M046: EVIDENCE FOR ASIALOGM1 AS A GLYCOLIPID RECEPTOR FOR PSEUDOMONAS AERUGINOSA ADHERENCE TO REPAIRING RESPIRATORY EPITHELIUM.
S. Girod de Bentzmann. Roger P., Bajolet-Laudinat O. and Puchelle E. (INSERM U314, CHR Maison Blanche, Reims France).

Gangliotetraosylceramide (asialoGMI) is described as a major superficial epithelial receptor containing GalNAcBl-4Gal sequence for Pseudomonas aeruginosa (PA) adherence to epithelia. Recently, Saiman et al. (J. Clin. Invest., 1994, 92, 1875) demonstrated that asialoGMI was significantly more expressed on the surface of cystic fibrosis (CF) respiratory epithelial cells than on non-CF cells. Since we have observed an increase of PA adherence to repairing respiratory epithelial cells, we speculated whether asialoGM I could be implicated in the increased PA affinity for these cells from CF and non-CF patients. Human respiratory epithelial cells obtained from nasal polyps of non-allergic patients, non-CF (n=8) and CF (n=8) patients homozygous for the F508 mutation, were cultured according to the explant-outgrowth model. At the periphery of the outgrowth, we observed flattened cells spreading over the collagen I matrix with lamellipodia, which were identified during respiratory epithelial wound repair. A non-CF piliated strain of PA was added to the cultures at a concentration Of 108 CFU/ml for I hour at 37°C. In vitro competitive binding inhibition assays were performed by incubation with rabbit polyclonal antibody against asialoGM I for I hour at 37 °C. Controls were performed by omitting incubation with anti-asialoGM1 or by incubating with non-immune IgG at 10, 50 and 1001lg/rnl prior to PA addition. Quantitation of PA adherence was performed by computer assisted SEM observation. In situ distribution of asialoGMI was revealed by immunofluorescence. PA adherence to repairing respiratory epithelial cells was found to be significantly higher in the F508 homozygous CF patients (5.7xl0-2 2 bact/m2 versus 2.9xl0-20.5 bact/m2, p<O.OI). The anti-asialoGMI antibody significantly reduced PA adherence to repairing respiratory epithelial cells by 75% in 2 CF patients (p<0.01) and by 58% in 2 non-CF patients (p<0.01). Preincubation of cultures with non-immune IgG (whatever the concentrations used) did not reduce PA adherence, excluding an aspecific phenomenon. In situ immunolocalization provided evidence that asialoGM1 was identified in CF and in non-CF repairing epithelial cells. All these results suggest that during respiratory epithelial wound repair, specific PA adherence for repairing cells may be partly due to apical asialoGM1 receptors.

This work was supported by the Association Francaise de Lutte contre la Mucoviscidose (AFLM).

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