LPS-induced NF-kappa B activation and TNF-alpha release in human monocytesare protein tyrosine kinase dependent and protein kinase C independent

Background. Tumor necrosis factor alpha (TNF-alpha) is an important mediato r of septic shock. Endotoxin (LPS) signal transduction in human monocytes l eads to activation of nuclear factor-kappa B (NF-kappa B) and TNF-alpha rel ease. Previous studies have implicated activation of both protein kinase C (PKC) and protein tyrosine kinases (PTK) in LPS-induced NF-kappa B activati on and TNF-alpha production. We hypothesized that inhibition of either PKC or PTK would decrease LPS-induced NF-kappa B DNA binding and TNF-alpha y re lease in human monocytes. Materials and methods. Human monocytes were stimulated with PMA (50 ng/ml) alone or LPS (100 ng/ ml) with and without a nonspecific serine/threonine p rotein kinase inhibitor staurosporine (Stauro), a specific pan-PKC inhibito r bisindolylmaleimide (Bis), or an inhibitor of PTK genistein (Gen). TNF-al pha release in culture supernatants was measured by an ELISA. NF-kappa B DN A binding was evaluated by electrophoretic mobility shift assay. Results. LPS increased NF-kappa B DNA binding and TNF-alpha release in huma n monocytes. Nonspecific protein kinase inhibition inhibited NF-kappa B act ivation and TNF-alpha release, while specific PKC inhibition with Bis had n o effect on LPS-induced NF-kappa B DNA binding or TNF-alpha release. PTK in hibition with Gen attenuated both LPS-induced NF-kappa B DNA binding and TN F-alpha production in human monocytes. Direct activation of PKC with PMA in duced both NF-kappa B activation and TNF-alpha production by human monocyte s. Conclusions. These results suggest that LPS-induced NF-kappa B activation a nd TNF-alpha release in human monocytes are independent of PKC activity, Fu rthermore, our results provide evidence that PTK plays a role in LPS-induce d NF-kappa B activation and TNF-alpha release in human monocytes and thus c ould be a potential therapeutic target inflammatory states. (C) 1999 Academ ic Press.