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*
Department of Medicine, Department of Microbiology and Molecular Genetics, and Molecular Biology Institute, and
Department of Pathology, University of California, Los Angeles, CA 90095; and
Department of Medicine, Division of Rheumatology, Veterans Affairs Medical Center, Sepulveda, CA 91343
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The MRL/lpr mouse, which carries the lpr mutation, has been shown to have many features of generalized autoimmune disease, including extensive production of anti-DNA Abs, glomerulonephritis, vasculitis, and massive lymphadenopathy, closely resembling the immunopathologic features of human SLE. The lpr mutation was identified as the defective Fas apoptotic gene (8). This mutation leads to a breakdown of the central and/or peripheral tolerance, which results in the failure of proper clearance of CD4/CD8-negative T cells. The MRL/+ strain differs genetically by <1% from the MRL/lpr strain and develops a late-onset form of autoimmune disease. Furthermore, when the lpr mutation was transferred to other mouse backgrounds, relatively mild autoimmune manifestations were observed (9). For example, relatively little autoimmune disease occurs when the lpr mutation is transferred onto a strain C3H background. Taken together, these results point to the requirement of other background genes contributing to the autoimmune manifestations in MRL/lpr mice. Previously, in a backcross between strains MRL/lpr and CAST/Ei, genes other than the Fas locus regulating glomerulonephritis and autoantibody production were identified on chromosomes (Chr) 7 and 12 (10), although the QTLs obtained for these loci were of marginal significance, with Chrlod scores of 3.0 and 2.9, respectively. In an effort to understand the multigenic predisposition underlining MRL/lpr autoimmune manifestations, we established an intercross mouse model generated from the MRL/lpr and BALB/cJ parental strains. Here we report the identification of two gene-containing intervals on Chr 2 and 11 in addition to the Fas locus that contribute to autoantibody production in this intercross mouse model. We also examined the effects of these QTLs on the development of vasculitis. The results indicate that vasculitis is determined by the Fas gene mutation and by the Chr 11 QTL, indicating shared genetic determinants with autoantibody levels.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MRL/lpr and BALB/cJ mice were obtained from The
Jackson Laboratory (Bar Harbor, ME). Inbred strains MRL/lpr
and BALB/cJ were crossed to produce F1 mice, and brother-sister mating
of F1 produced a total of 272 F2 animals. After weaning at 21 days
after birth, mice were housed two or three per cage with free access to
food and water and a 12-h light, 12-h dark cycle. All animals, parental
as well as F2 mice, were maintained on Purina mouse chow diet (Ralston
Purina, St. Louis, MO) containing 4.5% fat until they were 3.5
mo of age. Plasma samples were collected after an overnight fast. All
F2 mice were then fed a high fat, high cholesterol diet (HF) for
another 2 mo. The HF diet contains 7.5% cocoa butter, 1.25%
cholesterol, and 0.5% cholic acid with a total fat content of 15%
(Teklad 9022, Teklad Premier Laboratory Diets, Madison, WI). Parental
mice were separated into two groups. One group was kept on chow diet,
while the other group was given the HF diet for another 2 mo. The
animals were then fasted overnight, bled, and sacrificed. Plasma
samples were again taken, and certain relevant tissues were stored
frozen. The atherogenic diet was used for the last 2 mo because we were
also interested in examining the interaction of autoimmune disease and
atherogenesis in this cross. As shown in Table I
, 2 mo of the atherogenic diet did not
significantly change the autoantibody levels in either parental strain
compared with those in age- and sex-matched control mice receiving the
chow diet.
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IgG Abs to ssDNA, dsDNA, and cardiolipin were assayed by ELISA using calf thymus DNA or cardiolipin (Sigma, St. Louis, MO), respectively, coated on 96-well polystyrene microtiter plates (Dynatech, Chantilly, VA) as described by Weisbart et al. (11). Coated plates were stored at 4°C until use. Briefly, sera were added in a 1/100 dilution in 0.5% hen egg albumin/PBS/Tween. One hundred microliters of the dilution was added to each well in duplicate. Plates were incubated at 4°C overnight and were washed three times with PBS/Tween. One hundred microliters of alkaline phosphatase-conjugated goat anti-mouse IgG (Southern Biotechnology Associates, Birmingham, AL; lot J194-Y894), diluted 1/3000 in 0.5% hen egg albumin/PBS/Tween, was added to each well and incubated at room temperature for 1 h. The wells were then washed three times in PBST and developed by adding phosphatase substrate in diethanolamine buffer (Sigma; 104). The absorbances at 405 nm were determined on a plate reader. In previous studies, monoclonal anti-DNA Abs were shown to bind DNA, but not cardiolipin. In the present studies three anti-DNA Abs, mAbs 3E10, 5C5, and 5C6, were assayed simultaneously with the mouse serum to establish the separate specificities of the anti-DNA and anti-cardiolipin assays.
Vasculitis
When the animals were sacrificed, the heart and proximal aorta were dissected and washed to remove blood. The basal portion of the heart and root of the aorta were embedded in OCT compound (Miles, Elkhart, IN) and frozen quickly on dry ice as previously described (12). For a semiquantitative assessment of vasculitis in the coronary arteries, every fifth 10-µm heart section was collected from the lower portion (the site of the first cut) to the aortic root, stained with oil red O and hematoxylin, and counterstained with fast green FCF (12). Vasculitis was defined by clear vessel wall destruction, cellular infiltration, and abnormal lipid accumulation as previously described (12). A total of about 70 sections were evaluated for each animal. The semiquantitation of vasculitis was determined by the number of positive sections that contained vasculitis as previously described (12).
Genotype analysis
Genomic DNA was isolated from mouse tails. Genotyping was performed by PCR amplification of simple sequence repeat (microsatellite) markers (13, 14) using PCR primer pairs (MapPairs) purchased from Research Genetics (Huntsville, AL). Primer pairs were first screened for polymorphic bands between MRL/lpr and BALB/cJ parental strains (data not shown). A total of >100 markers were used to construct a complete linkage map in 189 (MRL/lpr x BALB/cJ)F2 mice. A list of these markers as well as the linkage data are available from A. J. Lusis.
All genomic regions scored were examined for the possibility of segregation distortion. Four regions, linked to markers D2Mit227, D7Mit71, D1Mit47, and D19Mit93, exhibited evidence of a non-Mendelian distribution of alleles (data not shown), raising the possibility of effects on prenatal or postnatal viability. However, none of these overlapped with the three autoimmune QTLs described above.
Statistical analysis
Phenotypic values are presented as the mean ± SD. Analysis of variance, regression analysis, and correlation analysis were performed on Macintosh computers using the StatView (Abacus Concept, Berkeley, CA) application. Linkage analysis among the microsatellite markers used was performed using the MAPMAKER (15) and Map Manager (16) programs. The MAPMAKER/QTL and QT Manager subprograms were used for quantitative trait linkage analysis as described for F2 intercrosses (17, 18, 19). Both analyses yielded similar results. Phenotypes were normalized using either the log(trait) or sqr(trait) functions when necessary. A lod score >4.3 indicates significant linkage, while a lod score of 2.8–4.3 indicates suggestive linkage (also see Discussion), as determined by Lander and Kruglyak for the intercross free model analyses (18).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Parental MRL/lpr and BALB/cJ mice as well as
(MRL/lpr x BALB/cJ)F1 and F2 mice were examined for
quantitative traits associated with autoimmune disease at 24 wk of age.
MRL/lpr and BALB/cJ differed significantly
(p < 0.05 for all comparisons) in the levels
of anti-dsDNA Ab, anti-ssDNA Ab, and anti-cardiolipin Ab
for both sexes and both diets (Table I
). F1 female mice had
intermediate anti-dsDNA and anti-ssDNA Ab levels compared with
the parental strains and similar anti-cardiolipin Ab levels as the
MRL/lpr strain. They differed significantly from age- and
sex-matched parental BALB/cJ mice for all Ab levels
(p < 0.005 for all comparisons) and from
MRL/lpr mice for anti-ssDNA Ab levels
(p = 0.005). These autoantibody levels of F1
mice indicated the involvement of genes from MRL/lpr as well
as BALB/cJ mice. Anti-DNA mAbs, mAbs 3E10, 5C5, and 5C6, bound ssDNA
and dsDNA, but showed no reactivity to cardiolipin, indicating that the
anticardiolipin ELISA used in our study was not cross-reactive with DNA
epitopes (data not shown). As expected for a genetically determined
trait, the variance in autoantibody levels among F2 mice was greater
than that among F1 mice. The sex differences among both parental
strains and F2 mice were not significant. In addition to the
autoantibody levels, the two parental strains differed in their
susceptibility to the development of coronary vasculitis (Table I).
An F2 intercross consisting of 272 mice was constructed by intercrossing (MRL/lpr x BALB/cJ)F1 mice. At 24 wk of age, the F2 mice were examined for the above autoantibody traits associated with autoimmune disease. As a quantitative measure of autoimmune disease, we determined the levels of autoantibodies and spleen weight. As discussed below, we also performed semiquantitative analyses of coronary vasculitis to examine the relationship of vasculitis with generalized autoimmune disease.
As shown in Table II, we observed
significant correlations among autoantibody levels and spleen weight in
both male and female mice. Among them, the Spearman rank correlation
coefficients for anti-dsDNA Ab levels and anti-ssDNA Ab levels
were 0.78 (p < 0.0001) in male mice and 0.78
(p < 0.0001) in female mice. The Spearman rank
correlation coefficients for anti-dsDNA Ab levels and
anti-cardiolipin Ab levels were 0.80 (p <
0.0001) in male mice and 0.78 (p < 0.0001) in
female mice. Spleen weight correlated best with ssDNA Ab levels, with
Spearman rank correlation coefficients of 0.55
(p < 0.0001) in male mice and 0.46
(p < 0.0001) in female mice. Interestingly,
anti-cardiolipin Ab levels have higher correlation coefficients
with anti-dsDNA Ab levels (0.80 for males and 0.79 for females)
than with anti-ssDNA Ab levels (0.60 for males and 0.58 for
females).
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In an effort to identify loci contributing to autoantibody levels,
we performed QTL analysis on 189 (MRL/lpr x BALB/cJ)F2
intercross animals. A total of 109 polymorphic microsatellite markers
were used to construct a linkage map designed to cover the whole mouse
genome at intervals of 20 centimorgen (cM) or less. Due to a failure in
some cases to identify informative markers, gaps of >20 cM were
present in three chromosomal regions (These three gaps are located at
Chr 5 between D5Mit105 and D5Mit10 with a
distance of 20.5 cM, at Chr 8 between D8Mit155 and
D8Mit30 with a distance of 23.6 cM, and at Chr 16 between
D16Mit55 and D16 Mit173 with a distance of 31.5
cM). Each F2 mouse was genotyped for the above 109 markers and
phenotyped for the levels of anti-dsDNA, anti-ssDNA, and
anti-cardiolipin Abs. The distributions of these values are
presented in Fig. 1. The broad range of
Ab levels reflects the large differences in the parental strains. The
majority of F2 animals showed values between the parental extremes. The
individuals with values outside the parental strain ranges presumably
resulted from the recombining of genetic components in the F2 mice;
that is, certain F2 mice exhibited higher levels of autoantibodies than
did the parental strain MRL/lpr mice due to the inheritance
of unique combinations of MRL/lpr and BALB/cJ alleles. We
then performed statistical analysis combining both genotypes and
phenotypes. Analysis using the MAPMAKER-QTL statistical program and the
QT Manager program yielded similar results (only QTL results are
shown).
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and Fig. 2). This locus explained about 83% of
the variance in autoantibody levels observed among the F2 mice. As
expected, mice homozygous for the MRL/lpr alleles of the
Fas gene exhibited much higher autoantibody levels than
those heterozygous or homozygous for the BALB/cJ alleles, indicating
recessive inheritance with the MRL allele condoning high expression of
autoantibodies. The peak QTLs for all three autoantibodies exhibited
coincident locations (Fig. 2). Mice heterozygous for the
MRL/lpr allele at the Fas gene locus exhibited
somewhat elevated autoantibody levels compared with BALB/cJ mice (Fig. 3; p < 0.0001 for the
difference of dsDNA Ab levels between CC and MC groups), suggesting
that the Fas mutation is not completely recessive. This was
supported by QTL analysis of F2 mice when mice homozygous for the
mutation were eliminated (see below).
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). This locus
has not previously been reported in studies of autoimmune-related
traits in mice. It is noteworthy that among the F2 mice, those
heterozygous for the D11Mit70 marker tended to exhibit
higher autoantibody levels than those homozygous at the locus for
either the MRL/lpr or BALB/cJ alleles. This suggests an
interaction between BALB/cJ and MRL/lpr alleles in the
development of autoimmune disease, consistent with the observation that
a subset of F2 mice exhibited higher autoantibody levels than the
MRL/lpr parental strain.
In addition to the three QTLs reported here, we have carefully examined
in our cross any loci for the trends of association with autoantibody
levels. The only genomic region in which we observed a trend of
association was at marker D5Mit32 for ssDNA Ab (lod
score = 2.1; Table III), overlapping with a region reported
previously to be linked to mortality in New Zealand mice (20).
Since the lpr mutation has an overwhelming effect on the
autoimmune manifestations of the MRL/lpr mouse, and a major
interest was to identify other background genes, we performed QTL
analysis with lpr/+ and +/+ subpopulations. Specifically,
all the mice that were homozygous for the MRL/lpr genotype
at the Fas locus were removed, and a total of 144 mice were
left in this subpopulation. The results from MAPMAKER-QTL analysis are
presented in Table III. The Fas locus showed a reduced, but
still significant, contribution to autoantibody levels, indicating an
incomplete recessive phenotype of the lpr mutation as has
been previously reported (21). In addition, a locus on Chr 2 closely
linked to marker D2Mit12 showed a lod score of 4.3 for
anti-ssDNA Ab levels and a lod score of 2.6 for anti-dsDNA Ab
levels. The failure to identify this locus in the entire sample
presumably resulted from the overwhelming contribution of
lpr homozygous animals to the autoantibody distributions in
F2 mice. It is also noteworthy that the Chr 11 locus was not
significantly associated with autoantibody levels in this
subpopulation, suggesting that the Chr 11 locus interacts with the
Fas gene. The fact that QTL peaks for anti-dsDNA and
anti-ssDNA Abs on Chr 2 coincided (Fig. 2) indicates that they are
determined by the same gene. When separated by genotype at the
D2Mit12 marker, individuals with the MM genotype at the Chr
2 locus had increased anti-ssDNA autoantibody levels compared with
mice with CC and MC genotypes, which had similar levels of
anti-ssDNA autoantibodies. Thus, the Chr 2 locus represents one of
the genetic factors contributed by the MRL genetic background
predisposing to autoimmune disease. It acts in a recessive fashion to
control anti-DNA autoantibody levels (Fig. 3).
It is noteworthy the levels of anti-cardiolipin Abs in the F1 mice
slightly exceeded those in MRL mice, which were, in turn, higher than
those in BALB/c mice (Table I). Thus, there is a difference in the
genetic control of the specificity of anti-cardiolipin Abs compared
with anti-DNA Abs. Nevertheless, at the two QTLs that exhibited
linkage with anti-cardiolipin Ab levels (Chr 11 and Chr 19), the
inheritance pattern for anti-cardiolipin Ab levels resembled those
for anti-DNA Ab levels. Thus, at the Fas gene locus,
mice homozygous for the MRL allele exhibited elevated
anti-cardiolipin levels compared with heterozygous mice and mice
homozygous for the BALB/c allele. Similarly, at the Chr 11 locus,
heterozygous mice exhibited the highest levels of anti-cardiolipin
Ab, suggesting an interaction between the MRL and BALB/c alleles (data
not shown).
We also performed QTL analysis with spleen weight in the F2 mice. The only QTL observed with this trait is on Chr 19, coincident with the QTL for autoantibodies, with a peak lod score of 59.0. This locus for spleen weight exhibited the same pattern of inheritance as it did with the dsDNA Ab levels (data not shown). The failure to detect the Chr 2 and 11 loci using spleen weight presumably reflects the fact that spleen weight is a less precise measurement of autoimmune disease than autoantibody levels.
Common genetic control of autoantibody levels and vasculitis
MRL/lpr mice also develop severe vasculitis; some
evidence suggests that the vasculitis is mediated in part by the
autoantibodies (12, 22). In a previous study, we examined in detail
vasculitic lesion formation in the arteries of MRL/lpr mice.
We showed that when mice are fed a HF diet, concentric and transmural
lesions with a large amount of lipid accumulation occur in coronary
arteries of MRL/lpr mice, but not in a variety of other
strains examined. These lesions are prone to infiltration of
inflammatory cells and exhibit extensive deposition of Ig (9). In an
attempt to examine the relationship of vasculitis with autoantibodies,
we scored the vasculitic lesion formation in the coronary arteries of
the F2 mice. The severity of vasculitis was scored by determining the
number of sections positive for vasculitis as described in
Materials and Methods. As shown in Table IV, we observed significant associations
of vasculitis with the autoantibody levels in 252
(MRL/lpr x BALB/cJ)F2 mice. The correlation
coefficients were 0.35 (p < 0.0001) for both
dsDNA Ab and cardiolipin Ab, and 0.33 (p <
0.0001) for ssDNA Ab. We noticed that vasculitis correlated equally
well with dsDNA Ab and cardiolipin Ab levels, but less well with ssDNA
Ab levels.
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and Fig. 2). It is interesting that the
Chr 2 QTL for anti-DNA Abs did not contribute to either
vasculitis or anti-cardiolipin Ab levels. As shown in Fig. 4, both Chr 19 and 11 QTLs had
significant impact on vasculitis formation. Specifically, mice
homozygous for the MRL/lpr alleles of the Fas
gene exhibited much higher vasculitis scores than those heterozygous or
homozygous for the BALB/cJ alleles (p <
0.0001, by analysis of variance), indicating recessive inheritance. For
the Chr 11 QTL, mice heterozygous for the D11Mit70 marker
exhibited higher vasculitis scores than those homozygous at the locus
for either the MRL/lpr or BALB/cJ alleles
(p = 0.05, by analysis of variance), suggesting
an interaction between BALB/cJ and MRL/lpr alleles in the
development of vasculitis. Again, the Chr 2 QTL did not exhibit a
significant relationship with coronary vasculitis in either the whole
F2 population or the lpr/+ and +/+ subpopulations using
analysis of variance analysis. This raises the possibility that the Chr
2 locus contributes to autoimmune disease by a mechanism that has less
impact on vasculitis. Alternatively, the semiquantitative nature of the
scoring of coronary vasculitis may have observed a relationship,
although it is noteworthy that the Chr 11 locus, which exhibited a
lower lod score than the Chr 2 locus, was significantly associated with
vasculitis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The three mouse models that have been most commonly used to study the human autoimmune disease SLE are the MRL/lpr mouse model, the (NZB x NZW)F1 mouse model, and the B x SB mouse model, which carry the disease-accelerating Yaa gene on the Y Chr (5, 6, 7, 23). Most genetic studies of SLE have been conducted in NZB/W-related strains using either backcross or intercross approaches (20, 24, 25, 26, 27, 28, 29, 30). An elegant study by Morel et al. (24) using a backcross between NZM/Aeg2410 and C57BL/6 mice resulted in the identification of three chromosomal loci containing recessive glomerulonephritis susceptibility genes on Chr 1, 4, and 7. Each locus contained several potentially interesting candidate genes. Other studies confirmed these loci and revealed additional loci contributing to the autoimmune phenotype. Summaries of all chromosomal regions contributing to SLE-related phenotypes derived from studies of NZB-related strains and MRL mice were published recently (5, 6, 7). Overall, there are 12 loci from NZB/W-related strains in addition to the MHC gene, and two loci from the MRL strains in addition to the lpr (Fas) and gld (Fas ligand) genes, that have been shown to be associated with or to have a trend of association with lupus-related traits (6). These loci reside on 14 separate Chr, including Chr 1, 4–7, 9, 11–14, and 16–19. In our study of an (MRL/lpr x BALB/c)F2 cross, we identified two QTLs, on Chr 2 and 11, in addition to the Fas gene on Chr 19. The Chr 11 QTL centered around D11Mit70 (52 cM from the centromere) (31), which lies about 16 cM from D11Mit114/Lbw8 (36 cM from the centromere) (31). Lbw8 was previously linked to antichromatin Ab production (20). The same area was shown later in two other studies to overlap with regions associated with nephritis (28, 30). The Chr 2 QTL, however, did not overlap with any of the SLE loci identified before, although the same place was shown to contain a locus linked to type 1 diabetes, IDD13 (32).
Previously, a complete linkage study using (MRL/lpr x CAST/Ei)F1 x MRL/lpr backcross mice by Watson and colleagues (10) identified two QTLs contributing to the development of glomerulonephritis in addition to the lpr mutation. These two QTLs resided on Chr 7 (lod score = 3.0) and 12 (lod score = 2.9). Since the submission of this paper, two studies were published where the MRL strain was used as a model for the genetic analysis of autoimmunity (33, 34). In an (MRL-lpr x C57BL/6-lpr)F2 cross, four loci (Lmb1–4) with significant linkage to lymphadenopathy and/or splenomegaly were identified on Chr 4, 5, 7, and 10 (33). We failed to confirm the QTLs obtained in their studies. However, we used a different parental strain (BALB/cJ) than the CAST/Ei or C57BL/6 strains they used. Previous studies have shown that the genetic backgrounds of the nonautoimmune strains in these crosses can markedly alter the genes showing linkage with disease (27, 28, 30). C57BL/6 and CAST/Ei strains are phylogenetically very distantly related to BALB/cJ, and it is quite possible that the choice of partner strains contributed to the different findings of these studies. Further, our study also provides a better understanding of the relationships of anti-DNA Abs and anti-cardiolipin Abs with vasculitis.
The maximum lod score we obtained for the Chr 11 locus (3.1) is strongly suggestive and overlaps with previous identified loci, whereas the lod score for the Chr 2 locus appears to be significant, as judged by the proposed thresholds by Lander and Kruglyak (18). Due to the nature of the algorithms used for maximal likelihood estimation in QTL analysis, a high lod score may not necessarily reflect the true strength of the association of the locus with the trait, even though it is true with respect to the mathematical model. Factors such as map distance, species, and trait distribution also influence the significance threshold (19, 35). Therefore, we determined the maximal lod score peaks of 1000 randomly permutated trait data to test in our dataset how often a QTL could actually occur due to chance, permitting the definition of a realistic significance threshold for a given experimental dataset (19). Using the method of Churchill and Doerge (19), as implemented in the QT Manager program, each trait was permutated 500–1000 times. The resulting thresholds for significance were similar to those proposed by Lander and Kruglyak (18). That is, for the intercross free model analysis, a lod score of about 4.3 indicated significant linkage (the likelihood of a false positive being <5%), while a lod score of 2.8–4.3 indicated suggestive linkage.
The 95% confidence intervals (going 1 lod down from the maximum lod
score approximates the 95% confidence interval for the QTL) for the
Chr 2 and Chr 11 QTLs contain some interesting candidate genes. The
genes for IL-1
and IL-1ß lie about 4 cM proximal to marker
D2Mit46 on Chr 2. IL-1 was shown to be overexpressed in
MRL/lpr mice and may contribute to high levels of IgG
production in MRL/lpr mice (36, 37). Another candidate gene
in the Chr 2 confidence interval is the immune response-2 locus, which
is about 6 cM proximal to marker D2Mit46. This locus was
first characterized by the difference of response to erythrocyte
antigen-1 among different inbred strains of mice, with the BALB/cJ
strain being nonresponsive (38). In the confidence interval of Chr 11,
a family of small inducible cytokines (Scya), including Scya1–6, are
located about 5–7 cM proximal to marker D11Mit70. These
genes include a family of cytokines that function as proinflammatory
chemoattractants for CD4+ T cells, monocytes, and
eosinophils and as activators of basophils to release histamine.
Members of the ß-chemokine RANTES family have also been implicated in
a number of chronic inflammatory and autoimmune processes (39).
MHC genes influence the susceptibility to SLE both in humans (40, 41) and in NZB/NZW-related mouse strains (42). For example, replacing the H-2d haplotype of the NZB mouse with the H-2bm12 haplotype (a variant modifying the peptide binding groove of I-A), but not the H-2b haplotype, results in increased levels of anti-DNA Abs, whereas replacing the H-2b haplotype of the B x SB mouse with the H-2d haplotype leads to decreased autoimmune symptoms (42). Most likely, other genetic factors, such as the differences in TCR repertoire, may act together with MHC genes in controlling disease susceptibility. However, in the present study, we did not observe linkage to the MHC locus on mouse Chr 17. Linkage to the MHC locus was also not revealed in previous (MRL/lpr x CAST/Ei)F1 x MRL/lpr backcross (10) and MRL-lpr x C57BL/6-lpr intercross studies (33). Thus, either the MHC locus (H-2k for MRL/lpr, H-2d for BALB/cJ) is irrelevant to the MRL/lpr model, or the effects were obscured by other stronger genetic influences.
We also clearly observed that the lpr mutation functions to
a significant extent in the heterozygous state (Table III). After
removing the lpr/lpr homozygous individuals in our F2
population, we obtained lod scores of 13.7 for anti-dsDNA Abs and
10.9 for anti-ssDNA Abs, which still explains the majority of the
variance for these two traits. Our result is consistent with a previous
report (21). Since the influence of the lpr locus remains
extremely large in the heterozygous mice, it is of interest whether the
Chr 2 or 11 loci exert any effects in mice +/+ at the lpr
locus. However, the number of animals in this subpopulation was
insufficient for QTL analysis using the MAPMAKER-QTL program.
Anti-cardiolipin Abs shared the same Chr 19 and 11 QTLs with anti-DNA Abs, but not the Chr 2 QTL, indicating that the genetic controls of anti-cardiolipin Abs and anti-DNA Abs are not entirely the same. It is also noteworthy that anti-cardiolipin Ab levels of F1 mice were slightly higher than those of MRL/lpr mice, supporting a difference in the genetic control of this specificity compared with that of anti-DNA Abs. Nevertheless, the two loci influencing anti-cardiolipin Abs exhibited similar inheritance patterns for anti-DNA and anti-cardiolipin Ab levels. Abs detected by solid phase anti-cardiolipin immunoassays are heterogeneous. These Abs can bind to different epitopes of cardiolipin and can cross-react with other phospholipid and ß2-glycoprotein I-cardiolipin complexes (43, 44, 45). Further, since the cardiolipin coated on the ELISA plates was stored at 4°C before use, it was likely to be oxidized. Abs against oxidized cardiolipin were also reported to recognize oxidized low density lipoproteins (46, 47). The ability of anti-cardiolipin to recognize these diverse epitopes could influence its clearance from the circulation and may explain in part why there is differing genetic control of anti-cardiolipin compared with anti-DNA Abs. For example, genetic factors are likely to influence the oxidation of phospholipids (discussed in 12 , which could, in turn, affect the clearance of anti-cardiolipin Abs. However, the use of monoclonal anti-DNA Abs in these assays showed no evidence of cross-reactivity between DNA and cardiolipin.
Autoimmune vasculitis is characterized by the presence of autoantibodies in patient sera, such as anti-neutrophil cytoplasmic Abs, anti-nuclear Abs (48, 49, 50), and anti-dsDNA Abs (51). Although the mechanism of this interaction has yet to be clearly established, autoantibodies have been shown to substantially up-regulate the expression of cell adhesion molecules, an early phase in the development of an inflammatory vascular lesions (48). Recently, anti-cardiolipin Abs were also shown to be significantly associated with vasculitis in autoimmune-prone (NZW x B x SB)F1 mice, suggesting that anti-cardiolipin Abs, and their proposed thrombogenic and vascular injury consequences, contribute to development of microvasculitis in lupus-prone mice (52). It is of interest to note that the QTLs associated with elevated anti-cardiolipin Abs (Chr 11 and 19), but not the QTL associated only with anti-DNA Abs (and not anticardiolipin Ab; Chr 2) were significantly associated with coronary vasculitis. We have, in our (MRL/lpr x BALB/cJ) intercross, demonstrated common genetic controls of vasculitis with autoantibodies. Our results provided a genetic link between these two most common features of lupus that will help to clarify the mechanisms underlying their interaction.
In conclusion, using an (MRL/lpr x BALB/cJ) intercross we identified two loci, on Chr 2 and 11, in addition to the lpr mutation on Chr 19, controlling anti-DNA Ab levels. The Chr 2 QTL was also shown to contribute to anti-cardiolipin Ab levels. In addition, there are undoubtedly many loci that cannot be detected with confidence without using a much larger number of animals than that used in this study. We also demonstrated common genetic controls of both vasculitis and autoantibodies by Chr 19 and 11 QTLs. The localization of disease-modifying genes from our mouse model may be useful in the prediction of loci contributing to human SLE genes due to the chromosomal conservation of linked genes between mice and humans. More importantly, genetic loci for complex disease can be isolated in mice using breeding strategies similar to those employed in the identification of histocompatibility loci, thereby providing more detailed molecular information about the underlying genes (53, 54).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Aldons J. Lusis, Division of Cardiology, Department of Medicine, University of California School of Medicine, 47-123 CHS, Los Angeles, CA 90095-1679. E-mail address:
3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; MRL/lpr, MRL-lpr/lpr; Chr, chromosome; QTL, quantitative trait loci; HF, high fat; cM, centimorgan.
Received for publication February 24, 1997. Accepted for publication August 31, 1998.
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References |
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mRNA in the lymph nodes of lupus-prone mice. Mol. Immunol. 32:495.[Medline]
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