Defective lipopolysaccharide-dependent ERK 1/2 activation in endotoxin tolerant murine macrophages is reversed by direct protein kinase C stimulation

Lipopolysaccharide (LPS,) pretreatment inhibits TNF secretion in endotoxin- tolerant macrophages via alterations in signal transduction pathways of LPS activation (LPSa). Protein kinase C inhibitors prevent TNF release in resp onse to LPSa and direct protein kinase C activation with phorbol myristate acetate (PMA) restores TNF secretion after LPSp. In the current experiments the effect of protein kinase C modulation on LPSa-stimulated ERK 1/2 activ ation was investigated. Murine macrophage TNF production was determined aft er stimulation with 100 ng/mL of LPSa, +/- 24 h pretreatment with 10 ng/mL of LPSp. Direct protein kinase C activators (PMA or indolactam) or inhibito rs (H7 or bisindolylmaleimide) were added 1 h before LPSa. Diphosphorylated ERK1/2 was assayed after LPSa stimulation by Western blot. LPS tolerance a fter LPSp was characterized by inhibition of LPSa-stimulated TNF and accomp anied by impaired ERK 1/2 activation by LPSa. Protein kinase C activation w ith PMA or indolactam restored ERK 1/2 activation and TNF secretion. Inhibi tion of protein kinase C with H7 or bisinidolylmaleimide prevented TNF secr etion and ERK1/2 activation by LPSa, These findings suggest that both ERK 1 /2 and protein kinase C are required for TNF production in nontolerant macr ophages and that LPS tolerance may be associated with an inability to phosp horylate ERK 1/2.