Valia Verriere

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

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.

Activation of P2RY11 and ATP release by lipoxin A4 restores the airway surface liquid layer and epithelial repair in cystic fibrosis.

In cystic fibrosis (CF), the airway surface liquid (ASL) height is reduced as a result of impaired ion transport, which favors bacterial colonization and inflammation of the airway and leads to progressive lung destruction. Lipoxin (LX)A4, which promotes resolution of inflammation, is inadequately produced in the airways of patients with CF. We previously demonstrated that LXA4 stimulates an ASL height increase and epithelial repair. Here we report the molecular mechanisms involved in these processes. We found that LXA4 (1 nM) induced an apical ATP release from non-CF (NuLi-1) and CF (CuFi-1) airway epithelial cell lines and CF primary cultures. The ATP release induced by LXA4 was completely inhibited by antagonists of the ALX/FPR2 receptor and Pannexin-1 channels. LXA4 induced an increase in intracellular cAMP and calcium, which were abolished by the selective inhibition of the P2RY11 purinoreceptor. Pannexin-1 and ATP hydrolysis inhibition and P2RY11 purinoreceptor knockdown all abolished the increase of ASL height induced by LXA4. Inhibition of the A2b adenosine receptor did not affect the ASL height increase induced by LXA4, whereas the PKA inhibitor partially inhibited this response. The stimulation of NuLi-1 and CuFi-1 cell proliferation, migration, and wound repair by LXA4 was inhibited by the antagonists of Pannexin-1 channel and P2RY11 purinoreceptor. Taken together, our results provide evidence for a novel role of LXA4 in stimulating apical ATP secretion via Pannexin-1 channels and P2RY11 purinoreceptors activation leading to an ASL height increase and epithelial repair.

Physiological levels of lipoxin A4 inhibit ENaC and restore airway surface liquid height in cystic fibrosis bronchial epithelium

In cystic fibrosis (CF), the airway surface liquid (ASL) is depleted. We previously demonstrated that lipoxin A4 (LXA4) can modulate ASL height (ASLh) through actions on Cl− transport. Here, we report novel effects of lipoxin on the epithelial Na+ channel ENaC in this response. ASL dynamics and ion transport were studied using live‐cell confocal microscopy and short‐circuit current measurements in CF (CuFi‐1) and non‐CF (NuLi‐1) cell cultures. Low physiological concentrations of LXA4 in the picomolar range produced an increase in ASLh which was dependent on inhibition of an amiloride‐sensitive Na+ current and stimulation of a bumetanide‐sensitive Cl− current. These ion transport and ASLh responses to LXA4 were blocked by Boc‐2 an inhibitor of the specific LXA4 receptor ALX/FPR2. LXA4 affected the subcellular localization of its receptor and enhanced the localization of ALX/FPR2 at the apical membrane of CF cells. Our results provide evidence for a novel effect of low physiological concentrations of LXA4 to inhibit airway epithelial Na+ absorption that results in an ASL height increase in CF airway epithelia.

P. aeruginosa LPS stimulates calcium signaling and chloride secretion via CFTR in human bronchial epithelial cells

BACKGROUND Pseudomonas aeruginosa airway infection is associated with a high mortality rate in cystic fibrosis. Lipopolysaccharide (LPS), a main constituent of the outer membrane of P. aeruginosa, is responsible for activation of innate immune response but its role on airway epithelium ion transport, is not well known. The aim of this study was to determine the role for P. aeruginosa LPS in modulating chloride secretion and intracellular calcium in the human bronchial epithelial cell line, 16HBE14o-. METHODS We used intracellular calcium imaging and short-circuit current measurement upon exposure of cells to P. aeruginosa LPS. RESULTS Apical LPS stimulated intracellular calcium release and calcium entry and enhanced chloride secretion. This latter effect was significantly inhibited by CFTR(inh)-172 and BAPTA-AM (intracellular Ca(2+) chelator). CONCLUSIONS Our data provides evidence for a new role of P. aeruginosa LPS in stimulating calcium entry and release and a subsequent chloride secretion via CFTR in human bronchial epithelium.

Eosinophil peroxidase induces the expression and function of acid-sensing ion channel-3 in allergic rhinitis: in vitro evidence in cultured epithelial cells

Acid‐sensing ion channels (ASIC) are a family of acid‐activated ligand‐gated cation channels. As tissue acidosis is a feature of inflammatory conditions, such as allergic rhinitis (AR), we investigated the expression and function of these channels in AR.

WS15.2 Airway surface liquid layer height in cystic fibrosis bronchial epithelial cells is increased by lipoxin A4 via an apical ATP release activating a P2Y receptor pathway

WS15.1 Volume increase in alveolar type II cells following fusion dependent activation of P2X4 receptors on lamellar bodies − linking secretion and fluid transport in the lung? K. Thompson1, E. Hecht2, O. Wittekindt1, P. Miklavc1, C. Kranz2, P. Dietl1, M. Frick1. 1Institute of General Physiology, University of Ulm, Ulm, Germany; 2Institute of Analytical and Bioanalytical Chemistry, University of Ulm, Ulm, Germany