Am. J. Respir. Cell Mol. Biol., Vol 16, No. 4, 04 1997, 407-412.
Effects of exogenous nitric oxide on neutrophil oxidative function and viability
AH Daher, JD Fortenberry, ML Owens and LA Brown
Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, USA.
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 (O2), or NO at 20 or 5 ppm blended with room air
(NO20/RA, NO5/RA) or blended with 80% oxygen (NO20/O2) (NO5/O2).
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/O2 (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/O2 (6.5 +/- 1.2 nmol O2/ml/min/10(7)
neutrophils) as compared with RA (13.7 +/- 3.9 nmol O2/ml/min/10(6)
neutrophils) or O2 alone (11.6 +/- 3.1 nmol O2/ml/min/10(7) neutrophils) (P
< 0.002). MPO levels were significantly decreased with NO20/O2 (2.3 +/-
0.4 microg/ml) as compared with RA (4.0 +/- 0.4 microg/ml, P < 0.005),
and also with NO5/O2. Cell viability as reflected by trypan blue dye
exclusion was decreased with O2 (70 +/- 2.3%), NO20/RA (61 +/- 4%), and
NO20/O2 (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 O2 (80.8 +/- 2.8%), NO20/RA (62.8 +/- 6.1%), and NO20/O2 (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.
