Figure 2

Cigarette smoke downregulates TLR4 gene and protein expression in A549 cells resulting in relative hyporesponsiveness to LPS. A549 cells (3 × 10⁵) were seeded onto 6-well plates and grown to confluence. Cells were washed, placed in serum free medium or cigarette smoke condensates for 4 hours. Cigarette smoke condensates were prepared as described in the methods and numbers correspond to serial dilutions of the initial cigarette smoke extract. A. Following treatment, total RNA was extracted, reverse transcribed into cDNA and used as a template for semi-quantitative PCR reactions using TLR4 and GAPDH gene-specific primers. 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 anti-TLR4 antibody. Data are representative of three separate experiments. (CSE † cigarette smoke extract). Because actin is not compartmentalised to the membrane, equal protein loading is demonstrated with a panel from the Ponsceau Stain. C. Viability assay of A549 cells following treatment with CSE. Data are representative of three separate experiments. D. A549 cells (3 × 10⁵) were seeded onto 6-well plates and grown to confluence. Cells were washed, placed in low-serum (1% FCS) medium and were left untreated or incubated with serial dilutions of CSE × 4 hours. Following treatment with CSE cells were stimulated with LPS 10 μg/ml for a further 24 hours. Levels of IL-8 in supernatants were measured by ELISA and values are expressed as pg/ml. Assays were performed in duplicate a minimum of three times. Values are expressed as mean +/- S.E. (n = 3). (* signifies P ≤ 0.05 of observed effect vs. control, † signifies P ≤ 0.05 of observed effect vs. control plus LPS).