Abstract
Background
The incidence and characteristics of spontaneous ankylosis in the ankle of specific F1 mice descended from two Fas‐deficient strains were reported. Here the coincidence of synovial proliferation and ankylosis in the descendent F2 mice is reported.
Aim
To clarify whether the two distinct manifestations are genetically different.
Methods
An arthropathic group of mice (MCF2) were bred by intercrossing MRL/Mp.Faslpr‐sap–/sap– and C3H/He.Faslpr mice. All mice were killed by bleeding under anaesthesia when they were 6 months old. Pathological grades for synovial proliferation were determined by microscopical examination. To obtain a linkage locus, the whole genome of male MCF2 mice was scanned by using 73 microsatellite markers.
Results
Synovial proliferation was equally observed in male and female MCF2 mice. No correlation was observed between the grades of synovial proliferation and the ankylosis occurring in the MCF2 mice. A suggestive susceptibility locus was shown in the middle of chromosome 11. This locus was an MRL allele with a recessive inheritance mode.
Conclusion
The pathogenic mechanisms of synovial proliferation and ankylosis are genetically different. The present locus is overlapped with some loci associated with rheumatoid arthritis and with others associated with experimental arthritides.
An inflammatory joint disease is usually recognised as an admixture of destruction and proliferation of the joint components. An imbalance of such counterprocesses may result in a pathological remodelling of joint structures giving rise to a unique clinicopathological outcome of the disease. Rheumatoid arthritis is characterised by synovial proliferation and erosion of joint cartilage due to chronic inflammation. The remodelling process in rheumatoid arthritis is associated with the contraction and destruction of joints. On the other hand, an ankylosing disease, another type of inflammatory joint disease, involves a distinct remodelling process causing joint ankylosis. Although the remodelling process for ankylosis has not been completely characterised, recent studies using rodent ankylosis models have evaluated the pathological proliferation of a particular region, where a joint capsule or ligament attaches to a bone.1,2,3,4 The histological characteristics are proliferation, cartilage formation and, subsequently, replacement of cartilage by bone, a process typical of endochondral bone formation. The process is called ankylosing enthesitis. A comparative study on the distinct joint remodelling processes may shed new light on the distinct and essential mechanisms of rheumatoid arthritis and ankylosing diseases.
We recently reported a mouse model with spontaneous and male‐predominant onset of progressive ankylosis in ankle joints.5 It should be noted that this joint ankylosis occurred in a particular F1 generation of mice, which descended from two ankylosis‐free strains—MRL/Mp.Faslpr‐sap–/sap– (previously denoted as MRL/rpl)6 and C3H/He.Faslpr (C3H/lpr). The MRL/rpl strain spontaneously arose from a lupus‐prone MRL/Mp.Faslpr (MRL/lpr) strain. It is noteworthy that the mutation on signalling lymphocyte activation molecule (SLAM)‐associated protein (SAP) attenuated the development of autoimmune diseases as seen in MRL/Mp.Faslpr (MRL/lpr) mice.6 These mice develop spontaneous arthritis characterised by synovial proliferation; therefore, they are considered as a model for rheumatoid arthritis. The onset of ankylosis in (MRL/rpl×C3H/lpr) F1 mice was unexpected. A defect of SAP or a coupling of two predisposed genes descended from the two parental strains was a possible interpretation for this onset. To deal with this issue, we performed a linkage analysis using (MRL/rpl × C3H/lpr) F2 (MCF2) mice and arrived at two genetic mechanisms for the onset of ankylosis in these mice: the dissociation of the SAP defect and association of the single locus on chromosome 7 with the onset of ankylosis.5 Although the question is not completely answered, the results provided an opportunity to characterise a non‐major histocompatibility complex (MHC)‐linked locus associated with the pathogenesis of ankylosis.
In this study, we observed sporadic incidences of synovial proliferation and ankylosis (entheseal proliferation) in MCF2 mice. The relationship between the pathogenic mechanisms of synovial and entheseal proliferations has been discussed. We provide evidence that the two distinct joint manifestations independently develop and are controlled differently by distinct genetic loci.
Methods
Mice
C3H/lpr mice were purchased from The Jackson Laboratory (Bar Harbor, Maine, USA) and bred under specific pathogen‐free conditions in the Animal Research Institute, Tohoku University Graduate School of Medicine, Sendai, Japan. MRL/rpl, MCF1 and MCF2 mice were housed in the same condition. In all animal experiments, we observed the Tohoku University guidelines for animal experimentation.
Histopathological grading of ankylosis
All mice were killed at 6‐months of age under ether anaesthesia. Ankle joints were fixed in 10% formalin buffered with 0.01 M phosphate (pH 7.2), decalcified in 5% formic acid and 5% formalin and embedded in paraffin wax. Tissue sections were stained with haematoxylin and eosin. The pathological grade of the synovial proliferation was determined according to the following criteria: normal (grade 0), multiplication of synovial lining layer (grade 1) and grade 1 with villous proliferation (grade 2). The grade of an individual mouse was defined as a maximal grade determined in six longitudinal sections of bilateral hind paws. Samples inappropriate for microscopical examination, mainly because of the absence of synovium in accountable sections, were precluded from further analysis. The number of female and male MCF2 mice accounted in this study was 111 and 81, respectively. The grade of ankylosis was determined previously.5
Genomewide screening for susceptibility loci to synovial proliferation
We searched association loci in the whole genome using 81 male MCF2 mice and 73 microsatellite markers, which were all able to distinguish the parental genotypes and provided a coverage of the mouse genome with an average distance of 23.3 cM apart. The genetic positions of microsatellite markers and genes were determined on the basis of the Mouse Genome Informatics provided by The Jackson Laboratory.
Statistical analysis
A correlation between the grades of synovial proliferation and ankylosis was evaluated by Fisher's exact test with standard 3×4 contingency matrices. An association of the onset of synovial proliferation and the genotype at each marker position was evaluated by the χ2 test with standard 2×3 contingency matrices. A significant or suggestive association was estimated by p<0.000052 or 0.0016, respectively, as recommended by Lander and Kruglyak.7
Results
Incidence of synovial proliferation
In addition to the onset of ankylosis, we observed a thickening of the synovial lining layer, with mild lymphocytic infiltration and proliferation of small vessels in MCF2 mice (fig 1A,B). Villous formations were evident in the advanced stage of the synovial proliferation. Neither lymphoid aggregation in the synovial lesion nor bone erosion with pannus‐like formation was detected in all the mice examined. The microscopical incidence of synovial proliferation was 12/25 (48%) in MRL/rpl, 12/26 (46.2%) in C3H/lpr, 14/69 (20.3%) in MCF1 and 69/192 (35.9%) in MCF2 mice. The synovial lesion was detected equally in male and female MCF2 mice (p = 0.973) (fig 1C). No significant correlation was obtained between the progression of ankylosis and synovial proliferation in male‐only (p = 0.258, fig 1D) and all MCF2 mice (data not shown).
Figure 1 Specific manifestations of the synovitis or enthesitis developed in MCF2 mice. Representative microscopic manifestations of synovitis (A) and enthesitis (B) in aged MCF2 mice. Asterisk in (A) indicates a synovial cavity; arrows in (B) indicate the entheseal proliferation of ankle‐associated tendon sheath (haematoxylin and eosin). Bars indicate 200 μm. (C) Percentage distribution of grades of synovitis and enthesitis in aged MCF2 mice. The number of the mice analysed and p values provided by the χ2 test are indicated. (D) The bar graph indicates the number distribution of grades of enthesitis and synovitis in the 81 male MCF2 mice. No correlation is shown between the onsets of synovitis and enthesitis. p Value was determined by Fisher's exact test with standard 3×4 contingency matrices. Cub, cuboidal bone; Met, metatarsal bone; Nav, navicular bone; Cal, calcaneus; Cun, cuneiform bone.
Mapping of susceptibility loci associated with synovial proliferation
In this mapping study, we analysed the same MCF2 mice that were used in the previous mapping study on ankylosis. The genomewide search using 81 male mice suggested a single marker D11Mit263 (55.6 cM) on chromosome 11 that was significantly associated with the onset of synovial proliferation (p<0.001; table 1). The incidence of synovial proliferation in the MRL homozygote group (17/26, 65.4%) in association with D11Mit263 was higher than that in the MRL/C3H heterozygote (8/40, 20%) and C3H homozygote (4/15, 26.7%) groups. This indicates that this MRL‐derived locus regulates the onset of synovial proliferation in a recessive inheritance manner. No significant association was observed between synovial proliferation and any marker on the X chromosome where the SAP gene exists.
Table 1 Association of microsatellite genotype and the incidence of synovitis in MCF2 male mice.
| Marker | Position | Incidence of synovitis | χ2 test | p Value | |||||
|---|---|---|---|---|---|---|---|---|---|
| Negative | Positive | ||||||||
| (CM) | MM | MC | CC | MM | MC | CC | |||
| D11Mit71 | 1.1 | 13 | 25 | 14 | 7 | 16 | 6 | 0.48 | 0.79 |
| D11Mit263 | 55.6 | 9 | 32 | 11 | 17 | 8 | 4 | 14.8 | 0.001 |
| D11Mit334 | 68 | 11 | 29 | 12 | 12 | 13 | 4 | 3.92 | 0.14 |
| D11Mit338 | 75 | 10 | 30 | 12 | 11 | 12 | 6 | 3.51 | 0.17 |
Genotypes MM, MC and CC indicate MRL/MRL homozygote, MRL/C3H heterozygote and C3H/C3H homozygote, respectively.
Discussion
Recent studies have reported several mouse models with spontaneous ankylosis. DBA/1 is a spontaneous ankylosis model and an induced synovitis model with immunisation of type II collagen. The two DBA/1‐related arthopathies develop similarly in a male‐predominant fashion, although they are histopathologically different. It has been of particular interest to determine whether the pathogenic mechanisms of the distinct arthropathies in the DBA/1 mice are overlapped.
A cross mating of the DBA/1 strain with a particular lupus‐prone strain such as BXSB or MRL/Mp resulted in an acceleration of the onset and increase in the severity of ankylosis in F1 mice.2,4 These facts may suggest a correlation between lupus susceptibility and the pathogenesis of ankylosis. The MCF2 mice used in this study also shared lupus‐prone and synovitis‐prone genetic backgrounds with MRL/lpr mice to varied extents. Previous studies have shown some MRL alleles significantly linked to the onset of spontaneous synovitis in the lpr constraint.8,9 Therefore, there is a reason for the onset of synovitis in MCF2 mice. This study showed the onset of synovitis in MCF2 mice, although inflammation and erosive changes were present to a lesser extent than those in the original MRL/lpr mice. We thought that this was a countereffect of ankylosis on synovitis; however, this notion turned out to be erroneous. This may be partly due to the difference in bleeding condition and genetic effects derived from the C3H/lpr strain. Our results totally revised these considerations and enforced a notion that proliferating enthesitis entail specific mechanisms for the development of ankylosis.
Our results suggest a susceptibility locus in the middle of chromosome 11. As the number of markers and samples were not sufficient to detect complex loci, some loci may have remained undetected. However, the detected locus has a major effect on the development of synovial proliferation. This locus contains several genes that are highly conserved in a particular interval of human chromosome 17. To date, a high‐resolution linkage and association mapping study identified a susceptibility locus for rheumatoid arthritis in this region.10 The central part of mouse chromosome 11 also contains several loci associated with experimental autoimmune encephalomyelitis,11,12 collagen‐induced arthritis13 and proteoglycan‐induced arthritis.14 This region is noteworthy when considering the genetic predisposition of rheumatoid arthritis.
A previous study using a larger number of backcross mice prepared from the same parental strains of MRL/Mp and C3H/He indicated that the five linkage loci are associated with the onset of arthritis.9 None of these loci overlap with the present linkage region. This discrepancy is possibly due to a difference in the mouse generation used (N2 or F2), a difference in the housing condition or a suppressive effect of the Y chromosome derived from C3H/lpr as discussed previously while purporting a possible reason for the onset of new cases of ankylosis.5
Acknowledgements
We thank Ms N Yamaki, Dr N Tanda and Dr H Umeda for technical assistance, Mrs N Fujisawa for secretarial assistance and Dr M Nose for critical comments.
Abbreviations
MCF2 - (MRL/rpl×C3H/lpr)F2
SAP - signalling lymphocyte activation molecule‐associated protein
SLAM - signalling lymphocyte activation molecule
Footnotes
Funding: This study was supported by Grants‐in‐Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan to SM (number 15390607) and MO (number 16390113).
Competing interests: None.
References
- 1.Nordling C, Karlsson‐Parra A, Jansson L, Holmdahl R, Klareskog L. Characterization of a spontaneously occurring arthritis in male DBA/1 mice. Arthritis Rheum 199235717–722. [DOI] [PubMed] [Google Scholar]
- 2.Holmdahl R, Jansson L, Andersson M, Jonsson R. Genetic, hormonal and behavioural influence on spontaneously developing arthritis in normal mice. Clin Exp Immunol 199288467–472. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Lories R J, Matthys P, de Vlam K, Derese I, Luyten F P. Ankylosing enthesitis, dactylitis, and onychoperiostitis in male DBA/1 mice: a model of psoriatic arthritis. Ann Rheum Dis 200463595–598. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Oishi H, Miyazaki T, Mizuki S, Kamogawa J, Lu L M, Tsubaki T.et al Accelerating effect of an MRL gene locus on the severity and onset of arthropathy in DBA/1 mice. Arthritis Rheum 200552959–966. [DOI] [PubMed] [Google Scholar]
- 5.Mori S, Zhang M C, Tanda N, Date F, Nose M, Furukawa H.et al Genetic characterization of spontaneous ankylosing arthropathy with unique inheritance from Fas‐deficient strains of mice. Ann Rheum Dis 2006281273–1278. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Komori H, Furukawa H, Mori S, Ito M R, Terada M, Zhang M C.et al A signal adaptor SLAM‐associated protein regulates spontaneous autoimmunity and Fas‐dependent lymphoproliferation in MRL‐Faslpr lupus mice. J Immunol 2006176395–400. [DOI] [PubMed] [Google Scholar]
- 7.Lander E, Kruglyak L. Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results. Nat Genet 199511241–247. [DOI] [PubMed] [Google Scholar]
- 8.Hang L, Theofilopoulos A N, Dixon F J. A spontaneous rheumatoid arthritis‐like disease in MRL/l mice. J Exp Med 19821551690–1701. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Kamogawa J, Terada M, Mizuki S, Nishihara M, Yamamoto H, Mori S.et al Arthritis in MRL/lpr mice is under the control of multiple gene loci with an allelic combination derived from the original inbred strains. Arthritis Rheum 2002461067–1074. [DOI] [PubMed] [Google Scholar]
- 10.Barton A, Eyre S, Myerscough A, Brintnell B, Ward D, Ollier W E.et al High resolution linkage and association mapping identifies a novel rheumatoid arthritis susceptibility locus homologous to one linked to two rat models of inflammatory arthritis. Hum Mol Genet 2001101901–1906. [DOI] [PubMed] [Google Scholar]
- 11.Karlsson J, Zhao X, Lonskaya I, Neptin M, Holmdahl R, Andersson A. Novel quantitative trait loci controlling development of experimental autoimmune encephalomyelitis and proportion of lymphocyte subpopulations. J Immunol 20031701019–1026. [DOI] [PubMed] [Google Scholar]
- 12.Butterfield R J, Blankenhorn E P, Roper R J, Zachary J F, Doerge R W, Teuscher C. Identification of genetic loci controlling the characteristics and severity of brain and spinal cord lesions in experimental allergic encephalomyelitis. Am J Pathol 2000157637–645. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Liljander M, Sallstrom M A, Andersson S, Andersson A, Holmdahl R, Mattsson R. Identification of collagen‐induced arthritis loci in aged multiparous female mice. Arthritis Res Ther 20068R45. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Adarichev V A, Nesterovitch A B, Bardos T, Biesczat D, Chandrasekaran R, Vermes C.et al Sex effect on clinical and immunologic quantitative trait loci in a murine model of rheumatoid arthritis. Arthritis Rheum 2003481708–1720. [DOI] [PubMed] [Google Scholar]