Effects of exogenous nitric oxide on neutrophil oxidative function and viability

Previous studies have suggested that nitric oxide (NO) can modulate neutrophil function. Exposure to inhaled NO for pulmonary vasodilation could thus potentially affect neutrophil involvement in lung inflammation and infection. We evaluated the effect of exogenous NO gas exposure at clinically relevant concentrations in vitro on the oxidative function of human neutrophils. Isolated neutrophils were exposed for 2 h to either room air (RA), 80% oxygen (O-2), or NO at 20 or 5 ppm blended with room air (NO20/RA, NO5/RA) or blended with 80% oxygen (NO20/O-2) (NO5/O-2). Neutrophils were then evaluated for superoxide anion generation with the cytochrome c reduction assay, for oxygen consumption with the Clark oxygen electrode technique, and for myeloperoxidase (MPO) release by enzyme-linked immunosorbent assay (ELISA). Neutrophil viability was determined by both trypan blue dye exclusion and fluorescence viability/cytotoxicity assay. Neutrophils exposed to NO at 20 ppm demonstrated a significant decrease in superoxide anion generation in both NO20/RA (97 +/- 46 nmol/10(6) neutrophils) and NO20/O-2 (102 +/- 54 nmol/10(6) neutrophils) groups as compared with RA (190 +/- 41 nmol/10(6) neutrophils) (mean +/- SEM, P < 0.005 by analysis of variance [ANOVA] and the Student-Newman-Keuls test). No significant difference was seen at 5 ppm NO exposure. Neutrophil oxygen consumption was decreased with NO20/O-2 (6.5 +/- 1.2 nmol O-2/ml/min/10(7) neutrophils) as compared with RA (13.7 +/- 3.9 nmol O-2/ml/min/10(6) neutrophils) or O-2 alone (11.6 +/- 3.1 nmol O-2/ml/min/10(7) neutrophils) (P < 0.002). MPO levels were significantly decreased with NO20/O-2 (2.3 +/- 0.4 mu g/ml) as compared with RA (4.0 +/- 0.4 mu g/ml, P < 0.005), and also with NO5/O-2. Cell viability as reflected by trypan blue dye exclusion was decreased with O-2 (70 +/- 2.3%), NO20/RA (61 +/- 4%), and NO20/O-2 (58 +/- 2.5%) exposure as compared with RA control (84.4 +/- 0.9%) (P < 0.0001). Decreased neutrophil viability was confirmed by live/dead assay for O-2 (80.8 +/- 2.8%), NO20/RA (62.8 +/- 6.1%), and NO20/O-2 (31.7 +/- 5.6%) groups as compared with RA control (95.8 +/- 1.4%, P < 0.0001). Adjusting neutrophil superoxide anion generation, oxygen consumption, and MPO values for cell viability abolished differences between exposure groups. We conclude that exogenous NO exposure at clinically relevant concentrations decreases neutrophil oxidative function, primarily as a result of reduced cell viability. Further studies are necessary to determine if these effects serve an in vivo immunoregulatory or immunosuppressive role in neutrophil response to lung injury and infection.