Mouse model of chronic inflammatory polyarthritis
D1BC mice express murine B7.1 in their chondrocytes and synovial membranes . A low dose of bColII (10 mg per mouse) was used to induce chronic inflammatory arthritis in D1BC mice, and a dose of 150 mg per mouse was used for conventional CIA in DBA/1J mice . Briefly, 7–8-weeks-old D1BC or DBA/1J mice were housed in a pathogen-free animal care facility at Nagoya City University Medical School in accordance with institutional guidelines. Mice were anesthetized with isoflurane and administered bColII emulsified with an equal volume of complete Freund’s adjuvant (BD Biosciences, Sparks, MD, USA). The same protocol was used for secondary induction with bColII, except for the use of incomplete Freund’s adjuvant (BD Biosciences).
Anti-CTLA-4 antibody treatment
B-cell hybridoma for anti-CTLA-4 monoclonal antibody (clone UC10-4F10-11, Hamster IgG) was provided by Dr. J. Bluestone from the University of California, San Francisco. B-cell hybridoma was cultured in DMEM (Sigma-Aldrich, Steinheim, Germany) with 10% fetal bovine serum (Gibco, Life Technologies, Carlsbad, CA, USA), 0.15% sodium bicarbonate (Gibco, Life Technologies), 0.01 mM non-essential amino acids (Gibco, Life Technologies), 0.05 mM 2-mercaptethanol (Fujifilm-Wako, Tokyo, Japan), and 500 U/ml penicillin-streptomycin (Gibco, Life Technologies). The monoclonal antibody was purified using a Protein A Sepharose 4 Fast Flow system (GE Healthcare, Uppsala, Sweden). Anti-CTLA-4 antibodies (0.1 mg) were injected intraperitoneally (ip) three times at 6 weeks in RA-induced D1BC mice. The same amount of saline was injected as the negative control.
CTLA-4 Ig treatment
CTLA-4-Ig (abatacept,100 mg/ 0.1 ml/ mouse, Bristol-Myers Squibb, Princeton, NJ, USA) and human IgG (hIgG, 100 mg/0.1 ml/mouse, supplied by Pharma Foods International, Kyoto, Japan) as a control, were injected ip three times a week, from weeks 3 to 10 after the first induction (Fig. 1a).
Serum RF-IgG and RF-IgM concentrations were measured using ELISA, according to the manufacturer’s instructions (Fujifilm-Wako-Shibayagi, Gunma, Japan). Serum obtained before bColII induction was used as the control.
Each joint and lymph node was fixed in 4% paraformaldehyde/PBS, decalcified in 2.5% EDTA, and stored in 7% sucrose at 4°C for one month. Paraffin sections (2 mm) were stained with hematoxylin and eosin (H&E; Muto, Tokyo, Japan) for immunohistochemical analysis. The sections were stained with the following primary antibodies: rat anti-F4/80 (Bio-Rad), rabbit anti-CD3 (Genemed Biotechnologies, South San Francisco, CA, USA), rat anti-PTPRC/CD45R (Aviva Systems Biology, San Diego, CA, USA), rabbit anti-syndecan 1 (Bioss Antibodies, Woburn, MA, USA), anti-CD4, and anti-CD8 (Cell Signaling Technology, Danvers, MA, USA). Histofine simple stain mouse MAX-PO secondary antibodies and the Opal multiplex fluorescent immunohistochemistry system (Akoya Biosciences, Marlborough, MA, USA) were used according to the manufacturers’ protocols. All images were captured using a fluorescence microscope (BZ-X710; Keyence, Osaka, Japan).
Flow cytometric analysis of mouse lymph nodes
Cells were separated by density gradient centrifugation using Ficoll-Paque Plus (GE Healthcare) and resuspended in flow cytometry buffer (Hanks’ Balanced Salt Solution supplemented with 2% heat-inactivated fetal calf serum, 0.05% sodium azide, and 0.5% EDTA). The cells were stained with the following fluorochrome-labeled monoclonal antibodies: anti-CD3-PE-Cyamine7 (Clone 17A2, Invitrogen, Waltham, MA, USA), anti-CD4-FITC (Clone RM4-5, Invitrogen), anti-CD8-eFluor 450 (Clone 53-6.7, Invitrogen), anti CD11c-PE (Clone N418, BD Biosciences), and anti-CD45R(B220)-eFluor®780 (Clone RA3-6B2, Biolegend, Hercules, USA), and incubated at 4°C for 30 min in the dark. These cells were then analyzed using a FACSCanto II (BD Biosciences).
In situ hybridization
In situ hybridization for CD80 and PD-L1 (CD274) was performed using the RNAscope Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostics, Newark, CA, USA), according to the manufacturer’s instructions.
Synovial cell isolation and flow cytometry analysis of the cells
Synovial cells were isolated from the pannus and incubated in dissection buffer, Hanks’ Balanced Salt Solution (HBSS, Gibco) supplemented with 48.1 mg/ml DNase I (Worthington Biochemicals, Lakewood, NJ, USA), 1.92 mg/ml Collagenase type III (Worthington Biochemicals), and 1.77 U/ml Dispase (Gibco) at 37°C for 30 min. After centrifugation at 100×g for 3 min, the cells were resuspended in DMEM with 10% FBS supplemented with penicillin-streptomycin, and cultured at 37°C in 5% CO2 for five days. For flow cytometry analysis, approximately 1×105 cells of isolated fibroblast-like synoviocytes (FLSs) blocked FCgR with anti CD16/CD32 antibody (BD Biosciences) at 4°C for 5 min. After washing with PBS containing 2% FBS, the cells were analyzed using the following primary antibodies: CD11b-PE (Clone M1/70, BD Biosciences), podoplanin-BV421 (Clone 8.1.1, BioLegend), Pdgfra (CD140a)-APCvio770 (Clone REA637, Miltenyi Biotec, NRW, Germany), CD45-APC (Clone, 30-F11, BioLegend), MHC class II-PE (Clone REA813, Miltenyi Biotec), CD80-BV421 (Clone 16-10A1, BD biosciences), CD274 (PD-L1)-PE (Clone 10F.9G2, BioLegend), and 7-Aminoactinocynin D (7-AAD) (BD Biosciences). These cells were analyzed using a FACSCanto II (BD Biosciences). To block CD80 internalization by the endocytosis inhibitor, the synovial cells were pre-incubated with 80 mM Dynasore hydrate (Sigma-Aldrich) in DMEM without FBS for 30 min, and then CTLA-4 Ig (1,000 ng/ml) was added and incubated at 37°C for 2 h. This was followed by flow cytometry analysis as described above.
Gene microarray in synovial cells
Isolated synovial cells were cultured in DMEM supplemented with 10% FBS overnight at 37°C in a 5% CO2 atmosphere. Thereafter, abatacept (100 mg/ml), human IgG (100 mg/ml), and a vehicle (DMEM with 10% FBS as a control) were added to the medium and the cells were incubated at 37°C, under 5% CO2 for 48 h. Total RNA extracted from the synovial cells was used as the sample in the ReliaPrep RNA Tissue miniprep system (Promega, Madison, WI, USA). Toray’s 3D-Gene Mouse Oligo chip 24k (Toray Industries, Tokyo, Japan) was used for RNA expression profiling according to the manufacturer’s instructions. Briefly, for efficient hybridization, this microarray adopted a columnar structure to stabilize spot morphology and enable microbead agitation. Total RNA was labeled with Cy5 using the Amino Allyl Message AMP II amplified RNA (aRNA) Amplification Kit (Applied Biosystems, Waltham, MA, USA). Cy5-labeled aRNA pools were mixed with hybridization buffer and hybridized for 16 h. Hybridization was performed according to the manufacturer’s instructions (www.3d-gene.com). Hybridization signals were obtained using a 3D-Gene Scanner (Toray Industries) and processed using 3D-Gene Extraction software (Toray Industries). The detected signals for each gene were normalized using the global normalization method (the median of the detected signal intensity was adjusted to 25). Gene expression data have been archived as Minimum Information About a Microarray Experiment (MIAME) and are publicly available as record GSE200403 in the NCBI’s Gene Expression Omnibus database.
undefinedData are expressed as the mean ± standard error (SE). Differences between CTLA-4-Ig and human IgG were evaluated using Student’s t-test for clinical scores and lymphoid cell populations. Serum RF-IgG and RF-IgM levels were measured and evaluated using one-way analysis of variance (ANOVA), followed by Dunnett’s test for parametric data. Statistical significance was set at P < 0.05.