Genetic Dissection of the Murine Lupus Susceptibility Locus Sle2: Contributions to Increased Peritoneal B-1a Cells and Lupus Nephritis Map t
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免疫学杂志 2005年第14期
Abstract
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 x 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.
Introduction
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) x 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.
Materials and Methods
Mice
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 x B6.Sle2)F1 x 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.
Flow cytometry
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.
Autoantibody measurement and spleen immunofluorescence
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.
Renal pathology
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 -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.
Statistics
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.
Results
Sle2a and Sle2c both contribute to perC B-1a cell expansion
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. 2A). In 8- to 11-mo-old mice, B6.Sle2c mice presented a significantly higher percentage of perC B-1a cells (Fig. 2B), a higher B-1a/B-2 ratio (Fig. 2C), and a 2-fold increase in absolute number (Fig. 2D). 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).
Discussion
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 Ifn 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--blocking Ab, or decreased with IFN- 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).
Acknowledgments
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.
Disclosures
The authors have no financial conflict of interest.
Footnotes
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.
2 Address correspondence and reprint requests to Dr. Laurence Morel, Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, 1600 Archer Road, Gainesville, FL 32610-0275. E-mail address: morel@ufl.edu
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.
Received for publication March 17, 2005. Accepted for publication May 10, 2005.
References
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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 x 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.
Introduction
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) x 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.
Materials and Methods
Mice
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 x B6.Sle2)F1 x 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.
Flow cytometry
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.
Autoantibody measurement and spleen immunofluorescence
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.
Renal pathology
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 -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.
Statistics
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.
Results
Sle2a and Sle2c both contribute to perC B-1a cell expansion
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. 2A). In 8- to 11-mo-old mice, B6.Sle2c mice presented a significantly higher percentage of perC B-1a cells (Fig. 2B), a higher B-1a/B-2 ratio (Fig. 2C), and a 2-fold increase in absolute number (Fig. 2D). 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).
Discussion
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 Ifn 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--blocking Ab, or decreased with IFN- 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).
Acknowledgments
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.
Disclosures
The authors have no financial conflict of interest.
Footnotes
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.
2 Address correspondence and reprint requests to Dr. Laurence Morel, Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, 1600 Archer Road, Gainesville, FL 32610-0275. E-mail address: morel@ufl.edu
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.
Received for publication March 17, 2005. Accepted for publication May 10, 2005.
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