Arthritis in MRL/lpr mice is under the control of multiple gene loci with an allelic combination derived from the original inbred strains

Authors


Abstract

Objective

To clarify the mode of inheritance and the genome origins of arthritis in a lupus-prone strain of mice, MRL/MpJ, bearing a Fas deletion mutant gene, lpr (MRL/lpr).

Methods

Using non–lupus-prone strains of mice, C3H/HeJ-lpr/lpr (C3H/lpr), (MRL/lpr × C3H/lpr)F1 intercross and MRL/lpr × (MRL/lpr × C3H/lpr)F1 backcross mice were prepared. Arthritis in individual mice was analyzed by histopathologic grading, and the genomic DNA of the backcross mice was examined by simple sequence-length polymorphism analysis to determine the polymorphic microsatellite markers highly associated with arthritis.

Results

Arthritis-susceptibility loci with significant linkage were mapped between D15Mit111 and D15Mit18 (map position 17.8–18.7 cM) on chromosome 15 and between D19Mit112 and D19Mit72 (map position 43.0–55.0) on chromosome 19 (logarithm of odds scores 3.5 and 4.3, respectively). Three other loci, one mapped to each of chromosomes 1, 2, and 7, showed suggestive linkage. Loci homozygous for MRL alleles on chromosomes 1 and 19 enhanced arthritis in both sexes, whereas other loci on chromosomes 2 and 15 selectively affected males. A locus homozygous for MRL alleles on chromosome 7 inhibited arthritis in both sexes. Three of these loci were found to originate from an LG/J strain and 1 from an AKR/J strain. Some combinations of these loci showed an additive effect in a hierarchical manner on the development of arthritis.

Conclusion

Arthritis in MRL/lpr mice is a complex pathologic manifestation resulting from the cumulative effect of multiple gene loci with an allelic combination derived from the original inbred strains.

MRL/MpJ-lpr/lpr (MRL/lpr) mice (H-2k), which originated from crosses of the inbred strains AKR/J (H-2k), C57BL/6J (H-2b), C3H/Di (H-2k), and LG/J (H-2d), spontaneously develop arthritis in association with other collagen diseases, such as systemic vasculitis, glomerulonephritis, sialadenitis, and interstitial pneumonia (1). They exhibit many of the serologic characteristics of rheumatoid arthritis (RA), including increased serum levels of antinuclear antibodies, circulating immune complexes, and IgM and IgG rheumatoid factors (2–5). The histopathologic features of the articular lesions in these mice resemble RA with respect to synovial cell proliferation, inflammatory cell infiltration, mesenchymoid transformation in synovial sublining tissue, and pannus formation (3, 6, 7). Moreover, in recent immunohistochemical studies, we observed infiltration of CD4-positive T cells and high expression of class II antigens on synovial cells, including the lining cells and the sublining fibroblastic cells, in arthritic lesions (Mori S, et al: unpublished observations). These serologic and histopathologic characteristics have made the MRL/lpr mouse a useful model for studying RA in humans, even though the incidence of arthritis in these mice is slightly less than 30%.

The lymphoproliferation gene, lpr, which is a mutant autosomal-recessive gene from an MRL/MpJ strain of mice (1, 8), was identified as a Fas antigen deletion mutation (9, 10). However, other strains of mice carrying the lpr gene, such as C3H/HeJ (C3H) and C57BL/6J (B6), rarely develop arthritis and other lesions (11), although they have increased rheumatoid factor, anti-gp70, and/or anti-DNA antibodies and abnormal cytokine production (12–14). This information, together with evidence that old MRL/MpJ mice that do not have the lpr gene also develop arthritis (15), suggests that the lpr gene acts as an accelerant for arthritis in the presence of an MRL background and that the development of arthritis requires susceptibility genes in MRL/MpJ mice. In fact, in our previous genome-wide screening for collagen disease susceptibility loci in MRL/lpr mice using MRL/lpr × (MRL/lpr × C3H/lpr)F1 backcross mice, the susceptibility loci for each of the diseases, such as vasculitis, glomerulonephritis, arthritis, and sialadenitis, seemed to be located on different regions of different chromosomes (16).

Herein we report the precise map positions for arthritis in MRL/lpr mice, and we clarify the mode of inheritance of arthritis and the genome origins. We present evidence indicating that a particular allelic combination of multiple genes derived from the original inbred strains of mice is a cause of arthritis in MRL/lpr mice.

MATERIALS AND METHODS

Mice

MRL/MpJ-lpr/lpr (MRL/lpr), C3H/HeJ-lpr/lpr (C3H/lpr), LG/J (LG), AKR/J (AKR), and C57BL/6J (B6) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Using MRL/lpr and C3H/lpr strains, we prepared (MRL/lpr × C3H/lpr)F1 intercross mice and MRL/lpr × (MRL/lpr × C3H/lpr)F1 backcross (N2) mice. A total of 77 F1 mice (33 females and 44 males) and 179 N2 progeny (76 females and 103 males) ages 5.0–5.5 months were used. Parent strains of the same age were also used in this study (108 MRL/lpr and 113 C3H/lpr mice).

Histopathologic evaluation

Mice were killed under ether anesthesia, and their hind limbs were processed for histopathology. Whole ankle joints were fixed in 10% formalin in 0.001M phosphate buffer, pH 7.2, decalcified in 10% formic acid, and embedded in paraffin. Serial sections 2–3-μm thick were taken sagittally through the talus and stained with hematoxylin and eosin for examination by light microscopy. The lesions, including the calcaneus bone and anterior and posterior synovial tissue at the ankle joints, were evaluated histopathologically.

Each joint was graded on a scale of 0–3, where 0 = normal, 1 = thickening and proliferation of the synovial lining, with slight inflammatory cell infiltration, 2 = grade 1 changes plus granulomatous lesions in the synovial sublining tissue, and 3 = grade 2 changes plus pannus formation and bone destruction (Figure 1). To map gene loci that affect the progression of arthritis in the association study, we categorized mice with grade 0 or 1 changes as arthritis-negative, and mice with grade 2 or 3 changes as arthritis-positive.

Figure 1.

Representative sections showing the grades of histopathologic manifestations of arthritis in the ankle joints of MRL/lpr × (MRL/lpr × C3H/lpr)F1 mice. A, Normal joint (grade 0), showing no proliferation of the synovial lining cells or inflammatory cell infiltration. B, Villous proliferation and thickening of synovial lining layers associated with slight inflammatory cell infiltration (grade 1). No remarkable changes of the synovial lining adipose tissue are seen. C, Extensive proliferation of synovial lining cells and granulomatous changes of synovial sublining tissue, associated with severe inflammatory cell infiltration (grade 2), but without pannus formation. D, Granulomatous lesions in the synovial sublining tissue, extending to the bone, showing pannus formation (grade 3). There is bone destruction associated with the accumulation of osteoclasts (arrowheads). (Hematoxylin and eosin stained; original magnification × 93.)

Genotyping, chromosome mapping, and statistical analysis

The genotype of each microsatellite in the N2 progeny was determined by simple sequence-length polymorphism analysis using microsatellite markers (Research Genetics, Huntsville, AL), in which a total of 273 informative polymorphic markers were used. Briefly, amplifications were performed using germline DNA (200–300 ng) obtained from each mouse tail or liver as a template in a polymerase chain reaction (PCR) mixture (20 μl) containing 25 pmoles of each primer, 25 mM dNTPs, and 5.0 units of Taq polymerase (Takara Shuzo, Shiga, Japan). The PCR conditions were 30 seconds at 94°C, 30 seconds at 55°C, and 30 seconds at 72°C for 40 cycles. The PCR products were separated by electrophoresis in 2–5% agarose or polyacrylamide gels.

Each N2 mouse was classified as an MRL/MRL (M/M) homozygote or an MRL/C3H (M/C) heterozygote. In a preliminary whole-genome scan using 139 polymorphic microsatellite markers covering a segment of ∼10–20 cM of each autosome, 5 regions of interest were mapped, one each on chromosomes 19, 15, 7, 2, and 1. We performed the association study between a marker genotype and the histopathologic phenotype by chi-square analysis using a standard 2 × 2 contingency matrix, and we determined the P values. As recommended by Lander and Kruglyak (17), P < 0.0034 (logarithm of odds [LOD] score >1.9; χ2 > 8.58 with 1 degree of freedom) and P < 0.0001 (LOD score >3.3; χ2 > 15.13 with 1 degree of freedom) were the thresholds for suggestive and significant linkage, respectively.

Relative risks (odds ratios) and 95% confidence intervals were calculated according to the method described by Morris and Gardner (18). Map positions of microsatellite markers were based on information from the Mouse Genome Database, The Jackson Laboratory (available at http://www.informatics.jax.org). The quantitative trait loci (QTL) involved in the disease were analyzed using MapMaker/QTL software (19), based on the histopathologic grades of arthritis. The LOD threshold for significant linkage was 3.3 (17). For determining phenotypic differences between two groups, chi-square test, Fisher's exact test, and the Bonferroni test were used as indicated. P values less than 0.05 were considered significant.

Sequence analysis of complementary DNA (cDNA) of the positional candidate genes

We prepared cDNA from total RNA isolated from the spleens of 16–20-week-old MRL/lpr and C3H/lpr mice. The cDNA sequences of the Ang1, Sdc2, Csf2ra, Hoxd9, Itga4, Itgav, Fcgr2b, and Fcgr3 genes were determined by the dye-terminator cycle sequencing reaction of the PCR products. Primers were synthesized to amplify the cDNA-translated regions. The cDNA were sequenced using a DNA Sequencing kit (Perkin-Elmer Applied Biosystems, Foster City, CA) with an ABI Prism 310 Genetic Analyzer (Perkin Elmer), according to the manufacturer's specifications.

RESULTS

Incidence and histopathologic grading of arthritis in MRL/lpr, C3H/lpr, F1 intercross, and N2 backcross mice. The numbers of mice with each histopathologic grade of arthritis in each generation are shown in Table 1. Arthritis-positive mice (grade 2 or 3 changes) accounted for 21.3% of the MRL mice, while only 5 of the C3H mice were arthritis-positive (4.4%). Among F1 mice, 22.1% were arthritis-positive. The incidence of arthritis-positive mice was significantly higher in the N2 progeny than in MRL/lpr (P < 0.0001) and the F1 progeny (P < 0.001), with 45.8% of the N2 mice developing arthritis (35 females and 47 males). Moreover, among these positive mice, 10 (5.6%) developed severe pannus, corresponding to grade 3 changes. These results indicate that recessive genes that are inhibitory for arthritis may exist in the MRL strain or, alternatively, that dominant C3H genes may accelerate arthritis.

Table 1. Incidence and histopathologic grade of arthritis in MRL/lpr, C3H/lpr, (MRL/lpr × C3H/lpr)F1 intercross (MC F1), and MRL/lpr × (MRL/lpr × C3H/lpr)F1 backcross (MC N2) mice*
 Arthritis gradeArthritis incidence
0123
  • *

    Values are the number of mice or the number of arthritis-positive mice (grades 2 and 3)/total number examined (%).

  • P < 0.0001 versus total MRL/lpr mice, and P < 0.001 versus total MC F1 mice, by chi-square test.

MRL/lpr
 Female154414317/76 (22.4)
 Male719606/32 (18.8)
 Total226320323/108 (21.3)
C3H/lpr
 Female2629303/58 (5.2)
 Male3815022/55 (3.6)
 Total6444325/113 (4.4)
MC F1
 Female921303/33 (9.1)
 Male131712214/44 (31.8)
 Total223815217/77 (22.1)
MC N2
 Female132832335/76 (46.1)
 Male253140747/103 (45.6)
 Total3859721082/179 (45.8)

Chromosome mapping of arthritis-susceptibility loci. Five loci for arthritis susceptibility showing a recessive mode of inheritance were mapped, one on each of chromosomes 15 and 19, with significant linkage, and on chromosomes 7, 2, and 1, with suggestive linkage (Table 2). The locus at D15Mit111 (map position 17.8 cM) on chromosome 15 conferred susceptibility only in males. QTL analyses also showed significant linkage between D15Mit111 and D15Mit18 in males (LOD score 3.5) (Figure 2A). The locus on chromosome 19 was determined by QTL analysis to be at a position between D19Mit112 and D19Mit72 on chromosome 19 (LOD score 4.3) (Figure 2B). In this area, there were no additional informative markers for the association study. We designated these two loci Paam1 (progression of autoimmune arthritis in MRL mice 1) and Paam2, respectively.

Table 2. Loci linked with arthritis in MRL/lpr × (MRL/lpr × C3H/lpr)F1 mice*
ChromosomeMarkerPosition (cM)DesignationArthritisχ2POR95% CIOrigin of MRL allele
PositiveNegative
M/MM/CM/MM/C
  • *

    * M/M = MRL/MRL homozygote; M/C = MRL/C3H heterozygote; OR = odds ratio; 95% CI = 95% confidence interval; LG = LG/J mouse; AKR = AKR/J mouse.

  • † Locus of interest.

  • ‡ Locus showing significant linkage.

  • §

    § Locus showing suggestive linkage.

15Female
 D15Mit13014.5161920210.070.790.88
 D15Mit18015.6161919220.0030.960.98
 D15Mit11117.8151919230.010.920.96
 D15Mit1818.7171818230.20.681.21
 D15Mit15220.2171818230.20.681.21
15Male
 D15Mit13014.53611223414.50.000145.062.2–11.7
 D15Mit18015.63611223414.50.000145.062.2–11.7
 D15Mit11117.83710213517.60.0000276.172.6–14.4LG
Paam1
 D15Mit1818.73611213515.80.0000705.452.4–12.6
 D15Mit15220.23512233311.60.000664.181.8–9.5
19Total
 D19Mit8941.0513150472.00.15LG or AKR
D19Mit11243.0493347502.30.13LG or AKR
Paam2
 D19Mit7255.0542846516.10.013LG
7Total
 D7Mit8123.0354764339.80.00182.60
 D7Mit36423.53448643310.80.00102.741.5–5.0
 D7Mit6924.53448653211.70.00061§2.871.6–5.2LG
 D7Mit8225.0344863349.90.00172.621.4–4.8
 D7Mit8326.5344863349.90.00172.621.4–4.8
2Female
 D2Mit9242.6241122191.80.181.88
 D2Mit3844.0241122191.80.181.88
 D2Mit3745.0241122191.80.181.88
 D2Mit27249.0221321201.00.311.61
 D2Mit12850.3241121202.40.122.08
2Male
 D2Mit9242.6321522348.50.00363.30
 D2Mit3844.0311621358.30.00403.23
 D2Mit3745.0311620369.30.0022§3.491.6–7.8AKR
 D2Mit27249.0301724324.50.0342.35
 D2Mit12850.3291826302.40.121.86
1Total
 D1Mit3381.6523146503.90.0481.82
 D1Mit14386.6542945516.00.0152.11
 D1Mit11599.7572546518.90.0029§2.531.4–4.7LG
 D1Mit358100.0572546518.90.0029§2.531.4–4.7LG
 D1Mit291101.5562744528.50.00362.45
 D1Mit362106.3483538585.90.0152.09
Figure 2.

Plots for quantitative trait loci (QTL) on A, chromosome 15 and B, chromosome 19, control arthritis. The y-axis represents the logarithm of odds (LOD) scores. The x-axis represents the genetic distance, at 10-cM intervals, using the MapMaker/QTL program (19).

Loci with suggestive linkages were found at D7Mit69 (map position 24.5 cM) on chromosome 7, at D2Mit37 (map position 45.0 cM) on chromosome 2, and at D1Mit115 and D1Mit358 (map position 99.7 and 100.0 cM, respectively) on chromosome 1 (Table 2A). One locus on chromosome 1 was associated with arthritis susceptibility in both sexes, and 1 locus on chromosome 2 was associated with arthritis susceptibility only in males. The locus on chromosome 7 was associated with resistance to arthritis in both sexes.

Interactions between the arthritis-susceptibility loci. Next, we studied the interactions between 3 gene loci that highly affect arthritis in males in relation to the incidence of arthritis in order to identify possible additive genetic effects. Since there were no informative markers between D19Mit112 and D19Mit72 (Paam2) on chromosome 19, we analyzed Paam1 (D15Mit111), D2Mit37, and D7Mit69, in 8 combinations as shown in Table 3.

Table 3. Additive and hierarchical effects of 3 candidate arthritis loci in male mice of the N2 progeny
No. of susceptiblity lociGroupGenotype at susceptibility loci*Arthritis incidence
Paam1 (Chr. 15)D2Mit37 (Chr. 2)D7Mit69 (Chr. 7)
  • *

    * Genotypes susceptible for arthritis at Paam1, D2Mit45, and D7Mit69 are M/M, M/M, and M/C, respectively. Chr. = chromosome; M/M = MRL homozygote; MC = MRL/C3H heterozygote.

  • † Values are the number of arthritis-positive mice/total number examined (%).

  • P < 0.05 versus groups D, F, and G, and P < 0.01 versus group E, by Bonferroni test.

3AM/MM/MM/C17/22 (77.3)
BM/MM/MM/M10/13 (76.9)
2CM/MM/CM/C4/5 (80.0)
DM/CM/MM/C4/11 (36.4)
EM/MM/CM/M6/18 (33.3)
1FM/CM/MM/M0/5
GM/CM/CM/C3/10 (30.0)
0HM/CM/CM/M3/19 (15.8)

Mice with susceptible genotypes at all loci, that is, M/M homozygosity at Paam1 and D2Mit37 and M/C heterozygosity at D7Mit69, had a highly increased incidence of arthritis (77.3%) (group A, Table 3), while those with susceptible genotypes at only 1 locus developed arthritis at a significantly lower incidence (33.3%, 0%, and 30.0% in groups E, F, and G, respectively). However, among mice with susceptible genotypes at 2 loci, those with the combination of Paam1 (M/M) and D2Mit37 (M/M) (group B) and Paam1 (M/M) and D7Mit69 (M/C) (group C) had a 76.9% and 80.0% incidence of arthritis, respectively, incidence rates that were as high as the rate in mice with the combination of all 3 loci (group A). Only 36.4% of mice with D7Mit69 (M/C) and D2Mit37 (M/M) (group D) developed arthritis. Mice with the combination of Paam1 (M/C), D2Mit37 (M/C), and D7Mit69 (M/M) (group H) had the lowest arthritis incidence (15.8%).

Allelic origin of the arthritis-susceptibility loci. The genome composition of an MRL strain is derived from LG/J (75.0%), AKR/J (12.6%), C3H/Di (12.1%), and C57BL/6J (0.3%) strains (1). The genotype of each arthritis-susceptibility locus in an MRL strain was classified into the genotype of C57BL/6J, AKR/J, and LG/J strains by using polymorphic microsatellite markers. As shown in Table 2, alleles in 3 of the 5 loci were derived from an LG strain, whereas the locus at D2Mit37 on chromosome 2 was from an AKR strain.

Allelic polymorphism of the positional candidate genes. Candidate genes for arthritis, located on the susceptibility loci with significant linkage (Paam1 and Paam2), were investigated with respect to differences in cDNA polymorphism between the MRL/lpr and C3H/lpr strains. Ang1 (angiopoietin 1) cDNA from the MRL strain had 1 nucleotide substitution causing an amino acid change, 262Val→Ile, compared with that from a C3H strain. Both amino acids are hydrophobic. The cDNA sequences of Sdc2 and Csf2ra, located in the Paam1 and Paam2 regions, respectively, were identical in both strains. In addition, regarding candidate genes of the susceptibility loci with suggestive linkage on chromosomes 2 and 1, cDNA sequences of at least Hoxd9, Itga4, Itgav, Fcgr2b, and Fcgr3 were the same in both strains.

DISCUSSION

In this study, we focused on clarifying the inheritance of arthritis in MRL/lpr mice and the gene loci that affect its progression as defined by histopathologic manifestations. To our knowledge, there are only a few reports of linkage analyses using histopathologic evaluations in animal models of arthritis.

It is worth noting that the incidence of arthritis in N2 backcross mice was increased compared with that in MRL/lpr mice (P < 0.0001), indicating that the development of arthritis in the N2 progeny may be under the control of C3H-susceptible or MRL-resistant alleles. This would mean that a new genome combination has the potential for increasing the incidence and severity of arthritis. In the association study and QTL analysis, we mapped 5 gene loci affecting arthritis, Moreover, in some combinations, interactions between these loci clearly showed an additive effect (Table 3), and Paam1 was hierarchical to the loci at D7Mit69 and D2Mit37, with Paam1 conferring higher susceptibility than D7Mit69 and D7Mit69 conferring higher susceptibility than D2Mit37. These results suggest that arthritis in MRL/lpr mice results from complex genetic traits encoded by a set of multiple genes, which are composed of different alleles derived from the LG/J and AKR/J strains. This may indicate that arthritis develops in a polygenic manner.

The development of RA is generally considered to be initiated by systemic or in situ activation of T and B cells, macrophages, and synovial cells, followed by progression, which is characterized by granulomatous inflammation of synovial sublining regions and mesenchymoid transformation extending to pannus formation. Some variations involve spontaneous regression before the onset of progression. In a preliminary study, when we classified mice with grade 0 histopathologic changes as arthritis-negative and mice with grade 1 changes as arthritis-positive, a different candidate gene locus (on chromosome 14) was mapped, but the significance was low (data not shown). This suggests that, compared with the progression of arthritis, a different gene might affect its initiation. In this study, we focused on mapping gene loci that affect the progression of arthritis, as manifested histopathologically by granulomatous inflammation of synovial sublining regions.

In this regard, several genes, such as Ang1, Syd2, and ank, located in the region of Paam1, are interesting since they seem to be relevant to the development of granulomatous inflammation. Angiopoietin 1 (Ang1) (20) is an endothelium-specific ligand that is also essential for postnatal angiogenesis, alone or in combination with vascular endothelial growth factor (21). Recently, it was reported that angiopoietin 1 is responsible for the neovascularization of synovial tissue in juvenile rheumatoid arthritis (22). Although the allelic polymorphism of angiopoietin 1 between the MRL and C3H strains consists of only a single amino acid substitution, 262Val→Ile, which may not cause a conformational change in the polypeptide, it might contribute to the development of arthritis associated with the other two susceptibility loci. Recently, the mouse ankylosis locus, ank, was shown to encode a multipass transmembrane protein (ANK) that controls pyrophosphate levels (23). This gene may also be a candidate for arthritis susceptibility in MRL/lpr as well as ank mice, with respect to the mechanisms of tissue calcification and joint destruction. The region of Paam2 also contains several candidate genes relevant to the progression of arthritis, such as Fgf8, Nfkb2, Col7a1, and Csf2ra, although we did not find allelic polymorphism in Csf2ra cDNA in this study.

In addition to this study, there are several important reports about arthritis-associated loci in experimental murine models of RA. Using another strain of mice, Otto et al (24) identified a total of 12 separate QTLs associated with proteoglycan-induced arthritis. Among them, Pgia8 on chromosome 15 and Pgia12 on chromosome 19 are located near Paam1 and Paam2, respectively. Some of the major loci controlling collagen-induced arthritis on chromosome 2, Cia2 and Cia4 (25), are only 10 cM proximal to the marker position D2Mit37. These results indicate that some gene loci may be responsible for susceptibility to both spontaneously developing and experimentally induced arthritis.

Analyses of mouse–human synteny, based on a comparative map of the mouse and human genomes (26), will be important for the use of mouse models in the understanding of human diseases. The human chromosomal region corresponding to Paam1 is well conserved on chromosome 8q22-q23. It should be noted that this region is consistent with 1 of 3 RA susceptibility loci, D8S556 (RA2), which was detected by Shiozawa et al (27) in genome-wide mapping studies of 41 Japanese RA families with at least 2 affected siblings. D8S556 locates close to ANGPT1 (angiopoietin 1) in humans, which corresponds to the mouse gene Ang1. This region may be relevant in the development of arthritis in both mice and humans. Thus, further studies of the human synteny of our mouse susceptibility loci will be important for gaining new insights into the genetic control and pathogenesis of RA.

Acknowledgements

The authors are indebted to the medical students of Ehime University, and to Mr. H. Komori and Mr. H. Mitsui for technical help with the simple sequence-length polymorphism analysis. We also thank Dr. L.-M. Lu for analyzing the data with MapMaker/QTL software, Dr. H. Schulman for reviewing the manuscript, and Mr. M. Arita and Ms M. Aibara for preparing the manuscript.

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