Bovine cii

All animal experiments were performed conform national guidelines and the study protocol was approved by the Ethical Committee for Animal Experimentation (Dier Experimentele Commissie; DEC) of Leiden University. DBA/1J mice were obtained from our own breeding colony (originally obtained from Charles River). C57Bl/6 mice were purchased from Charles River. CIA was induced in 8–10 week old mice by injection at the tail base with 100 µg of CII emulsified in complete Freunds adjuvant (CFA). On day 21 the mice received a subcutaneous boost with 100 µg of CII in incomplete Freunds adjuvant (IFA; Sigma-Aldrich). For DBA/1J mice bovine CII (Chondrex) and CFA containing 0.5 mg/ml of M.tuberculosis (Difco) were used and for C57Bl/6 mice chicken CII (Chondrex) and CFA containing 2.5 mg/ml of M.tuberculosis (Chondrex) were used. For the CFA only immunizations PBS was used instead of CII. Arthritis severity was monitored as described earlier using a clinical score with a maximum of 15 per paw [17] (link). For the kinetics experiments, mice were bled before immunization, on day 7, 14, 21, 28, 35, 49 after immunization and at the end of follow up. Blood was centrifuged and serum was harvested and stored at −80°C until use.

Antibodies against CII were detected by coating 96-well plates with bovine CII (Chondrex) at the concentration 1 ug/ml. Samples were serial diluted and incubated for 2 h in RT following blocking with 0.5% BSA. Secondary antibodies were purchased from Jackson Immunoresearch. Plates were developed with TMB substrate reagent set (BD), stopped with 1 M H₂SO₄ and read at 450 nm.

We injected DBA/1 mice intradermally at the tail base with 200 µg of bovine CII (Chondrex Inc., Woodinville, WA, USA) emulsified in CFA (Sigma Aldrich, St. Louis, MO, USA). Twenty-one days later, the mice were equally boosted with CII emulsified in IFA. After a random division of the immunized mice, we injected the mice i.p. with either TAPBPL-Ig or control Ig protein (25, or 50 µg) every 3 days for 30 days beginning from day 21. We recorded the onset of CIA and individual clinical scores. The swelling of four paws was scored from 0 to 4: grade 0, no sign of erythema and swelling; grade 1, erythema and slight swelling limited to tarsals or ankle joint; grade 2, erythema and slight swelling from ankle to tarsals; grade 3, erythema and moderate swelling from ankle to metatarsal joints; and grade 4, erythema and severe swelling involving the ankle, foot, and digits, or ankylosis of the limb [5 (link)]. Grading was performed for each paw, and the four scores were summed; the maximum score/mouse was 16. We euthanized the CIA mouse if (1) weight loss was ≥25%, (2) they were unable to eat or drink, (3) there was evidence of joint swelling >50% of baseline, (4) there was evidence of self-trauma to the joint, (5) excessive skin breakdown occurred in arthritic joints, or (6) the general health of the mouse was at a level requiring euthanasia for humane reasons.

The induction of CIA was described previously [58 (link)]. Briefly, 6 week-old DBA/1 J male mice (Shanghai SLAC Laboratory Animal Co.,Ltd) were immunized at the base of the tail with 200 µg bovine CII (Chondrex, USA) emulsified with CFA (Chondrex, USA), and boosted by immunization on day 21 in the same manner. The animal experiments were performed in accordance with the guidelines approved by Institutional Animal Care and Use Committee of Nanjing Medical University. The arthritis of mice was optically evaluated by using laser speckle imaging equipment (moorFLPI V5.0, Moor Instruments Ltd, UK).

Serum samples were collected on days 7, 14, and 21 post immunization, and the titers of anti-CII IgG Abs were measured by ELISA. Bovine CII (1 μg/mL, Chondrex, Inc., Redmond, WA, USA) was coated onto microtiter plates (Maxisorp; Nunc, Roskilde, Denmark) overnight at 4 °C. After blocking with 1% BSA in PBS, serially diluted serum samples were added and incubated for 1 h at room temperature. After washing, HRP-conjugated rabbit anti-mouse IgG1, IgG2a, or IgG2b Ab (Zymed Laboratories, San Francisco, CA) was added and incubated for 2 h at 37 °C. After washing, Ab binding was visualized using o-phenylenediamine (Sigma-Aldrich). A standard serum composed of a mixture of sera from arthritic mice was added to each plate in serial dilutions, and a standard curve was constructed. The standard serum was defined as 1 U, and the Ab titers of serum samples were determined by the standard curve.

Collagen-induced arthritis was performed as referred in Kim et al. [21 (link)]. At day 0, DBA/1 male mice (age 6 weeks), obtained from Janvier (Le Genest Saint Isle) were injected subcutaneously at the tail base with 100 μg of bovine CII (Chondrex) emulsified in complete Freund’s adjuvant (CFA). A booster injection of 100 μg of CII in incomplete Freund’s adjuvant (IFA) was performed subcutaneously at day 21. All animal experiments were conducted in accordance with institutional guidelines. The agreement numbers of the animal facilities are B76-450-05, B76-540-7 and experimentator is A.76-91. This study has been approved by the ethics committee (Comité Regional d’éthique en Expérimentation Animale Normandie) with the following numbers N/02-12-10/24/12-13.

The induction of CIA was described previously26 (link). Briefly, male 6 wk-old DBA/1J (purchased from purchased from the Shanghai Laboratory Animal Center, Chinese Academy of Science) mice were injected intradermally at the base of the tail with 200 μg of bovine CII (Chondrex, 20021) emulsified with complete Freund adjuvant, and their arthritis manifestations were graded and scored as described26 (link). For standard histologic assessment, hind paws were fixed overnight in 4% paraformaldehyde and decalcified using EDTA. The material was then embedded in paraffin, cut and 1–2 μm sections were stained with H&E. The histological arthritis score was determined in a blinded fashion for changes in synovial lining, cellular infiltrate, cartilage damage, and pannus, as previously described27 (link).