Figure 7.

Roflumilast N-oxide (RNO) protected from loss of ciliated bronchial epithelial cells induced by CSE. In (A, left panel), differentiated bronchial epithelial cells (D-BECs) were exposed to CSE at different concentrations (0–10%) and times (0–7 days). Medium was replenished with fresh CSE every 24 h. Cells with cilia motility were identified based on DHSV as described in Methods. At each of the indicated times and concentrations of CSE exposure, the average number of cells with cilia motility per field was then calculated based on captures from a minimum of 10 separate fields and expressed as % of corresponding cilia motility obtained for non-treated cells at day 0 (= 100%) (control). In (A, right panel), D-BECs were pre-incubated with RNO at 2 nM or 1 µM or vehicle for 30 min and then exposed to CSE at 10% for 3 days. Culture medium with fresh CSE and RNO was replaced every 24 h. The average number of cells with cilia motility per field was determined as in (A), left panel. In (B), D-BECs were pre-incubated with RNO and then exposed to CSE at 10% for 3 days as described in (A). β-tubulin IV was identified by immunofluorescent labelling with a primary anti-β-tubulin IV antibody followed by a secondary FITC-conjugated antibody as detailed in Methods. Representative microphotographs are depicted in the upper, left panel (scale bar 20 µm, original magnification × 400) with zoomed pictures in the lower, left panel. Results of the morphometric evaluation are shown in the right panel as the percentage of cells that expressed β-tubulin IV of total cells. Results are given as means ± SEM of cultures from two donors and three experiments per donor. *P < 0.05 versus control, ^#P < 0.05 versus CSE.