EFFECTS OF EXOGENOUS NITRIC-OXIDE ON NEUTROPHIL OXIDATIVE FUNCTION AND VIABILITY

Previous studies have suggested that nitric oxide (NO) can modulate ne utrophil function. Exposure to inhaled NO for pulmonary vasodilation c ould thus potentially affect neutrophil involvement in lung inflammati on and infection. We evaluated the effect of exogenous NO gas exposure at clinically relevant concentrations in vitro on the oxidative funct ion 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 genera tion with the cytochrome c reduction assay, for oxygen consumption wit h the Clark oxygen electrode technique, and for myeloperoxidase (MPO) release by enzyme-linked immunosorbent assay (ELISA). Neutrophil viabi lity was determined by both trypan blue dye exclusion and fluorescence viability/cytotoxicity assay. Neutrophils exposed to NO at 20 ppm dem onstrated a significant decrease in superoxide anion generation in bot h 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 [ANO VA] 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 comp ared with RA (13.7 +/- 3.9 nmol O-2/ml/min/10(6) neutrophils) or O-2 a lone (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/m l) 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 as say 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 consum ption, 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, prim arily as a result of reduced cell viability. Further studies are neces sary to determine if these effects serve an in vivo immunoregulatory o r immunosuppressive role in neutrophil response to lung injury and inf ection.