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.

Genetic factors involved in central nervous system/immune interactions.

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.

Genetic analysis of collagen-induced arthritis in rats: a polygenic model for rheumatoid arthritis predicts a common framework of cross-species inflammatory/autoimmune disease loci.

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.

Genetic dissection of a rat model for rheumatoid arthritis: significant gender influences on autosomal modifier loci.

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.

Genetic dissection of collagen-induced arthritis in Chromosome 10 quantitative trait locus speed congenic rats: evidence for more than one regulatory locus and sex influences.

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 size < or =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 size < or = 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.

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.

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.

An integrated genetic linkage map with 1,137 markers constructed from five F2 crosses of autoimmune disease-prone and -resistant inbred rat strains.

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.

Susceptibility to autoimmune disease and drug addiction in inbred rats. Are there mechanistic factors in common related to abnormalities in hypothalamic-pituitary-adrenal axis and stress response function?

DA and LEW inbred rats are extraordinarily susceptible to a wide range of experimental autoimmune diseases. These diseases include rheumatoid arthritis models such as collagen-induced arthritis (CIA) and adjuvant-induced arthritis (AIA), multiple sclerosis models such as myelin-basic-protein (MBP)-induced experimental autoimmune encephalomyelitis (MBP-EAE), and autoimmune uveitis models such as retinal S antigen (SAG) and interphotoreceptor-retinoid-binding-protein (IRBP)-induced experimental autoimmune uveitis (SAG-EAU and IRBP-EAU, respectively). DA and LEW rats are also addiction-prone to various drugs of abuse, such as cocaine. Moreover, they exhibit a variety of behavioral and biochemical characteristics that appear to be related to their susceptibility to addiction. By contrast, F344 and BN rats show quite different phenotypes. They are relatively resistant to CIA, AIA, MBP-EAE, SAG-EAU, and IRBP-EAU, and they are relatively resistant to addiction. Interestingly, both DA and LEW rats, in contrast to F344 and BN rats, have abnormalities in hypothalamic-pituitary-adrenal (HPA) axis function. For example, circadian production of corticosteroids is very abnormal in DA and LEW rats; that is, they exhibit minimal circadian variation in corticosterone levels. Since corticosteroids potentially have significant influences on immune function and autoimmune disease susceptibility and may also influence sensitivity to drugs of abuse, we have begun to dissect genetic control of these various phenotypic differences, focusing initially on the regulation of autoimmune disease expression. Using genomewide scanning techniques involving F2 crosses of DA x F344 (CIA and AIA), DA x BN (CIA), and LEW x F344 [IRBP-EAU and streptococcal-cell-wall arthritis (SCWA)], we have identified, to date, 14 genomic regions [quantitative trait loci (QTL)] that regulate disease expression in these crosses. Development and analysis of QTL-congenic rats involving these loci are in progress and should permit us to address the relationships among autoimmune disease susceptibility, drug addiction, and HPA axis and stress response function. These initial data, however, indicate that the genetic control of the autoimmune disease traits is highly complex.

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.

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.

Identification of a new quantitative trait locus on chromosome 7 controlling disease severity of collagen-induced arthritis in rats.

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.

Animal models of rheumatoid arthritis and related inflammation.

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.

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.