A2 b受体）过表达GFP-A2 b以研究：i）使用结构域特异性生物素化和免疫荧光将A2 b受体募集至顶膜与基底外侧膜，ii）将使用缺失构建体和位点特异性免疫荧光研究A2 b受体与E3 KARP相互作用的分子机制。定向诱变iii）A2 b受体募集在受体信号传导和功能以及脱敏中的作用。具体目标2：我们将调查：i）使用具有各种核因子结合位点的缺失和点突变的IL-6启动子构建体的腺苷介导的IL-6诱导的机制，ii）通过检查STAT-3和NF-κ B对IL-6对肠上皮细胞的影响。B活化和ICAM-1和金属蛋白酶的表达，这些蛋白由STAT-3和/或NF-？已知B在肠道炎症的发病机制中起关键作用。具体目标3：我们将研究：i）IL-6对嗜中性粒细胞脱粒的作用和ii）IL-6在嗜中性粒细胞粘附和迁移中的作用。