BAL and blood analysis

The patients underwent BAL, as described previously in the reports from our laboratory [19,20]. Briefly, the affected segmental bronchus was identified on the chest computed tomography (CT) scan, and was lavaged 3 times using 50-mL aliquots (total volume, 150 mL) of sterile 0.9% saline at room temperature through a wedged flexible fiberoptic bronchoscope (Olympus 1T-200 or 1T-240, Tokyo, Japan). The BALF obtained was separately filtered through several layers of gauze to remove excess mucus and debris, and was subsequently centrifuged at 1500 rpm for 5 min at 4°C to separate the supernatant from the cells. Aliquots of the supernatant (1 mL) were immediately frozen and stored at -80°C prior to the assay. Cell pellets were counted in a hemocytometer, and Diff-Quik^™ (International Reagents, Kobe, Japan)-stained smears were used to identify the differential profiles after cytospin preparation. Differential counts were performed by examining 300 cells using a standard light microscope. The T lymphocyte subpopulations were determined using flow cytometry. Whole blood samples were incubated with fluorescent conjugated monoclonal antibodies CD3, CD4, and CD8 (Beckman Coulter, Tokyo, Japan). The BAL samples were incubated with fluorescent monoclonal antibodies CD3, CD4, CD8, and CD45 (Beckman Coulter). The samples were subsequently stained at room temperature in the dark for 15 min. Red blood cell (in whole blood samples) lysis was performed using the Q-Prep lysing kit (Beckman Coulter). Cells were then washed and resuspended in PBS. Cells were analyzed with a flow cytometer (Cytomics FC500, Beckman Coulter, Tokyo, Japan). Venous blood was withdrawn 30 minutes before the BAL. After collection, serum samples were immediately frozen and stored at -80°C until the assay.