Induction of collagen-induced arthritis
Mice of the DBA/1 strain were bred in the Experimental Animal Centre of the Katholieke Universiteit Leuven (Leuven, Belgium). The experiments were performed in 8- to 12-week-old male mice that were age-matched within each experiment.
CII from chicken sternal cartilage (Sigma-Aldrich Co., St Louis, MO, USA) was dissolved at 2 mg/ml in PBS containing 0.1 M acetic acid by stirring overnight at 6°C. The CII solution was emulsified with an equal volume of complete Freund's adjuvant (CFA; Difco Laboratories, Detroit, MI, USA) with added heat-killed Mycobacterium butyricum (Difco), reaching a final Mycobacterium content of 750 μg/ml emulsion. Mice were injected intradermally with 100 μl emulsion at the base of the tail on day 0.
Mice were examined daily for signs of arthritis. The disease severity was recorded for each limb, as described in : score 0, normal; score 1, redness and/or swelling in one joint; score 2, redness and/or swelling in more than one joint; score 3, redness and/or swelling in the entire paw; score 4, deformity and/or ankylosis.
All animal experiments were approved by the local ethical committee (University of Leuven).
Treatment with AMD3100
AMD3100 was provided by AnorMED (Langley, British Columbia, Canada). For the treatment with AMD3100, Alzet osmotic minipumps model 2002 (DURECT corporation, Cupertino, CA, USA) were subcutaneously implanted at the dorsolateral part of the body. During the procedure, the mice were anaesthetized with a solution of PBS containing 0.2% (v/v) Rompun (Bayer, Brussels, Belgium) and 1% (v/v) Ketalar (Parke-Davis, Zaventem, Belgium). The minipumps delivered AMD3100 at a constant rate of 600 μg/day for 14 days.
Fore and hind limbs (ankles and interphalanges) were fixed in 10% formalin and decalcified with formic acid. Paraffin sections were haematoxylin stained. Severity of arthritis was evaluated blindly using three parameters: infiltration of mono- and polymorphonuclear cells; hyperplasia of the synovium; and bone destruction. Each parameter was scored on a scale from 0 to 3: score 0, absent; score 1, weak; score 2, moderate; score 3, severe.
Serum anti-collagen type II ELISA
Individual sera were tested for the amount of anti-CII antibody by ELISA, as described previously . Briefly, ELISA plates (Maxisorp, Nunc, Wiesenbaden, Germany) were coated overnight with chicken CII (1μg/ml; 100 μl/well; Sigma-Aldrich Co, St Louis, MO, USA) in coating buffer (50 mM Tris-HCL, pH 8.5; 0.154 mM NaCl) followed by a 2 h incubation with blocking buffer (50 mM Tris-HCl, pH 7.4; 154 mM NaCl and 0.1% (w/v) casein). Serial twofold dilutions of the sera and the standard were incubated overnight in assay buffer (50 mM Tris-HCl; pH 7.4; 154 mM NaCl and 0.5% Tween-20). The quantification of total IgG was done by ELISA making use of a standard with known IgG concentration. For determination of the IgG2a, IgG2b and IgG1 antibody concentrations, a standard of arbitrary U/ml was used (standard = 1,000 U/ml). Plates were then incubated for 2 h with biotinylated rat antibody to mouse total IgG, IgG2a, IgG2b or IgG1 (Zymed Laboratories, San Francisco, CA, USA). Plates were washed and incubated for 1 h with streptavidin-peroxidase. Finally, the substrate 3,3'-5,5'-tetramethyl-benzidine (Sigma-Aldrich Co.) in reaction buffer (100 mM sodium acetate/citric acid, pH 4.9) was added. Reaction was stopped using 50 μl H2SO4 2 M and absorbance was determined at 450 nm.
Delayed-type hypersensitivity experiments
For evaluation of DTH reactivity, CII/CFA-immunized mice were subcutaneously injected with 10 μg of CII/20 μl PBS in the right ear and with 20 μl PBS in the left ear. DTH response was calculated as the percentage swelling (the difference between the increase of thickness of the right and the left ear, divided by the thickness of the ear before challenge, multiplied by 100).
Assays for in vivo leukocyte migration and for in vitrochemotaxis
For the in vivo assay, mice were treated with AMD3100 or PBS as described above. The assay was performed on the last day of the treatment. Six days before, mice were subcutaneously injected at the dorsolateral site of the body with 2.5 ml of sterile air, creating a subcutaneous air pouch. At day three before the assay, injection with 2.5 ml sterile air was repeated at the same location. The chemotactic assay was performed by injecting 1 ml 0.9% (w/v) NaCl/CXCL12 2 μg or 0.9% (w/v) NaCl alone into the air pouch (human CXCL12 was provided by Dr I Clark-Lewis, University of British Columbia, Vancouver, BC, Canada). Two hours later, cells were washed out of the air pouch by 2 ml PBS/FCS 2% (v/v) and cells were immediately counted with a light microscope in the Burker chamber.
In vitro chemotactic assays were performed at day 21 post immunization. Spleens were isolated and passed through cell strainers to obtain a single cell suspension. Erythrocytes were removed by lysis with NH4Cl (0.83% (w/v) in 0.01 M Tris-HCl, pH 7.2; two consecutive incubations of 5 and 3 min, 37°C). Splenocytes of three mice were pooled and incubated with AMD3100 at different concentrations in assay buffer (HBSS, 20 mM Hepes, 0.2% (w/v) BSA, pH 7.2). Transwell filter membranes (5 μm pore; Costar, Boston, MA, USA) were placed in the wells of a 24-well plate, each containing 600 μl buffer with or without CXCL12 at a concentration of 100 ng/ml (human CXCL12 was provided by Dr I Clark-Lewis). 106 cells were loaded on each Transwell filter. The plate was then incubated for 3.5 h at 37°C, whereupon the filter inserts were carefully removed. The migrated cells were collected and counted in a flow cytometer (FACScalibur; Becton Dickinson, San Jose, CA, USA) as described [18–20]. The number of cells is represented as the number of counts registered during a two-minute acquisition (number of cells/2 minutes).
Migrated cells were incubated with anti-CD16/CD32 Fc-blocking antibodies (BD Biosciences Pharmingen, San Diego, CA, USA) and washed with PBS. After washing, the cells were stained for 30 minutes with anti-CD4-PE, anti-CD8-FITC, anti-CD19-PE or anti-CD11b-FITC (BD Biosciences Pharmingen). Cells were washed, fixed with 0.37% formaldehyde in PBS, and analysed by a FACScalibur flow cytometer (Becton Dickinson).
The chemotactic index was calculated as the number of migrated cells obtained with 100 ng/ml CXCL12 divided by the number of cells in the negative control without CXCL12.
Flow cytometric analysis of cells from joint cavities
Cells from joint cavities were obtained by inserting a 25-gauge needle into the ankle joint. Cold PBS (800 μl) was injected into the joint cavity. Fluid exiting spontaneously from the opening was collected and was only used when it was found to contain <5% of erythrocytes. Cells were washed and resuspended in cold PBS. Cells were incubated with anti-CD16/anti-CD32 Fc-receptor-blocking antibodies (BD Biosciences Pharmingen). After washing, the cells were stained for 30 minutes with anti-CD11b-FITC and anti-CXCR4-PE or isotype control rat IgG2b (BD Biosciences Pharmingen). Cells were washed, fixed with 0.37% formaldehyde in PBS, and analysed by a FACScalibur flow cytometer (Becton Dickinson).
Polymerase chain reaction
Synovial tissues from the ankle joints were carefully isolated under a stereomicroscope. Total RNA was extracted with Trizol reagent (Invitrogen, Paisley, Scotland, UK), in accordance with the manufacturer's instructions. cDNA was obtained by reverse transcription with a commercially available kit (Thermoscript; Invitrogen) with oligo(dT)20 as primer.
For PCR reactions we used a TaqMan® Assays-on-Demand™ Gene Expression Product from Applied Biosystems (Foster City, CA, USA; assay ID Mm00445552_m1). Expression levels of the gene were normalized for 18S RNA expression.
Cytokine detection in serum and cultured medium
Control-treated and AMD3100-treated mice were bled both before and 6 h after intraperitoneal injection with 10 μg anti-CD3. Sera were collected and pooled. This allowed us to determine the concentrations of the following cytokines: IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, tumour necrosis factor-α and IFN-γ.
Spleens of three mice were isolated on day 21 after immunization and were passed through cell strainers to obtain a single cell suspension. Erythrocytes were removed by lysis with NH4Cl (0.83% (w/v) in 0.01 M Tris-HCl, pH 7.2; two consecutive incubations of 5 and 3 minutes, 37°C). Splenocytes of the mice were pooled and cultured in a 96-well plate. 105 cells were cultured in one well in Roswell Park Memorial Institute (RPMI) medium alone, RPMI with mouse CXCL12 (0.1 μg/ml) (PeproTech, London, UK), or RPMI with mouse CXCL12 and AMD3100 (25 μg/ml). Supernatant was collected after 48 h. Detection of cytokine concentrations in serum and cultured medium was done with the Endogen SearchLight™ array (Pierce Boston Technology, Woburn, MA, USA).
In vitroinduction of osteoclast formation by splenocytes
Spleens were isolated on day 21 after immunization and were passed through cell strainers to obtain a single cell suspension. Erythrocytes were removed by lysis with NH4Cl (0.83% (w/v) in 0.01 M Tris-HCl, pH 7.2; two consecutive incubations of 5 and 3 minutes, 37°C). Leukocytes from the blood were obtained by lysis of red blood cells by two incubations (5 and 3 minutes at 37°C) with NH4Cl solution (0.083% (w/v) in 0.01 M Tris-HCl; pH 7.2). Remaining cells were washed two times with ice-cold PBS.
Splenocytes were suspended in Minimal Essential Medium alpha Medium (α-MEM) containing 10% (v/v) FCS (GIBCO, Invitrogen corporation, Paisley, Scotland, UK). Cells (2.5 × 105) in a total volume of 400 μl were seeded in chamber slides (LAB-TEK Brand Products, Nalge Nunc International, Naperville, IL, USA). Cells were incubated with macrophage colony stimulating factor (M-CSF; 20 ng/ml) + CXCL12 (0.1 or 0.5 μg/ml; AnorMED), with M-CSF + RANKL (100 ng/ml) + CXCL12 or with M-CSF + RANKL + CXCL12 + AMD3100 (25 μg/ml; AnorMED). M-CSF and RANKL were obtained from R&D Systems Europe (Abingdon, UK). On day 4, supernatants were removed and cultures were provided with fresh media and stimuli. On day 7, media were removed and cells were stained for the presence of tartrate-resistant acid phosphatase (TRAP) (described below).
Splenocyte suspensions were obtained as described above and resuspended in α-MEM containing 10% (v/v) FCS (GIBCO, Invitrogen Corporation). 106 cells were cultured for 5 days with M-CSF (20 ng/ml) and RANKL (100 ng/ml), both from R&D systems Europe, on transparent quartz slides coated with a calcium phosphate film (BioCoat Osteologic Discs; BD Biosciences Pharmingen). On day 6, media were removed and replaced with media containing M-CSF, M-CSF + CXCL12 (0.5 μg/ml), M-CSF + CXCL12 + AMD3100 (25 μg/ml) or M-CSF + AMD3100. Another two days later, cells were removed and resorption pits were quantified using a Leitz DM RBE microscope equipped with a colour video camera (Optronics Engineering, Goleta, CA, USA) and attached to a computer-aided image analysing system (Bioquant, R&M Biometrics, Nashville, TN, USA). Quantification and size determination of the pits was performed at a magnification of ×20 in 15 areas of constant size, positioned adjacent to one another and spanning the whole quartz slide. In all slides, the minimum threshold of a pit surface area was set to 50 μm2. Upon thresholding, the number and square surface of plaques are determined automatically.