CpG-A (ODN 2216) was purchased from InvivoGen (Toulouse, France), and CpG-C from HyCult Biotechnology (Uden, the Netherlands). RPMI 1640 and FCS were obtained from Gibco-BRL Life Technologies (Breda, Netherlands). LY294002, as PI3-K α inhibitor was purchased from Calbiochem (VWR International B.V. Amsterdam, The Netherlands).
Preparation of CSE
Cigarette smoke extract (CSE) was prepared as described before. CSE was generated by the burning of commercially available Lucky Strike cigarettes without filter (British-American Tobacco, Groningen, The Netherlands), using the TE-10z smoking machine (Teague Enterprises, Davis, CA), which is programmed to smoke cigarettes according to the Federal Trade Commission protocol (35-ml puff volume drawn for 2 s, once per minute . Briefly, this machine was used to direct main and side stream smoke from one cigarette through 5 ml PBS. Hereafter, absorbance was measured spectrophotometrically and the buffer was standardized to a standard curve of CSE concentration against absorbance at 320 nm. The pH of the resultant extract was titrated to pH 7.4, and diluted with medium. This solution is considered to be 100% CSM. Solutions ranging from 0.75% to 1.5% were used in the present study following preliminary experiments, which indicated that these were nontoxic concentrations (viability ≥ 96%).
Isolation of human plasmacytoid dendritic cells (pDCs)
Trisodium citrate (0.4% (w/v) (pH 7.4)) anti-coagulated fresh blood was obtained from healthy volunteers at the donor service of the University Medical Center (Utrecht, the Netherlands). Peripheral blood mononuclear cells (PBMC) were collected after centrifugation over isotonic Ficoll (Pharmacia, Uppsala, Sweden), and plasmacytoid dendritic cells were isolated from PBMCs by Diamond Plasmacytoid Dendritic Cell Isolation Kit by using CD304 MicroBeads (Magnetic cell sorting/MACS/; Miltenyi Biotech, Utrecht, the Netherlands). The purity was higher than 95% assessed by the flow cytometric analysis of CD123 expression (BD Biosciences, San Jose, CA, USA) in a pilot study. Cells were cultured in RPMI 1640 supplemented with 10% FCS.
Activation of pDCs
Human pDCs were cultured with various stimuli at 37°C under a humidified atmosphere with 5% CO2 in a V-bottom 96-well plate for 5 h for real-time PCR analysis, 8 h for assessing cytokine production (106/ml) and 90 min in the p-Akt assay (80,000 cells/well). Cells were stimulated with CSE (1.5%) alone and, in order to test the effect of CSE on activated pDCs, cells were simultaneously activated with TLR9 (2 μM CpG-A or 1 μM CpG-C) agonists. For the real time PCR analysis, cells were spun for 5 min at 1200 rpm at 4°C, washed once in cold PBS, the reverse transcription protocol was immediately, and cDNA was stored at -20°C till the real-time PCR reaction. For cytokine and chemokine measurements, supernatants were collected by spinning for 5 min at 1200 rpm at 4°C and stored at -20°C till analysis.
Assessing cell viability
Cell viability was assessed on fresh cells at the end of each treatment with propidium idodide (PI, Sigma-Alderich) staining. 10,000 events were counted in the cell gate by FACS (FACS Calibur, BD Bioscience, Becton Dickinson).
Measurement of cytokines
IFN-α was measured by sandwich ELISA (PBL Biomedical Laboratories, Piscataway, NY, USA) while IL-6, IL-8, TNF-α, IL-10 and IL-1β concentrations were measured with the Cytometric Bead Array Human Inflammation Kit (BD Bioscience) on a flow cytometer (FACS Calibur).
cDNA synthesis and Q-PCR were performed using the Superscript III Platinum CellsDirect Two-step qRT-PCR Kit with SYBR Green (Invitrogen, Toulouse, France) according to the manufacturer's instructions. Briefly, after incubation cells were washed once and resuspended in 10 μl cold PBS and 2 μl (i.e. 10,000 cells) was lysed in 11 μl lysis buffer. Then the cell lysate was treated with DNase I to degrade any contaminating DNA. During first strand cDNA synthesis, the reaction mix contained 2 μl RT Enzyme Mix (100 units/μl SuperScript III RT, 20 units/μl RNaseOUT Recombinant Ribonuclease Inhibitor), 20 μl 2× RT Reaction mix (2.5 μM oligo(dT)20, 2.5 ng/μl random hexamer, 10 mM MgCl2 and dNTPs) in 40 μl reaction volume. The reaction was allowed to proceed (incubation: 25°C, 10 min, RT: 50°C, 20 min; PCT-100 Programmable Thermal Controller, MJ Research Inc., Watertown, MA, USA), and then it was halted by heating the samples to 85°C for 5 min. Afterwards 1 μl of RNase H (2 U/μl) was added to each well and incubated at 37°C for 20 min. The reaction was chilled on ice.
Primers for Q-PCR were synthesized by Invitrogen and were as follows: β2-microglobulin (forward, 5'-CTCCGTGGCCTTAGCTGTG-3'; reverse, 5'-TTTGGAGTACGCTGGATAGCCT-3'), IL-8 (forward 5'-CTGGCCGTGGCTCTCTTG-3'; reverse 5'-CCTTGGCAAAACTGCACCTT-3'), IFN-α (forward 5'-GGTGCTCAGCTGCAAGTCAA-3'; reverse, 5'-GCTACCCAGGCTGTGGGTT-3'). The Q-PCR reactions were performed in 25 μl reaction volume with 4 μl cDNA, 12.5 μl Platinum SYBR Green qPCR SuperMix-UDG and 200 nM sense and antisense primers. Cycling conditions were 50°C for 2 minutes, 95°C for 2 minutes and then 50 cycles of 95°C for 15 seconds and 60°C for 30 seconds (ABI PRISM 7000 Sequence Detection System, Applied Biosystems, Foster City, CA, USA). PCR amplicon efficiency for primers was calculated using serially diluted pooled cDNA. The relative level of mRNA expression of a specific gene was calculated based on ΔΔCT method, and normalized to mRNA level for the housekeeping gene β2-microglobulin.
Measurement of Nitric Oxide
As an indicator of NO production, nitrite concentration was measured in the supernatants of samples. Equal volumes of the sample and Griess reagent (1% sulphanilamide in 5% phosphoric acid and 0.1% N-[1-naphthyl]-ethylendiamine) were mixed and the absorbance was read at 450 nm as described before . The amount of nitrite was obtained by an extrapolation from a standard curve with sodium nitrite and was expressed as μmol/mg tissue.
Detection of p-Akt by flow cytometry
The method was adopted from Guiducci et al . 8 × 104 pDCs were pretreated with LY294002 (10 μM) and then stimulated with 1 μM CpG-C, CSE or the combination of the two stimulants for 90 min in a V-bottom 96-well plate. Then cells were fixed with 4% paraformaldehyde for 15 min at 37°C. Cells were then washed with incubation buffer (1% FCS in PBS), and permeabilized with 90% ice-cold methanol for 30 min on ice. After washing cells were blocked for 10 min in incubation buffer, and then stained with Alexa Fluor 647 anti-human AKT (pS473) or with Alexa 647 isotype control antibodies according to the manufacturer's instructions. All antibodies were purchased from Cell Signaling Technologies (Bioke, Leiden, the Netherlands).
For assessing the effect of CSE on IFN-α production induced by different stimulants, unpaired t-test was used. In other comparisons, one-way ANOVA was applied with the Newman-Keuls Multiple Comparison post-hoc test (GraphPad Prism 4.0; GraphPad Software Inc., San Diego, California, USA). Data are expressed as mean ± SEM, p < 0.05 was considered to be significant.