We have shown that bacterial products (proteoglycan-polysaccharide, PG- APS) found in the lower bowel produce chronic granulomatous inflammation similar to Crohn's disease (regional ileitis) in genetically susceptible rats as well as associated systemic inflammation. Activation of the contact system (CS) in plasma produces plasma kallikrein which activates neutrophils, cleaves high molecular weight kininogen (HK) and releases bradykinin which induces pain, swelling, diarrhea, and muscle contraction, all of which are characteristic symptoms of intestinal inflammation. We have shown that the CS activates mediates acute and chronic phases of intestinal inflammation in susceptible Lewis rats and is selective activated in these rats, but not in the resistant Buffalo rats. A specific kallikrein inhibitor decreases CS activation, acute inflammatory changes (edema, neutrophil infiltration), chronic intestinal inflammation and the systemic complications (arthritis, splenomegaly, hepatomegaly, leukocytosis, and the acute phase reaction. We have recently shown that there is a molecular difference between the plasma kininogen from Lewis rats which results in more rapid cleavage to yield bradykinin than in Buffalo rats. This proposal will test two hypotheses: (1) genetic differences between kininogen in susceptible and resistant rats result in selective activation of the Plasma Cs mediating certain of the pathological changes; (2) locally, intestinal tissue kallikrein is released and contributes to inflammatory changes. We will define the relationship of the single amino acid change to the functional consequences. In the view of the efficacy of plasma kallikrein inhibitors in blocking enterocolitis, we will use recombinant HK derivatives and peptides derived from HK which can distinguish in vitro whether plasma kallikrein stimulation of neutrophils or bradykinin actions are responsible. In vivo, we will use kinin receptor blockers to define the mechanisms responsible for the enterocolitis. If bradykinin (BK) is responsible, then BK receptor blockers already used in clinical trials may merit evaluation in the therapy of chronic granulomatous enterocolitis. If plasma kallikrein is responsible, the development of plasma kallikrein inhibitors should be pursued. We will study the effect of total kininogen deficiency on the development of acute and chronic enterocolitis and systemic inflammation. Our recent studies show that tissue kallikrein (TK) is localized in macrophages of chronic granulomas and that TK may be secreted from inflamed intestinal cells. We will assay low molecular weight kininogen, the substrate of Tk using a newly designed assay as well as the natural protease inhibitor of TK. We will study the behavior of the TK system in intestinal cell lines and macrophages stimulated with PG-APS or inflammatory cytokines. In vivo, we will use a new specific TK inhibitor to attempt to modulate intestinal and systemic inflammation. These studies should demonstrate important mechanisms in the pathogenesis of inflammatory bowel disease. Assays of the CS and/or TK systems could distinguish active form inactive disease. In addition, the inhibitors used alone or in combination could serve in the future as potential therapeutic agents of human disease.
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