The MHC Haplotype H2b Converts Two
Pure Nonlupus Mouse Strains to Producers of
Antinuclear Antibodies
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J Immunol 2009; 183:3542-3550; Prepublished online 5
August 2009;
doi: 10.4049/jimmunol.0900579
http://www.jimmunol.org/content/183/5/3542
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References
Kristian Hannestad and Helge Scott
The Journal of Immunology
The MHC Haplotype H2b Converts Two Pure Nonlupus
Mouse Strains to Producers of Antinuclear Antibodies1
Kristian Hannestad2* and Helge Scott†
T
he classical spontaneous mouse lupus models are the New
Zealand Black (NZB),3 New Zealand White (NZW), and
their inbred recombinant NZM2410 strains, males of the
BXSB strain, and the MRL/lpr strain. Usually, Abs to chromatin
(nucleosomes) appear first (1, 2), followed by hypergammaglobulinemia, splenomegaly, and fatal glomerulonephritis mediated at
least in part by the deposition of harmful immune complexes (IC)
containing antinuclear Abs (ANA) typically directed to intact
chromatin, dsDNA, and ssDNA (3). By activating the CpG DNAbinding TLR9 in B cells, DNA of chromatin acts as a built-in
autoadjuvant that, in combination with BCR signals, recruits and
expands chromatin-specific B cells during the autoimmune response (reviewed in Ref. 4). This dual engagement of BCR and
TLR9 likely plays a critical, but not necessarily sufficient, role for
the initiation and predominance of chromatin-reactive Abs in the
early stage of mouse lupus.
Many genes acting together cause mouse lupus, complicating
the task of sorting out those genes that have a major impact on
autoimmunity to chromatin. The complexity can be reduced by
breeding small chromosomal intervals, identified in advance by
genetic mapping of lupus traits, onto the nonlupus-prone C57BL/6
(B6) background. In this way it was shown that intervals located
on distal chromosome 1 such as Sle1 and its high penetrance sublocus Sle1b from NZW, Nba2 from NZB, and Bxs1– 4 from BXSB
*Institute of Immunology and †Institute of Pathology, University of Oslo and Rikshospitalet University Hospital, Oslo, Norway
Received for publication February 20, 2009. Accepted for publication July 5, 2009.
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 a grant from Helse Sør (to K.H.).
2
Address correspondence and reprint requests to Dr. Kristian Hannestad, Institute for
Immunology, Rikshospitalet, Sognsvannsveien 20, N-0027 Oslo, Norway. E-mail address: [email protected]
3
Abbreviations used in this paper: NZB, New Zealand Black; AHGG, aggregated
human IgG; ANA, antinuclear autoantibody; AP, alkaline phosphatase; cM, centimorgan; IC, immune complex; NZW, New Zealand White; p-NPP, p-nitrophenyl
phosphate.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900579
mice are sufficient to induce production of ANA (reviewed in Refs.
5 and 6). The Sle1b interval contains a number of polymorphic
candidate genes (7), among which the Ly108 gene has been considered a strong candidate for mediating the Sle1b phenotype (8).
Another lupus susceptibility locus is the MHC of both mice
(known as H2) and humans (HLA) that contains numerous genes
including the polymorphic MHC class I and II genes (reviewed
in Refs. 9 –11). The H2 region of the NZW strain also contains
the Sles1 lupus-resistance locus, which is located in an interval
that also contains the class II genes (12). From the standpoint of
the current report, relevant past studies of B6/lpr, BXSB, and
NZB.H2bm12 lupus mice have shown that the MHC H2b and
H2bm12 haplotypes are associated with more severe lupus serology
and nephritis when compared with the H2d and H2k haplotypes
(13–17). Because the Ea gene of the H2b locus is defective, expression of the I-E ␣-chain (E␣-chain) and functional I-E heterodimers are absent in H2b mice (18). Furthermore, a peptide
derived from the E␣-chain, E␣52– 68, binds to I-Ab molecules
with high affinity, so that in mice expressing both I-Ab and I-Eb
this self-peptide is present in ⬃12% of I-Ab molecules (19). Analyses of Ea-transgenic BXSB and related lupus mice have provided
evidence that the E␣-chain inhibits lupus autoimmunity but not
immune responses against several foreign Ags. Thus, transgeneencoded E␣-chains suppress serological and renal manifestations
of lupus and prolong survival, most pronounced for mice that carry
an unphysiologically high number (50 –100) of transgene copies
(20). These data have led to the proposal that the E␣52– 68 selfpeptide (which is missing in H2b mice) partially blocks I-A molecules of B cells from presenting putative pathogenic self-peptides
to Th cells, resulting in the lowering of autoantibody levels (10,
20). According to this hypothesis, production of ANA depends on
Th cells, and E␣ is a lupus resistance gene.
The H2b haplotype by itself, in the context of pure nonautoimmune backgrounds, has to date not been associated with loss of
tolerance to chromatin (21). In this study we demonstrate that,
upon aging, H2b/b (called H2b here) haplotype-congenic BALB/c
and C3H mice spontaneously produce anti-chromatin Abs dominated by the IgG2a isotype. These findings indicate that the H2b
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Studies of mouse lupus models have linked the MHC H2b haplotype with the earlier appearance of antinuclear autoantibodies and
the worsening of nephritis. However, it is unknown whether H2b by itself, in the context of pure nonlupus strains, is “silent” or
sufficient with regard to loss of tolerance to chromatin (nucleosomes). In this study we show that, beginning ⬃6 –9 mo of age,
H2b-congenic BALB/c (denoted BALB.B) mice, unlike BALB/c (H2d) and H2k-congenic BALB/c (denoted BALB.K) mice, develop
strikingly increased serum levels of anti-chromatin Ab dominated by the IgG2a subclass, along with minor increase of Abs to DNA
and moderately increased total serum IgG2a. The BALB.B mice did not have glomerulonephritis or an increased mortality rate.
H2b-congenic C3H/He mice (designated C3.SW mice), unlike C3H/He (H2k) mice, showed low but measurable serum levels of
chromatin-reactive IgG2a Abs and minor but significant hypergammaglobulinemia. By immunofluorescence, IgG2a of sera from
both H2b-congenic strains stained HEp-2 cell nuclei, confirming the presence of antinuclear autoantibodies. Thus, in the context
of two pure nonlupus genomes, the MHC H2b haplotype in homozygous form is sufficient to induce loss of tolerance to
chromatin. The Journal of Immunology, 2009, 183: 3542–3550.
The Journal of Immunology
haplotype contains one or more genes that play a major role in
inducing ANA production.
Materials and Methods
Mice
Preparation of soluble chromatin
To prepare nucleosome core particles, chicken erythrocytes were lysed
and the nuclei were washed and digested with micrococcal nuclease
(Worthington Biochemical) as described by Ausio et al. (23). We used
100 U of enzyme per 100 OD260 U of DNA for 15 min at 37°C. The
digestion was terminated by adding EDTA to a final concentration of 10
mM, the suspension was spun down at 12,000⫻ g in a SS-34 rotor at
4°C for 5 min, and the supernatant was collected, aliquoted, and stored
at ⫺70°C. The preparation contained a single broad DNA band of ⬃150
bp, and electrophoresis in 18% polyacrylamide gel (SDS-PAGE) revealed the characteristic band pattern of the four core histones H2A,
H2B, H3, and H4 (data not shown). The same preparation was used
throughout the study.
ELISA for specific Abs
Anti-chromatin. Ninety-six-well Nunc Maxisorp plates (Nalge Nunc)
were coated overnight with 5 ␮g/ml nucleosome core particles in PBS.
After washing with PBS, the wells were blocked with 100 ␮l of PBS
with1.8% BSA for 1 h at 37°C. Sera typically diluted 1/200, 1/1000, and
1/5000 in PBS, Tween 20, and 0.5% BSA were added to the wells for 1 h.
Then, 1/2000 diluted affinity-purified biotinylated goat secondary Abs
against mouse Fc␥ (Jackson ImmunoResearch Laboratories) or the relevant
IgG isotype (SouthernBiotech) were added for 30 min. Finally, bound secondary Abs were detected with 1/3000 diluted streptavidin-alkaline phosphatase (AP) conjugate (GE Healthcare catalog no. RPN1234V1) followed
by p-nitrophenyl phosphate (p-NPP) substrate. After 30 min, plates were
analyzed at 405 nm in a Victor3 V MultiLabel plate reader (PerkinElmer).
The wells were washed four times with PBS with 0.05% Tween 20 between each step.
Anti-DNA. Plates precoated with methylated BSA (5 ␮g/ml) were coated
with 10 ␮g/ml dsDNA or boiled ssDNA from salmon sperm. Serum from
a patient with active systemic lupus erythematosus served as positive control for coating.
Anti-total histones. Wells were coated with calf thymus histones (Roche
Applied Science catalog no. 10 223 565) at 2.5 ␮g/ml in 0.1 M carbonate
buffer (pH 9.6). An in-house IgG2a mAb (3F7-A10) derived from a
BALB.B mouse served as positive control for histone coating.
Anti-cardiolipin. Cardiolipin-precoated wells (Varelisa kit) were purchased from Phadia.
Anti-U1RNP, -Sm, -Ro, and -La. Precoated wells (Varelisa Recombi ANA
Screen; Phadebas) were purchased from Pharmacia. Other Ag-specific
ELISAs were performed by coating with 1 ␮g/ml heat-aggregated human
IgG (AHGG), 10 ␮g/ml boiled LPS, and 5 ␮g/ml chicken gizzard actinin.
Unless specified, all reagents were obtained from Sigma-Aldrich.
Standard curves established by sandwich ELISA
Wells were coated with 2 ␮g/ml unlabeled IgG subclass-specific polyclonal goat anti-mouse capture Abs (SouthernBiotech) that were blocked
and the corresponding serially diluted purified mouse IgG subclasses
(SouthernBiotech) were added at known concentrations. After washing, the
standards were revealed with the corresponding biotinylated isotype-specific secondary Abs (SouthernBiotech) diluted 1/2000, AP-streptavidin diluted 1/3000, and p-NPP substrate. The serum Ab level of a given isotype
was calculated from the OD of a serum dilution that fell on the steep slope
of the standard curve for that isotype. The background OD was subtracted
to obtain the final OD values.
Sandwich ELISA of total serum IgM, IgG1, and IgG2a
To wells coated with unlabeled, isotype-specific, goat capture Abs (2.5
␮g/ml) were added appropriate dilutions (determined by pilot experiments) of sera or known concentrations of purified mouse Ig standards
for 1 h. After washing, bound Igs were revealed by incubation with
AP-conjugated, isotype-specific, goat anti-mouse Abs (SouthernBiotech) and
p-NPP substrate. The amount of bound Ig isotype was calculated from
OD values of sera dilutions that fell on the steep slope of the standard
curve.
Urine analysis
Proteinuria was measured using urinalysis dipsticks (Multistix 8 SG; Bayer
Diagnostics) and a Clinitek 500 reader and was graded as follows: ⫹/⫺,
trace; 1⫹, 0.3 g/L; 2⫹, 1 g/L; and 3⫹, 3 g/L.
Statistics
All statistics were calculated using GraphPad Prism version 4 for Windows. A two-tailed Mann-Whitney rank sum U test was applied to the data
to assess statistical significance ( p ⬍ 0.05 is considered significant).
Results
Serological analysis of BALB/c and H2 congenic BALB/c strains
To investigate the impact of the H2 haplotype in the context of
pure nonlupus strains on serum Abs to chromatin, we bled mice of
the BALB.B (H2b) (n ⫽ 17–18), BALB/c (H2d) (n ⫽ 12–17), and
BALB.K (H2k) (n ⫽ 18) strains at 3, 6, 9, 12, and 14 mo of age.
ELISA showed that the serum IgG reactivity of BALB.B mice
with immobilized nucleosome core particles increased gradually in
an age-dependent fashion, indicating a developing autoimmune response to chromatin (Fig. 1). By contrast, the autoantibody levels
of most age- and gender-matched BALB.K and BALB/c mice remained low throughout. The difference was significant at all ages
for BALB.B vs BALB.K mice, and at 9 and 12 mo of age for
BALB.B vs BALB/c mice (Fig. 1). At 9 mo of age, 100% of
BALB.B mice were deemed positive for IgG anti-chromatin (OD
value 4 SD above the mean level of 18 BALB.K mice of the same
age). The average IgM anti-chromatin activity was also higher for
the BALB.B group: significant by 9 and 12 mo of age for BALB.B
vs BALB.K mice ( p ⬍ 0.0001), and by 12 mo of age for BALB.B
vs BALB/c mice ( p ⫽ 0.0003) (data not shown). These results
indicate that BALB.B mice are susceptible to late-onset augmentation of chromatin-reactive IgM and IgG Abs.
Isotypes, specificity, and age and gender dependence of
autoantibodies
We assessed IgG subclass levels of chromatin-specific Abs of 14mo-old BALB.B mice (n ⫽ 17). ELISA showed that IgG2a was
the dominant subclass; its average concentration was 138 ⫾ 28
␮g/ml compared with 13 ␮g/ml for IgG2b, 5.4 ␮g/ml for IgG3,
and 1.9 ␮g/ml for IgG1 (Fig. 2A). Another cohort of 12-mo-old
BALB.B mice (n ⫽ 14) had average anti-nucleosome levels of
14.8 ⫾ 5.8 ␮g/ml for IgG2a, 7.2 ⫾ 3.8 ␮g/ml for IgG2b (not
significantly different vs IgG2a), 0.69 ⫾ 0.2 ␮g/ml for IgG3 ( p ⫽
0.0006 vs IgG2a), and 0.12 ⫾ 0.02 for IgG1 ( p ⬍ 0.0001 vs
IgG2a) (data not depicted). Thus, the IgG2a/IgG1 ratio of Abs to
chromatin ranged from ⬃70 –120. A third sample of 12-mo-old
BALB.B mice (n ⫽ 12) had an average serum level of chromatinspecific IgG2a and IgM of 81 ⫾ 23 ␮g/ml and 19 ⫾ 7 ␮g/ml,
respectively, i.e., an IgG2a/IgM ratio of ⬃4. These results demonstrate that the autoantibodies of BALB.B mice have a distinct
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BALB/cOlaHsd (H2d) mice and the H2b haplotype-congenic BALB.B/
OlaHsd (also known as C.B10-H2b) and H2k haplotype-congenic BALB.K/
OlaHsd mice were obtained from Harlan. The H2b congenic interval of the
BALB.B strain is externally delimited by the microsatellite markers
D17Mit80 (at 12.9 centimorgans (cM)) and D17Mit232.1 (at 20.37 cM),
implying that the size of the congenic interval is 7.47 cM (Ref. 22 and
correction from C. Penha-Goncalves, unpublished observations). This interval includes the MHC class I, II, and III genes. Endotoxin sensitive
C3H/HeSnJ (H2k) mice and H2b-congenic C3.SW-H2b/SnJ mice were purchased from Jackson ImmunoResearch Laboratories. Sera from 9-mo-old
B6.Sle1b mice were kindly provided by Dr. E. Wakeland (University of
Texas Southwestern Medical Center, Dallas, TX). Unless indicated, all
mice were female. Sentinel mice were examined according to recommendations from the Federation of European Laboratory Animal Science Associations and tested negative for parasitical, bacterial, and viral agents. An
in-house expanded virus test program revealed that approximately onethird of the sentinel mice tested positive for Abs to mouse norovirus. Animal care was in accordance with national legislation and institutional
guidelines.
3543
3544
H2b HAPLOTYPE AND ANA
FIGURE 1. Effect of the H2 haplotype on production of IgG autoantibodies to chromatin. Sera from BALB.B (n ⫽ 17–18), BALB/c (n ⫽ 12–17),
and BALB.K (n ⫽ 18) mice were diluted 1/100 and incubated in ELISA wells coated with 5 ␮g/ml avian nucleosome core particles. Bound Ab was
detected by using biotinylated goat anti-mouse Fc␥, AP-conjugated streptavidin, and p-NPP substrate. Shown are OD values at 405 nm. Each symbol
represents one mouse. Horizontal bars indicate mean. The values were compared by the two-tailed Mann-Whitney U test. ns, Not significant.
gender on anti-chromatin IgG2a of BALB.B mice. By 12 mo of
age, the average IgG2a autoantibody level of the female cohort
(n ⫽ 15) was 45 ⫾ 19 ␮g/ml, compared with 0.7 ⫾ 0.35 ␮g/ml
for the males (n ⫽ 8; p ⫽ 0.003 by Mann-Whitney U test) (data
not depicted). This 64-fold difference suggests a female bias. It
should be noted that the BALB.B males were aggressive and
mutilated each other, so that 12 of 20 mice either died or were
euthanized before 12 mo of age.
Taken together, we conclude that the bulk of IgG2a autoantibodies of BALB.B mice is directed to intact nucleosome core particles and is not polyreactive. The low level IgG2a reactivity with
dsDNA, ssDNA, and LPS found in 30% of BALB.B mice could
represent a minor polyreactive fraction (25, 26).
Serum levels of total IgM, IgG1, and IgG2a
We also measured total serum concentrations of IgM, IgG1, and
IgG2a (IgG2b and IgG3 were not measured). The most striking
finding was that the total IgG2a levels of BALB.B mice rose
steeply from 3 mo through to14 mo of age, whereas those of
BALB/c and BALB.K mice rose more slowly and hit a lower
plateau (Fig. 3). By 14 mo of age, BALB.B mice had ⬃2.6-fold
more total IgG2a (4.01 ⫾ 0.35 mg/ml) compared with BALB.K
(1.52 ⫾ 0.11 mg/ml; p ⬍ 0.0001) and BALB/c (1.48 ⫾ 0.14
mg/ml; p ⬍ 0.0001) mice (Fig. 3). As the average level of
chromatin-specific IgG2a in this sample of 14-mo-old mice was
⬃0.14 mg/ml (Fig. 2A), the autoantibodies represent a small
fraction (3.5%) of total IgG2a. The total serum levels of IgM
and IgG1 of BALB.B mice did not differ markedly from those
of age-matched BALB/c and BALB.K mice (Fig. 3). Thus, the
hypergammaglobulinemia of BALB.B mice mainly presents as
a moderate elevation of IgG2a. This result is consistent with
that of a previous study showing that H2b mice with B10 or
BALB/c backgrounds have higher serum levels of IgG2a than
H2k and H2d congenic strains (27).
Induced Ag-specific Ab responses of BALB.B mice
To analyze the IgG subclass of the response induced by a foreign
protein Ag, 10-wk-old BALB.B (n ⫽ 5) and BALB/c (n ⫽ 5) mice
were injected once s.c. with 200 ␮g of AHGG, and the mice were
bled 15 days later. ELISA showed that all mice of both strains
produced similar levels of IgG1 anti-AHGG Abs, but only a single
BALB/c mouse had a good IgG2a anti-AHGG response (Fig. 4).
Thus, young BALB.B mice have normal responsiveness and produce predominantly IgG1 Abs to AHGG.
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profile with respect to isotype distribution: IgG ⬎ IgM, and
IgG2a ⬎ IgG2b ⬎ IgG3 ⬎ IgG1. For the remaining analyses we
focus mainly on chromatin-specific IgG2a.
From 3 to 12 mo of age, the average anti-chromatin IgG2a level
of BALB.B mice (n ⫽ 17–18) increased ⬃90-fold, from 0.49 ⫾
0.06 ␮g/ml at 3 mo, 1.67 ⫾ 0.47 ␮g/m at 6 mo ( p ⬍ 0.0001 vs
BALB.K and p ⬍ 0.017 vs BALB/c), 22.1 ⫾ 13.9 ␮g/ml at 9 mo
( p ⬍ 0.0001 vs BALB.K and BALB/c), and 45.4 ⫾ 11.6 ␮g/ml at
12 mo ( p ⬍ 0.0001 vs BALB.K and BALB/c) (Fig. 2B). By comparison, BALB.K and BALB/c mice were typically negative or
low for IgG2a Abs to chromatin (Fig. 2B). Thus, in these cohorts
the average serum IgG2a anti-chromatin level of 12-mo-old
BALB.B mice was ⬃35- and ⬃110-fold higher as compared with
BALB/c and BALB.K mice, respectively. The same sera were assayed for ANA activity by indirect immunofluorescence with
HEp-2 cells. A homogenous nuclear staining pattern was produced
by IgG2a of sera from 15 of 16 BALB.B mice (Fig. 2E), two of 16
BALB/c mice (the same two mice that had anti-chromatin IgG by
ELISA; see Fig. 1), and none of 18 BALB.K mice. Serum IgG1
produced no nuclear staining, and no IgG2a staining of HEp-2
cytoplasm was observed.
The average anti-dsDNA IgG2a levels of the sera from 12mo-old mice were very low: 0.62 ␮g/ml for BALB.B mice, 0.09
␮g/ml for BALB/c mice ( p ⬍ 0.0001 vs BALB.B), and 0.02
␮g/ml for BALB.K mice ( p ⬍ 0.0001 vs BALB.B) (Fig. 2C).
Thus, despite the low BALB.B serum levels of anti-dsDNA
Abs, they were significantly higher than those of BALB/c and
BALB.K mice. Of 17 sera from 14-mo-old BALB.B mice, 5
(⬃30%) stood out with respect to elevated levels of IgG2a Abs
against both ssDNA (mean of 3.2 ␮g/ml) and dsDNA (mean of
4.5 ␮g/ml); four of the five sera also contained ⬃1.5 ␮g/ml
LPS-reactive IgG2a (Fig. 2D). Of note, the abundance of antidsDNA IgG2a of the five sera was 8-, 15-, 62-, 83-, and 94-fold
lower compared with anti-chromatin IgG2a. No IgG2a reactivity was detected by ELISA against cardiolipin, actinin, human
IgG, U1RNP, Sm, SS-A/Ro, and SS-B/La (our unpublished
data). Sera from another sample of 12-mo-old BALB.B mice
(n ⫽ 12) were analyzed for IgG2a Abs to total histones. ELISA
showed that the average level was 0.093 ␮g/ml, which is 870fold lower than the average concentration (81 ␮g/ml) of antichromatin IgG2a of the same sera (data not depicted).
Compared with males, the lupus of (NZB ⫻ NZW)F1 females
is strongly accelerated (3), and the IgG anti-H2A/H2B/DNA
serum levels are higher in 5-mo-old, but not 7- to 9-mo-old,
B6.Sle1 female mice (24). We therefore assessed the impact of
The Journal of Immunology
3545
Impact of increased NaCl concentration on autoantibody
binding avidity
Increasing NaCl concentrations have been used to gauge the avidity of Abs to dsDNA (28). We applied this method to assess the
avidity of serum IgG2a from 14-mo-old BALB.B mice (n ⫽ 5) for
immobilized nucleosome core particles. The results are shown in
Fig. 5. The average concentration of NaCl that gave 50% of maximal absorbance was 0.32M. In the presence of 0.5M NaCl, the
amount of polyclonal IgG2a Ab bound to nucleosomes was reduced by 75–90%, whereas that of three lupus IgG2ab mAbs derived from (NZB ⫻ BXSB)F1 mice with full-blown lupus (29) was
reduced by a modest 25–30%. A likely explanation for the high
sensitivity of the BALB.B autoantibodies to moderately increased
NaCl concentration is that they depend heavily on electrostatic
compared with other forces for binding avidity. This interpretation
is consistent with the highly cationic IgH CDR3 regions of antinucleosome mAbs derived from NZM2410 lupus mice (30).
Spleen weight
Renal studies and survival
The Multistix scores of urine from 12- to 14-mo-old BALB.B mice
(n ⫽ 12) did not exceed 1⫹ (0.3 g/L), showing that the mice did
not have clinically significant proteinuria. By light microscopic
analysis (Fig. 6), histopathological abnormalities were not found in
the kidneys of any of 10 14-mo-old BALB.B mice of the cohort
shown in Fig. 2A. The survival of BALB.B mice through to 14 mo
of age (95%) did not differ significantly from that of BALB/c and
BALB.K mice (92% and 100%, respectively).
Analysis of C3H and C3.SW mice
FIGURE 2. Analysis of IgG autoantibodies. A, Isotype profile of antichromatin Abs. Sera from 14-mo-old BALB.B mice (n ⫽ 17) were diluted
5-fold starting at 1/200 and incubated in wells coated with core particles.
Bound Ab was detected with biotin-labeled goat anti-IgG subclass Abs and
AP-conjugated streptavidin. Ab levels were calculated by comparison with
IgG subclass standard curves generated for each plate by sandwich ELISA.
The numbers beside the horizontal bars indicate mean (␮g/ml). Each symbol represents one mouse. B, Anti-chromatin IgG2a levels as a function of
age and H2 haplotype. See legend to A for details of the assay. Shown are
means and SEM (n ⫽ 17–18). C, Effect of H2-haplotype on levels of IgG2a
anti-dsDNA Abs at 1 year of age. See the legend to A for details of the
assay. Each symbol represents one mouse. Horizontal bars represent
means. Sera from BALB.B (n ⫽ 17), BALB/c (n ⫽ 16), and BALB.K (n ⫽
17) mice were analyzed. The groups were compared by Mann-Whitney U
test. D, Serum levels of IgG2a Abs to ssDNA, dsDNA, and LPS. The
ELISA was conducted with sera from 14-mo-old BALB.B mice (n ⫽ 17)
as described in the legend to A. Note the low Ab levels compared with
IgG2a anti-chromatin Abs of A. Each symbol represents one mouse. E,
Typical nuclear staining of HEp-2 cells by IgG2a of BALB.B sera. Serum
samples from 1-year-old mice were diluted 1/100 and incubated with
HEp-2 cells (Immuno Concepts), followed by biotin-labeled goat antimouse IgG2a (2.5 ␮g/ml), and FITC-labeled streptavidin (10 ␮g/ml).
The foregoing results prompted us to assess the nonautoimmune
C3H/HeSnJ (H2k) strain and its H2b-congenic C3.SW/SnJ strain.
ELISA showed that the mean anti-chromatin IgG2a reactivity of
1/200 diluted sera from C3.SW mice (n ⫽ 18) was significantly
higher at all ages compared with the C3H control mice (n ⫽ 12)
(Fig. 7A). The same C3.SW mice at 12 mo of age tested negative
for chromatin-specific IgG1 (Fig. 6A) and IgG2b Abs (data not
shown; IgM and IgG3 were not measured). At 9 and 12 mo of age,
83 and 72%, respectively, of the C3.SW mice were deemed positive for anti-chromatin IgG2a (OD value 4 SD above C3H mean).
The average anti-chromatin IgG2a concentration among the eight
12-mo-old C3.SW mice with the highest ODs was 0.56 ␮g/ml, i.e.,
⬃25– 80-fold lower compared with the mean levels of 15 ␮g/ml
and 45 ␮g/ml found for two independent samples of age-matched
BALB.B mice (see above). The sera from 12-mo-old C3.SW mice
had low, but highly significant, levels of IgG2a reactivity to total
histones (Fig. 7B) and dsDNA (Fig. 7C) compared with sera from
the C3H group; 39 and 27% of the C3.SW mice were considered
positive for anti-histone and anti-dsDNA, respectively (OD value
4 SD above C3H mean). The same sera were examined at 1/50
dilution for reactivity of IgG2a with nuclei of HEp-2 cells.
Whereas all 12 C3H sera were ANA negative, eight of 17 C3.SW
sera (47%) were ANA positive. Of these, four exhibited homogenous staining patterns with more bright fluorescence of mitotic
chromatin (Fig. 7D, lower panel), indicative of specificity for
DNA/histone. The other four sera showed weak speckled nuclear,
but not cytoplasmic, staining (Fig. 7D, upper panel). Collectively,
these results indicate that the H2b haplotype, when recombined on
a C3H background, converts the mice to producers of ANA.
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Compared with age-matched BALB/c mice (n ⫽ 17), the average
postmortem spleen weight of 12- to14-mo-old BALB.B mice (n ⫽
15) was slightly, but significantly, increased (170 mg vs 140 mg;
p ⫽ 0.0011).
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H2b HAPLOTYPE AND ANA
FIGURE 3. Total serum IgM, IgG1, and
IgG2a concentrations. The levels of BALB.B
(n ⫽ 17–18), BALB/c (n ⫽ 12–18), and
BALB.K (n ⫽ 18) mice were determined by
sandwich ELISA and calculated by comparison with serially diluted standards. Mean ⫾
SEM are shown. The values of BALB/c and
BALB.K mice were compared with those of
BALB.B mice by Mann-Whitney U test.
ⴱⴱⴱ, p ⬍ 0.001, ⴱⴱ, p ⬍ 0.01; ⴱ, p ⬍ 0.05.
Anti-chromatin IgG2a/c and IgG1 levels of BALB.B vs B6.Sle1b
mice
The spontaneous autoimmunity to chromatin displayed by
BALB.B female mice is not unlike that reported for B6.Sle1b
mice. We therefore did a side-by-side comparison of levels of antichromatin (nucleosome) autoantibodies in sera from 12-mo-old
BALB.B (n ⫽ 8) and 9-mo-old B6.Sle1b (n ⫽ 8) mice. The sera
were diluted 2-fold for seven serial steps starting at 1/200. For
BALB.B mice, the individual levels (␮g/ml) of IgG2a Abs were
153, 22.4, 13.6, 4.4, 2.1, 1.3, 1.2, and 0.64 (mean 24.8 ⫾ 18.5
␮g/ml). For B6.Sle1b mice, the levels of anti-chromatin IgG2c
(also known as IgG2ab) Abs were 86.4, 70.4, 27.2, 10.4, 7.6, 4.8,
FIGURE 4. IgG1 and IgG2a Ab responses induced by AHGG. Ten
week-old BALB.B (n ⫽ 5) and BALB/c (n ⫽ 5) mice were injected s.c.
with 200 ␮g of AHGG, sera were harvested just before and 15 days later,
diluted 1/400, and incubated in wells coated with 1 ␮g/ml AHGG. Bound
IgG1 or IgG2a Ab was determined with biotin-labeled goat anti-mouse
IgG1 or IgG2a adsorbed with human IgG (SouthernBiotech) followed by
AP-conjugated streptavidin and substrate. IgG1 and IgG2a anti-AHGG Ab
levels were calculated by comparison with IgG subclass standard curves
generated by sandwich ELISA. Horizontal bars indicate means. The preimmune sera did not contain anti-AHGG activity (not depicted). ND, Not
detectable.
and 0.5 (mean 26 ⫾ 11.9 ␮g/ml; not significantly different vs
IgG2a levels of BALB.B mice). Anti-chromatin IgG1 was below
detection in four of eight BALB.B sera and 0.58, 0.54, 0.5, and
0.26 ␮g/ml in the other four. For B6.Sle1 mice, anti-chromatin
IgG1 was 0.32 ␮g/ml in one and below detection in the other seven
sera. Thus, sera from these cohorts of BALB.B and B6.Sle1b mice
displayed similar levels of anti-chromatin IgG2a/c and minimal
anti-chromatin IgG1.
Discussion
In this work we demonstrated the following: 1) H2b in the context
of two nonlupus-prone genomes (BALB/c and C3H) is sufficient to
induce production of ANA; 2) the bulk of autoantibodies is specific for intact nucleosome core particles; and 3) the most prominent IgG isotype is IgG2a, with some contribution of IgG2b in
BALB.B mice. Abs with similar characteristics are conspicuous
among the major spontaneous mouse lupus models (3, 31, 32).
However, BALB.B and C3.SW mice differ from these models by
minimal representation of the IgG1 isotype, low levels of antiDNA and anti-total histone reactivity, moderate hypergammaglobulinemia, mild splenomegaly, and no renal disease. The average anti-chromatin Ab levels are 25- to 80-fold higher in BALB.B
compared with C3.SW mice, indicating that the penetrance also
depends on non-MHC loci.
Unlike the spontaneous anti-chromatin response, IgG1 Abs prevailed over IgG2a in the response of young BALB.B mice to immunization with adjuvant-free AHGG. This dichotomy may reflect
different compositions of the Ags driving these responses. Thus,
FIGURE 5. Effect of NaCl concentration (conc.) on interaction of
IgG2a autoantibodies with chromatin. Wells coated with 5 ␮g/ml nucleosome core particles were incubated with 1/800 diluted sera from 14-mo-old
BALB.B mice (n ⫽ 5) in the presence of NaCl ranging from 0.15 to 0.5M
(dashed lines). For comparison, three IgG2ab (IgG2c) mAbs (60 ng/ml)
derived from (NZB ⫻ BXSB)F1 mice with full-blown lupus were analyzed
in parallel (solid lines). Bound Abs were detected with AP-conjugated
anti-IgG2a (for sera) or anti-IgG2c (for mAbs). Maximum OD is OD at 405
nm in the presence of 0.15 M NaCl. Shown is a pool of two experiments.
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The total serum levels of IgM, IgG2a, and IgG1 were moderately (⬍2-fold) elevated in C3.SW compared with C3H mice at all
ages (IgG2b and IgG3 were not measured) (Fig. 8). Thus, at 12 mo
of age the C3.SW vs C3H levels were mean ⫾ SEM 0.98 ⫾ 0.08
vs 0.73 ⫾ 0.04 mg/ml ( p ⫽ 0.044) for IgM, 0.8 ⫾ 0.13 vs 0.43 ⫾
0.05 mg/ml ( p ⫽ 0.0059) for IgG1, and 2.23 ⫾ 0.24 vs 1.25 ⫾
0.18 mg/ml ( p ⫽ 0.0031) for IgG2a. The average spleen weight of
C3.SW and C3H mice at 12 mo of age was 178 and 150 mg,
respectively (not significantly different). All C3.SW and C3H mice
survived through to 1 year of age and appeared healthy.
The Journal of Immunology
switching to IgG2a is promoted indirectly by Th1 cells producing INF-␥ (33–35) or by direct stimulation of TLR9/MyD88 in
B cells by DNA rich in nonmethylated CpG motifs (36, 37).
However, CpG DNA can also induce Th1-like responses via
TLR9 (38) and may therefore activate both pathways. The direct pathway was responsible for the switching of spontaneous
anti-DNA Abs from IgM to IgG2a/2b in B6.Fc␥RIIB⫺/⫺ mice
transgenic for the high affinity IgH anti-DNA gene 3H9/56R
(denoted B6.56R.Fc␥RIIB⫺/⫺ mice) (39) and for the induced
IgG2a response against a T-dependent nonself Ag mixed with
CpG (40). Taken together with the chromatin specificity, these
findings suggest that the Ag driving the IgG2a class switch of
autoantibodies of BALB.B and C3.SW mice contains CpG-rich
DNA, likely in the form of nucleosomes.
The benign humoral autoimmunity of BALB/c and C3.SW mice
is not unlike that reported for B6.Sle1 and B6.Sle1b congenic mice
that produce IgG autoantibodies preferentially targeted to H2A/
H2B/DNA subnucleosomes, with little “spreading” of the specificity to histone-free DNA (41, 42). In this study we show that two
cohorts of BALB.B and B6.Sle1b female mice aged 12 and 9 mo,
respectively, had similar levels of anti-chromatin (nucleosome)
IgG2a/2c and minimal expression of IgG1 autoantibodies. One difference between the strains appears to be the age of onset and the
progression of autoimmunity. Thus, for BALB.B mice this type of
ANAs is usually clearly detectable by 9 mo of age, and the levels
continue to increase through to 14 mo of age. B6.Sle1 female mice,
by contrast, have detectable ANA as early as 5 mo of age but the
levels peak at 7–9 mo of age and wane thereafter (41).
The lupus susceptibility alleles at the Sle1b locus of NZW mice
are present in many standard mouse strains, including BALB/c,
C3H/He, and 129, but not B6, mice (7). Are these alleles also
involved in the autoimmunity of H2b haplotype-congenic BALB.B
and C3.SW mice? Because the vast majority of strains that carry
the Sle1b susceptibility alleles are not autoimmune (7), it seems
likely that these alleles must interact with polymorphic genes of
the normal B6 genome to break tolerance to chromatin. This notion
is supported by the finding that, unlike nonautoimmune 129 mice,
B6 mice congenic for a 129-derived interval on distal chromosome
1 (denoted 129chr1b) encompassing the Sle1b locus also produce
ANA (43). Hence, the genetic backgrounds of BALB.B and
C3.SW mice may not be compatible with a role for Sle1 alleles in
the ANA responses of these mice. Another question raised by our
data, whether the H2b interval of the B6 background is needed for
the loss of tolerance to chromatin occurring in B6.Sle1b mice, has
not yet been studied.
The lupus of another model, that of Fc␥RIIB-null B6 mice, does
depend on the H2b haplotype. The null mutation of these mice was
generated in 129 embryonic stem cells (44) and then backcrossed
to B6. The fact that Fc␥r2 is tightly linked to Sle1b (45) and that
a Sle1b-containing chromosome 1 segment derived from the 129
FIGURE 7. Analysis of autoantibodies of C3.SW and C3H mice. Serum samples diluted 1/200 were incubated in ELISA wells containing immobilized
Ag. Bound Ab was detected using biotinylated goat Abs to IgG2a or IgG1 and AP-conjugated streptavidin. Shown are OD405 values. Horizontal bars
indicate means. Each symbol represents one mouse. A, Reactivity of IgG2a and IgG1 with nucleosome core particles; B and C, Reactivity of IgG2a with
total histone (B) and dsDNA (C) at 1 year of age. The values of C3.SW mice (n ⫽ 18) and C3H mice (n ⫽ 12) were compared by the Mann-Whitney U
test. ⴱⴱ, p ⫽ 0.0025; ⴱⴱⴱ, p ⬍ 0.0001; ns, not significant. D, Representative immunofluorescence staining of HEp-2 cells by IgG2a of 1/50-diluted sera
from two C3.SW mice at 1 year of age. For details, see legend to Fig. 2D. Upper panel shows speckled nuclear staining. Lower panel shows homogenous
staining with bright fluorescence of mitotic chromatin.
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FIGURE 6. Representative photomicrograph of kidney sections. Kidneys from 14-mo-old BALB.B mice were fixed in 4% formaldehyde, embedded in paraffin, sections were cut, and slides were stained with H&E
plus safran. The image was taken at ⫻400 total magnification.
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3548
genome mediates autoimmunity to chromatin in B6 mice despite carrying a normal Fc␥r2 gene (43, 46) complicates the
interpretation of the phenotype of Fc␥RIIB-null mice. Nonetheless, B6.Fc␥RIIB⫺/⫺ (H2b), but not BALB/c.Fc␥RIIB⫺/⫺
(H2d), mice develop IgG Abs to dsDNA and chromatin as well
as glomerulonephritis (44), and spontaneous production of IgG
anti-DNA and anti-IgG2a rheumatoid factors was almost eliminated in B6.Fc␥RIIB⫺/⫺ mice made congenic for H2d (47). Are
BALB/c.Fc␥RIIB⫺/⫺ mice protected from lupus because of their
H2d haplotype? When this hypothesis was tested by breeding the
H2b haplotype onto BALB/c.Fc␥RIIB⫺/⫺ mice in homozygous
form, the mice were still tolerant to chromatin (45).
The inability of H2b to convert the BALB/c.Fc␥RIIB⫺/⫺ strain
to ANA producers is contradictory to our own observations of
Fc␥RIIB-sufficient BALB.B mice, opening the intriguing possibility that BALB.B mice actually need Fc␥RIIB for the production of ANA. The role of Fc␥RIIB might be to enable cells
within secondary lymphoid organs to capture chromatin-containing IC and present the autoantigen of the IC in a multivalent, membrane-bound form to BCR, leading to immunological
synapse formation with B cells (48). The ensuing strong activation of BCR could overcome the inhibitory signals mediated
by Fc␥RIIB and result in highly efficient delivery of DNAcontaining IC to endosomes. In this way, DNA may reach
threshold concentrations required for TLR 9 activation (49).
This idea is supported by the following: 1) the observation that
IC-loaded dendritic cells can recycle native Ag to the cell surface in an Fc␥RIIB-dependent manner and present it to B cells
(50); 2) our previous work showing that Fc␥RIIB of viable
splenic B cells binds IC containing IgG2ab (IgG2c) antinucleosome mAbs in vivo (29); and 3) a recent report indicating
that follicular dendritic cells depend on Fc␥RIIB for IC loading
and strong B cell stimulation (51).
Another explanation for why BALB/c.Fc␥RIIB⫺/⫺.H2b mice
remain tolerant may be that they carry a recently discovered allele
on BALB/c chromosome12 (designated sbb2a) that eliminates antinuclear IgG2 production in an otherwise B6.Fc␥RIIB⫺/⫺ background (52). Our own data showing that Fc␥RIIB-sufficient
BALB.B mice produce ANA despite carrying the sbb2a suppressor
locus suggest that the mechanism underlying their autoimmunity is
distinct from that operating in B6.Fc␥RIIB⫺/⫺.H2b mice.
The 7.47 cM H2b-congenic interval of BALB.B mice (see the
paragraph titled “Mice” in Materials and Methods) contains numerous genes, including genes encoding the polymorphic Ag-presenting MHC class I and II proteins, complements C2, C4, and B,
and the cytokines TNF-␣, LT␣, and LT␤ (where LT is lymphotoxin). Thus, the gene(s) in this interval responsible for triggering
ANA is unknown. Inasmuch as the present study has revealed a
critical impact of the H2b haplotype on an autoantibody response,
and given the normal (nonlupus) genetic background of the mice
as well as the role of class II H2 allotypes in regulating Ab responses to protein Ag (53), it is tempting to consider I-Ab molecules as critical mediators of the BALB.B and C3.SW phenotypes.
In this scenario, the role of the I-Ab allotype is visualized as dependent on its peptide-binding properties, i.e., the higher affinity of
the I-Ab molecules of B cells for certain peptides (as yet uncharacterized) when compared with the MHC II molecules encoded by
H2d and H2k, leading Th cells specific for those peptides to be
strongly activated and aid ANA-producing B cells. This hypothesis, like that advanced by Iwamoto et al. to explain the protective
effect of transgene-encoded E␣ chains in BXSB and related lupus
mice (see Introduction) (20), assumes that the anti-chromatin Ab
responses depend on Th cells and autoantigen presentation on
I-Ab. In this context, it has been proposed that genetic defects of
lupus-prone mice lower TCR activation thresholds for autoantigen
(20) (54, 55). However, the normal genetic backgrounds of
BALB.B and C3.SW mice suggest that this putative mechanism
does not apply to these mice.
With respect to the need for Th cells, a recent study of B6 mice
transgenic for the high affinity IgH anti-DNA gene 56R reported
that, except in young mice, T cells made little difference for the
spontaneous development and isotype switch of anti-dsDNA Abs
(56). In contrast, as cited by Tsao et al. (56), earlier studies of
mouse lupus models with more normal B cell repertoires, such as
MRL/lpr, indicated that T cells play important roles in the production of anti-DNA Abs. In line with this conclusion, clinically
healthy MHC II-deficient B6.Sle1 and B6.Nba2 mice showed minimal levels of IgG Abs to chromatin, histones, and dsDNA, indicating the need for Ag presentation to Th cells (57). Furthermore,
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FIGURE 8. Total serum Ig levels of C3.SW and C3H mice. Top panel,
IgM level. Middle panel, IgG1 level. Bottom panel, IgG2a level. The levels
were determined by sandwich ELISA and calculated by comparison with
serially diluted standards. The data points represent mean ⫾ SEM. The
values of C3.SW (n ⫽ 18) and C3H (n ⫽ 12) mice were compared by the
Mann-Whitney U test. ⴱⴱ, p ⬍ 0.01; ⴱ, p ⬍ 0.05.
H2b HAPLOTYPE AND ANA
The Journal of Immunology
Acknowledgments
We thank Edward Wakeland (University of Texas Southwestern Medical
Center) for providing sera from B6.Sle1b mice.
Disclosures
The authors have no financial conflict of interest.
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although TCR␣␤-deficient B6.Sle1 mice produced a high abundance of IgM Abs to ssDNA and chromatin, the amount of chromatin-reactive IgG was minimal, in sharp contrast to TCR␣␤-sufficient B6.Sle1 mice (58). Thus, despite the importance of TLR9
signals for eliciting production of anti-chromatin IgG in vivo (59 –
61), it seems that in the presence of a normal B cell repertoire with
low-avidity anti-DNA BCRs, Th cells are also required.
Previous work has shown that inoculation of 3- to 4-wk-old
mice with the Moloney leukemia virus induced a higher proportion
of BALB.B mice to become ANA positive by indirect immunofluorescence and to develop higher ANA titers when compared
with 5-mo-old BALB/c mice. Because this difference was not observed in nonviremic mice, it was concluded that high viral production was required for ANA triggering (62). In the present study,
spontaneous anti-chromatin IgG became prominent beyond 6 mo,
which likely explains why 5-mo-old virus-nonreplicating BALB.B
mice tested negative for ANA. Nonetheless, the findings by Piatier
et al. highlight the link between environmental provocation, the
H2b haplotype, and ANA responses (62). In this context it has been
reported that, after LPS stimulation, B cells from BALB.B mice
display higher cell activation, cell proliferation, and IgM secretion
when compared with B cells from BALB/c mice, suggesting that
genetic variation at the MHC region contributes to the control of B
cell responsiveness to LPS stimulation (22).
Autoimmunity to chromatin has been suggested to involve several mechanisms, including altered apoptosis, reduced clearance of
apoptotic material or IC, altered cytokine environment, disruption
of the balance between activation and inhibitory signaling pathways, inefficient BCR editing, and TLR9 activation. Our observation that the H2b haplotype triggers ANA even in the context of
genetic backgrounds not prone to lupus indicates that the H2b haplotype contains one or more major lupus susceptibility genes or is
lacking genes that confer resistance to ANA responses. Thus,
BALB.B and C3.SW mouse strains may be important tools for the
dissection of yet other mechanisms involved in loss of tolerance to
chromatin.
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H2b HAPLOTYPE AND ANA