The importance of genetic factors in rheumatoid arthritis (RA) is well characterized by the aggregation of the disease in families and the high risk of the disease, both in siblings and in genetically identical twins of affected subjects, compared with that in the general population. In addition to previous studies focusing on the contribution of the HLA–DR locus to RA, recent genome-wide screening has shown linkage of many non–major histocompatibility complex (non-MHC) regions to the disease (1, 2). Despite decades of research, however, the genes controlling RA susceptibility have not been precisely identified.
In mouse models, a strong influence of the MHC region was seen in collagen-induced arthritis (CIA) (3), as observed in patients with RA. Polymorphic non-MHC genes, such as C5, Fcgr2b, Ncf1, and Il1b, were also suggested to affect susceptibility and severity in CIA and in the K/BxN serum–transfer model of arthritis in mice (4–7). In murine models of spontaneously occurring RA, such as MRL/lpr mice, SKG mice, IL-1 receptor antagonist (IL-1Ra)–deficient mice, and gp130-mutated mice, aberrant signals to immune cells due to mutated Fas (8), ZAP-70 (9), IL-1R (10), and IL-6R gp130 (11), respectively, have been shown to contribute to pathogenesis. However, none of these RA mouse models shares such mutated genes. These findings illustrate the genetic heterogeneity of RA susceptibility, in which the effects of different sets of susceptibility genes induce the same RA phenotypes. Thus, further studies to identify additional susceptibility genes could contribute to a more thorough understanding of the genetic basis of RA.
The presence of several autoantibodies, such as rheumatoid factors (RFs) and antibodies against type II collagen (CII) and cyclic citrullinated peptide (CCP), is a characteristic feature of RA patients, and immune complexes composed of these autoantibodies have been suggested to be involved in the pathogenesis of joint inflammation. Thus, molecular mechanisms responsible for the activation of RA-specific autoantibody-producing B cells are a matter of intense investigation. Among a number of molecules controlling B cell activation, Fcγ receptor type IIb (FcγRIIb) is one of the major regulators that negatively controls B cell receptor (BCR)–mediated activation signals (12). FcγRIIb-deficient C57BL/6 (B6) mice exhibit marked serum RF activities (13), indicating the critical role of FcγRIIb in RF induction. It is also reported that the FcγRIIb deficiency renders mice highly susceptible to CIA (14, 15). However, the reports by Bolland and Ravetch (16) and Nimmerjahn and Ravetch (17) indicated that FcγRIIb-deficient B6 mice did develop systemic lupus erythematosus (SLE), but not RA.
In the present study, we established 2 lines of FcγRIIb-deficient B6 congenic strains of mice. Surprisingly, one of them spontaneously developed severe RA, but not SLE, and the other failed to develop either disease. The genomic difference between the 2 strains resides in the ∼6.3-Mb interval distal from the null-mutated Fcgr2b gene, and the interval of the RA-prone strain is of the 129 strain origin and the interval of the other is of the B6 strain origin. This 129 strain–derived interval is located within the Sle16 locus, an ∼9.3-Mb interval (18, 19), which has been shown to induce susceptibility to the production of high levels of autoantibodies and the development of mild glomerulonephritis when transferred on a B6 background (18). Our studies show evidence that the 129 strain–derived interval within the Sle16 locus confers the predisposition to RA in mice with the FcγRIIb-deficient B6 genetic background.
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In the current study, we showed evidence that the 129 strain–derived ∼6.3-Mb interval distal from the null-mutated Fcgr2b gene plays a pivotal role in the susceptibility to RA in FcγRIIb-deficient KO1 mice, suggesting that combined effects of the null-mutated Fcgr2b gene and the gene(s) in the 129 strain–derived ∼6.3-Mb interval are involved in the genetic regulation of RA. Because the 129 strain–derived interval in the telomeric region of chromosome 1 contains several reported candidate genes for susceptibility to SLE (18, 19), our model is useful for clarifying the genetic mechanisms that control the outcome of the separate autoimmune diseases SLE and RA.
The 129 strain–derived ∼6.3-Mb interval in KO1 mice is included in the locus Sle16 that contains several candidate genes for susceptibility to SLE, such as Fcgr2b, Fcgr3, Slam family genes, and IFN-inducible genes (Figure 1). There have been several reports indicating that the polymorphic Fcgr2b gene (21, 23, 28) and Slam family genes (19, 29) are the most plausible candidate genes for susceptibility to SLE in mice.
We previously found that autoimmune-prone mice, such as NZB, MRL, BXSB, and NOD mice, share autoimmune-type Fcgr2b polymorphism, which has nucleotide deletion in the activating enhancer binding protein 4 binding site in the promoter region (23). Because of this type of polymorphism, the level of FcγRIIb expression on activated B cells is markedly suppressed, leading to B cell activation and enhanced pathogenic autoantibody production (21). A significant association has been reported between the polymorphism of FCGR3A/B and human SLE (30). However, it is still unknown whether the polymorphism of Fcgr3 contributes to susceptibility to SLE in murine models. Slam family genes include Cd244, Cd229, Cs1, Cd48, Cd160, Cd84, and Ly108 (29). The 129 strain carries autoimmune-type, haplotype 2 Slam family genes, the same haplotype as that reported in the autoimmune-susceptible NZB, NZW, MRL, and BXSB strains (29), which have been shown to be involved in the control of B cell tolerance (19). The IFN-inducible gene Ifi202 was reported to be a possible candidate (31); however, recent congenic dissection studies revealed that this gene had no significant effects on susceptibility to SLE (32).
In the current study, since both KO1 and KO2 carry the null mutation of the Fcgr2b gene, the most plausible candidate conferring susceptibility in the locus Sle16 is a cluster of Slam family genes. Although the exact susceptibility gene(s) in multiple Slam family genes remains undetermined, Ly108 may be the strongest candidate. Ly108 produces 2 splice isoforms, Ly108.1 and Ly108.2, and the autoimmune-type Ly108 allele preferentially expresses the former in immature B cells, while the B6 type expresses the latter (29). Kumar et al (33) reported that upon BCR stimulation, immature B cells expressing Ly108.1 exhibited reduced calcium flux and decreased cell death, as compared with those expressing Ly108.2. This suggests that the Ly108.1 isoform expressed in lupus-prone mice is less effective in the induction of clonal anergy and the deletion of immature autoreactive B cells.
Recent case–control studies in a Japanese population identified a linkage disequilibrium segment associated with RA in the chromosome 1q region containing multiple Slam family genes (34). Association peaks were seen at 2 functional single-nucleotide polymorphisms in Cd244. Investigators in that study also identified a cohort with SLE that had a genotype distribution similar to that in the RA cohorts, suggesting that CD244 plays a role in the autoimmune process shared by RA and SLE. Related to this are genome-wide linkage studies in UK and Canadian families, showing that another nearby Slam family gene for LY9 (Cd229) has risk variants for SLE (35). Further investigations in different ethnic groups are needed to clarify the roles of variants of Slam family genes in patients with RA and SLE.
Bolland and Ravetch (16) and Nimmerjahn and Ravetch (17) reported the development of SLE, but not RA, in their FcγRIIb-deficient B6 mouse strains. In striking contrast, not only our KO1 and KO2 mice, but also the parental mouse lines failed to develop any sign of lupus-like disease, despite the fact that our FcγRIIb-deficient B6 mice and those described by Bolland and Ravetch (16) were obtained by backcrossing the FcγRIIb-deficient mice originally constructed on a hybrid (129 × B6) background into a B6 background (20). The reason for this discrepancy remains unknown; however, identification of the reasons for this difference is extremely important in our understanding of the genetic and/or environmental mechanisms that control the outcome of the separate autoimmune diseases SLE and RA.
With regard to environmental factors, it is possible that B6 mice obtained from different commercial vendors may have different commensal intestinal bacteria, which could play an important role. Ivanov et al (36) reported that this difference affects the immune system, most strikingly, IL-17 production, which may possibly affect the severity and specificity of autoimmune disease. In addition, we cannot exclude the possibility that the development of RA, but not SLE, in KO1 mice can be influenced by additional environmental factors that are unique to our animal facility. Alternatively, a spontaneous mutation that occurred in the 129 strain–derived interval during the establishment of the KO1 strain may promote the development of RA-like joint lesions rather than lupus nephritis. Clearly, identification of the susceptibility gene(s) for RA located in the Slam-linked distal Sle16 locus is of paramount importance for shedding light on the genetic mechanisms that control not only RA, but also SLE.
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All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Hirose had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Shirai, Hirose.
Acquisition of data. M. Ohtsuji, Nishikawa, Sudo, Ono, Izui, Takai, Nishimura, Hirose.
Analysis and interpretation of data. Sato-Hayashizaki, M. Ohtsuji, Lin, Hou, N. Ohtsuji, Nishikawa, Tsurui.