PEROXYNITRITE IS NOT A MAJOR MEDIATOR OF ENDOTHELIAL-CELL INJURY BY ACTIVATED NEUTROPHILS IN-VITRO

Objective: Human polymorphonuclear leukocytes (PMN) produce nitric oxi de (NO), superoxide (O-2(.-))and peroxynitrite (ONOO-) upon stimulatio n. We investigated the role of ONOO- in PMN-induced injury to cultured bovine aortic endothelial cells (BAEC). Methods: BAEC were cocultured with phorbol 12-myristate 13-acetate (PMA)-activated human PMN (effec tor-to-target ratio, 10:1) and injury to BAEC was evaluated at interva ls by Cr-51 release assay. The levels of NO, O-2(.-), ONOO- and nitrot yrosine, a reaction product of ONOO-, were also measured, and the infl uence of NO synthase inhibitors, O-2(.-) and hydroxyl radical scavenge rs and other effecters was examined. Results: In BAEC cocultured with PMA-activated PMN, Cr-51 release was significantly increased [14.6+/-2 .2% at 2 h (p<0.05) and 42.6+/-2.7% at 4 h (p<0.01); control (nonactiv ated PMN), <4%]. Superoxide dismutase (100 U/ml) reduced Cr-51 release to 4.6+/-2.2% at 2 h (p<0.05). N-Iminoethyl-L-ornithine (L-NIO, 0.1 m M) potentiated Cr-51 release (30.6+/-3.8% at 2 h, p<0.01), and the pot entiation was eliminated by anti-CD18 monoclonal antibody. The Cr-51 r elease was completely prevented by dimethyl sulfoxide or by deferoxami ne. Treatment of PMN with L-NIO inhibited NO generation and increased O-2(.-) production. The nitrotyrosine level did not increase in BAEC c ocultured with PMA-activated PMN. Conclusion: NO-derived ONOO- is not a major cytotoxic mediator in BAEC injury by activated PMN. NO may hav e a cytoprotective effect by inhibiting PMN adherence to endothelial c ells. (C) 1998 Elsevier Science B.V. All rights reserved.