Leukocyte adherence to endothelial cells is a rate-limiting event in the overall pathogenesis of ischemia/reperfusion (I/R)-induced injury to the intestinal microvasculature. The propensity for leukocytes to adhere within the postischemic intestinal microvasculature is principally determined by adhesive forces generated by membrane glycoproteins expressed on the surface of activated leukocytes and endothelial cells. Although both direct and indirect approaches have been used to demonstrate an increased surface expression of adhesion endothelial cell adhesion molecule (ECAMs) in postischemic tissues. We have recently developed a dual- radiolabeled monoclonal antibody (MAb) technique that provides a high resolution, quantitative measure of endothelial cell surface expression of P-selectin, E-selectin, ICAM-1 and VCAM-1 in mouse intestine and other tissues. The studies outlined in this project will employ the dual radiolabeled MAb technique to test the central hypothesis that cytokines, released as a consequence of an imbalance between the formation of reactive oxygen metabolites and nitric oxide, and in response to gut-derived endotoxin modulate athe level of expression of endothelial cell adhesion molecules after I/R.
Four specific aims are proposed; 1) to characterize the time-course and magnitude of expression of endothelial cell adhesion molecules (ICAM-1, VCAM-1, E-selectin and P-selectin) in intestines exposed to either I/R or cytokine (tumor necrosis factor or interleukin-1) stimulation;' 2) to define the contribution of reactive oxygen metabolites (ROM), derived from either activated endothelial cells (xanthine oxidase) or granulocytes, to I/R- or cytokine-induced expression of ECAMs; 3) to define the contribution of nitric oxide to the ECAM expression elicited by I/R or cytokine stimulation, and 4) to determine whether cytokines (TNFalpha and IL-1beta) and/or endotoxin contributes to the induction of ECAM expression elicited by I/R. In addition to ECAM expression, the following variables will be monitored in the postischemic and cytokine- treated mouse intestine: ECAM mRNAs, activation of the nuclear transcription factor NFkB, granulocyte accumulation, as well as plasma cytokine and endotoxin concentrations and blood leukocyte counts. The proposed experiments should also allow us to gain a better understanding of the critical contribution of ECAMs to the pathobiology of intestinal ischemia/reperfusion injury.
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