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 I kappaB-alpha. What is not known, however, is the mechanism by which IL-10 exerts its effect on I kappaB-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 I kappaB-alpha degradation in macrophages. Treatment of the cells with lipopolysaccharide (LPS) caused activation of NF-kappaB and rapid proteolysis of I kappaB-alpha as determined by the electrophoretic mobility shift assay, gene transfection, and Western blot. IL-10 pretreatment inhibited both NF-kappaB activation and I kappaB-alpha degradation. Both of these processes were also inhibited by ROS scavengers, catalase (H2O2 scavenger), and sodium formate (. OH scavenger) but were minimally affected by superoxide dismutase (O-2(-) scavenger). These results suggests that . OH radicals, formed by an H2O2-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 I kappaB-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.