Figure 5

Human rMUC1-ED exhibits flagellin-targeting decoy receptor function, and enhances the PMN phagocytic response. (A) PAK was incubated for 1 h at 4 °C with 25 µM of human rMUC1-ED. The bacteria were washed with PBS, pH 7.4 and incubated for 1 h at 4 °C with mouse anti-MUC1-ED antibody (panel i) or nonimmune mouse IgG control (panel ii), each at 1:5,000 dilution, followed by 1 h at 4 °C with gold-labeled goat anti-mouse IgG secondary antibody at 1:10,000 dilution. Bacterial flagella were examined by transmission immunoelectron microscopy. Each section was photographed at 44,000X. Scale bar, 25 nm. (B, C) The indicated concentrations of human rMUC1-ED or the PBS vehicle control were incubated with PAK, and the bacteria were washed and assayed for (B) motility and (C) biofilm formation. (D, E) The indicated concentrations of human rMUC1-ED or the PBS vehicle control were incubated with PAK, and (D) the bacteria were assayed for adhesion to A549 cells and (E) cell culture supernatants were assayed for IL-8 levels by ELISA. (F) The indicated concentrations of human rMUC1-ED or the PBS vehicle control were incubated with WT-PAK or the PAK/fliC¯ flagellin-deficient strain and the bacteria were assayed for phagocytosis by PMNs. Bars represent mean ± S.E. values (n = 3). Open circles represent individual values. **, significantly decreased Pa motility, biofilm, adhesion, or IL-8 levels compared with the PBS control, or phagocytosis of PAK/fliC¯ compared with WT-PAK, at p < 0.05. *, significantly increased WT-PAK phagocytosis compared with the PBS control at p < 0.05. (G) PAK were cultured with 100 µM of human rMUC1-ED or the PBS vehicle, cultured over time in LB medium, and assayed for bacterial growth. Error bars represent mean ± S.E. A₆₀₀ values (n = 3). The results are representative of 2 or 3 independent experiments.