The inflammatory bowel diseases (IBD) Crohn's disease and ulcerative colitis are debilitating diseases of unknown etiology that affect 1-2 million individuals in the U.S. The current objective of this project is to further define the role that mucosal T cells play in the pathogenesis of IBD. The specific focus is to determine the mediators released by T cells in lesions, and to determine the T cell receptor specificity of lymphocytes in lesions. Previous studies of lymphokine production by IBD T cells have depended on in vitro activation of lymphocytes isolated from enzymatic digests of intestinal tissues, and there is no information regarding lymphokine production by T cells in situ. In current studies RNA was isolated from colonoscopic biopsies from patients with inflammatory bowel disease and noninflammatory controls (colon cancer or suspected cancer). We developed a sensitive method for quantitation of lymphokine mRNA expression in endoscopic biopsies. RNA was extracted from intestinal biopsies, reverse transcribed into cDNA, and amplified by PCR for IL-2. Dilutions of synthetic IL-2 mRNA transcripts that were slightly longer than native IL-2 were added to RNA samples as internal standards. IL-2 mRNA was detectable in as few as 100 activated lymphocytes or about 1 pg of mRNA, and dose response curves showed that there was a quantitative relationship between input mRNA and PCR products over a multiple log range. Using this method, we have found that expression of IL-2 was increased in biopsies of IBD patients who have active inflammation. This is the first demonstration of an abnormal increase in lymphokine expression in IBD. The proposed course will take advantage of this technology to define the T cell mediators expressed in inflammatory lesions and identify the T cell receptors expressed by T cells in IBD lesions. These results may serve as the basis for novel immunotherapies.
Showing the most recent 10 out of 37 publications
