EPS06.3 Neutrophil elastase activity on the surface of sputum neutrophils is associated with severity of cystic fibrosis lung disease

Abstract

Objective Previous studies identified free neutrophil elastase (NE) in bronchoalveolar lavage fluid and sputum of patients with cystic fibrosis (CF) as a key risk factor of early bronchiectasis and decline in lung function. However, the pathophysiologic relevance of membrane-bound NE in CF lung disease is not well understood. We therefore used a ratiometric Foerster resonance energy transfer reporter (NEmo-2) to determine NE activity on sputum neutrophils and correlated NE levels with lung function measurements. Methods Inflammatory cells isolated from spontaneous or induced sputum of CF patients (n = 37) and healthy non-smokers (control, n=9) were incubated with NEmo-2. To control for NE-specific cleavage of NEmo-2, cells were preincubated 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 T0. Results Membrane-bound NE activity (CF: 1.57±0.29 vs. control: 1.07±0.13, p 1 % predicted (R = –0.33, p 1 /VC (R = –0.44, p Conclusion We conclude that NE activity is elevated on the surface of neutrophils in CF airways. This may contribute to severity of lung disease and serve as valuable biomarker in CF.