A Genome Scan Using a Novel Genetic Cross Identifies New Susceptibility Loci and Traits in a Mouse Model of Rheumatoid Arthritis

Abstract

Proteoglycan-induced arthritis (PGIA) is a murine model for rheumatoid arthritis (RA) both in terms of its pathology and its genetics. PGIA can only be induced in susceptible mouse strains and their F2 progeny. Using the F2 hybrids resulting from an F1 intercross of a newly identified susceptible (C3H/HeJCr) and an established resistant (C57BL/6) strain of mouse, our goals were to: 1) identify the strain-specific loci that confer PGIA susceptibility, 2) determine whether any pathophysiological parameters could be used as markers that distinguish between nonarthritic and arthritic mice, and 3) analyze the effect of the MHC haplotype on quantitative trait loci (QTL) detection. To identify QTLs, we performed a genome scan on the F2 hybrids. For pathophysiological analyses, we measured pro- and antiinflammatory cytokines such as IL-1, IL-6, 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. We have identified four new PGIA-linked QTLs (Pgia13 through Pgia16) and confirmed two (Pgia5, Pgia10) from our previous study. All new MHC-independent QTLs were associated with either disease onset or severity. Comprehensive statistical analysis demonstrated that while soluble CD44, IL-6, and IgG1 vs IgG2 heteroantibody levels differed significantly between the arthritic and nonarthritic groups, only Ab-related parameters colocalized with the QTLs. Importantly, the mixed haplotype (H-2b and H-2k) of the C3H × C57BL/6 F2 intercross reduced the detection of several previously identified QTLs to suggestive levels, indicating a masking effect of unmatched MHCs.

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.…”

“…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. Sera were applied at increasing dilutions, and both total anti-PG antibodies and isotypes of PG-specific antibodies were determined using peroxidase-conjugated goat anti-mouse IgG (Accurate, Westbury, NY) or rat anti-mouse IgG1 or IgG2a (Zymed, San Francisco, CA) as secondary antibodies (23,24,37,39). Serum antibody levels were calculated relative to the corresponding mouse IgG isotype standards (all from Zymed) or purified mouse IgG (Sigma-Aldrich) (23,24,37,39).…”