Scientific Abstract

Proposal No.   IBD-0080R
Principal Investigator:  Robert W. Colman, M.D.
Applicant Organization:   Temple University (Philadelphia, Pennsylvania, U.S.A.)
Project Title:  Kallikrein-kinin system in inflammatory bowel disease
Period of Award:  January 1, 2004 – December 31, 2006

We have shown that experimental enterocolitis, a model for Crohn’s disease, is associated with in vivo activation of the kallikrein-kinin system (KKS), which generates the enzyme, kallikrein, the inflammatory mediator, bradykinin (BK) and cleaved kininogen (HKa).  Kallikrein is chemotactic for neutrophils and stimulates the release of elastase, a potent effector of tissue injury.  We have also shown that contact system activation mediates acute and chronic phases of granulomatous intestinal inflammation in Lewis rats and is selectively activated in these susceptible Lewis rats, but not in resistant Buffalo rats.  Further, the changes are not specific to the PG-APS model, since indomethacin-induced ulceration in the Lewis rats and DSS-induced colitis are also characterized by contact system activation.  A specific kallikrein inhibitor (P8720) decreased both the KKS activation and acute inflammatory changes (edema, neutrophil infiltration) and prevented chronic intestinal inflammation and the systemic complications (arthritis, splenomegaly, hepatomegaly, leukocytosis, and the acute phase reaction) in the PG-APS model.

An important recent discovery from our laboratories is that there is a genetic difference in kininogen between the resistant Buffalo rat and the susceptible Lewis rat, resulting in increased cleavage of high molecular weight kininogen (HK) in the latter associated with a single nucleotide polymorphism in the kininogen gene that determines glycosylation and rate of cleavage.  This supports our previous work demonstrating that chronic intestinal inflammation occurs in genetically predisposed hosts challenged with bacterial products.  A second major observation is that purified HKa stimulates TNF production by isolated rat splenocytes.

This application explores two major hypotheses.  First, we will test whether the increased bradykinin or HKa formed due to more rapid HK cleavage leads to increased production of inflammatory cytokines by splenocytes.  We will explore the molecular requirements for the release of inflammatory cytokines including which domain of HK is responsible and which cells are involved, in particular the relationship of lymphocytes to macrophages.  Secondly, we will study in vitro which receptors are involved using antibodies to uPAR and MAC-1 as well as cells from mice deficient in these HKa receptors.  We will study the effect of inflammation on these receptors.  Finally, we will use murine knockouts of uPAR, MAC-1 and bradykinin receptors 1 and 2 to explore the role of these receptors in acute and chronic colitis, the latter performed in double knockout uPAR-/- or MAC-1-/- X IL-10-/- mice.  These studies should help define the mechanisms by which the KKS contribute to the pathogenesis of inflammatory bowel diseases and suggest entirely new modes of treatment.