Neutrophil-epithelial interaction mediated by adenosine

描述（由申请人提供）：肠道炎症性疾病急性发作的特征是中性粒细胞穿过肠上皮迁移到腔内形成“隐窝脓肿”。腺苷是在活跃的肠道炎症中由上皮5‘外核苷酸酶将中性粒细胞衍生的5’AMP转化为腺苷而产生的。腺苷通过A2b受体起作用，不仅介导电致氯离子分泌，还诱导促炎细胞因子白介素-6 （IL-6）的顶端极化分泌。反过来，IL-6诱导中性粒细胞中的钙通量，从而向中性粒细胞提供旁分泌信号。在本R01申请中，我们提出研究A2b受体在肠道炎症反应的腹泻和炎症成分中发挥核心作用的假设。如果是这样，对A2b受体的药理学操作可能对肠道炎症有治疗作用。本提案的总体目标是表征肠道A2b受体（特异性目的1），研究腺苷诱导的IL-6分泌的调节（特异性目的2），并确定IL-6对中性粒细胞功能的影响（特异性目的3）。具体目的1：我们将使用表达天然A2b受体的模型肠细胞系T84和过表达GFP-A2b的Caco2-BBE（不表达A2b受体）进行研究：i)利用结构域特异性生物素化和免疫荧光技术将A2b受体募集到根尖膜和基底膜；ii)将利用缺失构建和定点诱变技术研究A2b受体与E3KARP相互作用的分子机制；iii) A2b受体募集在受体信号传导、功能和脱敏中的作用。具体目的2：我们将研究：i)利用含有各种核因子结合位点缺失和点突变的IL-6启动子构建的腺苷介导的IL-6诱导机制；ii)通过检测STAT-3和NF-？B ICAM-1和金属蛋白酶的激活和表达，stat3和/或NF-？已知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对中性粒细胞脱颗粒的影响以及ii) IL-6在中性粒细胞粘附和转运中的作用。

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.