Lupus pathogenesis in the NZM2410 mouse model results from the expression of multiple interacting susceptibility loci. Sle2 on chromosome 4 was significantly linked to glomerulonephritis in a linkage analysis of a NZM2410 × B6 cross. Yet, Sle2 expression alone on a C57BL/6 background did not result in any clinical manifestation, but in an abnormal B cell development, including the accumulation of B-1a cells in the peritoneal cavity and spleen. Analysis of B6.Sle2 congenic recombinants showed that at least three independent loci, New Zealand White-derived Sle2a and Sle2b, and New Zealand Black-derived Sle2c, contribute to an elevated number of B-1a cells, with Sle2c contribution being the strongest of the three. To determine the contribution of these three Sle2 loci to lupus pathogenesis, we used a mapping by genetic interaction strategy, in which we bred them to B6.Sle1.Sle3 mice. We then compared the phenotypes of these triple congenic mice with that of previously characterized B6.Sle1.Sle2.Sle3, which express the entire Sle2 interval in combination with Sle1 and Sle3. Sle2a and Sle2b, but not Sle2c, contributed significantly to lupus pathogenesis in terms of survival rate, lymphocytic expansion, and kidney pathology. These results show that the Sle2 locus contains several loci affecting B cell development, with only the two NZW-derived loci having the least effect of B-1a cell accumulation significantly contributing to lupus pathogenesis.

Systemic lupus erythematosus (SLE)3 is an autoimmune disease in which abnormal B cell function and development have long been recognized (1). We have used the NZM2410 mouse model to perform a genetic analysis of SLE susceptibility (2), and found that all three major susceptibility loci, Sle1, Sle2, and Sle3, resulted in the production of autoantibodies (3). Although both Sle1 and Sle3 affect multiple cell subsets (4, 5, 6), the effects of Sle2 expression have been confined to date to the B cell compartment (7). When expressed on a C57BL/6 (B6) background, the chromosome (Chr.) 4 interval carrying the Sle2 locus is associated with B cell hyperactivity and polyclonal activation, and with an expansion of the B-1a subset, especially in the peritoneal cavity (perC). Although Sle2 is not sufficient by itself to induce any autoimmune pathology, its contribution to lupus pathogenesis was clearly demonstrated by comparing B6.Sle1.Sle3 with B6.Sle1.Sle2.Sle3 congenic mice (8). The combination of Sle1 and Sle3 resulted in the production of pathogenic nephrophyllic autoantibodies (9). The addition of Sle2 significantly increased B cell activation, which was concomitant with an earlier onset of autoantibodies, and a complete penetrance of lupus nephritis (8). T cell phenotypes were similar in B6.Sle1.Sle3 and B6.Sle1.Sle2.Sle3 mice, suggesting that Sle2 contribution to autoimmune pathology occurred through the B cell lineage.

Expansion of the B-1a cell compartment is the most characteristic phenotype associated with Sle2 expression on a B6 background. We have shown that this expansion requires Sle2 expression in B cells, and depends on multiple mechanisms, which include greater initial output from the fetal liver, increased proliferation and decreased apoptosis, and continuous output from adult lymphoid organs (10). The B-1a cells are the major source of serum IgM, and positive selection by autoantigens has been shown to play an important role in their development (11). Several lines of evidence have suggested a role of B-1 cells in lupus pathogenesis, through the production of low affinity Abs, diminished negative regulation and recruitment to germinal center reactions, or production of IL-10 (12). The most compelling evidence for their involvement was that the deletion of perC B-1 cells by hypotonic shock reduced disease severity in (New Zealand Black (NZB) × New Zealand White (NZW))F1 (BWF1) mice (13). The same group also showed that transgenic expression of osteopontin resulted in simultaneous increased perC B-1a cells and anti-dsDNA Ab production on a nonautoimmune genetic background (14). B-1a cells display enhanced Ag presentation capabilities (15). It has been suggested that B-1a cells may activate autoreactive T cells and produce autoantibodies in target organs as the consequence of their increased number and altered migration pattern toward nonlymphoid tissue in BWF1 mice (16, 17). It is possible, however, that the accumulation of B-1 cells represents a bystander consequence of a dysregulated B cell development, and that these cells by themselves do not play a specific role in lupus pathogenesis. Supporting this latter hypothesis, it has been shown that B-1a cells do not contribute to autoantibody production in FAS-deficient mice (18). Furthermore, transgenic overexpression of IL-5 in the BWF1 model greatly increased the number of B-1a cells, but, surprisingly, significantly reduced anti-dsDNA Ab production and incidence of nephritis (19). Finally, the NZB-derived Nba2 locus was shown to induce disease when crossed to NZW without affecting the size of the B-1 cell compartment (20), and the same observation was made for Sle1 (L. Morel, unpublished observations). Overall, these results imply that an increase in the size of the B-1a cell compartment is not necessary or sufficient for lupus pathogenesis.

The purpose of this study was first to refine the Sle2 genetic map using the expansion of the B-1a compartment as the selecting phenotype. With the same approach that we have used successfully for S1e1 (21), we have produced a series of congenic recombinant lines and showed that three loci (Sle2a, Sle2b, and Sle2c) independently contribute to elevated perC B-1a cells. The NZB-derived Sle2c produced by itself the largest effect on B-1a cell numbers, while NZW-derived Sle2a and Sle2b impact both B-1a and B-2 cells, and at a later age than Sle2c. To assess the respective contribution of these three loci to lupus pathogenesis, we have used an interactive mapping strategy by breeding these loci to B6.Sle1.Sle3 and comparing the resulting triple congenic mice with both B6.Sle1.Sle3 and B6.Sle1.Sle2.Sle3. Surprisingly, Sle2c expression did not increase or accelerate lupus pathogenesis. However, Sle2a and Sle2b were shown to significantly increase lymphocytic expansion and kidney pathology in B6.Sle1.Sle3 mice. These results show that Sle2 corresponds, as Sle1, to a cluster of functionally related genes, and that selective enhancement of B-1a cell expansion does not, by itself, contribute to autoimmune pathology.

The B6.Sle2 congenic mice carry a Chr. 4 NZM2410-derived 26-cM segment that represents the 95% confidence interval flanking Sle2 on a C57BL/6 (B6) background (22). This NZM2410 segment originated from NZW and NZB on the centromeric and telomeric portions, respectively (Fig. 1). The production of the bicongenic B6.Sle1.Sle3 and triple congenic B6.Sle1.Sle2.Sle3 (TC) has been described previously (8, 23). A series of five overlapping homozygous recombinant congenic lines were generated from a (B6 × B6.Sle2)F1 × B6 cross (Fig. 1). Three separate regions delineated by these recombinants were named Sle2a, Sle2b, and Sle2c. Sle2a, defined by D4MIT139 and D4MIT80, and Sle2b, defined by D4MIT144 and D4MIT205, are both entirely NZW derived, and potentially overlap in the D4MIT80-D4MIT144 region. Sle2c, defined by D4MIT278 and D4MIT72, is NZB derived. A smaller recombinant, named Sle2c(124–72), was obtained within that interval at D4MIT124. Finally, a recombinant that carries Sle2b and Sle2c, plus the intervening region, was named Sle2bc. Three B6.Sle2 recombinants were bred to B6.Sle1.Sle3 to generate triple congenic strains B6.Sle1.Sle3.Sle2a (1/3/2a), B6.Sle1.Sle3.Sle2bc (1/3/2bc), and B6.Sle1.Sle3.Sle2c (1/3/2c), which were then compared with B6.Sle1/Sle3 and the complete triple congenic TC strain. We tried repeatedly to generate the B6.Sle1/Sle3/Sle2b strain without any success, i.e., we never obtained enough adult homozygous mice to establish the line. Unless specified, all experimental and age-matched control groups contained an equal number of males and females. All experiments were conducted according to protocols approved by the University of Florida Institutional Animal Care and Use Committee.

FIGURE 1.

Genetic map of the Sle2 locus on mouse Chr. 4. From top to bottom, this figure shows the locations in cM of: 1) candidate genes for this locus; 2) MIT microsatellite markers (such as 6 stands for D4MIT6) polymorphic between NZM2410 and B6; 3) the recombinant intervals, in which the gray rectangles indicate the NZM2410 homozygous segments, and the flanking lines show the areas of recombination between the NZM2410 and B6 genomes; and 4) the NZW or NZB origin of the corresponding regions.

FIGURE 1.

Genetic map of the Sle2 locus on mouse Chr. 4. From top to bottom, this figure shows the locations in cM of: 1) candidate genes for this locus; 2) MIT microsatellite markers (such as 6 stands for D4MIT6) polymorphic between NZM2410 and B6; 3) the recombinant intervals, in which the gray rectangles indicate the NZM2410 homozygous segments, and the flanking lines show the areas of recombination between the NZM2410 and B6 genomes; and 4) the NZW or NZB origin of the corresponding regions.

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Flow cytometric analysis was performed, as previously described (8). Briefly, cells were blocked first with saturating amounts of anti-CD16/CD32 for 15 min in staining buffer (PBS, 5% horse serum, and 0.09% sodium azide). Cells were then stained with allophycocyanin-, FITC-, PE-, or biotin-conjugated mAbs, followed by streptavidin Quantum Red conjugate (Sigma-Aldrich). In some cases, Abs directly conjugated to CyC (BD Pharmingen) were used. mAbs to CD5 (53-7.3), B220 (RA3-6B2), IgMb (AF6-78), CD23 (B3B4), CD3e (145-2C11), CD4 (RM4-5), CD8 (Ly-2), CXCR5 (2G8), and their isotype controls were purchased from BD Pharmingen. The stained cells were analyzed on a FACScan or FACSCalibur (BD Biosciences). Nonviable cells were excluded on the basis of forward and side scatter characteristics. At least 10,000 events were acquired per sample.

Anti-chromatin and anti-dsDNA IgG serum levels were quantified by ELISA, as previously described (24). Frozen sections (8 μM) of spleen were stained with 1/100 dilutions of FITC-conjugated anti-B220, PE-conjugated anti-CD5, and allophycocyanin-conjugated CD11b.

Proteinuria was determined semiquantitatively with Albustix strips (Bayer), with scores of 1–4 ranging from 30 to >2000 mg/dl protein in the urine. The extent of glomerular lesions was evaluated semiquantitatively on H&E- and periodic acid-schiff (PAS)-stained sections, as previously described (8). Briefly, the percentage of affected glomeruli was scored on a 0–4 scale (1, 1–10%; 2, 11–25%; 3, 26–49%; 4, ≥50%). In addition, the dominant pattern, mesangial, hyaline, or proliferative, was recorded. The presence of immune complexes in the kidneys was evaluated on 7 μM frozen sections stained with FITC-conjugated anti-C3 (Valeant Pharmaceuticals), anti-IgG γ-chain (Jackson ImmunoResearch Laboratories), or IgM (Igh-6b; BD Pharmingen). Staining intensity was evaluated in a blind fashion on a semiquantitative 0–4 scale. The number of CD68+ (biotinylated FA-11, from Serotec; revealed with streptavidin-HRP, from Vector Laboratories) macrophages was averaged for 10 glomeruli per mouse.

Statistical analyses were performed with the GraphPad Prism 4 software. Unless specified, Mann-Whitney tests were used and levels of significance are indicated in the figures as ∗, p < 0.05; ∗∗, p < 0.01; and ∗∗∗, p < 0.001.

We have previously shown that one of the most robust Sle2 phenotypes was a significant increase in number and proportion of perC CD5+ B220low B-1a cells (7, 10). We assessed the contribution of the Sle2 recombinants to this B cell compartment as compared with B6 (Fig. 2,A). In 8- to 11-mo-old mice, B6.Sle2c mice presented a significantly higher percentage of perC B-1a cells (Fig. 2,B), a higher B-1a/B-2 ratio (Fig. 2,C), and a ∼2-fold increase in absolute number (Fig. 2,D). In terms of B-1a cells as percentage of perC lymphocytes, Sle2c was equivalent to the entire Sle2 interval, suggesting that Sle2c is a strong contributor to this phenotype. Interestingly, these values for the B6.Sle2c(124–72) mice were identical with that of B6 (Fig. 2). These results map a major locus for increased perC B-1a cells to the 1.3-cM (∼2-Mb) segment between D4MIT278 and D4MIT 37, although it could extend up to the 6.6-cM (∼20-Mb) segment between (but excluding) D4MIT331 and D4MIT124 (Fig. 1).

FIGURE 2.

Increased perC B-1a cell compartment maps to Sle2c. A, Representative FACS plots showing the CD5+B220low B-1a and CD5 B220high B-2 populations in 8- to 11-mo-old B6.Sle2 recombinants and B6 controls. Comparisons of perC B-1a cells as percentage of lymphocytes (B), B-1a/B2 ratio (C), or absolute numbers (D). Each symbol represents a different mouse, and the horizontal lines represent the mean values. Data for each Sle2 recombinant interval were compared with B6, and significance levels are shown on the top of each column of data. Recombinants that were significantly different from B6 were then compared with Sle2, and significant values from these comparisons were indicated with the brackets.

FIGURE 2.

Increased perC B-1a cell compartment maps to Sle2c. A, Representative FACS plots showing the CD5+B220low B-1a and CD5 B220high B-2 populations in 8- to 11-mo-old B6.Sle2 recombinants and B6 controls. Comparisons of perC B-1a cells as percentage of lymphocytes (B), B-1a/B2 ratio (C), or absolute numbers (D). Each symbol represents a different mouse, and the horizontal lines represent the mean values. Data for each Sle2 recombinant interval were compared with B6, and significance levels are shown on the top of each column of data. Recombinants that were significantly different from B6 were then compared with Sle2, and significant values from these comparisons were indicated with the brackets.

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The B-1a/B2 ratio and the perC B-1a absolute number were lower in B6.Sle2c than in B6.Sle2 mice. Moreover, expansion of the perC B-1a compartment occurred later in B6.Sle2c than in B6.Sle2 mice. Indeed, no difference was observed between 2-mo-old B6.Sle2c and B6 (data not shown), while a significant difference exists between B6.Sle2 and B6 (10). These results indicate the involvement of other loci in the Sle2 interval. The absolute number of perC B-1a cells at 8–9 mo was significantly higher in both Sle2a and Sle2b than in B6 (Fig. 2,D). This was also true for B-2 cells in these two strains, as illustrated by a normal B-1a/B2 ratio (Fig. 2 C), suggesting that Sle2a and Sle2b globally increase perC B cell numbers. In older (12- to 14-mo-old) mice, the size of the perC B-1a compartment was significantly increased in B6.Sle2a mice as compared with B6 (57.45 ± 3.98 vs 20.84 ± 2.59% of lymphocytes, 3.61 ± 0.74 vs 0.60 ± 0.09 B-1a/B-2 ratio for B6.Sle2a and B6, respectively; p < 0.0001). This indicated that Sle2a expression also affects the size of the perC B-1a compartment, although with a later onset than Sle2c.

PerC B-1a cells express high levels of surface IgM (12). We have shown previously that Sle2 was associated with the accumulation of a population of CD19+B220lowCD11b+CD5+ IgMlow perC B-1a cells (10). Examination in the Sle2 recombinants showed this phenotype maps primarily to the Sle2a locus (Fig. 3,A). Some B6.Sle2a mice displayed up to three populations of perC B1-a cells based on IgM expression (Fig. 3,B). Although some low numbers of CD5+ IgMintermediate (low) cells exist in the other strains observed in this study (Fig. 3,A), the CD5+ IgMlow population was found only in the strains carrying Sle2a (B6.Sle2, B6.Sle2a, 1/3/2a, and B6.TC). It was recently reported that IgM expression is lower on splenic than on perC B-1a cells (25). We have shown that the expansion of the perC B-1a population in B6.Sle2 mice was due in part to an influx from splenic cells (10), which would correspond to B-1a cells with reduced IgM expression. B-1a cell homing to the peritoneal cavity is mediated by CXCL13 (26). Interestingly, the Sle2 and Sle2a IgMlow/int perC B-1a cells express higher levels of CXCR5, the CXCL13 ligand, than the IgMhigh perC B-1a cells (Fig. 3 C). CXCR5 expression was similar between the IgMint and the IgMlow populations; moreover, no difference of CXCR5 expression on B-2 and IgMhigh B-1a cells was observed between the B6.Sle2, B6.Sle2a, and B6 strains (data not shown). This result suggests that the IgMlow/int population represents B-1a cells from splenic origin, and that the Sle2a locus may control this phenotype.

FIGURE 3.

The perC IgMlow CD5+ B-1a population maps to Sle2a. A, Representative FACS plots showing the IgMlow B-1a population in 9-mo (upper panels) and 12-mo-old (lower panels) B6.Sle2 recombinants and B6 mice (n ≥ 10 per strain). B, Up to three populations of perC B-1a cells are represented in mice carrying Sle2a based on the level of IgM expression. The histogram shows a representative sample perC CD5+ IgM+ population from a 1/3/2a 9-mo-old mouse. C, IgMlow/int perC B-1a cells (thick line) express higher CXCR5 levels as compared with their IgMhigh counterparts (thin line), or B-2 cells (gray dash line). The filled histogram corresponds to the isotype control. Representative perC cells from a 9-mo-old B6.Sle2 mouse.

FIGURE 3.

The perC IgMlow CD5+ B-1a population maps to Sle2a. A, Representative FACS plots showing the IgMlow B-1a population in 9-mo (upper panels) and 12-mo-old (lower panels) B6.Sle2 recombinants and B6 mice (n ≥ 10 per strain). B, Up to three populations of perC B-1a cells are represented in mice carrying Sle2a based on the level of IgM expression. The histogram shows a representative sample perC CD5+ IgM+ population from a 1/3/2a 9-mo-old mouse. C, IgMlow/int perC B-1a cells (thick line) express higher CXCR5 levels as compared with their IgMhigh counterparts (thin line), or B-2 cells (gray dash line). The filled histogram corresponds to the isotype control. Representative perC cells from a 9-mo-old B6.Sle2 mouse.

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Finally, both Sle2a and Sle2c contribute to a significant increase in the percentage of splenic B-1a cells as compared with B6 (Fig. 4,A). Interestingly, values for Sle2a were similar to that of Sle2, while the percentage of splenic B-1a cells was significantly lower in B6.Sle2c than in B6.Sle2 mice. Absolute numbers of splenic B-1a cells showed a similar distribution as the percentages (data not shown). Spleen weights and total splenocyte numbers were similar between B6, B6.Sle2 and its recombinants (data not shown). The distribution of splenic B cell subsets (T1, T2, marginal zone, and follicular) in B6.Sle2 mice or any of its recombinants was similar to that of B6 (Fig. 4 B). Finally, no difference was observed between the Sle2 subcongenic strains and B6 regarding the number and distribution of T cells and non-T cells/non-B cells in the spleen (data not shown).

FIGURE 4.

Sle2a and Sle2c increase the size of the splenic B-1a compartment, but do not affect the other B cell subsets. A, Percentage of CD5+B220low splenic lymphocytes in 8- to 11-mo-old Sle2 recombinant mice, B6 and B6.Sle2 controls. See Fig. 2 for details. B, Distribution of splenic IgM+ into T1 (CD21lowCD23low), T2 (CD21highCD23high), marginal zone (MZ: CD21high CD23negative/low), and follicular (Fo: CD21lowCD23high) in 8- to 11-mo-old Sle2 recombinant mice, B6 and B6.Sle2 controls (mean and SD, n ≥ 10 per strain).

FIGURE 4.

Sle2a and Sle2c increase the size of the splenic B-1a compartment, but do not affect the other B cell subsets. A, Percentage of CD5+B220low splenic lymphocytes in 8- to 11-mo-old Sle2 recombinant mice, B6 and B6.Sle2 controls. See Fig. 2 for details. B, Distribution of splenic IgM+ into T1 (CD21lowCD23low), T2 (CD21highCD23high), marginal zone (MZ: CD21high CD23negative/low), and follicular (Fo: CD21lowCD23high) in 8- to 11-mo-old Sle2 recombinant mice, B6 and B6.Sle2 controls (mean and SD, n ≥ 10 per strain).

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We have previously shown that the addition of Sle2 to the combination of the Sle1 and Sle3 loci significantly accelerated disease progression and aggravated clinical manifestations (8). To map this phenotype, we bred Sle2a, Sle2bc, and Sle2c to B6.Sle1.Sle3 to produce triple congenic strains 1/3/2a, 1/3/2bc, and 1/3/2c and compared them with B6.Sle1.Sle3 and B6.Sle1.Sle2.Sle3 (TC). The contribution of the Sle2b locus was assessed indirectly by comparing the 1/3/2bc and 1/3/2c strains, because we have not been able to produce the B6.Sle1.Sle3.Sle2b strain. In cohorts followed up to 12 mo of age, 1/3/2c mice showed a significantly better survival than TC mice, while there was no difference between 1/3/2a or 1/3/2bc and TC (Fig. 5,A). B6.Sle1/Sle3 mice are very poor breeders, and our cohort was not large enough for meaningful statistical comparisons of their survival. Proteinuria was, however, significantly higher in 1/3/2a than in B6.Sle1/Sle3 mice (Fig. 5,B, p = 0.013). Splenomegaly and increased lymphocyte numbers have been consistently associated with murine lupus, including in the NZM2410 model (2, 8, 9). The expression of the Sle2a or Sle2bc loci, but not Sle2c alone, resulted in a significant increased spleen weight and splenocyte number in B6.Sle1.Sle3 mice (Fig. 5,C, and data not shown). The 1/3/2a and 1/3/2bc values were, however, significantly lower than TC, suggesting that Sle2a and Sle2b, and possibly Sle2c, have additive effects. Similarly, 78% of 1/3/2a and 25% of 1/3/2bc 8- to 12-mo-old mice developed extensive hyperplasia of the cervical lymph nodes. Lymph node hyperplasia is also found in the majority of old NZM2410 and TC mice (B. Croker, S. Chattopadhyay, J. Ward, H. Morse, and L. Morel, manuscript in preparation). In contrast, none of the aged-matched B6.Sle1.Sle3 or 1/3/2c mice presented enlarged lymph nodes (data not shown). Old mice (8–12 mo old) from all five strains produced similar amounts of anti-dsDNA Abs (data not shown). Younger 1/3/2c mice showed, however, a significant reduction of serum anti-dsDNA Abs as compared with TC (Fig. 5 D, p < 0.01). Overall, these results suggest that Sle2a and Sle2b to a lesser extent, but not Sle2c, significantly increased lupus pathogenesis in B6.Sle1.Sle3 mice.

FIGURE 5.

Sle2a and Sle2b, but not Sle2c, enhance lupus phenotypes in combination with the Sle1 and Sle3 loci. A, 1/3/2a, 1/3/2bc, and TC mice showed a comparable survival rate, but 1/3/2c mice showed a significantly increased survival as compared with TC (log rank test, p = 0.02, n ≥ 50 mice per strain, aged up to 12 mo of age). B, Proteinuria in 8- to 12-mo mice was significantly increased by the expression of Sle2a, but not of Sle2b or Sle2c in combination with Sle1 and Sle3. C, Sle2a and Sle2bc, but not Sle2c expression resulted in a significant increase in spleen weight in 8- to 12-mo-old B6.Sle1.Sle3 mice. Splenomegaly in 1/3/2a and 1/3/2bc mice was, however, significantly lower than in TC mice. D, Anti-dsDNA IgG Ab production was significantly lower in 1/3/2c mice than in TC at 3–5 mo of age. See Fig. 2 for details.

FIGURE 5.

Sle2a and Sle2b, but not Sle2c, enhance lupus phenotypes in combination with the Sle1 and Sle3 loci. A, 1/3/2a, 1/3/2bc, and TC mice showed a comparable survival rate, but 1/3/2c mice showed a significantly increased survival as compared with TC (log rank test, p = 0.02, n ≥ 50 mice per strain, aged up to 12 mo of age). B, Proteinuria in 8- to 12-mo mice was significantly increased by the expression of Sle2a, but not of Sle2b or Sle2c in combination with Sle1 and Sle3. C, Sle2a and Sle2bc, but not Sle2c expression resulted in a significant increase in spleen weight in 8- to 12-mo-old B6.Sle1.Sle3 mice. Splenomegaly in 1/3/2a and 1/3/2bc mice was, however, significantly lower than in TC mice. D, Anti-dsDNA IgG Ab production was significantly lower in 1/3/2c mice than in TC at 3–5 mo of age. See Fig. 2 for details.

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Examination of kidney pathology confirmed the results presented above (Fig. 6,A). C3, IgG, and IgM deposits tended to be more intense in 1/3/2a and 1/3/2bc glomeruli, without reaching statistical significance. The distribution of these deposits was, however, different, with a striking predilection for the basement membrane in 1/3/2a, 1/3/2bc, and TC mice (Figs. 6,A and 7,A). Interestingly, these two strains presented a significantly higher number of CD68+ macrophages in the glomeruli as compared with B6.Sle1.Sle3 (p = 0.004 for 1/3/2a and p = 0.016 for 1/3/2bc; Figs. 6,A and 7,B). Very few T or B cells were found in the glomeruli from lupus mice (1–2 per glomerulus on average; data not shown), which were too low to perform meaningful comparisons between strains. As expected for B6.Sle1.Sle3 mice (9), all mice examined presented some degree of glomerulonephritis (GN). However, the extent of renal lesions assessed by H&E and PAS stains was different among strains (Fig. 7,C). All B6.TC mice obtained a GN score of 4 (≥50% affected glomeruli), which was similar to the results obtained with 1/3/2a and 1/3/2bc mice (85 and 88% with a GN score of 4, respectively). Conversely, the GN scores of 1/3/2c mice were similar to that of B6.Sle1.Sle3 mice. The type of lesions was not affected by the expression of Sle2a or Sle2b, however (Fig. 7 D). The high penetrance of proliferative/hyaline lesions that is highly correlated with clinical nephritis in our model (8) was found only in B6.TC mice. Overall, these results show that Sle2a and Sle2b contribute to renal pathology by promoting the deposition of immune complexes of the basement membrane and the infiltration of macrophages in the glomeruli, which result in more extensive and severe renal lesions. The expression of the entire Sle2 locus is, however, necessary to reach the level of severe proliferative lesions characteristic of B6.TC GN.

FIGURE 6.

Impact of Sle2a and Sle2b expression on kidney pathology and splenic architecture in B6.Sle1.Sle3 mice. A, Representative glomeruli from B6.Sle1.Sle3, 1/3/2a, 1/3/2bc, 1/3/2c, and TC (from top to bottom), stained with anti-C3, anti-IgG, and anti-CD68 Abs (from left to right). ×100 amplification. B, Representative splenic frozen section from 8- to 12-mo mice from the indicated strains stained with PE-conjugated anti-CD5 (red), FITC-conjugated anti-B220 (green), and allophycocyanin-conjugated anti-CD11b (blue); ×50 amplification.

FIGURE 6.

Impact of Sle2a and Sle2b expression on kidney pathology and splenic architecture in B6.Sle1.Sle3 mice. A, Representative glomeruli from B6.Sle1.Sle3, 1/3/2a, 1/3/2bc, 1/3/2c, and TC (from top to bottom), stained with anti-C3, anti-IgG, and anti-CD68 Abs (from left to right). ×100 amplification. B, Representative splenic frozen section from 8- to 12-mo mice from the indicated strains stained with PE-conjugated anti-CD5 (red), FITC-conjugated anti-B220 (green), and allophycocyanin-conjugated anti-CD11b (blue); ×50 amplification.

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FIGURE 7.

Semiquantitative assessment of the effects of Sle2a and Sle2b expression on renal pathology in 8- to 12-mo-old B6.Sle1.Sle3 mice. A, Relative intensity scores of glomerular basement membrane staining with anti-C3. B, Average number of CD68+ macrophages in 10 randomly selected glomeruli per mouse. ∗, Indicates significance levels in comparison with B6.Sle1.Sle3 values. C, Semiquantitative assessment of nephritis severity on a 1 (<10% of glomeruli affected) to 4 (>50% glomeruli affected scale). D, Qualitative assessment of renal lesions. Mesangial lesions, white; proliferative and/or hyaline lesions, gray. Fifteen mice per strain were represented in C and D.

FIGURE 7.

Semiquantitative assessment of the effects of Sle2a and Sle2b expression on renal pathology in 8- to 12-mo-old B6.Sle1.Sle3 mice. A, Relative intensity scores of glomerular basement membrane staining with anti-C3. B, Average number of CD68+ macrophages in 10 randomly selected glomeruli per mouse. ∗, Indicates significance levels in comparison with B6.Sle1.Sle3 values. C, Semiquantitative assessment of nephritis severity on a 1 (<10% of glomeruli affected) to 4 (>50% glomeruli affected scale). D, Qualitative assessment of renal lesions. Mesangial lesions, white; proliferative and/or hyaline lesions, gray. Fifteen mice per strain were represented in C and D.

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To decipher the mechanisms by which Sle2a and Sle2b, but not Sle2c, were associated with a significant amplification of the lupus phenotypes, we compared the cell distribution in the perC and spleen among the triple congenic strains and B6.Sle1.Sle3. Unexpectedly, as shown by Fig. 8, the number and percentage of perC B-1a cells were not enhanced by Sle2c, but by Sle2a (p < 0.001 for both absolute numbers and B1a/B2 ratio) and Sle2bc (p = 0. 0023 for absolute numbers, but not significant for B1a/B2 ratio). In addition, perC B-1a cells in 1/3/2a mice presented the characteristic IgMlow/int population that we have associated with Sle2a expression (Fig. 3,B). In the spleen, no significant differences were found in the distribution of the major cell subsets (data not shown). Their follicular organization was, however, strikingly different (Fig. 6 B). The white pulp organization was fairly normal with defined B and T cell zones, and the majority of CD11b+ located in the red pulp in B6.Sle1.Sle3 and 1/3/2c mice. In B6.TC, 1/3/2a, and 1/3/2bc mice, however, the boundaries were far less defined, and a large number of CD11b+ cells was found in the T and B cell zones. This phenotype was not observed in B6.Sle2, B6.Sle2a, or B6.Sle2b spleens (data not shown). These results show that interactions resulting from the coexpression of Sle1, Sle3, Sle2a, or Sle2b lead to a significant remodeling of the splenic architecture, which most likely increases the amount of interaction between T cells, B cells, and APCs.

FIGURE 8.

Sle2a and Sle2b expression increase the number of perC B1-a cells (A) and B-1a/B2 ratios (B) in 8- to 12-mo-old mice. See Fig. 2 for details.

FIGURE 8.

Sle2a and Sle2b expression increase the number of perC B1-a cells (A) and B-1a/B2 ratios (B) in 8- to 12-mo-old mice. See Fig. 2 for details.

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The indispensable role of B cells in lupus pathophysiology has been formally demonstrated when the genetic ablation of that lymphocyte lineage largely abrogated disease manifestations in the MRL/lpr model (27). Currently, the most promising therapeutic approach for lupus patients uses a mAb against CD20 to eliminate B cells (28). Defects in multiple B cell subsets may contribute to lupus pathogenesis. Marginal zone B cells are enriched in autoreactive B cells in some models (29, 30), but not in others (20, 31). We have recently described in the NZM2410 model a defect in plasma cells that accumulate in the spleen instead of migrating to the bone marrow (32). The size of the marginal zone B cell compartment and the plasma cell defect do not map to any single Sle locus (32) (this study, and L. Morel and B. Duan, unpublished results). In contrast, a large increase in the number and proportion of B-1a cells in the perC and in the spleen represents the single most consistent phenotype specifically associated with Sle2 (7). Significant differences in the number of perC B-1a cells were observed as early as 1-mo-old B6.Sle2 mice, and are accentuated with age (10). Adoptive transfers have shown that this phenotype was intrinsic to Sle2-expressing B cells (10). Considering the recurrent association of B-1 cells with autoreactivity (12), we selected this phenotype to refine the Sle2 genetic map and assess its contribution to pathogenicity.

Multiple factors control the size of the perC B-1 compartment. Numerous transgenic models have shown that B cell intrinsic factors, such as the antigenic specificity of the BCR, positive selection, and the strength of the BCR signal, were important (12). Genetic factors are also undeniably involved (33), and both NZB and NZW are among the strains with the highest numbers of perC B-1a cells. A number of quantitative trait loci linked to B1-a cells expansion have been mapped in NZB (34, 35), and a gain-of-function polymorphism in the receptor-type protein tyrosine kinase lymphocyte tyrosine kinase was identified as the causative allele for the Chr. 2 locus (35). On Chr. 4, an NZB locus, Mott-1, has been linked to an increased ability of B-1 cells to become Mott cells and secrete Ab (36). Mott-1, however, was mapped to a more telomeric region relative to Sle2c (66 cM for Mott1 and 57.4 cM for D4MIT124, the most telomeric marker on the Sle2c critical interval), and most likely corresponds to a different gene. We found in this study that three nonoverlapping genomic intervals on NZM2410 result in elevated perC B-1a when expressed on a B6 background. None of these loci produced a phenotype equivalent to that of the entire Sle2 locus, implying additive or multiplicative effects between these loci. Therefore, Sle2 corresponds to a cluster of functionally related loci, all contributing to an enhanced development of the B-1a compartment. This result offers a remarkable parallel with the Sle1 cluster of loci resulting in the loss of tolerance of B and T cells toward nuclear Ags (21). Interestingly, the Sle2a, Sle2b, and Sle2c critical intervals exclude two genes that may have played a role in B-1 cell development (see Fig. 1): Mycbp, a c-Myc-binding protein, and Lck, which has been shown recently to be required for BCR signal transduction in B-1 cells (37). It is early to speculate on the candidacy of genes that are located within the congenic intervals corresponding to each of the Sle2 loci. Three genes, however, have been shown to have an effect on B-1a cells. Cd72, which is potentially in the Sle2a interval, is a BCR-negative regulator, and CD72 deficiency results in an increased number of B-1a cells (38). The role of type I IFN in lupus has received a lot of attention (39). The Ifnα gene cluster, which encodes for type I IFNs, is located in the Sle2b interval. We have recently shown that B6.Sle2 mice produce lower levels of type I IFN, and that the number of B-1a cells could be increased by anti-IFN-α-blocking Ab, or decreased with IFN-α injections.4 Finally, PDE4 (encoded by Pde4b, which is potentially in the Sle2b interval) is a phosphodiesterase that has been implicated in B cell chronic lymphocytic leukemia proliferation (40). Further refinement of the genetic maps through the generation of additional recombinants will be necessary to reduce significantly the list of candidate genes, and specifically address the contribution of these three genes. The expansion of the B-1a cell compartment in B6.Sle2 results from at least three mechanisms: greater input from fetal liver, increased proliferation/decreased apoptosis, and production of B-1a cells from adult lymphoid organs (10). A detailed study of the B6.Sle2 congenic recombinants will be necessary to address which locus is responsible for each of these three phenotypes. The association of Sle2a with high levels of IgMlow B-1a cells that express high levels of CXCR5 suggests that Sle2a is responsible for a substantial influx of B-1a cells from the spleen.

Because Sle2 does not by itself result in any clinical manifestation, a mapping strategy by genetic interaction was necessary. This approach, in which recombinants from a locus with a weak phenotype are mapped in the context of other interacting loci that are kept constant, was successfully used in the NOD model of type 1 diabetes (41). We have shown previously that the addition of Sle2 to the combination of Sle1 and Sle3 significantly increased pathogenicity (8). We therefore evaluated the respective effect of Sle2a, Sle2b, and Sle2c on the Sle1.Sle3 combination comparatively to B6.Sle1.Sle3 and B6.Sle1.Sle2.Sle3 (TC). Surprisingly, the Sle2c locus did not enhance disease by any of the parameters measured (survival, spleen weight, anti-dsDNA Ab, proteinuria, or kidney lesions). Interestingly, the number and proportion of perC B-1a cells in B6.Sle1.Sle3.Sle2c mice were lower than in the other triple congenic mice, suggesting complex interactions regulating this phenotype within the Sle2 locus, but also with yet unknown loci in Sle1 and/or Sle3. Nonetheless, this result demonstrates clearly that a mere increased number or percentage of B-1 cells is not sufficient to affect lupus pathogenesis.

Sle2a increased significantly lupus pathogenesis of B6.Sle1.Sle3 mice, resulting in similar survival curve, splenic architecture, proteinuria, and renal pathology in B6.Sle1.Sle3.Sle2a as in TC mice. Interestingly, Sle2a expression was associated with a significant switch from a mesangial to basement membrane staining pattern of immune complexes, and a significant increase in the number of infiltrating macrophages in the glomeruli. Renal infiltrating macrophages are predictive of a poor diagnosis in human lupus nephritis (42), and their central role has been demonstrated in lupus nephritis in the MRL/lpr model (43). The NZW strain is highly susceptible to nephrotoxic serum-induced nephritis, and the resulting glomerular lesions correlate with a significant increase in infiltrating macrophages (44). Sle2a is of NZW origin (22), and therefore could contribute to this phenotype. Unmanipulated kidneys from B6.Sle2 or B6.Sle2a mice do not show renal lesions or infiltrates, indicating that this phenotype results from interactions among the Sle1, Sle3, and Sle2a loci.

The contribution of Sle2b was evaluated indirectly by comparing the outcome of the B6.Sle1.Sle3.Sle2bc and B6.Sle1.Sle3.Sle2c strains. It is not possible at this time to conclude that our failure to breed the B6.Sle1.Sle3.Sle2b strain is due to the extreme pathogenicity of this locus combination or to fortuitous events. Alternative breeding strategies are being used to address this question. Nonetheless, data presented in this study strongly suggest that Sle2b, possibly in association with Sle2c, results in decreased survival, increased renal pathology, and abnormal splenic architecture in B6.Sle1.Sle3 mice. Interestingly, both Sle2a and Sle2b enhanced the number and percentage of perC B-1a cells when expressed in combination with Sle1 and Sle3. This observation associated again the enlargement of the B-1a pool with increased lupus autoimmune pathology, but also demonstrated complex synergistic effect between Sle loci that will have to be further clarified.

In conclusion, our study has identified one region of NZM2410 Chr. 4 with a strong effect on the size of the B-1a pool and no discernible impact on renal pathology, and two regions with modest individual effects on B-1a cells, but a strong impact on renal pathology. Our results also suggest a synergistic effect of the Sle2 subloci as none of them can recapitulate the range of the entire Sle2 phenotype. We have shown that strong and complex interactions exist between the Sle1 subloci (45), which is likely to be also the case for Sle2. Finally, the pathogenic loci Sle2a and Sle2b are both from NZW origin, while the major B-1a cell-promoting locus was NZB derived (22). This later result coincides with the fact that NZB mice have a larger pool of B-1a cells than NZW (35). We have already determined that both Sle1 and Sle3 are from NZW origin (22). Therefore, this study shows that on a B6 background, the combination of NZW loci in the absence of any NZB contribution is sufficient to induce a strong lupus nephritis phenotype. This illustrates again the complex interplay between susceptibility and resistance loci (46) and the propensity of the B6 genomic background to allow expression of autoimmune loci (47, 48).

We thank Daniel Perry for excellent technical help, and Amanda and Jessica Merritt for their outstanding work with the mouse colony. We also acknowledge the contribution of Eric Sobel and Chandra Mohan through numerous discussions about this work.

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 by National Institutes of Health Grant PO1 AI39824 to E.K.W.

3

Abbreviations used in this paper: SLE, systemic lupus erythematosus; NZB, New Zealand Black; NZW, New Zealand White; Chr., chromosome; GN, glomerulonephritis; int, intermediate; perC, peritoneal cavity; TC, triple congenic B6.sle1.sle2.sle3.

4

J. Li, Y. Liu, C. Xie, J. Zhu, D. Kreska, L. Morel, and C. Mohan. Submitted for publication.

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