Investigation of the pro-oxidative and pro-inflammatory interactions of manganese with cells of the innate immune system

Abstract

Manganese (Mn2+) is an essential nutrient required in trace amounts for human health; however excessive exposure to the metal, predominantly reported in adults exposed occupationally via inhalation, has been associated with adverse central nervous system effects and respiratory symptoms. Pro-oxidative interactions with cells of the innate immune system may play a role in mediating these effects. The aim of the current study was to investigate the pro-oxidative and proinflammatory interactions of Mn2+ with cells of the innate immune system, specifically neutrophils and monocyte-derived macrophages. The primary objectives of the current study were to investigate:i) the pro-oxidative interactions of MnCl2 (1.56-100μM) with isolated human blood neutrophils stimulated with the chemoattractant, N-Formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP), or the phorbol ester, phorbol 12-myristate 13-acetate (PMA), according to effects on the generation of superoxide, hydroxyl radical, hypohalous acids and hydrogen peroxide;ii) the effects of MnCl2 on the generation of the reactive oxygen species (ROS), superoxide, hydrogen peroxide and nitric oxide by PMA-activated human blood monocyte-derived macrophages;iii) the effects of MnCl2 on the production of the pro-inflammatory cytokines, interleukin 1β (IL-1β), interleukin 6 (IL-6), interleukin 8 (IL-8), granulocyte colony-stimulating factor (G-CSF), interferon gamma (IFN لا), and tumor necrosis factor (TNF) by unstimulated and lipopolysaccharide (LPS)-activated monocyte-derived macrophages; and iv) the effects of MnCl2 on signal transduction pathways involved in pro-inflammatory cytokine production. The generation of ROS was measured using a series of laboratory procedures designed to precisely characterize the pro-oxidative properties of MnCl2. These included measurement of oxygen consumption, lucigenin/luminol-enhanced chemiluminescence, spectrofluorimetric detection of oxidation of 2,7-dichlorodihydrofluorescein, and radiometric assessment of myeloperoxidase-mediated protein iodination, while a spectrophotometric v method was used for measurement of nitric oxide (NO). In addition, several cellfree ROS-generating systems were used. The Bio-Plex Pro TM assay kit was used to measure/ detect the production of pro-inflammatory cytokines, while inhibitors (NFκB activation inhibitor, a p38MAP kinase inhibitor and dithiothreitol) were used to probe the signal transduction pathways utilized by MnCl2 to modulate the production of the pro-inflammatory cytokines by monocyte-derived macrophages. The findings of the study demonstrated that treatment of neutrophils and macrophages with Mn2+ markedly increasedthe production of the ROS, H2O2 and HOCl in the setting of decreased reactivity of superoxide, while cellular O2 consumption and generation of NO were unaffected. Taken together with the findings of experiments using cell-free ROS-generating systems, these observations are compatible with a mechanism whereby Mn2+,by acting as a superoxide dismutase mimetic, increases the formation of H2O2 by activated phagocytes. Mn2+ also potentiated the production of pro-inflammatory cytokines by monocytederived macrophages, apparently by affecting H2O2-mediated redox-sensitive signaling pathways. Importantly, this is the first study which has: i) conclusively established that exposure of human neutrophils and macrophages to MnCl2potentiates the generation of the relatively stable, cell-permeable, potentially damaging ROS, H2O2; and ii) that H2O2, in turn, via activation of redox-regulated intracellular signaling mechanisms in macrophages, initiates and potentiates the generation of proinflammatory cytokines by the cells. These findings not only provide a mechanism for Mn2+-mediated toxicity, but also underscore the requirement for strictly controlled exposure to this metal in both vi the occupational and environmental settings, as well as the potential for H2O2- neutralizing, anti-oxidative preventive and therapeutic strategies.