Materials and Methods

Nasal Epithelial Cells: Harvest and Culture The method for harvesting nasal epithelial cells was based on the procedure suggested by Bridges and co-workers (15). Nasal brushings were obtained from normal volunteers by scraping the nasal mucosa with a cytological brush (gastroscope cytology brushes; BARD Diagnostics, Ontario, Canada). Nasal scrapings were then resuspended vigorously in LHC-8 medium (Biofluids, Rockville, MD) containing 10 ng/ml epidermal growth factor (EGF; Collaborative Research, Boston, MA); 1ng/ml cholera toxin; 10Itg/ml transferrin (Collaborative Research); 50 nM glutamine; 100 D/ml penicillin; 1 ILg/ml streptomycin; and 1 Itg/ml fungizone. Tubes were then centrifuged at 300 g for 10 min, and the cell pellet was plated in 400 Itl of complete LHC-8 medium on collagen disks (ICN Biochemicals, Cleveland, OH) inserted in 24-well microtiter plates. Cells were incubated for 24 h in a humidified atmosphere of 5% CO2/95% air, after which fresh medium was added to the wells. In selected experiments, supernatant fluids were obtained for cytokine analysis immediately after overnight adherence. After a further incubation of 5 to 7 days, adherent epithelial cells were trypsinized, washed in medium, and plated directly on 24-well plates at approximately 104 cells/well in complete medium. Cells were cultured for an additional 7 days at 37 C in 5% CO2/95% air. Cells were trypsinized again and usually split 1: 2 in 24-well plates. Semiconfluent cultures are obtained after 5 to 7 days of incubation and were used at this stage. Approximately 106 cells were obtained from the nasal brushing of one subject after culture.

Epithelial Cell Lines Epithelial cell lines derived from the nasal scrapings of normal (13.21) and CF subjects (56.8, 56.12, and 56.13) were obtained as a gift from Dr. M. Buchwald (Hospital for Sick Children, Toronto, Ontario, Canada) after being transformed as described by other investigators (16). Briefly, retroviruses were used to transduce SV-40 large T antigen cDNA and the 3'phosphotransferase gene, which confers G418 resistance into nasal airway epithelial cells (16). After infection, cells were selected for G418 resistance by adding 0.5 mg/ml G418. Resulting transformed cell lines were helper virus-free, as shown in reverse transcriptase activity. Cells were grown in plastic dishes (3071; Falcon, Oxnard, CA) in a-MEM and DMEM (both from GffiCO, 50% vol! vol) supplemented with 10 Itg/ml of EGF, 1 mM glutamine, 15 ILg/ml insulin, 10 Itg/ml transferrin, and 10 ILg/ml Na selenite (all from Sigma Chemical Co., St. Louis, MO).