A genome scan localizes five non-MHC loci controlling collagen-induced arthritis in rats.

Identification of specific genetic loci that contribute to susceptibility to rheumatoid arthritis (RA) in humans has been hampered by several factors, including: i) multiple interacting genetic loci contributing to susceptibility; ii) complex interactions of environmental and genetic factors; iii) genetic heterogeneity; and iv) low penetrance. We have, therefore, mapped quantitative trait loci (QTLs) that control inflammatory arthritis susceptibility and/or severity in progeny of two inbred rat strains with significantly different susceptibilities to collagen-induced arthritis (CIA), an animal model for RA. Not surprisingly, we identified a major susceptibility factor, Cia1, on chromosome 20 in the vicinity of the rat major histocompatibility complex (MHC). However, by limiting the analysis to animals with arthritis-susceptible MHC genotypes and using genome-wide QTL analytic techniques, we also found four non-MHC QTLs-Cia2, 3, 4 and 5-on chromosomes 1, 4, 7 and 10, that contributed to disease severity. In addition, a QTL on chromosome 8 was suggestive for linkage. Characterization of the genes underlying these QTLs will facilitate the identification of key biochemical pathways regulating experimental autoimmune arthritis in rats and may provide insights into RA and other human autoimmune diseases. These genes may also represent novel targets for therapy.

HLA-DQ8 transgenic mice are highly susceptible to collagen-induced arthritis: a novel model for human polyarthritis.

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 in T cell receptor V beta congenic B10.Q mice.

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.

Protective role of major histocompatibility complex class II Ebd transgene on collagen-induced arthritis.

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.

Vaccination with collagen-pulsed dendritic cells prevents the onset and reduces the disease severity in the mouse model of spontaneous polychondritis.

Immature 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)-DQ6alphabeta8alphabeta transgenic mouse model. Bone marrow-derived iDCs were pulsed in vitro with CII and transferred into 6-week-old HLA-DQ6alphabeta8alphabeta 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.

Cytotoxic activity of rheumatoid and normal lymphocytes against allogeneic and autologous synovial cells in vitro.

The possibility that lymphocytes from patients with rheumatoid arthritis (RA) might be sensitized to RA synovial cell antigens was investigated with a 51Cr release cytotoxicity assay. Peripheral blood lymphocytes from rheumatoid and normal donors were tested for cytotoxic activity against their own synovial cells and against allogeneic rheumatoid and nonrhemuatoid synovial cells. In the allogeneic studies, the degree of cytotoxicity was significantly influenced by the age in culture (passage number) of the synovial target cells (P less than 0.001). When the passage number of the target cells was considered in the analysis, rheumatoid lymphocytes were found to have greater cytotoxic activity than normal lymphocytes against young cultures (low passage number) of both RA and non-RA synovial cells (P = 0.0042). Differences in susceptibility to lysis between RA and non-RA synovial cells were more susceptible to both RA and normal lymphocyte-induced lysis than were non-RA synovial cells (P = 0.0048). No evidence of cytotoxicity was detected when lymphocytes from nine RA patients and two osteoarthritis patients were reacted against their own synovial cells. Although the data demonstrated an increased cytotoxic activity of peripheral blood lymphocytes from some RA patients against allogeneic synovial cells, the fact that this reactivity was seen against both non-RA and RA synovial cells and was not demonstrated against autologous synovial cells argues against the presence of an immunospecific response of RA lymphocytes to RA synovial cell antigens.

HLA-DQB1 polymorphism determines incidence, onset, and severity of collagen-induced arthritis in transgenic mice. Implications in human rheumatoid arthritis.

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.

Mapping autoimmunity genes.

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.

An HLA-DRB1*0402 derived peptide (HV3 65-79) prevents collagen-induced arthritis in HLA-DQ8 transgenic mice.

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 DRB 1*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 DRB 1*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.

Complementation between HLA-DR4 (DRB1*0401) and specific H2-A molecule in transgenic mice leads to collagen-induced arthritis.

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.

HLA-B27 transgenic mice are susceptible to collagen-induced arthritis: type II collagen as a potential target in human disease.

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 + m beta 2 m% (HLA-B27) transgenic mice lacking mouse beta 2m with and without human beta 2m) 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 beta 2m. Our data suggest that beta 2m 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.

Human leukocyte antigen-DRB1*1502 (DR2Dw12) transgene reduces incidence and severity of arthritis in mice.

A strong correlation exists between susceptibility to RA in humans and some DRB1 alleles of the MHC region, such as DRB1*0401 and DRB1*0101. Meanwhile, incidences of other DR specificities, such as DR2, DR5, or DR7 have often been found reduced among RA patients. Like RA, susceptibility to mouse CIA is influenced by the MHC class II loci. To analyze the effect of a DRB1 molecule associated with low incidence of RA on mouse CIA, a human DRB1*1502 (DR2Dw12) transgene was introduced into CIA-susceptible B10.RQB3 (H2Aq) mice. Transgene-positive DRB1*1502 mice showed a significant reduction in the incidence and severity of arthritis. Moreover, the clinical reduction of arthritis correlated with the T-cell proliferative response of B10.RQB3-DRB1*1502 mice against a self-derived DRB1 peptide from the third hypervariable region. Our results suggest that the DRB1*1502-mediated protection against CIA can be explained by the DRB1 molecule acting as a source of self-antigenic peptide which interferes with the T-cell response against immunodominant regions(s) of the arthritogenic type II collagen molecule. By analogy, a similar mechanism might play a critical role in influencing the class II-associated predisposition to RA.

Immunogenetics of collagen-induced arthritis in rats.

CIA can be viewed as a multifactorial animal model of experimentally-induced autoimmunity that is targeted to joint tissues and under multiple gene control. Thus, although induction of CIA requires immune reactivity to type II collagen, a high immune response to type II collagen is not pathognomonic of CIA, indicating that determinant specificity is of crucial importance. Also, both RT1-linked and non-RT1-linked gene directed functions are involved in the final clinical response to immunization with type II collagen. RT1-linked control is likely exerted at the level of Class II (Ia) molecules (as it is in mice) with inherent selectivity of arthritogenic vs non-arthritogenic epitopes for presentation to the immune response system; non-RT1-linked control may reflect genes controlling T-cell receptors, immunoglobulin subtypes or complement components. There is also evidence that the effects of potentially pathological anti-collagen autoimmunity may in some strains be muted or even obviated by other non-RT1 gene controlled traits that are not directly related to the immune system. These general conclusions are in close accord with those of other investigators who have carefully conducted extensive and in-depth studies of the immunogenetics of CIA in mice. CIA is obviously not an exact model of any one of the more common rheumatic diseases, such as rheumatoid arthritis or systemic lupus erythematosus. In fact, it is more closely analogous to polychondritis and some of the other sero-negative connective tissue diseases. However, CIA remains an extremely useful model in attempts to understand the genetic and environmental factors which influence a specific and definable autoimmune process--anti-collagen reactivity. In turn, autoimmunity to collagen, and to other autoantigens, is a contributing or complicating aspect of most of the diverse human rheumatic disease syndromes which have been identified to date. The characteristics of the CIA model in rats which have been discussed in this article, i.e., genetically controlled variations in incidence, severity, rate of progression and expression of clinical disease, are also characteristic of the human rheumatic disease patient population. Likewise, the probable contribution of multiple genes to these syndromes is recognized. Continued investigation of the CIA model can be expected to yield important information that can be used to better understand its human counterparts.

Passive transfer of adjuvant-induced arthritis into irradiated DA recipient rats.

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.

Type XI and II collagen-induced arthritis in rats: characterization of inbred strains of rats for arthritis-susceptibility and immune-responsiveness to type XI and II collagen.

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.

Identification of a cyanogen bromide fragment of porcine type II collagen capable of modulating collagen arthritis in B10.RIII (H-2r) mice.

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.

Altered development of collagen induced arthritis in T cell receptor V beta congenic B10.RIII mice.

Analysis of the mouse T cell receptor (TCR) V beta genome has revealed the existence of two distinct genotypes which bear deletions of certain V beta genes. Mice bearing the V beta a genotype lack approximately 50% of the V beta genome while V beta c mice lack 70% of the known V beta genes. Studies of the experimental model collagen induced arthritis (CIA) have indirectly suggested that the presence of truncated V beta genotypes may influence susceptibility to this autoimmune disease. In order to confirm the influence of V beta a and V beta c genotypes on CIA, we derived mice congenic for the known V beta haplotypes in the CIA susceptible B10.RIII (H-2r) background. Flow cytometric analysis of splenic lymphocytes revealed normal T cell levels in both B10.RIII-V beta congenic lines. Expectedly, a generalized increase in the expression of some non-deleted V beta genes was detected. In addition, the mice were immunized with porcine type II collagen and monitored for CIA. B10.RIII-V beta a mice showed little difference in arthritis incidence or severity versus B10.RIII, but a significant delay in the onset of CIA was seen. In contrast, B10.RIII-V beta c mice showed a marked decrease in arthritis incidence versus B10.RIII and the severity of CIA in arthritic mice was also significantly lower (p < 0.01). Thus, in the B10.RIII strain, the presence of truncated TCR V beta genotypes alters the development of CIA. These findings may shed light on the influence of TCR genotypes in the induction and development of human rheumatoid arthritis.

T-cell receptors and collagen induced arthritis in H-2r mice.

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%; p < 0.005) 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)

Exacerbation of collagen-induced arthritis in rats by rat cytomegalovirus is antigen-specific.

Collagen-Induced Arthritis (CIA) is an experimentally induced and genetically controlled animal model of chronic joint inflammation. In rats, there are informative strain differences in susceptibility to CIA. DA rats (RT1avl) develop severe CIA after immunization with bovine (BII), chick (CII), or homologous rat (RII) type II collagens. In contrast, the MHC-congenic DA. 1N(BN) and WF.1N(BN) rats (RT1n) are relatively resistant to CIA and develop moderate CIA in response to immunization with CII but not BII or RII. We previously found that simultaneous infection with rat cytomegalovirus (RCMV) greatly exacerbates the severity of arthritis that develops in BII-immunized DA rats. To examine the mechanism of RCMV amplification of CIA, the effect of simultaneous infection with RCMV on arthritis and autoimmunity to type II collagen was determined in WF.1N and DA.1N rats after immunization with BII, CII and RII. RCMV increased the incidence of CIA and the level of autoimmunity to type II collagen (skin-testing and IgG antibody titer) selectively in DA.1N and WF.1N rats immunized with CII, but not in littermates immunized with BII, although the transient reversal of CD4+/CD8+ mononuclear cell ratios in peripheral blood that is associated with RCMV infection occurred equally in both BII- and CII- immunized DA.1N rats. Likewise, RCMV infection moderately increased the levels of anti-RII autoimmunity and arthritis in DA rats sub-optimally immunized with RII but had no consistent effect on either anti-RII immunity or arthritis in RII-immunized DA.1N and WF.1n rats. The data show that RCMV augments arthritis only in rats that are genetically susceptible to CIA and that are appropriately immunized with a species of type II collagen that is arthritogenic for the MHC-haplotype being tested. Two possible mechanisms are suggested by these data: RCMV-associated increases in anti-RII autoimmunity in rats with CIA may result from amino acid sequence homologies between RCMV and type II collagen; alternatively, virus-induced pro-inflammatory cytokines may activate RII-reactive lymphocytes thereby potentiating autoimmunity and arthritis.

Collagen-induced arthritis in mice: synergistic effect of E. coli lipopolysaccharide bypasses epitope specificity in the induction of arthritis with monoclonal antibodies to type II collagen.

DBA/1 mice develop a chronic peripheral arthritis after immunization with type II collagen termed collagen-induced arthritis. We have localized the main arthritogenic determinants of CB11, a CNBr-generated arthritogenic fragment of chick type II collagen (CII), using 3 smaller peptide fragments of CB11 generated by endoproteinase LysC, LysC1 (CII 124-290), LysC2 (CII 291-374) and LysC3 (CII 375-402) and a panel of monoclonal antibodies (mAb) specific to CB11. MAb specific to the arthritogenic region of CB11 were also used to study the synergistic effect of E. coli lipopolysaccharide (LPS) on antibody-mediated arthritis in naive DBA/1 mice. LysC2 contained a minimum essential arthritogenic fragment of type II collagen: LysC2 induced arthritis by active immunization, also, a combination of four mAb specific to LysC2 passively transferred arthritis to naive mice. A single i.p. injection of LPS (50 micrograms/mouse) reduced the threshold values of the arthritogenic dose of mAb from 1 mg to 50 micrograms/clone per mouse, and decreased the number of mAb required for inducing arthritis from 4 to 2 clones. These observations suggest that LysC2, an 84 amino acid residue fragment, contains the main arthritogenic determinants within chick CB11. Importantly, LPS, a strong inducer of pro-inflammatory cytokines, negates the required multiple epitope specificity of autoantibodies in the passive transfer model and acts synergistically in the induction of arthritis by autoantibody.