Materials
HL-60 (CCL-240) and Porphyromonas gingivalis (33277) cells were purchased from ATCC (Manassas, VA, USA). Penicillin and streptomycin were purchased from Gibco Laboratories (Grand Island, NY, USA). Fetal bovine serum and Hema-3 staining kits were from Fisher Scientific (Nepean, ON, Canada). Bromophenol blue, CaCl2, cotinine, Escherichia coli K12 LPS, gelatin, gentamicin, Giemsa-Wright, metronidazole, NaCl, nicotine, nitroblue tetrazolium (NBT), paraformaldehyde, phorbol 12-myristate 13-acetate (PMA), RPMI 1640 medium, Triton X-100, and trypan blue were bought from Sigma-Aldrich (St. Louis, MO, USA). Glacial acetic acid, glycerol, and methanol were supplied by Fisher Scientific (Ottawa, ON, Canada). OsO4, Epon, lead citrate/uranyl acetate were bought from Electron Microscopy Sciences (Hatfield, PA, USA). Anti-α7 nicotinic acetylcholine receptor antibodies (AChRα7 [H-302]), HRP- or FITC-conjugated goat anti-rabbit IgG secondary antibodies, and α7 protein positive controls (mouse frontal brain extractions) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Nitrocellulose membranes were from BioRad Laboratories (Mississauga, Canada). Amersham Biosciences Corp (Piscataway, NJ, USA) enhanced chemiluminescence (ECL) western detection kits and x-ray films were used for visualization of western blots. PE-conjugated anti-CD11b (clone ICRF44) and Mouse IgG1, K (MOPC-21) isotype control antibodies were purchased from BD Biosciences Pharmingen (San Diego, CA, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively. Matrix metalloproteinase (MMP)-2, MMP-9, and Quantikine human MMP-9 (total) immunoassays were bought from R and D Systems (Minneapolis, MN, USA). Gifu anaerobic medium (GAM) broth was obtained from Nissui Pharmaceutical Co., Ltd, Tokyo, Japan.
Subjects
The study population has been previously described [45]. Briefly, twenty smokers (10 female/10 male; 33.8, s.d. 8.8 years) and twenty age (32.6, s.d. 9.1 years) and gender matched non-smokers were recruited from among the patients and staff of Guy's, King's and St. Thomas' Dental Institute and King's College London. Smokers were required to report consumption of ≥ 10 cigarettes per day, whereas age and gender-matched non-smokers were required not to have smoked for ≥ 3 years. A medical history was taken for each subject and those with diabetes, pregnancy, on anti-inflammatory drug regimens including aspirin and other NSAID's, and with any history of chronic inflammatory disease or leukocyte dysfunction were excluded. Ethical approval was granted by the local Health Research Ethics Board, and written, informed consent was obtained from each subject. Serum samples from each subject were stored at -80°C until required.
Growth of HL-60 cells
HL-60 (ATCC CCL-240) cells were cultured in RPMI 1640 medium with 0.3 g dm-3 L-glutamine and 2.0 g dm-3 sodium bicarbonate supplemented with 10% (v/v) heat-inactivated fetal calf serum, penicillin (50 U ml-1) and streptomycin (50 μg ml-1). The cells were cultured at 37°C in an atmosphere of 5% CO2 and 100% humidity.
Expression of nicotine receptors by HL-60 cells
Total cell extracts (40 μg of protein) were electrophoresed through 10% PAGE gels and transferred onto nitrocellulose membranes (0.45 μm). Membranes were blocked with 5% milk casein. A primary polyclonal antibody (AChRα7 [H-302], 1:200 dilution in 3% milk casein) recognizing amino acids 367–502 mapping to the C terminus of α-7 subunit of the human receptor. The secondary antibody (HRP-conjugated goat anti-rabbit IgG) was used at a dilution of 1:1000 in 3% milk casein. Binding sites were visualized by ECL and application to X-ray film. Quantitation was done using Quantiscan v3.0 (Biosoft, Cambridge, UK) with background set to correct as an interpolated minimum. α7AChRs were also visualized by fluorescent immunocytochemistry using primary antibody (AChRα7 [H-302]), a FITC-conjugated secondary anti-rabbit IgG antibody, an inverted fluoresence microscopic (Nikon Eclipse TS100 with Nikon Plan Fluor objectives and Omega Optical Fluor filter set) and an inverted confocal microscopic (Olympus IX70 with Nomarski DIC optics). Images were captured by InCytIm 1 software (Intracellular Imaging, Cincinnati, OH) and FluoView software (Olympus), respectively. Control cells were treated similarly, but without the primary antibodies.
Differentiation of HL-60 cells
Granulocytic differentiation was initiated by addition of 1.25% DMSO (final concentration) for 5 days with or without various doses of nicotine (10-7 to 10-4 M). Multiple aspects of neutrophil differentiation were monitored. Gross morphological examination of the HL-60 cells was performed microscopically following Hema-3 staining, with ultrastructure characteristics examined by electron microscopy. For electron microscopy, cells were fixed in 4% paraformaldehyde, postfixed in OsO4, dehydrated, embedded in Epon, cut into thin sections, stained with lead citrate/uranyl acetate and viewed on a Philips transmission CM-10 electron microscope (Philips Electronics, Eindhoven, Netherlands). The ability to reduce NBT (1.0 mg ml-1) after incubation with phorbol 12-myristate 13-acetate (PMA, 200 ng ml-1) for 30 mins, 37°C was determined by microscopically evaluating formazan deposition in 200 randomly selected cells. The percentage of cells in which formation of intracellular formazan deposits occurs increases with maturation [46]. Cell density was measured using a hemocytometer. Viability was determined by 0.4% trypan blue staining. Expression of the terminal differentiation marker CD11b was determined by flow cytometry, as described below. Finally, the re-distribution of key cellular components during differentiation was determined by infrared spectroscopy.
Expression of CD11b (integrin αM) by HL-60 cells
Cells were stained with PE-labeled anti-CD11b monoclonal antibody (ICRF44 at 20 μl per 1 million cells in 100 μl total staining volume). An isotype-matched antibody (Mouse IgG1) was used as a negative control. Data from 10000 cells were collected using an EPICS Altra-fluorescence-activated cell sorter (Beckton Dickinson, San Jose, CA, USA).
Conformational changes in differentiating HL-60 cells
FT-IR spectroscopy can monitor structural alterations to key cellular components, including membranes, proteins, and nucleic acids. Therefore, conformational changes in nicotine exposed differentiating HL-60 cells or unexposed cells were examined by FT-IR spectroscopy, as described previously [47]. Essentially, HL-60 cells were washed in 0.9% NaCl, loaded onto BaF2 windows and vacuum-dried (25 Torr). Infrared spectra were recorded using a Bio-Rad FTS-40A IR spectrometer (Bio-Rad Laboratories) at a nominal resolution of 2 cm-1. Two separate films from each subject sample were measured, each spectrum consisting of 256 co-added interferograms, apodized with a triangular smoothing function before Fourier transformation. All IR spectra were baseline corrected and area normalized between 900 – 1800 cm-1 using WIN-IR software (Bio-Rad Laboratories, Cambridge, MA) and analyzed using WIN-IR and in-house FT-IR spectroscopy software (Molecular Spectroscopy Group, National Research Council, Winnipeg, Canada). Alterations to DNA were determined by qualitative analysis of the DNA-specific spectral bands at 965 cm-1 (C-C/C-O stretching vibration), 1087 cm-1 (vsPO2- stretching vibrations), 1240 cm-1 (vasPO2- stretching vibrations), and 1713 cm-1(characteristic of base-paired DNA strands). Qualitative lipid changes were assessed by analysis of the spectral band at 1740 cm-1originating from lipid ester C = O group. Qualitative membrane protein changes were assessed by analysis of the spectral components in the amide I band arising from the amide C = O stretching vibration of the peptide groups in all proteins such as α-helix (1 657 cm-1), or parallel and antiparallel β-sheets (1640 and 1680 cm-1 respectively).
Apoptosis assays
The percentage of apoptotic cells in control and nicotine-treated (10-4M), differentiated (5 day), HL-60 cells was determined by propidium iodine (PI) DNA staining. Briefly, 3.0 × 106 cells were washed in PBS, 4°C, centrifuged (200 × g, 5 min, 4°C), and resuspended in 0.5 ml of cold PBS. Cells were fixed in ice cold 70% ethanol. Fixed cells were washed in PBS, centrifuged (300 × g, 5 min, 4°C) and resuspended in cold PBS with 100 U ml-1 RNase A and incubated at room temperature for 30 min. The cells were further incubated in 25 μg ml-1 PI (final concentration, 30 min, room temperature) in the dark. PI staining was analyzed by flow cytometery (EPICS Altra-Fluroscence-Activated Cell Sorter). The ability of nicotine to block camptothecin (10 μM)-induced apoptosis in differentiated (5 day) HL-60 cells was also examined.
Quantification of the oxidative burst
The ability of HL-60 cells, differentiated for 5 days in the presence or absence of nicotine or cotinine, to mount an oxidative burst in response to PMA was quantified colorimetrically. 2 × 106 cells were treated for 1 h with 0.5 mg ml-1 NBT and 200 ng ml-1 PMA (both final concentration), at 37°C and the reaction stopped by adding 100% acetic acid. After removal of supernatant (12000 × g, 2 mins, RT), any formazan in the cell pellet was extracted using 50% acetic acid and sonication. Cellular debris was pelleted by centrifugation (12 000 × g, 5 mins, RT) and reduced NBT quantified colorimetrically at 560 nm using a Bio-Rad model 550 microplate reader (Bio-Rad Laboratories).
Intracellular bacterial survival assay
Porphyromonas gingivalis cultures were grown anaerobically in Gifu anaerobic medium (GAM) broth in a Coy AALC anaerobic chamber (Coy Laboratory Products, Grass Lake, MI, USA). Treated (nicotine differentiated) and control 5-day, DMSO-differentiated HL-60 cells were incubated at 37°C with P. gingivalis for one hour (MOI = 10). Extracellular, non-adherent bacteria were removed by washing, while extracellular adherent bacteria were killed by addition of gentamicin (300 μg ml-1) and metronidazole (200 μg ml-1) for one hour. After washing, internalized bacteria were released by lysis of the mammalian cells in sterile distilled water for 20 min, a treatment that does not affect bacterial viability. Serial dilutions of the lysates were than plated on GAM and cultured anaerobically for CFU enumeration.
Matrix Metalloproteinase (MMP) secretion
Five-day DMSO-differentiated (with or without various doses of nicotine (10-7 to 10-4 M) were stimulated with 1.0 μg ml-1 (final concentration) purified LPS for 1 or 24 hr and cell-free supernatants collected. The importance of the α7 nAChR in nicotine-induced MMP-9 release was determined by using the α7 nAChR antagonist α-BTX (200 ng ml-1). MMP secretion was measured by gelatin zymography and densitometry. Briefly, 20 μl aliquots of cell-free supernatant (corresponding to 10000 cells) were mixed with 10 μl of zymogram sample buffer (62.5 mM Tris-HCl pH 6.8, 25% Glycerol, 4% SDS, and 0.01 % Bromophenol Blue) and run on 8.0% SDS-polyacrylamide gels complemented with 0.1% gelatin type I. SDS was removed from the gels by washing twice with 2.5% Triton X-100, 30 min, RT, allowing the separated proteins to renature. Subsequently, the gels were immersed into TCS buffer (50 mM Tris-HCl, pH 7.4, 0.2 M NaCl, 5 mM CaCl2) and incubated for 18–24 hr, 37°C then stained with 0.25% (w/v) Coomassie Blue, 60 min, RT. Active MMP-9 and MMP-2 were noted as clear bands against the blue background after destaining (30 mins, RT, in 10% glacial acetic acid and 30% methanol). MMP-9 and MMP-2 bands were quantified by densitometry, as described earlier.
Quantification of serum cotinine
Serum cotinine levels were determined by gas-liquid chromatography, as previously described [48]. Smokers can be reliably differentiated from non-smokers by serum cotinine concentration at an optimal cut-off point of 13.7 ng ml-1 [17]. Accordingly, smokers and non-smokers were required to present with serum cotinine levels of = 20 ng ml-1 and < 13.7 ng ml-1, respectively.
Circulating MMP-9 in smokers and non-smokers
The circulating concentrations of total MMP-9 were determined in smokers and non-smokers by Quantikine ELISA, according to the manufacturer's instructions.
Statistical analyses
Where appropriate, statistical analysis was done by post hoc application of Duncan's New Multiple Range Test, assuming a significant difference at p < 0.05. Regression lines were fitted to the raw data using SigmaPlot v7.101 (SPSS Incorporated, Chicago, ILL). Non-parametric tests (Mann-Whitney U) were required for analyses of in vivo cotinine and MMP-9 concentrations.
