Bacterial Lipopolysaccharide Induces Early and Late Activation of Protein Kinase C in Inflammatory Macrophages by Selective Activation of PKC-ϵ

Experiments from our and other laboratories have shown that specific inhibitors of protein kinase C (PKC) inhibited the secretion of nitric oxide, TNFα, and IL-1β from lipopolysaccharide (LPS)-stimulated macrophages, suggesting an important role for PKC in the inflammatory response. The present study was designed to investigate the mechanism whereby LPS stimulates PKC activity in inflammatory macrophages. Mouse macrophages were stimulated with 0-1 μg/ml LPS for 0-18 hours, and PKC activity was detected in cell lysates. PKC isoform specificity was determined by blocking PKC activity with isoform-specific antibodies. Treatment of macrophages with 1 μg/ml LPS induced a two-fold increase in PKC activity within 15 minutes and an additional more significant peak of PKC activity appeared 3 hours post-LPS stimulation. A lower dose of LPS (10 ng/ml) induced the later peak only. The enhancement in PKC activity induced by LPS occurred in both the cytosol and membrane fractions, but the enhancement in the membrane fraction was significantly greater than in the cytosol. The increase in PKC activity in both peaks was abolished only by the addition of anti-PKC-ϵ antibody. The present experiments suggest that PKC activation is an important pathway in the LPS-induced secretory response of macrophages and that PKC-ϵ is the major isoform involved.