The goal of this project is to elucidate the molecular mechanisms of leukocyte recruitment and leukocyte-epithelial cell interactions in inflammatory bowel disease (IBD). We have recently reported the novel finding that colonic epithelial cells may be induced to express the cell adhesion molecule ICAM1. This proposal addresses the hypothesis that surface expression of ICAM1 and of other leukocyte adhesion counter- receptors by intestinal epithelial cells plays a central part in the regulation of leukocyte activation and trafficking in intestinal inflammation. A combination of in vivo human and animal studies and in vitro cell culture experiments will be performed. Human colonic tissue from IBD patients and controls will be studied immunohistochemically for expression of the cell adhesion molecules (CAMs), ICAM1, VCAM1 and ELAM1. We will also study the regulatory effects of the cytokines IFNgamma, TNFalpha and IL-1beta on epithelial cell CAM expression in short term organ culture of colonic biopsies. Our preliminary data demonstrates that the HT29 human colon cancer cell line displays cytokine-regulated ICAM1 expression and supports ICAM1 and CD11/CD18- dependent adhesion of PMNs and of lymphocytes. Further studies in this in vitro model will examine cytokine regulation of CAM (ICAM1, VCAM1 & ELAM1) expression by intestinal epithelial cells by a combination of ELISA (to quantify surface expression) and Northern blot analysis (to study transcriptional regulation). CAM-specific blocking monoclonal antibodies will be used to determine the individual CAMs which support leukocyte-HT29 cell adhesion and transmigration. Lymphocyte-HT29 cell adhesion and migration will be compared in peripheral blood and lamina propria lymphocytes from patients with IBD and from controls. In other experiments CAM-specific blocking monoclonal antibodies will be used to test the hypothesis that PMN-mediated epithelial (HT29) cell damage is adhesion-dependent. the essential role of CAM interactions in leukocyte activation and recruitment suggest a potential for the therapeutic application of CAM blockade in IBD. This suggestion is supported by our preliminary data showing that in vivo treatment with a blocking monoclonal antibody to the adhesion molecule CD18 substantially reduced Clostridium difficile toxin A induced enteritis in the rabbit. Further studies will define the mechanisms of CAM upregulation in this experimental model of intestinal inflammation.
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