Superoxide Mediates Human Endothelial Cell Damage by Stimulated Granulocytes

Abstract

The events that lead to irreversible neutrophil-mediated cell damage are poorly understood. Evidence indicates that the mechanism of injury varies with regard to cell type and the means of neutrophil (PMN) stimulation. The molecular basis of neutrophil-mediated cell injury and inhibitors of this process were evaluated in two in vitro assays: a neutrophil-mediated cytotoxicity assay, which measures the release of endothehal bound radiolabel and detects both cell injury and detachment; and a cytochrome c reduction assay which measures neutrophil-derived superoxide (O₂-) activity. Human aortic endothelial (HAE) and human dermal microvascular endothelial (HDME) cells were found to differ in susceptibility to neutrophil-mediated injury. The amount of HAE cell injury correlated well with the amount of Or generated by stimulated PMN. HDME cells were injured in the presence of lower concentrations O₂-than HAE cells and by PMN stimulated with inflammatory mediators (LPS, PAF, and fMLP) which appeared to generate little or no O₂-. Neutrophil-mediated injury to both HAE and HDME cells was found, however, to be inhibitable by superoxide dismutase (SOD) in a dose dependent manner. The injury was not abrogated by catalase of the iron chelator, deferoxamine mesylate (desferol). When the amount of nitric oxide (NO) released from endothelial cells and unstimulated PMN (measured as nitrite) was examined, HDME cells and PMN were found to release significantly more NO than HAE cells. Stimulation of PMN to generate O₂-resulted in significant decreases in basal nitrite levels. Furthermore, induction of NO synthesis by bradykinin or addition of exogenous NO significantly enhanced neutrophilmediated HAE cell injury. This injury was abolished by SOD. These results indicate that the superoxide anion is directly involved in the neutrophil-mediated killing of HAE and HDME cells. These data also suggest a role for peroxynitrite (formed upon interaction of NO and O₂-) in oxidative damage. Differential release of NO from cell types may explain variation in susceptibility to neutrophil-mediated injury.