Interferon‐γ/lipolysaccharide‐treated mouse embryonic fibroblasts are killed by a glycolysis/l‐arginine‐dependent process accompanied by depression of mitochondrial respiration

Abstract

Normal mouse embryo fibroblasts (MEF) are killed by treatment with low doses of interferon γ (IFN‐γ) in combination with lipopolysaccharide (LPS). This cytotoxicity has previously been shown to represent an active suicidal reaction. Here we show that the time period between first contact with IFN‐γ/LPS (t= 0 h) and cell death (t= 48 h) can be separated into two distinct periods, during which glycolytic metabolism of glucose either has a positive (8–24 h) or a negative (30–48 h) effect on cytotoxicity. During the first period (8–24 h), withdrawal of glucose from the culture medium, or inclusion in the medium of the glycolytic inhibitors deoxy‐d‐glucose, NaF or iodoacetate, prevented later cell death. During the second period (30–48 h), withdrawal of glucose or supplementation of the culture medium with glycolytic inhibitors was no longer protective; instead it was a requirement for cell suicide to occur. Glycolytic activity during the first period was found to be increased twofold in LPS‐treated MEF and almost threefold in IFN‐γ/LPS‐treated MEF. A variety of agents were found both to protect cells against IFN‐γ/LPS‐induced cytotoxicity and to inhibit increased glycolysis in these cells: glucocorticoids, the serine‐type protease inhibitor N‐acetyl‐dl‐phenylalanine‐β‐naphthyl ester, the ADP‐ribosylation inhibitors 3‐aminobenzamide and nicotinamide, and the transcription and translation inhibitors actinomycin and cycloheximide. Mitochondrial function, although normal in LPS‐treated cells, was markedly depressed in IFN‐γ/LPS‐treated MEF. Specifically, malate‐ and succinate‐driven respiration was found to be impaired. Furthermore, IFN‐γ/LPS‐treated MEF contained one‐third of the ATP level of LPS‐treated MEF. Withdrawal of l‐arginine from the culture medium prevented cell death in IFN‐γ/LPS‐treated MEF. N‐Methyl‐l‐arginine, which is an inhibitor of nitric oxide (NO·) biosynthesis from l‐arginine, also inhibited cell death. In conclusion, we propose that cell death in our experiments is due to an l‐arginine/glycolysis‐dependent impairment of mitochondrial respiration. Copyright © 1991, Wiley Blackwell. All rights reserved