B E Drysdale

Stimulation of rat astrocytes in vitro by calcium ionophore A23187 and/or lipopolysaccharide results in the generation of a cytotoxic factor that is functionally similar to the previously described macrophage-derived cytotoxic factor, tumor necrosis factor. Like the macrophage product, the astrocyte cytotoxic factor kills murine L 929 cell targets. In addition, it kills rat oligodendrocytes, the myelin-producing cells of the central nervous system. Human recombinant tumor necrosis factor also has cytotoxic activity directed against rat oligodendrocytes.

The mechanism of TNF-mediated cytotoxicity was studied in several cell lines, including L929 murine fibroblasts. TNF caused a time- and dose-dependent increase of ADP-ribosylation in L929 target cells parallel to cell death. During the course of TNF-mediated cytotoxicity in the presence of actinomycin D, an increase in ADP-ribosylation became apparent between 4 and 6 h after exposure to TNF. Intracellular NAD+ and ATP levels decreased parallel to but not preceding cell death. Two inhibitors of ADP-ribosylation, namely 3-aminobenzamide and nicotinamide, prevented TNF-mediated cytotoxicity. Another target, the human cervical carcinoma cell line ME-180, showed an increase in ADP-ribosylation when treated with TNF, and the cytotoxic action of TNF on this target cell was inhibited by these two inhibitors. In the absence of actinomycin D, treatment of L929 cells with TNF also increased ADP-ribosylation, and the cytotoxic action of TNF was inhibited by nicotinamide. These results indicate that ADP-ribosylation may be involved in the TNF-mediated cytotoxic reaction.

Peritoneal macrophages from C3HeB/FeJ mice became cytotoxic for 6C3HED lymphosarcoma cells, P815 mastocytoma cells, and L-929 fibroblasts when treated with the calcium ionophore, A23187, at concentrations ranging from 1.0 to 20 microM. The effect of A23187 on other activation processes was also tested. It was found that A23187 and lipopolysaccharide (LPS) acted synergistically, but no consistent synergy with macrophage-activating factor (MAF) was observed. Cytotoxic activity (M phi-CF) was found in cellfree supernatants from M phi activated by A23187 or LPS. Furthermore, these two activating agents synergize in the production of M phi-CF. The cytotoxic activity of the crude material was not blocked by catalase or protease inhibitors. Fractionation of supernatants by high pressure liquid chromatography has shown that there was a peak of cytotoxic activity with a m.w. of approximately 45,000. Interestingly, L-929 cells were 30-fold more sensitive to M phi-CF than a lymphotoxin-resistant subline of L-929.

Guinea pig peritoneal macrophages, when activated for cytotoxicity by the calcium ionophore A23187 or lipopolysaccharide, produce a cytotoxic factor [macrophage cytotoxic factor (M phi-CF)] that is not blocked by catalase or protease inhibitors. Fractionation of culture supernates containing M phi-CF by gel filtration revealed one peak of cytotoxic activity of Mr approximately 45,000, the same as guinea pig lymphotoxin (LT). Antiserum prepared against purified guinea pig LT completely neutralized the cytotoxic activity of M phi-CF. In addition, the cytotoxic factor in guinea pig tumor necrosis serum was found to have a Mr of 45,000 and was neutralized by anti-LT. Thus, M phi-CF is physicochemically and immunochemically similar to LT and tumor necrosis factor, if not identical. To investigate the role of M phi-CF in macrophage-mediated cytotoxicity, anti-LT was added to A23187- or lipopolysaccharide-activated macrophages before addition of L-929 target cells. In 10 of 16 experiments, the inhibition of macrophage-mediated cytotoxicity was 100%. In the others, cytotoxicity was blocked partially, the lowest inhibition being 49%. The effectiveness of inhibition appeared to be inversely related to the intensity of macrophage activation. These results indicate that M phi-CF plays a significant role in macrophage-mediated cytotoxicity but involvement of another mechanism cannot be excluded.

Yeast cells almost completely deficient in all cytochromes were obtained by introducing two defective nuclear genes, cyd1 and cyc4, into the same haploid strain. The action of the two mutant genes is synergistic, since either gene acting singly results in only partial cytochrome deficiency. Normal synthesis of all cytochromes can be restored in the double mutant by adding delta-aminolevulinic acid to the growth medium. The optimum concentration of delta-aminolevulinate for restoration of cytochrome synthesis is about 40 muM; when higher concentrations are used, synthesis of cytochromes is partially suppressed, particularly that of cytochrome a.a3. Growth yield of the double mutant is stimulated by ergosterol and Tween 80, a source of unsaturated fatty acid. Methionine stimulates further. None of these nutrients is required for growth when sufficient delta-aminolevulinic acid is present in the growth medium. With respect to nutritional responses, the single-gene, cytochrome-deficient mutant, ole3, behaves like the double mutant. The frequency of the p-mutation in the double mutant grown in the absence of ergosterol, Tween 80, and delta-aminolevulinic acid is at least 15%. The frequency can be reduced to less than 1% by either delta-aminolevulinic acid or Tween 80. Ergosterol alone does not decrease the p- frequency. The ole3 mutant does not exhibit increased p-frequency under similar conditions of unsaturated fatty acid deficiency.