Mechanism of human lymphotoxin and tumor necrosis factor induced destruction of cells in vitro: phospholipase activation and deacylation of specific-membrane phospholipids.
The role of phospholipase (PLase) activation and lipid metabolism in lymphotoxin (LT)- and tumor necrosis factor (TNF)-mediated destruction of murine L929 cells was examined. At the levels of LT and TNF employed, cell destruction began at 4-6 h and was 99% complete by 30 h. Cell membrane phospholipids (PL), labelled in situ at the C2 position with 14C arachidonic acid, were analyzed by two-dimensional thin-layer chromatography and quantitated over a 30 h time course after LT or TNF treatment. The ratio of radiolabel incorporation relative to the actual amount of each PL present was determined by inorganic phosphate analysis. Radiolabelled arachidonic acid, eicosanoids, and neutral lipids were released into the medium prior to the onset of cell death (4-6 h) and continued to accumulate linearly throughout the destructive reaction. There was a quantitative relationship between the appearance of radiolabelled metabolites in the media and the loss of radiolabelled cellular PL. Cellular phosphatidylethanolamine was the primary PL deacylated by PLase action, showing a 75% reduction in radiolabel. The PLase inhibitors--quinacrine, hydrocortisone, dexamethasone, and indomethacin--were potent inhibitors of LT- and TNF-mediated cell destruction, suggesting that selective deacylation of specific membrane PL by PLase activation is an important step in the events that lead to LT- and TNF-mediated cellular destruction in vitro.
Knauer
M F
MF
Department of Molecular Biology and Biochemistry, College of Medicine, University of California, Irvine 92717.
Longmuir
K J
KJ
Yamamoto
R S
RS
Fitzgerald
T P
TP
Granger
G A
GA
D000818:Animals; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects*; D003907:Dexamethasone / Q000494:pharmacology; D004789:Enzyme Activation; D006801:Humans; D006854:Hydrocortisone / Q000494:pharmacology; D007213:Indomethacin / Q000494:pharmacology; D008233:Lymphotoxin-alpha / Q000494:pharmacology*; D008563:Membrane Lipids / Q000378:metabolism*; D051379:Mice; D010740:Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D011796:Quinacrine / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D001095:Arachidonic Acids; D008233:Lymphotoxin-alpha; D008563:Membrane Lipids; D010743:Phospholipids; D014409:Tumor Necrosis Factor-alpha; D016718:Arachidonic Acid; D003907:Dexamethasone; D010740:Phospholipases; D011796:Quinacrine; D006854:Hydrocortisone; D007213:Indomethacin