Bronchoscopy and Lymphocyte Isolation: Following topical lidocaine anesthesia, an Olympus BF-10 bronchoscope was passed into the airways transnasally and, after inspection of the airways, the bronchoscope was wedged into a segmental orifice of an affected lobe (as assessed by chest roentgenogram). BAL was performed by infusing approximately 150 ml of sterile 0.9 percent NaCl solution in 20- to 30-ml aliquots, with gentle aspiration until a 100-ml fluid return was achieved. At initial bronchoscopy, transbronchial biopsies were obtained from segments different from those used for BAL using standard techniques.
The BAL fluid was centrifuged at 275 x g for 10 min and the cell pellet was washed three times with Hanks’ balanced salt solution without CA + 2 and Mg+ 2 (HBSS, Gibco) and resuspended in 2 to 5 ml of RPMI-1640 medium supplemented with 15 percent fetal calf serum, 2 mM glutamine, 5xl0-5 M 2-mercaptoethanol, 100 IU penicillin, and 100 p-g/ml streptomycin (Gibco). Total and differential cell counts were determined by using a hemocytometer after addition of 40 fxl of 0.1 percent crystal violet (in 0.1 M citric acid) to 40 μl of the cell suspension. Viable cell counts were determined by eosin Y dye exclusion. other
Isolation of blood mononuclear leukocytes on Ficoll-Hypaque cushions were performed. Further purification of BAL and peripheral blood lymphocytes was performed by using nylon fiber columns by the method of Greaves and Brown to yield a monocyte/macrophage- and B-cell-depleted, nonadherent lymphocyte preparation. Briefly, using aseptic technique, prewashed and presoaked, scrubbed nylon fiber, 3-denier, type 200 (Fenwal Laboratories), was loaded into disposable 10- or 20-mi syringes under pyrogen-free water. The nylon column was then washed with 4 vol of Hanks’ balanced salt solution, followed by 2 vol of RPMI-complete. BAL or peripheral blood mononuclear cells were placed in the column at concentrations of 1 x 107 cells/ml and then incubated at 37°C in 5 percent COa and 95 percent air. The cells were collected by washing the column with 40 to 50 ml of prewarmed RMPi-complete. These nylon column nonadherent cells were concentrated into a volume of 2 to 5 ml, and total and differential cell counts were determined as described above. Lymphocyte recovery from the column is usually greater thdn 85 percent. These nonadherent cells contained <1 percent monocytes/macrophages.