Polymorphonuclear leukocytes consume oxygen in sputum from chronic Pseudomonas aeruginosa pneumonia in cystic fibrosis

Mette Kolpen¹, Christine R Hansen², Thomas Bjarnsholt¹, Claus Moser¹, Louise D Christensen³, Maria van Gennip³, Oana Ciofu³, Lotte Mandsberg³, Arsalan Kharazmi¹, Gerd Döring⁴, Michael Givskov³, Niels Høiby¹, Peter Ø Jensen^1,*

¹ Department of Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark;
² Copenhagen CF center, Rigshospitalet, Copenhagen, Denmark;
³ Institute of International Health, Immunology, and Microbiology, University of Copenhagen, Cope, Denmark;
⁴ Institute of Medical Microbiology and Hygiene, University of Tübingen, Tü, Denmark

Correspondence to: Peter Østrup Jensen, Department of Clinical Microbiology 9301, Rigshospitalet, Juliane Mariesvej 22, Copenhagen, 2100, Denmark; pojensen{at}excite.com

Accepted 29 September 2009

Background: Chronic lung infection with Pseudomonas aeruginosa is the most severe complication for patients with cystic fibrosis (CF). This infection is characterized by endobronchial mucoid biofilms surrounded by numerous polymorphonuclear leukocytes (PMNs). The mucoid phenotype offers protection against the PMNs, which are in general assumed to mount an active respiratory burst leading to lung tissue deterioration. An ongoing respiratory burst by the PMNs has, however, not been demonstrated previously in endobronchial secretions from chronically infected CF patients.

Objective: Based on the accumulating evidence for depletion of molecular oxygen (O₂) in the mucus in infected CF bronchi,we hypothesized that the O₂ depletion in the mucus in infected CF bronchi may be accelerated by the respiratory burst of the PMNs due to the reduction of O₂ to the superoxide anion (O₂^-) by the phagocyte NADPH-oxidase (Phox).

Methods: We established methods to isolate the O₂ consumption by the respiratory burst from aerobic respiration in freshly expectorated sputum from chronically infected CF patients.

Results: Inhibition of the Phox with diphenylene iodonium (DPI) delayed O₂ depletion, nearly abolished staining of O₂^- producing PMNs with hydroethidine and inhibited the rapid luminol-enhanced chemiluminescence in sputum. Furthermore, the total O₂ consumption was correlated to the concentration of PMNs in the sputum samples.

Conclusion: Our results demonstrate that CF sputum contains PMNs with an active consumption of O₂ for O₂^- production and suggest that the respiratory burst is ongoing and causes accelerated O₂ depletion due to formation of O₂^- in the lungs of chronically infected CF patients.

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