Regulation of macrophage eicosanoid generation is dependent on nuclear factor kappaB.
BACKGROUND
Prostaglandin E2 (PGE2) is a major contributor to the production and maintenance of immunosuppression after overwhelming injury, leading to increased infectious morbidity and mortality in trauma patients. Elucidation of the cellular pathways involved in PGE2 production could lead to potential therapeutic interventions. The purpose of this study was to determine the role of cyclooxygenase II (COX-2) in PGE2 production by Mphi and to investigate the cellular mechanism of COX-2 gene activation.


METHODS
Mouse macrophages (Mphi), RAW 264.7, were exposed to Escherichia coli lipopolysaccharide (LPS) in the presence of cyclooxygenase inhibitors (ibuprofen or NS398). COX-1 and COX-2 mRNA expression and PGE2 production were measured by Northern blot assay and enzyme-linked immunosorbent assay, respectively. Nuclear factor kappaB (NFkappaB) activity was measured by electrophoretic mobility shift assay. To elucidate the role of NFkappaB in LPS-induced COX-2 gene activation, Mphi were exposed to LPS in the presence of an NFkappaB inhibitor, TPCK.


RESULTS
LPS increased Mphi COX-2 mRNA expression but had no effect on COX-1 mRNA expression. Both ibuprofen and NS398 inhibited COX-2 mRNA as well as PGE2 production by LPS-stimulated Mphi. In addition, LPS-induced NFkappaB activity was attenuated by these agents. Inhibition of NFkappaB with TPCK reduced COX-2 but not COX-1 gene expression and decreased PGE2 production by LPS-stimulated Mphi.


CONCLUSIONS
Our data indicate that COX-2 gene expression by LPS-stimulated Mphi is dependent on NFkappaB. Cyclooxygenase inhibitors reduced PGE2 production by inhibiting both COX-2 mRNA expression and preventing NFkappaB activation.
Department of Surgery, University of California, Los Angeles, USA. clo@surgery.medsch.ucla.edu
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D000818:Animals; D002460:Cell Line; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D015232:Dinoprostone / Q000378:metabolism*; D004926:Escherichia coli; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D007052:Ibuprofen / Q000494:pharmacology; D008070:Lipopolysaccharides; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D009104:Multiple Trauma / Q000150:complications / Q000201:enzymology / Q000378:metabolism*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D009578:Nitrobenzenes / Q000494:pharmacology; D011451:Prostaglandin-Endoperoxide Synthases / Q000187:drug effects / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology; D015533:Transcriptional Activation
D016861:Cyclooxygenase Inhibitors; D008070:Lipopolysaccharides; D016328:NF-kappa B; D009578:Nitrobenzenes; D013449:Sulfonamides; C080955:N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide; C036871:methanesulfonamide; D014108:Tosylphenylalanyl Chloromethyl Ketone; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone; D007052:Ibuprofen