The long-term goal of the investigators is to examine how the epithelium functions as both a target and a participant in intestinal inflammation. In this proposal, studies will focus primarily on the intestinal epithelium as a site of arachidonic acid metabolism, the role of these biochemical pathways in mediating the effect of inflammatory mediators on chloride secretion, and the regulation of epithelial arachidonic acid metabolism by cytokines. The underlying hypotheses to be tested are that 1) the intestinal epithelium is a site of intracellular and transcellular arachidonic acid metabolism; 2) adenosine and related inflammatory mediators stimulate chloride secretion by activating novel signal transduction mechanism involving arachidonic acid; and 3) cytokines upregulate epithelial arachidonic acid metabolism, with consequences of inflammatory mediator generation and/or enhanced chloride secretion. The hypotheses will be tested by addressing four specific aims. These will be 1) to determine the profile of arachidonic acid metabolites produced by epithelial cell lines; 2) to determine the involvement of arachidonic acid mobilization/metabolism in the secretory effects of adenosine and other agonists; 3) to study the effect of cytokines on the expression of enzymes responsible for arachidonic acid mobilization and metabolism; and 4) to determine whether such enzymes are induced in tissue from patients with inflammatory bowel disease. The experimental strategy will be primarily to examine responses of human intestinal epithelial cells growth in culture. Cells will be studied under basal conditions, following stimulation with adenosine or other chloride secretagogues, and after chronic exposure to specific cytokines or media conditioned by mast cells or lymphocytes. The experiments will employ biochemical assays of arachidonic acid metabolism with HPLC analysis of generated products, Ussing chamber studies of ion transport, analysis of cellular signal transduction mechanisms, and molecular biological approaches to examine enzyme expression. These in vitro approaches will be complemented by immunohistochemical analyses of enzyme expression in the epithelium of biopsy specimens from patients and controls. The significance of the studies lies in their potential to delineate pathogenic mechanisms in intestinal inflammation, inflammatory diarrhea, and possibly, secretory diarrhea in general. It is the goal of the investigators to provide information that will be useful in improving the therapy of patients with inflammatory bowel disease.
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