Inhibition of Phosphatidylinositol-3′-kinase Prevents Induction of Endotoxin Tolerancein Vitro

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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 anin vitromodel 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. coli0111: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-α (TNF-α) and nitrite production were measured as indicators of ET induction. Macrophage TNF-α production decreased significantly while nitrite production increased significantly following ET induction. Specific inhibition of PI-3′-K prevented this decrease in TNF-α and increase in nitrite production in ET macrophages. Production of each mediator returned to levels not different than in non-tolerant macrophages. In thisin vitromodel of macrophage ET, pharmacologic inhibition of the PI-3′-K signaling pathway prevented the induction of LPS tolerance as measured by the pro- duction of two inflammatory mediators.