Proceedings of The Physiological Society

University College Dublin (2009) Proc Physiol Soc 15, C134

Oral Communications

Effect of Lipoxin A4 in Modifying the Airway Surface Liquid Layer

V. Verriere1, M. Al-Alawi1, O. Mc Cabe1, V. Urbach3, R. W. Costello2, B. J. Harvey1

1. Molecular Medicine, RCSI, Dublin, Ireland. 2. Respiratory Medicine, RCSI, Dublin, Ireland. 3. U661, INSERM, Montpellier, France.

A key aspect of the lung’s innate defence system is the ability of the epithelium to regulate the Airway Surface Liquid (ASL) volume. The ASL is tightly autoregulated by ion and water transport regulation which need the control ion transport. The transporters involve in ion transport are required for effective ciliary beating and muco-ciliary clearance quality. Na+ transport is closely controlled to maintain an appropriate fluid layer on the alveolar surface through the Amiloride-sensitive sodium channel (ENaC). Lipoxin A4 (LXA4) is an endogenous anti-inflammatory molecule produced from arachidonic acid. LXA4 has been reported to be reduced in inflammatory Cystic Fibrosis(CF)lung1. CF is a severe genetic disease due to the mutation of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR)gene. The consequence of the mutation is Na+ hyper absorption and a defect in Cl- secretion, resulting in dehydration of the airway lumen. This limited mucociliary clearance favours chronic infection and inflammation. One of the therapeutic avenues in CF is to explore a way to inhibit the Na+ hyperabsorption. Our hypothesis is that LXA4 treatment increases the height of ASL in CF and non-CF cell lines. Materials and Methods: CF (CuFi-1) and non CF (NuLi-1) cell line are grown to confluency on semi-permeable filters in order to obtain a well differentiated polarised epithelium showing high transepithelial resistance. Cells are loaded with fluorescent dye Calcein AM (5μM). A small volume of a dextran solution containing the Texas Red fluorochrome is applied on the apex of the epithelium in order to label the ASL compartment. The ASL height is measured using a laser scanning confocal microscopy in live cells in response to LXA4. Results: ASL stabilisation is obtained between 12 and 24 hours after the application of the dextran solution on the apex of the cells. The ASL of the CF epithelium is reduced when compared to non-CF epithelium. In CuFi and NuLi cell lines, a LXA4 (1nM) basolateral pretreatment significantly increased the ASL stabilized height from a physiological baseline height of 5±0.28 µm (n=18) and 7±0.21 µm (n=27) respectively to 15.25±1.18 µm at 15 minutes (n=19) in CuFi cells. This effect is also observed to a level of 9.23±0.56 µm (n=28) at 30 min, and 7.86±0.92 µm (n=10) at 45 minutes and appears to be maintained at 48 hours of stimulation in CuFi-1 (n=9). LXA4 effect on ASL height is abolished using the lipoxin receptor antagonist, boc2 (10nM) on a 15 and 30 minutes subsequent exposure of LXA4 (1nM, n=27). Discussion: LXA4 treatment resulted in an increase of the ASL volume and may provide a novel avenue in complementing existing therapy inflammatory in CF. Future Perspectives: The role of the ENaC channel in modulating the ASL via LXA4 will be investigated in addition to its downstream cell signalling cascades.

Where applicable, experiments conform with Society ethical requirements