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*
Departments of Pediatrics and Medicine, National Jewish Medical and Research Center, Denver, CO 80206;
Departments of Medicine and Immunology, University of Colorado Health Sciences Center, Denver, CO 80262;
Department of Immunology, Mayo Clinic and Mayo Graduate School of Medicine, Rochester, MN 55905;
§
Basel Institute for Immunology, Basel, Switzerland; and
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Department of Pathology, Centre Medical Universitaire, Geneva, Switzerland
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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 genes encoded within H2z that account for the genetic contribution to lupus susceptibility are not known. However, a number of studies have suggested that MHC class II genes, either H2-Az (Az) or H2-Ez (Ez), are likely candidates. For example, studies with mAbs to CD4 12 and I-Az 13 have shown that (NZB x NZW)F1 disease is dependent on CD4+ T cells and class II MHC-bearing cells, respectively. In addition, studies of NZB mice congenic for H2bm12 vs H2b indicated a role for the bm12 mutation of the I-Aß chain in disease susceptibility 14 . Studies also have shown that increased expression of I-E molecules can suppress lupus-like disease in New Zealand hybrid mice 15 . Finally, the MHC class II hypothesis has been supported by studies of other murine models of autoimmunity, such as NOD mice with spontaneous diabetes or strains induced to develop experimental autoimmune encephalomyelitis, experimental myasthenia gravis, or collagen-induced arthritis 16, 17, 18, 19, 20, 21, 22 . In all these models of organ-specific autoimmunity, class II MHC genes have been shown to be important for disease susceptibility. However, some investigators have questioned the paramount importance of MHC class II genes in the development of murine lupus and have suggested that MHC class I or class III genes (e.g., Tnfa) may at least partially account for the MHC contribution to disease 23, 24 .
We recently studied the potential role of Ez genes in New Zealand murine lupus by comparing C57BL/6 (B6) mice transgenic for Ez genes (designated B6.Ez mice) and B6 mice congenic for the entire H2z interval (B6.H2z mice) in a backcross analysis 25 . The development of nephritis in approximately 30% of (B6.H2z x NZB)F1 x NZB backcross mice was strongly linked with inheritance of H2z 8, 25 . In contrast, none of the similarly backcrossed B6.Ez mice with the Ez transgene developed nephritis. Although a subset of the (B6.Ez x NZB)F1 x NZB backcross mice produced moderate levels of autoantibodies, this production was not linked with inheritance of the Ez transgenes 25 . IgG autoantibody production was, however, linked with MHC heterozygosity determined by inheritance of H2b from the B6 background of the B6.Ez mice.
In the current study, Az transgenes were examined in a similar fashion. C57BL/10 (B10) mice transgenic for Aaz and Abz genes (designated B10.Az mice) were crossed with NZB mice and then studied as (B10.Az x NZB)F1 x NZB backcross mice. A subset of these mice produced high levels of autoantibodies and developed severe proteinuria; however, no autoimmune phenotype was linked with inheritance of the transgenes. In the same mice, inheritance of H2b contributed strongly to the development of autoimmune disease. Taken together with our previous Ez studies, the present work suggests that class II genes may not underlie the MHC contribution to lupus susceptibility in this model.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Parental NZB/BINJ, C57BL/10 (B10), and C57BL/6J (B6) mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and were maintained in the animal care facility at the National Jewish Medical and Research Center (Denver, CO). All congenic, transgenic, F1, and backcross mice were bred and maintained at the National Jewish Medical and Research Center. Only female mice were studied for expression of disease.
B6 mice were made congenic for H2z by mating these
mice with NZW mice and backcrossing the progeny to B6 8, 25 .
Inheritance of H2z was monitored by
immunofluorescence analysis of I-Az expression and by
screening for a simple sequence length polymorphism in the TNF-
gene
(Tnf). The congenic strain (designated B6.H2z)
was made homozygous for H2z after 12 generations.
Congenic mice were analyzed for the length of the NZW chromosome 17
interval bred onto the recipient B6 strain. In relation to the MHC,
analysis of markers approximately 1 cM proximal to MHC on chromosome 17
(D17 Mit16; 
18.1 cM from the centromere), within the MHC
(Tnf or H2z Alu repeat; 19
cM from the centromere), and about 4 cM distal to MHC (D17
Mit49 or D17 Mit50; 23.2 cM from the centromere)
showed alleles inherited from NZW in the congenic mice.
In preparation for the generation of transgenic mice, genomic fragments encoding the Aaz and Abz coding regions were isolated from an NZW splenic DNA cosmid library (see below). Transgenic mice were generated in the laboratories of Chella S. David using methods previously described 26, 27 . (CBA/J x B10.M)F1 or (SWR x B10.M)F2 eggs were coinjected with Aaz and Abz genomic DNA and reimplanted into foster mothers. Tails from the resulting offspring were analyzed by Southern blotting for integration of the injected DNA. Three founders were bred, of which two were found to have both Aaz and Abz genes. These lines were perpetuated by repeated backcrossing with B10 mice for seven generations. Inheritance of the transgenes was determined by PCR analysis of genomic DNA. Primer sequences (5'-3') to detect the Aaz transgene were GTA GGC TCC TAT GGT ATA GT (forward) and GTC AAA GCT TCT CAG TTG AG (reverse), and primer sequences to detect the Abz transgene were CCT TGA GGG CCA CGG TTG TC (forward) and TAA GAG GCT CTG GGG GTA TC (reverse). Occasional offspring were also analyzed by immunofluorescence staining for expression of I-Az on peripheral blood cells (see below). One of the lines with both Aaz and Abz transgenes was subsequently designated B10.Az; the other had lower levels of surface I-Az expression and was designated B10.Azlo. Integration sites for each of these lines were not linked to MHC or to loci on distal chromosome 1 and were not studied further. Integration of the Az genes had no noticeable effect on the health of the B10 recipients, and there was no evidence of autoimmune disease or autoantibody production in the transgenic strains.
Evaluation of renal disease and collection of tissue
Mice were studied from 4–12 mo of age, and were evaluated for
proteinuria at bimonthly intervals using
tetrachlorophenol-tetrabromosulfophthalein paper (Chemstrip, Boehringer
Mannheim, Indianapolis, IN) as previously described 4 . A scoring
system of 0–3+ was used, as follows: 0/trace, <30 mg/dl; 1+, 30
mg/dl; 2+, 100 mg/dl; and 3+, >300 mg/dl. A score of 2+ or greater
was considered indicative of severe proteinuria, and mice exhibiting
severe proteinuria on two or more successive occasions, or at the final
evaluation before death or sacrifice, were considered positive for
renal disease. A negative phenotype was ascribed to mice that did not
exhibit proteinuria during the 12 mo of follow-up, and these mice
appeared healthy at the time of sacrifice. A correlation between severe
proteinuria and death from renal failure was demonstrated previously
4, 25 , and a strong correlation with histological severity of
glomerulonephritis has been more recently confirmed (T.J.V. and B.L.K.,
unpublished observations), supporting the validity of utilizing high
levels of proteinuria as a sole indicator of severe and progressive
glomerulonephritis. The development of proteinuria also predicted early
mortality in the current study. For example, 36 of 43 mice (85%) with
high grade proteinuria before 9 mo of age died by 12 mo of age. In
contrast, during the entire study only 5 of 65 mice (8%) with no
proteinuria died, and several of these mice showed evidence for a cause
of death unrelated to nephritis.
One hundred and twenty (B10.Azlo x NZB)F1 x NZB and 86 (B10.Az x NZB)F1 x NZB female mice were followed for the development of severe proteinuria for 12 mo. The tip of the tail from all backcross mice was excised at 4 mo of age. The liver and kidneys were collected at the time of death or elective sacrifice at 12 mo of age. All tissues were stored at -70°C, and DNA was extracted as previously described 28 . The study mice were also bled (from the tail) at monthly intervals from the age of 5 mo. The blood was allowed to clot at room temperature, and the serum was stored at -20°C until analyzed for autoantibody levels.
Generation of NZW splenic DNA cosmid library
DNA extracted from NZW spleen cells was used to generate a
cosmid library as previously described 29 . Splenic DNA was partially
digested with MboI to generate 35- to 45-kb fragments and
ligated into BamHI-digested pCV 107 cosmid vector, packaged
(Gigapack Gold, Stratagene, La Jolla, CA), and grown in
Escherichia coli. The library was plated at approximately
10,000 colonies/filter, and 4.1 x 105 total colonies
were screened. Probes were generated from mRNA expressed by
LPS-stimulated B cell blasts from NZW mice, PCR amplification of
segments of the Aaz and Abz
genes, and cloning of PCR fragments into pEMBL. Before generation of
the transgenic mice, the selected cosmid clones (see Fig. 1
) were shown to mediate expression of
I-A after transfection into A20 cells.
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Spleen cells from the different parental strains and backcross
mice were prepared and stained as previously described 30 . The
fluoresceinated mAbs used included 3F12 (anti-I-Az
31 , obtained from Dr. John Freed, National Jewish Medical and
Research Center), 10-2.16 (anti-I-Aßz 32 , hybridoma
cells obtained from American Type Culture Collection, Manassas, VA),
and HB35 (anti-I-Ab,d, hybridoma cells obtained from
American Type Culture Collection). Splenic B cells were also double
stained using a biotinylated mAb to B220 (RA3-6B2, PharMingen, San
Diego, CA) followed by avidin-phycoerythrin (PharMingen). In some
experiments PBL were double stained with fluoresceinated 3F12 and
biotinylated 10-2.16 followed by avidin-phycoerythrin. Fluorescence
intensity was analyzed on an EPICS C flow cytometer (Coulter, Hialeah,
FL). Viable mononuclear cells were gated by scatter analysis, and
1 x 104 cells were collected for each Ab combination.
Analysis of thymus expression of I-Az
Thymus cells were also analyzed for the expression of I-Az by direct immunohistochemistry. Briefly, thymus tissue was frozen in embedding medium (O.C.T. compound, Miles, Elkhart, IN). Four-micron sections were cut and fixed, and endogenous peroxidase activity was blocked using 0.5% H2O2. The sections were incubated at 4°C overnight with biotinylated primary Ab followed by incubation with streptavidin-conjugated horseradish peroxidase for 1 h at room temperature. Finally, the sections were incubated with a solution of 3,3'-diaminobenzidine tetrahydrochloride and peroxide and counterstained with hematoxylin for 1 min. Sections were studied using light microscopy.
Typing for inheritance of H2 haplotype
Inheritance of H2b vs H2d haplotypes in backcross mice was determined by analysis of genomic DNA for a simple sequence length polymorphism in Tnf 33 . Oligonucleotide primers flanking the Tnf microsatellite were synthesized in the Molecular Resource Center at the National Jewish Medical and Research Center using an Applied Biosystems model 392 DNA synthesizer (Foster City, CA). Primer nucleotide sequences and the methods for SSLP mapping have been previously described 8 .
Serological assays
Abs to chromatin were determined by ELISA as previously described 9, 11 . Briefly, wells of microtiter plates were coated with calf thymus chromatin at 2.5 µg/ml and postcoated with gelatin. Serum samples were diluted 1/300 before adding them to Ag-coated wells for 90 min. After wells were incubated with peroxidase-conjugated Ab for mouse IgG, substrate was added, and OD was determined with an automated spectrophotometer. The results were plotted against a standard curve obtained using control (NZB x NZW)F1 sera as previously described 9 . IgG subclass anti-chromatin autoantibody levels were assayed using the same anti-chromatin ELISA, but IgG subclass-specific second step Abs were used as detecting reagents as previously described 9 .
The production of autoantibodies to gp70 was quantitated as serum levels of gp70-anti-gp70 immune complexes (gp70 IC), since the relative excess of gp70 in sera makes free anti-gp70 Abs difficult to detect 34 . These complexes were measured by ELISA after precipitation of the serum with polyethylene glycol (average m.w., 6000) as previously described 35 . The results are expressed as micrograms per milliliter of gp70 complexed with anti-gp70 Abs. Although gp70 is detectable in the serum of nearly all murine strains, only lupus-prone strains produce autoantibodies to gp70 and form gp70 IC 36 .
For certain comparisons, mice were separated into groups based on their serum levels of a particular autoantibody. The cut-offs used to group mice in the current study were originally determined in (NZB x NZW)F1 x NZW backcross mice by dividing the frequency distribution of autoantibody levels on the basis of tertiles. This separation into autoantibody phenotypes identified one-third of mice with low/negative levels and one-third of mice with high levels for each autoantibody measured. Backcross mice with intermediate levels were defined as the middle third. The cut-offs for anti-chromatin and gp70 IC autoantibodies correlated well with low levels of production in NZW and nonautoimmune strains and high levels of production in (NZB x NZW)F1 mice 9, 25 .
Statistical analysis
The linkage of the Az transgene or MHC type with
nephritis was quantified by 
2 analysis, using a standard
(2 x 2) contingency matrix. Evidence that these genes are linked
with autoantibody levels as quantitative trait loci (QTL) was
determined by using the linkage program, MAPMAKER/QTL 37, 38 . The
autoantibody levels were log10 transformed before analysis
with MAPMAKER/QTL because this tended to normalize their frequency
distribution and improve the accuracy of MAPMAKER/QTL 37 . It is
emphasized that these analyses were directed at MHC genes or transgenes
and were not part of a genome-wide screen for linked loci. The
statistical threshold used for significant linkage was
p < 0.01, based on recommendations that this cut-off
be used to confirm linkage in a new dataset 39 .
In separate analyses the frequency of nephritis was compared in H2z-congenic B6 mice and Az-transgenic B10 mice by Fisher’s exact test. The mean values for particular autoantibodies in different backcrosses were compared using the nonparametric Dunn procedure of the Kruskal-Wallis test (two-tailed).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To study the roles of Az genes in the
lupus-like disease of (NZB x NZW)F1 mice, cosmid
clones of Aaz and Abz were
isolated from an NZW genomic library. Restriction maps of both clones
are shown in Fig. 1
. Double-transgenic mice were then prepared by
coinjecting both clones into (CBA x B10.M)F2 or
(SWR x B10.M)F2 eggs and selecting for founder mice
that expressed both Aaz and
Abz transgenes. After backcrossing the
transgenes onto a B10 background, two lines that differed in
I-Az expression levels were selected for use in the present
studies. These were named B10.Az and B10.Azlo
based on relative levels of both Aaz and
Abz mRNA expression and relative levels of splenic B
cell surface expression of I-Aßz. As shown in Fig. 2, I-Aßz expression on
splenic B cells of the B10.Az and B10.Azlo
lines was approximately twofold and 10%, respectively, compared with
H2z-congenic B6 mice. As expected, both transgenic
lines expressed levels of endogenous I-Ab similar to that
expressed in normal B10 mice, whereas MHC-congenic B6.H2z
mice did not express I-Ab.
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z and
Aßz mRNA in transgenic mice was also associated
with expression of I-Az and I-Aßz surface
proteins. The mAb recognizing I-Az cross-reacts with
I-Ab (present in the B10 transgenic lines) but not with
I-Ad. We therefore studied (B10.Az x
NZB)F1 x NZB backcross mice and selected progeny that were
H2d/d by genotyping. Peripheral blood cells from
transgene-positive and transgene-negative mice were then double stained
with mAbs directed to I-Az and I-Aßz. Fig. 3 shows that both proteins were expressed
on the surface of the same cells in transgene-positive mice, as in
B6.H2z congenic controls. Cells from backcross animals
genotyped as transgene-negative failed to stain positive with either
I-Az reagent.
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(B10.Az x NZB)F1 x NZB and
(B10.Azlo x NZB)F1 x NZB backcross mice
(collectively referred to as Az backcross mice) were
bred to examine the contribution of Az to lupus-like
disease. PCR amplification of genomic DNA using primers capable of
distinguishing Aaz and Abz
products was used to analyze transgenic lines and backcross mice for
inheritance of the transgenes. Fig. 4
shows representative results for H2z-positive and
-negative control strains, transgene-positive and -negative strains,
and backcross mice. As expected, neither Aaz nor
Abz PCR products were observed for DNA from NZB
(H2d) or B10 mice (H2b).
Alternatively, both NZW (H2z) and
H2z-congenic B6 mice showed the presence of both
genes, as did mice from transgenic B10.Az and
B10.Azlo lines. In backcross mice constructed using either
the B10.Az or B10.Azlo strains, inheritance of
both transgenes or lack thereof was always concordant, consistent
with integration of the transgenes into the same chromosomal position.
Backcross mice were also screened for expression of
Abz mRNA and splenic B cell surface expression of
I-Aßz using RT-PCR analysis and flow cytometry,
respectively. Agreement among all these analyses for transgene
inheritance and expression was consistently observed.
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Previous studies have shown that (B6.H2z x
NZB)F1 mice do not develop severe lupus-like disease, but
that a subset of (B6.H2z x NZB)F1 x NZB
backcross mice produces high levels of autoantibody production and die
from lupus nephritis within 12 mo 8, 40 . Disease development was
strongly linked to inheritance of the H2z congenic interval
8 . For the current study we bred two similar backcrosses using the
Az-transgenic B10 lines to study the influence of
Az on disease. Female mice with severe nephritis or
no nephritis after 12 mo of follow-up were included in the analysis. As
shown in Fig. 5A, 62% of
(B10.Azlo x NZB)F1 x NZB mice and 61%
of (B10.Az x NZB)F1 x NZB developed severe
proteinuria, and the kinetics of disease expression were nearly
identical in the two Az backcrosses. This incidence of
severe nephritis in the B10.Az backcross mice was
significantly greater than that in previous backcrosses with
B6.H2z (p < 2.5 x
10-13) or B6.Ez (p <
7.0 x 10-19) mice (Fig. 5A) 8, 25 .
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B).
Furthermore, differences in I-Az expression between the two
backcrosses were not important for development of lupus-like disease,
and results for the Az backcrosses are pooled for
the remainder of this report. Interestingly, B10.Az
backcross mice (transgene-positive or transgene-negative) demonstrated
a significantly increased frequency of lupus nephritis compared with
H2z-positive and H2z-negative
(B6.H2z x NZB)F1 x NZB backcross mice
(p < 2.7 x 10-4 and
p < 3.5 x 10-11, respectively).
In the (B10.Az x NZB)F1 x NZB backcrosses,
mice differed based on their Az genotype as well as
inheritance of H2b from the B10 strain. Thus,
backcross mice were either homozygous for H2d or
were heterozygous H2b/d. Table I shows a linkage analysis of nephritis
with all the different MHC genotypes. As predicted from the data shown
above, there was no trend for linkage with the Az
genotypes. This is irrespective of whether mice were
H2d or H2b/d (data not shown). In
contrast, inheritance of H2b showed significant
linkage with nephritis (p < 1 x
10-4), with an odds ratio of 4.45.
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To further study whether the Az transgenes
contributed to autoimmunity in New Zealand mice, we quantitated serum
levels of IgG autoantibodies to chromatin and gp70 immune complexes.
Previous studies have shown that IgG antinuclear autoantibody
production is coordinately controlled and that serum levels of
anti-chromatin autoantibodies are highly correlated with levels of
IgG autoantibodies to dsDNA and to histones 11 . The linkages of these
serological traits with the different MHC genotypes were analyzed as
quantitative trait loci. The results in Table II are strongly concordant with the
results shown above for nephritis. The Az genotype
showed no linkage or trend for linkage with any serological trait. In
contrast, in the same mice, H2b/d vs
H2d was strongly linked with total IgG
anti-chromatin levels and gp70 IC. Linkage with each of the IgG
subclass anti-chromatin autoantibodies was also apparent, with the
strongest linkage for IgG2a anti-chromatin Abs. No significant
difference was observed between (B10.Azlo x
NZB)F1 x NZB and (B10.Azhi x
NZB)F1 x NZB mice in the amounts of any of the measured
autoantibodies.
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2 analysis, a strong association with
nephritis was found for gp70 IC (2 = 16.6;
p < 2.5 x 10-4) and less so for IgG
anti-chromatin Abs (2 = 7.9; p <
0.02). Compared with healthy (B10.Az x NZB)F1
x NZB mice, age-matched (B10.Az x NZB)F1 x
NZB mice with severe proteinuria produced significantly greater amounts
of autoantibodies (p < 6.0 x
10-4 for total IgG anti-chromatin, p
< 2.0 x 10-4 for IgG2a anti-chromatin,
p < 5 x 10-4 for IgG3
anti-chromatin, and p < 1.0 x
10-4 for gp70 IC). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our experimental design was based on transgenic expression of I-Az and an attempt to mirror expression of I-Az in mice with an intact H2z haplotype. Transgenic strains were derived by injection of genomic Az clones with wild-type promoter and enhancer elements. We developed two different lines of Az transgenic mice, one with twofold higher and another with considerably lower expression of surface I-Az on B cells compared with H2z-positive mice. Neither transgene showed a trend for influencing disease expression in the respective backcrosses. For peptide presentation in the peripheral lymphoid tissues, cells in transgenic mice with higher expression should have functioned similarly to those in wild-type animals, especially considering that autoimmunity in older (NZB x NZW)F1 mice correlates with higher expression levels of class II MHC molecules 41 . We also compared Az transgenic mice with H2z-positive or -negative control strains for expression of I-Az in the thymus, since this is the major site where class II MHC expression affects T cell development. The results indicated normal patterns of thymic I-Az expression in the transgenic mice.
The lack of effect of Az genes on autoimmunity also
indicates that mixed haplotype I-Ad/I-Aßz
and mixed isotype I-Ed/I-Aßz molecules do
not explain the effect of H2z on disease in
(NZB x NZW)F1 mice as previously suggested 42 .
Since H2d-encoded molecules were present in the
backcross animals, I-Aßz should have been equally likely
to pair with I-Ad or I-Ed as in wild-type
H2z backcross mice. This is consistent with a
previous report showing that an Abz transgene alone
expressed in H2d homozygous (NZB x
NZW.H2d)F1 mice resulted in no greater
autoantibody production and nephritis than was found in (NZB x
NZW)F1 mice 43 . In our studies, the lack of effect of the
transgene on lupus-like disease was also not influenced by inheritance
of H2b/d vs H2d/d and therefore
was not related to competition from I-Ab or
I-Aßb for pairing.
Although the Az transgenes had no effect on autoimmune disease, inheritance of H2b from the normal B10 background greatly enhanced IgG autoantibody production and nephritis in the same backcross. These results are consistent with a large body of evidence indicating that MHC heterozygosity is important for full expression of disease in New Zealand hybrid mice (2–11; reviewed in Refs. 1 and 25). Previous studies have consistently shown that New Zealand hybrid or backcross mice that are H2d/z have increased IgG autoantibody production and increased incidence of nephritis compared with genetically similar mice with a double dose of either H2d or H2z genes. The mechanism by which this H2 heterozygosity confers greater disease susceptibility than H2 homozygosity is unknown. Consistent with our current results, recent studies have shown that inheritance of H2b, in the context of H2d or H2z, also enhances IgG autoantibody production and nephritis 10, 25 . It may be important that heterozygous H2b/d mice have only one copy of H2d genes, like H2d/z mice, which is not recapitulated by transgenic expression of individual class II genes.
Inheritance of H2b in our previous B6.Ez backcrosses 25 and in the current B10.Az backcrosses was strongly linked with the production of IgG autoantibodies, especially IgG2a anti-chromatin Abs. IgG2a subclass antinuclear Abs have been regarded as strongly nephritogenic. However, nephritis was not observed in the previous B6.Ez backcrosses. Interestingly, H2b in the current B10.Az backcrosses showed much stronger linkage with gp70 IC compared with B6.Ez backcross mice. In some genetic analyses, gp70 IC vs antinuclear Abs have been implicated as the major pathogenic autoantibody in this model of lupus 11, 36 . Consistent with this hypothesis, levels of gp70 IC showed a stronger association with severe nephritis in the current study than did IgG anti-chromatin Abs.
The linkage of H2b with IgG autoantibody production
in the current backcross analysis raises questions similar to those
that prompted the current studies. For example, is this effect mediated
by class II genes or by other genes encoded with the MHC? Although the
answer is unknown at this time, it is of interest that the effect of
H2b was not specific for one type of autoantibody.
Thus, increased serum levels of IgG autoantibodies to chromatin and to
gp70 were both linked with H2b. Furthermore, linkage
was most apparent for the IgG2a and IgG3 subclasses of IgG
anti-chromatin autoantibodies. The results therefore suggest that
genes influencing immune responses in general, such as via cytokine
production, may be more likely to underlie the H2b
contribution. In this regard, the Tnf gene has been
previously proposed as a gene that may underlie the
H2z contribution to lupus in (NZB x
NZW)F1 mice 24 . A Tnf polymorphism, which was
shown to correlate with decreased TNF- production, is present in
H2b and H2z, but not in the
H2d, haplotype 24, 44, 45 . Although cytokine genes
may be involved in the H2 contribution to disease,
considering that there are influences from each haplotype, additional
contributing genes seem likely.
The differences in autoantibody production and incidence of nephritis in (B10.Az x NZB)F1 x NZB mice compared with (B6.Ez x NZB)F1 x NZB may provide a new approach to dissect the genetic control of murine lupus. These backcrosses differ genetically in only two ways: 1) the transgenes present in the two systems, which are not linked with any autoimmune phenotype measured in either cross; and 2) approximately 1% of the genome, which differs between C57BL/6 and C57BL/10 strains 46, 47 . The genetic differences between these strains have been mapped by others, including one study in which a whole genome scan showed differences limited to small regions on chromosomes 2, 4, 11, 12, 13, and 16 47 . Additionally, these strains are not histocompatible due to differences at the minor histocompatibility locus, H9, which has not been mapped 48 . The limited genetic differences in these closely related strains thus offer a novel approach to identify the disease-enhancing locus (or loci) in the B10 background. Preliminary studies suggest a disease susceptibility B10 allele (not present in B6) at a locus on distal chromosome 13 close to a previously mapped NZB susceptibility allele (S.J.R., S.I., and B.L.K., unpublished observations).
In summary, the current studies provide evidence that Az genes do not underlie the contribution of H2z to IgG autoantibody production and nephritis in New Zealand hybrid mice. Together with our other studies showing no disease enhancement from Ez genes, the present studies also suggest that class II MHC genes are not mainly responsible for this MHC contribution. This conclusion may therefore separate the role of MHC in lupus compared with that in NOD mice and type 1 diabetes, in which class II genes appear to be most important 16, 17, 18, 19 . In support of this idea is the observation that MHC heterozygosity enhances disease susceptibility in murine lupus 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , whereas homozygosity for high risk alleles enhances disease susceptibility in NOD mice 16, 17, 18, 19 . Although the present studies did not address whether class II genes, in conjunction with other H2z-encoded genes, are involved in disease susceptibility, together the results implicate other MHC genes in lupus susceptibility.
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Acknowledgments |
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Footnotes |
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2 Current address: Imperial College School of Medicine, Hammersmith Campus, London, W12 ONN, U.K.
3 Address correspondence and reprint requests to Dr. Brian L. Kotzin, Division of Clinical Immunology (B-164), University of Colorado Health Sciences Center, 4200 East Ninth Ave., Denver, CO 80262. E-mail address:
4 Abbreviations used in this paper: NZB, New Zealand Black; NZW, New Zealand White; gp70 IC, gp70-anti-gp70 immune complexes; QTL, quantitative trait locus.
Received for publication May 27, 1998. Accepted for publication November 17, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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: relevance to genetic predisposition to systemic lupus erythematosus. Proc. Natl. Acad. Sci. USA 87:1233. and Aß polypeptides. J. Immunol. 136:2953.[Abstract]
promoter identifies an allele unique to the NZW mouse strain. J. Exp. Med. 171:2141.This article has been cited by other articles:
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  S. C. Schweitzer, A. M. Reding, H. M. Patton, T. P. Sullivan, C. E. Stubbs, E. Villalobos-Menuey, S. A. Huber, and M. K. Newell Endogenous versus exogenous fatty acid availability affects lysosomal acidity and MHC class II expression J. Lipid Res., November 1, 2006; 47(11): 2525 - 2537. [Abstract] [Full Text] [PDF]
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 M. R. Gubbels, T. N. Jorgensen, T. E. Metzger, K. Menze, H. Steele, S. A. Flannery, S. J. Rozzo, and B. L. Kotzin Effects of MHC and Gender on Lupus-Like Autoimmunity in Nba2 Congenic Mice J. Immunol., November 1, 2005; 175(9): 6190 - 6196. [Abstract] [Full Text] [PDF]
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S. Subramanian, Y.-S. Yim, K. Liu, K. Tus, X. J. Zhou, and E. K. Wakeland Epistatic Suppression of Systemic Lupus Erythematosus: Fine Mapping of Sles1 to Less Than 1 Mb J. Immunol., July 15, 2005; 175(2): 1062 - 1072. [Abstract] [Full Text] [PDF]
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C. Laporte, B. Ballester, C. Mary, S. Izui, and L. Reininger The Sgp3 Locus on Mouse Chromosome 13 Regulates Nephritogenic gp70 Autoantigen Expression and Predisposes to Autoimmunity J. Immunol., October 1, 2003; 171(7): 3872 - 3877. [Abstract] [Full Text] [PDF]
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Z. SM. Rahman, S.-K. Tin, P.-N. L. Buenaventura, C.-H. Ho, E. P. H. Yap, R. Y. Y. Yong, and D.-R. Koh A Novel Susceptibility Locus On Chromosome 2 in the (New Zealand Black x New Zealand White)F1 Hybrid Mouse Model of Systemic Lupus Erythematosus J. Immunol., March 15, 2002; 168(6): 3042 - 3049. [Abstract] [Full Text] [PDF]
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S. Xie, S. Chang, P. Yang, C. Jacob, A. Kaliyaperumal, S. K. Datta, and C. Mohan Genetic Contributions of Nonautoimmune SWR Mice Toward Lupus Nephritis J. Immunol., December 15, 2001; 167(12): 7141 - 7149. [Abstract] [Full Text] [PDF]
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 A. DUGUET, H. IIJIMA, S.-Y. EUM, Q. HAMID, and D. H. EIDELMAN Eosinophil Peroxidase Mediates Protein Nitration in Allergic Airway Inflammation in Mice Am. J. Respir. Crit. Care Med., October 1, 2001; 164(7): 1119 - 1126. [Abstract] [Full Text] [PDF]
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T. Paisansinsup, A. N. Vallejo, H. Luthra, and C. S. David HLA-DR Modulates Autoantibody Repertoire, But Not Mortality, in a Humanized Mouse Model of Systemic Lupus Erythematosus J. Immunol., October 1, 2001; 167(7): 4083 - 4090. [Abstract] [Full Text] [PDF]
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