Oxidative induction of pro-inflammatory cytokine formation by human monocyte-derived macrophages following exposure to manganese in vitro

Abstract

Manganese (as Mn2+), a superoxide dismutase mimetic, catalyzes the formation of the relatively stable membrane-permeable reactive oxygen species (ROS) hydrogen peroxide (H2O2), a mediator of intracellular redox signaling in immune and inflammatory cells. The goal of this study was to investigate the potential for Mn2+, via its pro-oxidative properties, to activate production of pro-inflammatory cytokines/chemokines IL-1β, IL-6, IL-8, IFNγ, TNFα, and G-CSF by human monocyte-derived macrophages in vitro. For these studies, the cells were isolated from peripheral blood mononuclear leukocytes and matured to generate a population of large CD14/CD16 co-expressing cells. The monocyte-derived macrophages were then exposed to bacterial lipopolysaccharide (LPS, 1μg/ml) or MnCl2 (25-100μM)-alone or in combination-for 24h at 37°C, after which cell-free supernatants were analyzed using a multiplex cytokine assay procedure. Exposure of the cells to LPS caused modest statistically insignificant increases in cytokine production; MnCl2 caused dose-related increases in production of all six cytokines (achieving statistical significance of p<0.0171-<0.0005 for IL-1β, IL-6, IL-8, IFNγ, and TNFα). In the case of LPS and MnCl2 combinations, the observed increases in production of IL-1β, IL-6, IL-8, IFNγ, and G-CSF were greater than those seen with cells exposed to the individual agents. The Mn2+-mediated induction of cytokine production was associated with increased production of H2O2 and completely attenuated by inclusion of the H2O2-scavenger dithiothreitol, and partially by inhibitors of NF-κB and p38MAP kinase. The findings from the studies here help to further characterize the pro-inflammatory mechanisms that may underpin clinical disorders associated with excess exposure to Mn2+, particularly those disorders seen in the central nervous and respiratory systems.