Figure 5
SP-D expression in monolayer epithelial cultures treated by IL-13. SP-D mRNA was examined in non-asthmatic monolayer cultures treated with IL-13 (10 ng/mL) alone and in cultures pre-treated with either goat IgG isotype control (20 μg/mL), IL-13Rα1 neutralizing antibody (20 μg/mL), or IL-13Rα2 neutralizing antibody (2 μg/mL) one hour prior to IL-13 exposure. To obtain fold changes, ΔΔCT values were obtained by normalizing SP-D mRNA to HPRT mRNA and normalizing treatment groups to untreated control. A significant difference was observed between the IgG pre-treated group and IL-13Rα1 neutralizing antibody pre-treated group (n = 3, one-way ANOVA p = 0.01) [A]. SP-D protein levels in non-asthmatic monolayer cultures under the same treatment conditions were detected via Western blotting. A significant difference was found between the IL-13 treated group and IL-13Rα1 neutralizing antibody pre-treated group. Quantification was performed by densitometry and SP-D protein expression was normalized to untreated control (n = 6, one-way ANOVA p = 0.04) [B]. Phosphorylated-STAT6 (pSTAT6) expression in non-asthmatic monolayer cultures from the same experiment as panel [B] was examined via Western blotting. IL-13 treatment significantly increased the phosphorylation of STAT6 compared to control while pre-incubation with IL-13Rα1 neutralizing antibody significantly reduced the phosphorylation of STAT6 compared to IgG (n = 5, one-way ANOVA p = 0.005) [C]. pSTAT6 to STAT6 protein ratio in non-asthmatic ALI cultures treated with IL-13 (10 ng/mL) over the course of two weeks show significant increases relative to untreated control (n = 3, one-way ANOVA p = 0.001) [D]. IL-13 treatment resulted in a significant decrease in SP-D protein expression in non-asthmatic monolayer culture (n = 5, t-test p = 0.03) whereas no significant change was detected in asthmatic monolayer culture (n = 4) [E].