Patients
Samples of synovial tissue were isolated from 5 patients with RA (mean age 55.2 ± 3.8 years; range 44–64 years) and 5 with osteoarthritis (OA) patients (mean age 57.8 ± 3.0 years; range 50–68 years), who were undergoing total knee replacement surgery. Synovial fluid was obtained from patients with RA, who fulfilled the revised criteria of the American College of Rheumatology, 1987 (formerly the American Rheumatism Association), and from patients who had symptomatic knee OA. Informed consent was obtained from all patients, and the experimental protocol was approved by the Konkuk University School of Medicine Human Research Ethics Committee (KUH1010186).
Isolation of FLS
FLSs were isolated by enzymatic digestion of synovial tissues obtained from patients with RA, who were undergoing total knee replacement surgery, as described previously [13].
Reagents
IL-22, IL-25, RANKL, and macrophage colony-stimulating factor (M-CSF) were obtained from R&D Systems (Minneapolis, MN, USA).
Enzyme-linked immunosorbent assay (ELISA) of IL-22, IL-25, and sRANKL
In brief, a 96-well plate (Nunc, Roskilde, Denmark) was coated with 4 µg/ml monoclonal antibodies against IL-22, IL-25, and sRANKL (R&D Systems, Minneapolis, MN, USA) at 4 °C overnight. After blocking with phosphate-buffered saline/1% bovine serum albumin (BSA)/0.05% Tween 20 for 2 h at room temperature (22–25 °C), the test samples and the standard recombinant IL-22, IL-25, and sRANKL (R&D Systems) were added to the 96-well plate and incubated at room temperature for another 2 h. The plates were washed four times with phosphate-buffered saline/Tween 20, and then incubated with 500 ng/ml biotinylated mouse monoclonal antibodies against IL-22, IL-25, and sRANKL (R&D Systems) for 2 h at room temperature. After washing, streptavidin-alkaline phosphate–horseradish peroxidase conjugate (Sigma, St Louis, MA, USA) was incubated for 2 h, followed by another wash, and incubated with 1 mg/ml p-nitrophenyl phosphate (Sigma) dissolved in diethanolamine (Sigma) to develop the colour reaction. The reaction was stopped by the addition of 1 M NaOH and optical density of each well was measured at 405 nm. The lower limit of IL-22, IL-25, and sRANKL detection was 10 pg/ml. Recombinant human IL-22, IL-25, and sRANKL, diluted in culture medium, were used as calibration standards, ranging from 10 to 2000 pg/ml. A standard curve was drawn by plotting optical density against log of the concentration of recombinant cytokines, and the curve was used for determining IL-22, IL-25, and sRANKL concentrations in test samples.
Immunohistochemistry of RA synovium
Immunohistochemical staining for IL-25 was performed with sections of synovium. Briefly, synovial samples were obtained from patients with RA and OA, fixed with 4% paraformaldehyde solution overnight at 4 °C, dehydrated with alcohol, washed, embedded in paraffin, and sectioned into 7-∝m thick slices. Sections were depleted of endogenous peroxidase activity by adding methanolic H2O2 and blocked with normal serum for 30 min. After overnight incubation with polyclonal anti-human IL-25 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C, the samples were incubated with a secondary antibody, biotinylated anti-rabbit IgG, for 20 min, and then with streptavidin-peroxidase complex (Vector Laboratories, Peterborough, UK) for 1 h, followed by a 5-min incubation with 3,3′-diaminobenzidine (Dako, Glostrup, Denmark). The sections were counterstained with haematoxylin. Samples were finally photographed using an Olympus (Tokyo, Japan) photomicroscope.
Expression of RANKL mRNA by real-time polymerase chain reaction (PCR)
FLSs were stimulated with various concentrations of IL-22 (0, 1, 10 ng/ml). They were incubated in the presence or absence of IL-25 (10, 50, 100 ng/ml) for 4 h before the addition of IL-22. After 72 h, mRNA levels were measured using real-time PCR, as reported previously [14].
Western blot analysis
FLSs were incubated with IL-22 in the presence or absence of IL-25. After incubation for 1 h, whole-cell lysates were prepared from approximately 2 ⋅ 105 cells, by homogenisation in the lysis buffer, and then centrifuged at 14,000 rpm for 15 min. Protein concentration in the supernatant was determined using the Bradford method (Bio-Rad, Hercules, CA, USA). Protein samples were separated by 10% sodium dodecyl sulphate-polyacrylamide electrophoresis (SDS–PAGE), and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Uppsala, Sweden). For western blotting, the membrane was pre-incubated with 0.5% skim milk in 0.1% Tween 20 and Tris-buffered saline (TTBS) at room temperature for 2 h. The primary antibodies to phospho-stat3, stat3, phospho-p38, p38, phospho-IκB-α, and IκB-α, (Cell Signaling Technology Inc., Danvers, MA, USA), diluted 1:1000 in 5% BSA-0.1% Tween 20/TBS, were added and incubated overnight at 4 °C. The membrane was washed 4 times with TTBS, followed by the addition of horseradish peroxidase-conjugated secondary antibody and incubation for an hour at room temperature. After TTBS washing, hybridised bands were detected using the ECL detection kit and Hyperfilm-ECL reagents (Amersham Pharmacia).
Osteoclast formation
PBMCs were collected from healthy blood by density gradient separation, and monocytes (osteoclast precursors: pre-OC) were prepared from them. Human monocytes were seeded in 48-well plates at 5 × 104 cells/well with 1 mL of medium. Monocytes were pre-treated with IL-25 for 4 h, following which they were added to each well along with IL-22. RANKL was used as the positive control. On day 21, tartrate-resistant acid phosphatase (TRAP)-positive cells were identified, as described previously [14].
Statistical analysis
All data are expressed as the mean ± standard error of the mean (SEM). Statistical analysis was performed using one-way analysis of variance and Bonferroni’s multiple comparisons test. In all analyses, P < 0.05 indicated statistical significance.