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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Objective

Fcγ receptor type IIb (FcγRIIb) is a major negative regulator of B cells, and the lack of FcγRIIb expression has been reported to induce systemic lupus erythematosus (SLE) in mice of the C57BL/6 (B6) genetic background. The 129 strain–derived Sle16 locus on the telomeric region of chromosome 1 including polymorphic Fcgr2b confers the predisposition to systemic autoimmunity when present on the B6 background. We undertook this study to examine the effect of the Sle16 locus on autoimmune disease in FcγRIIb-deficient B6 mice.

Methods

We established 2 lines of FcγRIIb-deficient B6 congenic mouse strains (KO1 and KO2) by selective backcrossing of the originally constructed FcγRIIb-deficient mice on a hybrid (129 × B6) background into a B6 background. Although both lack FcγRIIb expression, the KO1 and KO2 strains carry different lengths of the 129 strain–derived telomeric chromosome 1 segment flanked to the null-mutated Fcgr2b gene; the KO1 strain carries a 129 strain–derived ∼6.3-Mb interval distal from the null-mutated Fcgr2b gene within the Sle16 locus, while this interval in the KO2 strain is of B6 origin.

Results

Unexpectedly, both strains failed to develop SLE; instead, the KO1 strain, but not the KO2 strain, spontaneously developed severe rheumatoid arthritis (RA) with an incidence reaching >90% at age 12 months.

Conclusion

The current study shows evidence that the epistatic interaction between the Fcgr2b-null mutation and a polymorphic gene(s) in the 129 strain–derived interval located in the distal Sle16 locus contributes to RA susceptibility in a new mouse model with the B6 genetic background, although the participation of other genetic polymorphisms cannot be totally excluded.

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.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Mice.

Two FcγRIIb-deficient B6 congenic lines, KO1 and KO2, were obtained by backcrossing the FcγRIIb-deficient mice originally constructed on a hybrid (129 × B6) background (20) into a B6 background for 12 generations using C57BL/6NCrJ mice (Charles River Japan). Genotyping of these strains in the telomeric chromosome 1 region around the knockout FcγRIIb gene was done, taking advantage of gene polymorphisms or microsatellite markers polymorphic between the 129 and B6 strains (Figure 1). All mice were housed under identical specific pathogen–free conditions, and all experiments were performed in accordance with our institutional guidelines.

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Figure 1. Genetic map of the telomeric region on chromosome 1 (Chr. 1) in two different Fcγ receptor type IIb (FcγRIIb)–deficient C57BL/6 (B6) congenic strains of mice (KO1 and KO2; see Results for description of establishment of strains). The gene segment shown by the solid bar was introduced into the B6 background from the originally constructed FcγRIIb-deficient mice on a hybrid (129 × B6) background. Genotypes were determined by analysis of microsatellite marker polymorphisms and allele polymorphisms of Fcgr2b, Fcgr3, and 2 members of the Slam family (Cd229 and Cd84). The shaded bar represents the region of recombination between the B6 strain–derived interval (open bar) and the 129 strain–derived interval (solid bar). The hatched bar represents the reported 129 strain–derived Sle16 locus (18, 19).

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Genotyping.

DNA was extracted from mouse tail tissue. Genotyping of microsatellite markers was carried out as described elsewhere (21). Genotyping of the Fcgr2b-null mutation was performed using polymerase chain reaction (PCR) with the forward primers 5′-AAACTCGACCCCCCGTGGATC-3′ (wild-type) and 5′-CTCGTGCTTTACGGTATCGCC-3′ (Fcgr2b-null) and with the reverse primer 5′-TTGACTGTGGCCTTAAACGTGTAG-3′ (common). These primer pairs give rise to a 161-bp product for the wild-type allele and a 232-bp product for the Fcgr2b-null allele. Polymorphism of Fcgr3 in the B6-unique insertion and deletion sites of the α-chain was determined by length difference of the PCR products between strains 129 and B6, using the forward primer 5′-TCCATCTCTCTAGTCTGGTACC-3′ and the reverse primer 5′-AAAAGTTGCTGCTGCCACC-3′.

Polymorphisms for Cd229 and Cd84 were examined using single-strand conformational polymorphism. Primers for PCR were designed to amplify fragments encompassing the second exon of Cd229 including amino acid position 130 and the second exon of Cd84 including amino acid position 27. The forward and reverse primers used were 5′-GCAGACTCAAAGTCAGCGAAG-3′ and 5′-TGGTGAGGATAACATTCTTTTGG-3′, respectively, for Cd229 and 5′-AAAACAATTCAACAGTGTGATGG-3′ and 5′-AAGTCCAGGCAATGTTGTCA-3′, respectively, for Cd84. PCR products were denatured at 98°C for 10 minutes, loaded on a 15% polyacrylamide gel, and run in 0.5× Tris–borate–EDTA using a constant-temperature control system (AB-1600 and AE-6370; Atto) at 17°C under a constant current of 20 mA/gel for 3 hours. Single-stranded DNA fragments in the gel were visualized by silver staining (Daiichi Pure Chemicals).

Scoring of arthritis.

Ankle joint stiffness was examined by inspection and arbitrarily scored as follows: 0 = none, 1 = mild, 2 = moderate, and 3 = severe. Scores for both ankle joints were totaled for each mouse.

Radiography.

Whole skeletal specimens were placed on shielded x-ray film and exposed to low-energy x-rays (Softex-CMB).

Histopathology and tissue immunofluorescence.

For histologic examination, tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Sections were stained with hematoxylin and eosin or periodic acid–Schiff and hematoxylin. Joint tissues were decalcified in 10% EDTA in 0.1M Tris buffer (pH 7.4). For immunofluorescence, tissues were embedded in Tissue-Tek OCT compound and frozen in liquid nitrogen. Frozen kidney sections were stained with fluorescein isothiocyanate (FITC)–labeled goat antibodies to IgG for 60 minutes at room temperature. For analysis of splenic tissues, frozen sections were 3-color stained for 30 minutes at room temperature with Alexa 488–labeled anti-CD4 and anti-CD8 monoclonal antibodies (mAb), Alexa 647–labeled anti-B220 mAb, and Alexa 546–labeled peanut agglutinin (PNA). Antibodies and PNA were purchased from BD PharMingen and Vector, respectively, and the labeling of these reagents was done in our laboratory. Color images were obtained using laser scanning microscopy (LSM 510 META; Carl Zeiss).

Serum levels of antibodies.

Serum levels of RF were measured using an enzyme-linked immunosorbent assay (ELISA), as previously described (22). Briefly, an ELISA plate precoated with mouse IgG Fc fragment (OEM Concepts) was incubated with appropriately diluted mouse serum samples, washed, and then incubated with peroxidase-conjugated rat anti-mouse κ-chain antibodies (BD PharMingen). RF activities were expressed in units by reference to a standard curve obtained by serial dilution of a standard serum pool from 4–6-month-old MRL/lpr mice containing 1,000 unit activities/ml. Serum levels of anti-CII antibodies were measured using an ELISA plate precoated with bovine CII (Sigma-Aldrich). Serum levels of IgG anti-CCP antibodies were measured with a commercially available kit (Cosmic Corporation) using anti-mouse IgG second antibodies and were expressed as relative units according to the manufacturer's instructions. Serum levels of IgG antibodies against double-stranded DNA (dsDNA), histone, and chromatin were measured using ELISA, as previously described (23–25). Serum levels of binding activities against dsDNA, histone, and chromatin were expressed in units by reference to a standard curve obtained by serial dilution of a standard serum pool from (NZB × NZW)F1 mice ages >8 months, containing 1,000 unit activities/ml (23–25).

Assays for cytokines.

An aliquot of 1 × 106 cells/well obtained from popliteal lymph nodes was cultured for 3 days in 96-well flat-bottomed plates precoated with 10 μg/ml of anti-CD3 (145-2C11; eBioscience) and anti-CD28 (37.51; eBioscience) mAb in RPMI 1640 culture medium supplemented with 10% fetal calf serum, 45 μM 2-mercaptoethanol (Invitrogen), 100 units/ml penicillin (Invitrogen), and 100 μg/ml streptomycin (Invitrogen). The levels of cytokines in the culture supernatants were measured using a standard sandwich ELISA according to the manufacturer's instructions (BD PharMingen for IL-6, interferon-γ [IFNγ], tumor necrosis factor α [TNFα], IL-4, and IL-10; eBioscience for IL-17).

Flow cytometric analysis.

For the analysis of splenic lymphocytes, aliquots of 1 × 106 spleen cells were 3-color stained with phycoerythrin (PE)–labeled anti-CD4 or anti-CD8, FITC-labeled anti-B220 (6B2), and biotin-labeled anti-CD69 mAb, followed by staining with streptavidin–allophycocyanin (APC). For plasma cell analysis, spleen cells were stained with FITC-labeled anti-B220 and PE-labeled anti-CD138 mAb. For germinal center B cell analysis, cells were stained with FITC-labeled anti-B220 mAb and biotinylated PNA, followed by staining with streptavidin-APC. For follicular T helper cell phenotype analysis, spleen cells were 2-color stained with FITC-labeled anti-CD4 and PE-labeled anti–inducible costimulator (anti-ICOS) or anti–programmed death 1 (anti–PD-1) mAb. For intracellular cytokine staining, lymph node cells or cells obtained from ankle joint synovial tissues digested with 30 μl of Liberase TH (Roche Applied Science) for 1 hour at 37°C were stimulated with 0.2 μg/ml phorbol myristate acetate (PMA) and 2 μg/ml ionomycin in the presence of Golgi Plug (BD PharMingen) for 5 hours, stained with APC-labeled anti-CD4 mAb, and fixed/permeabilized using BD Cytofix/Cytoperm solution (BD PharMingen), followed by staining with PE-labeled anti–IL-17 and FITC-labeled anti-IFNγ or anti-TNFα mAb. Data were analyzed using a FACSAria flow cytometer (Becton Dickinson) and FlowJo software (Tree Star).

Measurement of urinary protein.

Five microliters of urine was diluted 20 times with 0.1% Triton X-100 in distilled water and mixed with 100 μl of 2.5 × 10−5M bromphenol blue solution in sodium acetate buffer, pH 3.2. The optical density at 605 nm (OD605 nm) of the mixture was measured, and the amounts of protein were calculated according to a standard curve obtained using bovine serum albumin solution.

Statistical analysis.

Statistical analysis was carried out using the Mann-Whitney U test. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Establishment of 2 lines of FcγRIIb-deficient B6 congenic mice.

Two lines of FcγRIIb-deficient B6 congenic strains of mice, designated KO1 and KO2, carry different lengths of the 129 strain–derived telomeric chromosome 1 region flanked to the null-mutated Fcgr2b gene (Figure 1). The KO1 strain carries the 129 strain–derived interval from microsatellite marker D1Mit501 to D1Mit359, and the KO2 strain bears the 129 strain–derived interval from D1Mit501 to null-mutated Fcgr2b. In the KO2 strain, recombination occurred between the Fcgr2b and Fcgr3 genes, and the region downstream of Fcgr3 including Slam family genes is derived from B6 mice, thus lacking the ∼6.3-Mb interval distal from the null-mutated Fcgr2b gene, which corresponds to the distal region of the 129 strain–derived Sle16 locus (Figure 1). Microsatellite analysis confirmed that other autoimmune-linked 129 strain–derived intervals on chromosomes 3, 7, and 13 reported in studies using crosses between 129 and B6 strains (26) are of B6 origin in both KO1 and KO2 strains.

Spontaneous development of severe RA in KO1 mice, but not in KO2 mice.

We found that KO1 mice, but not KO2 mice, spontaneously developed severe arthritis, with swelling and limited mobility of both ankle and wrist joints symmetrically after age 4 months. Severity of arthritis increased with increasing age, and the incidence was >90% at age 12 months (Figures 2A and B). There was no sex difference in the incidence of disease. Although the arthritis score tended to be higher in females than in males, the difference was not statistically significant. Radiographic examination revealed the deformity of finger joints associated with the osteoporosis and destruction/fusion of subchondral bones, particularly in the carpal and tarsal bones (Figure 2B). Large joints such as the knee and shoulder were also affected (Figure 2C), but vertebral joints were not affected. Histopathologic examination revealed severe synovitis with remarkable mononuclear cell and neutrophil infiltration and the destruction of cartilage and bone due to pannus formation in KO1 mice, but not in KO2 mice (Figure 2D). These findings clearly indicate that the distal region of the 129 strain–derived Sle16 locus, which is absent in KO2 mice, is essential for the development of RA in KO1 mice.

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Figure 2. Comparisons of arthritis changes. A, Incidence of arthritis and mean ± SEM arthritis scores in KO1 mice (33 females and 30 males) and KO2 mice (15 females and 11 males). B, Representative macroscopic and radiographic findings in forepaws and hind paws of KO1 and KO2 mice at age 10 months. KO1 mice show marked swelling and stiffness of the wrist and ankle joints. Radiographs show the deformity of palmar and tarsal bones and finger joints. C, Representative radiographic findings in knee and shoulder joints of female KO1 and KO2 mice at age 10 months. The complete loss of joint space with marked destruction and resorption of bone is observed in KO1 mice. D, Representative histopathologic changes in finger and ankle joints of KO1 and KO2 mice (circles in B) at age 10 months. KO1 mice show marked synovitis with inflammatory cell infiltration and the destruction of bone due to pannus formation. Boxed areas in middle panels are shown at higher magnification in right panels. Results are representative of those obtained from 6 female mice in each strain. Pn = pannus; Tib = tibia; Tal = talus. Hematoxylin and eosin stained; original magnification × 100 in finger joint; × 40 and × 200 in tarsal joint. See Results for description of establishment of KO1 and KO2 strains.

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Figure 3A compares serum levels of RA-associated autoantibodies, namely, RF and IgG antibodies against CII and CCP, and of SLE-associated autoantibodies, such as those against dsDNA, histone, and chromatin, among female KO1, KO2, and B6 mice at age 8 months. The levels of RA- and SLE-associated autoantibodies were significantly increased in KO1 mice compared with those in KO2 mice and in B6 mice. As for sex difference, serum RF levels in male KO1 mice were significantly lower than those seen in female KO1 mice (mean ± SEM 542 ± 43 units/ml versus 857 ± 114 units/ml; P < 0.01); however, differences in the mean ± SEM levels of other autoantibodies between male and female KO1 mice were not statistically significant (for anti-CII, 0.04 ± 0.01 OD492 nm versus 0.07 ± 0.01 OD492 nm; for anti-CCP, 362 ± 30 reference units [RU]/ml versus 401 ± 28 RU/ml; for anti-dsDNA, 204 ± 81 units/ml versus 409 ± 100 units/ml; for antihistone, 164 ± 29 units/ml versus 208 ± 19 units/ml; and for antichromatin, 250 ± 29 units/ml versus 336 ± 32 units/ml in males versus females).

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Figure 3. Comparisons of serum levels of autoantibodies and cytokine-producing potential among female KO1, KO2, and C57BL/6 (B6) mice. A, Serum levels of rheumatoid factor (RF) and IgG antibodies to type II collagen (CII), cyclic citrullinated peptide (CCP), double-stranded DNA (dsDNA), histone, and chromatin were compared at age 8 months. Horizontal and vertical bars represent the mean ± SEM. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001. B, Lymphocytes from inguinal and popliteal lymph nodes from 10-month-old mice were cultured in the presence of antibodies to CD3 and CD28 for 3 days, and the amounts of interleukin-6 (IL-6), tumor necrosis factor α (TNFα), interferon-γ (IFNγ), IL-17, IL-4, and IL-10 in the culture supernatants were measured using enzyme-linked immunosorbent assay. Horizontal and vertical bars represent the mean ± SEM. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001. C and D, Lymph node cells from arthritic KO1 mice and nonarthritic KO2 mice (C) or synovial tissue from the ankle joints of arthritic KO1 mice (D) at age 10 months were stimulated with phorbol myristate acetate/ionomycin and stained with anti-CD4 and anticytokine monoclonal antibodies, and intracellular cytokines in CD4+ T cells were analyzed. Numbers in each comparison are the percentage of total CD4+ T cells producing the cytokines (IL-17, TNFα, or IFNγ). Results are representative of those obtained from 3 independent experiments. See Results for description of establishment of KO1 and KO2 strains. OD = optical density; RU = reference units.

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To examine the cytokine milieu contributing to the development of arthritis, popliteal and inguinal lymph node cells from 10-month-old mice were cultured for 3 days in plates precoated with anti-CD3 and anti-CD28 mAb, and the amounts of IL-6, TNFα, IFNγ, IL-17, IL-4, and IL-10 in culture supernatants were measured using ELISA. Lymphocytes from arthritic KO1 mice exhibited a higher potential to produce all these cytokines, including the antiinflammatory cytokines IL-4 and IL-10, than those from nonarthritic KO2 and B6 mice (Figure 3B). These data were not consistent with the possibility that inflammatory responses were counteracted by an increased production of antiinflammatory cytokines in KO2 mice. We then analyzed proinflammatory cytokine–producing CD4+ T cells by staining intracellular cytokines of PMA/ionomycin-stimulated lymph node cells. As shown in Figure 3C, in arthritic KO1 mice, >40% of cells produced TNFα, and a lesser but considerable population produced IFNγ. Frequencies of IL-17–positive cells were low, and the majority of them were included in TNFα producers. In contrast, in nonarthritic KO2 mice, frequencies of cells positive for these cytokines were minimal. The analysis of the frequencies of cytokine-producing CD4+ T cells in the inflamed synovial tissues in KO1 mice showed that TNFα- and IFNγ-producing cells were again predominant (Figure 3D).

Lymphocyte activation in KO1 mice, but not in KO2 mice.

KO1 mice, but not KO2 or B6 mice, displayed splenomegaly (Table 1). Flow cytometric analysis revealed that while there were no differences in the frequencies of B220+ B cells among the 3 strains of mice, the percentages of CD69+ activated B cells, PNA+ germinal center B cells, and CD138+ plasma cells were significantly higher in KO1 mice than in KO2 and B6 mice (Table 1). Examination of spleen sections under immunofluorescence revealed that the formation of germinal centers with abundant PNA+B220+ B cells was prominent in KO1 mice compared with KO2 and B6 mice (Figure 4A). As for T cells, while there was no difference in the frequencies of CD4+ T cells, KO1 mice showed higher frequencies of CD69+ activated T cells with elevated CD4:CD8 T cell ratios (Table 1). In addition, CD4+ T cells in KO1 mice displayed phenotypes characteristic of follicular T helper cells, with increased expression levels of ICOS and PD-1 molecules, as compared with those in KO2 and B6 mice (Figure 4B). These findings are consistent with the report by Subramanian et al (27) demonstrating that CD4+ T cells with the follicular T helper cell phenotype were markedly expanded in autoimmune-prone B6.Sle1.Yaa mice, but not in nonautoimmune B6.Yaa mice. Because B6.Sle1.Yaa mice carry the autoimmune-type Slam family genes derived from NZW mice, the expansion of follicular T helper cells in these mice can be attributed to the autoimmune-type Slam family genes (27), the same types that are present in the telomeric region of chromosome 1 of KO1 mice.

Table 1. Spleen weight and splenic lymphocyte subpopulations in KO1, KO2, and C57BL/6 (B6) mice*
 KO1KO2B6
  • *

    Values are the mean ± SEM of at least 10 female mice ages 9–10 months. See Results for description of establishment of KO1 and KO2 strains. PNA = peanut agglutinin.

  • P < 0.01 versus KO2 mice.

  • P < 0.005 versus KO2 mice.

  • §

    P < 0.05 versus KO2 mice.

Spleen weight, gm0.26 ± 0.020.12 ± 0.010.11 ± 0.01
Spleen cell populations, %   
 B220+ B cells/total cells48.8 ± 3.451.8 ± 3.452.2 ± 2.8
 CD69+B220+ B cells/total B cells11.7 ± 0.65.2 ± 0.75.1 ± 0.4
 PNA+B220+ B cells/total B cells6.9 ± 0.42.1 ± 0.64.0 ± 0.3
 CD138+ plasma cells/total cells2.2 ± 0.40.8 ± 0.20.4 ± 0.1
 CD4+ T cells/total cells16.9 ± 1.317.6 ± 0.618.1 ± 0.8
 CD69+CD4+ T cells/total T cells37.4 ± 4.8§18.1 ± 0.715.6 ± 0.5
CD4:CD8 T cell ratio3.0 ± 0.7§1.3 ± 0.031.4 ± 0.1
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Figure 4. Spontaneous germinal center formation and increased numbers of CD4+ T cells with the follicular T helper cell phenotype in spleens of KO1 mice. A, Frozen sections of spleens of 10-month-old mice were triple-stained with a mixture of anti-CD4 and anti-CD8 monoclonal antibodies, anti-B220 monoclonal antibody, and peanut agglutinin (PNA) to examine the extent of germinal center formation. Results are representative of those obtained from 6 female mice in each strain. Original magnification × 200. B, Representative histograms of inducible costimulator (ICOS) and programmed death 1 (PD-1) expression on splenic CD4+ T cells from 10-month-old mice are shown at the left. The gray zone represents autofluorescence. The mean ± SEM mean fluorescence intensity (MFI) of ICOS and PD-1 on CD4+ T cells in KO1, KO2, and C57BL/6 (B6) mice is shown at the right. Results were obtained from 6 female mice in each group. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001. See Results for description of establishment of KO1 and KO2 strains.

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No signs of lupus nephritis in either KO1 or KO2 mice.

Bolland and Ravetch (16) reported that FcγRIIb-deficient B6 mice developed severe lupus nephritis with marked immune complex deposition in glomeruli. To examine the development of lupus-related glomerular lesions in our FcγRIIb-deficient B6 mice, kidney sections were stained with periodic acid–Schiff/hematoxylin or anti-IgG antibodies. As shown in Figure 5, there were no significant alterations in glomerular size and cellularity in KO1 and KO2 mice as compared to B6 mice. Minimal amounts of IgG deposits were observed only in mesangial areas of KO1 and KO2 mice at the extent comparable to those found in B6 mice. Mean ± SEM urinary protein levels remained in the normal range (<100 mg/dl) in all strains of mice, even at age 10 months, with no significant difference between strains (for KO1 mice, 76.1 ± 24.0 mg/dl; for KO2 mice, 58.2 ± 6.7 mg/dl; and for B6 mice, 42.1 ± 10.0 mg/dl).

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Figure 5. Histopathologic findings in glomeruli of KO1, KO2, and C57BL/6 (B6) mice at age 10 months. Top, Formalin-fixed sections were stained with periodic acid–Schiff/hematoxylin. Bottom, Frozen sections were stained with anti-mouse IgG to evaluate the deposition of IgG in renal glomeruli. Results are representative of those obtained from 6 female mice in each strain. Bars = 50 μm. See Results for description of establishment of KO1 and KO2 strains.

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The development of RA is related to the 129 strain–derived interval on the telomeric region of chromosome 1 in KO1 mice.

To further confirm that the development of RA is related to the 129 strain–derived interval on the telomeric region of chromosome 1 in KO1 mice, we produced (KO1 × B6)F1 × KO1 backcross mice and examined the association of RA with the genotypes either homozygous or heterozygous for the 129 strain–derived interval on the telomeric region of chromosome 1. At age 12 months, 14 of 15 mice with the 129/129 homozygous genotype (93%) developed RA, but none of 12 mice with the 129/B6 heterozygous genotype developed RA. These results support our finding that the 129 strain–derived interval on the telomeric region of chromosome 1 in KO1 mice plays a pivotal role in RA susceptibility, although this analysis does not exclude the contribution of other genetic polymorphisms to the arthritis phenotype.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

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.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

We thank Shogo Yamamoto, Yukari Aizawa, and Kanami Fukunaga for their excellent technical assistance.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES