The current hypothesis regarding the pathogenesis of ischemia/reperfusion injury to the small intestine implicates a role for neutrophils in the resultant microvascular and parenchymal dysfunction. We have recently developed an in vitro model of anoxia/reoxygenation (A/R; an in vitro correlate of ischemia/reperfusion) to test various aspects of this hypothesis. We propose to exploit this well-defined and controllable in vitro model by testing various mechanisms which cannot be addressed with in vivo models. We have recently demonstrated that exposure of endothelial cells to A/R results in the production of a proinflammatory substance(s) which 1) increases surface expression of the adherence complex, CD11/CD18, on neutrophils and 2) promotes neutrophil adherence to naive endothelial cells. In order to gain further insights regarding the pathogenesis of intestinal ischemia/reperfusion microvascular dysfunction, we will increased the complexity of the system by introducing additional elements which may modify neutrophil-endothelial cell interactions (i.e., neutrophil adherence to endothelium and neutrophil-mediated endothelial cell injury). Specifically, we will determine whether platelets enhance neutrophil-endothelial cell interactions and identify the platelet-derived products which are responsible by using immunoneutralization approaches (antibodies) or antagonists of serotonin, platelet activating factor, or leukotriene B4. We will also assess whether plasma and relevant concentrations of its constituent antioxidant proteins or antiproteases can modify neutrophil-endothelial cell interactions during A/R. Endothelial cells will also be exposed to acute and chronic shear stresses to address the potential mechanisms by which neutrophil-endothelial cell adhesive interactions are able to withstand higher shear stresses in vivo than in vitro. Finally, we will assess whether epithelial barrier function is impaired by neutrophils which have been activated by factors released from endothelial and/or epithelial (Caco-2) cells. In these studies we will address whether neutrophil adherence to Caco-2 cell monolayers is a prerequisite for A/R-induced neutrophil mediated barrier disruption by using monoclonal antibodies against neutrophil (CD11/CD18) and or Caco-2 cell (ICAM- 1) adherence glycoproteins. Inhibitors of myeloperoxidase and scavengers of hypochlorous acid will be used to assess the importance of oxidants generated by neutrophilic myeloperoxidase in the neutrophil-mediated barrier dysfunction. A role for neutrophil-derived proteases in this barrier disruption will be assessed by using protease inhibitors and antibodies directed against neutrophilic proteases. The results of these studies should allow us to construct a comprehensive model of the mechanisms involved in ischemia/reperfusion (and transplantation)-injury to the small intestine and provide a basis for the design of various therapeutic regimens.
