Tumour Necrosis Factor-Alpha and Nitric Oxide Mediate Apoptosis by D-Galactosamine in a Primary Culture of Rat Hepatocytes: Exacerbation of Cell Death by Cocultured Kupffer Cells

Abstract

BACKGROUND: Prostaglandin E1(PGE1) reduces cell death in experimental and clinical liver dysfunction.OBJECTIVES: Whether PGE1protects against d-galactosamine (D-GalN)-associated hepatocyte cell death by the regulation of tumour necrosis factor-alpha (TNF-alpha) and/or nitric oxide (NO) in hepatocytes or cocultured Kupffer cells was examined.METHODS: Anti-TNF-alpha antibodies were used to evaluate the role of TNF-alpha during d-GalN cytotoxicity and its protection by PGE1in cocultured hepatocytes and Kupffer cells. Cell apoptosis and necrosis were assessed by DNA fragmentation and lactate dehydrogenase release, respectively. Nitrite+nitrate (NOx), as NO end products, and TNF-alpha concentrations were measured in the culture medium. The role of NO was determined by measuring inducible NO synthase (iNOS) expression and the effect of its inhibition during d-GalN cytotoxicity and its protection by PGE1.RESULTS: d-GalN enhanced hepatocyte cell death associated with high TNF-alpha and NOx levels in a culture medium. Anti-TNF-alpha and iNOS inhibition suggested that TNF-alpha was mediating apoptosis, but not necrosis, through the stimulation of NO production. The antiapoptotic activity of PGE1was associated with a reduction of NO production, but was blocked by iNOS inhibition. This apparent contradiction was explained by the ability of PGE1to enhance iNOS expression shortly after its administration and inhibit it later during d-GalN treatment. Anti-TNF-alpha antibodies did not reduce the exacerbation ofD-GalN-associated cell death in hepatocytes by cocultured Kupffer cells.CONCLUSION: TNF-alpha mediatesD-GalN-induced apoptosis via NO production in cultured hepatocytes. The protective effect of PGE1againstD-GalN-induced apoptosis is probably through the induction of low iNOS expression that was followed by a reduction of iNOS expression and NO production induced by the hepatotoxin. The exacerbation of hepatocyte cell death by Kupffer cells was not related to TNF-alpha and NO.