Reactive oxygen species and L-type calcium channels mediates hypoxia-dependent CCL2 secretion in mast cells

Abstract

Mast cells (MCs) are well-known cells for their important role in initiating allergic response through of cross-linking of FcɛRI receptor with IgE-Ag complexes. As participants on inflammatory reactions, they are exposed to environmental conditions such as hypoxia, which on MC-dependent cytokine secretion has not been described. Here, we show that bone marrow derived MCs (BMMCs) exposed to hypoxia, produce important amounts of CCL2 mRNA and protein, that is maximal secreted between 12 and 24 hours. CCL2 production induced by hypoxia was sensitive to the antioxidant trolox and L-type Ca2+ channel blocker nifedipine. In order to analyze the possible mechanism involved on hypoxia-induced CCL2 production, we found that this conditions caused an increase on reactive oxygen species (ROS) that was maximal after one hour. We also observed an increase on [Ca2+]i two hours after hypoxia, which was sensitive to trolox. With the aim to confirm this mechanism, BMMCs were treated with H2O2 and as expected, H2O2 caused an increase on ROS and [Ca2+]i in BMMCs. Ca2+ increase produced by H2O2 was sensitive to nifedipine. CCL2 mRNA accumulation and CCL2 protein secretion induced by H2O2 was sensitive to trolox and nifedipine too. When trolox and nifedipine were used in the B16-F1 murine model of melanoma, a close relationship between ROS, Ca2+ rise and CCL2 production was found, since those compounds were able to prevent pimonidazole-positive areas and CCL2 accumulation inside tumors. Our data suggest that conditions prevalent in tumor microenvironment contribute to the generation of ROS and L-type Ca2+ channel-dependent Ca2+ entry in MCs, causing the secretion of the potent immunosuppressor chemokine CCL2.