! *&!
2. Materials and Methods
2.1 Monocyte isolation from peripheral blood
Human monocytes were obtained from peripheral blood buffy coats of healthy donors (n=3) by two-step gradient density centrifugation with Ficoll-Paque PLUS (GE Healthcare, Bio-Sciences AB, Uppsala, Sweden) and then separated using Monocyte Isolation kit II (Miltenyi Biotec, Bergisch-Gladbach, Germany) according to the manufacturer’s protocol.
The purity of isolated cells (> 98%) was determined microscopically after cytocentrifugation and staining with a modified Wright Giemsa dye (Diff Quick®, Medion Diagnostics AG, Düdingen, Switzerland). Viability was determined by trypan blue dye exclusion. Monocytes were also analyzed for cell surface CD14 and CD16 antigen expression by flow cytometry (FACScan, Becton Dickinson, Rutherford, NJ, USA). The CD14dimCD16+ subset in the purified monocytes was about 8%, the same as in the blood before purification.
2.2 Culture condition and monocyte in vitro activation
Monocytes were cultured at a density of 5x106 cells/well in 6-well culture plates (Corning Incorporated, Costar®, NY, USA) in 2 ml of RPMI 1640+Glutamax-I Medium (GIBCO® by Life Technologies, Paisley, UK) supplemented with 50 µg/ml Gentamicin (GIBCO®), 5% heat-inactivated human AB serum (Sigma-Aldrich Inc., St. Louis, MO, USA) in moist air with 5% CO2. During culture, monocytes were
sequentially exposed to inflammatory and anti-inflammatory stimuli at different time points to mimic the micro-environmental conditions of an inflamed tissue. All synthetic nucleic acids were obtained from InvivoGen (San Diego, CA, USA), IFNs and human recombinant cytokines were obtained from R&D Systems (Minneapolis, MN, USA), LPS (from E.coli serotype 055:B5) was from Sigma-Aldrich Inc., while adenovirus type 5 was kindly provided by Davide Lucchesi.
Briefly, the in vitro stimulation was performed as follows: at time 0 monocytes were exposed to CCL2 (20 ng/ml) at 37°C. After 2 h, CCL2 was removed, cells were washed with PBS, fresh medium was added containing viral inflammatory stimulus, i.e. a mixture of synthetic polyuridines (500 ng/ml) (CL097), CpG motifs (1 µM) (ODN 2395), polyI:C (50 ng/ml) (Poly(I:C)LyoVec) and human recombinant adenovirus serotype 5, AdV5 (50 MOI). The temperature was increased to 39°C. IFN-" (10000 U/ml) and IFN-! (1000 U/ml) were added at time 3 h and IFN-# (25 ng/ml) at 7 h. Temperature was maintained at 39°C. At time 14 h, all inflammatory stimuli were removed, monocytes were washed with PBS, fresh medium was added
! *'!
containing IL-10 (20 ng/ml), and temperature brought back to 37°C until the end of experiment. At time 24 h, medium containing IL-10 was removed, fresh medium containing TGF-! (10 ng/ml) was added, and the culture prolonged until 48 h.
In the bacterial model after 2 h fresh medium was added containing LPS (5 ng/ml), while TNF-" (10 ng/ml) was added at time 3 h.
Freshly isolated monocytes were taken as time 0. Cells were harvested in 700 µl of Qiazol (Qiagen, Hilden, Germany) at times 0, 4, 8, 14, and 24 h. Supernatants were collected at times 0, 4, 14, 24, 48 h. Samples were stored at -80°C until analysis.
2.3 Evaluation of gene expression by qPCR
For evaluation of gene expression by qPCR, cDNAs were reverse transcribed from total RNA (100 ng) according to the QuantiTect Reverse Transcription Kit (Qiagen) instructions, with oligo-dT and random primers, to allow for high cDNA yield. Three separate reverse transcriptions were performed for each samples and an identical reaction without the reverse transcriptase was run, as negative control. Taqman polymerase chain reaction was performed by a Rotor-Gene™ 3000 (Corbett Research, Doncaster Victoria, Australia), using the QuantiTect SYBR Green PCR master Mix (Qiagen). The final reaction contained 12.5 µl 2x QuantiTect SYBR Green PCR Master Mix, 0.3 µM of each primer and 2.5 µl of cDNA in a total volume of 25 µl. PCR conditions were 95°C for 15 min, followed by 45 cycles of 95°C for 15 s, 50-60°C for 30 s and 72°C for 30 s. Primers were supplied by PrimerDesign (PrimerDesign Ltd, Southampton, United Kingdom)(IL1B and IL-18), and by Qiagen as pre-made solutions, both for target (IL1RN, IL1R2 and IL18BP) and housekeeping (HPRT, ACTB) genes. Relative gene expression was calculated using the efficiency correction method, which calculates the relative expression ratio from the qPCR efficiencies and the Ct between the target gene and the endogenous control, relative to a calibrator sample (0 h) [93].
2.4 Protein detection by ELISA
IL-1!, IL-1Ra, sIL-1RII, IL-18, IL-18BP, IL-6, TNF-", CXCL8 and CCL5 proteins were measured on supernatants collected at different stimulation times (4, 14, 24 and 48 h), by enzyme-linked immunosorbent assay (ELISA). ELISA kits for IL-1!, IL-1Ra, sIL-1RII, IL-18BP, IL-6, TNF-", CXCL8 and CCL5, were purchased from R&D Systems, while the kit for IL-18 was obtained from MBL (Nagoya Aichi, Japan). ELISA assays were performed according to the manufacturers’ instructions.
! *(!
Each sample was assayed in duplicate, and detection carried out with a JUPITER microplate spectrophotometer (Asys Hitech GmbH, Eugendorf, Austria).
After measuring the concentration of both IL-18 and IL-18BP in each sample, the law of mass action was used to calculate free-IL-18 (i.e., the fraction of cytokine not bound to its inhibitor IL-18BP) as previously described [94, 95]. Briefly, the calculation was based on 1:1 stoichiometry in the complex IL-18 and IL-18BP, a molecular weight of 18.4 kDa for IL-18 and 17.6 kDa for IL-18BP, and a dissociation costant (Kd) of 0.4 nM [70].
2.5 Statistical data analysis
The qPCR and ELISA results are expressed as mean values ± SEM. Differences between groups were analyzed using ANOVA and Fisher’s test. A P value of less than 0.05 was considered to be statistically significant.
