To clarify the mode of inheritance of the tissue distribution of vasculitis in MRL/Mp-lpr/lpr (MRL/lpr) lupus-prone mice and to identify the susceptibility loci.
To clarify the mode of inheritance of the tissue distribution of vasculitis in MRL/Mp-lpr/lpr (MRL/lpr) lupus-prone mice and to identify the susceptibility loci.
Vasculitis in individual MRL/lpr, C3H/HeJ-lpr/lpr (C3H/lpr), (MRL/lpr × C3H/lpr)F1, and (MRL/lpr × C3H/lpr)F2 intercross mice was analyzed by histopathologic grading of main branches of the aorta and of medium-sized arteries in the lower limbs. Genomic DNA samples from F2 intercross mice were examined by simple sequence-length polymorphism analysis, and the polymorphic microsatellite markers highly associated with vasculitis in each tissue were determined as vasculitis susceptibility loci.
A susceptibility locus with significant linkage to vasculitis of main branches of the aorta was mapped on chromosome 4 at D4Mit213 (map position 13.3cM) selectively in males, while vasculitis of medium-sized arteries in the lower limbs was mapped to different chromosomes: at D8Mit31 on chromosome 8 (map position 33.0) selectively in females and at D5Mit36 on chromosome 5 (map position 65.0). All of these were different from the previously defined loci governing susceptibility to vasculitis involving the kidneys.
Systemic vasculitis in MRL/lpr mice is genetically controlled with cumulative effects of multiple gene loci, each of which has tissue specificity.
Systemic vasculitis is a chronic disease associated with syndromes such as Takayasu arteritis, polyarteritis nodosa, Wegener's granulomatosis, and microscopic polyangiitis (1). It is also often a complication of other collagen diseases such as systemic lupus erythematosus, rheumatoid arthritis, and Sjögren's syndrome (2). Vasculitic lesions in these diseases show variation in tissue distribution, involving, for example, aortic branches, extremity arteries, renal arteries, or soft tissue. However, the mechanisms responsible for the tissue or organ distribution of vasculitis have not been well studied.
MRL/Mp-lpr/lpr (MRL/lpr) mice are a useful model of systemic vasculitis because they spontaneously develop systemic vasculitis with glomerulonephritis, sialoadenitis, and arthritis (3, 4). The vasculitic lesions are distributed mainly over kidneys, pancreas, main aortic branches, coronary arteries, tongue, and extremities; in all of these areas the lesions are histopathologically characteristic of granulomatous arteritis (5). Thus, this strain of mice may be useful for clarifying whether the tissue distribution of vasculitis is under genetic control. In previous studies of the genetics of vasculitis in MRL/lpr mice (6, 7), we identified 2 distinct recessive susceptibility loci to kidney vasculitis on chromosome 4, designated Arvm1 and Arvm2, and 1 resistant locus on chromosome 3 (7). However, it is not clear whether these loci are involved in vasculitis in other tissues.
To investigate the tissue and organ specificity of vasculitis susceptibility loci, we analyzed vasculitis in main branches of the aorta and lower limbs and compared the susceptibility loci with those described previously for kidneys (7). Herein we present evidence that vasculitic lesions in different tissues are under the control of different susceptibility loci.
MRL/lpr and C3H/HeJ-lpr/lpr (C3H/lpr) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and bred under specific pathogen–free conditions in the Animal Research Institute of Tohoku University School of Medicine. Using MRL/lpr and C3H/lpr strains, (MRL/lpr × C3H/lpr)F1 and (MRL/lpr × C3H/lpr)F2 intercross mice were produced and housed at the Animal Research Institute of Tohoku University School of Medicine and some were also produced at the animal laboratory of Kawashima Co., Ltd.
At 16–20 weeks of age, each mouse was killed under ether anesthesia. The aorta including main branches and the lower limbs were fixed with 10% formalin in 0.001M phosphate buffer (pH 7.2) and were then treated with 5% formic acid for decarboxylation. Specimens were stained with hematoxylin and eosin and elastica–Masson's trichrome for examination by light microscopy. Vasculitic lesions in aortic branches and lower limbs, including femoral, tibial, and fibular arteries, were graded as follows: 0 = normal to minimal perivascular lymphocyte infiltration; 1 = moderate perivascular cell infiltration associated with destruction of external elastic lamina; 2 = the above plus intimal thickening with destruction of internal elastic lamina (Figure 1). An animal with 1 or more vascular lesions corresponding to grade 1 or 2 was considered to have vasculitis.
The genotypes of F2 intercross mice (MRL/MRL, MRL/C3H, and C3H/C3H) were determined by polymerase chain reaction (PCR) of DNA from the liver or tail, using 103 polymorphic microsatellite markers between the MRL and C3H strains, all of which were defined as described previously (6). This provided full coverage of the mouse autosomes, with the markers spaced an average of 10 cM apart and a maximum distance of 37 cM between any 2 markers. For linkage analysis in the candidate chromosomal regions, the density of polymorphic microsatellite markers was increased and then susceptibility loci were determined. There were no informative polymorphic markers between the MRL/lpr and C3H/lpr strains in the telomeric region of chromosome 8, especially telomeric to 53 cM.
PCR was performed using standard reagents and the following conditions: 94°C for 2 minutes, 40 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, and final extension at 72°C for 5 minutes. After electrophoresis, PCR products were visualized on 2–5% agarose gels or 10% polyacrylamide gels, by staining with ethidium bromide or SYBR Green (FMC BioProducts, Rockland, ME), respectively. The map positions of microsatellite loci were based on information from the Mouse Genome Database of The Jackson Laboratory (http://www.informatics.jax.org).
The association of each polymorphic microsatellite marker with vasculitis in the F2 progeny was evaluated by chi-square test for independence of marker genotypes between positive and negative groups, using standard 2 × 2 or 2 × 3 contingency matrices. P values were determined and, as recommended by Lander and Kruglyak (8), the thresholds used to determine suggestive and significant linkage in the F2 progeny were P < 0.0024 and P < 0.00072 (1 degree of freedom [df] either recessive or dominant), respectively, or P < 0.0016 and P < 0.000052 (2 df). Relative risks (odds ratios) and 95% confidence intervals were calculated according to the method described by Morris (9). Quantitative trait loci (QTLs) for vasculitis were analyzed using MapMaker/QTL software (10), based on histopathologic grades of vasculitis as the indicator of phenotype. The logarithm of odds (LOD) thresholds for suggestive and significant linkage in the F2 progeny were 1.9 (1 df, additive) and 2.0 (1 df, either recessive or dominant) and 3.3 (1 df, additive) and 3.4 (1 df, either recessive or dominant), respectively. Student's t-test was used to determine the significance of differences between groups; P values less than 0.05 were considered significant.
Vasculitis in main aortic branches and lower limbs of MRL/lpr mice was manifested by granulomatous arterial lesions associated with infiltration of mononuclear cells in perivascular regions and destruction of external elastic lamina followed by destruction of the internal lamina. Some animals showed significant internal thickening of the arterial wall. These histopathologic features of vasculitis resembled those in kidneys (5, 7).
The incidence of vasculitis (grade 1 or 2) in aortic branches and lower limbs in each progeny is summarized in Table 1. In MRL/lpr mice, the incidence of vasculitis in the aortic branches and in the lower limbs was remarkably lower (22.9% and 16.7%, respectively) than in kidneys (82.9%) (7). In the F2 progeny, the incidence of vasculitis in aortic branches was significantly reduced (9.0%; P = 0.0046) while that in lower limbs was not (17.3%; P = 0.92), suggesting that the latter may involve the susceptibility gene(s) with a dominant mode of inheritance. Among the 266 F2 mice studied, overlap of vasculitis between aortic branches, lower limbs, and/or kidneys (see ref. 7, in which the same animals as in this study were used) was observed, to various degrees; i.e., the number of mice with all 3 types of vasculitis was 3 (1.1%), and the numbers with 2 types were 10 (3.8%) with vasculitis of the aortic branches and lower limbs, 8 (3.0%) with vasculitis of the lower limbs and kidneys, and 5 (1.9%) with vasculitis of the aortic branches and kidneys. In addition, in the F1 progeny only, there was a striking sex difference in the incidence of vasculitis in aortic branches and lower limbs. The former was male dominant (P = 0.01), while the latter was female dominant (P = 0.035). This might suggest the existence of a susceptibility locus for each vasculitis lesion, with a sex-linked mode of inheritance (see below).
|Tissue, progeny, sex||Vasculitis grade||Vasculitis incidence (grade 1 or 2), no. (%)|
In a genomewide search for vasculitis in aortic branches, we identified 2 loci of interest, which were located on chromosomes 4 and 5. As shown in Table 2, 1 of them showed significant linkage to vasculitis, with a recessive-susceptible mode of inheritance (P = 0.000052) and the strongest effect at the D4Mit213 (map position 13.3 cM) marker position. This locus, which was designated “autoimmune aortitis in MRL mice 1” (Aaom1), conferred susceptibility only in males. The position of the locus was identical to that determined by QTL analysis (Figure 2), in which the highest LOD score was 3.9 with a recessive mode. The other locus, on chromosome 5, was found around D5Mit23 (54.0 cM) with suggestive linkage in QTL analysis (LOD score 2.2 with a recessive mode), although chi-square analysis showed neither suggestive nor significant linkage at this position (P = 0.0040) (Table 2).
|Tissue, chromosome/sex, marker||Position, cM||Designation||Vasculitis||χ2, 1 df||P||OR||95% CI||Mode of inheritance||Origin of MRL allele|
|D4Mit213||13.3||Aaom1†||10||2||3||22||72||22||16.4||0.000052‡||8.55||2.7–27.5||AKR/J or LG/J mouse|
Susceptibility loci to vasculitis in the lower limbs were mapped on chromosomes 8 and 5 (Table 2). On chromosome 8, we recognized significant linkage at the D8Mit31 (33.0 cM) marker position in females only, with a recessive-susceptible mode of inheritance (P = 0.000027). On chromosome 5, the locus at D5Mit136 (map position 65.0 cM) showed significant linkage in both sexes in a dominant-susceptible mode (P = 0.00069). These loci at the D8Mit31 and D5Mit136 marker positions were designated “autoimmune extremity vasculitis in MRL 1 and 2” (Aevm1 and Aevm2), respectively. These results were supported by QTL analysis, in which the highest LOD score on chromosome 8 was 4.5 at marker position D8Mit31, in an additive mode. On chromosome 5 it was 2.3 at D5Mit136 in a dominant mode, which was limited, however, to a suggestive linkage (data not shown).
To examine why specific vasculitic lesions (at least Aaom1, Aevm1, and Aevm2) are under the control of different susceptibility loci, we analyzed the allelic origin of the 3 loci in an MRL strain, using polymorphic microsatellite markers. The genome of the MRL strain originates from the genomes of LG/J, AKR/J, C3H/Di, and C57BL/6J strains (3). We found that the D4Mit213 (Aaom1) locus in the MRL strain came from an AKR/J or LG/J strain, and the D8Mit31 (Aevm1) and D5Mit136 (Aevm2) loci originated from an AKR/J and an LG/J strain, respectively (Table 2). These findings were similar to those for the 2 susceptibility alleles for vasculitis in kidneys in an MRL strain, Arvm1 and Arvm2, which originate from an LG/J and an AKR/J strain, respectively (7).
The MRL/lpr strain of mice has been used to study systemic lupus erythematosus. However, this strain of mice also provides one of the few animal models of systemic vasculitis, in which vasculitic lesions are distributed almost throughout the body. In the present study, we found that this wide distribution of vasculitic lesions in MRL/lpr mice, at least in aortic branches, lower limbs, and kidneys, is a result of the cumulative effects of multiple gene loci, each of which restricts the tissue distribution of the lesions. We found that the loci governing susceptibility to vasculitis in each tissue are located in different chromosomal positions. As summarized in Table 3, vasculitis of the aortic branches is affected mainly by at least 1 locus at D4Mit213 (Aaom1), while that of the lower limbs is under the control of 2 loci, at D8Mit31 (Aevm1) and D5Mit136 (Aevm2). Moreover, 2 loci governing susceptibility to vasculitis in kidneys, Arvm1 and Arvm2 (7), are located at marker positions around D4Mit89 and D4Mit147, respectively. To our knowledge this is the first report of a genomewide analysis of the tissue specificity of vasculitis.
|Tissue, chromosome||Marker||Position, cM||Designation||P||LOD score||Mode of inheritance in MRL alleles (QTL analysis)||Sex|
|4||D4Mit213||13.3||Aaom1||5.2 × 10−5†||3.9†||Recessive-susceptible||Male|
|5||D5Mit23||54.0||4.0 × 10−3||2.2‡||Recessive-susceptible||Both|
|8||D8Mit31||33.0||Aevm1||2.7 × 10−5†||4.5†||Additive-susceptible||Female|
|5||D5Mit136||65.0||Aevm2||6.9 × 10−4†||2.3‡||Dominant-susceptible||Both|
|4||D4Mit89||19.8||Arvm1||1.1 × 10−4†||5.1†||Recessive-susceptible||Both|
|4||D4Mit147||57.6||Arvm2||3.8 × 10−5†||5.2†||Recessive-susceptible||Both|
|3||D3Mit14||64.1||8.4 × 10−4‡||Recessive-resistant||Both|
Interestingly, Aevm2 in an MRL allele, which governed susceptibility to vasculitis in the lower limbs with significant linkage, also showed a weak association with vasculitis in aortic branches (with a suggestive linkage in QTL analysis [LOD score 2.2]). Thus, it seems that Aevm2 might be a susceptibility locus common to vasculitis both in lower limbs and in aortic branches, although it may dominantly affect vasculitis in lower limbs. Moreover, it is worth noting that Aaom1 (13.3 cM) and Arvm1 (19.8 cM), which are the susceptibility loci to vasculitis in aortic branches and kidneys, respectively, are located close to one another on chromosome 4. This might suggest that development of vasculitis in these tissues is closely associated. However, the number of F2 mice tested in this study (n = 266) is insufficient to evaluate the statistical difference between genotype and phenotype overlap of vasculitis in each combination of tissue distribution.
We also found sex differences in the vasculitis susceptibility loci. Aaom1 on chromosome 4 affected vasculitis only in males, while Aevm1 on chromosome 8 did so only in females. This might be reflected in the results in Table 1 showing that the incidence of vasculitis in aortic branches was higher in males than in females, while the reverse was true of lower limbs in the F1 progeny only. Although the results suggest that sex hormone–related genes are involved in vasculitis susceptibility loci, these 2 loci should be analyzed further, together with studies of castrated or oophorectomized MRL/lpr mice. In this regard, several studies indicate that estrogen accelerates murine lupus. For example, estrogen receptor selectively modulates the progression of glomerulonephritis in MRL/lpr mice (11), and acceleration of murine autoimmune disease by prolactin appears to be accentuated by estrogen stimulation of prolactin secretion and to be independent of the immunosuppressive effects of androgens such as testosterone (12). Theofilopoulos and Dixon (13) argued that estrogen is critical for the development of systemic lupus erythematosus in (NZB × NZW)F1 mice. However, the present study may be the first to indicate that the tissue distribution of vasculitis has a sex difference which is under the control of gene loci with sex specificity.
The vasculitis susceptibility loci identified here, Aaom1, Aevm1, and Aevm2, were not associated with loci identified for other murine phenotypes such as glomerulonephritis, arthritis, and sialoadenitis (14), even in examinations of the same MRL/lpr and C3H/lpr intercross strains (15, 16). Several positional candidate genes of interest for such novel vasculitis susceptibility loci may be postulated. For example, the Aaom1 locus on chromosome 4 involves the interleukin-11 receptor (IL-11R) gene (IL11R; 12.7 cM), which modulates antigen-specific antibody responses in vitro and in vivo (17), although we could not find any allelic polymorphism, at least in the coding region, between the 2 strains.
In the Aevm1 locus, the IL-12Rβ1 component gene (IL12Rβ1; 33.5 cM), which codes for a receptor subunit common to IL-12 (18) and IL-23 (19), is involved. It is an essential component of functional mouse IL-12R, although this subunit by itself is not related to IL-12–mediated signaling (20, 21). It is known that inhibition of nitric oxide synthesis by anti–IL-12 antibody can ameliorate autoimmune disease in MRL/lpr mice (22). In addition, Takahashi et al (23) argued that an imbalance toward Th1 predominance, possibly induced by IL-12, might play a significant role in the acceleration of lupus-like autoimmune disease in MRL mice. Thus, IL-12Rβ1 might be substantially involved in the pathogenesis of vasculitis in the lower limbs. In fact, we recently found allelic polymorphism of IL12Rβ1 in the coding region, resulting in amino acid substitutions (Yamada A, et al: unpublished observations). Further studies are needed to examine the functional differences related to the polymorphism and to elucidate why this gene may be associated with vasculitis in the lower limbs but not in other tissues.
The different allelic origins of the susceptibility loci in an MRL strain may explain why MRL mice seem to develop vasculitis over almost their entire bodies; i.e., it may result from the cumulative effect of the susceptibility alleles derived from AKR/J and LG/J strains, each of which corresponds to vasculitis in a specific tissue. From these findings we can speculate that variations in the tissue or organ distribution of vasculitis in humans with vasculitis syndromes and other collagen diseases might be caused, at least in part, by a combination of susceptibility alleles resulting from genome crosses.
The authors are indebted to Dr. Koji Morimoto for technical help. We also thank Dr. Herbert M. Schulman for reviewing the manuscript.