Protein kinase C regulates TNF-alpha production by human monocytes

Tumor Necrosis Factor alpha (TNF alpha) is a cytokine mediator that is produced primarily by activated monocytes/macrophages in response to endotoxin/lipopolysaccharide (LPS) as well as other stimuli. The second messenger systems that regulate the synthesis and release of TNF alpha are not clearly defined. In the present study, the role of protein kinase C (PKC) in the production of TNF alpha was investigated in human peripheral blood monocytes stimulated with either LPS or zymosan. Two broad spectrum protein kinase inhibitors (staurosporine and K252a) and two PKC specific inhibitors (calphostin C and chelerythrine), were used as probes to delineate the involvement of PKC in the production of TNF alpha. The results indicate that inhibition of PKC diminished LPS- or zymosan- induced TNF alpha production in a concentration-dependent manner. The IC50 values for the inhibition of TNF alpha production were 0.2 nM for staurosporine, and 20 nM for K252a, Calphostin C and chelerythrine. Furthermore, long term PMA treatment of these cells (to abrogate PKC-mediated responses) resulted in a significant reduction of stimuli-induced TNF alpha production. LPS and zymosan also induced an increase in membrane associated PKC activity in human monocytes, which could be inhibited by pretreatment of the cells with calphostin C. Finally, western blot analysis with PKC isoform-specific antibodies demonstrates that the alpha and xi are the predominent isoforms expressed in human monocytes. These data strongly suggest that an initial step in TNF alpha production by human monocytes challenged with physiological stimulants, such as LPS and zymosan, involves a PKC-dependent mechanism.