Figure 5

Salmeterol upregulates TLR4 membrane protein expression in respiratory epithelial cells. A549 cells (3 × 10⁵) were seeded onto 6-well plates and grown to confluence. Cells were washed, placed in serum free medium and were left untreated or incubated with salmeterol over the dose ranges 10^-9 to 10^-6 M for 6 hours. Beta-agonist effect was examined by pre-treatment of cells with Butoxamine 0.5 M × 30 minutes prior to salmeterol treatment. A. Real time PCR was performed as described in the methods. Data is expressed as mean +/- SEM of 7 independent experiments with TLR4/actin given an arbitrary value of 1 in control cells. B. Western blot analysis of total cell extracts (t) and cytosolic extracts (c) (10 μg) from A549 cells probed with an anti-TLR4 or anti-Actin antibody. Data are representative of three independent experiments. C. Western blot analysis of membrane extracts (10 μg) from A549 cells probed with an anti-TLR4. Densitometry was performed and corrected for corresponding Ponsceau S staining density. Data are expressed as mean +/- S.E. and are obtained from three experiments. (* P = 0.05 compared to control). D. A459 cells were incubated with an isotype control (clear) or anti-TLR4 (solid) antibody and fluorophore-conjugated detection antibodies. HBD2 expression was quantified by laser scanning cytometry, as described and data from three experiments is presented. HBD2 expression is expressed as Median Channel Fluorescence (MCF) ± SEM. (* P < 0.05 vs. control, ** P < 0.005 vs. control).