Primary human bronchial epithelial cells (NHBE) were obtained from Cambrex Bio Science (Walkersville, Inc., Maryland, USA). NHBE cells were grown according to the suppliers instructions in serum-free and hormone supplemented bronchial epithelial growth media (BEGM) which included supplements of 13 g/l bovine pituitary extract, 0.5 g/l hydrocortisone, 0.5 mg/l human recombinant epidermal growth factor, 0.5 g/l epinephrine, 10 g/l transferrin, 5 g/l insulin, 0.1 mg/l retinoic acid, 6.5 mg/l 3,3',5-triiodo-L-thryonine, 50 g/l gentamicin, and 50 mg/l amphotericin B (Cambrex Bio Science, Walkersville, Inc., Maryland, USA). Medium was changed daily starting from the day after seeding. Cells reached confluency in approximately 9 to 10 days, and nearly confluent cells were subcultured using trypsin/EDTA (Cambrex) at a ratio of 1:5. Experiments were carried out on the same batch of cells at passage 3 to 4. The cells were incubated in a humidified atmosphere (5% CO2, 37°C) under liquid-covered conditions.
Primary human alveolar epithelial cells (type II pneumocytes) were isolated from human non-tumor lung tissue obtained from 13 patients (mean age 65 yr [range, 46–77 yr], 10 males and 3 females) undergoing lung resection in Grantham Hospital, Hong Kong. The research protocol was approved by the ethics committee of the University of Hong Kong and Hospital Authority Hong Kong West Cluster. Human type II pneumocytes were isolated using a modification of the methods previously described [19, 23]. Briefly, after removing visible bronchi, the lung tissue was chopped into pieces of >0.5 mm thickness using a tissue chopper, washed with balanced salt solution (BSS, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 10 mM HEPES, 5.5 mM glucose, pH 7.4) for 30 min at 37°C three times to partially remove macrophages and blood cells. The tissue was digested using a combination of trypsin (0.5%, GIBCO BRL, Gaithersburg, MD, USA) and elastase (2 units/ml, Worthington Biochemical Corporation, Lakewood, NJ, USA) twice for 15 min at 37°C in a shaking water-bath. The partially digested tissue was minced in the presence of 40% fetal bovine serum (FBS) in DMEM/F12 medium and DNase I (350 units/ml) (GIBCO BRL, Gaithersburg, MD, USA), and cell clumps dispersed by repeatedly pipetting the cell suspension for 10 minutes. After filtration through gauze and a 40 μm cell strainer to ensure a single cell suspension, the cells were incubated with a 1:1 mixture of DMEM/F12 medium and small airway growth medium (SAGM, Cambrex Bio Science Walkersville, Inc., Maryland, USA) containing 5% FBS and 350 units/ml DNase I, on tissue-culture treated plastic Petri dishes in a humidified incubator (5% CO2, 37°C) for 2 hours in order to let macrophage attach on the plastic surface. The non-adherent cells were layered on a discontinuous Percoll density gradient (densities 1.089 and 1.040 g/ml) and centrifuged at 25 × g for 20 min. The cell layer at the interface of the two gradients was collected and washed four times with BSS to remove the Percoll. To remove remaining alveolar macrophages, the cell suspension was incubated with magnetic beads coated with anti-CD-14 antibodies at room temperature for 20 min under constant mixing. After the removal of the beads using a magnet and assessment of cell viability by trypan-blue exclusion, the purified type II pneumocyte suspension was suspended in SAGM supplemented with 1% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin, and plated at a cell density of 300,000 cells/cm2. The cells were maintained in a humidified atmosphere (5% CO2, 37°C) under liquid-covered conditions, and growth medium was changed daily starting from 60 hours after plating the cells.
Characterization of human type II pneumocytes
Staining for alkaline phosphatase
Human type II pneumocytes were identified by staining for alkaline phosphatase. Freshly isolated cells were spun down on glass slides, air-dried, and stained for 20 min at room temperature. The stain was prepared by dissolving 10 mg naphthol AS bi-phosphate (Sigma) in 40 μl DMSO and was diluted in 10 ml of 0.125 M 2-amino-2-methyl propanol buffer (pH 8.9, Sigma) containing 10 mg fast red (Sigma). The slide was washed and counterstained in 1% methylene green (Sigma) for 30 seconds and was mounted in aqueous medium .
Transmission electron microscopy
Cells were fixed in 2% glutaraldehyde (Electron Microscopy Sciences, Washington, PA, USA), washed three times in phosphate buffered saline and serially dehydrated in acetone. The tissue was post-fixed in 1% osmium tetroxide and embedded in an Araldite resin (Polysciences, Inc., Washington, PS, USA). Semi-thin sections (1 μm) were cut using an ultra-microtome (Reichert Ultracut S, Leica Aktiengesellscharft, Wien, Australia) with a diamond knife and were stained with toluidine blue for light microscopic examination. Ultra-thin sections (80 nm) mounted on copper grids were electron contrasted with uranyl acetate (1.5 hours, 30°C, Electron Microscopy Sciences) and lead citrate (40 minutes, 20°C, Electron Microscopy Sciences, Washington, PA, USA), and were examined with a transmission electron microscope (EM 208S, FEI Company, Hillsboro, Oregon, USA).
The expression of cell surface antigen was measured by staining purified type II pneumocytes with optimal dilution of rabbit anti-human surfactant protein-C (SP-C) (Upstate, Lake Placid, NY, USA) monoclonal antibodies (24°C, 30 minutes) followed by a fluorescein isothiocyanate (FITC-conjugated goat anti-mouse IgG antibody; Sigma, F-0257, 24°C, 30 minutes). Each cell preparation was also stained with antibody specific for monocyte/macrophage surface antigen (CD14 conjugated with FITC, MCA2185F; Serotec. Oxford, UK). The cells were examined by the flow cytometry (FACSSCalibur; Becton Dickinson), and the FITC-stained cells were detected by measuring green light emitted at 530 nm (FL1 channel). The percentage of cells expressing the epithelial and macrophage makers were determined.
Influenza virus infection of type II pneumocytes and bronchial epithelial cells
Human type II pneumocytes and bronchial epithelial cells (seeded at 1 × 106 cells per well in 24-well tissue-culture plates) were infected at a multiplicity of infection (MOI) of two unless otherwise indicated. After 60 min of virus adsorption, the virus inoculum was removed, and the cells were washed with warm culture medium (SAGM for type II pneumocytes and BEBM for bronchial epithelial cells) and incubated in medium supplemented with 0.6 mg/L penicillin, 60 mg/L streptomycin, and 2 mg/L N-p-tosyl-L-phenylalanine chloromethyl ketone-treated-trypsin (Sigma, St Louis, MO, USA). Aliquots of culture supernatant were collected and frozen at -80°C for subsequent virus titration and cytokine analysis. The supernatants were titrated on MDCK cells and the viral titre was quantitated as log10TCID50/ml. RNA was extracted from cells for analysis of cytokine gene expression. Ten hours after infection, replicate cell monolayers were fixed and analyzed by immuno-fluorescent staining specific for influenza virus nucleoprotein (DAKO Imagen, Dako Diagnostics Ltd, Ely, UK) to determine the proportion of cells infected.
Quantification of cytokine mRNA by real-time quantitative RT-PCR
DNase-treated total RNA was isolated by means of RNeasy Mini kit (Qiagen, Hilden, Germany). The cDNA was synthesized from mRNA with poly(dT) primers and Superscript II reverse transcriptase (Life Technologies, Rockville, MD, USA) and quantified by real-time PCR analysis with a LightCycler (Roche, Mannheim, Germany). The mRNA for IP-10, interferon beta, IL-6, RANTES and tumor necrosis factor (TNF) alpha were quantitated using real-time RT-PCR. The oligonucleotide primers and methods used for real-time quantification of cytokines, viral matrix gene and the housekeeping gene product γ-actin mRNA have been described previously [12, 24].