Mice and experimental conditions
The generation and the basic characteristics of the mutant mouse strain (129/Sv/Ev) with a disruption in the gene coding for the α-chain of the IFN-γ receptor (IFN-γR KO) have been described . These IFN-γR KO mice were backcrossed with DBA/1 wild-type mice for 10 generations to obtain the DBA/1 IFN-γR KO mice used in the present study. The homozygous IFN-γR KO mice were identified by PCR as described . Wild-type and IFN-γR KO DBA/1 mice were bred in the Experimental Animal Centre of the University of Leuven. The experiments were performed in mice 6 to 10 weeks old, but in each experiment the mutant and wild-type mice were age-matched within 5-day limits. The male : female ratio was kept between 0.8 and 1.3 in each experiment group, unless otherwise mentioned. All animal experiments were approved by the local ethical committee (University of Leuven).
Induction and clinical assessment of arthritis
Native chicken CII (Sigma-Aldrich, St Louis, MO, USA) was dissolved at 2 mg/ml in PBS containing 0.1 M acetic acid by stirring overnight at 6°C and emulsified in an equal volume of complete Freund's adjuvant (CFA; Difco Laboratories, Detroit, MI, USA) with added heat-killed Mycobacterium butyricum (0.5 mg/ml). IFN-γR KO and wild-type mice were sensitised with a single intradermal injection at the base of the tail with 100 μl of the emulsion on day 0. From day 0 after immunisation, mice were examined for signs of arthritis five times a week. The disease severity was recorded with the following scoring system for each limb: 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.
Media, reagents and antibodies
All cells were grown in RPMI 1640 (Bio Whittaker Europe, Verviers, Belgium), supplemented with 10% heat-inactivated FCS (Gibco, Paisley, UK), penicillin (100 IU/ml; Continental Pharma, Brussel, Belgium), streptomycin (100 μg/ml; Continental Pharma), 2 mM L-glutamine, 10 mM Hepes (Gibco), 0.1 mM nonessential amino acids (ICN, Asse Relegem, Belgium), 1 mM sodium pyruvate (Gibco) and 50 μM 2-mercaptoethanol (Fluka, AG, Switzerland).
Anti-CD25 IL-2Rα monoclonal antibody was produced by hybridoma PC61 in an INTEGRA CELLine CL1000 (Elscolab, Kruibeke, Belgium) and is a rat IgG1 antibody. The hybridoma supernatant was purified by Protein G-Sepharose chromatography (Amersham Biosciences, Roosendaal, The Netherlands) for administration in vivo.
The hamster monoclonal antibody, directed against the mouse CD3 complex, was prepared from the culture supernatant of 145-2C11 hybridoma cells . The antibodies were purified by affinity chromatography with Protein A-Sepharose (Amersham Biosciences). Batches of anti-CD3 antibody were tested for endotoxin content with the Limulus amebocyte lysate QCL-1000 kit (Bio Whittaker) and were found to contain less than 3 ng/ml endotoxin.
Lymph nodes (axillary, inguinal and mesenteric) and spleens were harvested from mice 6 to 8 weeks old. Lymph nodes and spleens were gently cut into small pieces and passed through cell strainers (Becton Dickinson Labware, Franklin Lakes, NJ, USA). Red blood cells were lysed by two consecutive incubations (5 and 3 min at 37°C) of the suspension in NH4Cl (0.83% in 0.01 M Tris-HCl, pH 7.2). Remaining cells were washed, resuspended in cold PBS and counted. Lymph node preparations were then enriched for CD4+ T cells with the Mouse T cell CD4 Subset Column Kit (R&D systems, Abingdon, UK). To purify CD4+CD25+ and CD4+CD25- cells, the enriched CD4+ T cells were incubated for 20 min at 4°C with FITC-conjugated anti-CD25 and phycoerythrin (PE)-conjugated anti-CD4 antibodies (10 μg per 108 cells) in PBS containing 2% FCS. They were sorted by flow cytometry on a FACS Vantage (Becton Dickinson, San Jose, CA, USA). The resultant purity of the CD4+CD25- population was 99%, whereas the purity of the CD4+CD25+ population varied from 96% to 99%. Alternatively, CD4+ T cells were labelled with PE-conjugated anti-CD25 monoclonal antibody, followed by incubation with magnetic-activated cell sorting (MACS) anti-PE beads (CD25 Microbead Kit; Miltenyi Biotec, Bergisch Gladbach, Germany). CD4+CD25+ T cells were selected on an LS column in a magnetic field and the flow-through was collected as CD4+CD25- T cells. After removal of the column from the magnetic field, CD4+CD25+ T cells were flushed out by a plunger. The purity of the CD4+CD25- population was 99% and the purity of the CD4+CD25+ population varied from 90% to 95%.
T cell-depleted spleen suspensions were prepared by MACS (Miltenyi Biotec) and used as accessory cells (ACs). For MACS separation, the cell suspension was magnetically labelled with CD90 (Thy1.2) microbeads and passed through a CS separation column, placed in a magnetic field. The unlabelled CD90- cells ran through.
Single-cell suspensions (5 × 105 cells) were incubated for 15 min with the Fc-receptor-blocking antibodies anti-CD16/anti-CD32 (CD16/CD32; BD Biosciences Pharmingen, San Diego, CA, USA). Cells were washed with PBS containing 2% FCS and stained with the indicated FITC-conjugated antibodies (0.5 μg) for 30 min, washed twice and incubated for 30 min with the indicated PE- or biotin-conjugated antibodies. For the biotin-conjugated antibodies, a third staining step with streptavidin conjugated with peridinin chlorophyll a protein (PerCP) was performed. After washing, propidium iodide (Sigma-Aldrich) was added at a final concentration of 4 μg/ml to distinguish dead cells from living cells. Biotin-conjugated anti-CD25 (7D4), FITC-conjugated anti-CD25 (7D4), FITC-conjugated CD69 (H1.2F3), PE-conjugated anti-CD4 (RM4-5) and PerCP-conjugated streptavidin were purchased from BD Biosciences Pharmingen. FITC-conjugated anti-CD62L (MEL-14) and anti-CD44-FITC (IM7.8.1) were from CALTAG Laboratories (Burlingame, CA, USA).
For intracellular staining with anti-CTLA-4-PE (UC10-4F10-11; BD Biosciences Pharmingen), 106 cells were first labelled with anti-CD25-FITC as described above. Then, cells were fixed, permeabilised and stained with anti-CTLA-4-PE using the Cytofix/Cytoperm™ Kit (BD Biosciences Pharmingen) according to the recommendations of the manufacturers.
Flow-cytometric analysis was performed on a FACScan flow cytometer with Cell Quest software (Becton Dickinson).
CD4+CD25- cells (5 × 104 per well) were cultured in U-bottomed 96-well plates (200 μl) with ACs (5 × 104 per well, 30 Gy γ-irradiated or treated with mitomycin-C (Sigma-Aldrich)), 3 μg/ml anti-CD3 and the indicated numbers of CD4+CD25+ cells for 48 hours at 37°C in 7% CO2. Cultures were pulsed for the last 16 hours with 1 μCi of [3H]TdR and harvested. The suppressive activity of the Treg cells can be presented by plotting the percentage of inhibition (100 × (Radioactivity in condition without Treg cells – Radioactivity in condition with Treg cells)/Radioactivity in condition without Treg cells) against the number of Treg cells.
DBA/1 mice were immunised with CII in CFA; 13 days after immunisation, the mice were treated every second day with 0.25 mg of anti-CD25 (PC61) or control IgG antibodies, for 4 weeks (injected intraperitoneally).
Forelimbs and hindlimbs were fixed in 10% formalin and decalcified with formic acid (31.5% (v/v) formic acid and 13% (w/v) sodium citrate). The paraffin sections were stained with haematoxylin and eosin.
Measurement of serum anti-CII antibodies
Blood samples were taken from the orbital sinus and were allowed to clot at room temperature for about 1 hour, and at 4°C overnight. Individual sera were tested by ELISA for antibodies directed against chicken CII. In brief, ELISA plates (Maxisorb; Nunc, Wiesbaden, Germany) were coated overnight at 4°C with native CII (1 μg/ml; 100 μl per well) in coating buffer (50 mM Tris-HCl, pH 8.5, 0.154 mM NaCl), followed by incubation for 2 hours with blocking buffer (50 mM Tris-HCl, pH 7.4, 0.154 mM NaCl and 0.1% caseine) to saturate non-specific binding sites. Serial twofold dilutions of the sera in assay buffer (50 mM Tris-HCl, pH 7.4, 154 mM NaCl and 0.05% Tween 20) were added and incubated for 2 hours at room temperature. The plates were then incubated for 2 hours with peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Finally, the substrate 3,3',5,5'-tetramethylbenzidine (Sigma-Aldrich) in reaction buffer (100 mM sodium acetate/citric acid, pH 4.9) was added for a 10 min incubation and absorbance was determined at 450 nm. Plates were washed five times between each step with PBS containing 0.05% Tween 20. A serial twofold dilution series of a purified standard was included to permit a calculation of the antibody content of each sample. The standard was purified by affinity chromatography from pooled sera obtained from various arthritic wild-type and IFN-γR KO mice.
Isolated CD4+CD25+ and CD4+CD25- cells were pelleted and directly used for total RNA isolation, using the Micro-to-Midi Total RNA Purification System (Invitrogen Life Technologies, Carlsbad, CA, USA). Total RNA (1 μg) was used for random primed cDNA synthesis with RAV-2 reverse transcriptase (Amersham, Aylesbury, Bucks., UK). The reaction mixture was incubated for 80 min at 42°C and the reverse transcriptase was inactivated by incubating the cDNA samples for 5 min at 95°C.
The cDNA samples were then subjected to real-time quantitative PCR, performed in the ABI prism 7700 sequence detector (Applied Biosystems, Foster City, CA) as previously described . The sequences of the forward (-FW) and reverse (-RV) primers and probes (-TP) for β-actin and Foxp3 were as follows: β-actin-FW, AGA GGG AAA TCG TGC GTG AC; β-actin-RV, CAA TAG TGA TGA CCT GGC CG T; β-actin-TP, CAC TGC CGC ATC CTC TTC CTC CC; Foxp3-FW, CCC AGG AAA GAC AGC AAC CTT; Foxp3-RV, TTC TCA CAA CCA GGC CAC TTG; Foxp3-TP, ATC CTA CCC ACT GCT GGC AAA TGG AGT C; TGF-β-FW, TGA CGT CAC TGG AGT TGT ACG G; TGF-β-RV, GGT TCA TGT CAT GGA TGG TGC; TGF-β-TP, TTC AGC GCT CAC TGC TCT TGT GAC AG. Probes were dual-labelled with 5'-FAM and 3'-TAMRA.
All primers and probes were designed with the assistance of the computer program Primer Express (AB) and were purchased from Eurogentec (Seraing, Belgium). The 5'-nuclease activity of the Taq polymerase was used to cleave a nonextendable dual-labelled fluorogenic probe. Fluorescent emission was measured continuously during the PCR reaction. PCR amplifications were performed in a total volume of 25 μl containing 5 μl of cDNA, 12.5 μl of Universal PCR Master Mix, no AmpErase UNG (AB), each primer at 100 to 300 nM, and the corresponding detection probe at 200 nM. Each PCR amplification was performed in triplicate wells under the following conditions: 94°C for 10 min, followed by 40 or 45 cycles at 94°C for 15 s and 60°C for 1 min. cDNA plasmid standards, consisting of purified plasmid DNA specific for each individual target, were used to quantify the target gene in the unknown samples, as described . All results were normalised to β-actin and/or hypoxanthine–guanine phosphoribosyltransferase (HPRT) to compensate for differences in the amount of cDNA in all samples. Results were similar whether β-actin or HPRT was used as the housekeeping gene.