title: Nitric oxide production by polymorphonuclear leucocytes in infected cystic fibrosis sputum consumes oxygen.
abstract: Chronic Pseudomonas 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.
authors.affiliation: Department of Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark; Department of International Health, Immunology and Microbiology, Faculty of Health Sciences, University of Copenhagen, Helsingør, Denmark.
authors.forename: M
authors.initials: M
authors.lastname: Kolpen
authors.forename: T
authors.initials: T
authors.lastname: Bjarnsholt
authors.forename: C
authors.initials: C
authors.lastname: Moser
authors.forename: C R
authors.initials: CR
authors.lastname: Hansen
authors.forename: L F
authors.initials: LF
authors.lastname: Rickelt
authors.forename: M
authors.initials: M
authors.lastname: Kühl
authors.forename: C
authors.initials: C
authors.lastname: Hempel
authors.forename: T
authors.initials: T
authors.lastname: Pressler
authors.forename: N
authors.initials: N
authors.lastname: Høiby
authors.forename: P Ø
authors.initials: PØ
authors.lastname: Jensen
mesh_terms: D000328:Adult; D002478:Cells, Cultured; D002908:Chronic Disease; D003550:Cystic Fibrosis / Q000150:complications / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008168:Lung / Q000276:immunology / Q000378:metabolism* / Q000382:microbiology; D008297:Male; D008875:Middle Aged; D009504:Neutrophils / Q000276:immunology* / Q000382:microbiology; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D010101:Oxygen Consumption; D011552:Pseudomonas Infections / Q000150:complications / Q000276:immunology / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000276:immunology*; D020545:Respiratory Mucosa / Q000473:pathology*; D013183:Sputum / Q000378:metabolism*; D055815:Young Adult; D019323:omega-N-Methylarginine / Q000494:pharmacology
chemical_list: D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase
keywords: Pseudomonas aeruginosa; cystic fibrosis; neutrophil biology; nitric oxide; pneumonia