Interferon-gamma/lipopolysaccharide-treated mouse embryonic fibroblasts are killed by a glycolysis/L-arginine-dependent process accompanied by depression of mitochondrial respiration.
Normal mouse embryo fibroblasts (MEF) are killed by treatment with low doses of interferon gamma (IFN-gamma) 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-gamma/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-gamma/LPS-treated MEF. A variety of agents were found both to protect cells against IFN-gamma/LPS-induced cytotoxicity and to inhibit increased glycolysis in these cells: glucocorticoids, the serine-type protease inhibitor N-acetyl-DL-phenylalanine-beta-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-gamma/LPS-treated MEF. Specifically, malate- and succinate-driven respiration was found to be impaired. Furthermore, IFN-gamma/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-gamma/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.
Laboratory of Immunobiology, Rega Institute, University of Leuven Medical School, Belgium.
R
R
Dijkmans
A
A
Billiau
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001120:Arginine / Q000494:pharmacology*; D016923:Cell Death / Q000187:drug effects; D004622:Embryo, Mammalian; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology; D006019:Glycolysis / Q000187:drug effects; D007371:Interferon-gamma / Q000494:pharmacology*; D007700:Kinetics; D008070:Lipopolysaccharides; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D010101:Oxygen Consumption
D008070:Lipopolysaccharides; D007371:Interferon-gamma; D000255:Adenosine Triphosphate; D001120:Arginine; D005947:Glucose