Pneumologie 2015; 69 - A14
DOI: 10.1055/s-0035-1556606

Neutrophil elastase activity on the surface of sputum neutrophils is associated with severity of airflow obstruction in cystic fibrosis

AS Dittrich 1, 2, 3, N Heath 1, 4, M Wiebel 3, FJ Herth 2, 3, C Schultz 2, 4, MA Mall 1, 2
  • 1Department of Translational Pulmonology, University of Heidelberg, Germany
  • 2Translational Lung Research Center Heidelberg (TLRC), Member of the German Center for Lung Research (DZL), Heidelberg, Germany
  • 3Department of Pneumology and Critical Care Medicine, Thoraxklinik at the University Hospital Heidelberg, Germany
  • 4Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany

Introduction: Neutrophilic airway inflammation is a hallmark of cystic fibrosis (CF) and previous studies identified free neutrophil elastase (NE) in bronchoalveolar lavage fluid and sputum as a key risk factor of early bronchiectasis and decline in lung function. However, the relevance of membrane-associated NE activity in the pathophysiology of CF lung disease is not well understood. In this study, we used a ratiometric Foerster resonance energy transfer (FRET) reporter (NEmo-2) to determine NE activity on the surface of sputum neutrophils and correlated NE levels with measurements of pulmonary function.

Methods: Inflammatory cells were isolated from spontaneous and induced sputum of patients with CF (n = 43) and healthy non-smokers (control, n = 9). 20,000 – 30,000 cells were incubated with NEmo-2 for 10 min (T10). To control for NE-specific cleavage of NEmo-2, cells were pre-incubated with the NE inhibitor sivelestat (T0). NE activity was calculated as the ratio of donor and acceptor fluorescence measured by confocal microscopy and normalized to samples treated with sivelestat (T10/T0).

Results: Membrane-associated NE activity (CF: 1.59 ± 0.28 vs. control: 1.07 ± 0.13, p < 0.001) and differential count of sputum neutrophils (CF: 94.34 ± 4.06 [%] vs. control: 42.12 ± 24.22 [%], p < 0.001) were significantly increased in CF compared to control samples. However, membrane-bound NE activity did not correlate with the percentage of neutrophils in CF or control sputum (CF: R = 0.17, p = 0.26 and control: R = 0.11, p = 0.78). In CF, levels of NE activity were inversely correlated with FEV1% predicted (R = –0.37, p < 0.05) and FEV1/VC (R = –0.51, p < 0.001) and directly correlated with residual volume (R = 0.54, p < 0.01) and intrathoracic gas volume (R = 0.55, p < 0.01).

Discussion: We conclude that NE activity is elevated on the surface of neutrophils in CF airways. Our findings suggest that membrane associated NE activity may contribute to severity of lung disease and serve as a valuable biomarker in patients with CF.

*Presenting author