MACROPHAGE TNF MESSENGER-RNA EXPRESSION IS MODULATED BY PROTEASE INHIBITORS

Background: Nuclear factor kappa B (NF kappa B) is an important transc riptional activator protein and is a crucial component of the host's r esponse to infection. The activation of NF kappa B is correlated with the phosphorylation of inhibitory kappa B (I kappa B) and its subseque nt degradation. We hypothesized that protease inhibitors which prevent ed I kappa B degradation could inhibit the macrophage gene activation and reduce the production of inflammatory cytokines. Methods: Rabbit a lveolar macrophages (M phi) were obtained by bronchoalveolar lavage. M phi were exposed to Escherichia coli lipopolysaccharide (LPS) (10 ng/ ml) in the presence of various concentrations of protease inhibitors, either N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) or N-benzoyl -L-tyrosine ethyl ester (BTEE). Total RNA was extracted for Northern b lot assay of tumor necrosis factor (TNF) mRNA expression using a rabbi t genomic DNA probe. Total nuclear extracts were also obtained for the measurement of the NF kappa B activity with the electrophoretic mobil ity shift assay. The TNF production in the M phi supernatant was measu red by L929 bioassays. Results: NF kappa B activity induced by LPS was inhibited by either BTEE or TPCK. Inhibition of NF kappa B activity b y these agents also prevented TNF mRNA expression and TNF production i nduced by LPS. The cellular mechanism leading to NF kappa B activation was further studied. TNF mRNA expression and NF kappa B activation we re inhibited by D609, a phospholipase C (PLC) inhibitor, as well as by protein kinase C (PKC) inhibitors. In addition, direct stimulation of PKC led to NF kappa B activation and TNF mRNA expression. Conclusions : These data suggest that TNF mRNA expression of LPS-stimulated M phi is mediated through NF kappa B. NF kappa B activation is intimately re gulated by the PLC signaling pathway. (C) 1997 Academic Press.