Endotoxin induced cytotoxicity of macrophages is due to apoptosis caused by nitric oxide production

Abstract

Bacterial LPS is one of the most powerful immunostimulators in nature and causes a wide spectrum of acute pathophysiological effects recognized as toxic properties. At the cellular level, LPS also is cytotoxic for a variety of cells. However, the molecular mechanisms responsible for LPS cytotoxicity are not well understood. In this study, possible mechanisms of LPS cytotoxicity for mouse macrophages were examined in comparison with the effect of detoxified LPS (DeLPS). Mouse peritoneal exudate macrophages cultured with 10 μg/ml of LPS for 48 h evinced approximately 40% cell death as measured by the tetrazolium dye reduction assay. Co-treatment of the macrophages with IFNγ enhanced LPS-associated toxicity so that as little as 0.1 μg/ml of LPS induced a significant toxicity. In contrast, neither DeLPS alone nor the combination of DeLPS and IFNγ induced any cytotoxicity even at a high concentration of DeLPS, such as 100 μg/ml, and a relatively long incubation period such as 72 h. Examination of nitric oxide (NO) production demonstrated a dose-response production of NO in culture supernatants of LPS treated macrophages but not in DeLPS macrophages. However, when the macrophages were primed with IFNγ and stimulated with LPS, more NO production was observed as compared to stimulation with LPS alone. Macrophages were cultured with LPS in the presence of anti-IFNβ; cytotoxicity as well as NO production was significantly reduced. Treatment with the NO inhibitor, NG-monomethyl-L-arginine, completely blocked production of NO and, furthermore, diminished the cytotoxicity induced by either the combination of IFNγ and LPS or LPS alone. Investigation of the possible mechanism of LPS induced cytotoxicity implicated the involvement of apoptosis as shown by DNA fragmentation. Furthermore, treatment with the NO inhibitor completely blocked DNA fragmentation induced by LPS and IFNγ. The data obtained suggest that LPS-induced cytotoxicity of macrophages may be due to apoptosis caused by excessive production of NO.