BLOOD MONONUCLEAR CELL PRODUCTION OF TNF-ALPHA AND IL-8 - ENGAGEMENT OF DIFFERENT SIGNAL-TRANSDUCTION PATHWAYS INCLUDING THE P42 MAP KINASEPATHWAY

Recent studies of human peripheral blood mononuclear cells (PBMC) stim ulated with IgG subclasses have suggested that tumor necrosis factor a lpha (TNF-alpha) and interleukin-8 (IL-8) production proceed along dif ferent signal transduction pathways. To investigate this possibility i nhibitors of signal transduction pathways were employed. Human PBMC we re pretreated with various inhibitors before being added to IgG2-coate d wells and 4-h supernatant fluids evaluated for cytokine content. The effects of various inhibitors on MAP kinase activation were determine d. Inhibitors of protein tyrosine kinases, phosphatases, and phospholi pase C decreased TNF-alpha and IL-8 production, suggesting that all th ree enzyme pathways are involved in cytokine generation. Inhibitors of G-proteins had differing effects: pertussis toxin inhibited IL-8 but not TNF-alpha production, whereas cholera toxin inhibited TNF-alpha bu t not IL-8 production, Pretreatment of PBMC with pertussis toxin resul ted in reduced IgG2-induced calcium mobilization, whereas cholera toxi n had no effect, correlating with the effects of pertussis toxin on IL -8 expression, Inhibitors of protein kinase C (PBC) completely blocked TNF-alpha generation but had no effect on IL-8 production. Go6976, wh ich inhibits certain isoforms of PKC, inhibited production of both IL- 8 and TNF-alpha. Isoforms of PKC may have opposing effects on cytokine production. PD 98059, a compound that specifically inhibits the activ ation of mitogen-activated protein kinase kinase (MEK1), inhibited TNF -alpha production, but had insignificant effects on IL-8 production. P retreatment of PBMC with either PD 98059 or genistein reduced the exte nt of phosphorylation of p42 MAP kinase in cells activated on contact with IgG2, These findings suggest distinct signal transduction pathway s for cytokine production in PBMC stimulated with IgG2.