Alveolar macrophages (AM) are known to play an essential role in lung defense through their ability to remove the foreign matters reaching the lung alveoli. Cigarette smoke (CS) is a critical risk factor for many lung diseases. CS is inhaled into the lung by respiretion and affects AM. It has been previously reported that CS induces inhibition of cytokine production, cell surface receptor expression and antigen presentation in AM. However, the relationship of immune suppression and DNA damage caused by CS in AM is still unclear. Therefore, in this study, we investigated AM immune function and DNA damage in CS-exposed mice. Mice were exposed to CS of 20 cigarettes/day during 10 days using a HambrugⅡsmoking machine. After exposure, AM were obtained by bronchoalveolar lavage. The number of AM was significantly increased in CS-exposed mice compared with non-CS-exposed mice. Phagocytic activity of AM was significantly inhibited by CS exposure. Percentage of CD11b-, CD14-, Toll-like receptor (TLR)2- or TLR4-positive cells was significantly decreased in CS-exposed mice compared with non-CS-exposed mice. Interleukin-1β mRNA expression in lipopolysaccharide-stimulated AM was significantly inhibited by CS exposure. Intracellular reactive oxygen species (ROS) (, H2O2) production of AM was significantly increased, and DNA damage was induced by CS exposure. These results suggest that impaired immune functions by CS exposure may be related to DNA damage via excessive ROS induced by CS. These alterations of AM caused by CS could be associated with infection and development of pulmonary diseases.
Alveolar macrophages (AM) are a main population of the cells in alveolar space and are constantly exposed to inhaled foreign materials and microorganisms [
Cigarette smoke (CS) is well known to be a critical risk factor for many lung diseases including chronic obstructive pulmonary disease (COPD). CS contains more than 7,000 chemicals and components, with many of them are toxic, carcinogenic and mutagenic chemicals, as well as free radicals [6,7]. CS enters the lung through the airway, and would directly contact with AM and impact them.
Previously, there have been some reports that CS impaired AM immune functions such as phagocytosis, antigen presentation and production of inflammatory cytokines [8-11]. In addition, it has been reported that CS increases ROS production in AM [12,13], but few studies regarding DNA damage in CS-exposed AM have been demonstrated [14,15]. The relationship of immune suppression, ROS production and DNA damage caused by CS in AM is still unclear. Therefore, we investigated phagocytic activity, cell surface receptor expression, IL- 1β mRNA expression, ROS production and DNA damage of AM in CS-exposed mice.
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 [
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.
The number of AM was significantly (p < 0.001) increased in CS-exposed mice ((4.36 ± 0.13) × 105 cells/ mouse) compared with non-CS-exposed mice ((2.81 ± 0.14) × 105 cells/mouse) (
Phagocytic activity of AM was assessed by percentage of AM ingesting fluorescent beads. The percentage of AM ingesting fluorescent beads in non-CS-exposed mice was 77.08% ± 2.54% while that in CS-exposed mice was 60.56% ± 2.96% (