Inhibition of phosphatidylinositol-3'-kinase prevents induction of endotoxin tolerance in vitro.
Previous studies have shown an increase in the expression of phosphatidylinositol-3'-kinase (PI-3'-K) in macrophages from endotoxin tolerant (ET) rats. This implicates PI-3'-K cell signaling in attenuated macrophage responsiveness to lipopolysaccharide (LPS). These experiments examined the effects of selective pharmacologic inhibition of PI-3'-K in an in vitro model of ET. To induce ET, RAW 264.7 macrophages cultured in RPMI 1640 with 10% fetal calf serum were initially exposed to 10 ng/ml LPS (E. coli 0111:B4) for 19 hr. Non-tolerant cells received an equal volume of phosphate buffered saline. Some cultures were also incubated with the specific PI-3'-K inhibitor wortmannin (10 nM) during this tolerizing period. Cells were then washed and re-challenged with 100 ng/ml LPS for 24 hr. Next, macrophage tumor necrosis factor-alpha (TNF-alpha) and nitrite production were measured as indicators of ET induction. Macrophage TNF-alpha production decreased significantly while nitrite production increased significantly following ET induction. Specific inhibition of PI-3'-K prevented this decrease in TNF-alpha and increase in nitrite production in ET macrophages. Production of each mediator returned to levels not different than in non-tolerant macrophages. In this in vitro model of macrophage ET, pharmacologic inhibition of the PI-3'-K signaling pathway prevented the induction of LPS tolerance as measured by the production of two inflammatory mediators.
Bowling
W M
WM
Department of Surgery, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Flye
M W
MW
Qiu
Y Y
YY
Callery
M P
MP
D000730:Androstadienes / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line; D004361:Drug Tolerance; D004731:Endotoxins / Q000633:toxicity*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D007700:Kinetics; D016018:Least-Squares Analysis; D008264:Macrophages; D008954:Models, Biological; D009573:Nitrites / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors*; D051381:Rats; D012044:Regression Analysis; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis; D000077191:Wortmannin
D000730:Androstadienes; D004731:Endotoxins; D004791:Enzyme Inhibitors; D009573:Nitrites; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D014409:Tumor Necrosis Factor-alpha; D000077191:Wortmannin