Induction of oral tolerance in DBA/1 mice
DBA/1 mice used in this study were fed either with 100 μg bovine CII (a kind gift from Prof. Andrew Kang, University of Tennessee) dissolved in 0.05 N acetic acid at 2 mg/ml (50 μl solution plus 150 μl acetic acid) or with an equal volume of phosphate-buffered saline (PBS) using an oral Zonde needle (Natsume, Japan) every 2 days for 2 weeks. All experimental procedures were examined and approved by the Animal Research Ethics Committee at The Catholic University of Korea.
Induction of CIA and evaluation of arthritis
Bovine CII was dissolved in 0.05 N acetic acid at 2 mg/ml and emulsified with an equal volume of Freund's complete adjuvant (CFA). As primary immunization, 0.1 ml of the emulsion, containing 100 μg CII, was injected into the tails of DBA/1 mice (both tolerized mice and nontolerized control mice; n
= 6 per group). Two weeks later, a booster injection consisting of 200 μg CII similarly dissolved and emulsified 1:1 with incomplete Freund's adjuvant was injected into a hind leg. Starting from 2.5 weeks (18 days) after primary immunization, three independent observers examined the degree of arthritis three times a week for up to 11 weeks. The severity of arthritis was represented as mean arthritic index on a 0–4 scale according to the following criteria [11
]: 0 = no oedema or swelling; 1 = slight oedema and erythema limited to the foot and/or ankle; 2 = slight oedema and erythema from the ankle to the tarsal bone; 3 = moderate oedema and erythema from the ankle to the tarsal bone; and 4 = oedema and erythema from the ankle to the entire leg. The sum of values from three legs, excluding the hind leg into which CII/incomplete Freund's adjuvant was injected, was determined at the time of the second injection. The final values presented in the Results section represent an average of the indices recorded by three independent observers. All experimental procedures were examined and approved by the Animal Research Ethics Committee at The Catholic University of Korea.
Analysis of IgG antibody subtypes
Blood samples obtained from each mouse at 3, 5 and 7 weeks after primary immunization were used to investigate IgG antibody subtype concentrations using the mouse IgG1/IgG2a ELISA quantitation kit (Bethyl Lab Co., Montgomery, TX, USA). Levels of IgG1 and IgG2a were measured in mice sera diluted 50,000- to 400,000-fold.
Determination of collagen-specific T-cell proliferative response
The draining lymph nodes and the spleen were removed from each mouse and washed twice with PBS. Tissues were minced and the cells were filtered through a cell strainer and centrifuged at 1500 rpm at 4°C for 10 min. The cell pellet was resuspended in RPMI-1640 medium to a concentration of 1 × 105 cells/ml. Cells were than plated in 96-well microtitre plates at a concentration of 2 × 105 cells/well concentration and cultured with 40 μg/well CII in 0.3 ml Click's medium supplemented with 0.5% mouse serum for 3 days. The same amount of ovalbumin was added instead of CII to control wells. Eighteen hours before the termination of culture, 0.5 μCi [3H]thymidine (NEN, Boston, MA, USA) was added to each well. Cells were harvested onto glass fibre filters and counted on a Matrix-96 direct ionization β counter (Packard Instrument Co., Downers Grove, IL, USA). The degree of T-cell proliferation is presented as the stimulation index, which is calculated by dividing the counts/min in the presence of CII by the counts/min in the presence of ovalbumin.
Analyses of cytokine production by ELISA
Mononuclear cells were isolated from the Peyer's patch and spleen, and cultured at a density of 0.5 × 106 cells/ml in flat-bottomed, 48-well tissue culture plates (Corning, Corning, NY, USA). After 2 days, culture supernatants were harvested and stored at -70°C. To determine the amount of IL-10 and TGF-β in each supernatant, 96-well ELISA plates were coated with rat antimurine IL-10 and TGF-β monoclonal antibodies (R&D Systems, Minneapolis, MN, USA) for 24 hours at 4°C. After incubating with blocking agents, the plates were incubated with previously collected supernatants for 1 hour at room temperature. The plates were then washed and incubated for 1 hour at room temperature with biotin-conjugated rat antimurine IL-10 and TGF-β monoclonal antibodies, followed by an alkaline phosphatase-conjugated goat antibiotin monoclonal antibody (R&D Systems). The fluorescent substrate for alkaline phosphatase (R&D Systems) was used for colour development, and fluorescence was measured using a microtitre plate reader (Dynex, Chantilly, VA, USA) at excitation and emission wavelengths of 450 nm. The amounts of cytokines present in test samples were determined from standard curves established with serial dilutions of recombinant murine IL-10 and TGF-β.
Fluorocytometric analysis of T cells
Single-cell suspensions were prepared from each lymphoid organ and cultured in 24-well plates at a concentration of 1 × 106/well with or without 40 μg/well CII for 3 days. Golgi Stop (Pharmingen, San Diego, CA, USA) was added 4 hours before the termination of culture. Cells were subsequently washed and resuspended in fluorescence-activated cell sorting (FACS) staining buffer (PBS plus 0.1% bovine serum albumin plus 0.09% sodium azide), and probed with anti-CD4-perCP and/or anti-CD25-FITC (Pharmingen) for 30 min at 4°C. Next, cells were fixed with cytoperm/cytofix (Pharmingen) for 20 min and probed for intracellular cytokines using phycoerythrin-labelled anti-IL-10 antibody, anti-IFN-γ antibody, or isotype control antibody (Pharmingen) for 30 min at room temperature. Finally, cells were washed with PBS and analyzed on a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA, USA).
Experimental findings are presented as mean ± standard deviation. Statistical significance was determined by Student's t-test using the SPSS program (version 10.0; SPSS Inc, Chicago, IL, USA). P < 0.05 was considered statistically significant.