Alcoholic liver disease (ALD) is an important public health problem. Patients with alcoholic hepatitis (AH) frequently have endotoxemia and elevated plasma tumor necrosis factor (TNF) levels which correlate with clinical indicators of liver dysfunction and mortality. Hepatic neutrophil (PMN) infiltration is an early manifestation of AH. Interleukin-8 (IL-8), a cytokine also known as neutrophil chemotactic factor, is an 8 kDa polypeptide produced by monocytes and hepatocytes in response to endotoxin (LPS) and TNF. We recently reported elevated IL-8 levels in acute AH that decreased as patients improved clinically. The grade of immunohistochemical staining for IL-8 in liver tissue correlates with the degree of hepatic PMN infiltration in AH. A positive effect of treatment with corticosteroids in AH can be predicted by hepatic PMN infiltration, and corticosteroids downregulate IL-8 production. Ethanol metabolism produces reactive oxygen intermediates and chronic alcohol abuse is associated with decreased levels of nutritionally dependent free radical scavengers such as glutathione. NF(kappa)B is a transcription factor for several cytokines including IL-8. NF(kappa)B is activated by reactive oxygen intermediates and its activation can be blocked by antioxidants. It is our working hypothesis that IL-8 plays an etiologic role in the neutrophilia, hepatic PMN infiltration and PMN-induced liver damage in AH. The overall research goals of our laboratory are to further define mechanisms and modulatory pathways whereby cytokines such as IL-8 induce metabolic disturbances and liver injury in ALD, with the ultimate goal being to develop a specific and anti-cytokine therapy for ALD. In this proposal, we will evaluate the role of oxidative stress and antioxidants in regulating IL-8 gene expression. We will determine whether plasma IL-8 levels increase in normal volunteers given alcohol or LPS, and if so, can this response be decreased by giving antioxidants such as vitamin E. We will also determine whether the moderately increased plasma levels of IL-8 present in alcohol-dependent patients without clinical liver disease normalized with abstinence. In vitro, we will determine whether or not Hep G2 liver cells cultured in alcohol, have increased NF(kappa)B activity, mRNA for IL-8 and secreted IL-8 protein in response to recombinant human TNF or monocyte supernatants (containing TNF) from patients with AH. We will also study Hep G2 cells transfected to overexpress cytochrome P450 2E1 to determine if they have increased production of reactive oxygen intermediates, NF(kappa)B activity and IL-8 gene expression when cultured with alcohol. These studies should provide new insights into the mechanism(s) of the increased IL-8 activity seen in ALD, both in vivo and in vitro on the transcription factor level. The knowledge gained should be useful in the development of """"""""anticytokine"""""""" therapy for ALD.
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