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: 6670116
• 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/6670116
• 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（由申请人提供）：肠道炎症性疾病的急性耀斑的特征是中性粒细胞遍布肠上皮的中性粒细胞到管腔中，形成了“隐窝脓肿”。通过上皮5'ectonucleotidase将中性粒细胞衍生的5'AMP转化为腺苷，在活性肠炎期间产生腺苷。通过A2B受体作用的腺苷不仅介导了氯化物的分泌，而且还诱导了促炎性细胞因子的根尖分泌，介导性细胞因子Interleukin-6（IL-6）。 IL-6反过来诱导中性粒细胞中的钙通量，从而为中性粒细胞提供了旁分泌信号。在此R01应用中，我们建议研究A2B受体在策划肠炎症反应的腹泻和炎症成分中起着核心作用的假设。如果是这样，A2B受体的药理操作可能是治疗肠炎的治疗方法。该提案的总体目标是表征肠道A2b受体（特定目标1），以检查腺苷诱导的IL-6分泌的调节（特定目标2），并确定IL-6对中性粒细胞功能的影响（特定目标3）。具体目的1：我们将使用模型的肠道细胞系T84，表达天然A2b受体和CACO2-BBE（不表达A2B受体）过表达GFP-A2B的过表达：i i）A2b受体使用域基于基底膜的基底膜和免疫化的II相互作用A2B受体募集具有E3KARP的受体将使用缺失构建体和定点诱变III研究）A2B受体募集在受体信号传导以及功能以及功能和脱敏化中的作用。具体目的2：我们将研究：i）使用IL-6启动子构建体的腺苷介导的IL-6诱导的机制，具有各种核因子结合位点的缺失和点突变，ii）IL-6通过通过检查肠道上皮的影响，通过检查Stat-3和NF-的stat-3和NF-型和NF-型和NF-型和NF-的表达和统计量和蛋白蛋白蛋白蛋白和蛋白质的蛋白酶蛋白酶，或蛋白质酶，蛋白酶蛋白酶，蛋白酶蛋白酶，蛋白酶蛋白酶，蛋白酶蛋白酶，蛋白酶和核蛋白酶酶，蛋白酶和蛋白酶蛋白酶酶（NOT）蛋白酶蛋白酶酶（蛋白酶）和ote蛋白（在肠道炎症的发病机理中起关键作用。具体目的3：我们将研究：i）IL-6对中性粒细胞脱粒的影响，ii）IL-6在中性粒细胞粘附和移民中的作用。