Marie M. Griffiths

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.

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.

IDENTIFICATION OF FOUR NEW QUANTITATIVE TRAIT LOCI REGULATING ARTHRITIS SEVERITY AND ONE NEW QUANTITATIVE TRAIT LOCUS REGULATING AUTOANTIBODY PRODUCTION IN RATS WITH COLLAGEN-INDUCED ARTHRITIS

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 x 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 x BN)F1 rats (7 of 60) and 31% of (DA x 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 x F344) and (DA x 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 x 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.

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.

CD4 and CD8 T Cells in Susceptibility/Protection to Collagen-Induced Arthritis in HLA-DQ8-Transgenic Mice: Implications for Rheumatoid Arthritis

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.

The Non-MHC Quantitative Trait Locus Cia5 Contains Three Major Arthritis Genes That Differentially Regulate Disease Severity, Pannus Formation, and Joint Damage in Collagen- and Pristane-Induced Arthritis

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.

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.

Delineating the Role of the HLA-DR4 “Shared Epitope” in Susceptibility versus Resistance to Develop 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.

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.

Immunogenetic control of experimental collagen-induced arthritis in rats. II. ECIA susceptibility and immune response to type II collagen (CALF) are linked to RT1.

The segregation of genes controlling ECIA susceptibility and the level of immune response to native calf type II collagen were determined in the F2 progeny of matings between WF (RTIu/u; ECIA‐susceptible; high responders) and LEW.B3 (RT1n/n; ECIA‐resistant; low to intermediate responders). RT1n/n F2 progeny showed resistance to ECIA, low skin test reactivity to type II collagen and intermediate levels of IgG anti‐collagen antibodies (‐log2 of 6.2 ± 2.6; mean ± SD, n= 10). RT1u/u and RT1u/u F2 progeny were susceptible to ECIA and were high responders to type II collagen by skin testing and IgG antibody titres (‐log2 of 12.1 ± 1.3, mean ± SD, n= 26). Although all rats that developed arthritis were also high responders to type II collagen one group of immature F2 progeny, RT1u/u and RT1u/n, showed high anti‐collagen immune responses in the absence of detectable arthritis. The data indicate that genes linked to RT1.A control susceptibility to ECIA and at least part of the immune response to native calf type II collagen in WF and LEW.B3 rats.