EXOGENOUS NITRIC-OXIDE ENHANCES NEUTROPHIL CELL-DEATH AND DNA FRAGMENTATION

Inhaled nitric oxide (NO) is an important new therapeutic agent used t o treat pulmonary arterial hypertension in a variety of disease states . However, the effects of NO on cells in the lung are uncertain. Previ ously, we have shown that NO gas depresses neutrophil oxidative cell f unction and increases neutrophil cell death. The purpose of this in vi tro study was to determine the mechanism of neutrophil death. We hypot hesized that NO hastened cell death by inducing apoptosis. To mimic th e clinical environment of patients with respiratory failure, we also s tudied the effects of hyperoxia on neutrophil cell viability and apopt osis. Isolated human neutrophils were exposed to 80% O-2 (O-2), NO at 20 ppm in room air (NO/PA), 20 ppm NO blended with 80% O-2 (NO/O-2), o r RA alone (control) for 2 to 24 h. Experiments were repeated with NO concentrations of 5 and 50 ppm and with 20 ppm in the presence of supe roxide dismutase (SOD). Neutrophils were also incubated in the absence or presence of neutrophil stimulant fMLP (10 nM). Neutrophil cell via bility was measured by fluorescence viability/cytotoxicity assay. Neut rophil apoptosis was assessed by cell death detection ELISA for histon e-associated DNA fragments, TdT transferase-mediated fluorescence-labe led dUTP nick end labeling (TUNEL) assay, and DNA-fragmentation gel el ectrophoresis. NO/O-2-exposed neutrophils showed decreased viability a t 2 h (31.7 +/- 3.7%, mean % viability +/- SD) compared with control ( 94.7 +/- 4.7%), O-2 (75.6 +/- 9.3%), and NO/RA (62.8 +/- 14.9%; P < 0. 05 by ANOVA; n = 9). Although control neutrophils demonstrated marked apoptosis at 24 h, there was no significant apoptosis at 2, 4, or 6 h (P < 0.001 by Kruskal-Wallis, n = 20) as assessed by ELISA and TUNEL a ssays. When compared with RA controls at 2 h, neutrophils exposed to N O/O-2 showed significantly more apoptosis (292% of control, range: 106 to 2,488%, P < 0.001 by ANOVA and Kruskal-Wallis) but not with exposu re to NO/RA or O-2 alone. These findings were confirmed by TUNEL assay (n = 4, P < 0.05). NO/RA and NO/O-2-exposed neutrophils demonstrated both evidence of necrosis and enhanced DNA fragmentation at 2 h by gel electrophoresis (n = 2). Fifty parts per million NO produced similar findings, but exposure to 5 ppm NO did not induce significant DNA frag mentation. Coincubation with SOD inhibited NO/O-2-associated apoptosis , suggesting peroxynitrite contributed to cell death. Stimulation with fMLP did not alter apoptosis induced in neutrophils exposed to NO/RA or NO/O-2. We conclude that exogenous NO gas, at clinically relevant c oncentrations under hyperoxic conditions, induces cell death in neutro phils in paa by enhancing DNA fragmentation.