Antigen, mice and immunization
High-density cartilage proteoglycan (aggrecan) was purified from human cartilage, by CsCl gradient centrifugation, and depleted of glycosaminoglycan side chains as described previously . Female BALB/c mice (National Cancer Institute, Friedrich, MD, USA, or The Jackson Laboratory, Bar Harbor, ME, USA) and CD28-deficient mice (The Jackson Laboratory) were immunized intraperitoneally with cartilage proteoglycan (100 μg protein) in complete Freund's adjuvant on day 0, and boosted with proteoglycan in incomplete Freund's adjuvant on days 21 and 42. Female SCID mice on a BALB/c background (NCI/NCrC.B-17-scid/scid), aged 8–12 weeks or young retired breeders, were purchased from the National Cancer Institute and maintained under germ-free conditions.
Assessment of arthritis
A standard scoring system, based on swelling and redness of each paw, was used for the assessment of the severity of disease. The first clinical symptom of swelling was recorded as the time of onset of arthritis. Joint swelling was scored (ranging from 0 to 4 in each paw) and expressed as acute cumulative arthritis score, resulting in a possible maximum severity score of 16. Typically, in the primary form of PGIA, BALB/c mice developed swelling and redness in one or more limbs 7–14 days after the third injection with proteoglycan and adjuvant. In the transfer system, recipient SCID mice developed a more uniform disease from day 10 after transfer, with involvement of nearly all peripheral joints.
Flow cytometry analysis
The CD4+CD25+-expressing T cells were identified by staining spleen cells with fluorescein isothiocyanate (FITC)-labeled anti-CD4 and biotin-labeled anti-CD25 followed by CyChrome-labeled streptavidin (BD PharMingen, San Diego, CA, USA), and analyzed on a flow cytometer (Beckton Dickinson, San Jose, CA, USA). For analysis of intracellular CTLA-4, spleen cells were first stained with the above fluorescence-labeled antibodies, fixed and rendered permeable, and stained with R-phycoerythrin (PE)-labeled anti-CTLA-4 using a Cytofix/Cytoperm kit (BD PharMingen).
Purification of CD4+CD25+ and CD4+CD25- T cells and cell transfer in SCID mice
The CD4+CD25+ T cells were purified using a protocol described previously . Briefly, CD4+ T cells were isolated from spleens of 10- to 12-week-old, naïve, BALB/c mice by negative selection using CD4-enrichment columns (R&D System, Minneapolis, MN, USA). Purified CD4+ T cells were incubated with PE-labeled anti-CD25, and the stained cells were incubated with anti-PE microbeads (Miltenyi Biotech Inc., Auburn, CA, USA). The CD25+ cells were selected on an LS+ column (Miltenyi Biotech Inc.). The purity of the CD4+CD25+ T cells was approximately 90%. The CD4+CD25- T cells, which did not bind to magnetic beads, were collected from the flow through the washing steps (purity >98%). A total of 5 × 105 CD4+CD25+ or CD4+CD25- T cells were injected intraperitoneally, along with 1 × 107 spleen cells from arthritic animals that were depleted of CD25+ cells and 100 μg proteoglycan, into SCID mice.
Depletion of CD4+CD25+ T cells and cell transfer
Spleen cells from arthritic animals were depleted of CD25+ cells using negative selection as described above (0.5 μg PE-labeled anti-CD25 per 1 × 106 cells). The depletion of CD4+CD25+ cells was detected by flow cytometry. Typically, less than 0.3% of the depleted cells expressed CD25. a total of 1 × 107 CD25+ and CD25- spleen cells, together with 100 μg proteoglycan, were injected intraperitoneally into SCID mice.
In vitro suppression assay
CD4+CD25+ T cells (1 × 104) isolated from naïve BALB/c mice were mixed with CD4+CD25- T cells (5 × 104) from arthritic mice in the presence of irradiated syngenic splenocytes as antigen-presenting cells (7.5 × 104) with 1 μg/ml soluble anti-CD3 for 3 days or 50 μg/ml proteoglycan for 5 days, and T-cell proliferation was determined by [3H]thymidine incorporation.
Measurements of antigen-specific T-cell responses, antibodies, and cytokine production
Antigen-specific T-cell responses were measured in quadriplicate samples of spleen cells (3 × 105 cells/well) cultured in the presence of 50 μg proteoglycan protein/ml. T-cell proliferation was assessed on day 5 by [3H]thymidine incorporation [14, 22]. Antigen (proteoglycan)-specific interferon-γ, IL-10, and IL-4 production by T cells was determined in media collected on day 4 using capture enzyme-linked immunosorbent assays (ELISAs) from BioSource International, Inc. (Camarillo, CA, USA). For induction of TGF-β1, spleen cells were cultured in serum-free medium X-Vivo-20 (BioWhittaker, Inc., Walkersville, MD, USA) in the presence of proteoglycan. Total TGF-β1 was measured after acidification to activate latent TGF-β, followed by neutralization using an ELISA kit from Promega (Madison, WI, USA).
Maxisorp immunoplates (Nunc, Rochester, NY, USA) were coated with human or mouse cartilage proteoglycans (0.1 μg protein/100 μl per well) for ELISA, and free binding capacity of the wells was blocked by 1% fat-free milk in phosphate-buffered saline [23–25]. Sera were applied at increasing dilutions and isotypes of proteoglycan-specific antibodies were determined using peroxidase-conjugated rat antimouse IgG1 or IgG2a secondary antibodies (Zymed, South San Francisco, CA, USA) as previously described [24, 26]. Serum antibody levels were calculated according to mouse IgG1 and IgG2a standards. Mouse IgG1 and IgG2a standards were purified by sepharose-coupled protein G from irrelevant (non-proteoglycan-specific) monoclonal antibody-containing ascites fluids and coated directly onto the microplate's surface.
Statistical analysis was performed using SPSS v7.5 (SPSS, Chicago, IL, USA). The Mann–Whitney and Wilcoxon tests were used for intergroup comparisons. P < 0.05 was considered statistically significant.