Lipoxin A4 stimulates calcium-activated chloride currents and increases airway surface liquid height in normal and cystic fibrosis airway epithelia

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 LXA(4) is a member of the newly identified eicosanoids playing a key role in ending the inflammatory process. Levels of LXA(4) are reported to be decreased in the airways of patients with CF. We have previously shown that in normal human bronchial epithelial cells, LXA(4) produced a rapid and transient increase in intracellular Ca(2+). We have investigated, the effect of LXA(4) 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 LXA(4) stimulated a rapid intracellular Ca(2+) increase in all of the different CF bronchial epithelial cells tested. In non-CF and CF bronchial epithelia, LXA(4) stimulated whole-cell Cl(-) currents which were inhibited by NPPB (calcium-activated Cl(-) channel inhibitor), BAPTA-AM (chelator of intracellular Ca(2+)) but not by CFTRinh-172 (CFTR inhibitor). We found, using confocal imaging, that LXA(4) increased the ASL height in non-CF and in CF airway bronchial epithelia. The LXA(4) effect on ASL height was sensitive to bumetanide, an inhibitor of transepithelial Cl(-) secretion. The LXA(4) stimulation of intracellular Ca(2+), 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 LXA(4) in the stimulation of intracellular Ca(2+) signalling leading to Ca(2+)-activated Cl(-) secretion and enhanced ASL height in non-CF and CF bronchial epithelia.

Figure 1. LXA₄ effect on intracellular Ca²⁺ activity in non-CF and CF bronchial epithelial cells.

(A) Typical effect of LXA₄ (100 nM) on the cytosolic Ca²⁺ (ratio F340/F380) measured in Nuli-1 and CuFi-3 cell lines in control (plain line) and in external Ca²⁺ -free conditions (dotted line). (B) Typical effect of LXA₄ (100 nM) on the cytosolic Ca²⁺ in normal and CF primary cultures of bronchial epithelial cells (NHBE and CFBE). (C) Mean values of the maximum increase in Ca²⁺ (peak) and measured 2 and 5 minutes after the peak, in Nuli-1 (n = 6), CuFi-1 (n = 6), CuFi-3 (n = 6), CuFi-4 (n = 4) cell lines and in NHBE (n = 4) and CFBE (n = 4) bronchial epithelial cells in primary culture (* p<0.05, ** p<0.01).

Figure 2. Effect of Boc-2 on the intracellular Ca²⁺ signal induced by LXA₄.

(A) Representative effect of LXA₄ (100 nM) and ATP (100 µM) on cytosolic Ca²⁺ (ratio F340/F380) in NuLi-1 cells in control conditions (upper panel) and after 24 hours of pre-treatment with Boc-2 (10 µM) a specific inhibitor of ALX/FPR2 (lower panel). (B) Mean values corresponding to the effect of Boc-2 on the Ca²⁺ response to LXA₄ in Nuli-1 (n = 5) and in CuFi-3 (n = 4) cell lines (*** p<0.001).

Figure 3. Time dependency of the effect of LXA₄ on whole-cell currents in normal and CF bronchial epithelial cells in primary culture.

Typical I-V relationships and corresponding current records obtained before and after 5, 10 and 15 min exposure to LXA₄ (100 nM) in NHBE (A) and CFBE (B) isolated bronchial epithelial cells in primary culture. (C) Mean values corresponding to the time dependency of the LXA₄ effect onwhole cell inward conductances in Nuli-1 (n = 8) and in CuFi-3 (n = 4) cell lines and NHBE (n = 8) and CFBE (n = 6) primary cultures (**p<0.01).

Figure 4. Dose dependency of the effect of LXA₄ on whole-cell currents of normal (Nuli-1) and CF (CuFi-3) bronchial epithelial cell lines.

Typical I-V relationships obtained before and after 10 min exposure to 1 pM (n = 6 Nuli-1, n = 6 CuFi-3), 1 nM (n = 4 Nuli-1, n = 4 CuFi-3), 10 nM (n = 3 Nuli-1, n = 3 CuFi-3) and 100 nM (n = 6 Nuli-1, n = 6 CuFi-3) LXA₄ in Nuli-1 (A) and CuFi-3 (B) cell lines. (C) Mean inward conductance changes normalized to control values (gi/gic obtained without LXA₄) as a function of LXA₄ concentration in Nuli-1 (open bars) and CuFi-3 (black bars) cells in control conditions and obtained upon exposure to Boc-2 (10 µM) alone (10 min, 100 nM, n = 4 Nuli-1, n = 4 CuFi-3) or with Boc-2 (10 µM) and LXA₄ (10 min, 100 nM, n = 6 Nuli-1, n = 6 CuFi-3) and after BAPTA-AM pre-treatment alone (n = 6 Nuli-1, n = 6 CuFi-3) or with BAPTA-AM and LXA₄ (10 min, 100 nM, n = 4 Nuli-1, n = 6 CuFi-3).

Figure 5. Effects of Cl⁻ channel inhibitors on whole-cell currents stimulated by LXA₄.

(A) Representative I-V relationship of the effect of NPPB (1 µM) after the stimulation of primary cultures of bronchial epithelial cells with LXA₄ (100 nM). Corresponding histogram of the inhibitory effect of NPPB on the inward conductance obtained in non-CF primary NHBE cells (n = 4), CuFi-3 cells (n = 3) and CF primary CFBE cells (n = 3) after stimulation with LXA₄ (100 nM, 10 min). (B). Typical I-V relationships of the effect of LXA₄ (100 nM) obtained in NuLi-1 cells after treatment with the CFTR channel inhibition CFTR-inh172 (5 mM) and histogram showing the absence of inhibitory effect in Nuli-1 cells (n = 3).

Figure 6. LXA₄ (100 nM) effects on airway surface liquid height in NuLi-1 (A), CuFi-3 (B) and CuFi-1 epithelial cell line (C) and CFBE primary cultures (D).

Epithelial cells were stained with calcein green, and the ASL labelled with dextran-conjugated Texas red™ fluorochrome. For each cell preparation, typical z-plane confocal sections showing ASL responses to LXA₄ (top) and mean ASL height changes (bottom) in control conditions or following LXA₄ (100 µM) exposure in NuLi-1,CuFi-3, CuFi-1 and CFBE epithelia treated or not with Boc-2 (10 µM) and with bumetanide (10 µM).