Figure 6From: Epithelial expression of TLR4 is modulated in COPD and by steroids, salmeterol and cigarette smokeSalmeterol reverses the inhibitory effect of dexamethasone on TLR4 membrane protein expression despite downregulation of mRNA. 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 10-6 M dexamethasone (Dex), 10-7M salmeterol (Sal) or both drugs in combination (Sal + Dex) for 16 hours. Numbers indicate Molar doses of drug. Following treatment, total RNA or membrane protein was extracted for PCR and Western blot analysis. For IL-8 expression analysis, cells were further stimulated with LPS 10 μg/ml × 24 hours. A. Real-time PCR analysis of TLR4 mRNA expression as a factor of β-actin expression. TLR4 expression was given an arbitrary value of 1 in control cells. Data are expressed as mean +/- S.E. and are obtained from three experiments. (* P = 0.05 compared to control). B. Western blot analysis of membrane extracts (10 μg) from A549 cells probed with an anti-TLR4. Densitometry was performed and corrected for corresponding ponsceau staining density. Data are expressed as mean +/- S.E. and are obtained from three experiments. (* P = 0.05 compared to control). C. Levels of IL-8 in supernatants were measured by ELISA and values are expressed as fold change compared to unstimulated control. Assays were performed in duplicate a minimum of three times. Values are expressed as mean +/- S.E. (n = 3). (** signifies P ≤ 0.005 of observed effect vs. LPS alone; †signifies P ≤ 0.05 of observed effect vs. LPS + Dex).Back to article page