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SummaryImmature dendritic cells (iDCs) have a tolerogenic potential due to low expression of important co-stimulatory cell surface molecules required for antigen presentation and induction of an effective immune response. We report here that injection of iDCs pulsed with chick type II collagen (CII) delayed the onset significantly and suppressed the severity of spontaneous polychondritis (SP) in the human leucocyte antigen (HLA)-DQ6αβ8αβ transgenic mouse model. Bone marrow-derived iDCs were pulsed in vitro with CII and transferred into 6-week-old HLA-DQ6αβ8αβ transgenic mice. Mice receiving CII-pulsed iDCs did not display any clinical signs of disease until 5·5 months of age, indicating the ability of the DC vaccine to delay significantly the onset of SP. Control groups receiving unpulsed iDCs or phosphate-buffered saline (PBS) developed polyarthritis at 3·5 months, as we have reported previously. The severity and incidence of disease was reduced in mice injected with CII-pulsed iDCs. Proinflammatory cytokines were in low to undetectable levels in the serum and tissue in the CII-pulsed iDC mice, correlating with the protection. This is the first evidence of iDC therapy controlling SP and suggests that iDC vaccination may provide a tool to reducing clinical manifestations in human inflammatory autoimmune disease such as relapsing polychondritis and rheumatoid arthritis.

In humans, HLA-DR alleles sharing amino acids at the third hypervariable region with DRB1*0401(shared epitope) are associated with a predisposition to rheumatoid arthritis, whereas DRB1*0402 is not associated with such a predisposition. Both DRB1*0402 and DRB1*0401 occur in linkage with DQ8 (DQB1*0302). We have previously shown that transgenic (Tg) mice expressing HLA-DRB1*0401 develop collagen-induced arthritis. To delineate the role of “shared epitope” and gene complementation between DR and DQ in arthritis, we generated DRB1*0402, DRB1*0401.DQ8, and DRB1*0402.DQ8 Tg mice lacking endogenous class II molecules, AE°. DRB1*0402 mice are resistant to develop arthritis. In double-Tg mice, the DRB1*0401 gene contributes to the development of collagen-induced arthritis, whereas DRB1*0402 prevents the disease. Humoral response to type II collagen is not defective in resistant mice, although cellular response to type II collagen is lower in *0402 mice compared with *0401 mice. *0402 mice have lower numbers of T cells in thymus compared with *0401 mice, suggesting that the protective effect could be due to deletion of autoreactive T cells. Additionally, DRB1*0402 mice have a higher number of regulatory T cells and show increased activation-induced cell death, which might contribute toward protection. In DRB1*0401.DQ8 mice, activated CD4+ T cells express class II genes and can present DR4- and DQ8-restricted peptides in vitro, suggesting a role of class II+ CD4 T cells locally in the joints. The data suggest that polymorphism in DRB1 genes determines predisposition to develop arthritis by shaping the T cell repertoire in thymus and activating autoreactive or regulatory T cells.

Objective To generate a mouse model that can mimic human rheumatoid arthritis (RA). A major difference between RA in humans and collagen-induced arthritis (CIA) in mice is the lack of sex bias and autoantibodies in the animal model. We used DRB1*0401-transgenic mice to understand the role of DR4 in susceptibility and sex bias in RA. Methods A transgenic mouse was generated that lacked all endogenous mouse class II genes (AEo) and expressed the RA susceptibility allele HLA–DRB1*0401. These transgenic mice were tested for incidence, severity, and sex distribution of CIA. Results DRB1*0401.AEo mice developed CIA predominantly in females and produced rheumatoid factors, similar to the features of human RA. Another feature similar to human RA is the expression of class II molecules on antigen-presenting cells as well as T cells. Activated and sorted CD4+ T cells can present DR4-restricted type II collagen (CII)–derived peptide in vitro, but cannot process the antigen. This suggests a role for these cells in epitope presentation locally in joints, which affects disease severity. After challenge with CII, female mice had higher cellularity and increased T cell proliferation and produced higher levels of proinflammatory cytokines than did the male mice. Conclusion DR4.AEo mice expressed HLA similar to humans and displayed increased arthritis susceptibility in females, thus mimicking RA in humans. This model may be valuable for studying sex differences observed in humans and for understanding why autoimmunity is increased in women. These mice may also be useful for developing future therapeutic strategies.

Cia5 is a locus on rat chromosome 10 which regulates the severity of collagen- and pristane-induced arthritis (CIA and PIA). To refine the region toward positional identification, Cia5 subcongenic strains were generated and studied in PIA and CIA. The protective effect of the telomeric locus Cia5a was confirmed in both models. A second arthritis severity locus (Cia5d) was identified within the most centromeric portion of Cia5. DA.F344(Cia5d) rats had a significantly lower median arthritis severity index in PIA, but not in CIA, compared with DA. On histologic analyses DA.F344(Cia5a) and DA.F344(Cia5d) congenics with PIA preserved a nearly normal joint architecture compared with DA, including significant reduction in synovial hyperplasia, pannus, angiogenesis, inflammatory infiltration, bone and cartilage erosions. Cia5 and Cia5a synovial levels of IL-1β mRNA were reduced. Although both DA.F344(Cia5) and DA.F344(Cia5a) rats were protected in CIA, the arthritis scores of DA.F344(Cia5) were significantly higher than those of DA.F344(Cia5a), suggesting the existence of a third locus where F344-derived alleles centromeric from Cia5a contribute to increased arthritis severity. The existence of the third locus was further supported by higher levels of autoantibodies against rat type II collagen in DA.F344(Cia5) congenics compared with DA.F344(Cia5a). Our results determined that Cia5 contains three major arthritis severity regulatory loci regulating central events in the pathogenesis of arthritis, and differentially influencing CIA and PIA. These loci are syntenic to regions on human chromosomes 17q and 5q implicated in the susceptibility to rheumatoid arthritis, suggesting that the identification of these genes will be relevant to human disease.

Objective To construct rats congenic for the chromosome 2 arthritis-regulatory quantitative trait locus Cia10, originally identified in a (DA × ACI)F2 intercross rat strain that had been assessed for collagen-induced arthritis (CIA), and to determine the effect of this congenic interval on arthritis severity, joint histologic structure, and cytokine transcription in rats with pristane-induced arthritis (PIA). Methods A 52.6-MB interval derived from the ACI (CIA- and PIA-resistant) strain and containing the Cia10 interval was introgressed into the DA (arthritis-susceptible) background through genotype-guided congenic breeding. Homozygous male and female DA.ACI(Cia10) congenic rats were studied for their susceptibility to and severity of PIA, and were compared with same-sex DA rats. Histologic analyses were done on hind paws collected on day 32 following the pristane injection. Levels of interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) messenger RNA (mRNA) were measured with real-time polymerase chain reaction on synovial tissues from day-32 ankles. Results Both male and female DA.ACI(Cia10) congenic rats developed a significantly milder form of arthritis, with a 95% and 92% reduction in the arthritis severity index compared with DA male and female controls, respectively (males P ≤ 0.001 and females P = 0.003). DA.ACI(Cia10) congenic rat synovial tissue was more likely to preserve its normal histologic architecture, including minimal to no cartilage and bone erosions, synovial hyperplasia, and pannus formation, and reduced numbers of vessels (angiogenesis), when compared with DA synovial tissue. There was a 2.7- and 2.4-fold reduction in the amount of IL-1β and TNFα mRNA, respectively, in the synovial tissue of DA.ACI(Cia10) congenic rats compared with DA rats. Sequencing analyses of complementary DNA for the Cia10-predicted candidate gene Ptpn8, the rat homolog of the rheumatoid arthritis (RA)–susceptibility gene PTPN22, revealed no polymorphisms between the DA and ACI strains. Conclusion This study determined that Cia10 harbors a major autoimmune arthritis–regulatory gene. This gene regulates clinical disease severity, histologic damage, and the levels of at least two central proinflammatory cytokines. We are in the process of narrowing down the critical region for positional cloning of the Cia10 gene. The identification of this gene will provide novel targets or pathways for focused candidate-gene studies in RA.

Relapsing polychondritis is a multisystem autoimmune disease involving cartilage destruction but no known causative antigen. HLA-DQ8 has been associated with various autoimmune diseases in humans. To study the role of DQ8in autoimmune diseases, we have generated transgenic mice expressing DQ8 (DQA1*0301, DQB1*0302) in a NOD background lacking endogenous class II molecules (Aβo). Upon immunization with type II collagen (CII), 85% of NOD.DQ8 mice develop severe experimental polychondritis, auricular chondritis, and polyarthritis, with clinical and histological similarities to relapsing polychondritis (RP) in humans. CII-immunized mice mount a T cell response and produce Ab's to type IX collagen (CIX) and self-CII. Transgene-negative littermates do not develop any serological and clinical manifestations following immunization. B10.DQ8 transgenic mice develop polyarthritis and Ab's to CII only. The susceptibility to auricular chondritis in NOD.DQ8 mice can be attributed to response to CIX. A higher number of activated cells, CD4 + CD44 h i CD62L l o , and lower regulatory cells CD4 + CD152 + CD25 + were observed in NOD.DQ8 mice compared with B10.DQ8 mice. The NOD.DQ8 mice provide a model of RP with a high disease incidence and multiple organ involvement to investigate putative autoantigen and regulatory cells involved in disease pathogenesis. An experimental model restricted by the human class II molecule will be valuable when studying the role of various collagens in immunologic and pathologic responses in human RP.

To investigate the role of HLA-DR4 in predisposition to arthritis, we generated transgenic mice carrying DRB1*0401 and DRB1*0402 genes. We have previously shown that DRB1*0401 molecule renders B10.RQB3 (H2Aq) mice susceptible to porcine and human type II collagen-induced arthritis. We report that the introduction of DRB1*0402 transgene does not lead to development of arthritis in mice when they are immunized with porcine and human type II collagen. In addition, DRB1*0402 protects B10.RQB3 mice against developing arthritis with bovine type II collagen. These data show that DRB1 can modulate the disease mediated by Aq. In vivo depletion of DRB1*0402 did not lead to induction of collagen-induced arthritis in transgenic mice. In vitro cytokine analysis shows that mice protected from collagen-induced arthritis produce lower amounts of Th1 and higher levels of Th2 type cytokines upon immunization with type II collagen. Protection of mice was also related to higher apoptosis in DW10 mice as indicated by higher amounts of BclII in response to type II collagen. On the basis of our observations in HLA transgenic mice, we hypothesize that DRB1 polymorphism can modulate disease by shaping the T cell repertoire in thymus and select autoreactive T cells.

To investigate the role of CD4 and CD8 T cells in arthritis, we generated transgenic mice deficient in CD4 and CD8 molecules expressing RA-susceptible gene HLA-DQ8. DQ8·CD4−/− mice were resistant to developing collagen-induced arthritis (CIA). However, DQ8·CD8−/− mice developed CIA with increased incidence and more severity than DQ8 mice. Both DQ8·CD8−/− and DQ8 mice produced rheumatoid factor. In addition, DQ8·CD8−/− mice produced antinuclear Abs. The B cell compartment and expression of DQ8 were normal in all the strains, although frequency of cells expressing DQ8 was less in CD4−/− mice. An increased frequency of CD3+ double-negative (DN) T cells was found in DQ8·CD8−/− compared with DQ8·CD4−/− and DQ8 mice. These CD3+ DN T cells produced high amounts of IL-10 in CD8-deficient mice. Analysis of cell division using a cell cycle tracking dye showed a higher rate of division of CD3+ and CD3+ DN T cells in DQ8·CD8−/− mice compared with DQ8·CD4−/− and DQ8 mice. Decreased apoptosis was seen in CIA-susceptible DQ8 and CD8-deficient mice, indicating a defect in activation-induced cell death. These observations suggest that CD4 cells are necessary for initiation of CIA in DQ8 mice. We hypothesize that CD8+ T cells are not capable of initiating CIA in DQ8-transgenic mice but may have a regulatory/protective effect.

Collagen-induced arthritis (CIA) is a useful model for dissecting the genetic patterns underlying susceptibility to rheumatoid arthritis (RA) and related chronic/inflammatory autoimmune diseases. CIA exhibits three phenotypes characteristic of autoimmune disease pathogenesis: abnormal levels of immune reactivity to self antigens; chronic inflammation of target organs expressing that specific autoantigen; activation and direct participation of invading mononuclear cells and resident tissue fibroblasts in organ damage. Over 25 different quantitative trait loci (QTL) regulating arthritis severity and autoantibody in rats with CIA are mapped. QTL-congenic strains show that certain CIA-QTLs can modulate arthritis independently These monogenic models are proving to be highly informative for fine mapping and function studies, revealing gender effects and evidence of gene clusters. Recent genome scans of RA populations identified RA-susceptibility loci in chromosome regions homologous to rat chromosomal segments housing CIA-QTLs. Also, CIA-QTLs frequently co-localize with susceptibility QTLs mapped in other rat arthritis models induced with non-immunogenic adjuvant oils and/or in rat autoimmune models of multiple sclerosis and diabetes. Common autoimmunity genes and inflammation genes important to several human diseases are likely being detected in the various rat disease models. Continued dissection of the genetic underpinnings of rat arthritis models should provide candidate genes for investigation in human patients and lead to a clearer understanding of the complex genetics of RA.

Rat Chromosome 10 (RNO10) harbors Cia5, a non-MHC quantitative trait locus (QTL) that regulates the severity of type II collagen-induced arthritis (CIA) in DAxF344 and DAxBN F2 rats. CIA is an animal model with many features that resemble rheumatoid arthritis. To facilitate analysis of Cia5 independently of the other CIA regulatory loci on other chromosomes, DA recombinant QTL speed congenic rats, DA.F344(Cia5), were generated. These QTL congenic rats have a large chromosomal segment containing Cia5 (interval sizeor =80.1 cM) from CIA-resistant F344 rats introgressed into their genome. Phenotypic analyses of these rats for susceptibility and severity of CIA confirmed that Cia5 is an important disease-modifying locus. CIA severity was significantly lower in the Cia5 congenic rats than in DA controls. We also generated DA Cia5 speed sub-congenic rats, DA.F344(Cia5a), which had a smaller segment of the F344 genome, Cia5a, comprising only the distal q-telomeric end (interval sizeor = 22.5 cM) of Cia5, introgressed into their genome. DA.F344(Cia5a) sub-congenic rats also exhibited reduced CIA disease severity compared with the parental DA rats. The regulatory effects in both congenic strains were sex influenced. The disease-ameliorating effect of the larger fragment, Cia5, was greater in males than in females, but the effect of the smaller fragment, Cia5a, was greater in females. We also present an improved genetic linkage map covering the Cia5/Cia5a region, which we have integrated with two rat radiation hybrid maps. Comparative homology analysis of this genomic region with mouse and human chromosomes was also undertaken. Regulatory loci for multiple autoimmune/inflammatory diseases in rats (RNO10), mice (MMU11), and humans (HSA17 and HSA5q23-q31) map to chromosomal segments homologous to Cia5 and Cia5a.

Objective Collagen-induced arthritis (CIA) is a polygenic model of experimentally induced autoimmunity and chronic joint inflammation. This study maps genetic loci that regulate CIA susceptibility in DA/Bkl (DA) and BN/SsNHsd (BN) rats. Methods Genome scans covering chromosomes 1–20 and interval mapping techniques using 159 simple sequence-length polymorphism markers were used to identify quantitative trait loci (QTLs) that regulate CIA in (DA × BN)F2 hybrids. Serum antibody titers to type II collagen were determined by enzyme-linked immunosorbent assay. Results DA rats were high responders to porcine type II collagen (PII) and developed severe CIA (100%). BN rats were low responders to PII and resistant to CIA (0%). BN genes strongly repressed PII-induced CIA. Only 12% of (DA × BN)F1 rats (7 of 60) and 31% of (DA × BN)F2 rats (307 of 1,004) developed CIA. Three new QTLs (Cia11, Cia12, and Cia13) with significant logarithm of odds (LOD) scores of 5.6, 4.6, and 4.5, respectively, plus a suggestive QTL (Cia14*, LOD 3.0) regulating arthritis severity were identified on chromosomes 3, 12, 4, and 19. A new QTL, Ciaa3, associating with anticollagen antibody titer (antibody to PII LOD 6.5; antibody to rat type II collagen LOD 5.2) mapped to chromosome 9. Of 10 CIA QTLs previously identified in (DA × F344) and (DA × ACI) rats, only Cia1 in the major histocompatibility complex and a region coincident to Cia5 on chromosome 10 (LOD >8.0) influenced CIA severity in (DA × BN)F2 rats. Conclusion Since CIA exhibits many of the pathologic features of rheumatoid arthritis, the data indicate that the variety of genetic elements regulating human autoimmune and rheumatic diseases may be much larger and more varied than originally envisioned.

Rat and mouse models for the major human autoimmune/inflammatory diseases are under intense genetic scrutiny. Genome-wide linkage studies reveal that each model is regulated by multiple genetic loci. Many of these loci colocalize to homologous genomic regions associated with several different autoimmune diseases of mice, rats and humans. Candidate genes are being identified. Polymorphic alleles associated with these chromosomal segments may represent predisposing genetic elements common to a number of human diseases with very different clinical presentations.

We generated transgenic mice with DRB1*0401 gene with mutation in the beta2 domain (aa 110 and 139) for better interaction with mCD4. The DR4 transgene was introduced into H2-Aq (B10RQB3) and H2-Af (B10RFB3) to examine the role of DR4 in collagen arthritis. The HLA-DR molecules in these mice were found to be functional on the basis of their positive/negative selection of the Vbeta T cell repertoire. H2-Aq mice are resistant to porcine CII-induced arthritis. The RQB3/DR4 mice (H2Aq/DR4) developed severe collagen induced arthritis (CIA) when immunized with Porcine type II collagen while the negative littermates were resistant. RQB3.DR4 mice were also highly susceptible to CIA induced by Human CII while negative littermates got only mild disease. However, RFB3/DR4 mice (H2Af/ DR4) did not get CIA with any type II collagen. Therefore, the DR4 gene in the context of H2-Aq predisposes to severe arthritis but not in the context of H2-Af. Antibodies to renatured cyanogen bromide (CB) cleaved fragments of PII in RQB3/DR4 mice and negative littermates suggest that the presence of DR4 does not result in any differences in specificity of antibody response to CB fragments. These results indicate that a specific gene complementation occurring between DR4 and H2.Aq but not DR4 and H2Af promotes the induction of arthritis with PII and HII in these mice. A similar interaction may be involved between DR and DQ molecules in human RA.

On the basis of our studies with HLA class II transgenic mice, we had proposed that complementation of HLA-DQ and HLA-DR alleles may determine both disease susceptibility and severity in rheumatoid arthritis (RA). According to our model, certain HLA-DQ alleles, such as HLA-DQ8, predispose individuals to RA, while a self-peptide derived from the third hypervariable region (HV3 65-79) of HLA-DR alleles, such as DRB1∗0402, can protect from disease if presented by the DQ molecule. To test this hypothesis, we examined the immunomodulatory effects of the DRB1∗0402 derived peptide (HV3 65-79) on collagen-induced arthritis (CIA) in HLA-DQ8 mice. Co-immunization of the DRB1∗0402 peptide significantly reduced the severity of arthritis (mean score = 1.5 ± 0.6 vs 5.2 ± 1.4 in controls), whereas multiple doses of the peptide reduced the incidence of disease (3.5% vs 35–60% in controls). Subsequent analysis revealed that the DRB1∗0402 peptide mediated protection may be due to the generation of a subset of regulatory cells, which down-regulate collagen-specific pro-inflammatory responses. These results provide additional insights towards understanding the role of MHC class II molecules in RA predisposition.

Objective To identify novel non-major histocompatibility complex (non-MHC) genetic loci controlling the severity of homologous rat type II collagen-induced arthritis (CIA). Methods We conducted a genome-wide scan to identify CIA regulatory quantitative trait loci (QTL) in an F2 cross between DA (CIA highly susceptible) and ACI (CIA resistant) inbred rats immunized with homologous rat type II collagen (RII). These strains share the MHC/RT1av1 haplotype required for susceptibility to RII-induced CIA. Results F2 females had higher median arthritis scores than did males. Relative resistance in the males was determined by inheriting either a DA or an ACI Y chromosome and was independent of the source of the X chromosome. In addition, a major QTL was localized on chromosome 2 (Cia7, logarithm of odds score 4.6). Cia7 is in a region that shows linkage conservation with chromosomal regions that regulate autoimmune diabetes and experimental autoimmune encephalomyelitis in mice and multiple sclerosis in humans. Conclusion Sex chromosomes and Cia7 play an important role in regulating CIA in response to RII. This rat model should facilitate positional cloning and functional characterization of regulatory genes that may play a role in several forms of autoimmune disease, including rheumatoid arthritis.

We have generated transgenic (tg) mice expressing HLA-DQ8αβ (DQA1*0301/DQB*0302) or HLA-DQ6αβ (DQA1*0103/DQB1*0601) molecules lacking endogenous murine class II expression (Aβ0) to investigate the ability of these HLA class II to present type II collagen (CII) and induce collagen-induced arthritis. The DQ8αβ tg mice responded strongly to CII, developing severe arthritis, while DQ6αβ tg mice were nonresponsive to CII. The addition of the mixed haplotype DQ8α6β molecule did not significantly influence CII reactivity. To examine the interaction of DQ6αβ and DQ8αβ molecules in vivo, we generated double tg DQ6αβ/8αβ (Aβ0) mice expressing both the α- and β-chains of DQ6 and DQ8 molecules by mating DQ6αβ (Aβ0) and DQ8αβ (Aβ0) tg mice. CII-immunized DQ6αβ/8αβ tg mice developed severe experimental polychondritis, exhibiting both polyarthritis and auricular chondritis. The clinical, serologic, and histologic manifestations of experimental polychondritis are similar to those symptoms in human relapsing polychondritis. The susceptibility of DQ6αβ/8αβ tg mice compared with resistance in the parental strains suggests that expression of both the DQ6αβ and DQ8αβ tgs, unique to the DQ6αβ8αβ tg strain, is important in susceptibility to experimental polychondritis. The DQ6αβ/8αβ tg mice provide a model to investigate putative autoantigens and the mechanisms of pathogenesis involved in relapsing polychondritis as well as the influence of the expression of multiple HLA class II molecules on the disease process.

Adjuvant-induced arthritis (AIA) in rats is a widely used autoimmune experimental model with many features similar to rheumatoid arthritis (RA). To identify potential genetic regulatory mechanisms in RA, we conducted genome-wide linkage analysis in F2 progeny of arthritis-susceptible Dark Agouti (DA) and relatively resistant Fischer 344 (F344) inbred rats. We compared the data with our previously reported investigation of collagen-induced arthritis (CIA), which was expanded in the follow-up study reported in this work. We found two quantitative trait loci (QTLs) in common, i.e., Aia1/Cia1 on chromosome 20, which includes the MHC, and Aia3/Cia3 on chromosome 4. We also identified a second unique QTL in AIA, Aia2, on chromosome 4. Interestingly, the QTL region on chromosome 4 (Aia3/Cia3), like the MHC, appears to be involved in several other autoimmune diseases in rats, including insulin-dependent diabetes, thyroiditis, and experimental autoimmune uveitis. Moreover, an analysis of conserved synteny among rats, mice, and humans suggested that Aia2 and Aia3/Cia3, like Aia1/Cia1, contain candidate genes for several autoimmune/inflammatory diseases in mice and humans, including diabetes, systemic lupus erythematosus, inflammatory bowel disease, asthma/atopy, multiple sclerosis, and RA. The rat models appear to provide a powerful complementary approach to identify and characterize candidate genes that may contribute to autoimmune diseases in several species.

Mouse class II-deficient HLA-DQB1*0302, DQA1*0301 (DQ8) transgenic mice are susceptible to severe collagen-induced arthritis (CIA), an animal model for rheumatoid arthritis. To examine whether polymorphism at the DRB1 locus can modulate DQ-restricted arthritis, we generated double-transgenic (DR/DQ) mice. HLA-DRB1*1502 (DR2) and DRB1*0301 (DR3) were introduced separately into CIA susceptible DQ8.Abeta transgenic mice to generate DQ8/DR2.Abeta and DQ8/ DR3.AbetaO mice. The HLA-DR molecules in these mice were found to be functional on the basis of their positive/negative selection of the Vbeta T cell repertoire. Introduction of the DR2 gene led to a significant decrease in disease incidence in DQ8.Abeta mice, while the DR3 transgene had no effect. In vitro T cell proliferative responses against bovine Cll collagen in primed mice were higher in DQ8/DR3 mice compared with DQ8/DR2 mice. Cytokine analysis showed a Th2 profile in DQ8/DR2 mice, while DQ8/DR3 mice showed a Th1 profile. These results suggest that DRB1 polymorphism can modulate the disease.

Certain HLA-DR alleles have been associated with predisposition to human rheumatoid arthritis (RA). There is also evidence that certain HLA-DQ alleles may also be important in determining susceptibility to RA. We have previously demonstrated that mice transgenic for HLA-DQ8, a DQ allele associated with susceptibility to RA, develop severe arthritis after type II collagen immunization. To investigate the influence of polymorphic difference at the DQ loci on susceptibility to arthritis, we generated mice transgenic for HLA-DQ6, an allele associated with a nonsusceptible haplotype. The DQ6 mice were found to be resistant to collagen-induced arthritis. We also assessed the combined effect of an RA-susceptible and an RA nonassociated DQ allele by producing double-transgenic mice expressing DQ6 and DQ8 molecules, representing the more prevalent condition found in humans where heterozygosity at the DQ allele is common. The double-transgenic mice developed moderate CIA when immunized with CII when compared with the severe arthritis observed in DQ8 transgenic mice, much like RA patients bearing both susceptible and nonsusceptible HLA haplotypes. These studies support a role for HLA-DQ polymorphism in human RA.

Transgenic mice expressing DQA1*0301 and DQB1*0302 (HLA-DQ8) molecules in class II-deficient Ab degree mice are susceptible to collagen-induced arthritis (CIA). To evaluate the role of the H2-E molecule (a homolog of HLA-DR) in DQ-restricted arthritis, the H2-E gene was introduced into DQ8.Ab degree mice to generate DQ8/E+.Ab degree mice. Expression of the E molecule protects DQ8.Ab degree mice against arthritis. In vitro studies using draining lymph nodes from mice primed with bovine type II collagen (BII) showed that the response to BII in both transgenics is DQ and CD4 restricted. Challenge with BII in vitro leads to production of high levels of IFN-gamma in DQ8 and IL-4 in DQ8/E+ mice. We have hypothesized that the H2-E molecule modulates the T cell repertoire and changes the cytokine balance, resulting in protection of disease.

Collagen-induced arthritis (CIA) is an animal model of auto-immune inflammatory polyarthritis which has features similar to rheumatoid arthritis (RA). Much like RA, susceptibility to mouse CIA is influenced by the major histocompatibility complex (MHC) and is restricted to theH2 haplotypesq andr. In previous experiments, we have found that the introduction of anH2-Eb d transgene in H2-Aq CIA-susceptible mice was able to protect these mice against disease development. More recently, we have proposed that the polymorphism of the first domain of the Eβ molecule modulates this protection, and that the presentation of a peptide from the third hypervariable region of the Eβ chain by the H2-Aq molecule plays an important role in this mechanism. In the present report, we investigated whether the H2-E-mediated protection is H2-Aq-specific and whether the source of collagen has any influence. While the source of collagen had no effect on the protection, our results showed that the H2-E molecule failed to protect B10.RIII (H2r) mice against CIA. Further, theH2 haplotyper exerted a negative effect on the Eβd-mediated protection in H2-Aq-restricted disease. Our results provide additional proof that self-MHC-derived peptides, such as Eβ peptides, may play an important role in the T-cell repertoire education and/or modulation of the T-cell response in the periphery.

The rapidly expanding use of implants for reconstruction and repair of diseased or traumatized tissues and organs has fueled the search for biomaterials that are stable, nontoxic, and biologically inert. Unfortunately, implants frequently cause acute or chronic inflammation resulting in tissue damage and rejection. Inflammation usually occurs at the biomaterial–tissue interface and reflects surface adsorption of plasma proteins, complement activation, neutrophil and macrophage infiltration, hyperplasia, and release of inflammatory mediators and proteolytic enzymes. If the biomaterial degrades, either spontaneously or due to biologic activity, components can leach into surrounding tissues and may enter the circulation, causing toxic effects systemically and in distant sites. Inflammation of connective tissues in genetically susceptible individuals can potentially activate the immune system and break tolerance to tissue-specific antigens. Experimentally induced animal models of arthritis chronicle the evolution of a chronic, destructive inflammation localized to peripheral joints. Models of autoimmune arthritis involve hyperimmunization with heterologous cartilage components, such as type II collagen or proteoglycan, leading to cross-reactivity with self-cartilage. Other apparently nonimmune models, oil-induced arthritis and pristane arthritis, may reflect granuloma formation and uncontrolled cytokine production. The antigen-induced arthritis model, developed by hyperimmunization with a foreign antigen that is subsequently injected into the joint cavity, is ideal for biocompatibility testing of cationic substances that may be immunogenic. This variety of models offers the means to understand adverse implant–tissue interactions and granuloma formation such that safer biomaterials can be developed in the future.

We have developed a new model of autoimmune arthritis in DBA/1 mice by feeding chick type II collagen (CII) for 2-3 week intervals over a 15 week period. Clinically evident arthritis occurred in 8/10 mice receiving native CII (nCII; 100 micrograms/mouse) alone at 9-13 weeks. Arthritis was aggravated by the further ingestion of CII, while remission occurred after withdrawal of the CII. Heat-denatured CII (dCII; 200 micrograms/mouse) was also arthritogenic if co-administered with ovoinhibitor (OVI; 2 mg/mouse), a proteinase inhibitor. Co-oral administration of lipopolysaccharide (LPS; 10 micrograms/mouse) with CII enhanced the antibody production and T-cell responses to CII, and induced a more chronic arthritis that progressed spontaneously without further administration of CII or LPS. Long-term oral administration of LPS alone also induced a mild arthritis characterized by destruction of bone rather than cartilage. These observations suggest that abnormal gastrointestinal absorption of dietary mimic antigens and intestinal bacterial toxins can potentially disrupt self-tolerance mechanisms, thereby precipitating or exacerbating autoimmune disease in genetically susceptible individuals.

Genetic studies have indicated that susceptibility to rheumatoid arthritis (RA) maps to the HLA-DR locus of the major histocompatibility complex. Strong linkage disequilibrium between certain HLA-DQ genes and HLA-DR genes associated with RA, however, suggests that HLA-DQ molecules may also play a role in RA susceptibility. To examine the role of HLA-DQ molecules in arthritis, we generated transgenic mice expressing the DQA1*0301 and DQB1*0302 genes from an RA predisposing haplotype (DQ8/DR4Dw4). The transgenes were introduced into mouse class II-deficient H-2Ab0 mice, and their susceptibility to experimental collagen-induced arthritis was evaluated. The HLA-DQ8+,H-2Ab0 mice displayed good expression of the DQ8 molecule, while no surface expression of endogenous murine class II molecules could be detected. The DQ8 molecule also induced the selection of CD4+ T cells expressing a normal repertoire of V beta T cell receptors. Immunization of HLA-DQ8+,H-2Ab0 mice with bovine type II collagen (CII) induced a strong antibody response that was cross-reactive to homologous mouse CII. Also, in vitro proliferative responses against bovine CII, which were blocked in the presence of an antibody specific for HLA-DQ and mouse CD4, were detected. Finally, a severe polyarthritis developed in a majority of HLA-DQ8+,H-2Ab0 mice, which was indistinguishable from the disease observed in arthritis susceptible B10.T(6R) (H-2Aq) controls. In contrast, HLA-DQ8-,H-2Ab0 fullsibs did not generate CII antibody and were completely resistant to arthritis. Therefore, these results strongly suggest that HLA-DQ8 molecules contribute to genetic susceptibility to arthritis and also establish a novel animal model for the study of human arthritis.

Collagen-induced arthritis (CIA) is an animal model of auto immune polyarthritis, sharing similarities with rheumatoid arthritis (RA). Paradoxally, susceptibility to mouse CIA is controlled by the H2A loci (DQ homologous) while RA is linked to HLA.DR genes (H2E homologous). We recently showed that the E beta d molecule prevents CIA development in susceptible H2q mice. We addressed the question of whether H2Eb polymorphism will influence CIA incidence as HLA.DRB1 polymorphism does in RA. In F1 mice, only H2Ebd and H2Ebs molecules showed protection. Using recombinant B10.RDD (Ebd/b) mice, we found that CIA protection was mediated by the first domain of the E beta d molecule. Using peptides covering the third hypervariable region of the E beta chain, we found a perfect correlation between presentation of E beta peptides by the H2Aq molecule and protection on CIA. Therefore, the mechanism by which H2Eb protects against CIA seems to rely on the affinity of E beta peptides for the H2Aq molecule.

Previous studies have illustrated the importance of T cells bearing alpha beta TCRs in the induction and development of collagen induced arthritis (CIA) in mice. However, the scope of TCR usage in CIA has yet to be clearly defined. Given the inherent diversity of the TCR repertoire, the relative flexibility of the arthritogenic TCR repertoire specific for type II collagen (CII) is not clear. Therefore, we chose to examine the influence of a highly skewed TCR repertoire on CIA. Arthritis susceptible B10.Q (H-2q) mice were mated with C57L (H-2b) animals expressing an ovalbumin-specific V beta 8.2 TCR transgene (Tg) and Tg+ offspring were further backcrossed to B10.Q. Homozygous H-2q/q, V beta 8.2 Tg+ mice displayed a high level of V beta 8.2+ T cells in peripheral blood. However, expression of some endogenous V beta TCR, such as V beta 14, was still detected. Upon immunization with bovine CII in adjuvant, V beta 8.2 Tg+ mice were highly resistant to CIA when compared with Tg- littermates. Analysis of sera demonstrated a marked reduction in antibody specific for homologous mouse CII as well as heterologous bovine CII in Tg+ animals. Interestingly, V beta 8.2 Tg+ mice still mounted good antibody responses following immunization with human thyroglobulin, indicating that the skewed TCR repertoire affected anti-CII but not antithyroglobulin responses. Thus, our findings show that constraints placed on the TCR repertoire inhibit pathogenic responses against CII and suggest that in H-2q mice the arthritogenic TCR repertoire bears only limited flexibility.

Previous studies directed towards identifying epitopes on type II collagen (CII) important in collagen induced arthritis (CIA) in mice have focused primarily on responses mounted in susceptible H-2q strains. However, the nature of T and B cell responses against CII in susceptible H-2r strains remains ill-defined. In an effort to identify regions on CII important in CIA in H-2r mice, we examined the cellular and humoral response of susceptible B10.RIII (H-2r) mice against cyanogen bromide (CB)-cleaved fragments of porcine CII. Following immunization with native porcine CII, LNC from B10.RIII mice mounted proliferative responses predominantly to peptide CB10, while negligible proliferation was detected against fragment CB9, 7, CB8, CB11 or CB12. In contrast, sera from arthritic B10.RIII mice displayed a heterogeneous pattern of reactivity against porcine CII, with strong antibody binding measured against the major fragments CB11, CB8 and CB10. To determine the in vivo significance of the dominant cellular response to CB10, B10.RIII mice received an i.v. injection of soluble CB10 seven days before immunization with native porcine CII. Mice pretreated with CB10 were highly resistant to CIA compared to control animals. Interestingly, B10.RIII mice pretreated with fragment CB11, a region of CII implicated in H-2q restricted CIA, remained susceptible to arthritis induction. Collectively, our findings indicate that the CB10 region of porcine C11 bears determinants which may be important in the induction and/or regulation of CIA in the H-2r haplotype.

To determine the relationship between susceptibility to bovine type XI and II (BXI and BII) collagen-induced arthritis, we immunized 14 inbred and one outbred strains of rats with BXI and BII. Susceptibility to BXI-arthritis corresponded largely with susceptibility, or resistance, to BII-arthritis. LEW, BB, WF, DA, and WKY were readily susceptible to BXI- and BII-arthritis. Likewise, BII-resistant F344 and BN rats were BXI-resistant. Some strains responded differently to BXI and BII. BUF and COP, which are moderately susceptible to BII, were BXI-resistant, whereas the BII-resistant rats, DA.1N and WF.1N, were partially susceptible to BXI. (F344 x BN) F1 hybrids responded to both collagens suggesting gene complementation. Arthritis occurred in all strains producing the highest titer antisera (LEW, WF and BB). Antibody responses to BXI and BII were generally commensurate within individual strains. DA were susceptible to arthritis but produced low levels of antibody comparable to BN rats which were arthritis-resistant. BXI and BII-susceptibility was variable in rats producing intermediate antibody responses. Antibodies to RXI were detected in all BXI-immunized rats, whereas antibodies to RV and RII were uniformly weaker. DTH to RXI and RII was strong in both groups of rats, correlating poorly with arthritis and antibody responses. These studies show that phenotypic susceptibility to BXI- and BII-arthritis are largely concordant among inbred rat strains but clear differences exist in certain strains; multiple genes are likely involved.

B10.Q (H-2q) mice congenic for the truncated T cell receptor (TCR) V beta a and V beta c haplotypes were derived to examine the influence of TCR V beta genomic deletions in murine collagen-induced arthritis (CIA). Previous studies using gene complementation and segregation analyses suggested that in SWR (H-2q) mice, possession of the V beta a gene deletion results in CIA resistance. However, other studies have suggested alternative hypotheses. Thus, analysis of TCR V beta congenic mice allows for direct examination of V beta genotypes in CIA control. After immunization with bovine type II collagen, B10.Q-V beta a mice showed no difference in arthritis susceptibility, onset, or severity when compared with prototype B10.Q mice. In contrast, B10.Q-V beta c mice, which lack the V beta 6, 15, 17, and 19 families in addition to the V beta a deletion, were highly resistant to CIA. In vivo depletion of V beta 6+ T cells in B10.Q-V beta a mice significantly delayed arthritis onset suggesting that, among those V beta genes present in V beta a but absent in V beta c, V beta 6+ T cells contribute to arthritogenesis. Our findings show that, in B10.Q-V beta congenic mice, while the V beta a genotype does not prevent CIA, the highly truncated V beta c genotype renders B10.Q mice resistant to CIA. Thus, deletions within the V beta TCR genome can indeed influence CIA and suggests that the TCR repertoire displays only marginal flexibility in response to arthritogenic stimuli.

Collagen-induced arthritis (CIA) is an animal model of autoimmune inflammatory polyarthritis that has features similar to rheumatoid arthritis (RA). Much like RA, susceptibility to mouse CIA is influenced by the major histocompatibility complex (MHC), H-2, and restricted to the H-2q and H-2r haplotypes. Whereas the role of the H-2A molecule in susceptibility to CIA is well established, little is known about the role of H-2E molecule in the disease. In this study, we analyzed the effect of a transgenic E beta d molecule on CIA susceptibility in a recombinant mouse B10.RQB3, which expresses the CIA susceptible Aq genes and an Eak gene, but does not produce an E molecule since Ebq is nonfunctional. In the presence of an Ebd transgene, a viable E molecule is generated. Whereas B10.RQB3 were susceptible to CIA, B10.RQB3-E beta d+ showed a dramatic reduction in the incidence of arthritis as well as a decrease in the level of anti-mouse and anti-bovine CII antibodies in their serum. No clear cut differences in the expression of T cell receptor (TCR) V beta was observed between E beta d+ and E beta d- transgenic mice. Mechanisms underlying the protective effect of E beta d transgenic molecule on CIA may shed light on how HLA-DR molecules influence human RA.

Objective. To test for genetic differences in susceptibility to homologous (rat) type II collagen-induced arthritis (RII-CIA). Methods. Nine inbred and RT1-congenic rat strains were immunized with native rat type II collagen and evaluated for arthritis and IgG anti-RII serum antibody titers. Results. Only RT1av1 strains developed a high incidence of severe RII-CIA and high titers of IgG anti-RII serum antibody. Rats having RII-CIA-resistant haplotypes, RT1u,n,1 (which are known to develop CIA after immunization with heterologous type II collagen), were shown to also be susceptible to passive transfer of CIA with immune serum concentrates. Clinical expression of RII-CIA was down-regulated by non-RT1 genes of BN origin. No strong gender differences in anti-RII autoimmune responses were observed. Conclusion. Arthritogenic, autoimmune reactivity to homologous RII is under strict genetic control but occurs readily in RT1av1 rats.

To define the effects of incomplete Freund's adjuvant (ICFA) on subsequent arthritis induced by complete Freund's adjuvant (CFA) and type II collagen (CII) in DA and Lewis rats.ICFA was injected into DA and Lewis rats before CFA or CII injection.DA rats previously injected with ICFA had significantly less severe arthritis induced by CFA compared with those not receiving ICFA pretreatment (P0.01). ICFA had no significant impact on CFA-induced arthritis in Lewis rats or on CII-induced arthritis in DA rats.The injection of ICFA alone specifically suppresses subsequent CFA-induced arthritis in DA rats, but not in Lewis rats.

The prophylactic and therapeutic effects of gold sodium thiomalate, cyclosporin A, cyclophosphamide, and placebo on collagen-induced arthritis (CIA) were evaluated in DA rats. Prophylactic treatment with cyclosporin A and cyclophosphamide suppressed the arthritis incidence, clinical inflammation, destructive bone changes, and development of anti-collagen antibody in DA rats subsequently injected with porcine type-II collagen. Therapeutic treatment with cyclosporin A and cyclophosphamide had a definite suppression on established CIA when started 21 days after the initial collagen injection, but the suppression was less marked than that of prophylactic treatment. Gold had no impact on CIA in DA rats when administered either prophylactically or therapeutically.

Adjuvant-induced arthritis (AIA) can be passively transferred in Dark Agouti (DA) rats by spleen and lymph node cells after culture with Concanavalin A (Con A). A model not requiring in vitro Con A expansion and activation would be important in investigations of anti-rheumatic drugs in AIA. A new model using irradiated recipients fills this need. Donor DA rats treated with 0.1 ml complete Freund's adjuvant (CFA) containing 7.5 mg M. butyricum/ml were sacrificed 11 days after CFA injection, donor spleen cells harvested, and donor spleen cells injected intravenously into recipient DA rats previously irradiated with 5 Gy. Recipient rats developed arthritis 4-14 days after spleen cell transfer. This model can now be used to further define the effects of anti-rheumatic drugs in the passive transfer of AIA by eliminating the need for the in vitro Con A-induced expansion and/or activation of donor cells.

Mouse strains B10, B10.RIII, RIIIS/J and the F1 and backcross progeny arising from them were tested for susceptibility to porcine type II collagen-induced arthritis (PII-CIA). The clinically severe arthritis of rapid onset that is characteristic of PII-immunized B10.RIII mice developed predominantly in hybrid offspring that had inherited at least one copy of wild type T cell receptor (TCR) genes (V beta b genotype) from the B10 or B10.RIII parent. The results indicate that, in the development of PII-CIA, mice expressing the H-2r/r haplotype preferentially utilize TCR V beta genes that are normally encoded within the TCR V beta genomic deletion region of RIIIS mice (V beta c). After aggressive immunization with PII, the use of alternative TCR V beta genes, encoded outside of the RIIIS deletion region, produced a high IgG antibody response that was cross-reactive with mouse type II collagen (MII) and equivalent to that of B10.RIII mice, but only a very mild, late onset arthritis of 56% (27/48) incidence in RIIIS male mice and 28% (10/35) incidence in RIIIS female mice. In comparison, B10.RIII mice routinely developed early onset of PII-CIA of significantly higher incidence (100%; p0.005) and four-fold greater severity, even after milder immunization protocols. The data are compatible with the proposal that the clinically weak CIA response of RIIIs mice may be primarily antibody driven while the severe CIA of B10.RIII mice reflects the added inflammatory effects of collagen-reactive effector-T cells in the joint.(ABSTRACT TRUNCATED AT 250 WORDS)

HLA class II allele DRB1*0401 is associated with predisposition to Rheumatoid Arthritis in humans as well as collagen-induced arthritis in mice. Predominantly females develop arthritis in humans and DR4 transgenic mice; however the mechanism of sex-bias is still unknown. We have investigated the molecular basis by which DR4 is associated with sex-bias of arthritis. Here we show that differential antigen-specific immune mechanisms in DR4 male and female mice lead to increased susceptibility in female mice. B cells are hyperactive and present DR-restricted peptides robustly in females compared to males. Antigen-specific response showed that females produced B cell modulating cytokines like IL-13 while males produced IFNgamma. Male transgenic mice have higher number of T and B regulatory cells. An exogenous supply of 17beta estradiol in male mice led to enhanced expression of DR4 and antigen-specific response to DR4-restricted peptides. On the other hand, castration increased the incidence of arthritis. We propose that sex-bias in arthritis involves B cells and presentation of antigen by HLA-DR4 leading to activation of autoreactive cells and autoantibodies production in females, while regulatory B cells in males protect them from pathogenesis. The transgenic mice expressing RA susceptible haplotype simulate human RA and may be valuable to study gender differences observed in patients.

Collagen-Induced Arthritis (CIA) is a complex model of autoimmune-mediated arthritis that is regulated by multiple genetic and environmental factors. CIA is induced in rats by immunization with native type II collagen and develops joint pathology similar to that of rheumatoid arthritis. This chapter details methods for the extraction and purification of native type II collagen from sternal and articular cartilage, an arthritis induction protocol that has resulted in reproducible CIA expression in several rat strains from year to year and criteria for measuring clinical, radiographic and immunological outcome parameters characteristic of CIA.

Rheumatoid arthritis and its animal model, collagen-induced arthritis, are known as a T and B cell dependent disease. To analyze the role of B cells in arthritis, we generated B cell deficient (microMT) mice carrying HLA-DQ8 as transgene, Abetao.DQ8.micromt mice. HLA-DQ8 transgenic mice (Abetao.DQ8) are susceptible to collagen induced arthritis, an animal model for inflammatory arthritis. Deletion of IgM gene led to the absence of B cells while T cells were comparable to Abetao.DQ8 mice. Arthritis and autoantibodies was completely abrogated in B cell deficient DQ8 mice. T cell response and proinflammatory cytokine production in response to type II collagen and its derived peptides in vitro was significantly decreased despite an increased number of Mac-1 positive cells in DQ8.micromt mice compared to DQ8 mice suggesting B cells could be important for antigen presentation as well. In vitro substitution of B cells from wild type mice restored the response in DQ8.micromt mice. B cells could also present CII-derived peptides to antigen-specific DQ8-restricted hybridomas reinforcing the role of B cells in presentation of antigens to T cells. The data suggest that B cells can be involved in pathogenesis of arthritis by producing autoantibodies and antigen presentation.

Collagen-Induced Arthritis (CIA) is a complex model of autoimmune-mediated arthritis that is regulated by multiple genetic and environmental factors. CIA is induced in rats by immunization with native type II collagen and develops joint pathology similar to that of rheumatoid arthritis. This chapter details methods for the extraction and purification of native type II collagen from sternal and articular cartilage, an arthritis induction protocol that has resulted in reproducible CIA expression in several rat strains from year to year and criteria for measuring clinical, radiographic and immunological outcome parameters characteristic of CIA.

Analysis of several inbred rat strains has led us to hypothesize that HPA axis abnormalities may contribute, in part, to susceptibility to both autoimmune disease and addiction. In this article we review the evidence for this hypothesis and describe our ongoing efforts to genetically characterize these traits. We have mapped the locations of 23 loci that regulate autoimmune disease in rats, and are currently constructing QTL congenic lines in which a genomic region from the resistant strain is transferred to the susceptible strain or vice versa. These QTL congenic lines will be valuable to test whether genes encoding autoimmune regulation also control neuroendocrine traits. Further genetic dissection and identification of the underlying genes will be necessary to infer a mechanistic link between autoimmune and neuroendocrine traits.

CD28 is required to achieve optimal T cell activation to an Ag. To determine the role CD28 costimulation plays in collagen-induced arthritis, we have generated DQ8 transgenic, CD28-deficient mice. DQ8 mice deficient for CD28 had comparable numbers of CD4 and CD8 T cells as DQ8.CD28+/+ mice. DQ8.CD28−/− mice develop collagen-induced arthritis with delayed onset and less severity than DQ8.CD28+/+ mice. T cells from DQ8.CD28−/− mice did not respond to type II collagen efficiently in vitro, although the response to DQ8-restricted peptides was similar to that in the parent mice. There was no functional defect in T cells as observed by proliferation with Con A. Cytokine analysis from in vitro study showed the production of high levels of the inflammatory cytokine, IFN-γ, in response to type II collagen. We observed an increase in CD4+CD28−NKG2D+ cells after immunization, suggesting an important role for cells bearing this receptor in the disease process. CD28−/− mice also have an increased number of DX5+ cells compared with CD28+/+ mice, which can lead to the production of high levels of IFN-γ. DQ8.CD28−/− mice had an increased number of cells bearing other costimulatory markers. Cells from DQ8.CD28−/− mice exhibited a lower proliferation rate and were resistant to activation-induced cell death compared with DQ8.CD28+/+ mice. This study supports the idea that CD28 plays a crucial role in the regulation of arthritis. However, in the absence of CD28 signaling, other costimulatory molecules can lead to the development of disease, thus indicating that the requirement for CD28 may not be absolute in the development of arthritis.

To identify additional sex-specific and epistatic quantitative trait loci (QTL) regulating collagen-induced arthritis (CIA) severity overall, as well as within different stages during the disease course, in an intercross between major histocompatibility complex-identical inbred rat strains DA/Bkl (susceptible) and ACI/Hsd (resistant).Arthritic male (DA x ACI)F2 intercross offspring (n = 143) were analyzed separately from the females (n = 184). Phenotypic extremes (maximum arthritis scores [MAS]) were genotyped and used for QTL analysis. All 327 rats were genotyped with the simple sequence-length polymorphism (SSLP) markers closest to the peak of Cia7 and Cia10, the major loci previously identified in this intercross, and with SSLPs covering chromosomes 12 and 18. Phenotypes studied were disease onset, arthritis severity scores on days 14-39, MAS, mean and cumulative arthritis scores, delayed-type hypersensitivity, and antibody responses to rat type II collagen.A new female-specific arthritis-severity recessive locus was identified on rat chromosome 12 (Cia25), with a maximum effect observed on day 28 (logarithm of odds [LOD] 4.7). The homozygous DA genotype at Cia25 was associated with a 45% higher median arthritis score in females. Sequencing analyses of the Cia25 candidate gene Ncf1 revealed polymorphisms between DA and ACI. The previously identified locus, Cia10, was found to be male-specific. A 2-locus interaction model analysis identified a novel recessive chromosome 18 QTL, Cia26, which was dependent on Cia7, with its maximum effect observed at later stages during the disease course (peak LOD score of 3.6 for arthritis scores on day 39).This study identified 2 novel female-specific loci, and 1 male-specific locus. Cia25 regulates MAS and disease severity during the mid-to-late stages of the disease course and may be accounted for by Ncf1 polymorphisms. Cia26 is in epistasis with Cia7 and regulates later stages of disease, suggesting an involvement in disease perpetuation and/or chronicity.

Objective Collagen-induced arthritis (CIA) is a model of inflammatory arthritis with many similarities to rheumatoid arthritis (RA). We previously mapped in F2 offspring of CIA-susceptible DA and CIA-resistant F344 rats, 5 quantitative trait loci (QTLs) for which F344 alleles were associated with reduced CIA severity. In the present study, we sought to characterize the independent arthritis-modulating effects of these 5 QTLs. Methods CIA-regulatory regions were transferred from the F344 genome to the DA background or vice versa by repeated backcrossing. The arthritis-modulating effects of the transferred alleles were determined by comparing the severity of experimentally induced arthritis in congenic rats with that in DA rats. Results Congenic lines with either the F344 major histocompatibility complex (MHC) on the DA background or the DA MHC on the F344 background were resistant to CIA, confirming both MHC and non-MHC contributions to the genetic regulation of CIA. F344 alleles at the Cia3 and Cia5 regions of chromosomes 4 and 10 reduced CIA severity relative to that observed in DA rats. F344 Cia4 and Cia6 regions of chromosomes 7 and 8 failed to significantly alter CIA severity. Arthritis-modifying effects of Cia4 and Cia6 were, however, detected in pristane-induced and/or Freund's incomplete adjuvant oil–induced arthritis. The arthritis-modifying effects of the non-MHC CIA-regulatory loci differed in males and females. Conclusion These congenic lines confirmed the existence and location of genes that regulate the severity of experimental arthritis in rats. Mechanisms responsible for the sex-specificity of individual arthritis-regulatory loci may explain some of the sex differences observed in RA and other autoimmune diseases in humans.

The major, extensively studied, experimentally-induced rat and mouse models of arthritis with features resembling rheumatoid arthritis are reviewed here. Etiopathogenetic studies that were recently published are emphasized. In summary, multiple triggering stimuli can induce disease in genetically-prone strains of inbred rats and mice. Multiple genetic loci, including both MHC and non-MHC, regulate disease expression in these animals. By comparison with other models of autoimmune disease, clustering of regulatory loci within and among species is increasingly becoming evident. At the cellular level, both innate and acquired immune systems are involved in the disease manifestations. At the molecular level, unbalanced chronic production of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1, IL-6 and IL-12, as opposed to IL-4 and IL-10, is correlated with arthritis disease susceptibility and severity.

Rheumatoid arthritis (RA) is a common, chronic, autoimmune, inflammatory disease that is influenced by genetic factors including gender. Many studies suggest that the genetic risk for RA is determined by the MHC, in particular class II alleles with a 'shared epitope' (SE), and multiple non-MHC loci. Other studies indicate that RA and other autoimmune diseases, in particular insulin-dependent diabetes mellitus (IDDM) and autoimmune thyroid disease (ATD), share genetic risk factors. Rat collagen-induced arthritis (CIA) is an experimental model with many features that resemble RA. The spontaneous diabetes-resistant bio-breeding rat, BB(DR), is of interest because it is susceptible to experimentally induced CIA, IDDM and ATD, and it has an SE in its MHC class II allele. To explore the genetics of CIA, including potential gender influences and the genetic relationships between CIA and other autoimmune diseases, we conducted a genome-wide scan for CIA regulatory loci in the F(2) progeny of BB(DR) and CIA-resistant BN rats. We identified 10 quantitative trait loci (QTLs), including 5 new ones (Cia15, Cia16*, Cia17, Cia18* and Cia19 on chromosomes 9, 10, 18 and two on the X chromosome, respectively), that regulated CIA severity. We also identified four QTLs, including two new ones (Ciaa4* and Ciaa5* on chromosomes 4 and 5, respectively), that regulated autoantibody titer to rat type II collagen. Many of these loci appeared to be gender influenced, and most co-localized with several other autoimmune trait loci. Our data support the view that multiple autoimmune diseases may share genetic risk factors, and suggest that many of these loci are gender influenced.

HLA-B27 is highly linked with a group of human diseases called spondyloarthropathies (SpA). Many of these disorders begin after an infection with an enterobacteria. The symptoms seen in patients with spondyloarthropathies are inflammatory pain in the spine and asymmetrical arthritis of lower limbs. Additional symptoms related to SpA include inflammation in the eyes, bowel, and skin. The autoantigen(s) in SpA are not known. Proteins such as collagen and proteoglycans have been thought to be potent autoantigens in arthritidis including B27-associated human diseases. Type II collagen is a common denominator among eyes and joints, affected tissues in B27-linked diseases. Moreover, a few reports indicated CII specific T cells and antibodies in patients with spondyloarthropathies. We and others have previously described development of spontaneous arthritis and nail disease in HLA-B27 transgenic animals. To determine whether CII may be a target antigen in the B27-linked diseases, B27 + mβ 2 m o/o (HLA-B27 transgenic mice lacking mouse β 2 m with and without human β 2 m) mice were immunized with type II collagen inside the barrier facility. Male HLA-B27 transgenic mice developed collagen-induced arthritis compared to transgene negative littermates or female counterparts. There was no difference in the incidence of arthritis in HLA-B27 transgenic mice with and without human β 2 m. Our data suggest that β 2 m free heavy chain of HLA-B27 may present soluble antigens such as type II collagen to trigger specific T cells contributing in the development of arthritis. Our data also suggest that CII may be a potential target antigen in the cartilage during the disease process.

The rat (Rattus norvegicus) is an important experimental model for many human diseases including arthritis, diabetes, and other autoimmune and chronic inflammatory diseases. The rat genetic linkage map, however, is less well developed than those of mouse and human. Integrated rat genetic linkage maps have been previously reported by Pravenec et al. (1996, Mamm. Genome 7: 117-127) (500 markers mapped in one cross), Bihoreau et al. (1997, Genome Res. 7: 434-440) (767 markers mapped in three crosses), Wei et al. (1998, Mamm. Genome 9: 1002-1007) (562 markers mapped in two crosses), Brown et al. (1998, Mamm. Genome 9: 521-530) (678 markers mapped in four crosses), and Nordquist et al. (1999, Rat Genome 5: 15-20) (330 markers mapped in two crosses). The densest linkage map combined with a radiation hybrid map, reported by Steen et al. (1999, Genome Res. 9: AP1-AP8), includes 4736 markers mapped in two crosses. Here, we present an integrated linkage map with 1137 markers. We have constructed this map by genotyping F2 progeny of five crosses: F344/NHsd x LEW/NHsd (673 markers), DA/Bkl x F344/NHsd (531 markers), BN/SsN x LEW/N (714 markers), DA/Bkl x BN/SsNHsd (194 markers), and DA/Bkl x ACI/SegHsd (245 markers). These inbred rat strains vary in susceptibility/resistance to multiple autoimmune diseases and are used extensively for many types of investigation. The integrated map includes 360 loci mapped in three or more crosses. The map contains 196 new SSLP markers developed by our group, as well as many SSLP markers developed by other groups. Two hundred forty genes are incorporated in the map. This integrated map should allow comparison of rat genetic maps from different groups and thereby facilitate genetic studies of rat autoimmune and related disease models.

Autoimmune diseases, such as rheumatoid arthritis, Crohn's disease, and multiple sclerosis, are regulated by multiple genes. Major histocompatibility complex (MHC) genes have the strongest effects, but non-MHC genes also contribute to disease susceptibility/severity. In this paper, we describe a new non-MHC quantitative trait locus, Cia8, on rat Chromosome (Chr) 7 that controls collagen-induced arthritis severity in F2 progeny of DA and F344 inbred rats, and present an updated localization of Cia4 on the same chromosome. We also describe the location of mouse and human genes, orthologous to the genes in the genomic intervals containing Cia4 and Cia8, and provide evidence that the segment of rat Chr 7 containing Cia4 and Cia8 is homologous to segments of mouse Chr 10 and 15 and human Chr 8, 12, and 19.