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Objective: To study the immunologic function and determine the fine epitope structure of a synthetic peptide p135H ((2373)TTYKRRLQKRSSRHP) of the G3 domain of human cartilage proteoglycan (aggrecan), which contains a highly homologous sequence motif of the shared epitope (QKRAA), the most common sequence motif in HLA-DR4 alleles, which predispose humans to the development of rheumatoid arthritis (RA)., Methods: Synthetic p135 peptides with altered sequences were used for (hyper)immunization of arthritis-susceptible BALB/c mice and then challenged with a single dose of cartilage proteoglycan. Human p135 (p135H) and mouse p135 (p135M) synthetic peptides of the G3 domain of aggrecan were used to prime lymphocytes, which were then used for adoptive transfer of arthritis into "presensitized" SCID mice, determining cross-reactivity among p135 peptides and their analogous sequences, and generating T cell hybridomas. T cell hybridomas were also used for arthritis transfer into SCID mice and for characterizing the fine epitope structure of T cell receptor (TCR) and major histo-compatibility complex (MHC) binding sites of the immunogenic/arthritogenic p135H sequence., Results: While p135H peptide-(hyper)immunized mice became sensitized, they developed arthritis only after injection of a single dose of cartilage proteoglycan aggrecan. An altered peptide sequence (p135H-AA) carrying the shared epitope motif (QKRAA) was as effective as the natural peptide p135H sequence for inducing arthritis. Mouse p135M-specific lymphocytes induced arthritis with a lower incidence, but synthetic peptides to Escherichia coli heat-shock protein (DnaJ) or HLA-DR4 allele (both having the shared epitope sequence with different flanking regions) were also positive. Fine epitope sequence recognition of an arthritogenic T cell hybridoma derived from p135H-primed lymphocyte population was determined. Interestingly, in the most central position, a basic amino acid triplet of p135H peptide was found to be the MHC-binding motif, whereas the flanking amino acids bound to the TCR., Conclusion: Peptide p135H, corresponding to the peptide sequence in the G3 domain of human cartilage proteoglycan aggrecan, is immunogenic/arthritogenic in BALB/c mice. Peptide p135H includes a highly homologous motif of the shared epitope, a sequence that is overrepresented in bacterial heat-shock proteins, envelope protein of human JC polyomavirus, and numerous HLA-DR4 alleles. Since the G3 domain of cartilage proteoglycan aggrecan with the p135 sequence is "lost" during the normal metabolic turnover of cartilage proteoglycan or in pathologic conditions, an antigenoriented T cell migration into joints of presensitized (susceptible) individuals may contribute to the organ-specificity of RA.

Objective: To explore the effect of sex on clinical and immunologic traits in major histocompatibility complex-matched (H-2d) F(2) hybrid mice with proteoglycan (PG)-induced arthritis and to identify how the quantitative trait locus (QTL) on the X chromosome influences the onset QTL of another chromosome., Methods: (BALB/c x DBA/2)F(2) hybrid mice were immunized with cartilage PG, and a genome-wide linkage analysis was performed using >200 simple sequence-length polymorphic markers. The major clinical traits (susceptibility, onset, and severity) were assessed, and PG-specific T and B cell responses, and the production of proinflammatory and antiinflammatory cytokines (tumor necrosis factor alpha, interleukin-1 [IL-1], IL-6, interferon-gamma, IL-4, IL-10, and IL-12) were measured in 133 arthritic and 426 nonarthritic female and male F(2) hybrid mice. The major clinical and immunologic traits were linked to genetic loci, and potential linkages among these QTLs and the effect of sex were analyzed., Results: Thirteen QTLs reported in previous studies were confirmed. Binary traits (susceptibility to arthritis) and disease onset were female specific and were identified on chromosomes 3, 7, 10, 11, 13, and X. QTLs for disease severity were mostly male specific and were located on chromosomes 1, 4, 5, 8, 14, 15, and 19. In addition, we identified 4 new QTLs for the onset of arthritis on chromosomes 3, 4, and 11, and 1 new QTL for severity on chromosome 14; all showed a strong gender association. A locus on the X chromosome interacted with a QTL on chromosome 10, and these 2 loci together seemed to control disease incidence and onset. Most of the clinical traits (QTLs) shared common regions with the immunologic traits and frequently showed a locus-locus interaction., Conclusion: Numerous immunologic QTLs overlap with clinical QTLs, thus providing information about possible mechanisms underlying QTL function. Disease susceptibility and onset showed predominant linkage with the female sex, under the control of a QTL on the X chromosome, while the severity QTLs were more strongly linked to the male sex.