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The Journal of Immunology, 2002, 168: 3042-3049.
Copyright © 2002 by The American Association of Immunologists

A Novel Susceptibility Locus On Chromosome 2 in the (New Zealand Black x New Zealand White)F1 Hybrid Mouse Model of Systemic Lupus Erythematosus1

Ziaur SM. Rahman2,*, Soe-Kyaw Tin*, Pia-Nina L. Buenaventura*, Chiu-Han Ho*, Eric P. H. Yap{dagger}, Rita Y. Y. Yong{dagger} and Dow-Rhoon Koh3,*

* Department of Physiology, and {dagger} Defense Medical Research Institute, Clinical Research Center, National University of Singapore, Singapore


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Systemic lupus erythematosus (SLE) is inherited as a complex polygenic trait. (New Zealand Black (NZB) x New Zealand White (NZW)) F1 hybrid mice develop symptoms that remarkably resemble human SLE, but (NZB x PL/J)F1 hybrids do not develop lupus. Our study was conducted using (NZW x PL/J)F1 x NZB (BWP) mice to determine the effects of the PL/J and the NZW genome on disease. Forty-five percent of BWP female mice had significant proteinuria and 25% died before 12 mo of age compared with (NZB x NZW)F1 mice in which >90% developed severe renal disease and died before 12 mo. The analysis of BWP mice revealed a novel locus ({chi}2 = 25.0; p < 1 x 10-6; log of likelihood = 6.6 for mortality) designated Wbw1 on chromosome 2, which apparently plays an important role in the development of the disease. We also observed that both H-2 class II (the u haplotype) and TNF-{alpha} (TNFz allele) appear to contribute to the disease. A suggestive linkage to proteinuria and death was found for an NZW allele (designated Wbw2) telomeric to the H-2 locus. The NZW allele that overlaps with the previously described locus Sle1c at the telomeric part of chromosome 1 was associated with antinuclear autoantibody production in the present study. Furthermore, the previously identified Sle and Lbw susceptibility loci were associated with an increased incidence of disease. Thus, multiple NZW alleles including the Wbw1 allele discovered in this study contribute to disease induction, in conjunction with the NZB genome, and the PL/J genome appears to be protective.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Two of the most common features in both human and mouse models of systemic lupus erythematosus (SLE)3 are: 1) Abs against various nuclear constituents (ssDNA, dsDNA, histone, and chromatin); and, 2) glomerulonephritis (GN) from the deposition of immune complexes onto the glomerulus basement membrane in the kidney. Even though the etiology and the specific contributions of particular gene(s) to this disease remain unsolved, substantial evidence from multiple studies in murine and human SLE has suggested that SLE has a strong genetic basis and is a complex genetic trait.

The F1 hybrid of New Zealand Black (NZB) and New Zealand White (NZW) mice has been studied most extensively and is one of the best animal models for SLE mice. More than 90% of these hybrid mice spontaneously develop a severe renal disease that resembles the symptoms that are associated with human SLE. The prevalence of the disease in both NZB and NZW strains is <5% in the first year (1, 2), which suggests that genes from both NZW and NZB are required for full expression of the disease. Through classical backcross studies, many research groups have implicated a dominant NZW MHC locus on chromosome 17 and a dominant NZB locus on distal chromosome 4 (2, 3, 4, 5). Subsequently, from the backcross study of a recombinant strain called New Zealand mixed, a mixture of NZW and NZB, three chromosomal intervals containing lupus-susceptibility alleles were identified on chromosomes 1, 4, and 7 and designated Sle1, Sle2, and Sle3/Sle5, respectively (6). Eight other susceptibility loci (Lbw1 to Lbw8) were also identified in (NZB x NZW)F2 intercross mice (4).

PL/J is a nonautoimmune strain and has the same H-2 class II as NZW (I-Au and I-Eu). It has been shown that (NZB x PL/J)F1 hybrids do not develop lupus (7). This suggests that an H-2 class II (u haplotype) contribution alone is not sufficient to induce SLE and that there must be other genes from H-2 or non-H-2 loci contributing to the disease. It is also possible that dominant alleles from PL/J are protecting. In the present study, our approach was to determine the NZW contribution from both H-2 and non-H-2 loci and the effect of PL/J alleles using (NZW x PL/J)F1 x NZB (designated BWP) mice. Because the interaction of both NZB and NZW alleles is required for full expression of the disease in the BWF1 hybrid, the introduction of nonautoimmune PL/J alleles would preclude the interaction of a particular allele from both NZB and NZW strains and subsequently prevent the disease. Thus, identifying the chromosomal positions of the NZW alleles from both H-2 and non-H-2 loci and their effects on the disease phenotypes in BWP mice can serve as a model for dissecting the complex genetic interactions in the BWF1 hybrid mouse. The contribution of NZB alleles in (B x W)F1 hybrid mice has been studied before (8), but in the present study, we focused on the NZW contribution and identified a novel locus that plays a very important role in the development of murine lupus.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

NZB/BINJ, NZW/LacJ, and PL/J mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and subsequently bred and maintained in a conventional facility at the Physiology Department, National University of Singapore (Singapore). Control and experimental mice were bred and housed under the same conditions and fed an identical diet. Control mice were observed concomitantly for over 18 mo for the expression of disease and mortality. Only female mice were considered.

Mice were followed for 18 mo and survival was charted (Fig. 1Go). A protein assay kit (Bio-Rad, Hercules, CA) was used for the evaluation of proteinuria every 3 mo up to 12 mo of age. A value of 200 mg/dl or greater on two consecutive occasions was considered a positive phenotype and a value of 100 mg/dl and lower was considered a negative phenotype. The validity of using proteinuria to assess the severity of GN has been previously described (5, 9).



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  		FIGURE 1. Survival of control and experimental mice. P, W, and B represent PL/J, NZW and NZB, respectively. Closed and open triangles represent positive control (NZB x NZW)F1 and experimental (PL/J x NZW)F1 x NZB mice, respectively. Open and closed circles represent parental NZB and (PL/J x NZW)F1 mice.

 
Genotypic analysis

DNA was extracted from the mouse-tail using a standard method as described (10). Fluorescent-labeled primers for SSR (short sequence repeat) markers were obtained from Research Genetics (Huntsville, AL). Information about SSR markers can be obtained from the mouse genome database (MGD) at http://www.jax.org. Amplification of the markers was achieved by PCR in a PTC-100 thermal cycler (MJ Research, Watertown, MA) following the protocol from Research Genetics (mouse map pairs 20517.2). PCR products were analyzed on an automated sequencer (ABI Prism 377, PE; Applied Biosystems, Foster City, CA). The (PL/J x NZW)F1 x NZB mice were genotyped as BW (heterozygous for NZB and NZW) and BP (heterozygous for NZB and PL/J). We genotyped the mice for candidate regions and attempted to scan for all 19 autosomes using 75 informative SSR markers covering >70% of the mouse genome (calculated by MapManager/QT; Roswell Park Cancer Institute, Buffalo, NY).

Serological assay

Serum autoantibody (anti-dsDNA/anti-histone) levels were determined by ELISA. Abs to total histone and dsDNA were measured as previously described (2, 11, 12). All assays were performed in triplicate. Serum from lpr/lpr and NZB/W lupus mice was used as a positive control (11). Serum urea levels were also determined by ELISA using a kit (Bio-Rad).

Statistical analysis

The association of a particular locus with mortality and proteinuria was quantified by {chi}2 analysis, using a standard (2 x 2) contingency matrix as described (13), or by using the linkage program MapManager/QT (14) by scoring all progeny mice with a positive phenotype as 1 and those with a negative phenotype as 0 (6). To normalize the distributions of autoantibodies and serum urea levels, logarithmic transformation was done for serum levels of autoantibodies (anti-dsDNA and anti-histone IgG) and urea and was used as a quantitative trait for MapManager/QT analysis. Based on the recommendation of Lander and Kruglyak (15), and to avoid a false positive linkage by multiple hypotheses testing, a threshold for suggestive and probable linkage was set at log of likelihood (LOD) > 1.9 (p < 0.003; {chi}2 > 8.6; 1 df) and LOD > 3.3 (p < 1 x 10-4; {chi}2 > 15.1; 1 df), respectively. Markers were also considered to have probable linkage with a particular trait if a previously mapped marker (p < 0.01) was confirmed in the present study at p < 0.01 (15). Positions of proximal markers were obtained from the MGD map (The Jackson Laboratory).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of lupus-like renal disease and mortality in BWP mice

Forty-five percent of 164 female BWP mice generated had a high level of proteinuria at the end of 12 mo, 25% died in the first year, and 15% died between 12 and 15 mo of age (Fig. 1Go). For mortality, mice that died before 15 mo and mice that survived over 18 mo were considered positive and negative phenotypes, respectively. Mice with a high level (a value of 200 mg/dl or greater) of proteinuria before 12 mo of age on two consecutive occasions were scored positive for proteinuria. Nearly all the mice with high proteinuria died before 15 mo of age. Kidney histology was performed as previously described (16) for mice that were ill. The histology showed severe GN (data not shown) based on a 0–4 grading scale as previously described (17). Mice with a low level (a value of 100 mg/dl or lower) of proteinuria at the end of 12 mo were considered as having a negative phenotype. Nearly all the mice with low proteinuria survived over 18 mo. Therefore, there is a strong correlation between proteinuria and mortality.

Distribution of autoantibody production

Results from sera collected at 12 mo of age illustrated in Fig. 2Go, A and B, show that at this point, the frequency of the distribution of autoantibodies can be used to distinguish parental phenotypes from BWP mice. BWP mice with autoantibody levels lower than three SDs above the mean value (<0.6 OD for both anti-dsDNA and anti-histone) of female age-matched (PL/J x NZW)F1 were classified as low/negative and those with levels with a value of >0.8 OD were considered high/positive autoantibody phenotypes. To ascertain whether there was a correlation between autoantibodies with the development of severe proteinuria and mortality, both autoantibody levels were compared (Table IGo) and autoantibody production was found to be correlated with both proteinuria (p < 0.006 and p < 0.002) and mortality (p < 1 x 10-4 and p < 1 x 10-3). In addition, a number of BWP mice that died before 15 mo and were scored as positive for proteinuria did not produce high levels of autoantibodies and vice versa. Consistent with other research groups, these data indicate that the autoantibody production alone may not be sufficient as a cause of GN. It is also possible that a significant fraction of the "low antinuclear Ab" nephritis group may produce other pathogenic Abs, such as anti-gp70 that have been previously shown to associate with nephritis (18, 19). Because sera were collected at 12 mo, or at a terminal stage of the study, it is possible that some diseased mice may have decreased autoantibody production simply due to their advanced age and disease.



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  		FIGURE 2. Distribution of serum autoantibody titers in parental, ((PL/J x NZW)F1 and NZB), and experimental (PL/J x NZW)F1 x NZB mice. A, Anti-dsDNA and B, anti-histone IgG autoantibodies at 12 mo or terminal in individual BWP mice as compared with (PL/J x NZW)F1 mice. Each symbol represents the value obtained for one animal. The horizontal lines represent the mean plus three SDs for autoantibody levels in sex and age-matched (PL/J x NZW)F1 mice. The values for (NZB x NZW)F1 mice are all above the horizontal line (data not shown).

 

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  		Table I. Association of autoantibodies with proteinuria and mortality in (NZW x PL/J)F1 x NZB mice

 
Linkage of marker loci to mortality and proteinuria

The linkage analysis was done by {chi}2 analysis, using a standard (2 x 2) contingency matrix (13) or by using the linkage program MapManager/QT (14) in which the data are presented as LOD scores. Therefore, a threshold for suggestive and probable linkage was set at LOD > 1.9 (p < 0.003; {chi}2 > 8.6; 1 df) and LOD > 3.3 (p < 1 x 10-4; {chi}2 > 15.1; 1 df) respectively (15). Pearson’s {chi}2 analysis of the BWP mice for mortality revealed suggestive linkage to marker loci telomeric to the MHC locus on chromosome 17 and significant linkage to marker loci on chromosome 2, with the strongest effect at the D2 Mit285 locus ({chi}2 = 25.0; p < 1 x 10-6). We analyzed mortality as a quantitative trait (6) by using the linkage program MapManager/QT (14). Interval mapping with MapManager/QT revealed similar results (Fig. 3Go, Ch2 and Fig. 4Go, Ch2 only) for chromosome 2 with the highest peak direct to D2 Mit285 (LOD = 6.6) at {approx}86 cM (from the centromere) with a 3-LOD support interval (95% confidence value) of at least 35 cM (58–93 cM). We propose to designate this locus "Wbw1" (for NZW-NZB x NZW).



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  		FIGURE 3. {chi}2 analysis of linkage of the marker loci on chromosomes 1, 2, and 17. Only the chromosomes that showed at least suggestive linkage in (PL/J x NZW)F1 x NZB mice are shown. The x-axes represent the chromosomes and the estimated map distances (in cM) of the markers from the centromere (MGD map/JAX). The y-axes represent the p values. Suggestive (p = 1.0 x 10-3) and significant (p = 5.2 x 10-5) linkage values are indicated by the horizontal thin continuous and thick dotted lines, respectively.

 


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  		FIGURE 4. MapManager/QT interval mapping of chromosomes 1, 2, and the proximal part of 17 each containing the QTL for mortality, proteinuria, anti-dsDNA/anti-histone Abs, and serum urea. Markers are indicated on the x-axis, with the centromere at the xy junction. The distances between each marker were generated by MapManager/QT (14 ) using the Morgan function. The position of the proximal marker was obtained from the MGD map (JAX). LOD scores were calculated at 1-cM intervals by MapManager/QT and are represented on the y-axis. The horizontal broken line represents the accepted level of LOD score. Genotypic combinations revealed interaction of chromosome 2 and 17 (indicated in parentheses as fixed chromosome) for mortality (as discussed in the text). A 2-LOD support interval (95% confidence value) for each QTL is indicated by a horizontal bar below each plot. Only the values with a peak LOD score of 2.0 or above are shown.

 
The linkage of the marker loci from the MHC locus to mortality was weaker than that of chromosome 2, with the highest peak at D17 Mit177 ({chi}2 = 8.26; p < 0.005), telomeric to the MHC locus, meeting the criteria for suggestive linkage in the present study (14). The inheritance of the NZW D17 Mit177 allele provided an increase in the risk for early mortality. Of all H-2d/z mice (BW), 49% died before 15 mo compared with 29% of the H-2d/u (BP) mice. The effect of Wbw1 was highly significant in mice carrying the NZW D17 Mit177 allele ({chi}2 = 29.1; p < 1 x 10-7) compared with those carrying the PL/J allele ({chi}2 = 16.8; p < 1 x 10-4). Interval mapping with MapManager/QT revealed analogous results (Fig. 3Go, Ch17 and Fig. 4Go, Ch17) for this locus. The highest LOD score observed on chromosome 17 was 2.1 for the D17 Mit177 marker locus meeting the criteria for suggestive linkage (15). However, when the D2 Mit285 marker locus was fixed as a first quantitative trait loci (QTL) for mortality, the LOD score peak reached 2.9 and localized between marker loci D17 Mit177 and D17 Mit139 at {approx}26 cM (from the centromere) with a 2-LOD support interval of >10 cM. Therefore, the effect of Wbw1 appeared to be influenced by the NZW genotype at D17 Mit177 and we name this locus "Wbw2". Wbw2 mapped 7 and 2 cM telomeric to the H-2 and D17 Mit177 loci, respectively. In a genetic analysis of the NZB contribution to lupus-like autoimmune disease in (NZB x NZW)F1 mice, Drake et al. (5) previously reported a marker locus, D17 Mit10, (which is closely linked to Wbw2) having weak linkage (p < 0.05) with lupus-like disease. Linkage analysis of proteinuria to marker loci mapped the same two loci that had linkage with mortality: D2 Mit285 (LOD = 4.0; {chi}2 = 16.80; p < 1 x 10-4) and D17 Mit177 (LOD = 2.6; {chi}2 = 11.30; p < 1 x 10-3). There was no significant increase of LOD score at the D17 Mit177 marker locus when D2 Mit285 was fixed as a first QTL for proteinuria. These data may reflect the fact that Wbw2 is directly contributing to proteinuria whereas for mortality, Wbw2 is interacting with Wbw1. Wbw1, which has a stronger effect on early mortality compared with proteinuria and serum urea, may play a fundamental role in early death. Weak linkage was found for TNF-{alpha}. In the analysis of the MHC class II/TNF-{alpha} locus, TNFd/z (BW genotype) appears to contribute to both mortality and proteinuria. The mortality and proteinuria rates are increased (from 33 to 48% for mortality and 40 to 50% for proteinuria) in the mice carrying the Ad/u Ed/u TNFd/z allele (BW) compared with those carrying Ad/u Ed/u TNFd/u (BP). In linkage analysis of both mortality and proteinuria, no trend was found for previously identified non-MHC susceptibility loci (all Sle, Lbw, and Nba loci) in BWF1 hybrid mice. The marker loci, D1 Mit17 (represents Sle1c), D4 Mit17, and D7 Mit246, that were shown to have association to a certain extent with antinuclear autoantibody production in the present study were not found to have any trend to serum urea, proteinuria, and mortality.

Linkage of marker loci to autoantibody production and serum urea

The {chi}2 analysis of marker loci to anti-dsDNA autoantibody production revealed significant linkage to the telomeric part of chromosome 1 with the highest linkage ({chi}2 = 21.41; p < 1 x 10-5) at the D1 Mit17 marker locus. This marker locus was previously shown to have linkage (p < 0.01) with anti-ssDNA Ab production (20). However, this statistical analysis contrasts with the MapManager/QT analysis, which revealed only suggestive linkage with this locus, giving the highest peak (LOD = 2.78) at {approx}103 cM from the centromere (calculated by MapManager/QT) with a 2-LOD support interval of at least 30 cM. The discrepancy in these two statistical analyses of linkage for autoantibody production could be explained by the fact that {chi}2 analysis was done based on qualitative, but not quantitative, differences and the comparison was done between two extreme phenotypes. The other possibility is that some diseased animals may have a decrease in autoantibody production (because sera were collected at 12 mo, or at a terminal stage of the study) or may produce other pathogenic autoantibodies such as anti-gp70, which leads to such discrepancy in these two statistical analyses. Although this genomic interval meets the criteria for suggestive linkage in the present study, our results fulfill the criteria for "confirmed linkage" (15) because multiple susceptibility loci were previously identified in this region that mostly cause a loss of tolerance to nuclear constituents (4, 6). Through fine mapping analysis of the location of Sle1 locus, Morel et al. (21) have identified three loci within this genomic interval, termed Sle1a, Sle1b, and Sle1c and have demonstrated that each of these loci independently causes a loss of tolerance to chromatin. The locus identified in the present study overlaps with Sle1c. Weak linkage was found to marker locus D7 Mit246 ({chi}2 = 4.74; p < 0.03), which mapped between Sle3 and Sle5 loci on chromosome 7 (6). A similar locus was previously identified in Yaa (Y-linked autoimmune acceleration) gene-induced lupus-like nephritis in (NZW x C57BL/6)F1 mice (22). For anti-histone Ab production, we identified weak linkage to two loci: one colocalized with D1 Mit17 ({chi}2 = 7.66; p < 7 x 10-3), which had the strongest effect on anti-dsDNA autoantibody production, and the other was D4 Mit17 ({chi}2 = 7.26; p < 7 x 10-3) which is located 13 cM centromeric to the Sle2 locus. MapManager/QT analysis for anti-nuclear Ab production revealed two weak peaks (a LOD score of 1.8 for both anti-dsDNA and anti-histone Abs) at {approx}73 cM on chromosome 2 (data not shown). Wbw2 (D17 Mit177 NZW allele) on chromosome 17 that were shown to have linkage with proteinuria and mortality were not found to have any linkage with autoantibody production.

MapManager/QT analysis of serum urea (data not shown) revealed suggestive linkage (LOD = 2.72) with the highest peak closely linked to marker D2 Mit194 at {approx}82 cM from the Wbw1 genomic interval on chromosome 2 that had significant linkage for proteinuria and mortality. None of the other loci (Sle1c, Sle1, Sle2, Sle3, or Wbw2) had linkage with serum urea level.

Multigenic effect of NZW alleles to SLE susceptibility

As detailed above, we identified three loci that mainly contribute to the disease. The inheritance of NZW alleles at these three loci contributes to different magnitudes to SLE susceptibility. Mortality and proteinuria rates are higher, 52% (both) for Wbw1, 29 (mortality) and 42% (proteinuria) for Wbw2, and 12.5 (mortality) and 31% (proteinuria) for Sle1c. Even though Wbw1 appears to play a major role in both mortality and proteinuria for BWP mice carrying the NZW allele at Wbw1, mortality (52%) and proteinuria (52%) remained lower than those observed in (NZB x NZW)F1 hybrid mice (> 90%). This indicates the requirement of other genes.

The multigenic effect of NZW alleles to SLE susceptibility was analyzed by subgrouping BWP mice carrying 0, 1, 2, and 3 susceptibility genes identified in the present study (Table IIGo). None of the 17 mice carrying the PL/J allele at Wbw1, Wbw2, and Sle1c developed the disease. The average proteinuria and mortality rate with one gene was 52% (both) for Wbw1, 42 and 29% for Wbw2, and 31 and 12.5% for Sle1c, with two genes 56 and 53% for Wbw1 plus Wbw2, 50% each for Wbw1 plus Sle1c, and 35 and 30% for Sle1c plus Wbw2, respectively. More than 80% of mice carrying the NZW allele at these three loci had full expression of the disease. It appears that the combined effect of Wbw1, Wbw2, and Sle1c susceptibility loci is sufficient to induce the disease almost at the level that is observed in (NZB x NZW)F1 mice and none of these three loci alone is necessary for the disease. Even though the number is small, all eight mice carrying NZW alleles at all five loci including marker loci D4 Mit17 and D7 Mit246 that had weak linkage for antinuclear Ab production developed severe proteinuria and died early (data not shown). These data indicate that the frequency of high proteinuria and early mortality increases as the number of NZW alleles increases in individual BWP mice and the SLE susceptibility is incrementally increased by the presence of each susceptibility allele.


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  		Table II. The multigenic effect of SLE susceptibility in (NZW x PL/J)F1 x NZB mice

 
The combined effects of Wbw1 and previously described SLE susceptibility loci in BWP mice

The combined effects of Wbw1 and previously described SLE-susceptibility loci (Sle and Lbw loci) were also analyzed in multigenic combinations in a similar manner to that shown in Table IIGo. In Table IIIGo, the combined effects of Wbw1 and previously described Sle susceptibility loci are shown. Even though one mouse had high proteinuria, none of the seven mice carrying the PL/J allele at Wbw1, Sle1, Sle2, and Sle3 died early. In this combination, average proteinuria and mortality rate with one gene was 54 and 66% for Wbw1, 50and 40% for Sle1, 50 and 25% for Sle2, and 38 and 33% for Sle3, with a combination of Wbw1 and each of the Sle loci 60% (both) for Wbw1 plus Sle1, 56% (both) for Wbw1 plus Sle2, 54% and 63% for Wbw1 plus Sle3 respectively, and with combination of Wbw1 and two or three of the Sle susceptibility loci, the incidence of the disease remained almost the same. It is shown in Table IIIGo that in the presence of Wbw1, the contribution of Sle loci (Sle1, Sle2, and Sle3) is not obvious but in the absence of Wbw1 (or in the presence of the PL/J allele), the NZW alleles at all Sle susceptibility loci are contributing to the disease to certain extents, indicating a unique contribution of Wbw1 to the pathogenic process. Furthermore, none of the mice (carrying the PL/J allele at Wbw1 and Sle1 and the NZW allele at Sle2 and Sle3) developed the disease, suggesting the existence of protective PL/J alleles or the loss of recessiveness between NZB and NZW alleles at these two loci. Analogous results for the combined effects of Wbw1 and previously described (4) Lbw susceptibility loci were also observed (data not shown).


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  		Table III. The combined effect of Wbw1 with previously described SLE susceptibility loci in (NZW x PL/J)F1 x NZB mice

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Similar to other studies (2, 4, 20), we found the H-2 region contributing to the development of SLE in our studies. Our data for the H-2 locus are consistent with a previous study by Fujimura et al. (23). Using NZW H-2 congenic strains, these authors showed that compared with (NZB x NZW.H-2d)F1 (H-2d/d: Ad/d Ed/d TNFd/d) homozygous mice, (NZB x NZW.PL)F1 (H-2d/u: Ad/u Ed/u TNFd/d) heterozygous mice developed more severe disease, strongly suggesting the contribution of the u haplotype of H-2 class II genes. In the present study, the lack of association of the TNF locus (which is tightly linked to H-2) suggests the contribution of the u haplotype of a class II (I-A and I-E) gene shared between the NZW and PL/J strains. Furthermore, the incidence of disease is higher (48% for mortality and 50% for proteinuria) in mice carrying the Ad/u Ed/u TNFd/z alleles than in those with Ad/u Ed/u TNFd/u (33% for mortality and 40% for proteinuria), indicating the contribution of a unique polymorphism in the TNF-{alpha} NZW allele. Therefore, our data support the hypothesis that both H-2 class II and TNF-{alpha} gene polymorphisms act as H-2-linked predisposing genetic elements for the development of the disease. Our data also agree with the conclusions reached in previous studies (24, 25) in which both transgenes Ez and Az did not show any association with the development of nephritis but the inheritance of the entire H-2z locus (which includes both H-2 genes and TNF-{alpha}) was linked to nephritis. Some investigators proposed that mixed haplotype class II molecules such as A{alpha}dA{beta}z, E{alpha}dE{beta}z, or A{alpha}dE{beta}z are capable of a unique interaction with disease-inducing peptides and/or TCRs (26, 27, 28, 29). However, an A{beta}z transgene, in the mixed haplotype manner, was not linked to the disease in (NZB x NZW.H-2d)F1 mice (26). This indicates that mixed haplotype class II molecules alone may not be sufficient to initiate lupus-like renal disease, and strongly suggests the contribution of TNF-{alpha} polymorphism, which may act to modulate the initial steps of disease development. The contribution of TNF-{alpha} to lupus nephritis has been previously demonstrated (30, 31). It has been shown that increased TNF-{alpha} production and TNF-{alpha} injections reduce lupus-like nephritis whereas anti-TNF-{alpha} treatment resulted in increased disease (30). This was further supported by a recent study (32) in which it was shown that a reduced expression of the TNF-{alpha} gene in (NZBxB6, 129Tnf0)F1 hemizygous mice resulted in the development of autoimmunity, which was consistent with the study by Fujimura et al. (23). The Wbw2 locus (telomeric to H-2) which was not linked with autoantibody production might play a role in determining lupus susceptibility, suggesting the clustering of functionally related H-2 and non-H-2 genes in the H-2 region on chromosome 17.

The NZW alleles that we identified might be recessive to NZB alleles but we cannot rule out the possibility that these genes act in a dominant or codominant manner, because the BW genotype is associated with disease. The level of correlation with disease was particularly strong at Wbw1, which mapped to the distal part of chromosome 2. This is the most significant finding of our study and was not previously reported using the (NZB x NZW)F1 hybrid model. It does overlap well with an SLE locus recently identified in an (MRL/lpr x BALB/cJ)F2 intercross (33) although the highest peak in our study is >10 cM distal. The same region was also shown to contain a locus linked to type 1 diabetes, Idd13 (34). Previously, several groups failed to detect this locus in classical backcross or intercross studies of combinations of New Zealand mice, in the presence or absence of alleles from normal strains (SM/J, B6). Thus, it is possible that a unique genetic environment exists due to the presence of a PL/J allele or a set of alleles that reveal this locus. These data also argue for a dominant protective PL/J allele. IL-1, which encodes the pleiotropic cytokine (IL-1) involved in the maintenance of homeostasis as well as the progression of inflammatory disease, has been mapped in the Wbw1 genomic interval (Fig. 5Go, chromosome 2), which makes IL-1 a very interesting candidate gene for the locus on the distal part of chromosome 2. It has been reported previously that macrophages (M{phi}) from two genetically prone murine lupus models, (NZB x NZW)F1 and MRL+/+, have a defect in the production of IL-1 (35, 36). This was further supported by another study by the same group, in which it was shown that M{phi} from young (NZB x NZW)F1 and MRL+/+ mice had defects not only in the production of IL-1, but also in the production of other proinflammatory cytokines such as TNF-{alpha}. IL-6 and defective TNF-{alpha} production appeared to be responsible for defective IL-1 and IL-6 production (37).



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  		FIGURE 5. A map of mouse chromosome 1 and chromosome 2 showing the linkage for loci linked with murine SLE phenotypes. The distance of markers from the centromere in cM is shown at the left side of each figure. Numbers at the right side of the figure indicate the distance in cM between adjacent markers as calculated by MapManager/QT with a 95% confidence value. The probable candidate genes contained in the support interval are depicted at the right side of each figure (italicized).

 
The second non-H-2 locus identified by us and closely linked to D1 Mit17, mapped distal to all susceptibility loci (Sle1/Nba2/Lbw7) that had been identified previously on the telomeric part of chromosome 1 (Fig. 5Go, chromosome 1). This locus has a comparatively stronger effect on anti-dsDNA autoantibody production than on anti-histone Abs. The D1 Mit17 marker locus was previously shown to have linkage (p < 0.01) with anti-ssDNA (20). Lbw7 (dominant NZB) was shown to have linkage with anti-chromatin Ab production with the highest peak at D1 Mit36 (4). Distal to this was Nba2 (NZB-derived), which was shown to have linkage with nephritis (8, 9). An NZB locus at a similar position (Nba2) was also linked with nephritis (38). Proximal to Lbw7 was Sle1 (NZW origin), which was shown to have linkage with GN initially (6) and subsequently was shown to have association with selective loss of tolerance to a limited range of subnucleosomal (H-2A/H-2B/DNA) Ags (39). B6.NZMc1 congenic for Sle1 contains a genomic interval {approx}37 cM long (NZW origin), which basically includes all four susceptibility loci (Sle1, Lbw7, Nba2 and the locus identified in the present study) (40, 41). Accumulated data from different research groups and our study suggest that the telomeric part of chromosome 1 might contain multiple SLE-susceptibility genes, which are derived from both NZB and NZW. This hypothesis was confirmed recently by Morel et al. (21). Through fine mapping of the location of Sle1, they have identified a cluster of nonoverlapping SLE susceptibility loci (termed Sle1a, Sle1b, and Sle1c) that can independently cause a loss of tolerance to chromatin (21). However, these three loci differ with respect to their effect on various other cellular and serological phenotypes, which suggests that each of these susceptibility loci may contribute to a unique pathogenic process. The locus identified by us overlaps with Sle1c, which is consistent with the data published by Morel et al. (21). CR2, which is located at the telomeric end of chromosome 1 (close to the D1 Mit17 marker locus) and plays a very important role in humoral immune response, is a possible candidate gene for Sle1c because Sle1c is associated with autoantibody production in the present study. The expression of CR2 was shown to be significantly decreased in human SLE patients (42) and in MRL/lpr mice (43).

SLE is a complex polygenic trait. In the BWF1 hybrid mouse model of SLE, both NZB and NZW genomes contribute to lupus-like renal disease. Our approach to unravel the genetic contribution from the NZW strain has resulted in the discovery of a new susceptibility locus. We could observe the effects of the marker loci (genes) that were not observed in conventional backcross or intercross studies, probably due to the masking effect of dominant genes. Thus, we identified a novel locus (Wbw1) that plays a crucial role in the development of SLE in (PL/J x NZW)F1 x NZB mice. Neither Wbw1 on chromosome 2 nor Wbw2 on chromosome 17 had any influence on B cell tolerance, but they did have influence on kidney pathology and mortality. Therefore, we speculate that these alleles result in dysregulation of proinflammatory cytokines (such as IL-1 and TNF-{alpha}) and their synergistic effects, contributing to lupus-like nephropathy, and eventually death, in BWP mice. In contrast, Sle1c leads to the breakdown of tolerance. Different types of autoantibodies such as anti-dsDNA, anti-histone, or anti-gp70 all could combine with dysregulated cytokine effects to result in disease in the (NZB x NZW)F1 hybrid mouse model of SLE.


   Acknowledgments
 
We thank Dr. Timothy Manser (Kimmel Cancer Center, Jefferson Medical College, Philadelphia, PA) for his help in editing the manuscript. We also thank Dr. Chong Siew Meng from the Department of Pathology, National University Hospital, Singapore, for his kind support.


   Footnotes
 
1 This work was supported by the National Medical Research Council, Singapore, Grant R185000014112. Back

2 Current address: Kimmel Cancer Center, Jefferson Medical College, Bluemle Life Sciences Building 708, 233 South 10th Street, Philadelphia, PA 19107. Back

3 Address correspondence and reprint requests to Dr. Dow-Rhoon Koh, Department of Physiology, National University of Singapore, 2 Medical Drive, Singapore 117597. E-mail address: phskohdr{at}nus.edu.sg Back

4 Abbreviations used in this paper: SLE, systemic lupus erythematosus; GN, glomerulonephritis; SSR, short sequence repeat; MGD, mouse genome database; LOD, log of likelihood; QTL, quantitative trait loci. Back

Received for publication March 9, 2001. Accepted for publication January 8, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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