Inhibition of phosphatidyl inositol-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. (C) 1996 Academic Press, Inc.