Combined Autoimmune Models of Arthritis Reveal Shared and Independent Qualitative (Binary) and Quantitative Trait Loci

Abstract

Collagen-induced arthritis (CIA) and proteoglycan-induced arthritis (PGIA) are murine models for rheumatoid arthritis both in terms of their pathology and genetics. Using the F2 hybrids of the CIA-susceptible, but PGIA-resistant DBA/1 mice, and the CIA-resistant, but PGIA-susceptible BALB/c mice, our goals were to 1) identify both model-specific and shared loci that confer disease susceptibility, 2) determine whether any pathophysiological parameters could be used as markers that distinguish between nonarthritic and arthritic mice, and 3) analyze whether any immune subtraits showed colocalization with arthritis-related loci. To identify chromosomal loci, we performed a genome scan on 939 F2 hybrid mice. For pathophysiological analyses, we measured pro- and anti-inflammatory cytokines (IL-1, IL-6, TNF-α, IFN-γ, IL-4, IL-10, IL-12), Ag-specific T cell proliferation and IL-2 production, serum IgG1 and IgG2 levels of both auto- and heteroantibodies, and soluble CD44. In addition to multiple CIA- and PGIA-related loci identified in previous studies, we have identified nine new CIA- and eight new PGIA-linked loci. Comprehensive statistical analysis demonstrated that IL-2 production, T cell proliferation, and IFN-γ levels differed significantly between arthritic and nonarthritic animals in both CIA and PGIA populations. High levels of TNF-α, IFN-γ, IL-2, and Ab production were detected in F2 hybrids with CIA, whereas T cell proliferation, IL-2 and IFN-γ production, and a shift to IgG2a isotype were more characteristic of PGIA. Quantitative trait loci analysis demonstrated colocalization of numerous immune subtraits with arthritis-related traits. Quantitative trait loci on chromosomes 5, 10, 17, 18, and X were found to control arthritis in both models.

Induction of arthritis in HLA–DR4–humanized and HLA–DQ8–humanized mice by human cartilage proteoglycan aggrecan but only in the presence of an appropriate (non‐MHC) genetic background

“…Briefly, backcrossed hybrid males were selected first for the presence of both the HLA class II transgene and the Neo r gene, and then these positive males were screened for the BALB/c background using Ͼ150 polymorphic markers (17,24,37). Males with the most extensive BALB/c background were used for the next backcross with BALB/c females.…”

“…Antigen-specific T cell responses were measured in quadruplicate samples (3 ϫ 10 5 cells/well) cultured in the presence of 50 g of PG protein/ml or 25 g/ml of synthetic peptide. Antigen-specific IL-2 production was measured in 48-hour supernatants by the CTLL-2 bioassay, and T cell proliferation was assessed on day 5 by measuring the incorporation of 3 H-thymidine (23,24,(37)(38)(39). The antigen-specific T cell response (either direct T cell proliferation or the response of CTLL-2 cells to IL-2 in the supernatants) was expressed as a stimulation index, which represents the ratio of the counts per minute of incorporated 3 H-thymidine in antigen-stimulated cultures to the cpm in nonstimulated cultures.…”

“…The antigen-specific T cell response (either direct T cell proliferation or the response of CTLL-2 cells to IL-2 in the supernatants) was expressed as a stimulation index, which represents the ratio of the counts per minute of incorporated 3 H-thymidine in antigen-stimulated cultures to the cpm in nonstimulated cultures. Antigen-specific production of interferon-␥ (IFN␥), IL-4, and tumor necrosis factor ␣ (TNF␣) was measured in cell culture supernatants (3 ϫ 10 6 cells/ml) on day 4, using enzyme-linked immunosorbent assays (ELISAs; BD Biosciences, San Diego, CA, or R&D Systems, Minneapolis, MN) as described previously (23,24,37).…”

“…PG-specific antibodies were also measured by ELISA as described previously (23,24,37,39). Briefly, Maxisorp immunoplates (Nunc, Roskilde, Denmark) were coated with human or mouse cartilage PG (0.1 g of protein/well), and free binding sites were blocked with 1% fat-free milk in PBS.…”

“…17). As with RA, PGIA is polygenic, and both MHC and non-MHC components are critical to the development of arthritis (23,24). Many of the non-MHC susceptibility loci identified in PGIA correspond to human chromosome regions where quantitative trait loci have also been found in family studies of RA (17).…”