Specimens

Synovial tissue samples were obtained from seven RA patients, who fulfilled the ACR classification criteria [17] and three osteoarthritis (OA) patients undergoing total knee joint replacement. The RA patients were 62 (38-75) years old [median (range)], with a disease duration of 9 (3-15) years and C-reactive protein level of 4.6 (0.3-81) mg/l. Informed consent was obtained from all the patients. All experimental protocols were approved by the Ethics Committee of Tokyo Medical and Dental University.

Immunohistochemistry

Immunohistochemical analysis was conducted on formalin-fixed paraffin-embedded sections of synovial tissues. The sections were incubated overnight at 4°C with 2 μg/ml rabbit anti-CB 2 polyclonal antibody (pAb) (abcam, Cambridge MA) or normal rabbit IgG as a control. Subsequently, the samples were incubated with 2 μg/ml biotinylated goat anti-rabbit IgG (Santa Cruz Biotechnology, Dallas, TX) for 30 min at room temperature, and then incubated for 30 min with streptavidin–horseradish peroxidase (Dako, Glostrup, Denmark). Diaminobenzidine (Dako) was used for visualization. The sections were counterstained with hematoxylin. The CB 2 staining of three RA and three OA samples was semi-quantitatively evaluated by randomly selected three fields with scored as follows: 0 = none, 1 = focal, and 2 = diffuse. The maximum score was six for each sample.

For immunofluorescence double-staining with CD68, CD4, CD8, CD21 or vimentin, and CB 2 , the sections were incubated overnight at 4°C with 2 μg/ml rabbit anti-CB 2 pAb or normal rabbit IgG together with 1 μg/ml mouse anti-CD68 mAb (KP1; Dako), 1 μg/ml mouse anti-CD4 mAb (RPA-T4; eBioscience, San Diego, CA), 1 μg/ml mouse anti-CD8 mAb (HIT8a; BD Bioscience, San Diego, CA), 1 μg/ml mouse anti-CD21 mAb (1 F8; Dako) or 1 μg/ml mouse anti-vimentin mAb (V9; Dako). Subsequently, the samples were incubated with 2 μg/ml Alexa Fluor 488-conjugated goat anti-mouse IgG1 (Invitrogen, Grand Island, NY) and Alexa Fluor 568-conjugated goat anti-rabbit IgG1 (Invitrogen) for 30 min at room temperature. A nuclear stain was performed with 4’, 6-diamidino-2-phenylindole.

Protein detection in cultured FLS

FLS from the RA synovial tissues was cultured as was reported previously [18]. RA FLS was lysed with radioimmunoprecipitation assay buffer (Millipore, Billerica, MA, USA) for 30 min at 4°C. A total of 20 μg of protein were boiled in the presence of sodium dodecyl sulfate (SDS) sample buffer and separated on a 10% SDS-polyacrylamide gel (ATTO, Tokyo, Japan). Proteins were then electrotransferred onto a polyvinylidene fluoride microporous membrane (Millipore) in a semidry system. The membrane was blocked with Block Ace (Snow Brand Milk Products, Tokyo, Japan) for 1 h at room temperature, and then the immunoblots were incubated overnight with 1 μg/ml rabbit anti-CB 2 pAb in Can Get Signal Immunoreaction Enhancer Solution (Toyobo, Osaka, Japan) at 4°C. After washing, the immunoblots were incubated with 2 μg/ml biotinylated goat anti-rabbit IgG for 30 min at room temperature, and then incubated for 30 min with streptavidin–horseradish peroxidase. ECL Prime detection reagent and the ImageQuant LAS 4000 Mini Biomolecular Imager (both from GE Healthcare) were used to detect the bands.

RA FLS (1 × 105 cells/ml) was cultured in Dulbecco's Modified Eagle Medium (Sigma-Aldrich) + 10% fetal bovine serum (FBS) (Sigma-Aldrich) and stimulated with 5 ng/ml recombinant TNF-α (R&D Systems, Minneapolis, MN) for 24 h in the presence or absence of JWH133 (Tocris bioscience, Ellisville, MO) [14]. The concentrations of Interleukin (IL)-6, metalloproteinase-3 (MMP-3) and chemokine (C-C motif) ligand 2 (CCL2) in the culture supernatants were measured using enzyme-linked immunosorbent assay (ELISA) kits (DuoSet; R&D Systems).

Analysis of osteoclastogenesis

Peripheral blood mononuclear cells from healthy donors were collected using Ficoll-Conray (Imuuno-Biological Laboratories, Gunma, Japan) gradient centrifugation. Positive selection of CD14+ monocytes was performed using CD14 MicroBeads (Miltenyi Biotec, Auburn, CA). The purified peripheral blood CD14+ monocytes (1 × 106 cells/well) were incubated in 96-well plates in α-Minimum Essential Medium (Sigma-Aldrich) with 10% FBS, and incubated with 25 ng/ml macrophage colony-stimulated factor (M-CSF) (R&D systems) + 40 ng/ml receptor activator of nuclear factor kappa-B ligand (RANKL) (Peprotech, Rocky Hill, NJ). These cells incubated in the presence or absence of JWH133. The medium was replaced with fresh medium 3 days later, and after incubation for 7 days the cells were stained for tartrate-resistant acid phosphatase (TRAP) expression using a commercial kit (Hokudo, Sapporo, Japan). The number of TRAP-positive multinucleated cells (MNC: more than 3 nuclear) in a randomly selected field examined at ×40 magnification was counted under light microscopy. The CD14+ monocytes were seeded onto plates coated with calcium phosphate thin films (Osteo Assay Plate, Corning, NY, USA) and were incubated with 25 ng/ml M-CSF + 40 ng/ml RANKL for 7 days in the presence or absence of JWH133. The cells were then lysed in bleach solution (6% NaOCl, 5.2% NaCl). The resorption lacunae were examined under light microscopy. The viability of the cells treated with JWH133 (up to 50 μM) was more than 95% relative to the vehicle-treated cells.

Induction of collagen-induced arthritis (CIA)

Male 8-week-old DBA/1 J mice were purchased from Oriental Yeast (Tokyo, Japan) and were kept in the temperature of 23.5 ± 2 degrees Celsius with 40-50% humidity. Bovine collagen type II (CII; Collagen Research Center, Tokyo, Japan) was dissolved in 0.05 M acetic acid at 4 mg/ml and emulsified in equal volume of complete Freund’s adjuvant (CFA; Difco Laboratories, Detroit, MI). Mice were immunized with 100 μl of the emulsion injected intracutaneously at the base of the tail (day 1). After 21 days (day 22), the same amount of bovine CII emulsified in CFA was injected intracutaneously at the base of the tail as a booster immunization [19].

Treatment of collagen-induced arthritis (CIA) mice with JWH133

Twelve mice with CIA per group were twice daily injected intraperitoneally with JWH133, 1 mg/kg/day or 4 mg/kg/day in total volume of 200 μl/day of 20% dimethyl sulphoxide or vehicle alone from day 15 to day 35. To determine the therapeutic effects of JWH133, we also treated mice with 4 mg/kg JWH133 from day 28, after the development of arthritis, to day 35 and observed the mice for signs of arthritis. Disease severity for each limb was recorded as follows: 0 = normal, 1 = erythema and swelling of one digit, 2 = erythema and swelling of two digits or erythema and swelling of ankle joint, 3 = erythema and swelling of more than three digits or swelling of two digits and ankle joint, and 4 = erythema and severe swelling of the ankle, foot, and digits with deformity. The clinical arthritis score was defined as the sum of the scores for all 4 paws of each mouse. Thickness of each paw was measured using a pair of digital slide calipers. On day 36, the ankle joints were harvested and examined radiographically and histologically. The bilateral second-to-fourth metatarsophalangeal (MTP) joints were assessed radiographically as follows: 0 = not obvious, 1 = marginal osteoporosis, and 2 = erosion. This system yields a possible score between 0 and 4 per animal. The hind paw of each mouse was dissected and examined histologically after hematoxylin and eosin staining. The severity of arthritis was evaluated according to synovial inflammation, as follows: 0 = no inflammation, 1 = focal inflammatory infiltration, and 2 = severe and diffuse inflammatory infiltration. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University.

The harvested splenocytes (1 × 106 cells) on day 36 were cultured in 48-well plates in Roswell Park Memorial Institute 1640 medium (Sigma-Aldrich) with 10% FBS supplemented with 50 μg/ml denatured (100°C, 10 min) CΙΙ. After 72 h, the concentrations of interferon (IFN)-γ and IL-17 in the culture supernatant were measured using ELISA kits (DuoSet; R&D Systems).

Serum samples were obtained on day 36 for measurement of IgG1 anti-CII antibody by ELISA (normal, n = 4; vehicle, n = 12; 4 mg/kg JWH133, n = 12) as described previously [19].

Statistical analysis

All data are expressed as the mean ± standard error of the mean (SEM). Immunohistological score was analyzed by student’s t-test. Concentration of inflammatory mediators and osteoclastogenesis were analyzed by Kruskal-Wallis test and Dunnett’s test. Overtime analysis of arthritis score and paw thickness was performed by 2 way-ANOVA, and point-by-point analysis was followed by student’s t-test. Histological and radiographic score, and concentration of IgG1 were analyzed by Kruskal-Wallis test and student’s t-test.