Eight-week-old female C57BL/6N mice were purchased from Japan SLC (Shizuoka, Japan). Mice were housed in transparent plastic cages with stainless wire lids in the animal facility of Kyoto Sangyo University (Kyoto, Japan) and maintained under standard conditions with the dark cycle from 8 pm to 8 am. Water and food were provided ad libitum before, during and after exposure. Mice were used between 8 and 10 weeks of age. This study was approved by the Kyoto Sangyo University Committee for Animal Care and Welfare.
Mice were exposed to main stream smoke from 20 filtertipped cigarettes (Reference Cigarette: CORESTA APPROVED MONITOR No.6) per day during 10 days using a Hamburg II smoking machine (Borgwaldt KC, Hamburg, Germany). CS was diluted with air at a ratio of 7:3, and the puff volume was 35 ml/2 sec/1 puff. NonCS-exposed mice were treated under identical conditions as the CS-exposed mice, except for the CS exposure.
BAL was performed at the day after the last CS exposure. Mice were sacrificed under anesthetic. Each lung was washed 5 times with 1 ml phosphate-buffered saline (PBS; Nissui Pharmaceutical, Tokyo, Japan), and the BAL fluid (BALF) was collected. Recovered cells in BALF were separated by centrifugation (220 × g, 10 min, 4˚C) and resuspended in culture medium RPMI 1640 (Nacalai tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS; Nichirei Bioscience, Tokyo, Japan), 50 mM l-glutamine (Nacalai tesque), 100 µg/ml streptomycin (Meiji Seika, Tokyo, Japan) and 100 U/ml penicillin (Meiji Seika). The number and viability of recovered cells were determined by 0.2% trypan blue exclusion test, and the viability was more than 98%. The purity of AM separated from the BALF was found to be more than 98% by morphology and nonspecific esterase staining.
AM (5 × 104 cells/100 μl) were mixed with 100 µl of 0.025% Fluoresbrite™ Carboxylate YG 1.0 micron Micropheres (Polysciences, PA, USA) and cultured at 37˚C under the presence of 5% CO2 for 2 hours. After 2 hours, AM were centrifuged at 220 × g for 10 minutes and resuspended in 300 μl of FACS buffer [PBS containing 100 μg/ml CaCl2 (Nacalai tesque), 100 μg/ml MgCl2 (Nacalai tesque), 0.1% sodium azide (Nacalai tesque) and 1% FBS]. Percentage of cells ingesting fluorescent beads was analyzed by BD FACSCalibur™ (BD Biosciences, CA, USA).
AM (5 × 104 cells) were resuspended in 100 μl FACS buffer and stained with 0.5 μg of fluorescein isothiocyanate (FITC)-anti-CD11b, FITC-anti-CD16 (BD Biosciences), FITC-anti-Toll-like receptor (TLR)2 (e-Bioscience, CA, USA), phycoerythrin (PE)-anti-CD14 (BD Biosciences) or PE-anti-TLR4 (e-Bioscience) monoclonal antibodies at 4˚C for 45 minutes. After incubation, AM were washed twice and resuspended in 300 μl of FACS buffer. Percentage of surface antigen-positive cells was analyzed by BD FACSCalibur™.
Messenger RNA expression levels of IL-1β and β-actin (as a house keeping gene) were examined. AM (5 × 104 cells/well) were stimulated with 10 μg/ml lipopolysaccharide (LPS) in 96-well microplates at 37˚C in a 5% CO2 humidified atmosphere. After 24 h stimulation, total cellular RNA was extracted by the acid guanidinium thiocyanate-phenol-chloroform method. The extracted total RNA was transcribed to cDNA with murine leukemia virus reverse transcriptase (Invitrogen, CA, USA). PCR amplification was performed with Go-Taq® Green Master Mix (Promega, WI, USA) and following primer pairs: IL-1β sense (5’-AGCTACCTGTGTCTTTCCCG-3’) and IL-1β antisense (5’-GTCGTTGCTTGGTTCTCCTT-3’), β-actin sense (5’-GCATTGTTACCAACTGGGAC-3’) and β- actin antisense (5’-TCTCCGGAGTCCAT CACAAT-3’). PCR products were run on an 8% polyacrylamide gel, stained with ethidium bromide, and the density of each band was measured with Scion Image software (Scion, MD, USA). Expression ratio (IL-1β/β-actin) was used to evaluate relative gene expression.
Cellular oxidative stress was assessed by monitoring the oxidation of intracellular 2’, 7’-dichlorofluorescein diacetate (DCFH-DA) or hydroethidine (HE) as previously described [
production was analyzed by FACS Calibur™.
Evaluation of DNA damage was performed using the CometAssay™ kit (Trevigen, MD, USA) according to the manufacture’s protocol. Briefly, AM were mixed with the molten agar at 42˚C, and the mixture was spread onto CometSlide™. The slides were immersed in pre-chilled Lysis Solution (Trevigen) for 1 h at 4˚C and then immersed in alkaline solution (contains 1.2% NaOH in 1 mM EDTA) for 30 min at room temperature. After washing the slides with 1 × TBE (89 mM Tris-borate, 2 mM EDTA, pH 8.0) electrophoresis buffer, electrophoresis was carried out under neutral conditions for 10 min at 9 mA. The samples were air dried, fixed with 70% ethanol and stained with SYBR® Green I (Molecular Probes). The slides were observed under fluorescence microscopy (Olympus, Tokyo, Japan) with excitation at 494 nm and emission at 521 nm. Comet images were analyzed using the Comet Analyzer software (Youworks Co., Tokyo, Japan). Tail moment and tail length as a parameter for extent of DNA damage and fragment size of DNA strand, respectively, were used for evaluation of DNA damage.
Data are represented as means ± standard error (SE). Comparisons between non-CS-exposed mice and CSexposed mice were made by Student’s t test. Differences were considered significant when the P-value was <0.05.