Figure 1

AMø of current smokers show reduced expression of TLR3 mRNA transcripts and intracellular protein relative to AMø of never-smokers. A. mRNA transcripts. RNA was isolated from AMø, depleted of contaminating genomic DNA, reverse-transcribed and analyzed by quantitative real-time RT-PCR using Taqman chemistry and specific primer-probe sets, normalized to GAPDH transcripts. Data are expressed on the horizontal axis as mean ± SEM for relative quantity (dRn), calculated in comparison to a single never-smoker who was arbitrarily designated the reference sample. Never-smokers (n = 6), red bars; smokers (n = 11), blue bars. *, p < 0.05 by Mann–Whitney test. B. Intracellular TLR3 (top panel) and TLR9 (bottom panel) expression by flow cytometry. AMø were permeabilized, stained for TLR3 and TLR9 expression and analyzed by flow cytometry. Data are expressed as TLR-positive AMø as mean ± SEM, never-smokers (n = 10 for TLR3, n = 5 for TLR9), red circles; smokers (n = 13 for TLR3, n = 6 for TLR9), blue circles. *, p < 0.05 by Mann–Whitney test. C. Immunohistochemical staining. Cytospins from a representative smoker (right) and a never-smoker (left), stained for intracellular TLR3 expression using AEC (red product) and hematoxylin (original 20 X). Top row, isotype control staining, bottom row, specific TLR3 staining. Representative of three experiments with similar results.