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*
Rheumatology Section, Imperial College School of Medicine, London, United Kingdom;
Department of Histopathology, St Mary’s Hospital, London, United Kingdom; and
Department of Pathology, Centre Médical Universitaire, Geneva, Switzerland
B.J.M and M.J.W. and from the Swiss National Foundation for Scientific Research (to S.I.). M.E.K.H. was a recipient of an Arthritis Research Campaign PhD studentship, and T.J.V. is a Wellcome Trust Senior Clinical Research Fellow.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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4.0
µg/ml. The subdivision of the (BXSB x (C57BL/10 x
BXSB)F1)-backcrossed mice into those homozygous or
heterozygous for Bxs6 enabled a remarkable association
to be observed between high levels of gp70IC and severe nephritis in
the Bxs6 homozygote population. A further mapping study
in these two subgroups identified a previously unrecognized interval
associated with the production of
autoantibodies. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the autoimmune-prone strains, BXSB, MRL/Mp-lpr/lpr, and (New Zealand Black (NZB) x New Zealand White (NZW))F1, a spontaneous autoimmune response to serum xenotropic gp70 develops that is not seen in nonautoimmune strains such as C57BL/6 and BALB/c (8). This results in the production of immune complexes containing gp70 and anti-gp70 Abs (gp70IC),3 which can be detected not only in sera (8), but as immune deposits in the diseased glomeruli (2, 11). There is considerable support for a correlation between gp70IC and the development of proteinuria and nephritis, which has led to the conclusion that gp70IC is a more appropriate phenotypic marker than other serological markers for the development of systematic lupus erythematosus (12, 13, 14, 15). However, the correlation of gp70 and gp70IC with other widely accepted indicators of autoimmune disease is unclear. As disease progresses (measured by an increase in the severity of nephritis), the levels of both gp70 and gp70IC increase (15, 16). However, there is little evidence of correlation between the appearance in the circulation of gp70 or gp70IC and the production of anti-DNA Abs (17, 18).
We have recently completed two extensive linkage analyses (19, 20) using BXSB mice backcrossed to the nonautoimmune strain C57BL/10 (B10). Furthermore, we have extensively studied the derivation of the BXSB strain and identified intervals that are B6-derived and therefore identical with the corresponding B10 intervals resulting from the backcross (21). Therefore, these intervals will not segregate in the crosses. Using this information to exclude fixed regions, we estimate that 99.5% of the segregating genome was within 20 cM of one of the 97 microsatellite markers used in this study. We examined multiple subphenotypes for (BXSB x (B10 x BXSB)F1) (BXSB/BC) and (B10 x (B10 x BXSB)F1) (B10/BC) male mice, identifying four chromosome 1 loci (Bxs1-4) and one interval on chromosome 3 (Bxs5) that were linked to various aspects of disease. Using these same backcrossed animals, we analyzed the correlation between gp70 and gp70IC in the BXSB strain with other phenotypic traits and performed linkage analyses to identify the loci associated with gp70/gp70IC production. Our results indicate that genetic loci linked to higher gp70 levels, and hence gp70IC production, may represent a novel subset of lupus-susceptible loci in mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice were bred and maintained under identical conditions at the Imperial College School of Medicine (London, U.K.) from original stocks: BXSB (H2b) were obtained from The Jackson Laboratory (Bar Harbor, ME) and B10 (H2b) were obtained from Harlan Olac (Bichester, U.K.). All the backcrossed mice analyzed were male, Yaa+, and free from disease pathogens. X chromosome effects were not investigated because the X chromosome was fixed within each backcross. Mice were not selected for testing, and all available data was included in subsequent analyses.
Serological analyses
Mice were bled and analyzed at 8 mo (parental BXSB), 16 mo (parental B10), 12 mo ((C57BL/10 x BXSB)F1 and (BXSB/BC)), and 18 mo (B10/BC). The classification of individual mice as positive or negative for a phenotype was based on the range of results for parental mouse strains (19, 20).
Phenotypes were measured in a variety of ways as described previously
(19). Briefly, anti-nuclear Ab (ANA) levels were
measured by indirect immunofluorescence using Hep-2 cells and a
FITC-conjugated IgG Fc-specific anti-mouse Ab. Anti-dsDNA and
anti-ssDNA Abs were measured by ELISA using Nunc Maxisorp plates
(Nunc, Naperville, IL) sensitized with methylated BSA and coated with
calf thymus ssDNA or dsDNA. The level of IgG3, previously found to be
elevated in BXSB mice (19, 20), was measured by radial
immunodiffusion in 1.2% PBS agarose gel using polyclonal
anti-mouse IgG3 Ab. The results were calibrated against a serial
dilution of standard sera. Mice were categorized as positive or
negative on the basis of results obtained from B10 parental strain mice
(Table I
) (19). Gp70 and
gp70IC were measured by ELISA as described previously
(22).
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After sacrifice, the spleen and the inguinal, axillary, and cervical lymph nodes were removed. The wet weight of these tissues was expressed as a percentage of total body weight. Kidneys were removed and fixed in formal saline before histopathological analysis of glomerulonephritis as described previously (19). Coded kidney sections were scored with respect to the level of mesangial matrix increase and glomerular hypercellularity.
Genotyping and statistical analyses
Genotyping was conducted using microsatellite markers as
previously described (19, 20). The linkage program
MAPMAKER/QTL (available at http://www.genome.wi.mit.edu)
(23) was used to identify quantitative trait loci (QTL)
(1). Autoantibody levels were log10 transformed.
A 
2 analysis with 1 df was used to define the
association between loci and nephritis using a standard (2 x 2)
contingency matrix as described previously (19). Because
these are genome-wide searches and involve multiple hypothesis testing,
the level for suggestive linkage was set at log likelihood of the odds
(LOD) 
1.9 (p 
3.4 x
10-3 and 2 8.6), and
the level for significant linkage at LOD = 3.3
(p = 1.0 x 10-4
and 2 15.3) (24). Comparison
of data between cohorts of mice was conducted using a Mann-Whitney
U test. The correlation coefficient (r) was
determined using a nonparametric Spearman correlation test. Potential
skewing of microsatellite markers within each cohort was checked using
2 analysis of the distribution of homozygotes
and heterozygotes at every microsatellite marker.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The serum levels of gp70 were measured for BXSB (n
= 9), B10 (n = 10), (B10 x
BXSB)F1 (n = 14), BXSB/BC
(n = 246), and B10/BC mice (n = 210)
(Fig. 1a). There were
significantly greater levels of gp70 (p <
1.0 x 10-4) in the BXSB parental strain
than in any other cohort of mice. The level of gp70 in BXSB/BC mice was
much higher than that in B10, F1, or B10/BC
(p < 1.0 x 10-4
in all cases), whereas gp70 titers in the last three groups were all
comparable. The data for gp70IC in the sera of the mouse cohorts
followed an identical pattern (Fig. 1b). Notably, there was
a strong correlation between gp70 titers and levels of gp70IC in the
BXSB/BC mice (r = 0.75 and p <
1.0 x 10-4; Fig. 2). However, there appeared to be a
threshold of serum gp70, below which high titers of gp70IC were not
observed. Although it was difficult to assign a specific threshold
value, presumably due to confounding genetic and stochastic factors,
mice with a serum gp70 of 4 µg/ml or less failed to elicit titers of
gp70IC above 2.5 µg/ml. Levels of gp70 were not generally strongly
correlated with other autoantibody titers in BXSB/BC mice, although
there was evidence of significant correlation with histological
phenotypes (glomerulonephritis, lymphadenopathy, and splenomegaly;
Table II). However, gp70IC production was
significantly correlated with all phenotypic markers (Table II).
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We used MAPMAKER/QTL to identify loci associated with gp70 and/or
gp70IC production in both backcrosses of mice. A single gp70 locus was
identified located on chromosome 13 (Fig. 3) in a region of BXSB almost exclusively
derived from SB/Le (21). The peak LOD scores for gp70
linkage to this interval were 6.2 in the B10/BC mice
(p = 8.8 x 10-8)
and 36.7 in the BXSB/BC mice (p = 1.6 x
10-38) with a genetic contribution to phenotype
from this one locus (variance) of 0.85. We have termed this interval
Bxs6. The same interval was also strongly linked to the
production of gp70IC, but not to the production of other
autoantibodies, in the BXSB/BC mice, with a peak LOD of 19.9
(p = 1.2 x 10-21
and variance = 34.6%; Fig. 3a). No additional
significant gp70- or gp70IC-associated loci were identified. The
Bxs6 interval also showed linkage to glomerulonephritis in
the BXSB/BC mice but not in B10/BC mice (Table III). In fact, using these newly
identified markers, we have now demonstrated a highly suggestive
linkage in BXSB/BC mice between Bxs6 and glomerulonephritis
(2 = 14.1 and p = 1.7 x
10-4), supporting the argument that gp70IC play
an important role in the development of glomerulonephritis.
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15 cM (26), the number
of mice that may have undergone a double recombination event between
any two of the markers selected is negligible.
Analysis of the phenotype of these subcohorts demonstrated no
significant difference between heterozygotes and homozygotes for levels
of anti-DNA Abs, ANAs, IgG3, lymphadenopathy, or splenomegaly (data
not shown). However, with respect to serum gp70 levels, there were
significant differences (p < 1.0 x
10-4) between all of the categories of mice
(BXSB, Bxs6 homozygote, and Bxs6 heterozygote),
with the highest levels in the BXSB parental mice, intermediate levels
in the Bxs6 homozygotes, and lowest levels in the
Bxs6 heterozygotes (Fig. 4a). Given a threshold value
of 4 µg/ml for gp70 levels, as discussed earlier, the overwhelming
majority of Bxs6 homozygotes (74 of 76) exceeded this value,
whereas only a minority of Bxs6 heterozygotes fell into this
category (25 of 106) (Fig. 2
). In contrast, median levels of gp70IC
detectable in Bxs6 homozygotes were comparable to those seen
in the parental BXSB mice but higher than those of the Bxs6
heterozygotes (p < 1.0 x
10-4; Fig. 4b). When renal
histopathology was evaluated (Table IV),
the number of mice positive for nephritis, as defined previously
(19, 20), was significantly greater in the Bxs6
homozygote cohort (44 of 70) than in the Bxs6 heterozygotes
(47 of 104) (p = 2.2 x
10-2). Furthermore, the number of mice with
severe nephritis (>75% abnormal glomeruli) was greatly increased in
the Bxs6 homozygotes (27 of 70 compared with 24 of 104 for
the heterozygotes; p = 2.7 x
10-3). Thus, homozygosity for Bxs6
appears to be responsible for the vast majority of gp70IC in the sera
of BXSB mice, and this in turn is associated with an increase in
incidence and severity of lupus-like nephritis.
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We tested the distribution of microsatellite markers within each cohort to ensure there was no significant skewing that might introduce bias in the subsequent linkage analysis. Of 105 markers (excluding chromosome 13) tested for the two subcohorts, two markers, a different one in each subcohort, showed slightly significant skewing away from 50:50 homozygote:heterozygote (p = 0.03). Within the context of this analysis, this does not represent significant bias.
Two additional MAPMAKER/QTL analyses were performed using these two
subcohorts. The disease-associated loci for all phenotypic markers
identified for the Bxs6-heterozygous cohort were the same as
those identified for the entire BXSB/BC, namely chromosomes 1, 3, and 4
(19, 20). However, the levels of linkage were lower than
seen in the whole backcross analysis. For example, the subcohort
maximum LOD for chromosome 1 for anti-ssDNA Ab production was 3.5
vs a maximum LOD of 7.5 for the entire BXSB/BC. This probably resulted
from the lower number of mice in the subcohort (106) compared with the
number in the entire backcross (350). However, the QTL analysis for the
Bxs6-homozygous mice revealed a novel interval, which we
have named Gp1 (Table V and
Fig. 5). This locus was also associated
with the production of anti-dsDNA and anti-ssDNA Ab. We have
previously studied the derivation of the BXSB strain (21).
The additional disease-associated locus identified in this study,
Gp1, spans a region that is mainly derived from the B6
parental strain of BXSB.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An interval from D13 Mit10 (17.5 cM) to D13 Mit256 (25.1cM) that coincides with Bxs6 has been implicated in gp70/gp70IC production in NZW mice (25), although this is not a single-gene effect, with a variance of 43%. There is also good evidence for linkage of gp70/gp70IC to two more telomeric regions of chromosome 13. In a (B6.Az x NZB)F1 x NZB backcross, gp70 production was linked to an NZB-derived interval on chromosome 13 in addition to intervals on chromosomes 2 and 4 (27), whereas a similar backcross established to B10.Az indicated a B10 contribution linked to gp70IC production and nephritis (28). In the (NZB x NZW)F1 mouse model, the regulation of gp70 is obviously complex, involving multiple loci (29). Immune complex production in these strains is more closely associated with loci that control autoantibody production, presumably due to the multiple genes affecting both autoantigen production and autoantibody response. However, in BXSB, a single locus, Bxs6, directs gp70 serum levels and, therefore, the corresponding autoantibody response is linked to this locus. The retroviral regulatory locus Gv1 (30), which is responsible for the coordinate regulation of multiple endogenous retroviruses and mapping data for which was confusing (31), has recently been mapped to the Bxs6 interval (32). It is an intriguing possibility that Bxs6 and Gv1 are the same locus. The idea of an alteration to a control protein is consistent with the identification of Bxs6 as the only significant locus directing gp70 levels in BXSB mice, which is in marked contrast to the multiple genetic effects seen in other strains.
Bxs6 is the source of serum gp70 in BXSB mice and, thus, the major linkage for gp70IC was also to Bxs6, with the levels of autoantigen acting as a checkpoint for the production of autoantibodies. There appeared to be a threshold of serum gp70 concentration, below which high titer gp70IC were not produced. This threshold effectively divided these mice into two phenotypically defined subgroups that overlap with the genotypic subgroups. Thus, Bxs6 heterozygotes were predominantly subthreshold for gp70 with low titer gp70IC and limited nephritis, whereas Bxs6 homozygotes had high titer gp70 and, hence, high levels of gp70IC with an increased incidence of more severe nephritis. This provides excellent support for the contention that gp70IC play a major role in glomerulonephritis and that this process is Ag driven above a threshold Ag concentration.
MAPMAKER/QTL analysis identified an additional novel locus in
Bxs6 homozygotes that contributed to gp70IC production (Fig. 5 and Table V; Gp1). This interval showed a trend toward
linkage to the production of other autoantibodies in Bxs6
homozygotes (Table V) and in the genome-wide linkage analysis
(19, 20). This locus accounted for 20.4% of the variance
in the gp70IC phenotype. This interval has not been implicated in lupus
susceptibility in any other mouse model of the disease.
There is good evidence that gp70IC play a role in autoimmune renal
pathology in (NZB x NZW)F1 mice (2, 12, 13, 14, 33). However, gp70 autoantigen, in the absence of immune
complex formation, plays little if any direct role in disease.
Previously, no correlation of gp70 autoantigen production with disease
was reported in (NZB x SWR)F2 mice
(17). Moreover, there was no clear segregation of gp70
titers in these mice, indicating a complex genetic background
underlying autoantigen production. This is in contrast to the situation
in BXSB mice, in which gp70 and gp70IC are clearly segregating in the
backcrossed mice and a single major locus is implicated. In fact, using
these newly identified markers, we have now demonstrated a highly
suggestive linkage in BXSB/BC mice between D13 Mit253 and
glomerulonephritis(2 = 14.1 and
p = 1.7 x 10-4),
supporting the argumentthat gp70IC play an important role in the
development of glomerulonephritis.
Because gp70IC levels were significantly correlated with all phenotypic
markers (p < 1.0 x
10-4), the correlation observed between serum
gp70 and histological phenotypes (glomerulonephritis, lymphadenopathy,
and splenomegaly) is likely to be a secondary effect arising from the
intrinsic association between gp70IC production and gp70 autoantigen
availability (Fig. 2).
The present and previous studies have shown that in BXSB/BC mice, both gp70IC and anti-dsDNA Ab production were highly correlated with nephritis. Different genes appear to regulate the production of each of these autoantibodies; anti-dsDNA Ab production was strongly linked to Bxs2 and 3 on chromosome 1, whereas gp70IC expression was linked to Bxs6. However, there is some overlap between the loci contributing to these two pathways, as indicated by the linkage of both to Gp1. Therefore, we suggest that both gp70IC and anti-dsDNA Abs play important but independent roles in disease pathology, and these roles may be cumulative in their effects.
Lupus-like syndrome in BXSB mice is a complex genetic trait, but we have shown that a single locus, Bxs6, is responsible for the generation of gp70 autoantigen. This locus acts as a checkpoint for autoantibody production, subsequent formation of immune complexes, and hence, the development of severe nephritis. This locus can also be mapped in B10/BC mice, but gp70 serum levels are subthreshold in the B10/BC mice, and thus, there is very limited high titer gp70IC production. The identification of the single locus on chromosome 13, Bxs6, which directs gp70 and, by implication, gp70IC production in BXSB mice, will enable us to directly address the question of the importance of gp70 in disease. We are at present developing reciprocal congenic mice to examine this question, and further mapping studies are underway to identify the locus.
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Footnotes |
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2 Abbreviations used in this paper: gp70IC, gp70-anti-gp70 immune complexes; QTL, quantitative trait loci; LOD, log likelihood of the odds; ANA, anti-nuclear Ab.dlk(v)
Received for publication September 28, 2000. Accepted for publication May 29, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
Bxs6-homozygous mice. The dotted vertical line indicates
the threshold value of gp70 (4.0 µg/ml), below which high level
gp70IC are not formed.
