All biochemicals were of analytical grade and purchased from Sigma Chemical Co (St. Louis, MO) unless otherwise stated.
Penicillin, streptomycin and culture media (DMEM, RPMI 1640, F12K) were procured from Life technologies (Gaithersburg, MD, USA). Fetal bovine serum (FBS) was obtained from HyClone Laboratories (Logan, UT, USA). Rabbit polyclonal anti NF-κB Rel/p65 antibody (sc-372) was purchased from Santa Cruz Biotechnology Inc., (Santa Cruz, CA, USA).
Five different alveolar epithelial type II cell lines were used for this study along with the primary human small airway epithelial cells (SAEC). The sources of various cell lines were as follows: the human adenocarcinoma epithelial cells (A549) derived from lungs of adenocarcinoma patient, human lung epithelial cells from papillary adenocarcinoma patient (H441), human lung cancer cells from cancer patient (H1299), and rat lung epithelial cells (L2) were obtained from American Type Cell Collection (ATCC), Manassas, VA, USA. Murine type II epithelial cells (MLE-15) were derived from immortalized lung tumors of transgenic mice containing the simian virus 40 large T antigen under the transcriptional control of the regulatory sequences derived from the human surfactant protein (SP)-C promoter region [11, 12]. Cells were grown in culture media (A549 and H1299: Dulbecco's modified Eagle medium, H441: RPMI 1640 medium, MLE-15: DMEM/F12K medium and L2: F12K medium) supplemented with 10% FBS, 2 mM L-glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2.
SAEC derived from a single healthy non-smoker, and the basal media (SAGM) including all the growth supplements were purchased from Clonetics (San Diego, CA, USA). Cells were cultured according to the supplier's instructions. Passage number was kept to less than seven passages from original stocks. SAEC were maintained in SAGM supplemented with 52 μg/ml bovine pituitary extract, 0.5 ng/ml human recombinant epidermal growth factor (EGF), 0.5 μg/ml epinephrine, 10 μg/ml transferrin, 5 μg/ml insulin, 0.1 ng/ml retinoic acid (RA), 6.5 ng/ml triiodothyronine, 50 μg/ml Gentamicin/Amphotericin-B (GA-1000), and 50 μg/ml fatty acid-free bovine serum albumin (BSA). Polymyxin B sulfate, an endotoxin binding agent (10 μg/ml), was also included in the media to prevent lipopolysaccharide contamination .
Preparation of aqueous cigarette smoke extract
Research grade cigarettes (1R3F) were obtained from the Kentucky Tobacco Research and Development Center at the University of Kentucky, Lexington, KY, USA. The composition of 1R3F research grade cigarettes was: total particulate matter: 17.1 mg/cigarette, tar: 15 mg/cigarette and nicotine: 1.16 mg/cigarette. Cigarette smoke extract (10%) was prepared by bubbling smoke from one cigarette into 10 ml of culture media supplemented with 1% FBS at a rate of one cigarette/minute as described previously [9, 13], using a modification of the method described earlier by Carp and Janoff . The pH of the CSE was adjusted to 7.4, and was sterile filtered through a 0.45 μm filter (25 mm Acrodisc; Pall Corporation, Ann Arbor, MI). Cigarette smoke extract preparation was standardized by measuring the absorbance (OD 0.74 ± 0.05) at a wavelength of 320 nm. The pattern of absorbance (spectrogram) observed at λ320 showed a very little variation between different preparations of CSE. Cigarette smoke extract was freshly prepared for each experiment and diluted with culture media supplemented with 1% FBS immediately before use. Control medium was prepared by bubbling air through 10 ml of culture media supplemented with 1% FBS, and the pH was adjusted to 7.4, and sterile filtered as described above.
Epithelial cells (H1299, A549, H441, MLE-15 and L2) were seeded at a density of 1.5 million cells in 6-well plates containing culture media supplemented with 10% FBS in a final volume of 2 ml. The cells were grown to approximately 80–90% confluency, then changed to 1% FBS during the treatment. All treatments were performed in duplicate. The cells were treated with CSE (1.0–10%) for 24 hr at 37°C in a humidified atmosphere containing 5% CO2. 10 ng/ml tumor necrosis factor-α (TNF-α), was used as a positive control in selected experiments . After 24 hr treatment, cell supernatants were collected for LDH release and proinflammatory cytokines (interleukin-8 and interleukin-6) assays. Cell lysates were prepared for GSH and 4-HNE assays. Similarly, the epithelial cells were grown in 8-well chamber slides and treated with CSE (1.0–10%) for 24 hr and stained with a solution comprising of acridine orange and ethidium bromide dyes for apoptotic and necrotic studies.
Human SAEC were seeded in 12-well plates containing SAGM. After reaching 80% confluency, the cells were treated with either TNF-α (10 ng/ml) or CSE (0.2–1.0%); as higher doses (>1.0%) were cytotoxic to the cells. After the incubation period, the culture media was collected for LDH release and proinflammatory cytokines (IL-8 and IL-6) assay. Cell lysates were prepared for GSH, 4-HNE assays and western blotting for p65 protein. Primary cells were also grown in 8-well chamber slides, treated as described above, and were fixed with 4% paraformaldehyde for the detection of NF-κB nuclear translocation.
Cell toxicity was assessed by three separate methods: LDH release assay, trypan blue exclusion method and double staining with acridine orange and ethidium bromide.
Lactate dehydrogenase assay
LDH release, an indicator of membrane integrity and viability of alveolar epithelial cells, was measured in various treated samples, and compared with control (untreated) cultures using a commercially available LDH cytotoxicity assay kit (Roche Diagnostics, Indianapolis, USA). Following treatments, the culture medium was collected and centrifuged at 5000 rpm for 5 min prior to analysis. Assay was performed according to the manufacturer's instructions. LDH release was quantified by measuring the absorbance at 490 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). A 100% lysis control was prepared by adding 1% Triton-X-100 to control cell pellet to release all LDH. The absorbance value obtained was used for calculating percentage cytotoxicity.
Trypan blue exclusion assay
After 24 hr incubation, the culture medium was removed and replaced by 0.1% trypan blue solution in Ca2+/Mg2+-free phosphate buffered saline (PBS) for 3 min at room temperature. The cells stained blue were considered non-viable cells, whereas the cells that excluded the stain were considered viable.
Assay of apoptosis and necrosis
Morphological evidence of apoptosis and necrosis was obtained by means of acridine orange and ethidium bromide staining as described previously [16, 17]. In brief, after treatment, cells in 8-well chamber slides were stained with acridine orange (4 μg/ml) and ethidium bromide (4 μg/ml). Cells were examined by fluorescence microscopy (Olympus BX51 microscope, New Hyde Park, NY, USA), and photographed using a SPOT camera with SPOT RT software (Olympus). Acridine orange permeates throughout the cells and renders the nuclei green. Ethidium bromide is taken up by the cells only when cytoplasmic membrane integrity is lost, and stains the nuclei red. Viable (normal, green nuclei), early apoptotic (condensed, green nuclei), late apoptotic (condensed, red nuclei) and necrotic (normal, red nuclei) cells were quantified by counting a minimum of 100 cells in total in three independent experiments.
Measurement of intracellular 4-hydroxy-2-nonenal levels
4-HNE levels were measured in cell lysates by using lipid peroxidation assay kit (Calbiochem, San Diego, CA, USA). After the treatment period, cells were rinsed twice with ice-cold PBS and scraped off using cell scrapers (Sarsdet Inc. Newton, NC, USA). The pellet was resuspended in 200 μl of 20 mM Tris-HCl, pH 7.4, containing 5 mM butylated hydroxytoluene, and kept frozen at -70°C until assayed. To each sample, 650 μl of N-methyl-2-phenylindole and 150 μl of 15.4 M methanesulfonic acid were added. The reaction mixture was vortexed and incubated at 45°C for 60 min. After centrifugation at 15000 g for 10 min, the absorbance of the supernatant was determined at 586 nm. The levels of 4-HNE were determined from standard calibration curve constructed using 4-HNE diethylacetal in methanesulfonic acid. The values were expressed as μmol 4-HNE/mg protein.
Measurement of intracellular glutathione levels
Intracellular GSH levels in the cell extracts were measured by the 5,5'-dithiobis-2-nitrobenzoic acid DTNB-GSSG reductase recycling method described by Tietze  with slight modifications [8, 19, 20]. In brief, the cells were rinsed twice with ice-cold PBS, scraped off from the 6 well plate, suspended into 500 μl of ice-cold extraction buffer (0.1% Triton X-100 and 0.6% sulfosalicylic acid prepared in 0.1 M phosphate buffer with 5 mM EDTA, pH 7.5). The cells were vortexed for 20 seconds, followed by sonication (30 seconds) and centrifugation (2500 rpm for 5 min at 4°C). Twenty microlitres of the supernatant was added to 120 μl of 0.1 M phosphate buffer, 5 mM EDTA, pH 7.5, containing 100 μl of 5 mM DTNB and 0.5 units of glutathione reductase. Finally 60 μl of 2.4 mM NADPH was added and the rate of change in absorbance was measured for 1 min at 410 nm using a microplate reader (Bio-Rad, Hercules, CA, USA).
Protein levels were measured in the cell lysate supernatants in all samples using BCA kit (Pierce, Rockford, IL). Protein standards were obtained by diluting a stock solution of BSA. Linear regression was used to determine the actual protein concentration of each sample.
Proinflammatory cytokine assay
After treatment period, supernatants were removed and stored at -70°C. Pro-inflammatory cytokine (IL-8 and IL-6) levels were measured using an ELISA employing a biotin-streptavidin-peroxidase detection system with the respective duo antibody kits (R&D Systems) according to the manufacturer's instructions. Each sample was assayed in triplicate and the values were expressed as mean of three experiments.
Immunocytochemical analysis of NF-κB RelA/p65 localization
Activation of NF-κB in SAEC was assessed by immunocytochemical localization of RelA/p65 subunit of NF-κB. SAEC were seeded at 5000 cells/well in 8-well glass chamber slides and cultured overnight in SAGM at 37°C. Cells were then treated with CSE (1.0%) and TNF-α (10 ng/ml) as a positive control for 20 min. At the end of incubation, the cells were washed twice in PBS and fixed in 4% paraformaldehyde for 10 min at room temperature. The cells were permeabilized with 0.1% Triton X-100. The wash step was repeated and the cells were blocked with 10% normal goat serum for 1 hr. The cells were then incubated overnight in humidified chamber at 4°C, with rabbit polyclonal antibodies directed against the RelA/p65 subunit of NF-κB (Santa Cruz Biotechnology, USA), diluted at 1:200 in 1% goat serum in PBS. Furthermore, the cells were washed with PBS and incubated with FITC-labeled anti-rabbit IgG diluted 1:200 in 1% goat serum for 1 hr at room temperature in dark. After rinsing with PBS, the coverslips were mounted onto the slides and viewed under fluorescence microscope. Nuclear translocation of RelA/p65 was interpreted as a positive result from the fluorescence obtained.
Western blot analysis for NF-κB RelA/p65
Primary human SAEC were exposed to different concentrations of CSE (0.5 and 1.0%) for 1 hr. After treatment, the cells were washed with ice-cold PBS and resuspended in buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 0.5 mM PMSF). After 15 min of incubation, Nonidet P-40 was added and the samples were centrifuged to collect the supernatant containing cytosolic proteins. The pelleted nuclei were resuspended in buffer B (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF) and kept on ice. After 30 min of incubation, the cell lysates were centrifuged, and supernatants containing the nuclear proteins were collected. Twenty μg of isolated nuclear protein from each group was analyzed by SDS-PAGE and transferred onto nitrocellulose membrane (Amersham, Arlington Heights, IL, USA) using electro-blotting technique. The nitrocellulose membrane was blocked with 10% nonfat dry milk for 1 hr at room temperature, and subsequently incubated with rabbit polyclonal NF-κB RelA/p65 (1:1000) in 5% nonfat dry milk overnight at 4°C. After three washing steps of 15 min each, NF-κB RelA/p65 protein levels were detected using goat anti-rabbit antibody (1:20,000) linked to horseradish peroxidase (Dako, Santa Barbara, CA, USA), and bound complexes were detected using an enhanced chemiluminescence method.
Statistical analysis of significance was calculated using one-way Analysis of Variance (ANOVA) followed by Tukey's post-hoc test for multigroup comparisons using STATVIEW and Sigma plot statistical packages. The results were presented as the mean ± SEM of three independent experiments. *p < 0.05, #/**p < 0.01, and §/***p < 0.001.