BAL was performed as previously described54. Normally, the BAL fluid contains ~91% of AMΦ and ~9% of other cells, including PMN and lymphocytes. The immunomagnetic separation system was used to isolate AMΦ. Magnetic nanoparticle-conjugated antibody (CD11b MicroBeads, Miltenyi Biotec) was chosen to label and remove PMN and lymphocytes. The resulting cells consisted of >98% macrophages, and cell viability was >95%. AMΦ from wild type, TLR4−/−, TLR3−/− and MyD88−/− mice were cultured in DMEM containing 10% FBS and 10 μl/ml penicillin/streptomycin for 2 days, then they were washed three times with PBS and the medium was changed to low-serum medium (1% FBS). Cells were stimulated with LPS (NC, 0.01, 0.1, 1, 10 μg/ml) for 0–8 h in DMEM containing 10% FBS at a concentration of 1 × 106 cells/ml of medium. TLR3 expression in the AMΦ lysates was measured by Western blotting and RT-PCR.