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.
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