Nitric oxide production by polymorphonuclear leucocytes in infected cystic fibrosis sputum consumes oxygen

Summary Chronic P seudomonas aeruginosa lung infection in cystic fibrosis ( CF ) patients is characterized by persisting mucoid biofilms in hypoxic endobronchial mucus. These biofilms are surrounded by numerous polymorphonuclear leucocytes ( PMNs ), which consume a major part of present molecular oxygen ( O 2 ) due to production of superoxide ( O 2 − ). In this study, we show that the PMNs also consume O 2 for production of nitric oxide ( NO ) by the nitric oxide synthases ( NOS ) in the infected endobronchial mucus. Fresh expectorated sputum samples ( n  = 28) from chronically infected CF patients ( n  = 22) were analysed by quantifying and visualizing the NO production. NO production was detected by optode measurements combined with fluorescence microscopy, flow cytometry and spectrophotometry. Inhibition of nitric oxide synthases ( NOS ) with N G ‐monomethyl‐ L ‐arginine ( L ‐ NMMA ) resulted in reduced O 2 consumption ( P  < 0·0008, n  = 8) and a lower fraction of cells with fluorescence from the NO ‐indicator 4‐amino‐5‐methylamino‐2′,7′‐difluorofluorescein diacetate ( DAF‐FM ) ( P  < 0·002, n  = 8). PMNs stained with DAF‐FM and the superoxide indicator hydroethidine ( HE ) and host cells with inducible NOS ( iNOS ) were identified in the sputum. In addition, the production of the stable end‐products of NO in CF sputum was correlated with the concentration of PMNs ; NO 3 − ( P  < 0·04, r  = 0·66, n  = 10) and NO 2 − ( P < 0·006, r  = 0·78, n  = 11). The present study suggests that besides consumption of O 2 for production of reactive oxygen species, the PMNs in CF sputum also consume O 2 for production of NO .