Lipid peroxidation is involved in the activation of NF-kappaB by tumor necrosis factor but not interleukin-1 in the human endothelial cell line ECV304. Lack of involvement of H2O2 in NF-kappaB activation by either cytokine in both primary and transformed endothelial cells

Abstract

It has been proposed that reactive oxygen species, and in particular H2O2, may be involved in the activation of NF-kappaB by diverse stimuli in different cell types. Here we have investigated the effect of a range of putative antioxidants on NF-kappaB activation by interleukin-1 and tumor necrosis factor as well as the ability of H2O2 to activate NF-kappaB in primary human umbilical vein endothelial cells and the transformed human endothelial cell line ECV304. Activation of NF-kappaB and stimulation of IkappaBalpha degradation by H2O2 was only evident in the transformed cells and required much longer contact times than that observed with interleukin-1 or tumor necrosis factor. Furthermore, only H2O2 was sensitive to N-acetyl-L-cysteine, and no increase in H2O2 was detected in response to either cytokine. Pyrrolidine dithiocarbamate has been purported to be a specific antioxidant inhibitor of NF-kappaB that acts independently of activating agent or cell type. However, we found that tumor necrosis factor- but not interleukin-1-driven NF-kappaB activation and IkappaBalpha degradation were sensitive to pyrrolidine dithiocarbamate in transformed cells, while neither pathway was inhibited in primary cells. Phorbol ester-mediated activation was sensitive in both transformed and primary cells. Other antioxidants failed to inhibit either cytokine, while the iron chelators desferrioxamine and 2,2,6, 6-tetramethylpiperidine-1-oxyl mimicked the pattern of inhibition seen for the dithiocarbamate. This suggested that pyrrolidine dithiocarbamate was inhibiting NF-kappaB activation in endothelial cells primarily through its iron-chelating properties. Tumor necrosis factor, but not interleukin-1, was found to induce lipid peroxidation in ECV304 cells. This was inhibited by pyrrolidine dithiocarbamate and desferrioxamine. t-Butyl hydroperoxide, which induces lipid peroxidation, activated NF-kappaB. Finally, butylated hydroxyanisole, which inhibits lipid peroxidation but has no iron-chelating properties, inhibited NF-kappaB activation by tumor necrosis factor but not interleukin-1. Taken together, the results argue against a role for H2O2 in NF-kappaB activation by cytokines in endothelial cells. Furthermore, tumor necrosis factor and interleukin-1 activate NF-kappaB through different mechanisms in ECV304 cells, with the tumor necrosis factor pathway involving iron-catalyzed lipid peroxidation.