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Naturally occurring antibody response to DNA is associated with the response to retroviral gp70 in autoimmune New Zealand mice.

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242
NATURALLY OCCURRING ANTIBODY RESPONSE
TO DNA IS ASSOCIATED WITH THE RESPONSE
TO RETROVIRAL gp70 IN AUTOIMMUNE
NEW ZEALAND MICE
TOSHIKAZU SHIRAI, KIYOE OHTA, ATSUSHI KOHNO, FUKUMI FURUKAWA,
HARUYOSHI YOSHIDA, NAOKI MARUYAMA, and SACHIKO HIROSE
The spontaneous occurrence of retroviral gp70
immune complexes (ICs) in the blood of autoimmune
New Zealand black and (New Zealand black X New
Zealand white)F1 ([NZB X NZW]Fl) mice is determined
by a single dominant locus of the NZB strain (provisionally designated Agp-1). A combined effect of 2 unlinked
dominant NZB loci (Ads-1 and Ads-2) is required for the
production of anti-double-stranded DNA (anti-dsDNA)
antibodies in these mice. The present genetic studies
using (NZB x NZW)Fl x NZW backcross mice and
their second through fourth generation progeny revealed that Agp-1 and Ads-1 exist in common or are
closely linked on chromosome 17 of NZB mice and are
related to the occurrence of renal disease. The renal
disease and the serum level of both anti-dsDNA antibodFrom the Department of Pathology, Juntendo University
Scholol of Medicine, Tokyo, Japan, and the Department of Pathology, Faculty of Medicine, Kyoto University, Kyoto, Japan.
Supported by a grant for Intractable Diseases from the
Ministry of Health and Welfare and a grant for Cancer Research
from the Ministry of Education, Science and Culture, Tokyo, Japan.
Toshikazu Shirai, MD, PhD: Professor of Pathology,
Juntendo University School of Medicine; Kiyoe Ohta, PhD: Research Fellow, Department of Pathology, Faculty of Medicine,
Kyoto University (current address: Central Laboratories, National
Utano Hospital, Kyoto, Japan); Atsushi Kohno, MD, PhD: Research Fellow, Department of Pathology, Faculty of Medicine,
Kyot'o University; Fukurni Furukawa, MD, PhD: Assistant of
Dermatology, Faculty of Medicine, Kyoto University; Haruyoshi
Yoshida, MD, PhD: Assistant of Internal Medicine, Faculty of
Medi'cine, Kyoto University; Naoki Maruyama, MD, PhD: Assistant of Pathology, Faculty of Medicine, Kyoto University (currently
Director, Department of Pathology, Chiba Rosai Hospital, lchihara,
Japan); Sachiko Hirose, MD, PhD: Instructor of Pathology,
Juntendo University School of Medicine.
Address reprint requests to Toshikazu Shirai, MD, PhD,
Department of Pathology, Juntendo University School of Medicine,
2-1-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.
Submitted for publication March 18, 1985; accepted in
revised form July 19, 1985.
Arthritis and Rheumatism, Vol. 29, No. 2 (February 1986)
ies and gp70 ICs were more intense in (NZB x NZW)Fl
hybrids than in NZB mice, indicating the contribution of
NZW genes. In (NZB X NZW)FI mice, a single dominant locus from the NZW strain, Agp-3, intensified the
magnitude of gp70 IC formation, and a combined effect
of 2 unlinked dominant loci from the NZW strain, Ads-3
and Ads-4, acted to increase the serum titers of antidsDNA antibodies. This study clearly indicates that the
NZW loci Agp-3 and Ads-3 exist in common or are
tightly linked on chromosome 17, are closely linked to
the H-2 complex, and are associated with the severity of
renal disease in (NZB x NZW)Fl hybrids. Thus, it is
likely that the abnormalities involved in the antibody
response to gp70 are in part genetically related to the
production of autoantibodies to dsDNA as well as to the
occurrence of renal disease in NZB and (NZB X
NZW)F1 mice.
Viruses have been associated with a wide variety of immunologic abnormalities, including autoimmune diseases, in humans and animals (1). In New
Zealand black mice and in (New Zealand black X New
Zealand white)F1([(NZB X NZWIF,) mice, which are
characterized by the spontaneous occurrence of
autoimmune diseases associated with an immune complex (1C)-type glomerulonephritis resembling human
lupus nephritis, Yoshiki et a1 (2) found evidence that
the major glycoprotein constituent of the endogenous
retroviral envelope, gp70, was deposited, apparently
as an IC, in the renal glomeruli and vascular walls. The
sera from these mice contained significant amounts of
gp70-anti-gp70 ICs (3-5). Genetic studies revealed
that the appearance and the magnitude of gp70 ICs in
the serum were highly correlated with the renal disease in these mice (43). Since these New Zealand
243
gp70 AND ANTI-dsDNA IN MICE
strains carry a high serum level of free gp70, the
formation of gp70 ICs must reflect the production of
antibodies to gp70.
These New Zealand mice are also known to
produce autoantibodies to DNA. Our genetic studies
have shown that the appearance of antibodies to
double-stranded DNA (dsDNA) correlated highly with
the renal disease (6,7). Lambert and Dixon (8) demonstrated a significant concentration of antibodies to
DNA in the renal eluates in (NZB X NZW)FI mice,
thereby suggesting the pathologic importance of
DNA-anti-DNA immune complexes for the glomerular lesion.
Since the appearance of each renal disease
(5,9-11) and of naturally occurring antibody responses
to DNA (6,7,12) and of retroviral gp70 (43)are genetically determined in NZB and (NZB X NZW)FI hybrid
mice, the above findings suggest that some common
genetic mechanisms are operative among these 3 traits
(13). Correlation among these 3 traits is further suggested by 2 additional findings. First, all 3 traits are
more extensive in the (NZB x NZW)FI hybrid than in
the parental NZB strain, with involvement of NZW
gene actions which intensify the expression of autoimmune NZB predisposing genes responsible for these
traits (2,5,6,9,14). Second, the inheritance of the NZB
gene action for each of these traits in (NZB x NZW)FI
hybrids is significantly associated with the inheritance
of the H-2d haplotype, and the inheritance of the NZW
gene action for the intensification of all 3 traits is
associated with the inheritance of the H-2' haplotype
(4-6,%11,15).
We now report details of genetic studies which
suggest that both NZB and NZW strains contribute
common genes or clusters of tightly linked genes to the
naturally occurring antibody responses to both
retroviral gp70 and DNA, both of which, in turn, play
a role in the pathogenesis of renal disease in (NZB X
NZW)F1 hybrid mice.
MATERIALS AND METHODS
Mice. NZB and NZW mice were obtained from
colonies bred at our institution. Female (NZB x NZW)FI
hybrid mice were backcrossed to either NZW or NZB mice.
The second through fourth generations of backcrossed mice
were also used. To avoid the influence of sex hormones, only
female mice were used.
Measurement of anti-dsDNA antibodies. Measurement of antibodies to dsDNA was carried out by modified
F a r r a s s a y and Crithidia luciliae kinetoplast im-
munofluorescence (KIF) test (16), as previously described
(6,7). The results of the Farr assay were expressed as a
percentage of the 14C-dsDNA precipitated, and dsDNA
binding activity of 10% or more was regarded as positive.
Mouse sera that were positive on the KIF test at a dilution of
1: 10 or more were regarded as positive; thus, the heatinactivated sera were tested neat, at a 1:10 dilution and at
twofold dilutions beyond. The KIF titer was expressed as
the last tube number giving a positive KIF.
Measurement of retroviral gp70 and gp70 ICs.
Glycoproteins in the sera from NZB, NZW, and (NZB x
NZW)FI mice were first collected by using concanavalin
A-Sepharose affinity chromatography. Serum retroviral
gp70 was then isolated by immunosorbent column, prepared
by the covalent linkage of the IgG fractions of goat antiRauscher gp70 antiserum (kindly provided by Dr. J. c. Cole,
111, Division of Cancer Cause and Prevention, National
Cancer Institute, Bethesda, MD) to cyanogen bromideactivated Sepharose 4B (Pharmacia, Uppsala, Sweden).
Testing for gp70 activities in the samples was carried out by
inhibition radioimmunoassay, as previously described (5).
As controls, medium alone instead of test sample (Ab
control) and normal goat serum (NGS control) instead of
goat anti-Rauscher gp70 serum were used. Percent inhibition
was calculated as:
counts per minute of test sample - cpm of NGS control
1 =-
cpm of Ab control - cpm of NGS control
x 100
The circulating retroviral gp70 ICs were examined by
the polyethylene glycol (PEG) precipitation method (5). The
amount of serum gp70 ICs in the 3% PEG precipitates was
estimated by measuring the gp70 activity using the inhibition
radioimmunoassay, and was expressed as unitshl, according to the standard curve obtained by using pooled NZB
mouse serum containing 1,OOO units/ml (approximately 100
pglml) of free gp70 activity. Designation of positive or
negative gp70 ICs was based on the criterion that levels of
serum gp70 IC >17.8 unitshl, a value equal to 2 standard
deviations above the mean level in 10-month-old NZW mice
(9,were positive.
H-2 typing. H-2 typing was carried out by 2-stage dye
exclusion cytotoxicity testing, using peripheral blood lymphocytes as target cells. Anti-H-2d antibodies for typing the
H-2d antigen of the NZB strain were produced by immunizing B1O.BR mice (H-2k) with spleen cells from B10.D2 mice
(H-2d), and the antisera were absorbed with a mixture of
spleen cells and thymocytes from NZW mice (H-2').
Anti-H-2' antisera were obtained in the same manner, by
immunizing NZB mice with spleen cells from NZW mice,
and they were absorbed with NZB cells.
Assessment of proteinuria. The onset of renal disease
was monitored by biweekly measurement of urinary protein
(11). Mice with a level of 2 111 mg/100 ml were considered to
have the disease.
Statistical analysis. Statistical analyses were performed using chi-square, Yates' chi-square, and Student's
t-tests.
244
SHIRAI ET AL
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dsDNA binding ( O h )
dsDNA binding ( O h )
Figure 1. Association between the inheritance of positive serum gp70 immune complexes (IC) and
anti-double-stranded DNA (anti-dsDNA) antibodies in (New Zealand black x New Zealand white)F, X New
Zealand white backcross mice (A), and in the second through fourth generations of the backcross mice (B),
at 10 months of age.
serum gp70 ICs (2= 24.15, P < 0.001). Table 1 shows
that the inheritance of gp70 1Cs and the inheritance of
anti-dsDNA antibodies in these (NZB x NZW)FI x
NZW backcross mice were significantly associated
with the H-2d haplotype of the NZB strain.
For confirmation, we mated NZW mice with
(NZB X NZW)Fl X NZW backcross mice that were
positive for gp70 ICs, anti-dsDNA antibodies, and
H-2d haplotype, to produce the second generation of
backcrosses (BC2). This procedure of backcrossing
was repeated through the fourth generation (BC4). In
these 72 backcross mice, 24 (33.3%) were positive for
RESULTS
Associations between serum gp70 immune complexes and anti-dsDNA antibodies. We examined the
serum levels of both gp70 ICs and dsDNA binding
activities, as measured by Farr assay, in 156 (NZB x
NZW)F, x NZW backcross mice at 10 months of age.
As shown in Figure l A , positive anti-dsDNA antibodies were observed in 50 (32.1%) and positive gp70 ICs
in 77 (49.4%) of the backcross mice. A preponderance
of the backcross mice that were positive for the
anti-dlsDNA antibodies were also positive for the
Table 1. Association between the H-2" haplotype of the New Zealand black mouse strain and the
traits anti-double-stranded DNA (anti-dsDNA) antibodies and retroviral gp70 immune complexes
(ICs) in (New Zealand black x New Zealand white)F, X New Zealand white backcross ([NZB x
NZWIF, X NZW BC,) mice and in their progeny (BC2-BC4) at 10 months of age
No. positive
Strain and trait
(NZB X NZW)FI
Anti-dsDNA
gp70 ICs
BCz-BC4
Anti-dsDNA
gp70 ICs
X
H-2"/'
H-2"'
No. negative
H-2d'"
H-2'/'
Total
xz
P
NZW BCI
33
56
17
21
41
18
65
61
156
156
10.2
39.0
<0.001
<0.001
16
27
8
10
17
6
31
29
72
72
6.3
22.6
<0.001
<0.025
gp70 AND ANTI-dsDNA IN MICE
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IgM anti-dsDNA(KIF titer)
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4
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A
.... .......................................
0
1
2
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3
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lgGz anti-dsDNA(KIF titer)
Figure 2. Association between the inheritance of a high serum level of gp70 immune complexes (IC) and
IgG-class anti-double-stranded DNA (anti-dsDNA) antibodies in (New Zealand black X New Zealand
white)F, x New Zealand black backcross mice at 10 months of age, by kinetoplast immunofluorescence
(KIF). A, IgM-class; B, IgGl-class; C, IgG2-class anti-dsDNA antibodies.
anti-dsDNA antibodies and 37 (51.4%) were positive
for gp70 ICs, at 10 months of age (Figure 1B). The
incidence of the H-2d haplotype was 45.8% (33 of 72
mice). The incidence of all 3 traits in these mice was
comparable with the incidence in the first generation of
the backcross mice (BC,).
There was a highly significant association between the occurrence of gp70 ICs and anti-dsDNA
antibodies (2 = 34.05, P < 0.001). All the mice with
positive anti-dsDNA antibodies had positive gp70 ICs.
As shown in Table 1, the inheritance of both gp70 ICs
and anti-dsDNA antibodies was also associated with
the inheritance of H-2d haplotype.
The incidence of anti-dsDNA antibodies in the
parental (NZB x NZW)F, mice at 10 months of age
was 83.3% (25 of 30 mice), and 100% of these mice had
gp70 ICs. Among the NZW mice, 8.7% (2 of 23) had
anti-dsDNA antibodies and 4.5% (1 of 22) had gp70
ICs. Taking into account these incidences in the
parental mice, the 32.1% incidence of anti-dsDNA
antibodies in 156 (NZB x NZW)FI x NZW backcross
mice at 10 months of age was statistically consistent
with the idea that a combined effect of 2 unlinked
dominant loci of the NZB strain, Ads-1 and Ads-2,
determines the production of the antibodies (2= I .62,
P > 0.2). The same was true for the 72 backcross mice
of BC2 through BC4 (2 = 1.23, P > 0.2). Statistical
analyses also suggested that a single dominant locus of
NZB mice, Agp-1 , determines the appearance of gp70
ICs (2= 0.62, P > 0.3 for [NZB x NZW]FI x NZW
backcross mice, and ,$ = 0.04, P > 0.8 for the
backcross mice of BC2 through BC3. All these findings suggest that the Ads-1 and Agp-1 loci of the NZB
strain occur in common or are tightly linked, and are
located on chromosome 17.
Relationships between serum levels of gp70 immune complexes and IgG-class anti-dsDNA antibodies.
In studies using (NZB x NZW)FI x NZB backcross
mice, we previously found that a single dominant locus
of the NZW strain, Agp-3, acts to increase the serum
levels of gp70 ICs in these mice (9, and that a
combined effect of 2 unlinked dominant loci of the
NZW strain, Ads-3 and Ads-4, leads to an increase in
the serum anti-dsDNA antibody titer (7). The latter
effect of NZW genes is attributed to the gene action
which leads to conversion of the class of antibodies
from IgM of the NZB type to IgG of the (NZB x
NZW)F I type.
The findings in our study were in agreement
with the above observations. The incidence of high
serum levels of gp70 ICs (defined as >82.4 unitdml, a
value equal to 2 standard deviations above the mean
level in 10-month-old NZB mice) was 54.5% (48 of 88)
in 10-month-old (NZB x NZW)FI x NZB backcross
mice, while the incidence in (NZB X NZW)FI mice
was 90.6% (29 of 32 mice) and that in NZB mice
was 0% (0 of 25 mice). These incidences are consistent with the notion that a single dominant NZW
locus controls the serum level of gp70 ICs (2= 3.00,
P > 0.05).
As shown in Figure 2, the incidence of positive
IgG1 anti-dsDNA antibodies, as measured by the KIF
test, was 21.6% (19 of 88) in (NZB x NZW)F, x NZB
backcross mice. It was 84.6% (22 of 26) in (NZB x
NZW)FI hybrid mice and 7.1% (2 of 28) in NZB mice.
The incidence of IgG2 antibodies was 26.1% (23 of 88)
SHIRAI ET AL
246
TaMe 2. Association between the H-2' haplotype of the New
Zealand white mouse strain and the inheritance of IgG-class antidouble-stranded DNA (anti-dsDNA) antibodies and high serum
level of gp70 immune complexes (ICs) in (New Zealand black x
New Zealand white)F, x New Zealand black backcross mice at 10
months of age
No. positive
Trait
No. negative
H-2d/Z H-2d'd H-2d/' H-2d'd Total
x2
P
Anti-dsDN A
I@ 1
162
IgM
15
17
28
4
6
29
25
23
12
44
42
19
88
88
88
11.0 <0.001
10.2 C0.005
0.9 >0.3
High gp70 ICs
28
20
12
28
88
7.1 <0.01
in (NZB x NZW)FI x NZB backcross mice (Figure
2). It was 84.6% (22 of 26) in (NZB X NZW)FI hybrid
mice and 0% (0 of 28) in NZB mice. These findings are
in keeping with the idea that a combined effect of 2
unlinked dominant NZW loci leads, in concert with the
corresponding NZB loci, to the production of both
IgG1 (2= 1.08, P > 0.2) and IgG2 (2= 1.31, P > 0.2)
anti-dlsDNA antibodies in (NZB X NZW)FI hybrid
mice.
As shown in Figure 2, significant associations
existed between the occurrence of IgG-class (but not
of IgM-class) anti-dsDNA antibodies and high serum
levels of gp70 ICs in (NZB X NZW)FI X NZB
backcross mice (2= 0.17, P > 0.5 for IgM, 2 = 5.82,
P < 0.02 for IgG1, and 2 = 13.19, P < 0.001 for IgG2).
Linkage studies revealed that both the inheritance of
high serum gp70 IC levels and the inheritance of
IgG-class anti-dsDNA antibodies were significantly
associated with the H-2" haplotype of the NZW strain
in (NZB x NZW)FI x NZB backcross mice (Table 2).
Correlations between proteinuria and the presence of serum gp70 immune complexes and anti-dsDNA
antibodies. Figure 3A shows a comparison of the
age-associated incidences of proteinuria among the 4
groups of (NZB x NZW)FI x NZW backcross mice:
(a) mice that were positive for both serum gp70 ICs
and anti-dsDNA antibodies; (b) mice that were positive for gp70 ICs only: (c) mice that were positive for
anti-dsDNA antibodies only; and (d) mice that were
negative for both traits. As shown, the 2 groups of
progeny with positive gp70 ICs (groups a and b)
showed an accelerated onset and high incidence of
proteinuria, in marked contrast with the 2 groups with
negative gp70 ICs. These differences in incidence were
significant in mice over 10 months of age. As also
shown in Figure 3A, 50% of the mice in group a were
positive for proteinuria at 10.8 f 0.35 months of age,
and 50% of the group b animals were positive at 12.5
0.57 months. This difference was statistically significant ( t = 2.56, P < 0.02).
Almost the same findings were observed in the
BC2 through BC4 progeny of (NZB X NZW)F1 X
NZW backcross mice (Figure 3B). The differences in
the age-associated incidence of proteinuria between
groups a and b were more apparent, and the differences in the mean age at which 50% incidence occurred were statistically significant (8.8 k 0.92 months
in group a , 12.6 +- 0.69 months in group b; r = 2.81,
P < 0.01).
Figure 4 shows the age-associated incidence of
proteinuria in the 4 groups of (NZB X NZW)Fl X NZB
backcross mice: (a) mice with both a high serum level
of gp70 ICs and IgG2-class anti-dsDNA antibodies; (b)
mice with a high serum level of gp70 ICs and negative
IgG2-class anti-dsDNA antibodies; (c) mice with IgG2class anti-dsDNA antibodies and a low serum level of
gp70 ICs; and (d) mice with a low serum level of gp70
1Cs and negative IgG2-class anti-dsDNA antibodies.
Among these 4 groups, the earliest onset and the
highest incidence of proteinuria occurred in group a, in
marked contrast with group d. Compared with group
a, group b showed a lower incidence of proteinuria
throughout the ages tested, and the difference between
groups a and b in mean age at which 50% incidence
occurred was significant (7.8 -+ 0.57 months versus
11.0 2 1.47 months; t = 2.51, P < 0.02).
DISCUSSION
We have previously reported that a combined
effect of 2 unlinked dominant loci (provisionally designated Ads-1 and Ads-2) is required for the production of anti-dsDNA antibodies in NZB mice and their
progeny (6,7). Other studies revealed that a single
dominant locus of the NZB strain, Agp-1, determines
the occurrence of circulating retroviral gp70 ICs, and
that this effect represents the production of antibodies
to gp70 (5). In the present study, we found that the
majority of the (NZB X NZW)F1 x NZW backcross
mice with positive anti-dsDNA antibodies also had
serum gp70 ICs, indicating the close linkage between
Ads-I and Agp-1 .
Roughly one-fourth of the backcross mice were
positive for gp70 ICs only. This finding can be attributed to the presence of the Agp-1-Ads-1 complex and
the absence of Ads-2 in these mice. To confirm this
linkage, we selected the (NZB x NZW)F, X NZW
gp70 AND ANTI-dsDNA IN MICE
247
B
A
100.
A
A
(391
'gp70 IC (+),Anti-ds DNA?+)
:gp70 IC (+), Anti-ds DNA (-1
;gp70 lC(-1. Anti-ds DNA (-)
/
80.
(13 1
(24)
(38) 60
E
u
E
//
40
20
0 41
(351
I
I
6
8
Age (months)
I
14
10
12
Age (months
16
Figure 3. Cumulative incidence of proteinuria in (New Zealand black x New Zealand white)F1 x New
Zealand white backcross mice (A), and in the second through fourth generations of backcross mice (B),by
positivity or negativity for gp70 immune complexes (IC) and anti-double-stranded DNA (anti-dsDNA)
antibodies. Horizontal bars represent mean 2 SE age at which 50% incidence of proteinuria occurred. Values
in parentheses are number of progeny tested.
backcross progeny (BC1) that were positive for both
traits and mated them with NZW mice (BC2); this
procedure of backcrossing was repeated through the
fourth BC generation. This procedure eliminates the
possible involvement of minor genes which may to
some extent influence the disease phenotypes of BCI
progeny. Further, if the observations in BC1 mice are
attributed to chance, this procedure of backcrossing
may result in changes in inheritance patterns of the
traits and would thereby dissociate the two traits
under investigation. However, this was not the case in
the present study. Actually, there was no recombinant
progeny positive for anti-dsDNA antibodies and negative for serum gp70 ICs. Thus, it may be possible that
Ads-1 is Agp-1 per se. The association of the 2
above-mentioned traits with the H-2d haplotype in the
BCI as well as in BC2 through BC4 mice strongly
suggests that the Agp-1-Ads-1 complex is located on
chromosome 17 of the NZB strain and is, to some
extent, linked to the H-2 complex.
The present study also revealed that a single
dominant locus of the NZW strain (Agp-3), which acts
to intensify the magnitude of anti-gp70 antibody production, may exist in common with or be tightly linked
to Ads-3, which in concert with Ads-4, modifies the
expression of NZB Ads-1 and Ads-2. This modification represents the conversion of anti-dsDNA antibodies from IgM-class in NZB mice to IgG-class in (NZB
.;gp70
A; gp70
o;gp70
.;gp70
'"1
-
IC high. 1962 anti-dsDNA (4-1
IC high. 1962 anti-dsDNA (-1
IC low 1962 anti-dsDNA (+)
IClow , 1962 anti-dsDNA I-)
I
/
(19)
I
0
6
8
10
12
14
16
Age (months)
Figure 4. Cumulative incidence of proteinuria in (New Zealand
black x New Zealand white)F, x New Zealand black backcross
mice, by high or low serum level of gp70 immune complexes (IC)
and positivity or negativity for IgG2-class anti-double-stranded
DNA (anti-dsDNA) antibodies. Horizontal bars represent mean ?
SE age: at which 50% incidence of proteinuria occurred. Values in
parentheses are number of progeny tested.
248
x NZW)FI hybrid mice. The combination of Ads-1
and Ads-2 in NZB mice acts to produce only IgM-class
anti-cisDNA antibodies, while in the (NZB x NZW)FI
hybrid, a combined effect of Ads-3 and Ads-4 from the
NZPJ strain leads to the production of IgG-class
anti-&DNA antibodies (7).
The linkage of both Agp-3 and Ads-3 with the
H-2 complex of the NZW strain suggests that the
Agp-3-Ads-3 complex is located on chromosome 17.
Recently, we developed an NZW congenic line carrying the H-2d haplotype (ZWD/8), prepared a H-2d/H-2d
homozygous (NZB x ZWD/8)FI hybrid, and then
compared the autoimmune traits with those of H-2d/H2'heterozygous (NZB X NZW)FI hybrid mice (15). In
comparison with (NZB X NZW)FI hybrid mice, (NZB
x ZWD/8)FI hybrid mice showed markedly lower
serum levels of both anti-dsDNA antibodies and gp70
ICs. The decrease in the titer of anti-dsDNA antibodies was the result of decreases in IgG, but not IgM,
antibodies. These observations are consistent with
findings in the present study, and may imply that
Agp-3 is Ads-3 per se, and is located within or very
close to the NZW H-2 complex.
Braverman (17) crossed NZB and NZW mice,
exanlined the progeny for histologic changes of the
renal1 glomeruli, and observed renal disease of varying
severity in the Fz mice. These findings suggested that
the irenal disease of these mice is the result of a
complex phenotype. Nonetheless, using proteinuria as
a disease marker, genetic studies by Knight and coworkers (9,lO) as well as by us (5,Il) suggested that
the renal disease is largely controlled by rather limited
numlbers of major genes, with many more minor genes
possibly involved.
The data on (NZB x NZW)FI X NZW backcros!j mice were consistent with the idea that a single
domiinant locus or a cluster of closely linked loci of the
NZB, strain determines the development of renal disease. In the (NZB X NZW)FI hybrid, the disease is, to
a great degree, intensified either by the effect of a
single dominant locus, as we have previously reported
(3,
or by a combined effect of 2 unlinked dominant
loci derived from the NZW strain, as reported by
Knig,ht and Adams (9). The difference in the results of
these 2 studies may be due to the difference in the
criteria employed for the definition of severe renal
disease. In any case, the present studies strongly
suggest that the main effect of the NZB locus on the
pathogenesis of renal disease is the result of Agp-1
gene action, and the effect of the NZW locus is the
result of Agp-3 gene action.
SHIRAI ET AL
The question arises as to whether the significant
association between the inheritance of anti-dsDNA
antibodies and proteinuria in the crosses of NZB and
NZW mice is merely due to gene linkages between
Agp-1 and Ads-1 and between Agp-3 and Ads-3.
Actually, proteinuria developed in a considerable
number of the backcross progeny that were positive
for gp70 ICs but lacked the anti-dsDNA antibodies. In
the present study, however, we found that in (NZB x
NZW)Fl X NZW backcross mice with both antidsDNA antibodies and serum gp70 ICs, there was an
accelerated onset and an increased incidence of renal
disease, compared with findings in mice with serum
gp70 ICs alone. Thus, in addition to the major role of
gp70 ICs in the pathogenesis of renal disease, the
anti-dsDNA antibodies also appear to play a role in
promotion of glomerular disease.
This assumption was supported by additional
findings that even in the (NZB x NZW)Fl x NZB
backcross mice with high serum levels of gp70 ICs, the
progeny that also had IgG-class anti-dsDNA antibodies showed an earlier onset and a higher incidence of
proteinuria, compared with those without anti-dsDNA
antibodies. Therefore, it appears that in addition to the
effect of Agp-3, the combined effect of NZW Ads-3
and Ads-4 further leads to the promotion of renal
disease in (NZB x NZW)FI hybrids. All these findings
taken together suggest that the abnormalities involved
in the antibody response to retroviral gp70 are related
in part to the production of autoantibodies to dsDNA,
as well as to the occurrence of lupus nephritis in NZB
and (NZB x NZW)Fl hybrid mice.
Using recombinant inbred lines derived from
crosses of NIH Swiss and NZB strains, Miller et a1
(18) recently suggested that one family of NZB genes
leads to production of antibodies against murine leukemia virus (MuLV), single-stranded DNA (ssDNA),
and renal disease, as well as autoantibodies to erythrocytes. Although the antigens and the assay systems
used in that study were different from those used in the
present study, the association of anti-MuLV, antissDNA antibodies, and renal disease may be at least
partly related to the present findings. However, the
association of these 2 traits with anti-erythrocyte
autoantibodies contrasts with the results from our
progeny studies, as discussed below.
The importance of gp70 ICs in the pathogenesis
of renal disease has also been demonstrated in other
murine models of systemic lupus erythematosus and
periarteritis nodosa, using MRL/lpr, BXSB, and
SL/Ni mice (3,19). Dixon and coworkers (20) reported
gp70 AND ANTI-dsDNA IN MICE
strong associations among serum gp70 ICs, anti-DNA
antibodies, and survival time in the F2 progeny of
crosses of MRLApr and BXSB mice. Therefore, genetic mechanisms which are operative in New Zealand
mice may be at least partly operative eveti in MRLflpr
andlor BXSB mice.
Several genetic loci have been associated with
other autoimmune disease features in New Zealand
mice (see ref. 21). Among these, the main NZB and
NZW loci related to the production of erythrocyte
autoantibodies (Aia-1 and Aem-1) (22-24) and IgM
hypergammaglobulinemia (Imh- 1 and Imm-1) (25) are
located on chromosome 4 and thus are apparently
distinct from the Agp-1-Ads-1 complex of the NZB
strain and from the Agp-3-Ads-3 complex of the NZW
strain. On the other hand, we recently obtained evidence that a single dominant locus (Lbt-l) of the NZB
strain is responsible for the appearance of immunoglobulin deposition at the dermal-epidermal
junction in NZB and (NZB x NZW)Fl hybrid mice
(26). This skin lesion was shown to be significantly
associated wih the inheritance of H-2d antigen, renal
disease, and anti-dsDNA antibodies in (NZB x
NZW)FI x NZW backcross mice, indicating the linkage between Lbt-1 and the Agp-I-Ads-1 complex on
chromosome 17 of the NZB strain.
The viral pathogenesis of immunologic abnormalities is a complex process involving a wide variety
of biologic activities of viruses including the cytopathic effect, induction of polyclonal activatiori of lymphocytes, transformation of lymphocytes, formation of
virus-directed antigens, and derepression of certain
developmental antigens. Whether such effects of viruses can become manifest depends largely on the
genetic susceptibility of the host to the virus infection.
On the other hand, there is strong evidence that the
association of such viral susceptibility with immune
responsiveness to the virus-directed antigens in a
single host results in the development of autoimrhune
disease. Kuzumaki et a1 (27) induced autoimmune
disease by injecting the lymphatic leukemia virus of
the Friend retrovirus complex into newborn rats; they
subsequently found that with age, these rats developed
autoimmune hemolytic anemia associated with immune complex-type glomerulonephritis. Although it is
as yet unknown whether the same pathogenic mechanisms underlie the autoimmune disease of New
Zealand mice, the results of the present study strongly
suggest that the genetically determined immune responsiveness to the retrovirus, in association with an
intense expression of a retroviral antigen in NZB and
249
(NZB x NZW)F1 mice, is at least partly related to the
process of formation of antibodies to dsDNA in these
mice.
ACKNOWLEDGMENTS
We thank Professor Y. Hamashima, Department of
Pathology, Faculty of Medicine, Kyoto University, for support and advice, C. Katagishi and M. Nagayasu for expert
technical assistance, M. Mcirita for secretarial support, and
M. Ohara, Kyushu University, Fukuoka, Japan, for critical
reading of the manuscript.
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