Animals
The generation and characterization of transgenic hCD2-ΔkTβRII mice is described elsewhere [11]. In these mice, impaired TGFβ-signalling in T cells was shown to be similar to that in other models reported [13, 14]. All transgenic lines were established and maintained as heterozygotes on an FVB/N background. FVB/N mice are naturally resistant to the induction of CIA [15]. Therefore, hCD2-ΔkTβRII mice were crossed with DBA/1 mice (Charles River, Sulzfeld, Germany). The male F1 generation was genotyped using PCR as described elsewhere [11] and included in the experiments at 6 to 12 weeks of age. Nontransgenic male littermates were used as controls. In four separate experiments, 49 transgenic and 29 wild-type F1 mice were included in the analysis of acute arthritis. An additional 14 transgenic and 17 wild-type mice were included in the analysis of the chronic phase of disease. Animal care was in accordance with governmental and institutional guidelines.
Induction of CIA
Chicken collagen type II (CII) (Sigma, Deisenhofen, Germany) was dissolved and stored in 0.01 M acetic acid at 4 mg/ml. Wild-type and transgenic F1 mice were injected intradermally with 100 μg of CII emulsified in complete Freund's adjuvant (charge H37Ra) (Difco, Detroit, MI, USA) in both ears (25 μg each) and the base of the tail (50 μg). A booster injection of 100 μg CII in 100 μl PBS was given intraperitoneally 21 days later. Arthritis usually developed within the first week after the booster injection.
Clinical arthritis scoring
Mice were scored every two to three days in the acute phase and once a week in the chronic phase of arthritis, and grades ranging from 0 to 4 were allotted to each limb: grade 0, no visible abnormalities; grade 1, mild redness or swelling of wrist or up to three inflamed digits; grade 2, more than three inflamed digits or moderate redness and swelling of ankle or wrist; grade 3, severe ankle and wrist inflammation; grade 4, extensive ankle and wrist inflammation including all digits, or new bone formation with reduced motion. A maximum score of 16 could be achieved for each mouse.
Histological assessment
For the analysis of acute arthritis, anesthetized mice were killed by cervical dislocation when no further clinical deterioration occurred, which was within the first six weeks after the onset of arthritis. For the analysis of the healing phase of arthritis, mice were observed up to 24 weeks after arthritis induction. After removal of draining lymph nodes, all four limbs of mice with a clinical arthritis score of at least grade 1 were removed. Specimens were fixed in formalin and decalcified in 10% Tris-buffered EDTA (pH 7.3) for 24 to 72 hours using standard methods. Sections 5 μm thick were cut and stained with hematoxylin and eosin.
The histological arthritis score was determined in a blinded fashion for inflammatory and degenerative changes and graded from 0 and 3 for each limb as follows:
Synovial lining – grade 0, no changes; grade 1, localized monolayer cubical transformation; grade 2, localized multilayer cubical transformation; grade 3, multilayer synovial lining with extensive necrosis
Cellular infiltrate – grade 0, no changes; grade 1, few focal infiltrates; grade 2, extensive focal infiltrates; grade 3, extensive infiltrates invading the capsule with aggregate formation
Cartilage – grade 0, no changes; grade 1, superficial, localized cartilage degradation in more than one region; grade 2, localized deep cartilage degradation; grade 3, extensive deep cartilage degradation at several locations
Pannus – grade 0, no changes; grade 1, pannus formation at up to two sites; grade 2, pannus formation at up to four sites, with infiltration or flat overgrowth of joint surface; grade 3, pannus formation at more than four sites or extensive pannus formation at two sites.
Of the four limbs analyzed per animal, the maximum score for each category was used. Therefore, a maximum score of 12 could be reached per animal.
Cell culture and cell proliferation assay
Popliteal and axillary draining lymph nodes were removed and ground through a 40-μm nylon mesh. Cells were cultivated in RPMI 1640 medium (Biochrom, Berlin, Germany) containing 5% fetal calf serum supplemented with penicillin (100 U/ml) and streptomycin (100 μg/ml) (Life Technologies, Eggenstein, Germany). 2 × 106 cells/ml were plated and incubated with 50 μg/ml of CII or costimulated with anti-CD3/CD28 antibodies at 37°C in a water-saturated atmosphere with 5% CO2 in air. For costimulation, plates were precoated with 10 μg/ml antimouse CD3 monoclonal antibodies (BD Pharmingen, Heidelberg, Germany) in 0.1 M sodium phosphate buffer, pH 8.5, overnight at 4°C, and 10 μg/ml antimouse CD28 monoclonal antibodies (BD Pharmingen) was then added to the medium. Supernatants were collected after 48 hours and frozen in liquid nitrogen. For proliferation assays, cells were seeded at 5 × 105 cells per well in 96-well flat-bottomed plates (Greiner Bio-One, Frickenhausen, Germany) in RPMI medium. Cells were incubated for 48 hours and pulsed with 0.25 μCi/well 3H-thymidine (37 MBq/ml) for the last 16 hours of culture. Samples were harvested and counted in a Betaplate liquid scintillation counter (Wallac, Freiburg, Germany).
ELISA
Cytokine levels of IL-2, IL-4, IL-5, IL-6, IL-10, tumour necrosis factor α (TNFα), and IFNγ in supernatants were measured using Mouse BD OptEIA ELISA Sets (BD Pharmingen) in accordance with the manufacturer's instructions.
Statistical analysis
Means ± SEM are given. For comparison of groups, the two-sided Mann–Whitney rank sum test was applied. A value of P < 0.05 was considered significant.