Definition of a 1.06-Mb region linked to neuroinflammation in humans, rats and mice

Unbiased identification of susceptibility genes might provide new insights into pathogenic mechanisms that govern complex inflammatory diseases such as multiple sclerosis. In this study we fine mapped Eae18a, a region on rat chromosome 10 that regulates experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. We utilized two independent approaches: (1) in silico mapping based on sequence similarity between human multiple sclerosis susceptibility regions and rodent EAE quantitative trait loci and (2) linkage mapping in an F10 (DA x PVG.AV1) rat advanced intercrossed line. The linkage mapping defines Eae18a to a 5-Mb region, which overlaps one intergenomic consensus region identified in silico. The combined approach confirms experimentally, for the first time, the accuracy of the in silico method. Moreover, the shared intersection between the results of both mapping techniques defines a 1.06-Mb region containing 13 candidate genes for the regulation of neuroinflammation in humans, rats, and mice.     

Novel quantitative trait loci controlling development of experimental autoimmune encephalomyelitis and proportion of lymphocyte subpopulations

The B10.RIII mouse strain (H-2(r)) develops chronic experimental autoimmune encephalomyelitis (EAE) upon immunization with the myelin basic protein 89-101 peptide. EAE was induced and studied in a backcross between B10.RIII and the EAE-resistant RIIIS/J strain (H-2(r)), and a complete genome scan with microsatellite markers was performed. Five loci were significantly linked to different traits and clinical subtypes of EAE on chromosomes 1, 5, 11, 15, and 16, three of the loci having sex specificity. The quantitative trait locus on chromosome 15 partly overlapped with the Eae2 locus, previously identified in crosses between the B10.RIII and RIIIS/J mouse strains. The loci on chromosomes 11 and 16 overlapped with Eae loci identified in other mouse crosses. By analyzing the backcross animals for lymphocyte phenotypes, the proportion of B and T cells in addition to the levels of CD4(+)CD8(-) and CD4(-)CD8(+) T cells and the CD4(+)/CD8(+) ratio in spleen were linked to different loci on chromosomes 1, 2, 3, 5, 6, 11, and 15. On chromosome 16, we found significant linkage to spleen cell proliferation. Several linkages overlapped with the quantitative trait loci for disease phenotypes. The identification of subphenotypes that are linked to the same loci as disease traits could be most useful in the search for candidate genes and biological pathways involved in the pathological process.     

Identification and isolation of dominant susceptibility loci for pristane-induced arthritis

Rheumatoid arthritis is a chronic inflammatory autoimmune disorder, controlled by multiple genes as well as environmental factors. With animal models, like the pristane-induced arthritis (PIA) in rats, it is possible to reduce the environmental effects and the genetic heterogeneity to identify chromosomal regions harboring genes responsible for the arthritis development. The PIA model has proved to be useful for identifying gene regions controlling different phases of the disease based on intercrosses between the resistant E3 and the susceptible DA rat. We have now performed a high-powered backcross analysis that confirms previous intercross-based data but also identifies additional loci. Earlier identified PIA loci were reproduced with high significance; Pia1 (MHC region on chromosome 20), Pia4 (chromosome 12), and Pia7 (chromosome 4) are all major regulators of PIA severity and were also found to operate in concert. These three loci were verified in congenic strains using both disease- and arthritis-inflammatory-related subphenotypes as traits. We were also able to detect five new quantitative trait loci with dominant effects on PIA: Pia10, Pia12, Pia13, Pia14, and Pia15 on chromosomes 10, 6, 7, 8, and 18, respectively. These data highlight the usefulness of the statistical power obtained in a backcross of a complex disease like arthritis.     

Genome-wide linkage analysis of chronic relapsing experimental autoimmune encephalomyelitis in the rat identifies a major susceptibility locus on chromosome 9

The immunization of inbred Dark Agouti (DA) rats with an emulsion containing homogenized spinal cord and CFA induces chronic relapsing experimental autoimmune encephalomyelitis (EAE), a disease with many similarities to multiple sclerosis. We report here the first genome-wide search for quantitative trait loci regulating EAE in the rat using this model. We identified one quantitative trait locus on chromosome 9, Eae4, in a [DA(RT1av1) x BN(RT1n)]F2 intercross showing linkage to disease susceptibility and expression of mRNA for the proinflammatory cytokine IFN-gamma in the spinal cord. Eae4 had a larger influence on disease incidence among rats that were homozygous for the RT1av1 MHC haplotype (RT1av1 rats) compared with RT1n/av1 rats, suggesting an interaction between Eae4 and the MHC. Homozygosity for the DA allele at markers in Eae4 and in the MHC was sufficient for EAE. Thus, Eae4 is a major genetic factor determining susceptibility to EAE in this cross of DA rats. In addition, there was support for linkage to phenotypes of EAE on chromosomes 1, 2, 5, 7, 8, 12, and 15. The chromosome 12 region has been shown previously to predispose DA rats to arthritis, and the chromosome 2 region is syntenic to Eae3 in mice. We conclude that Eae4 and probably the other identified genome regions harbor genes regulating susceptibility to neuroinflammatory disease. The identification and functional characterization of these genes may disclose critical events in the pathogenesis of multiple sclerosis; understanding these events could be essential for the development of new therapies against the disease.     

Advanced intercross line mapping of Eae5 reveals Ncf-1 and CLDN4 as candidate genes for experimental autoimmune encephalomyelitis

Eae5 in rats was originally identified in two F(2) intercrosses, (DA x BN) and (E3 x DA), displaying linkage to CNS inflammation and disease severity in experimental autoimmune encephalomyelitis (EAE), respectively. This region overlaps with an arthritis locus, Pia4, which was also identified in the (E3 x DA) cross. Two congenic strains, BN.DA-Eae5 and BN.DA-Eae5.R1, encompassing the previously described Eae5 and Pia4, were established. DA alleles within the chromosome 12 fragment conferred an increase in disease susceptibility as well as increased inflammation and demyelination in the CNS as compared with BN alleles. To enable a more precise fine mapping of EAE regulatory genes, we used a rat advanced intercross line between the EAE-susceptible DA strain and the EAE-resistant PVG.1AV1 strain. Linkage analysis performed in the advanced intercross line considerably narrowed down the myelin oligodendrocyte glycoprotein-EAE regulatory locus (Eae5) to a approximately 1.3-megabase region with a defined number of candidate genes. In this study we demonstrate a regulatory effect of Eae5 on MOG-EAE by using both congenic strains as well as fine mapping these effects to a region containing Ncf-1, a gene associated with arthritis. In addition to structural polymorphisms in Ncf-1, both sequence polymorphisms and expression differences were identified in CLDN4. CLDN4 is a tight junction protein involved in blood-brain barrier integrity. In conclusion, our data strongly suggests Ncf-1 to be a gene shared between two organ-specific inflammatory diseases with a possible contribution by CLDN4 in encephalomyelitis.     

Inconsistent susceptibility to autoimmunity in inbred LEW rats is due to genetic crossbreeding involving segregation of the arthritis-regulating gene Ncf1

We recently identified a single-nucleotide polymorphism in the Ncf1 gene, a component of the NADPH oxidase complex, to be the cause of one of the strongest identified loci for arthritis severity in rats. This polymorphism was found to be naturally occurring in a collection of inbred rat strains as well as in wild rats. Among the inbred strains we found that different LEW substrains (LEW/Ztm and LEW/Mol), originating from different breeders, showed an allelic discrepancy in Ncf1, suggesting an impact on arthritis susceptibility between these substrains. In fact, the LEW/Mol strain was completely resistant to pristane-induced arthritis, in contrast to the LEW/Ztm strain, which was susceptible. Moreover, the LEW/Mol strain had higher production of radical oxygen species in peripheral blood leukocytes, a phenomenon most likely regulated by the polymorphisms in the Ncf1 gene. However, the phenotypic difference between LEW/Mol and LEW/Ztm is most likely a combination of several genes, of which Ncf1 is suggested to be the major regulating gene. This has also been confirmed by previous linkage analyses involving the LEW/Ztm strain which shows that a QTL on chromosome 12, most likely caused by polymorphism of Ncf1, is the major regulatory gene but that other loci are contributing. That more genes are likely to contribute was shown by a complete genome comparison of the LEW/Ztm and the LEW/Mol rat strains that uncovered an introduction of approximately 37% non-LEW genome into the LEW/Mol strain, which probably was caused by past crossbreeding. Therefore, the LEW/Mol should be regarded as a recombinant inbred strain.     

Myelin oligodendrocyte glycoprotein induces experimental autoimmune encephalomyelitis in the "resistant" Brown Norway rat: disease susceptibility is determined by MHC and MHC-linked effects on the B cell response.

Experimental autoimmune encephalomyelitis (EAE) induced by active immunization with the myelin oligodendrocyte glycoprotein (MOG) is an Ab-mediated, T cell-dependent autoimmune disease that replicates the inflammatory demyelinating pathology of multiple sclerosis. We report that disease susceptibility and severity are determined by MHC and MHC-linked effects on the MOG-specific B cell response that mediate severe clinical EAE in the EAE-resistant Brown Norway (BN) rat. Immunization with the extracellular domain of MOG in CFA induced fulminant clinical disease associated with widespread demyelination and with an inflammatory infiltrate containing large numbers of polymorphonuclear cells and eosinophils within 10 days of immunization. To analyze the effects of the MHC (RT1 system) we compared BN (RT1 n) rats with Lewis (LEW) (RT1 l) and two reciprocal MHC congenic strains, LEW.1N (RT1n) and BN.1L (RT1 l). This comparison revealed that disease severity and clinical course were strongly influenced by the MHC haplotype that modulated the pathogenic MOG-specific autoantibody response. The intra-MHC recombinant congenic strain LEW.1R38 demonstrated that gene loci located both within the centromeric segment of the MHC containing classical class I and class II genes and within the telomeric RT1.M region containing the MOG gene are involved in determining Ab production and disease susceptibility. This study indicates that the current T cell-centered interpretation of MHC-mediated effects on disease susceptibility must be reassessed in multiple sclerosis and other autoimmune diseases in which autoantibody is involved in disease pathogenesis.     

Genetic epidemiology of rheumatoid arthritis: what to expect from Latin America?

Rheumatoid arthritis is a chronic and systemic autoimmune disease characterized by inflammation and destruction of the synovial joints. It affects approximately 0.5% of the Latin-American population and is three times more common in women than in men. Evidence of familial aggregation (lambdas=2-17) was the first indication of a genetic susceptibility to disease. As in other autoimmune diseases, it has a complex genetic basis. Results from whole-genome scans indicate that the HLA region contains a significant and consistent set of linked loci. However, HLA accounts for only one-third of the genetic susceptibility of disease, indicating that non-HLA genes are also involved in the disease susceptibility. In Latin-America, association with HLA-DRB1*0404 and TNF -308A alleles has been uniformly established; however, many other candidate genes remain to be studied. The identification of genetic factors conferring susceptibility to rheumatoid arthritis will contribute to the knowledge of the pathogenic mechanisms, ability to predict its occurrence, the development of diagnostic tools, prognosis, and treatment. The genetic epidemiology of rheumatoid arthritis is herein reviewed; a set of recommendations is provided for the design, analysis and interpretation of genetic association studies in the context of Latin-American populations.     

Insight from genome-wide association studies in rheumatoid arthritis and multiple sclerosis

Autoimmune diseases are caused by multiple genes and environmental effects. In addition, genetic contributions and the number of associated genes differ among different diseases and ethnic populations. Genome-wide association studies (GWAS) on rheumatoid arthritis (RA) and multiple sclerosis (MS) show that these diseases share many genetic factors. Recently, in addition to the major histocompatibility complex (MHC) gene, other genetic loci have been found to be associated with the risk for autoimmune diseases. This review focuses on the search for genetic variants that influence the susceptibility to RA and MS as typical autoimmune diseases and discusses the future of GWAS.     

Genetic susceptibility to autoimmune disorders: clues from gene association and gene expression studies

Susceptibility to autoimmune disorders results from the interaction of multiple genetic factors that regulate the threshold of autoreactivity. Genome-wide microsatellite screens and large-scale single nucleotide polymorphism (SNP) association studies have identified chromosomal loci that are associated with specific disorders including systemic lupus erythematosus, rheumatoid arthritis, juvenile arthritis, multiple sclerosis, and diabetes. Numerous candidate gene association studies have in turn investigated the association of specific genes within these chromosomal regions, with susceptibility to autoimmune diseases (e.g. FcgammaReceptors, TYK2 and systemic lupus). More recently, large-scale differential gene expression studies performed on selected tissues from patients with autoimmune disorders, have led to the identification of gene signatures associated with the activation of specific pathways in these diseases (e.g. interferon signature in lupus). In the future, integrated analyses of gene (and protein) expression together with SNP data will allow us to sketch an intelligible picture of the genesis of autoimmunity in humans. This review sets out to illustrate how the most recent advances in the field of systemic lupus erythematosus, rheumatoid arthritis and juvenile arthritis have led to a better understanding of these disorders.     

New loci regulating rat myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis

Myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease in rats that closely mimics many clinical and histopathological aspects of multiple sclerosis. Non-MHC quantitative trait loci regulating myelin oligodendrocyte glycoprotein-induced EAE have previously been identified in the EAE-permissive strain, DA, on rat chromosomes 4, 10, 15, and 18. To find any additional gene loci in another well-known EAE-permissive strain and thereby to assess any genetic heterogeneity in the regulation of the disease, we have performed a genome-wide linkage analysis in a reciprocal (LEW.1AV1 x PVG.1AV1) male/female F(2) population (n = 185). We examined reciprocal crosses, but no parent-of-origin effect was detected. The parental rat strains share the RT1(av1) MHC haplotype; thus, non-MHC genes control differences in EAE susceptibility. We identified Eae16 on chromosome 8 and Eae17 on chromosome 13, significantly linked to EAE phenotypes. Two loci, on chromosomes 1 and 17, respectively showed suggestive linkage to clinical and histopathological EAE phenotypes. Eae16 and Eae17 differ from those found in previously studied strain combinations, thus demonstrating genetic heterogeneity of EAE. Furthermore, we detected a locus-specific parent-of-origin effect with suggestive linkage in Eae17. Further genetic and functional dissection of these loci may disclose critical disease-regulating molecular mechanisms.     

A comprehensive analysis of shared loci between systemic lupus erythematosus (SLE) and sixteen autoimmune diseases reveals limited genetic overlap

In spite of the well-known clustering of multiple autoimmune disorders in families, analyses of specific shared genes and polymorphisms between systemic lupus erythematosus (SLE) and other autoimmune diseases (ADs) have been limited. Therefore, we comprehensively tested autoimmune variants for association with SLE, aiming to identify pleiotropic genetic associations between these diseases. We compiled a list of 446 non–Major Histocompatibility Complex (MHC) variants identified in genome-wide association studies (GWAS) of populations of European ancestry across 17 ADs. We then tested these variants in our combined Caucasian SLE cohorts of 1,500 cases and 5,706 controls. We tested a subset of these polymorphisms in an independent Caucasian replication cohort of 2,085 SLE cases and 2,854 controls, allowing the computation of a meta-analysis between all cohorts. We have uncovered novel shared SLE loci that passed multiple comparisons adjustment, including the VTCN1 (rs12046117, P = 2.02×10⁻⁰⁶) region. We observed that the loci shared among the most ADs include IL23R, OLIG3/TNFAIP3, and IL2RA. Given the lack of a universal autoimmune risk locus outside of the MHC and variable specificities for different diseases, our data suggests partial pleiotropy among ADs. Hierarchical clustering of ADs suggested that the most genetically related ADs appear to be type 1 diabetes with rheumatoid arthritis and Crohn''s disease with ulcerative colitis. These findings support a relatively distinct genetic susceptibility for SLE. For many of the shared GWAS autoimmune loci, we found no evidence for association with SLE, including IL23R. Also, several established SLE loci are apparently not associated with other ADs, including the ITGAM-ITGAX and TNFSF4 regions. This study represents the most comprehensive evaluation of shared autoimmune loci to date, supports a relatively distinct non–MHC genetic susceptibility for SLE, provides further evidence for previously and newly identified shared genes in SLE, and highlights the value of studies of potentially pleiotropic genes in autoimmune diseases.     

Gene-Expression Profiling of Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a mouse model that serves as an experimental tool for studying the etiology, pathogenesis, as well as new therapeutic approaches of multiple sclerosis (MS). EAE is a polygenic chronic inflammatory demyelinating disease of the nervous system that involves the interaction between genetic and environmental factors. Previous studies have identified multiple quantitative trait loci (QTL) controlling different aspects of disease pathogenesis. However, progress in identifying new susceptibility genes outside the MHC locus has been slow. With the advent of new global methods for genetic analysis such as large-scale sequencing, gene expression profiling combined with classic linkage analysis and congenic and physical mapping progress is considerably accelerating. Here we review our preliminary work on the use of gene expression mapping to identify new putative genetic pathways contributing to the pathogenesis of EAE.     

DA rats from two colonies differ genetically and in their arthritis susceptibility

The arthritis-susceptible DA rat is one of the most commonly used rat strains for genetic linkage analysis and is instrumental for the identification of many genetic loci. Even though DA rats were kept as inbred lines at different institutes and suppliers, it became obvious that the various breeding stocks differed genetically. To be able to compare the results from different linkage studies it is very import to verify the genetic background of the substrains used in those studies. We performed a genetic and phenotypic analysis of two DA substrains, DA/ZtmRhd and DA/OlaHsd, and found several genetic differences. One of the allelic differences between the DA/ZtmRhd and the DA/OlaHsd strain was located at rat chromosome 3, a 17-Mb large fragment, including the peak marker of a previously identified quantitative trait locus (QTL) for collagen-induced arthritis, Cia11. In addition, the substrains exhibited a significant difference in the susceptibility to pristane-induced arthritis (PIA) and disease severity of collagen-induced arthritis and PIA. However, by generating and testing a congenic line, we could demonstrate that phenotypic differences were not due to the contaminating fragment on chromosome 3. Nevertheless, we conclude that DA substrains show distinct genetic differences and caution should be taken when comparing arthritis data from different DA substrains. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification of Genetic Determinants of the Sexual Dimorphism in CNS Autoimmunity

Multiple sclerosis (MS) is a debilitating chronic inflammatory disease of the nervous system that affects approximately 2.3 million individuals worldwide, with higher prevalence in females, and a strong genetic component. While over 200 MS susceptibility loci have been identified in GWAS, the underlying mechanisms whereby they contribute to disease susceptibility remains ill-defined. Forward genetics approaches using conventional laboratory mouse strains are useful in identifying and functionally dissecting genes controlling disease-relevant phenotypes, but are hindered by the limited genetic diversity represented in such strains. To address this, we have combined the powerful chromosome substitution (consomic) strain approach with the genetic diversity of a wild-derived inbred mouse strain. Using experimental allergic encephalomyelitis (EAE), a mouse model of MS, we evaluated genetic control of disease course among a panel of 26 consomic strains of mice inheriting chromosomes from the wild-derived PWD strain on the C57BL/6J background, which models the genetic diversity seen in human populations. Nineteen linkages on 18 chromosomes were found to harbor loci controlling EAE. Of these 19 linkages, six were male-specific, four were female-specific, and nine were non-sex-specific, consistent with a differential genetic control of disease course between males and females. An MS-GWAS candidate-driven bioinformatic analysis using orthologous genes linked to EAE course identified sex-specific and non-sex-specific gene networks underlying disease pathogenesis. An analysis of sex hormone regulation of genes within these networks identified several key molecules, prominently including the MAP kinase family, known hormone-dependent regulators of sex differences in EAE course. Importantly, our results provide the framework by which consomic mouse strains with overall genome-wide genetic diversity, approximating that seen in humans, can be used as a rapid and powerful tool for modeling the genetic architecture of MS. Moreover, our data represent the first step towards mechanistic dissection of genetic control of sexual dimorphism in CNS autoimmunity.     

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-1beta 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.     

Association of functional variants of PTPN22 and tp53 in psoriatic arthritis: a case-control study

Recent studies have implicated PTPN22 and tp53 in susceptibility to several autoimmune diseases, including rheumatoid arthritis, suggesting that these genes are important in maintaining immune homeostasis. Because autoimmune diseases may share similar susceptibility loci, investigation of these genes in psoriatic arthritis (PsA) is of potential relevance. As a result we investigated known coding polymorphisms in PTPN22 and tp53 in a homogenous Caucasian PsA cohort from Newfoundland, Canada and an admixed Caucasian PsA cohort from Toronto, Canada. We observed a moderate association of the R620W variant of PTPN22 with PsA in the Toronto population only. Because of the conflicting findings reported regarding the association of PTPN22 with PsA, further studies in other PsA populations are warranted.     

Selecting SNPs for association studies based on population frequencies: a novel interactive tool and its application to polygenic diseases

Common complex polygenic diseases as autoimmune diseases have not been completely understood on a molecular level. While many genes are known to be involved in the pathways responsible for the phenotype, explicit causes for the susceptibility of the disease remain to be elucidated. The susceptibility to disease is thought to be the result of genetic epistatic interactions between common polymorphic genes. This polymorphism is mostly caused by single nucleotide polymorphisms (SNPs). Human subpopulations are known to differ in the susceptibility to the diseases and generally in the distribution of single nucleotide polymorphisms. The here presented approach retrieves SNPs with the most divergent frequencies for selected human subpopulations to help defining properties for the experimental verification of SNPs within defined regions. A web-accessible program implementing this approach was evaluated for multiple sclerosis (MS), a common human polygenic disease. A link to a summary of data from "The SNP Consortium" (TSC) with sex-dependencies of SNPs is available. Associations of SNPs to genes, genetic markers and chromosomal loci are retrieved from the Ensembl project. This tool is recommended to be used in conjunction with microarray analyses or marker association studies that link genes or chromosomal loci to particular diseases.     

Two loci on chromosome 15 control experimentally induced arthritis through the differential regulation of IL-6 and lymphocyte proliferation

Using genetic linkage analysis of proteoglycan-induced arthritis (PGIA), a murine model for rheumatoid arthritis, we identified two loci, Pgia8 and Pgia9, on chromosome 15 (chr15) that appear to be implicated in disease susceptibility. Immunization of congenic strains carrying the entire chr15 and separately each of the two loci of DBA/2 arthritis-resistant origin in susceptible BALB/c background confirmed locations of two loci on chr15: the major Pgia9 and lesser Pgia8 locus. Distal part of chr15 (Pgia9) showed a major suppressive effect on PGIA susceptibility in females (40%, p < 0.001), whereas the effect of this locus in congenic males was still significant but weaker. Proximal part of chr15 (Pgia8) demonstrated mild and transient effect upon arthritis; this effect was PGIA-promoting in males and suppressive in females. Pgia8 and Pgia9 loci demonstrated an additive mode of inheritance, since when they were both incorporated in consomic chr15 strain, the total effect was a sum of the two loci. Using F(2) population of the intercross of wild-type and chr15 consomic strain, we confirmed and refined quantitative trait locus positions and identified a strong correlation between disease susceptibility and lymphocyte-producing cytokines of TNF-alpha and IL-6. Both Pgia8 and Pgia9 loci on chr15 appear to control IL-6 production in spleen cultures of arthritic mice, providing an important link to the mechanism of autoimmune inflammation.