The lupus-like disease that develops in hybrids of NZB and NZW mice is genetically complex, involving both MHC- and non-MHC-encoded genes. Studies in this model have indicated that the H2d/z MHC type, compared with H2d/d or H2z/z, is critical for disease development. C57BL/6 (B6) mice (H2b/b) congenic for NZB autoimmunity 2 (Nba2), a NZB-derived susceptibility locus on distal chromosome 1, produce autoantibodies to nuclear Ags, but do not develop kidney disease. Crossing B6.Nba2 to NZW results in H2b/z F1 offspring that develop severe lupus nephritis. Despite the importance of H2z in past studies, we found no enhancement of autoantibody production or nephritis in H2b/z vs H2b/b B6.Nba2 mice, and inheritance of H2z/z markedly suppressed autoantibody production. (B6.Nba2 × NZW)F1 mice, compared with MHC-matched B6.Nba2 mice, produced higher levels of IgG autoantibodies to chromatin, but not to dsDNA. Although progressive renal damage with proteinuria only occurred in F1 mice, kidneys of some B6.Nba2 mice showed similar extensive IgG and C3 deposition. We also studied male and female B6.Nba2 and F1 mice with different MHC combinations to determine whether increased susceptibility to lupus among females was also expressed within the context of the Nba2 locus. Regardless of MHC or the presence of NZW genes, females produced higher levels of antinuclear autoantibodies, and female F1 mice developed severe proteinuria with higher frequencies. Together, these studies help to clarify particular genetic and sex-specific influences on the pathogenesis of lupus nephritis.

Systemic lupus erythematosus (SLE)5 is a systemic autoimmune disease with diverse and variable clinical manifestations, including the development of an immune complex-mediated glomerulonephritis (reviewed in Refs.1 and 2). Although SLE can occur at nearly any age, women in their childbearing years show an increased incidence, suggesting that estrogens enhance disease development. The hallmark of lupus is elevated serum levels of IgG antinuclear Abs. F1 hybrids of NZB and NZW mice (BWF1 mice) develop severe glomerulonephritis associated with high serum levels of IgG antinuclear autoantibodies, remarkably similar to human SLE (3). Additionally, female BWF1 mice are more prone to disease than males, which is also similar to the sex-specific susceptibility observed in human SLE patients (4, 5, 6).

The genetic basis of disease in BWF1 mice is complex, and no particular gene is necessary or sufficient for disease expression (7, 8). However, the MHC and an NZB lupus susceptibility locus on distal chromosome 1 (named Nba2 for NZB autoimmunity 2) have shown consistent linkage with lupus traits in multiple mapping studies (7, 9, 10, 11, 12, 13, 14). In one cross, Nba2 in combination with the H2d/z MHC haplotype, accounted for >90% of the genetic contribution to IgG autoantibody production and lupus nephritis (kidney disease) (11). Genetic linkage and association studies have demonstrated that MHC heterozygosity is disease enhancing compared with homozygosity for either the H2d (NZB) or H2z (NZW) haplotype in BWF1 animals (7, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20).

The autoimmune phenotype in B6 mice congenic for the Nba2 interval (B6.Nba2 mice) is characterized by elevated serum levels of IgG antinuclear autoantibodies (21). However, these mice do not develop manifestations of severe lupus nephritis, including proteinuria or renal insufficiency. In contrast, (B6.Nba2 × NZW)F1 mice produce autoantibodies and develop nephritis, indicating that MHC heterozygosity, NZW-encoded non-MHC genes, or both are required in addition to the Nba2 interval for full disease penetrance. The studies described herein explore the effect of different MHC haplotypes and heterozygosity on disease expression in Nba2 congenic mice with and without the addition of NZW-encoded non-MHC genes. In addition, both male and female mice were evaluated to determine whether Nba2 congenic mice maintain the female sex-dependent susceptibility that has been observed in BWF1 mice.

The results indicate that H2b/z heterozygosity in the Nba2 congenic is not sufficient for the development of severe kidney disease. In addition, homozygosity for H2z in (B6.Nba2 × NZW)F1 mice prevented antinuclear Ab production and nephritis. Comparison of IgG autoantibody production, immune complex (IC), and complement deposition in the kidney and severe proteinuria in MHC-matched B6.Nba2 and (B6.Nba2 × NZW)F1 mice provided additional insight into the role of NZW-encoded, non-MHC genes. Furthermore, female mice produced greater amounts of autoantibodies and were more susceptible to lupus nephritis than male mice in each genetic system. These studies provide further insight into the impact of the MHC on the disease process and also provide a simplified genetic system for understanding sex-related differences in lupus susceptibility and lupus pathogenesis.

NZW (H2z/z), NZB (H2d/d), and B6 (H2b/b) mice were obtained from The Jackson Laboratory and were maintained in the animal care facility at University of Colorado Health Sciences Center (Denver, CO) in accordance with guidelines approved by the animal care and use committee. B6.Nba2 congenic mice were generated by backcrossing an NZB interval on distal chromosome 1 to the B6 strain as previously described (21). B6.H2d/d mice were obtained from The Jackson Laboratory, and B6 mice were made congenic for the NZW H2z/z (B6.H2z/z) as previously described (22).

Study mice were followed for the development of lupus nephritis by measuring proteinuria at monthly intervals as previously described (9, 11, 14). Mice with ≥2+ (100 mg/dl) on at least two consecutive occasions before 12 mo of age were designated as positive for high grade proteinuria and severe renal disease. The validity of using proteinuria to document severe renal disease and predict mortality from renal failure in New Zealand hybrid mice has been previously demonstrated (9, 11, 14).

Serum autoantibody levels to chromatin, dsDNA, ssDNA, and total histones were determined by ELISA as previously described (13, 14). Serum samples were tested at a dilution of 1/300. All assays were performed in duplicate, and OD determinations were converted to units per milliliter by comparison with a standard curve obtained with mAbs to the appropriate nuclear Ag as previously described (13, 14). Animals were considered positive for antinuclear Abs if levels were 2 SD above the median levels in age-matched B6 animals. Serum levels of total IgG were also determined by ELISA as previously described (13) using sera diluted 1/100,000. OD determinations were converted to units per milliliter by comparison with a standard curve obtained with known concentrations of polyclonal IgG (MP Biomedical).

Kidneys were snap-frozen in Tissue-Tek (OCT) and were kept at −70°C until sectioning. Sections were blocked with 10% nonimmune goat serum before staining. Five-micron sections were cut, fixed in acetone, and stained for total IgG deposition using FITC-conjugated rabbit anti-mouse IgG Abs (MP Biomedicals). Staining intensity was evaluated using ImagePro software (version 4.5; Media Cybernetics) on digitalized images acquired through a Pixera CL600 color CCD camera and an Olympus BX51 microscope. All pictures were taken at identical conditions. Blinded scores were determined for IgG and complement C3 staining using the following 0–4 scale: 0, no detectable staining; 0.5, trace staining in mesangium only; 1, staining in mesangium only (<50% of glomeruli); 2, staining in mesangium only (>50% of glomeruli); 3, strong staining in mesangium (>50% of glomeruli) with occasional staining of capillary loops; and 4, strong staining in mesangium (>50% of glomeruli) with widespread staining of capillary loops.

The statistical significance of differences in autoantibody levels and degree of IgG deposition between groups of mice was determined using the nonparametric Mann-Whitney U test. A survival curve comparison analysis was used to determine the statistical significance of proteinuria rates between different groups of mice.

Many studies have demonstrated the importance of MHC haplotype in mouse lupus, especially in the BWF1 model (7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). Therefore, we assessed autoantibody production in B6.Nba2 congenic mice bearing different relevant MHC haplotypes. Like H2b/b B6.Nba2 controls, H2b/z B6.Nba2 mice produced high levels of autoantibodies, with 100, 73, 50, and 45% of the mice positive for autoantibodies to chromatin, histone, ssDNA, and dsDNA, respectively. Importantly, heterozygosity at the MHC with H2z neither enhanced nor decreased autoantibody production, because there was no difference in autoantibody production between H2b/b and H2b/z B6.Nba2 mice (with the exception of anti-histone Abs) or between H2d/d and H2d/z B6.Nba2 mice (Fig. 1). A much smaller percentage of H2d/d and H2d/z mice were positive for autoantibodies, with overall levels similar to those in Nba2-negative mice. Serum levels of total IgG were slightly greater in H2b/b (3.9 ± 0.37 U/ml) and H2d/d (4.2 ± 0.31) compared with H2b/z B6.Nba2 mice (2.2 ± 0.17) and compared with Nba2-negative B6 mice (1.6 ± 0.21).

FIGURE 1.

MHC contribution to autoantibody production in B6.Nba2 mice. Serum samples from 7-mo-old B6.Nba2 and B6 mice bearing the indicated MHC haplotypes were analyzed for IgG autoantibodies to dsDNA (A), ssDNA (B), chromatin (C), and histone (D). H2b/z (n = 11) and H2d/z (n = 8) B6.Nba2 mice did not produce greater amounts of autoantibodies than H2b/b B6.Nba2 mice (n = 16). Comparisons between H2b/b B6.Nba2 and B6 were significantly different for IgG autoantibodies to dsDNA (p = 0.004), ssDNA (p = 0.004), chromatin (p = 0.0001), and total histones (p = 0.0001). H2d/d B6.Nba2 mice (n = 6), compared with B6 mice (n = 16), produced significantly higher levels of IgG autoantibodies to dsDNA (p = 0.01), chromatin (p = 0.01), and ssDNA (p = 0.02), but not to total histones.

FIGURE 1.

MHC contribution to autoantibody production in B6.Nba2 mice. Serum samples from 7-mo-old B6.Nba2 and B6 mice bearing the indicated MHC haplotypes were analyzed for IgG autoantibodies to dsDNA (A), ssDNA (B), chromatin (C), and histone (D). H2b/z (n = 11) and H2d/z (n = 8) B6.Nba2 mice did not produce greater amounts of autoantibodies than H2b/b B6.Nba2 mice (n = 16). Comparisons between H2b/b B6.Nba2 and B6 were significantly different for IgG autoantibodies to dsDNA (p = 0.004), ssDNA (p = 0.004), chromatin (p = 0.0001), and total histones (p = 0.0001). H2d/d B6.Nba2 mice (n = 6), compared with B6 mice (n = 16), produced significantly higher levels of IgG autoantibodies to dsDNA (p = 0.01), chromatin (p = 0.01), and ssDNA (p = 0.02), but not to total histones.

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B6.Nba2 (H2b/b) mice produced high levels of IgG antinuclear autoantibodies, yet did not develop lupus nephritis. However, when these congenic mice were crossed to NZW (H2z/z), severe lupus nephritis ensued. To determine whether the NZW contribution to lupus nephritis could be explained by inheritance of the H2z allele alone, we compared the incidence of severe proteinuria between H2b/b and H2b/z B6.Nba2 mice and (B6.Nba2 × NZW)F1 mice. Beginning at 5–6 mo, the percentage of (B6.Nba2 × NZW)F1 (H2b/z) mice with severe proteinuria progressively increased, and at 12 mo of age, 80% of these mice had developed severe proteinuria (Fig. 2). In contrast, by 12 mo of age, <10% of the H2b/b and H2b/z B6.Nba2 mice had developed severe proteinuria. These data indicate that inheritance of the H2b/z MHC haplotype without additional NZW-encoded genes is not sufficient for the development of lupus nephritis in this model.

FIGURE 2.

Development of glomerulonephritis in H2b/z (B6.Nba2 × NZW)F1 and MHC-matched B6.Nba2 mice. (B6.Nba2 × NZW)F1 (n = 10), H2b/z B6.Nba2 (n = 11), and H2b/b B6.Nba2 (n = 16) mice were followed monthly for evidence of severe proteinuria over a 12-mo follow-up period. The development of disease in F1 mice was significantly greater than that in MHC-matched (H2b/z) B6.Nba2 mice or H2b/b B6.Nba2 mice (p < 0.0001).

FIGURE 2.

Development of glomerulonephritis in H2b/z (B6.Nba2 × NZW)F1 and MHC-matched B6.Nba2 mice. (B6.Nba2 × NZW)F1 (n = 10), H2b/z B6.Nba2 (n = 11), and H2b/b B6.Nba2 (n = 16) mice were followed monthly for evidence of severe proteinuria over a 12-mo follow-up period. The development of disease in F1 mice was significantly greater than that in MHC-matched (H2b/z) B6.Nba2 mice or H2b/b B6.Nba2 mice (p < 0.0001).

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Potential mechanisms for how NZW-encoded non-MHC genes might enhance the development of glomerulonephritis in (B6.Nba2 × NZW)F1 mice include 1) enhanced autoantibody production leading to increased IC deposition; 2) production of additional pathogenic autoantibody specificities with increased IC deposition; 3) increased susceptibility to IC deposition in the kidney; and 4) enhanced damage-inducing inflammatory responses to deposited ICs (23). To help differentiate among these possibilities, we first compared the levels of IgG antinuclear autoantibodies in H2b/z B6.Nba2 and (B6.Nba2 × NZW)F1 mice. (B6.Nba2 × NZW)F1 mice produced more anti-chromatin (p = 0.003), anti-histone (p = 0.003), and anti-ssDNA (p = 0.05) Abs, but not anti-dsDNA Abs (Fig. 3 A). F1 mice also had elevated total IgG levels compared with MHC-matched B6.Nba2 mice (6.2 ± 0.43 vs 2.2 ± 0.17 U/ml).

FIGURE 3.

Autoantibody levels and immune complex deposition in H2b/z (B6.Nba2 × NZW)F1 and MHC-matched B6.Nba2 mice. A, IgG autoantibody levels at 7 mo of age in (B6.Nba2 × NZW)F1 (n = 8) and H2b/z B6.Nba2 (n = 11) mice. F1 mice produced significantly higher levels of autoantibodies to total histones (p = 0.003), chromatin (p = 0.003), and ssDNA (p = 0.05), but not to dsDNA. B, Examples of immunofluorescence analysis for glomerular IgG and C3 in the kidneys of different strains of lupus and control mice. B6 and B6.Nba2 kidney samples were taken at 1 year. (B6.Nba2 × NZW)F1 kidney samples were taken immediately after mice tested positive for severe proteinuria (ages ranged from 4 to 11 mo). B6.Nba2 mice were positive for IgG and C3 deposition in the kidney even though they do not go on to develop glomerulonephritis. C, Analysis of IgG deposition in kidneys of (B6.Nba2 × NZW)F1 and B6.Nba2 mice. The extent of IgG deposition in B6.Nba2 glomeruli was much greater than that in B6 mice (p = 0.02) and overlapped with that observed in (B6.Nba2 × NZW)F1 mice (p = 0.11).

FIGURE 3.

Autoantibody levels and immune complex deposition in H2b/z (B6.Nba2 × NZW)F1 and MHC-matched B6.Nba2 mice. A, IgG autoantibody levels at 7 mo of age in (B6.Nba2 × NZW)F1 (n = 8) and H2b/z B6.Nba2 (n = 11) mice. F1 mice produced significantly higher levels of autoantibodies to total histones (p = 0.003), chromatin (p = 0.003), and ssDNA (p = 0.05), but not to dsDNA. B, Examples of immunofluorescence analysis for glomerular IgG and C3 in the kidneys of different strains of lupus and control mice. B6 and B6.Nba2 kidney samples were taken at 1 year. (B6.Nba2 × NZW)F1 kidney samples were taken immediately after mice tested positive for severe proteinuria (ages ranged from 4 to 11 mo). B6.Nba2 mice were positive for IgG and C3 deposition in the kidney even though they do not go on to develop glomerulonephritis. C, Analysis of IgG deposition in kidneys of (B6.Nba2 × NZW)F1 and B6.Nba2 mice. The extent of IgG deposition in B6.Nba2 glomeruli was much greater than that in B6 mice (p = 0.02) and overlapped with that observed in (B6.Nba2 × NZW)F1 mice (p = 0.11).

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We next assessed the extent of IgG and C3 deposition in the kidneys of B6.Nba2 (H2b/b and H2b/z) mice compared with (B6.Nba2 × NZW)F1 mice. Surprisingly, both 12-mo-old proteinuria-free B6.Nba2 mice and 7-mo-old proteinuria-positive F1 mice showed extensive IgG and C3 deposition in glomeruli in both mesangial and capillary loop areas (Fig. 3,B). As expected, B6 (negative control) glomeruli exhibited only trace amounts of IgG or C3 deposition (Fig. 3,B). The extent of kidney IgG deposition was scored on a 0–4 scale. Overall, scores were greater for F1 mice (Fig. 3 C). However, scores overlapped between the two groups of mice, despite the nonoverlapping propensities to develop severe proteinuria.

The above-described studies indicated that NZW-encoded non-MHC genes are required for renal disease in the context of Nba2. We therefore studied the influence of H2b/z, H2d/z, and H2z/z MHC haplotypes on autoantibody production and kidney disease in (B6.Nba2 × NZW)F1 mice. As shown in Fig. 4,A, the frequency of severe proteinuria in H2b/z (B6.Nba2 × NZW)F1 mice progressed gradually, such that 80% of the mice were positive by 12 mo. In a similar pattern, disease progressively developed in H2d/z (B6.Nba2 × NZW)F1 mice, with 44% positive by 12 mo. In contrast, none of the H2z/z (B6.Nba2 × NZW)F1 mice developed disease (p = 0.002). No (B6 × NZW)F1 mouse in the control groups bearing any of these haplotypes developed severe renal disease during the 12-mo study period (Fig. 4 A), confirming that disease development in F1 mice was dependent on the presence of Nba2.

FIGURE 4.

Effect of MHC haplotype on the development of lupus nephritis and autoantibody production. A, (B6.Nba2 × NZW)F1 mice homozygous for the H2z MHC haplotype (n = 10) did not develop severe proteinuria compared with mice inheriting H2b/z (n = 8) and H2d/z (n = 9). Nba2-negative mice also did not develop lupus nephritis (H2b/z, n = 10; H2d/z, n = 12; H2z/z, n = 9). The difference in proteinuria between H2b/z and H2d/z (B6.Nba2 × NZW)F1 mice was not statistically significant (p = 0.13). Serum samples from 7-mo-old (B6.Nba2 × NZW)F1 and (B6 × NZW)F1 mice with the indicated MHC haplotypes were analyzed for IgG autoantibodies to dsDNA (B), ssDNA (C), chromatin (D), and histone (E). (B6.Nba2 × NZW)F1 mice homozygous for the H2z MHC haplotype did not produce significant levels of autoantibodies compared with heterozygous mice. (B6.Nba2 × NZW)F1 mice with the MHC haplotypes of H2d/z and H2b/z produced more anti-ssDNA (p = 0.001 and p = 0.0002), anti-chromatin (p = 0.0004 and p = 0.0001), and anti-histone (p = 0.0001 and p = 0.0001) autoantibodies than (B6.Nba2 × NZW)F1H2z/z mice.

FIGURE 4.

Effect of MHC haplotype on the development of lupus nephritis and autoantibody production. A, (B6.Nba2 × NZW)F1 mice homozygous for the H2z MHC haplotype (n = 10) did not develop severe proteinuria compared with mice inheriting H2b/z (n = 8) and H2d/z (n = 9). Nba2-negative mice also did not develop lupus nephritis (H2b/z, n = 10; H2d/z, n = 12; H2z/z, n = 9). The difference in proteinuria between H2b/z and H2d/z (B6.Nba2 × NZW)F1 mice was not statistically significant (p = 0.13). Serum samples from 7-mo-old (B6.Nba2 × NZW)F1 and (B6 × NZW)F1 mice with the indicated MHC haplotypes were analyzed for IgG autoantibodies to dsDNA (B), ssDNA (C), chromatin (D), and histone (E). (B6.Nba2 × NZW)F1 mice homozygous for the H2z MHC haplotype did not produce significant levels of autoantibodies compared with heterozygous mice. (B6.Nba2 × NZW)F1 mice with the MHC haplotypes of H2d/z and H2b/z produced more anti-ssDNA (p = 0.001 and p = 0.0002), anti-chromatin (p = 0.0004 and p = 0.0001), and anti-histone (p = 0.0001 and p = 0.0001) autoantibodies than (B6.Nba2 × NZW)F1H2z/z mice.

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The lack of lupus nephritis in the H2z/z (B6.Nba2 × NZW)F1 animals correlated with low IgG autoantibody production (Fig. 4, B–E). Only a few mice had anti-chromatin autoantibodies slightly above background, and none was positive for autoantibodies to dsDNA, ssDNA, or total histones. In contrast nearly all H2b/z and H2d/z (B6.Nba2 × NZW)F1 mice were positive for autoantibodies to chromatin (Fig. 4,D). In addition, 63 and 78% were positive for anti-dsDNA autoantibodies, 75 and 67% for anti-ssDNA autoantibodies, and 100 and 56% for anti-histone autoantibodies, respectively (Fig. 4, B–E). Only rare F1 mice without Nba2 developed elevated IgG autoantibody levels.

Past studies have shown that female BWF1 mice exhibit a greater susceptibility to autoantibody production and lupus nephritis than male mice (4, 5, 6). To determine whether the lupus-like disease associated with the Nba2 locus (derived from the NZB parent) still maintains this influence of gender, autoantibody levels and the incidence of proteinuria in both male and female animals were assessed. The current studies showed that female B6.Nba2 mice produced higher levels of anti-dsDNA (p = 0.03), anti-ssDNA (p = 0.02), anti-chromatin (p = 0.03), and anti-histone autoantibodies (p = 0.05) than male B6.Nba2 mice (Fig. 5).

FIGURE 5.

Autoantibody production in female vs male B6.Nba2 mice. Serum samples from 7-mo-old female and male B6.Nba2 mice were analyzed for IgG autoantibodies to dsDNA (A), ssDNA (B), chromatin (C), and histone (D). Female B6.Nba2 mice (n = 16) produced higher levels of anti-dsDNA (p = 0.03), anti-ssDNA (p = 0.02), anti-chromatin (p = 0.03), and anti-histone (p = 0.05) autoantibodies than B6.Nba2 male mice (n = 20).

FIGURE 5.

Autoantibody production in female vs male B6.Nba2 mice. Serum samples from 7-mo-old female and male B6.Nba2 mice were analyzed for IgG autoantibodies to dsDNA (A), ssDNA (B), chromatin (C), and histone (D). Female B6.Nba2 mice (n = 16) produced higher levels of anti-dsDNA (p = 0.03), anti-ssDNA (p = 0.02), anti-chromatin (p = 0.03), and anti-histone (p = 0.05) autoantibodies than B6.Nba2 male mice (n = 20).

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Enhancement of autoantibody production in females was also apparent in (B6.Nba2 × NZW)F1 mice (Fig. 6). Female (B6.Nba2 × NZW)F1 mice produced more anti-ssDNA (p = 0.0003), anti-chromatin (p < 0.0001), anti-dsDNA (p = 0.008), and anti-histone (p = 0.0003) autoantibodies than male (B6.Nba2 × NZW)F1 mice in the context of the H2b/z MHC haplotype (Fig. 6,A). Total IgG levels were also 2-fold higher in H2b/z female compared with male (B6.Nba2 × NZW)F1 mice (data not shown). In the context of the H2d/z MHC haplotype, female mice produced more anti-chromatin (p = 0.001), anti-dsDNA (p = 0.002), and anti-ssDNA (p = 0.03) autoantibodies than male mice (Fig. 6 B).

FIGURE 6.

Effect of MHC haplotype on autoantibody production and severe proteinuria in male and female (B6.Nba2 × NZW)F1 mice. Serum samples from 7-mo-old female and male (B6.Nba2 × NZW)F1 mice were analyzed for IgG autoantibodies to dsDNA, ssDNA, chromatin, and histone in the context of either the H2b/z (A) or H2d/z (B) MHC haplotypes. In H2b/z mice, female mice produced more autoantibodies to dsDNA (p = 0.008), ssDNA (p = 0.003), chromatin (p < 0.0001), and total histones (p = 0.0003) than male mice. In H2d/z mice, female mice produced more autoantibodies to dsDNA (p = 0.002), ssDNA (p = 0.03), and chromatin (p = 0.001) than male mice. Male and female (B6.Nba2 × NZW)F1 mice with the indicated MHC haplotypes were followed monthly for evidence of severe proteinuria for 12 mo (C). The incidence of proteinuria in H2b/z (B6.Nba2 × NZW)F1 female mice was greater than that in MHC-matched male littermates (p = 0.03). The difference in incidence between male and female H2d/z (B6.Nba2 × NZW)F1 mice did not reach statistical significance (H2b/z (B6.Nba2 × NZW)F1 females, n = 10; H2b/z (B6.Nba2 × NZW)F1 males, n = 10; H2d/z (B6.Nba2 × NZW)F1 females, n = 9; H2d/z (B6.Nba2 × NZW)F1 males, n = 10).

FIGURE 6.

Effect of MHC haplotype on autoantibody production and severe proteinuria in male and female (B6.Nba2 × NZW)F1 mice. Serum samples from 7-mo-old female and male (B6.Nba2 × NZW)F1 mice were analyzed for IgG autoantibodies to dsDNA, ssDNA, chromatin, and histone in the context of either the H2b/z (A) or H2d/z (B) MHC haplotypes. In H2b/z mice, female mice produced more autoantibodies to dsDNA (p = 0.008), ssDNA (p = 0.003), chromatin (p < 0.0001), and total histones (p = 0.0003) than male mice. In H2d/z mice, female mice produced more autoantibodies to dsDNA (p = 0.002), ssDNA (p = 0.03), and chromatin (p = 0.001) than male mice. Male and female (B6.Nba2 × NZW)F1 mice with the indicated MHC haplotypes were followed monthly for evidence of severe proteinuria for 12 mo (C). The incidence of proteinuria in H2b/z (B6.Nba2 × NZW)F1 female mice was greater than that in MHC-matched male littermates (p = 0.03). The difference in incidence between male and female H2d/z (B6.Nba2 × NZW)F1 mice did not reach statistical significance (H2b/z (B6.Nba2 × NZW)F1 females, n = 10; H2b/z (B6.Nba2 × NZW)F1 males, n = 10; H2d/z (B6.Nba2 × NZW)F1 females, n = 9; H2d/z (B6.Nba2 × NZW)F1 males, n = 10).

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Female (B6.Nba2 × NZW)F1 mice also developed more severe renal disease (Fig. 6 C). About 20% of female H2b/z (B6.Nba2 × NZW)F1 mice developed severe proteinuria by 6 mo, and by 12 mo, 80% were positive. In contrast, only 20% of male (B6.Nba2 × NZW)F1 mice developed severe proteinuria throughout the duration of the study (p = 0.01 compared with female frequency). The difference in the incidence of proteinuria between male and female H2d/z (B6.Nba2 × NZW)F1 mice was less dramatic, and by 12 mo of age, 44% of female H2d/z (B6.Nba2 × NZW)F1 mice had developed severe proteinuria compared with 22% of the males. Overall, regardless of MHC haplotype, females were nearly 7-fold more likely to develop severe disease than males.

The production of pathogenic IgG autoantibodies and the development of lupus nephritis in BWF1 mice are dependent upon both CD4+ T cells and MHC class II molecules (reviewed in Refs.1 and 2). Studies have also shown that heterozygosity for both parental MHC haplotypes (H2d/z), compared with H2d/d or H2z/z, is important for development of fatal lupus nephritis (7, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20). In addition, genetic linkage studies have shown that the most important dominant NZW contribution to disease is closely linked to the MHC (H2z) (7, 9, 11, 13, 18). The current investigations were designed to clarify the importance of H2z in the development of glomerulonephritis in Nba2 congenic mice, because (B6.Nba2 × NZW)F1 (H2b/z) mice develop glomerulonephritis, whereas B6.Nba2 (H2b/b) mice do not. Inheritance of H2d/z was not sufficient for the development of glomerulonephritis in B6.Nba2 mice. Furthermore, homozygosity for H2z was not permissive for either autoantibody production or glomerulonephritis, and H2b/b appeared to be the most autoantibody-promoting MHC type. In these studies we noted considerable deposition of IgG and complement in the glomeruli of B6.Nba2 mice, supporting the hypothesis that their complete absence of glomerulonephritis might be related to missing NZW-encoded non-MHC genes downstream of this process. In additional studies, we found that the female vs male enhancement of disease in BWF1 mice was also operative in B6.Nba2 and (B6.Nba2 × NZW)F1 mice regardless of MHC haplotype. Together, the current studies clarify the genes necessary for autoantibody production vs nephritis in BWF1 mice and provide a simplified system to understand the pathogenesis of disease.

The current studies did not support an important role of H2z from NZW in the B6.Nba2 or (B6.Nba2 × NZW)F1 model of disease. Mice heterozygous for H2z did not demonstrate enhanced IgG antinuclear Ab production or nephritis. In B6.Nba2 mice, H2b/b appeared to be the most disease-enhancing MHC haplotype, especially compared with H2d/d and H2d/z, whereas in the F1 model, H2z/z was completely suppressive of both autoantibody production and disease. The relative disease-promoting effect of H2b compared with other haplotypes has been noted in other lupus-prone mouse strains, such as B6-Faslpr mice (24, 25), BXSB mice (26, 27), certain backcross studies of NZB and NZW mice (16, 22, 28, 29), and even some knockout models of lupus (30). In contrast to H2z and H2d, the H2b haplotype does not allow expression of an IE-α protein and does not express an I-E molecule (31). With regard to this difference, other investigators have observed partial prevention of mouse lupus in mice with high levels of I-E expression (24, 32, 33). Studies have suggested that peptides derived from I-Ed and I-Ez can prevent autoantibody production, perhaps through blocking of I-A presentation of other self peptides (24, 32, 33, 34).

The marked prevention of autoantibody production and disease by homozygosity for H2z probably relates to mechanisms additional to I-E expression. For example, in the current studies, increased autoantibody production in H2d/z vs H2z/z F1 mice cannot easily be explained by levels of I-E, because both haplotypes encode functional I-E molecules about equally (22). Previous studies from our laboratory suggested that H2z alone may not be efficient at presenting certain types of self Ags compared with other haplotypes (18). Furthermore, in separate studies, B6.Nba2 mice heterozygous for deficiency of Abb (i.e., Ab+/−) mice produced higher amounts of autoantibodies than wild-type B6.Nba2+/+ (Ab+/+) mice, consistent with the idea that lower levels of class II molecules (as would be the case for each class II molecule in a mouse heterozygous for MHC) reduces the numbers of T cells that are negatively selected in the thymus (35).

The lack of antinuclear Abs and glomerulonephritis in our studies of H2z/z B6.Nba2 mice almost certainly relates to studies by Wakeland and colleagues (36) showing that the strongest disease-suppressor locus (Sles1) mapped to the H2z MHC region. In their studies, Sles1 also inhibited disease at the level of autoantibody production by congenic mice. Nevertheless, Sles1 is not sufficient to inhibit disease in every genomic context, because NZM2410 (H2z/z) mice produce autoantibodies and develop glomerulonephritis. Our study design also localizes the position of Sles1, because the congenic interval includes only 1 cM of NZW genomic DNA proximal and 4 cM distal to the H2z locus (22). Novel aspects of the H2z locus have been identified. For example, studies have described a novel allele of the factor B gene (class III region) in H2z vs other haplotypes that affects activation of the alternative complement pathway in the kidney (37).

Several explanations are possible for why B6.Nba2 mice do not develop glomerulonephritis. The current studies show that the levels of autoantibodies to chromatin and its constituents are lower in B6.Nba2 than in (B6.Nba2 × NZW)F1 mice, and it is possible that the B6.Nba2 levels do not achieve a threshold sufficient for disease. A second possibility is that different autoantibody specificities, critical for nephritis, are produced in F1, but not B6.Nba2 or H2b/z B6.Nba2, mice. Studies involving NZM.C57Lc4 mice, which develop a fatal glomerulonephritis in the absence of autoantibodies to dsDNA and other antinuclear Abs, support this possibility (38). Nevertheless, we observed prominent IgG and C3 deposition in the glomeruli of B6.Nba2 mice, suggesting that the block of fatal glomerulonephritis was at a level downstream of autoantibody production and IC deposition. Although the extent of deposition was not as diffuse as that observed in the F1 mice, and levels in F1 mice might continue to increase well beyond B6.Nba2 levels, our results suggest that at least one component of the NZW non-MHC contribution to disease in the F1 is related to mechanisms of inflammation or destruction after IC deposition and complement activation. The data are consistent with recent studies suggesting that NZW mice are more susceptible than other strains to nephrotoxic serum-mediated glomerulonephritis and glomerular damage (39). It has also been well demonstrated that genetic defects (and probably polymorphisms) can affect glomerulonephritis after the stage of IC deposition and complement activation (40).

These studies also showed that female mice, whether B6.Nba2 or (B6.Nba2 × NZW)F1, develop increased levels of autoantibodies and nephritis compared with male counterparts. Previous studies in humans and BWF1 mice suggested that the effects of gender are related to sex hormones, both disease-suppressive effects of androgens and disease-enhancing effects of estrogens (1, 4, 5, 6, 41, 42). One major candidate gene for Nba2 is IFN-inducible gene 202 (Ifi202), which encodes a protein that inhibits apoptosis (21, 43). Preliminary gene expression studies indicated an enhancing effect of estrogens on Ifi202 gene expression (D. Choubey, M. R. Gubbels, and B. L. Kotzin, unpublished observations), and this may be involved in their enhancing effect on disease. However, the consistent effects of sex hormones in different lupus models, including single-locus models such as Sle1 mice (36), suggest that the effects of sex hormones on gene expression involve separate pathways that can consistently interact with nearly any group of lupus susceptibility genes.

In summary, the current studies showed that the development of disease in (B6.Nba2 × NZW)F1 compared with B6.Nba2 mice depends on NZW-encoded non-MHC genes. Additionally, H2z homozygosity suppressed autoantibody production in these models, which may be dependent upon multiple genes within the MHC region. In these studies, the lack of nephritis in B6.Nba2 mice appeared to be partly independent of IC deposition in the kidney, suggesting that a component of the NZW genetic contribution required for kidney pathogenesis acts downstream of IC deposition. Whether this effect is dependent on sex hormones is unknown, because female mice exhibited both higher levels of autoantibodies and increased incidence of fatal lupus nephritis than male mice regardless of genetic background or MHC haplotype.

The authors have no financial conflict of interest.

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 in part by Grant AR37070 from the National Institutes of Health. M.R.G. was supported by an award from the American Heart Association.

5

Abbreviations used in this paper: SLE, systemic lupus erythematosus; IC, immune complex.

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