Figure 2.

IL13 increases MUC5AC expression and secretion. (A) In vitro protocol for IL13 treatment of human tracheal/bronchial epithelial cells (hTEC) differentiated using air-liquid interface conditions (ALI). Cells were assayed at the indicated times. (B) Representative images of hTEC treated with vehicle or IL13 for the indicated periods, then immunostained for MUC5AC and cilia marker acetylated tubulin (acetylated-TUB). Nuclei were stained with DAPI. MC, goblet cell; arrows, secreted MUC5AC on the cell surface. Scale bars: 10 μm. (C) IL13-induced MUC5AC mRNA levels (+) relative to vehicle (−) (n = 6 preparations/condition). (D) ROS production in hTEC cultured with or without IL13 during ALI d 14–21 d. Three h prior to loading the ROS probe DCF (CM-H2DCFDA), cells were treated with combinations of IL13 and the NOX inhibitor DPI (5 μM), or vehicle controls and the resulting mean fluorescent intensity signal was measured from 3 random fields. AU, arbitrary units. (E) MUC5AC secretion in hTEC preparations cultured with or without IL13 for 21 d as in (A) and pre-treated with DPI (5 μM) or DMSO vehicle prior to the MUC5AC secretion assay. Levels of secreted MUC5AC were measured by ELISA after performing a series of washes (to obtain baseline levels) followed by a 1 h incubation with IL13. Values were normalized to the baseline amount of MUC5AC for each condition and reported as fold change (n = 7 for DMSO vehicle pretreatment, n = 4 for DPI pretreatment). Measures in (C–E) were obtained from hTEC preparations obtained from at least 3 unique donors and independent experiments, displayed as the graph of the means ±SEM. In (C), means from each time point were compared to vehicle alone using the paired Student t test. In (D, E), means with different letters are significantly different by ANOVA with Bonferroni's correction.