Genetic regulation of anti-erythrocyte autoantibodies and splenomegaly in autoimmune hemolytic anemia-prone New Zealand Black mice

Kimiko Ochiai1, Shoichi Ozaki2, Akihiro Tanino1, Shinji Watanabe1, Tomoo Ueno1, Kenichi Mitsui3, Junichi Toei1, Yuji Inada1, Sachiko Hirose4, Toshikazu Shirai4 and Hiroyuki Nishimura1

1 Toin Human Science and Technology Center, Department of Biomedical Engineering, Toin University of Yokohama, 1614 Kurogane-cho, Aoba-ku, Yokohama 225-8502, Japan
2 Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, 54 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
3 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, 4-1-8 Honmachi, Kawaguchi, Saitama 332-0012, Japan
4 Department of Pathology, Juntendo University School of Medicine, 2-1-1 Hongo Bunkyo-ku,Tokyo 113-8421, Japan

Correspondence to: H. Nishimura


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
New Zealand Black (NZB) mice spontaneously produce anti-erythrocyte autoantibodies (AEA) in association with splenomegaly, thus serving as a model for autoimmune hemolytic anemia. Although these autoimmune traits are inherited as a dominant fashion, expression in F1 hybrids of NZB and most non-New Zealand strains is suppressed due to the contribution of wild-type modifying genes present in the latter strains. Using chromosomal microsatellite markers in the (C57BL/6 x NZB)F1 x NZB backcross progeny, we mapped C57BL/6 modifying loci for AEA production and splenomegaly. Generation of AEA was found to be down-regulated by a combined effect of two major independently segregating dominant alleles—one linked to D7MIT30 on chromosome 7 and the other linked to D10MIT42 on chromosome 10. Splenomegaly was modified mainly by a single C57BL/6 allele linked to D4MIT58 on chromosome 4. Thus, the autoimmune hemolytic anemia in the NZB strain is under multigenic control and a combined action of not only susceptibility but also modifying alleles with suppressive activities affects the outcome of disease features in the progeny. There are potentially important candidate genes which may be linked to the regulation of AEA and splenomegaly.

Keywords: anti-erythrocyte autoantibody, hemolytic anemia, microsatellite, New Zealand Black mice, quantitative trait locus, splenomegaly


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The New Zealand Black (NZB) strain of mouse spontaneously develops autoimmune hemolytic anemia, in association with anti-erythrocyte autoantibodies (AEA), decreases in hematocrit values, reticulocytosis, hemoglobinemia and splenomegaly, thus resembling the human counterpart of the disease (13). Earlier studies using crosses of NZB and other strains suggested that the AEA production is under multigenic control (49). Splenomegaly, which may be caused by both lymphoid hyperplasia and over-disposal of sensitized erythrocytes, has also been reported to be under multigenic control (10). Genetic studies using crosses of NZB and non-autoimmune New Zealand Chocolate (NZC) mouse strains revealed that 100% of (NZBxNZC)F1 and 74% of the F2 hybrids developed positive AEA (1,4), a finding consistent with the notion that a single dominant gene may control AEA. However, later studies demonstrated that F1 hybrids of NZB and non-autoimmune, non-New Zealand mouse strains had a much lower incidence and a later onset of positive AEA (57). These findings were interpreted, as a hypothetical model, that the NZB strain contributes a single dominant susceptibility allele Aia-1 (autoimmune anemia locus) loosely linked to the b locus of chromosome 4 (4) to the full expression of production of AEA, whereas the effect of this gene is suppressed to a varying degree by wild-type modifying dominant alleles present in most strains of mice, except for NZB and NZC (6,9), one of which (Aem-1, anti-erythrocyte autoantibody modifying gene) was mapped to the locus loosely linked to the Mup-1 on chromosome 4 (9).

Thus, it appears that the autoimmune hemolytic anemia in the NZB strain is regulated by a combined action of not only susceptibility but also modifying alleles with suppressive activity. Nonetheless, classic progeny studies have provided only limited information on the number, identity and chromosomal location of the quantitative trait loci (QTL) for multigenic diseases. Taking advantage of genome-wide analysis with microsatellite-based chromosomal maps (11), we mapped modifying alleles regulating the onset of AEA production and severity of splenomegaly, using (C57BL/6xNZB)F1xNZB backcross mice.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
NZB, C57BL/6 and their F1 mice were derived from colonies housed at Juntendo University. Female (C57BL/6xNZB)F1 mice at 2 months of age were crossed to male NZB mice to obtain (C57BL/6xNZB)F1xNZB backcross mice. Only female backcross mice were analyzed.

Test for AEA
Production of IgG AEA was examined by direct Coombs' test (12). Heparinized blood was obtained from the tail vein and erythrocytes were washed 4 times with isotonic PBS, pH 7.2. Aliquots of 50 µl of 4% erythrocytes suspensions were incubated with 50 µl of the 1:16 diluted rabbit anti-mouse Ig sera in test tubes for 30 min at room temperature and the mixture was then gently centrifuged. Macroscopically visible agglutination was regarded as a positive AEA. All mice 6–13 months of age were examined monthly. Onset of AEA production in an individual mouse was scored, based on the earliest month of age when positive AEA was detected. AEA score (S) was defined as S = –x + 14, in which x is the age (months) of onset of positive AEA. Thus, the highest score (S = 8) was given to mice that showed earliest onset (6 months of age) and the lowest score (S = 1) to mice of the most delayed (13 months of age) onset. Mice negative for AEA up to 13 months of age were scored as 0.

Genotyping of chromosomal microsatellite markers
Genomic DNA was extracted from liver tissues of mice that have been stored at –70°C before use, as described (13). Polymorphisms of microsatellite markers were analyzed using the modified method described by Dietrich et al. (11) PCR primers flanking chromosomal microsatellite markers (MapPair primers) were purchased from Research Genetics (Huntsville, AL). A sulfhydryl group (SH) was introduced at the 5' site of one of the PCR primer pair, using T4 polynucleotide kinase and {gamma}-S-ATP, and was subsequently reacted with N-iodoacetyl-N'-biotinylhexylenediamine, using an oligonucleotide biotin labeling kit (US Biochemical, Cleveland, OH). A PCR mixture (10 µl) contained 0.12 µM of one primer (biotin labeled), 0.12 µM of another primer (unlabeled), 0.25 mM of each dNTP, 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 1.3 µg/ml of DNA and 0.05 U/µl of Taq polymerase (Takara Shuzo, Kyoto, Japan), followed by overlay with mineral oil (14). The PCR amplifications were carried out using a DNA thermal cycler (Perkin-Elmer Cetus, Foster City, CA). The reaction consisted of initial denaturation at 94°C for 1 min, followed by 30 cycles of 94°C for 1 min, 56–58°C for 1.5 min and 72°C for 2 min, and finally followed by a single cycle of 72°C for 10 min. PCR products were diluted 4-fold with loading buffer consisting of xylene cyanol and bromophenol blue dyes (each 0.5%) in formamide (95%) containing 10 mM of EDTA (pH 8.0), denatured at 94°C for 10 min, and electrophoresed on 6% denaturing polyacrylamide gels (width; 300 mm, height; 200 mm, thickness; 0.2 mm) in 0.5xTBE buffer for 3–4 h at 1000 V. An electrophoretic apparatus (Nippon Eido, Tokyo, Japan) was attached to a hand-made device that enables the constant movement (15 cm/h) of Biodyne B nylon membrane (Pall, Port Washington, NY) at the bottom of the gel to obtain the blotting of DNA bands during electrophoresis. The blotted membrane was treated with SDS (5%) in PBS (1xblocking solution) and incubated with streptavidin–alkaline phosphatase conjugate (Boehringer Mannheim, Mannheim, Germany) at 37°C for 5 min. After extensive washings with 500 mM Tris–HCl (pH 9.5) containing 500 mM NaCl and 50 mM MgCl2, the membrane was soaked once in 0.1 mM diethanolamine (pH 10.0) containing 1.0 mM MgCl2 and 0.02 % sodium azide (assay buffer), and subsequently soaked in 0.125 mM CSPD (chemiluminescent substrate for phosphatases) (Boehringer) in assay buffer at 37°C for 5 min (15). The membrane with CSPD was sealed in a `Hybridization Bag' (Cosmo Bio, Tokyo, Japan) and the chemiluminescent image was obtained by exposure of the membrane to Kodak X-OMAT AR film.

Data analysis
Linkage analyses were done using both Pearson's {chi}2 test and interval mapping. Genomic interval mapping was done using a MAPMAKER/EXP and MAPMAKER/QTL software (16,17), kindly provided by Dr E. S. Lander (Whitehead Institute for Biomedical Research, Cambridge, MA) to identify the chromosomal location of quantitative trait loci (QTL). The likelihood ratio statistic (base-10 Lod score) of 1.9 and 3.3 was used as a threshold for statistically suggestive and significant linkage respectively (18). ANOVA was used to determine the difference in AEA scores among groups of backcross mice with different combinations of modifying suppressive alleles.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
AEA and splenomegaly in NZB, C57BL/6 mice and their crosses
Figure 1Go compares AEA scores (Fig. 1aGo) and spleen weights (Fig. 1bGo) of NZB, C57BL/6, (C57BL/6xNZB)F1 and (C57BL/6xNZB)F1xNZB backcross mice at 13 months of age. Observed incidences of AEA (AEA scores >=1) in NZB, C57BL/6, their F1 and the F1xNZB backcross mice were 100, 0, 0 and 61%, and those of splenomegaly (spleen weights >300 mg) were 86, 0, 0 and 48%, respectively.



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Fig. 1. Expression of the two autoimmune traits, AEA production (a) and splenomegaly (b) in NZB, C57BL/6 (B6), (C57BL/6xNZB)F1 (F1) and (C57BL/6xNZB)F1xNZB backcross (B.C.) mice. Mice were scored for the onset of AEA production as described in Methods (a). Spleen sizes were measured at the time of killing at 13 months of age (b).

 
Association of AEA and splenomegaly in (C57BL/6x NZB)F1xNZB backcross mice
A total 75 female progeny of (C57BL/6xNZB)F1xNZB backcross mice were examined to determine the association between AEA scores and spleen weights at 13 months of age (Fig. 2Go). AEA scores and spleen weights overall showed a low but a significant association (r = 0.327, P < 0.005). However, there were backcross mice that were positive for AEA (AEA scores >=1) and negative for splenomegaly, or vice versa, suggesting that these two autoimmune traits are not under identical genetic control. Compared to the parental NZB strain (AEA scores >=4), a proportion of backcross mice showed delayed onsets of AEA (AEA scores <4) and lower grades of splenomegaly findings which suggested that the suppressive effects of C57BL/6 modifying alleles for the two autoimmune traits appear to function in a quantitative rather than all-or-none fashion (Figs 1 and 2GoGo).



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Fig. 2. Association of the two autoimmune traits, production of AEA and splenomegaly in female progeny of the (C57BL/6xNZB)F1xNZB backcross mice. AEA scores of individual mice were determined by the earliest age (month) when macroscopically visible agglutination of erythrocyte was detected in direct Coombs' test (see Methods). Mice with spleens over 300 mg were regarded as having splenomegaly.

 
Mapping of modifying genes
To map the chromosomal loci for the regulation of AEA and splenomegaly, (C57BL/6xNZB)F1xNZB backcross mice were genotyped for 60 chromosomal microsatellite markers polymorphic between C57BL/6 and NZB strains (Fig. 3Go). Chi-square analyses of associations between incidences of these two autoimmune traits and microsatellites showed the presence of two potential C57BL/6 suppressive loci for AEA on chromosomes 7 and 10 [as a potential modifying locus on chromosome 4 was designated Aem-1 in our earlier studies (9), these two were provisionally designated Aem-2 and Aem-3 respectively; anti-erythrocyte autoantibody modifier, for convenience to describe the interaction of modifying alleles], and one locus for splenomegaly on chromosome 4 (Spm-1, splenomegaly modifier-1) (Table 1Go). Interval mapping of data from backcross mice at 13 months of age, using MAPMAKER/QTL, showed that the locus for Aem-2 on chromosome 7 is located between microsatellite markers D7MIT30 and D7MIT297 (Lod score = 3.1, suggestive linkage) (Fig. 4Go). On the other hand, the Lod score of another potential locus Aem-3 on chromosome 10, which was significantly linked to a marker D10MIT42, by using {chi}2 analysis, was not in the range of significant associations (data not shown). However, the existence of Aem-3 was suggested by ANOVA, as based on data from the backcross progeny, classified according to the combinations of genotypes for D7MIT30 and D10MIT42, i.e. Group A, NZB/NZB (NN) genotype for both D7MIT30 and D10MIT42 loci; Group B, NZB type for D7MIT30 and C57BL/6 type for D10MIT42 (NB); Group C, C57BL/6 type for D7MIT30 and NZB type for D10MIT42 (BN); and Group D, C57BL/6 type for both loci (BB) (Fig. 5Go). Among these four groups, the highest AEA scores (earliest onset) were observed in mice of Group A and the lowest in mice of Group D. Data on mice of Groups B and C were in between, indicating that the suppressive effects for AEA production were increased in a manner depending on the number of the corresponding C57BL/6 modifying alleles. Although the mean AEA scores of Groups A and B did not differ significantly, differences were evidently significant when Group D (C57BL/6 genotype for both loci) was compared with either Group C or Group B. Thus, it is reasonable to assume that the suppressive effect can be attributed to combined effects of Aem-2 and Aem-3.



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Fig. 3. Polymorphic microsatellite markers used. Linkage relationships for the 63 polymorphic markers were determined by MAPMAKER/EXE for 75 female (C57BL/6xNZB)F1xNZB backcross mice.

 

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Table 1. Linkage analysis of AEA production and splenomegaly in the backcross progeny
 


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Fig. 4. MAPMAKER/QTL scan of chromosome 7 for the presence of potential autoimmune-modifying locus in (C57BL/6xNZB)F1xNZB backcross progeny. A Lod score curve along chromosome 7 is shown on the right. Mapping positions and the loci of potential candidate genes are indicated on the left of the chromosome. AEA were scored, as described in Methods.

 


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Fig. 5. Analysis of variance in scores of AEA among groups classified by combined genotypes. (C57BL/6xNZB)F1xNZB backcross mice were sub-divided into four groups according to genotype combinations at microsatellite markers D7MIT30 and D10MIT42. Symbols `B' and `N' depict NZB/C57BL/6 heterozygous and NZB/NZB homozygous genotypes respectively. Scores of AEA production are expressed by means and SD. Significant differences are shown by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

 
Figure 6Go also shows the result of MAPMAKER/QTL scan for the C57BL/6 locus responsible for the suppression of splenomegaly on chromosome 4. The peak QTL was located most proximal to D4MIT58 (Lod score = 3.6, significant linkage).



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Fig. 6. MAPMAKER/QTL Scan of chromosome 4 for the presence of the potential quantitative trait locus regulating spleen weights at 13 months of age in female (C57BL/6xNZB)F1xNZB backcross progeny. Mapping positions and the loci of potential candidate genes are indicated on the left.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
At least two potential C57BL/6 loci probably function in a suppressive manner for the onset of AEA in NZB mice: one Aem-2 was located proximal to the marker D7MIT30 on chromosome 7 and the other Aem-3 proximal to D10MIT42 on chromosome 10. While the association of locus Aem-2 was significant in both {chi}2 and QTL scan analyses, the second locus Aem-3 was only significant by {chi}2 and not by QTL scan analyses. However, as the backcross progeny bearing both Aem-2 and Aem-3 showed a significantly late onset of AEA than was found in the progeny with either allele, Aem-3 appears to act in a combined manner with Aem-2 to suppress AEA. It is of note, in this respect, that the insulin-like growth factor receptor gene igf-1r is located close to Aem-2 on chromosome 7 (19) and the insulin-like growth factor-1 gene igf-1 to Aem-3 on chromosome 10 (20). These genes require study, because insulin-like growth factor is involved in the regulation of the transforming growth factor-ß activity that is responsible for the negative regulation of immune responses (21). Studies on polymorphisms and functions of igf-1 and igf-1r and the relation to the regulation of AEA production are underway in our laboratories.

In earlier studies, we found that a dominant suppressive locus Aem-1, loosely linked to Mup-1 on chromosome 4, is involved in the regulation of AEA production (9). In the present studies, there were no microsatellites on chromosome 4 which showed a significant association with the observed AEA regulation. It may be that chromosomal intervals between Aem-1 and the microsatellites used in the present studies are not close enough to show significant linkages. Alternatively, it may be due to the difference in evaluation of the disease trait in which the associations in earlier studies were analyzed based on the presence or absence of AEA irrespective of age, while the present analyses were made based on the time of AEA onset in order to evaluate the disease feature as a quantitative trait. It is also possible that the effect of Aem-1 is diluted or overridden by the effects of Aem-2 and/or Aem-3 in the present progeny studies.

On the other hand, splenomegaly was significantly regulated by a C57BL/6 allele Spm-1 proximal to a microsatellite D4MIT58 ~30 cM telomeric to the Mup-1 locus on chromosome 4. In early studies using recombinant inbred strains derived from (ALNxNZB)F2 or (NFSxNZB)F2 mice, Raveche et al. (10) suggested that at least two genes (one dominant and the other recessive) are involved in the splenomegaly in NZB mice and which is associated with chromosomal hyperdiploidy. Thus, it appears that C57BL/6 Spm-1 may be the wild-type allele of the NZB recessive gene that Raveche et al. (10) proposed. Also, the genotype distribution we noted strongly suggests that Spm-1 is the wild-type allele of NZB splenomegaly gene Sbw2 on chromosome 4, as reported by Kono et al. (22), which exhibited incomplete dominant inheritance in (NZBxNZW)F2 intercross mice. There is the possibility that Spm-1/Sbw2 and Aem-1 are a single polymorphic gene and contribute to the regulation of both splenomegaly and AEA, because AEA scores and spleen weights overall showed a significant association in the backcross progeny (Fig. 2Go). Our preliminary observation supports this idea in that the backcross progeny bearing C57BL/6 genotypes at both microsatellites D4MIT58 (linked to Spm-1) and D7MIT30 (linked to Aem-2) showed lower serum levels of AEA in later life than did the progeny with either one of the genotype (data not shown). Further studies are required to confirm this issue. The role of Spm-1/Sbw2 is unclear, but is most likely related to lymphocyte activation and hyperplasia. Candidate genes located in this vicinity are type I IFN genes (Ifa and Ifb), which are involved in regulating immune responses (23,24). Several types of autoimmune diseases can be induced by therapeutic administration of these IFN in patients with either cancer or viral hepatitis (25,26).

In addition to the above loci, previous genetic studies of autoimmune disease-prone mice mapped several autoimmune traits to loci on chromosome 4 (summarized in Fig. 7Go). Kono et al. (22) mapped Lbw2, one NZB allele for early mortality and glomerulonephritis, very close to Sbw2 and suggested them to be the same susceptibility locus (22). Morel et al. (27) mapped three genes responsible for the susceptibility to glomerulonephritis of NZM mice. One of these on chromosome 4, Sle2, was found in the vicinity of a Marker D4MIT9 that is closely linked to Spm1 and Sbw2. This allele was subsequently shown to be involved in the polyclonal activation of B cells (28). In genetic studies of (NZWxBXSB)F1 mice, a model of systemic lupus erythematosus-associated anti-phospholipid syndrome, Ida et al. (29) mapped Acla-2, one of the two susceptibility loci for anti-cardiolipin antibodies in BXSB mice, in the vicinity of Spm-1/Sbw2. Hirose et al. (29) mapped a dominant NZB allele for IgM hypergammaglobulinemia (Imh-1) on chromosome 4. This Imh-1 was closely linked to the dominant NZB allele Mott-1 for the formation of Mott cell, a pathologic state of plasma cells containing large amounts of IgM as intracytoplasmic inclusions (Russell bodies) (30). As both hyper-IgM and Mott cell formation represent aberrant hyper-differentiation of B cells, Imh-1 and Mott-1 are most likely the same allele. There is the possibility that Imh-1/Mott-1 is Spm-1/Sbw2 per se, but this is unlikely because the former is located more distal to the latter in the microsatellite genotype distribution analyses (Fig. 7Go). Drake et al.(31) mapped an allele (nba-1) responsible for glomerulonephritis of (NZBxNZW)F1 mice on the distal part of chromosome. It is possible that effect of nba-1 may be the combined effect of two distinct autoimmune-predisposing genes, Spm-1/Sbw2 and Imh-1/Mott-1. In the present studies, we also analyzed correlations of C57BL/6-type Spm-1, Aem-2 and Aem-3 alleles with hyper-IgM and IgM anti-DNA antibodies in the same backcross mice; however, results of QTL and {chi}2 analyses did not show any significant associations (data not shown). Thus, it appears that these alleles are not related to the regulation of the aberrant hyper-differentiation of IgM B cells (AEA we measured using direct Coombs' test are of IgG class).



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Fig. 7. Regions of mouse chromosome 4 associated with various autoimmune traits of New Zealand strains. The name of the allele is indicated at the position where it is most likely located, in relation to genetic markers used in each linkage study. Abbreviation, B6 = C57BL/6.

 
Aia-1 was a dominant NZB susceptibility allele which was considered to play a central role in the development of autoimmune hemolytic anemia (4). The chromosomal location, however, has been approximately defined on chromosome 4 (4). Further studies will be required for more precise localization, to analyze the correlation to other heretofore defined loci on chromosome 4, described above.

The results of the present studies provide insight into the genetic basis for the heterogeneity of disease traits observed in families of autoimmune diseases. Expression of autoimmune susceptibility genes is modified differently by combinations with different quantitative modifying genes. Identification of suppressive modifiers is of particular importance, because it has implications for developing therapeutic approaches to autoimmune diseases.


    Acknowledgments
 
We thank M. Ohara for critical comments. This work was supported by CREST/JST (Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation).


    Abbreviations
 
AEA anti-erythrocyte autoantibody
Aem anti-erythrocyte autoantibody modifier
Spm splenomegaly modifier
QTL quantitative trait locus

    Notes
 
The first two authors contributed equally to this work

Transmitting editor: T. Saito

Received 3 June 1999, accepted 14 September 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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