The overall objective of this proposal is to elucidate the mechanisms by which alcohol modulates several aspects of the biological activities and surface characteristics of neutrophils and mononuclear phagocytes, thereby contributing to the immunodeficiency of alcoholics. Specifically, this proposal will investigate the mechanisms responsible for injury after ethanol treatment or consumption by studying the cellular interactions between parenchymal and non-parenchymal cells (Kupffer, endothelial cells & neutrophils). During ethanol intoxication and endotoxemia, these cell types may interact to cause impairment of the immune response and to provoke tissue injury in the liver. This proposal will also investigate the modulation of neutrophil, Kupffer and endothelial cell function as they relate to the onset, development of cellular injury and inflammation during ethanol intoxication with or without endotoxemia. The mechanisms of leukocyte-endothelial cell-macrophage interaction and the release of relevant cytokines in vivo and in vitro will be examined. In order to achieve these goals, I will examine the release of chemotactic factors, such as C5a, leukotriene B4, interleukins 1 & 8, and tumor necrosis factor with or without endotoxemia. In vitro studies on the ability of phagocytes to interact with the above chemotaxins will be performed. In connection with these studies the modulation of receptors for selected ligands will be investigated during ethanol intoxication and endotoxemia. Consequences of neutrophil or Kupffer cell extravasation using an in vitro co-culture system will be used to complement the preceding studies. Furthermore the generation of superoxide anion, proteolytic enzymes such as cathepsin G and elastase will be determined. The destructive potential of these agents to endothelial cells and hepatocytes will be studied in a co-culture system. The thiobarbituric acid reaction for the determination of lipid peroxidation will be used to assess tissue or cell damage. For in vitro cytotoxicity assay, 51Cr-release and ASAT/ALAT assays will be employed. The above experimental models will be further manipulated with the use of 4-methypyrazole (inhibitor of alcohol dehydrogenase) acetaldehyde, alpha-1- protease inhibitor, ibuprofen (cyclooxygenase inhibitor), superoxide dismutase and allopurinol in vivo and in vitro. The purpose of these studies is to delineate the contribution of ethanol metabolites, oxygen- derived radicals, prostanoids and proteases on tissue injury and immunodeficiency. The mechanisms underlying the modulation of phagocyte function will be studied with the use of modulators of protein kinase C and NADPH oxidase in phagocytes.
