Paradoxical stimulation and inhibition by protein kinase C modulating agents of lipopolysaccharide evoked production of tumour necrosis factor in human monocytes.
Human blood monocytes were activated by bacterial lipopolysaccharide endotoxin (LPS) (10 ng/ml) for cytotoxicity of WEHI-164 mouse fibrosarcoma cells, determined by release of 51Cr from WEHI-164 tumour cells incubated with monocyte supernatants. The chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (FMLP) augmented LPS-induced cytotoxicity but had no effect alone. FMLP but not LPS stimulated phospholipase C (PLC), determined by the release of [3H]inositol phosphates. Addition of tumour promoter and protein kinase C stimulant, phorbol-12-myristate-13-acetate (PMA) at concentrations of 3 x 10(-10) M to 3 x 10(-9) M, resulted in an augmentation of 30-200% in LPS-evoked cytotoxicity. The effects of FMLP and PMA, like the effect of LPS alone, were completely blocked by antibody to recombinant human tumour necrosis factor-alpha (TNF-alpha), indicating that cytotoxicity induced by LPS, FMLP, and PMA were due solely to TNF release. Concentrations of PMA greater than 3 x 10(-9) M caused inhibition of TNF release. Okadaic acid (20 ng/ml), an inhibitor of phosphatases I and IIa, augmented the effects of LPS and the stimulatory effects of low levels of PMA, suggesting that phosphorylation was important in the actions of both LPS and PMA. The effects of LPS and of low levels of PMA were augmented by the protein kinase C (PKC) inhibitors H-7 (10-30 microM), staurosporine (2-10 nM) and calphostin C (0.1 microM). Higher concentrations of the inhibitors prevented LPS-evoked TNF release and its augmentation by low levels of PMA. However, they did not prevent the inhibition by high levels of PMA. One possible explanation for the results is that different isozymes of PKC may mediate the stimulatory as compared to the inhibitory effects of PKC on TNF production.
Coffey
R G
RG
Department of Pharmacology and Therapeutics, University of South Florida College of Medicine, Tampa 33612.
Weakland
L L
LL
Alberts
V A
VA
D002478:Cells, Cultured; D003602:Cytotoxicity, Immunologic / Q000187:drug effects / Q000276:immunology; D006801:Humans; D008070:Lipopolysaccharides / Q000276:immunology*; D009000:Monocytes / Q000276:immunology*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000276:immunology; D011493:Protein Kinase C / Q000276:immunology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*; D010738:Type C Phospholipases / Q000276:immunology
D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D009240:N-Formylmethionine Leucyl-Phenylalanine; D011493:Protein Kinase C; D010738:Type C Phospholipases; D013755:Tetradecanoylphorbol Acetate