Lipoxin A4 Stimulates Calcium-Activated Chloride Currents and Increases Airway Surface Liquid Height in Normal and Cystic Fibrosis Airway Epithelia

Valia Verrière,Paul Mcnally,Raphaël Chiron,Gerard Higgins,Richard W Costello,Brian J Harvey,Mazen Al-Alawi,Valérie Urbach

doi:10.1371/journal.pone.0037746

Abstract

Cystic Fibrosis (CF) is a genetic disease characterised by a deficit in epithelial Cl− secretion which in the lung leads to airway dehydration and a reduced Airway Surface Liquid (ASL) height. The endogenous lipoxin LXA4 is a member of the newly identified eicosanoids playing a key role in ending the inflammatory process. Levels of LXA4 are reported to be decreased in the airways of patients with CF. We have previously shown that in normal human bronchial epithelial cells, LXA4 produced a rapid and transient increase in intracellular Ca2+. We have investigated, the effect of LXA4 on Cl− secretion and the functional consequences on ASL generation in bronchial epithelial cells obtained from CF and non-CF patient biopsies and in bronchial epithelial cell lines. We found that LXA4 stimulated a rapid intracellular Ca2+ increase in all of the different CF bronchial epithelial cells tested. In non-CF and CF bronchial epithelia, LXA4 stimulated whole-cell Cl− currents which were inhibited by NPPB (calcium-activated Cl− channel inhibitor), BAPTA-AM (chelator of intracellular Ca2+) but not by CFTRinh-172 (CFTR inhibitor). We found, using confocal imaging, that LXA4 increased the ASL height in non-CF and in CF airway bronchial epithelia. The LXA4 effect on ASL height was sensitive to bumetanide, an inhibitor of transepithelial Cl− secretion. The LXA4 stimulation of intracellular Ca2+, whole-cell Cl− currents, conductances and ASL height were inhibited by Boc-2, a specific antagonist of the ALX/FPR2 receptor. Our results provide, for the first time, evidence for a novel role of LXA4 in the stimulation of intracellular Ca2+ signalling leading to Ca2+-activated Cl− secretion and enhanced ASL height in non-CF and CF bronchial epithelia.

Highlights

Cystic fibrosis is caused by the mutation of the gene coding for the Cystic Fibrosis Transmembrane conductance Regulator (CFTR), a cyclic AMP-dependent Cl2 channel
There was no significant difference in the maximum increase in calcium induced by lipoxin A4 (LXA4) (100 nM) between non-CF and CF bronchial epithelial cell lines (Figure 1A and 1C)
In the CF bronchial epithelial cells (CFBE primary cultures and CuFi cell lines), LXA4 induced a slower increase in Ca2+ and a delayed recovery toward basal values (Figure 1C)

Summary

Introduction

Cystic fibrosis is caused by the mutation of the gene coding for the Cystic Fibrosis Transmembrane conductance Regulator (CFTR), a cyclic AMP-dependent Cl2 channel. The major clinical features of CF are chronic pulmonary disease, exocrine pancreatic insufficiency and male infertility [1,2]. The lung disease is the main cause of morbidity and mortality in CF. The airway epithelium of patients with CF fails to transport Cl2 and water, resulting in a reduced ASL height and impaired mucociliary clearance. The hyper-absorption of Na+ observed in the CF bronchial epithelium may further exacerbate the dehydration of the ASL. It is generally accepted that the dehydration of the airway lumen favours chronic infection and inflammation leading to progressive destruction of the lung [3]. Identification of agents, natural endogenous biologicals, which stimulate alternative non-CFTR Cl2 secretory pathways and promote ASL hydration and recovery of optimal ASL height are likely to be of therapeutic benefit in improving mucociliary clearance in patients with CF

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Conclusion