Tumor necrosis factor production by Kupffer cells requires protein kinase C activation.
Tumor necrosis factor (TNF) has been proposed as a primary inflammatory mediator of septic shock. In vitro and in vivo studies indicate that endotoxin- or lipopolysaccharide (LPS)-activated macrophages are a principle source of TNF; however, membrane signal transduction and intracellular pathways by which LPS triggers TNF production in macrophages are unclear. Recent evidence indicates that specific protein phosphorylation via activation of protein kinase C (PKC) is an early, critical step in the signaling of macrophage TNF production by phorbol esters. We hypothesize that PKC activation is also required in LPS-signaled Kupffer cell (KC) TNF production. Murine KCs were obtained by liver perfusion and digestion and then stimulated with LPS (Escherichia coli O111:B4) or LPS in the presence of H-7, a selective PKC inhibitor. Conditioned media was collected at 3 hr for assay of TNF utilizing the L929 cytolysis bioassay standardized to murine-rTNF-alpha. We found that H-7 inhibited significantly LPS signaled TNF release at a concentration of 10 microM, while H-8 (a cyclic nucleotide specific inhibitor) had no effect. The effect of H-7 was dose dependent and present at varying concentrations of LPS. Down regulation of PKC activity by preincubation of KCs with phorbol myristate acetate (PMA, a direct activator of PKC) also resulted in significantly reduced TNF release after LPS stimulation. The inhibitor H-7 (10 microM) also significantly inhibited LPS signaled prostaglandin E2 release in Kupffer cells. Total and specific intracellular protein phosphorylation was determined by trichloroacetic acid precipitation and SDS-polyacrylamide gel electrophoresis after labeling stimulated Kupffer cells with 32Pi. Total protein phosphorylation was not significantly altered by LPS stimulation; however, autoradiograms from PMA- and LPS-stimulated KCs demonstrate enhanced phosphorylation of a 40-kDa protein (2.7 +/- 0.9-fold) and a 33-kDa protein (3.1 +/- 1.0-fold) which were inhibited by H-7. We conclude that activation of PKC and protein phosphorylation are required steps in the signal transduction pathway of LPS-stimulated TNF production in Kupffer cells.
Department of Surgery, University of Minnesota, Minneapolis 55455.
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P
Bankey
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Carlson
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Ortiz
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R
Singh
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Cerra
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000818:Animals; D002478:Cells, Cultured; D015232:Dinoprostone / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli; D007546:Isoquinolines / Q000494:pharmacology; D007728:Kupffer Cells / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation; D051379:Mice; D008807:Mice, Inbred BALB C; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*
D007546:Isoquinolines; D008070:Lipopolysaccharides; D010750:Phosphoproteins; D010879:Piperazines; D014409:Tumor Necrosis Factor-alpha; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D011493:Protein Kinase C; D015232:Dinoprostone; D013755:Tetradecanoylphorbol Acetate