GM-CSF,TNF-a and fas inhibit nitric oxide production by human neutrophils in culture

Neutrophils contribute to the inflammation associated with lung diseases such as cystic fibrosis, chronic bronchitis and asthma and production of nitric oxide (NO) may be an important functional response of neutrophils recruited into the lungs. The aim of this study was to investigate whether inflammatory mediators such as GM-CSF and TNF-a or induction of apoptosis with agonistic anti-Fas antibody, modulates NO production by neutrophils in culture. Methods: Neutrophils were purified (> 97%) from the peripheral blood of 12 healthy subjects by centrifugation on Percoll and erythrocyte lysis, followed by positive immunomagnetic selection. Neutrophils were cultured for 6 h or 16 h in RPMI medium with 10% FCS, supernatants were assayed for nitrite using a chemiluminescence analyser and the neutrophils were assessed for apoptosis by flow cytometric analysis of annexin V-FITC which binds to externalised phosphatidylserine on the membrane of apoptotic cells. Results: Mean NO production from untreated neutrophils was 3.0±1.0 (SEM, n=12) (iM at 6 h and 4.0±1.2 |xM at 16 h. Neutrophil NO production was significantly inhibited by both anti-Fas (400 ng/ml) (6 h: 1.9 ±0.5 l(i.M, p < 0.05; 16 h: 2.7 ±0.8 l(i.M, p<0.001) and TNF-a (500 U/ml) (6 h: 1.9 ±0.5 M, p < 0.01; 16 h: 2.7 ±0.7 (iM, p < 0.001 ) and this was associated with a significant enhancement of apoptosis at 6 h (p c 0.001 and p < 0.05, respectively). In contrast, although GM-CSF (100 ng/ml) significantly inhibited neutrophil apoptosis at 6 h (p < 0.01), it also reduced NO production at both 6 h (1.5 ±0.4 ,IJLM, p < 0.001) and 16 h (2.1 ±0.6 \M, p < 0.001). Conclusions: Stimuli likely to be present at sites of lung inflammation inhibit neutrophil NO production.