Nitric oxide modulates pro- and anti-inflammatory cytokines in lipopolysaccharide-activated macrophages.
BACKGROUND
Sepsis is a serious and life-threatening syndrome that occurs in intensive care unit patients. Lipopolysaccharide (LPS) has been implicated as one of major causes of sepsis. Nitric oxide (NO) and cytokines are involved in sepsis-induced inflammatory responses. This study is aimed at evaluating the effects of NO on the modulation of pro- and anti-inflammatory cytokines in LPS-activated macrophages and its possible mechanism.


METHODS
N-Monomethyl arginine (NMMA), an inhibitor of NO synthase, was used in this study to suppress NO production. Mouse macrophage-like Raw 264.7 cells were exposed to LPS, NMMA, or a combination of NMMA and LPS. Cell viability was determined by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide assay. The amounts of nitrite, an oxidative product of NO, in the culture medium were quantified according to the Griess reaction method. Enzyme-linked immunosorbent assay and reverse-transcriptase polymerase chain reaction were carried out to determine the expression of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, and IL-10 in macrophages.


RESULTS
Exposure of macrophages to LPS, NMMA, and a combination of NMMA and LPS for 24 hours did not affect cell viability. LPS significantly increased the amounts of nitrite in macrophages (p < 0.01). Treatment with NMMA decreased LPS-enhanced nitrite (p < 0.01) in a concentration-dependent manner. Analyses of enzyme-linked immunosorbent assays and reverse-transcriptase polymerase chain reaction revealed that LPS significantly induced TNF-alpha, IL-1 beta, and IL-10 proteins and mRNA (p < 0.01). A combined treatment with NMMA and LPS significantly blocked LPS-induced TNF-alpha and IL-1 beta (p < 0.01), but synergistically enhanced LPS-induced IL-10 (p < 0.05) protein and RNA.


CONCLUSIONS
This study has shown that NO suppression can inhibit LPS-induced TNF-alpha and IL-1 beta but enhance IL-10, and the modulation occurs at a pretranslational level.
D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004347:Drug Interactions; D004797:Enzyme-Linked Immunosorbent Assay; D007249:Inflammation / Q000378:metabolism; D007375:Interleukin-1; D016753:Interleukin-10 / Q000096:biosynthesis; D015850:Interleukin-6 / Q000096:biosynthesis; D008070:Lipopolysaccharides / Q000009:adverse effects / Q000037:antagonists & inhibitors*; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D009569:Nitric Oxide / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis; D019323:omega-N-Methylarginine / Q000494:pharmacology*
D007375:Interleukin-1; D015850:Interleukin-6; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D019323:omega-N-Methylarginine; D009569:Nitric Oxide