Macrophage TNF mRNA Expression Is Modulated by Protease Inhibitors

Abstract

Background: Nuclear factor κB (NFκB) is an important transcriptional activator protein and is a crucial component of the host's response to infection. The activation of NFκB is correlated with the phosphorylation of inhibitory κB (IκB) and its subsequent degradation. We hypothesized that protease inhibitors which prevented IκB degradation could inhibit the macrophage gene activation and reduce the production of inflammatory cytokines. Methods: Rabbit alveolar macrophages (Mφ) were obtained by bronchoalveolar lavage. Mφ were exposed toEscherichia colilipopolysaccharide (LPS) (10 ng/ml) in the presence of various concentrations of protease inhibitors, eitherN-tosyl-l-phenylalanine chloromethyl ketone (TPCK) orN-benzoyl-l-tyrosine ethyl ester (BTEE). Total RNA was extracted for Northern blot assay of tumor necrosis factor (TNF) mRNA expression using a rabbit genomic DNA probe. Total nuclear extracts were also obtained for the measurement of the NFκB activity with the electrophoretic mobility shift assay. The TNF production in the Mφ supernatant was measured by L929 bioassays. Results: NFκB activity induced by LPS was inhibited by either BTEE or TPCK. Inhibition of NFκB activity by these agents also prevented TNF mRNA expression and TNF production induced by LPS. The cellular mechanism leading to NFκB activation was further studied. TNF mRNA expression and NFκB activation were 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 NFκB activation and TNF mRNA expression. Conclusions: These data suggest that TNF mRNA expression of LPS-stimulated Mφ is mediated through NFκB. NFκB activation is intimately regulated by the PLC signaling pathway.