Macrophage TNF mRNA expression is modulated by protease inhibitors.
BACKGROUND
Nuclear factor kappa B (NF kappa B) is an important transcriptional activator protein and is a crucial component of the host's response to infection. The activation of NF kappa B is correlated with the phosphorylation of inhibitory kappa B (I kappa B) and its subsequent degradation. We hypothesized that protease inhibitors which prevented I kappa B degradation could inhibit the macrophage gene activation and reduce the production of inflammatory cytokines.


METHODS
Rabbit alveolar 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 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 NF kappa B activity with the electrophoretic mobility shift assay. The TNF production in the M phi supernatant was measured by L929 bio-assays.


RESULTS
NF kappa B activity induced by LPS was inhibited by either BTEE or TPCK. Inhibition of NF kappa B activity by these agents also prevented TNF mRNA expression and TNF production induced by LPS. The cellular mechanism leading to NF kappa B activation was further studied. TNF mRNA expression and NF kappa B 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 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 regulated by the PLC signaling pathway.
Lo
C J
CJ
Department of Surgery, University of California, Los Angeles 90095, USA.
Fu
M
M
Kim
B
B
D000818:Animals; D004789:Enzyme Activation / Q000187:drug effects; D015870:Gene Expression / Q000187:drug effects; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation / Q000187:drug effects; D016676:Macrophages, Alveolar / Q000502:physiology*; D008297:Male; D016328:NF-kappa B / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology*; D011493:Protein Kinase C / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D015398:Signal Transduction / Q000187:drug effects; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000235:genetics; D010738:Type C Phospholipases / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives / Q000494:pharmacology
D008070:Lipopolysaccharides; D016328:NF-kappa B; D011480:Protease Inhibitors; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; C006716:benzoyltyrosine ethyl ester; D014108:Tosylphenylalanyl Chloromethyl Ketone; D014443:Tyrosine; D011493:Protein Kinase C; D010738:Type C Phospholipases