Patients
This clinical study was approved by the University of Hiroshima Institutional Review Board, and written permission was obtained from all patients who participated in this study. Peripheral blood was taken from 6 RA patients (67.3 ± 8.02 years of age, mean ± standard deviation (SD)) and 6 OA patients (75.3 ± 5.0 years of age, mean ± SD). They were diagnosed according to the American Rheumatism Association Criteria for RA or OA[
28]. Synovial tissue specimens were obtained at the time of total knee arthroplasty on 4 RA patients and two RA patients (RA8,RA9) were obtained at the time of synovectomy (67.7 ± 10.4 years of age, mean ± SD). All OA synovial tissue samples were obtained by total knee arthroplasty (74 ± 5.1 years of age, mean ± SD). Patient RA3 and RA5 showed more erosive disease, with severe destruction in the large joints. They underwent total knee arthroplasty before, and they were subsequently well controlled. The disease in patients RA1, RA2 and RA4 were poorly controlled, but soft tissue swelling, juxta-articular osteopenia, and loss of joint space were observed. Patient RA6, RA7, RA10 and RA11 showed more erosive disease, with severe destruction in the large joints. RA8 and RA9 had the least erosive disease. In addition, 6 patients with knee osteoarthritis (OA) diagnosed according to typical clinical features. The demographics of the RA and OA patients are listed in Table .
| Table 1Demographic and clinical features of this study subjects. |
Cell isolation and expansion of IL-17 producing T cells
This clinical study was approved by the University of Hiroshima Institutional Review Board, and written permission was obtained from all healthy volunteers who participated in this study. Human peripheral blood was collected from 5 healthy volunteers (31.8 ± 1.1 years of age, mean ± SD) into DPBS-E (5 mM: 0.5 M EDTA) drop by drop and mixed well. This mixture was loaded slowly onto Histopaque
® (Sigma Chemical Co. CA) in another tube, and centrifuged at 1000 × g at room temperature for 10 min. PBMC accumulated as the middle white monolayer. After the supernatant was discarded, only the white monolayer cells were aspirated and put into DPBS-E, then centrifuged at 400 × g at 4°C for 10 min. The supernatant was discarded, DPBS-E and ammonium chloride (The cell Experts™) were added at the rate of 1:3, and the mixture was allowed to stand at room temperature for 10 min. After centrifuging at 400 × g at 4°C for 10 min, supernatant was discarded, and DPBS-E was added and mixed well. This final process was repeated several times. The remaining white cells were peripheral blood mononuclear cells (PBMC). CD4+ T cells were isolated from PBMC using
auto MACS (CD4+T cell Isolation kit, Miltenyi Biotec,). IL-17 producing cells were expanded from CD4+ T cells as previously described [
29]. PBMC or purified CD4+ T cells (1 × 10
6 per ml) were re-suspend in fresh culture medium containing requisite antibiotics and serum plus IL-2 (100 u/ml), IL -1 β, IL-2, IL -6 and IL -23 (R&D Systems; 10ng/ml). The cells were cultured in 24 well plates (2 ml per well) at 37°C for 4 days in 5% CO
2. Afterwards, IL-17 expression was confirmed with RT-PCR and ELISA, and the presence of IL-17 producing T cells was also confirmed (data is not shown).
Tissue samples
Three synovial tissue specimens per one patient were obtained from random sites during surgery. Each was visually inspected to minimize contamination with non-inflammatory tissue. Tissues were stored at -70°C until analysis. Total RNA for the PCR analysis was isolated from tissues homogenized with Trizol (Invitrogen) on ice for. The tissue specimens were fixed in 4% paraformaldehyde and paraffin-embedded for histopathological analysis.
Microarray analysis
miRNA microarrays (NCode microarray, Invitrogen) were performed for the identification of miRNA in differentiating IL-17 producing cells. The dual-color dye swap method was used to analyze differences of miRNA expression between expanded IL-17 producing cells and non-expanded cells. Five-hundred nanograms of the enriched miRNA was labeled with the NCode Rapid miRNA labeling kit (Invitrogen) and hybridized to NCode multispecies miRNA arrays as described earlier based on a loop design, balanced with respect to array, dye, and sample. The balanced design minimized sources of unwanted variation. The arrays were scanned, aligned, and median spot intensities were obtained using a GenePix 4000B scanner (Molecular Devices, Inc., Sunnyvale, CA, USA).
Synthesis of complementary DNA
One microgram of total RNA was reverse-transcribed using the QuantiTect® Reverse Transcription Kit (Qiagen, Chatsworth, CA) according to the manufacturer's protocol. The genomic DNA elimination reaction was carried out using 2 μl of gDNA wipeout buffer, 1 μg (1 μl) template RNA and 11 μl RNase-free water at 42°C for 2 min. Reverse transcription was performed in 1 μl quantiscript reverse transcriptase, 4 μl quantiscript RT buffer, 1 μl RT primer mix and 14 μl template RNA (the entire genomic DNA elimination reaction) at 42°C for 15 min and 95°C for 3 min and then the cDNA product was maintained at 4°C.
Quantitative (real time) PCR
Quantitative RT-PCR assays were performed using a TaqMan miRNA assay kit (Applied Biosystems, CA, USA) for the expression of miRNAs and SYBR Green (Invitrogen) for the expression of IL-17, Foxp3, retinoid-related orphan receptor γt (RORγt), IL-1 receptor-associated kinase 1(IRAK1) and suppressor of cytokine signaling 1(SOCS1). Reverse transcriptase reactions of mature miRNAs contained a sample of total RNA, 50 nM stem-loop RT primer, 10 × RT buffer, 100 mM each dNTPs, 50 U/μl MultiScribe reverse transcriptase, and 20 U/μl RNase inhibitor. 15 μl reactions were incubated in a thermo cycler (BioRad) for 30 min at 16°C, 30 min at 42°C, 5 min at 85°C, and held at 4°C. Real time PCR was performed using a Mini Opticon Real-time PCR System (BioRad, Hercules, CA) in a 10 μl PCR mixture containing 1.33 μl RT product, 2 × TaqMan Universal PCR Master Mix, 0.2 μM TaqMan probe, 15 μM forward primer, and 0.7 μM reverse primer. Each SYBR Green reaction was performed with 1.0 μl template cDNA, 10 μl SYBR Green mix, 1.5 μM primer, and water to adjust the final volume to 20 μl. Primer sequences were: IL-17, 5'- AAG ACC TCA TTG GTG TCA CTG CT AC-3'(forward), 5'- ATC TCT CAG GGT CCT CAT TGC G-3' (reverse); Foxp3, 5'- GAG AAG CTG AGT GCC ATG CA -3'(forward), 5'- AGA GCC CTT GTC GGA TGA T -3'(reverse); RORγt, 5'- TGA GAA GGA CAG GGA GCC AA-3'(forward), 5'- CCA CAG ATT TTG CAA GGG ATC A -3' (reverse); SOCS1, 5'- GAA CTG CTT TTT CGC CCT TA -3'(forward), 5'- CTC GAA GAG GCA GTC GAA G -3'(reverse); IRAK1, 5'- GCT CTT TGC CCA TCT CTT TG -3'(forward), 5'- GCT ACC ACG CCA GGC TAA TA -3'(reverse); GAPDH, 5'- AAG AAT TGC AAG TCT ACA TAT CAC CCA AG. -3'(forward), 5'- GGT CAT GGT CAC AGA GCC ACC-3'(reverse). All reactions were incubated in a 48 well plate at 95°C for 10 min, followed by 40 cycles of 95°C for 15 seconds, and 60°C for 1 min and performed in triplicate. The U18 or GAPDH gene was used as a control to normalize any differences in the total RNA levels in each sample. A threshold cycle (CT) was observed in the exponential phase of amplification, and quantification of relative expression levels was performed using standard curves for target genes and the endogenous control. Geometric means were used to calculate the ΔΔCT (delta-delta CT) values and expressed as 2-ΔΔCT. The value of each control sample was set at 1 and was used to calculate the fold-change of target genes.
Western Blotting
Ten μg of the protein were separated on NuPAGE® Novex® Bis-Tris Mini Gels (Invitrogen, Carlsband, CA) and transferred onto a nitrocellulose membrane (Invitrogen, Carlsband, CA). Mouse monoclonal antibody against a partial recombinant IRAK1 (Abnova, Taiwan) and rabbit polyclonal anti-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) were used as primary antibodies. Anti-mouse goat IgG (MP Biomedicals, LLC, Santa Ana, CA) for IRAK1 and anti-rabbit goat IgG (MP Biomedicals, LLC, Santa Ana, CA) for actin were used for secondary antibodies. Band detection was performed using the enhanced chemiluminescence reagent, ECL Western Blotting Detection Reagents (GE Healthcare UK Ltd, Little Chalfont, Buckinghamshire).
Immunohistochemistry
Paraffin sections were deparaffinized through xylene for three changes of five minutes each, followed by graded alcohol immersions to water and phosphate buffered saline solutions, the sections were treated with the retrieval solutions (DAKO, Dakocytomation Inc.,Copinteria,CA,USA) for 20 minutes at 95°C. Next, the sections were depleted of endogenous peroxidase by incubation with 0.3% H2O2 in absolute methanol for 15 minutes. After blocking nonspecific biding with blocking reagent for 30 minutes, the sections incubated with primary antibody at appropriate dilutions for overnight at 4°C. For primary antibodies, monoclonal rabbit anti-human antibody against IL-17 (Santa Cruz Biotechnology,Inc,CA,USA). The sections were washed and incubated with biotinylated goat anti-mouse (Sigma, Saint Louis, Missouri, USA) for 1 hour at room temperature, then, washed and incubated with avidin-biotinylated horseradish peroxidase complex (ABC) and diaminobenzidine tetrahydrochloride (DAB; Dakocytomation Inc.,Copinteria,CA), and counterstained with Mayer's hematoxylin. The negative control was prepared in the same manner except that the primary antibody was omitted.
Double staining combining in situ hybridization and immunohistochemistry
After deparaffinization, each section was fixed in 4% paraformaldehyde for 10 minutes at room temperature, washed 3 times in phosphate buffered saline (PBS) for 3 minutes, and subsequently treated with 600 g of proteinase K for 10 minutes at room temperature. After treatment in 0.2% glycine-PBS for 10 minutes, the sections were refixed in 4% paraformaldehyde for 10 minutes, washed 3 times in PBS for 3 minutes each, and acetylated with 0.25% acetic anhydride in 0.1M triethanolamine hydrochloride for 10 minutes. After washing in PBS for 30 minutes, sections were prehybridized for 1 hour at 65°C with prehybridization buffer (50% formamide and 5 saline-sodium citrate [SSC]). Hybridization with DIG-labeled riboprobes of miR-146a (B-Bridge International, Mountain View, CA) was performed overnight at 65°C in hybridization buffer (50% formamide, 5X SSC, 5X Denhardt's solution, and 250 g/ml of Baker's yeast transfer RNA). After hybridization, sections were washed in 5X SSC for 30 minutes at 65°C, 0.2 SSC for 2 hours at 65°C, and 0.2X SSC for 5 minutes at room temperature. Blocking was performed overnight at 4°C with 4% horse serum and alkaline phosphatase-conjugated Fab anti-DIG antibody (Roche) in 1% sheep serum. Staining was performed using BCIP and nitroblue tetrazolium (NBT; Roche). Thereafter, the sections were washed in PBS and then were treated for 20 minutes at 90°C with retrieval solutions (Dakocytomation Inc.,Copinteria,CA). After blocking for 30 minutes, the sections were incubated with primary antibody of IL-17 at appropriate dilutions for 1 hour at room temperature. After washing, the sections were incubated with Alexa Fluor 594 conjugate(Invitrogen, Carlsbad, CA) for 30 minutes at room temperature, washed, and then incubated with 4, 6-diamidino-2-phenylindole (Dojindo Laboratories, Kumamoto, Japan). The negative control was prepared in the same manner, but without the primary antibody.
Statistical analysis
The Mann-Whitney U test was used to compare the gene expression between two groups. A one-way analysis of variance (ANOVA) followed by Tukey's post hoc analysis was used to compare gene expression between the three groups. P values less than 0.05 were considered to be statistically significant. All statistical analyses were performed on a personal computer using the Stat View version 5.0 statistical software package (Abacus Concepts, Berkeley, CA).
1 Masakazu Ishikawa,1 Atsushi Okuhara,1 Bunichiro Izumi,1 Masataka Deie,1 Osami Suzuki,1 Nobuo Adachi,1 and Mitsuo Ochi1
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