Sepsis resulting from gram-negative bacterial infections is a major clinical problem associated with many pathophysiological alterations, including liver failure. Damage to the liver during sepsis is mediated by bacterial lipopolysaccharide (LPS). Activation of the PAR-1 thrombin receptor is required for LPS-induced liver injury, and this action of thrombin may stimulate the release of a chemotactic factor that promotes transendothelial migration of neutrophils (PMNs) from the liver sinusoids into the hepatic parenchyma. In other systems, thrombin promotes transendothelial migration of PMNs and stimulates the release of chemotactic factors (ie. CINC-1) necessary for PMN transmigration. Therefore, the studies in this proposal will test the hypothesis that thrombin stimulates the release of a chemotactic factor that is necessary for transmigration of PMNs from the sinusoid into the parenchyma during LPS-induced liver in injury and that this action of thrombin is mediated through the PAR-1 receptor. Studies will first determine which cell types in the liver express the thrombin receptor using immunohistochemical methods and/or electron microscopy. Morphologic techniques, including immunohistochemistry, will then be used to determine if inhibition of thrombin attenuates transendothelial migration of PMNs after exposure of rats to LPS in vivo and in the isolated, perfused liver. Western blot analysis, ELISA and quantitative PCR will be used to determine if thrombin is required for the induction and release of the chemotactic protein, CINC-1, during LPS-induced liver injury. Studies using primary cultures of liver cells will determine which liver cell types are required for thrombin-induced PMN transmigration and CINC-1 release. Results of these studies will elucidate the mechanism by which thrombin exerts its critical action during inflammatory liver injury and may lead to a useful strategy for clinical intervention during sepsis.
