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Analysis of MHC Class II Genes in the
Susceptibility to Lupus in New Zealand Mice
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J Immunol 1999; 162:2623-2630; ;
http://www.jimmunol.org/content/162/5/2623
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Copyright © 1999 by The American Association of
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References
Stephen J. Rozzo, Timothy J. Vyse, Chella S. David, Ed
Palmer, Shozo Izui and Brian L. Kotzin
Analysis of MHC Class II Genes in the Susceptibility to Lupus
in New Zealand Mice1
Stephen J. Rozzo,*† Timothy J. Vyse,2* Chella S. David,‡ Ed Palmer,§ Shozo Izui,¶ and
Brian L. Kotzin3*†
C
onsiderable evidence indicates that the development of
systemic lupus erythematosus has a strong genetic basis,
with contributions from MHC and multiple non-MHC
genes (1). New Zealand hybrid mice are considered to be an excellent model of this human systemic autoimmune disease, and F1
progeny of New Zealand black (NZB)4 and New Zealand white
(NZW) mice spontaneously develop a severe lupus-like glomerulonephritis associated with the production of antinuclear autoantibodies. In studies of genetic susceptibility, genes encoded within
or closely linked to the MHC have been shown to be important for
the development of disease in these F1 mice (1–11). Evidence supporting an important role for genes encoded within the H2z locus
of NZW mice has come from studies of (NZB 3 NZW)F1 3 NZB
backcross mice and (NZB 3 NZW)F2 intercross mice (2– 6),
crosses of NZB and NZW mice congenic for H2z and H2d, respectively (7), and backcross analyses of normal strains of mice (e.g.,
C57BL/6) congenic for H2z (8, 9).
*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 ¶Department of Pathology,
Centre Medical Universitaire, Geneva, Switzerland
Received for publication May 27, 1998. Accepted for publication November
17, 1998.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grants AR37070 (to
B.L.K.) and AI14764 (to C.S.D.) and by a grant from the Swiss National Foundation
for Scientific Research (to S.I.).
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: [email protected]
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.
Copyright © 1999 by The American Association of Immunologists
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 3
NZW)F1 disease is dependent on CD41 T cells and class II MHCbearing cells, respectively. In addition, studies of NZB mice congenic for H2bm12 vs H2b indicated a role for the bm12 mutation of
the I-Ab 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 –22). In all these models of organspecific 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 3
NZB)F1 3 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 3 NZB)F1 3 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.
0022-1767/99/$02.00
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Hybrids of New Zealand Black (NZB) and New Zealand White (NZW) mice spontaneously develop a disease similar to human
systemic lupus erythematosus. MHC and non-MHC genes contribute to disease susceptibility in this murine model. Multiple
studies have shown that the NZW H2z locus is strongly associated with the development of lupus-like disease in these mice. The
susceptibility gene(s) within H2z is not known, but different lines of evidence have pointed to class II MHC genes, either H2-E or
H2-A (Ez or Az in NZW). Recent studies from our laboratory showed that Ez does not supplant H2z in the contribution to lupus-like
disease. In the present work we generated C57BL/10 (B10) mice transgenic for Aaz and Abz genes (designated B10.Az mice) and
used a (B10.Az 3 NZB)F1 3 NZB backcross to assess the contributions of Az genes to disease. A subset of backcross mice produced
high levels of IgG autoantibodies and developed severe nephritis. However, no autoimmune phenotype was linked to the Az
transgenes. Surprisingly, in the same backcross mice, inheritance of H2b from the nonautoimmune B10 strain was strongly linked
with both autoantibody production and nephritis. Taken together with our previous Ez studies, the present work calls into question
the importance of class II MHC genes for lupus susceptibility in this model and provides new insight into the role of MHC in
lupus-like autoimmunity. The Journal of Immunology, 1999, 162: 2623–2630.
2624
MHC CLASS II GENES IN MURINE LUPUS
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 3 NZB)F1 3 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.
Materials and Methods
Mice
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 –31 was used, as follows:
0/trace, ,30 mg/dl; 11, ;30 mg/dl; 21, ;100 mg/dl; and 31, .300
mg/dl. A score of 21 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 devel-
FIGURE 1. Restriction maps of the H2-Aa (Aa) and H2-Ab (Ab) genes
isolated from an NZW genomic library. The shaded areas indicate the
coding region for each gene. The sizes of the genomic fragments are shown
in kilobases. Restriction enzyme abbreviations: Ba, BamHI; E, EcoRI; H3,
HindIII; Hp, HpaI; K, KpnI; N, Nci; P, PvuII; Ps, PstI; S, SalI; Sp, SphI;
and SS, SSpI.
opment 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 3 NZB)F1 3 NZB and 86 (B10.Az
3 NZB)F1 3 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 270°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 220°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 BamHIdigested 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 3 105 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.
Analysis of B cell surface I-A expression
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-Aaz (31), obtained from Dr. John Freed,
National Jewish Medical and Research Center), 10-2.16 (anti-I-Abz (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 102.16 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 3 104 cells were
collected for each Ab combination.
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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-a 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 3 B10.M)F1 or (SWR 3 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 (59-39) 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.
The Journal of Immunology
2625
Analysis of thymus expression of I-Az
Results
z
Thymus cells were also analyzed for the expression of I-A 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 3,39-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).
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 mg/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 3 NZW)F1 sera as previously described (9). IgG subclass antichromatin 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 3 NZW)F1 3
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 3 NZW)F1 mice (9, 25).
Statistical analysis
The linkage of the Az transgene or MHC type with nephritis was quantified
by x2 analysis, using a standard (2 3 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 H2zcongenic 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).
To study the roles of Az genes in the lupus-like disease of (NZB 3
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 3 B10.M)F2 or (SWR 3 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-Abz. As shown in Fig. 2, I-Abz 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.
We verified that expression of Aaz and Abz mRNA in transgenic
mice was also associated with expression of I-Aaz and I-Abz surface proteins. The mAb recognizing I-Aaz cross-reacts with I-Aab
(present in the B10 transgenic lines) but not with I-Aad. We therefore studied (B10.Az 3 NZB)F1 3 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-Aaz and I-Abz. 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.
We also stained thymus tissue sections and analyzed the expression pattern of I-Az or I-Ab,d by immunohistochemistry. Sections
from B10.Az transgenic mice stained positively for expression of
I-Az in a pattern indistinguishable from NZW and B6.H2z mice (data
not shown). B6, B10, and B6.Ez mice were negative for thymic Az
expression. B10.Az mice also showed appropriate staining for I-Ab/d,
similar to B6, B10, and B6.Ez controls. In contrast, NZW and B6.H2z
mice were negative for expression of thymic I-Ab/d.
(B10.Az 3 NZB)F1 3 NZB and (B10.Azlo 3 NZB)F1 3 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-Abz 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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Serological assays
Analysis of I-Az expression in transgenic mice
2626
MHC CLASS II GENES IN MURINE LUPUS
Analysis of backcross mice for contribution of MHC genes to
nephritis
Previous studies have shown that (B6.H2z 3 NZB)F1 mice do not
develop severe lupus-like disease, but that a subset of (B6.H2z 3
NZB)F1 3 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 3 NZB)F1 3 NZB mice
and 61% of (B10.Az 3 NZB)F1 3 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 3 10213) or B6.Ez
( p , 7.0 3 10219) mice (Fig. 5A) (8, 25).
Whereas inheritance of the entire H2z locus significantly increased disease susceptibility in (B6.H2z 3 NZB)F1 3 NZB backcross mice, inheritance of the Az transgenes had no influence on
disease incidence in either Az backcross (Fig. 5B). 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 3 NZB)F1 3 NZB backcross mice ( p , 2.7 3 1024 and
p , 3.5 3 10211, respectively).
In the (B10.Az 3 NZB)F1 3 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 3 1024), with an odds ratio of 4.45.
Analysis of backcross mice for the contribution of MHC genes
to autoantibody production
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 antichromatin 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 3 NZB)F1 3 NZB and (B10.Azhi 3 NZB)F1 3
NZB mice in the amounts of any of the measured autoantibodies.
The relevance of these autoantibody studies for development of
disease is supported by the association of gp70 IC and antichromatin levels with severe proteinuria in the Az backcross mice.
Mice were segregated according to their levels of gp70 IC, defined
as high (.3.5 mg/ml), intermediate (0.5–3.5 mg/ml), or low (,0.5
mg/ml), and whether they had severe proteinuria (9, 25). Similarly,
mice were categorized by their IgG anti-chromatin level, defined
as high (.4.6 U/ml), intermediate (1.0 – 4.6 U/ml), or low (,1.0
U/ml). Using a 3 3 2 contingency table and x2 analysis, a strong
association with nephritis was found for gp70 IC (x2 5 16.6; p ,
2.5 3 1024) and less so for IgG anti-chromatin Abs (x2 5 7.9; p ,
0.02). Compared with healthy (B10.Az 3 NZB)F1 3 NZB mice,
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FIGURE 2. B cell surface expression of I-Abz and I-Abb/d in Az transgenic and control strains. The I-A expression patterns are shown for B2201 B cells,
after staining and analyzing by two-color immunofluorescence. Using the gates shown at the left, the percentages of I-Abb/d positive cells (mean channel
of fluorescence) were: B6.H2z, 2.2% (2.9); B10.Azlo, 84% (63); B10.Az, 96% (95); and B6, 99% (100). At the right (I-Abz), the percentages of positive
cells (mean channel of fluorescence) were: B6, 2.3% (1.7); B10.Azlo, 7.4% (3.5); B6.H2z, 95% (46); and B10.Az, 97% (109).
The Journal of Immunology
2627
age-matched (B10.Az 3 NZB)F1 3 NZB mice with severe proteinuria produced significantly greater amounts of autoantibodies
( p , 6.0 3 1024 for total IgG anti-chromatin, p , 2.0 3 1024 for
IgG2a anti-chromatin, p , 5 3 1024 for IgG3 anti-chromatin, and
p , 1.0 3 1024 for gp70 IC).
Discussion
The present studies were based on the hypothesis that class II
MHC genes underlie the contributions of particular MHC haplotypes to increased lupus susceptibility in New Zealand hybrid
mice. This hypothesis appeared to be supported by different lines
of reasoning and by multiple studies, including the required role
for both CD41 T cells and class II-bearing cells in (NZB 3
NZW)F1 mice (12, 13). The present work focused on the strong
contribution of H2z from the NZW parent to IgG autoantibody
production and nephritis in (NZB 3 NZW)F1 mice (1–9). Recent
experiments from our laboratory showed that Eaz and Ebz genes
did not influence disease development. The present studies
strongly suggest that Az genes are also not the sole basis for this
effect of MHC.
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 3 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-Aad/I-Abz and mixed isotype I-Ead/I-Abz
molecules do not explain the effect of H2z on disease in (NZB 3
NZW)F1 mice as previously suggested (42). Since H2d-encoded
molecules were present in the backcross animals, I-Abz should
have been equally likely to pair with I-Aad or I-Ead as in wildtype H2z backcross mice. This is consistent with a previous report
showing that an Abz transgene alone expressed in H2d homozygous
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FIGURE 3. Analysis of PBL from transgenic and control mice for expression of surface
I-Aaz and I-Abz. mAb 3F12 (anti-I-Aa) crossreacts with I-Aaz and I-Aab, but not I-Aad (31).
Therefore, (B10.Az 3 NZB)F1 3 NZB backcross mice were first selected for the absence of
H2b by genotyping. PBL from transgene-positive and transgene-negative mice, determined
by genotyping, were then double stained with
anti-I-Aaz and anti-I-Abz. The percentage of
positive cells in each quadrant is indicated.
2628
(NZB 3 NZW.H2d)F1 mice resulted in no greater autoantibody
production and nephritis than was found in (NZB 3 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-Aab or I-Abb
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
FIGURE 5. Development of lupus nephritis in backcross mice followed
for 1 yr. A, The frequency of disease in all backcross mice, regardless of
transgene inheritance, is shown. The B6.Ez and B6.H2z backcrosses were
followed in the same animal facility but mostly before the B10.Az backcrosses, and the data for the earlier backcrosses are taken from Ref. 25. The
numbers of backcross mice studied were: B10Azlo, 86; B10.Az, 59;
B6.H2z, 133; and B6.Ez, 77. B, The frequency of disease in backcross mice
that are separated on the basis of transgene or H2z inheritance. The numbers of backcross mice studied were: B10.Az all (Tg1), 77; B10.Az all
(Tg2), 68; B10.Azlo (Tg1), 49; B10.Azlo (Tg2), 37; B10.Az (Tg1), 28;
B10.Az (Tg2), 31; B6.H2z (H2z1), 78; and B6.H2z (H2z2), 55.
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.
Table I. Linkage of nephritis with MHC and transgene genotypes
Linkage
Genotype
% with
Nephritis
x
p value
O.R.a
H2b/d (vs H2d/d)
Az Tg1 (vs Tg2)
80 (vs 47)
63 (vs 61)
15.2
0.6
1.0 3 1024
0.44
4.45
0.78
2
O.R., odds ratio, for H2b/d vs H2d/d or Tg1 vs Tg2 calculated as:
[(No. diseased mice with gene) 3 (no. healthy mice without gene]/[(no. diseased
mice without gene) 3 (no. healthy mice with gene)].
a
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FIGURE 4. Analysis of genomic DNA for the presence of Aaz and Abz
genes. A, Genomic DNA was amplified with primers specific for Aaz. Ø (no
DNA) or DNA from NZB (H2d) and C57BL/10 (H2b) do not encode Aaz
genes and are negative. In contrast, NZW (H2z), B6.H2z, and transgenic
strains all carry Aaz genes. Backcross 1 refers to (B10.Az 3 NZB)F1 3
NZB backcross mice, and backcross 2 refers to (B10.Azlo 3 NZB)F1 3
NZB backcross mice. Samples from transgene-positive mice and transgene-negative mice are shown. These backcross mice also showed coordinate positive or negative expression, respectively, of splenic Aaz mRNA
and B cell surface I-Az. B, Genomic DNA was amplified with primers
specific for Ab that surround an Alu repeat sequence. The upper band
represents Abz fragments, whereas the lower band is present in other H2
types and is therefore also present in the Az transgenic animals. The lower
band is present but faint for the B10.Az strain, most likely related to the
increased number of Abz genes present in this strain. The DNAs used for
amplification are identical with those described in A.
MHC CLASS II GENES IN MURINE LUPUS
The Journal of Immunology
2629
Table II. Linkage of MHC loci with IgG autoantibody production in
(B10.Az 3 NZB)F1 3 NZB backcross mice
Maximum lod Score (p value)a
Autoantibody
H2b/d
Az
IgG anti-chromatin
IgG1 anti-chromatin
IgG2a anti-chromatin
IgG3 anti-chromatin
gp70 IC
6.8 (2.2 3 1028)
2.3 (0.001)
6.6 (3.5 3 1028)
2.0 (0.002)
8.6 (3.1 3 10210)
0.11
0.40
0.02
0.53
0.04
a
p values are shown for p , 0.01.
Acknowledgments
We thank Virginia Appel and Ellen Roper for technical assistance.
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Inheritance of H2b in our previous B6.Ez backcrosses (25) and
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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
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The linkage of H2b with IgG autoantibody production in the
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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,
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The differences in autoantibody production and incidence of nephritis in (B10.Az 3 NZB)F1 3 NZB mice compared with (B6.Ez
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