Patients with RA or osteoarthritis (OA) were enrolled in this study from 2008 to 2013 in the Shanghai Guanghua Hospital of Integrated Traditional Chinese and Western Medicine (Shanghai, China). All the RA patients fulfilled the 1987 revised criteria of the American College of Rheumatology (formerly the American Rheumatism Association) ,, and the OA patients fulfilled the American College of Rheumatology criteria . Informed consent was obtained from each participant, and the experimental protocol was approved by the hospital’s Human Research Ethics Committee.
Exogenous IFN-β intervention in RA patients
Twenty RA patients were selected for an immune interference study with exogenous IFN-β (Rebif®, Merck Serono, Darmstadt, Germany) administered as in the MS and phase I clinical trials for RA patients ,. A clinical assessment was performed by evaluating the duration of morning stiffness (min), the number of painful joints and swollen joints, and the degree of pain (by Visual Analog Scale [VAS]) in RA patients both before and after exogenous IFN-β administration.
Enzyme-linked immunosorbent assay (ELISA)
Peripheral blood samples from 22 RA and 13 OA patients, as well as synovial fluid (SF) from 21 RA and 5 OA patients, were collected under aseptic conditions. The levels of inflammatory cytokines interleukin-17 (IL-17), interferon γ (IFN-γ), tissue inhibitor of metalloproteinases 1 (TIMP-1), matrix metalloproteinase 3 (MMP-3), osteoprotegerin (OPG), and receptor activator of nuclear factor κB (RANKL), as well as CII antibody, rheumatoid factor-IgM (RF-IgM), anti-cyclic citrullinated peptide antibody (CCP), and glucose-6-phosphate isomerase antibodies (GPI) were detected using Quantikine ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Thresholds of CII IgA/CII IgG >2.2 U/mL, CII IgM >2.4 U/mL, RF-IgM >20 U/mL, GPI >2.0 mg/L, and anti-CCP >5 U/mL were used to identify positive samples according to the standards of the clinical laboratory of Shanghai Guanghua Hospital of Integrated Traditional Chinese and Western Medicine.
BALB/c mice (20–23 g, 8–10 weeks) were purchased from the Chinese Academy of Sciences, Shanghai Laboratory Animal Center and housed following institutional guidelines. Experiments were conducted according to the guidelines of the Ethics Committee of Laboratory Animals Welfare of Shanghai Jiao Tong University School of Medicine.
Induction of CAIA and establishment of the treatment protocol
To induce the CAIA model, BALB/c mice were injected with 2 mg of collagen antibody cocktail (Chondrex, Redmond, WA, USA) intravenously on Day 1, and were then treated with 25 μg of lipopolysaccharide (LPS) intraperitoneally on Day 4. All the mice were monitored daily for arthritis. Each paw was scored for clinical signs of arthritis as follows: normal (0); erythema and edema in only one digit (0.5); erythema and mild edema of the footpad, ankle, or two to five digits (1); erythema and moderate edema of two joints (footpad, ankle, or two to five digits) (2); erythema and severe edema of the entire paw (3); reduced swelling and deformation leading to incapacitation of the limb (4). Each mouse arthritic score was obtained by summing the scores recorded for each paw. The clinical evaluations were performed by two blinded investigators, and the mean of both scores was calculated . On Day 4, after LPS injection, the intervention group CAIA model mice (n = 9) received 10,000 IU of exogenous mouse IFN-β (PBL interferon source, Piscataway, NJ, USA) every day by intraperitoneal injection for 4 days, while the control group (non-intervention group) CAIA model mice (n = 9) were similarly treated with sterile saline.
Molybdenum X-ray imaging
Prior to histology, molybdenum X-ray radiographs (Adobe Systems, Munich, Germany) of the knees and paws of each mouse were taken on day 12 after induction of arthritis. The limbs were extended to prevent joint buckling, and the bone mineral density was assessed.
At day 12 after induction of arthritis, the knees and paws were harvested and fixed in 4% paraformaldehyde, decalcified, and embedded in paraffin. Serial sections of the knees and paws were stained with hematoxylin and eosin (H&E, Sakura Finetek, Tokyo, Japan) or safranin-O with fast green counterstain. Inflammation and joint damage were scored on a scale of 0 (no inflammation) to 3 (severe inflammation) depending on the number of inflammatory cells. Cartilage destruction was scored on a scale of 0 (no loss) to 3 (complete loss of the articular cartilage). Scoring was performed by two blinded investigators, and the mean of both scores was calculated.
Quantitative real-time polymerase chain reaction (qRT-PCR)
The hind paws and joint bones of the CAIA model mice were pulverized in liquid nitrogen, and the total RNA was extracted using TRIzol® reagent (Invitrogen, Carlsbad, CA, USA). One μg of the total RNA was reverse transcribed using a reverse transcription kit (Promega, Madison, WI, USA). Quantitative real-time PCR (qRT-PCR) was performed with duplicate samples on the ABI7500 system (Applied Biosystems, Darmstadt, Germany) under the following conditions: 2 min of polymerase activation at 95°C followed by 45 cycles of 10 sec denaturation at 95°C and 30 sec annealing and extension at 60°C. The detection threshold was set to the log linear range of the amplification curve and kept constant (0.05) for all data analysis. Threshold cycle (C
) of each target product was determined and set in relation to the amplification plot of β-actin. Differences in the C
) between each gene and β-actin were used to calculate the relative expression (relative expression = 2−(CT of target genes−
CT of β-actin) =2−ΔCT). The mouse PCR primers (Sangon Biotech, Shanghai, China) used for RT-PCR were as follows: for IFN-β, sense: 5′-CGTTCCTGCTGTGCTTCTC-3′ and anti-sense: 5′-TGTAACTCTTCTCCATCTGTGAC-3′; TIMP-1, sense: 5′-GCCGCCATCATCGCAGAT-3′ and anti-sense: 5′- CCTTATGACCAGGTCCGAGTTG-3′; MMP-3, sense: 5′- AAGAGATCCAAGGAAGGCATCCT-3′ and anti-sense: 5′- GGTTCTGCCATAGCACATGCT-3′; TRAP, sense: 5′-AAATCACTCTTCAAGACCAG-3′ and anti-sense: 5′-TTATTGAACAGCAGTGACAG-3′; RANKL, sense: 5′-TGCCGCTACCGCAAGACAGA-3′ and anti-sense: 5′-GCAGGCTTACGTTGGCTCCC-3; TRAF-6, sense: 5′-GCTCAAACGGACCATTCGGA-3′ and anti-sense: 5′-GGGATTGTGGGTCGCTGAAA-3′; c-Fos, sense: 5′-CCCTTTGATGACTTCTTGTTTCCG-3′ and anti-sense: 5′-AATTGCTGTGCAGAGGCTCCC-3′; NFATc1, sense: 5′-TCTCGAAAGACAGCACTGGAGCAT-3′ and anti-sense: 5′-ACGGGATCTCCAGGAATTTGGTGT-3′; β-actin, sense: 5′-CTGTCCCTGTATGCCTCTG-3′ and anti-sense: 5′-ATGTCACGCACGATTTCC-3′.
Cell culture and differentiation
The murine macrophage cell line RAW 264.7 (generously provided by Dr. J. Luo, East China Normal University) was plated in 24-well plates (10,000 cells per well) containing α-minimum essential medium (α-MEM) supplemented with 10% fetal calf serum (FCS). The cells were stimulated with 50 ng/mL RANKL (R&D Systems) with or without exogenous mouse IFN-β (50 IU/mL) for 4 days. All cells were cultured in a 5% CO2/95% air incubator. The culture medium was replaced with fresh medium every day.
Tartrate-resistant acid phosphatase (TRAP) staining
The paraffin-embedded sections of the joint bones of the CAIA model mice and RANKL-induced osteoclastogenesis on the fourth day after induction were gently washed twice with pre-warmed, double-distilled water (37°C), fixed with stationary liquid for 20 sec, and stained with tartrate-resistant acid phosphatase (TRAP, Sigma, St. Louis, MO, USA) for 60 min at 37°C. The TRAP-stained cells were then gently washed, counterstained in the dark with hematoxylin or 100 μL/well of 300 nM diamidino-2-phenylindole (DAPI ) in phosphate buffer solution (PBS) containing 0.1% Triton X-100 at room temperature for 15 min, and examined with a ZEISS Vert.A1 microscope (Carl Zeiss, Oberkochen, Germany). TRAP-positive cells appeared dark red, and TRAP-positive multi-nucleated cells containing three or more nuclei were counted as osteoclasts. Osteoclasts were quantified by imaging five fields of view under 200× magnification and directly counting the number of TRAP-positive cells . All experiments were carried out in triplicate at least 3 times.
Statistical analyses were performed in Prism (GraphPad Software, La Jolla, CA, USA). Values are presented as mean ± standard deviation. Unpaired two-tailed Student’s t- tests were used for parametric outcomes to compare groups. The Kruskal–Wallis test for several group means followed by the Mann–Whitney U test for comparison of two groups  were also used. P-values <0.05 were considered statistically significant. *: P <0.05, **: P <0.01.