Nitrite generation and antioxidant effects during neutrophil apoptosis

Neutrophil apoptosis is important for the resolution of airway inflammation in a number of lung diseases. Inflammatory mediators, endogenous reactive oxygen and nitrogen species, and intracellular and extracellular antioxidan ts may all influence neutrophil apoptosis. This study investigated the invo lvement of these factors during apoptosis of neutrophils cultured in vitro. Neutrophils undergoing spontaneous apoptosis in culture as assessed by ann exin V binding generated significant amounts of nitrite. Incubation with ag onistic anti-Fas monoclonal antibody or tumor necrosis factor-alpha (TNF-al pha) enhanced neutrophil apoptosis at 6 h, although it decreased nitrite ac cumulation. Although granulocyte-macrophage colony-stimulating factor signi ficantly reduced neutrophil apoptosis, this was also associated with decrea sed nitrite accumulation. In contrast, inhibition of apoptosis at 16 h by d ibutyryl cyclic adenosine monophosphate was associated with increased nitri te accumulation. Exogenous glutathione (GSH) or N-acetylcysteine significan tly enhanced neutrophil apoptosis at 6 h and stimulated the production of H 2O2, which may mediate apoptosis through intracellular hydroxyl radical pro duction. Intracellular GSH concentrations decreased in neutrophils undergoi ng apoptosis, and this was more marked in neutrophils treated with anti-Fas or TNF-alpha. These results suggest a causal association between reduced e ndogenous nitric oxide production, reduced intracellular GSH, and Fas- and TNF-alpha-mediated neutrophil apoptosis, whereas enhanced neutrophil surviv al mediated by dibutyryl cyclic adenosine monophosphate is associated with increased nitrite generation and maintenance of intracellular GSH. The inte raction of endogenous reactive oxygen species with extracellular antioxidan ts such as GSH could also contribute to the complex processes regulating ne utrophil apoptosis and hence the resolution of inflammation in the lung. (C ) 2000 Elsevier Science Inc.