FIG 1.

Epithelial damage caused by neutrophil exposure to RSV-infected ciliated epithelial cells. The effect of respiratory syncytial virus (RSV) and neutrophil exposure on the number of α-tubulin-positive cells. (A) Bright-field image of an RSV-infected culture after 4 h of coculture with neutrophils, showing ciliated epithelial cells (*) and neutrophil clusters (#). Scale bar shown. (B) Representative confocal images of RSV-infected human nasal ciliated epithelial cells with and without neutrophil exposure for 1 h. Cells were stained with antibodies against ZO-1 to detect tight junctions and with 4′,6-diamidino-2-phenylindole (DAPI) to detect epithelial nuclei. (C) Mean fluorescence intensity of ZO-1 for ciliated airway epithelial cells mock infected (blue bars) or infected with RSV (red bars) for 24 h or 72 h and after 1 h with neutrophils (n = 6 epithelial donors, 6 heterologous neutrophils). Bars represent the mean ± SEM. P values show a significant difference. (D) The number of epithelial cells attached to membrane inserts after neutrophil exposure. Epithelial cells were quantified by counting the DAPI-stained nuclei of >50 μm² in area using ImageJ; the mean number (±SEM) of epithelial cells from all images is shown (n = 5 images per donor, 3 epithelial donors with heterologous neutrophils).