Interleukin-10-mediated inhibition of free radical generation in macrophages.
Interleukin-10 (IL-10) is a pleiotropic cytokine that controls inflammatory processes by suppressing the production of proinflammatory cytokines that are known to be transcriptionally regulated by nuclear factor-kappaB (NF-kappaB). Although still controversial, IL-10 has been shown to inhibit NF-kappaB activation through a process that involves proteolytic degradation of inhibitory subunit IkappaB-alpha. What is not known, however, is the mechanism by which IL-10 exerts its effect on IkappaB-alpha degradation. The present study investigates the possible role of reactive oxygen species (ROS) and their inhibition by IL-10 in NF-kappaB activation and IkappaB-alpha degradation in macrophages. Treatment of the cells with lipopolysaccharide (LPS) caused activation of NF-kappaB and rapid proteolysis of IkappaB-alpha as determined by the electrophoretic mobility shift assay, gene transfection, and Western blot. IL-10 pretreatment inhibited both NF-kappaB activation and IkappaB-alpha degradation. Both of these processes were also inhibited by ROS scavengers, catalase (H(2)O(2) scavenger), and sodium formate (.OH scavenger) but were minimally affected by superoxide dismutase (O scavenger). These results suggests that.OH radicals, formed by an H(2)O(2)-dependent, metal-catalyzed Fenton reaction, play a major role in this process. Electron spin resonance studies confirmed the formation of.OH radicals in LPS-treated cells. Addition of IL-10 inhibited both IkappaB-alpha degradation and generation of.OH radicals in response to LPS stimulation. These results demonstrate, for the first time, direct evidence for the role of IL-10 in ROS-dependent NF-kappaB activation.
Dokka
S
S
Department of Basic Pharmaceutical Sciences, West Virginia University, Morgantown, WV 26506, USA.
Shi
X
X
Leonard
S
S
Wang
L
L
Castranova
V
V
Rojanasakul
Y
Y
D000818:Animals; D015153:Blotting, Western; D002374:Catalase / Q000494:pharmacology; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005561:Formates / Q000494:pharmacology; D016166:Free Radical Scavengers / Q000494:pharmacology; D005609:Free Radicals / Q000378:metabolism; D017930:Genes, Reporter; D017665:Hydroxyl Radical / Q000378:metabolism; D020672:I-kappa B Proteins; D016753:Interleukin-10 / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation / Q000187:drug effects; D008264:Macrophages / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D051379:Mice; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D010101:Oxygen Consumption / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D019097:Spin Trapping; D013482:Superoxide Dismutase / Q000494:pharmacology; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
D004268:DNA-Binding Proteins; D005561:Formates; D016166:Free Radical Scavengers; D005609:Free Radicals; D020672:I-kappa B Proteins; D008070:Lipopolysaccharides; D016328:NF-kappa B; C000606205:Nfkbia protein, mouse; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; C030544:formic acid; D016753:Interleukin-10; D000072000:NF-KappaB Inhibitor alpha; D017665:Hydroxyl Radical; D002374:Catalase; D013482:Superoxide Dismutase