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The Journal of Immunology, 2005, 175: 1337-1345.
Copyright © 2005 by The American Association of Immunologists

Genetic Dissection of Systemic Lupus Erythematosus Pathogenesis: Partial Functional Complementation between Sle1 and Sle3/5 Demonstrates Requirement for Intracellular Coexpression for Full Phenotypic Expression of Lupus1

Masatoshi Wakui2,*, Laurence Morel*,{dagger}, Edward J. Butfiloski*, Chunsun Kim* and Eric S. Sobel3,*,{dagger}

* Department of Medicine and Division of Rheumatology and Clinical Immunology, and {dagger} Center for Mammalian Genetics and Department of Pathology, Immunology, and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, FL 32610


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Sle1 on chromosome 1 and Sle3/5 on chromosome 7 are two of the most critical lupus susceptibility loci of the New Zealand Black/White-derived NZM2410 mouse strain. In contrast to C57BL/6 mice congenic for either Sle1 (B6.Sle1) or Sle3/5 (B6.Sle3/5), strains that express only a modest lupus-related phenotype, the bicongenic B6.Sle1.Sle3/5 strain has a robust phenotype, suggesting a critical role for epistatic interactions in lupus pathogenesis. Mixed chimera experiments indicated that the two loci are functionally expressed by different cell populations and predicted that phenotypic expression of the phenotypic features of the B6.Sle1.Sle3/5 strain could be fully reproduced with a combination of B6.Sle1 and B6.Sle3/5 bone marrow. Contrary to our expectations, there was only a partial functional complementation in these mixed chimeras. Spleen enlargement, CD4:CD8 ratio elevation, and epitope spreading of autoantibodies were fully developed in B6+B6.Sle1.Sle3/5 but not in B6.Sle1+B6.Sle3/5 mixed chimeras. This study is the first to present evidence that the pathways mediated by two critical lupus susceptibility loci derived from the New Zealand White strain must be integrated intracellularly for epistatic interactions to occur. Our mixed chimera approach continues to provide novel insights into the functional genetic pathways underlying this important murine model of systemic autoimmunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Systemic lupus erythematosus (SLE)4 is a genetically complicated disease in both humans and in mouse models (1). All evidence gathered thus far supports a threshold liability model for disease expression (2). In this model, the likelihood of developing disease is a function of the number of susceptibility genes present in the genome of that individual (1). Additional complications that arise include the potential for different genetic combinations to give the same phenotype (genetic heterogeneity) and the potential for one genetic locus to influence the expression of another (epistatic interactions) (1). Both features are present in animal models and are very likely to occur in humans. The potential for epistatic interactions to occur is well demonstrated in the congenic dissection approach being used by our group (3, 4, 5, 6, 7) and others (8, 9, 10) to identify the pathogenesis of lupus.

By genome-wide scanning, three major genomic intervals were originally identified in the highly autoimmune NZM2410/Aeg strain (11, 12). Two of the most critical regions are Sle1 on chromosome 1 and Sle3/5 on chromosome 7. C57BL/6 (B6) mice congenic for Sle1 (B6.Sle1, previously called B6.NZMc1) spontaneously developed high titers of IgG specific to H2A/H2B/dsDNA subnucleosomes, autoreactive T cells responding to histone epitopes, and an increase in expression of the early cell activation marker CD69 but no renal disease (3, 4). C57BL/6 mice congenic for Sle3/5 (B6.Sle3/5, previously called B6.NZMc7) developed autoantibodies to a variety of nuclear Ags with a low titer and penetrance, an elevated CD4:CD8 ratio with an increase in activated CD4+ T cells that were relatively resistant to activation-induced apoptosis, and a low but significant incidence of glomerulonephritis (3, 5). The subsequent linkage study revealed that the Sle3/5 genetic interval consisted of two subintervals, Sle5 on centromeric chromosome 7 and Sle3 telomeric to Sle5 (13). Sle5 was linked to anti-dsDNA IgG production whereas Sle3 was linked to the development of glomerulonephritis as well as anti-ssDNA IgM and antithyroglobulin IgG production. The critical natures of Sle1 and Sle3/5 phenotypes were exemplified by the bicongenic strain B6.Sle1.Sle3/5, which showed a much more robust phenotype, including the development of severe glomerulonephritis and epitope spreading of autoantibody responses to include high titers of IgG anti-dsDNA and nephrophilic autoantibodies (6, 7). Epistatic interactions could occur via a number of pathways, including intercellular and extracellular. However, except for a limited number of cases in which induced or spontaneous mutations have resulted in complete loss of function, relatively little is known about epistatic interactions.

As part of our efforts to dissect individual gene or susceptibility loci contributions to SLE, we have been performing bone marrow adoptive transfer. In a previous report, we demonstrated by adoptive transfer of bone marrow that Sle1 was functionally expressed in bone marrow-derived cells, and that expression by B cells was essential to break tolerance to nuclear autoantigens and develop humoral autoimmunity (14). Subsequent experiments indicated that the increased percentage of CD4+CD69+ T cells was also an intrinsic property of Sle1-expressing T cells (15). In contrast, analogous experiments have shown that Sle3/5 was functionally expressed by non-T cells of hemopoietic origin, as T cells of normal origin showed the elevated CD4:CD8 ratio when coinfused with bone marrow from B6.Sle3/5 mice (16). Similarly, anti-chromatin Abs of normal B cell origin were present in these mixed chimeras, suggesting an important role for expression of Sle3/5 by dendritic cells (DCs) (16). These observations led us to predict that a combination of B6.Sle1 and B6.Sle3/5 bone marrow would be able to fully reconstitute the autoimmune phenotype seen in the B6.Sle1.Sle3/5 bicongenic strain. We now report the results of these experiments. Contrary to our expectations, there was only a partial functional complementation in these mixed chimeras. These results strongly suggest that the Sle3/5 susceptibility loci are also functionally expressed on B cells, and that the full phenotypic expression requires an intracellular integration of these loci.


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

The C57BL/6 (B6) and B6.PL-Thy1a/Cy (B6.Thy1a) strains were originally obtained from The Jackson Laboratory and bred in our animal colony. The B6.C20 strain (B6.Igha) was originally obtained from G. Bosma (Institute for Cancer Research, Philadelphia, PA). The development of the B6.Sle1 and B6.Sle3/5 congenic strains has already been described (5, 12). B6.Sle1 has a 37 cM interval derived from chromosome 1 of NZM2410 (12). This interval, defined by the markers D1 MIT101 and D1 MIT155, contains the 95% confidence limits for inclusion of Sle1, as determined previously and is of New Zealand White (NZW) origin (11, 12). The Sle3/5 interval is defined by the markers D7 MIT31 and D7 MIT178 and contains the 95% confidence limits for inclusion of Sle3 and is also of NZW origin (11, 12). The B6.Sle1.Sle3/5 bicongenic was derived as an intercross between B6.Sle1 and B6.Sle3/5 (6, 7). The B6.Sle1.Thy1a and B6.Sle1.Igha bicongenic strains were derived by intercrossing B6.Sle1 with B6.Thy1a and B6.Sle1 with B6.Igha, respectively. All experiments were conducted according to protocols approved by the University of Florida Institutional Animal Care and Use Committee.

Preparation of chimeras

In an attempt to reduce radiation-induced nephropathy as a confounder (17, 18), B6.Rag–/– mice were used as hosts in all experiments. This allowed the dose or radiation to be reduced to 2 Gy. Histological examination confirmed the absence of glomerulonephritis in a test group studied 3 mo after reconstitution (data not shown). Production of mixed bone marrow chimeras was performed at a 1:1 ratio for the two donors. In all other respects, the chimeras were prepared as previously described, including the use of mAb and complement to eliminate mature T cell and B cell (14).

Flow cytometry

Single cell suspensions of spleen cells, lymph node cells, and thymocytes were prepared, followed by lysis of RBC in 0.83% NH4Cl. For lymph node cells, only inguinal, axillary, and periaortic lymph nodes were used. Cells were first blocked with staining medium (PBS with 5% FCS and 0.05% NaN3) supplemented with anti-CD16/CD32 (clone 2.4G2; American Type Culture Collection). All reagents were from BD Pharmingen, unless otherwise stated. For mixed chimeras receiving bone marrow from two donors differentially marked by the Ig H chain allele, four-color flow cytometric analysis was conducted with allotype-type specific anti-IgM Abs. To identify B cell subsets by donor origin, either directly fluoresceinated anti-IgMa (clone DS-1) or anti-IgMb (clone AF6.78) was used. For phenotypic analysis of B cells, these reagents were combined with CyChrome C-conjugated anti-B220, PE-conjugated anti-CD23, and biotinylated anti-CD21/CD35 (19), followed by streptavidin-allophycocyanin (BD Pharmingen). To verify that there was minimal double-counting of cells, samples were also costained with fluoresceinated anti-IgMa and PE-conjugated anti-IgMb, along with anti-B220 CyChrome C. For mixed chimeras receiving bone marrow from two donors differentially marked by expression of allelic forms of CD90 (Thy-1), two different strategies were also used. In the first, fluoresceinated anti-CD90.2 was combined with biotinylated anti-CD90.1, followed by streptavidin-allophyocyanin. The second strategy was used to minimize systematic errors due to the effects of compensation. In this case, samples were treated with either directly fluoresceinated anti-CD90.2 (also called anti-Thy-1b) or anti-CD90.1 (also called anti-Thy-1a), along with with CyChrome C-conjugated anti-CD4 (clone H129.19) and anti-CD8 allophyocyanin (clone 53-6.7). For the remaining color, PE-conjugated anti-CD69 (clone H1.2F3) or biotinylated anti-CD134 (clone OX-86) was used, followed by streptavidin-PE. Cells were fixed in 1% paraformaldehyde. For detection and analysis of DCs, fluoresceinated anti-I-Ab (clone AF6-120.1), PE-conjugated anti-CD11c (clone HL3), and allophycocyanin-conjugated anti-CD11b (clone M1/70) were used. Cells were analyzed using a FACSCalibur (BD Biosciences). Dead cells were excluded by forward angle and side scatter profiles. At least 20,000 cells were collected per sample. Data were processed by FCS Express version 2.0 (De Novo software; Thornhill) and illustrations were created with WinMDI version 2.8 (<facs.scripps.edu/software.html>).

Ig assay by ELISA

Total IgM and IgG2a determinations were performed as previously described (14). The allotype nonspecific IgG2a anti-chromatin ELISA was performed as previously described for the subnucleosome assay (14), substituting chicken chromatin (20) for the dsDNA/H2A/H2B complex. Allotype nonspecific IgG2a anti-dsDNA were evaluated by ELISA as previously described (21). When necessary, allotype-specific IgM and IgG2a determinations were conducted for the above specificities, also as previously described (14). The allotype a and allotype b titers were compared with each other by using serial dilutions of standard sera from a B6/lpr-Igha (allotype a) and a B6/lpr mouse adjusted in dilution to give equivalent OD readings when developed by the allotype nonspecific reagent. The results of ELISA were expressed in arbitrary units.

Statistics

Comparisons between two groups were performed by Student’s t test. Where indicated, a paired t test was used. Comparisons among groups were by one-way ANOVA with pairwise testing of all combinations performed by the Tukey’s posttest. To test more conservatively, the data were also tested nonparametrically with the Kruskal-Wallis test, followed by Dunn’s posttest, with equivalent results. Correlations were determined by the Pearson Product correlation technique. Results were calculated with the software GraphPad Prism 4 for Windows (GraphPad). Values of p < 0.05 were considered statistically significant. The program corrects for multiple comparisons.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Splenomegaly in mixed chimeras

Splenomegaly is a feature of mouse lupus, which is associated with the disease progress and severity (6, 7). Although modest splenomegaly is present in both the B6.Sle1 and B6.Sle3/5 monocongenic strains, epistatic interactions between these two loci results in marked enlargement (6, 7). It was therefore of interest to determine whether a combination of bone marrow from B6.Sle1 and B6.Sle3/5 mice could reproduce this phenotype. B6.Rag–/– mice receiving various combinations of bone marrow were aged for 7–9 mo. In the case of mice receiving B6.Sle1.Sle3/5 bone marrow, there was a coinfusion of either B6.Igha or B6.Thy1a bone marrow to control for a dose effect. At the termination of the experiment, spleen weight was determined. The different groups are summarized in Table I. As shown in Fig. 1A and Table I, mice receiving bone marrow without Sle1 or Sle3/5 (group 1) had a mean spleen weight comparable to that of unmanipulated C57BL/6 (B6) mice (6, 7). Mice receiving bone marrow with either Sle1 (group 2) or Sle3/5 (group 3) alone also showed no spleen enlargement, whereas mice receiving a combination of B6.Sle1 and B6.Sle3/5 (group 4) bone marrow had a modest enlargement. By t test, this increase was statistically significant when compared with mice receiving neither Sle1 nor Sle3/5 (mean spleen weight, 0.121 vs 0.097 g; p = 0.026). In contrast, mice receiving B6.Sle1.Sle3/5 (group 5) bone marrow had marked splenomegaly (mean, 0.204 g; p < 0.001 by Kruskal-Wallis). These values are slightly lower than previously reported for unmanipulated, unirradiated female bicongenic mice, as previously reported (6) (mean, 0.27 ± 0.03 g) but are statistically different from the results for group 4 mice, clearly showing that integration of Sle1 and Sle3/5 within the same genome potentiated epistatic interactions.


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  		Table I. Allotype-nonspecific evaluation of splenic phenotypes in mixed chimeras

 


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  		FIGURE 1. Cellular profiles from spleens of mixed chimeras. A, The enlarged spleen characteristic of B6.Sle1.Sle3/5 bicongenic mice was seen only in mixed chimeras receiving B6.Sle1.Sle3/5 donor bone marrow. B, The percentage of splenic T cells, as defined by expression of CD90, was increased in group 5 chimeras. C, The percentage of splenic B cell, as defined by expression of IgM, was correspondingly decreased in group 5 chimeras. D, The perturbations in T cell and B cell populations resulted in a marked increase in the splenic T cell to B cell ratio in group 5 chimeras. In all cases, group 5 was different statistically from the other groups.

 
Alterations in the lymphoid compartments in mixed chimeras

To better understand how interactions of Sle1 and Sle3/5 affected cellular composition of secondary lymphoid organs, we performed flow cytometric analysis of spleens at the termination of the experiment (Fig. 1, B–D, and Table I). The relative proportion of CD3+ T cells was statistically higher in group 5 mice than in groups 1, 2, 3, or 4 (46 vs 33, 31, 30, or 28%, respectively, p < 0.0001) (Fig. 1B). Correspondingly, the percentage of B cells in the spleen was lower in group 5 chimeras than in any of the other groups (Fig. 1C). As shown in Fig. 1D, the elevated T cell and decreased B cell percentage seen in group 5 mice led to a marked increase in the T cell to B cell ratio that was statistically significant (p < 0.0001). Collectively, these data showed that the splenic lymphocyte composition was altered in the presence of an intracellular integration of both loci rather than in the coexistence of Sle1- and Sle3/5-expressed by different cell populations.

Elevated CD4:CD8 ratio in mixed chimeras

It has been reported that the CD4:CD8 ratio was more elevated in the B6.Sle1.Sle3/5 bicongenic strain than in the B6.Sle3/5 strain, whereas the B6.Sle1 strain did not exhibit this phenotype (6). Moreover, previous experience with mixed chimeras indicated that Sle3/5 could exert its influence on cells not expressing this susceptibility locus. Therefore, it was of great interest to assess the interactions of Sle3/5 with Sle1 in mixed chimeras (Fig. 2A and Table I). When Sle1 was integrated with Sle3/5 on the same donor cell (group 5), mixed chimeras exhibited the highest CD4:CD8 ratio (2.08, p < 0.0001 by one-way ANOVA). As expected, group 3 chimeras exhibited a higher CD4:CD8 ratio than either group 1 (1.67 vs 1.41, p = 0.005) or group 2 (1.67 vs 1.24, p = 0.002) chimeras. Surprisingly, the ratio seen in B6.Sle1+B6.Sle3/5 (group 4) chimeras was similar to that seen in B6+B6 (group 1) chimeras (1.37 vs 1.41) and lower than that of B6+B6.Sle3/5 (group 3) chimeras (1.37 vs 1.67, p = 0.0012). Thus, the presence of Sle3/5 increased CD4:CD8 ratio in mixed chimeras, whereas the presence of Sle1 seemed to decrease it.



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  		FIGURE 2. Full expression of the epistatic interactions between Sle1 and Sle3/5 for an elevated splenic CD4:CD8 ratio requires integration within the genome. A, CD4:CD8 ratio, CD90 allele-nonspecific. The splenic CD4:CD8 ratio was increased most markedly in mice receiving B6.Sle1.Sle3/5 bone marrow. B, The allele-specific CD4:CD8 ratio revealed that both donors contributed to the elevated CD4:CD8 ratios mediated by Sle3/5. When expressed on a different cell population, Sle1 appeared to decrease the ratio, an effect that was lost when integrated within the same cell (groups 5 and 6). C, The B6.Sle3/5 or B6.Sle1.Sle3/5 donor cell contribution determined the final CD4:CD8 ratio of the B6-derived T cells. The CD4:CD8 ratios shown are for B6.Thy-1a-positive (•) or B6.Sle1.Thy-1a-positive ({diamond}) T cells under the influence of B6.Sle3/5-derived bone marrow or B6.Thy-1a-positive T cells under the influence of B6.Sle1.Sle3/5-derived bone marrow ({circ}). Cells of B6.Sle1.Sle3/5 origin had a more profound effect on the ratio than B6.Sle3/5-derived cells, even when present in limited numbers.

 
To better understand how these loci were influencing the CD4:CD8 ratio based on donor origin, we examined the allele-specific CD4:CD8 ratio in mixed chimeras expressing two different alleles of CD90 (also known as Thy1). Representative contour plots are shown in Fig. 3, and the results of all mice are compiled, as shown in Fig. 2B. The CD4:CD8 ratio of B6.Thy1a-derived donor T cells was not significantly different from that of Thy1b donors when paired with either B6 (group 1) or B6.Sle3/5 (group 3) chimeras. Consistent with previous experiments, the presence of B6.Sle3/5-derived bone marrow raised the CD4:CD8 ratio in cells not expressing Sle3/5. The effect of B6.Sle3/5 bone marrow-derived donor cells on the T cells derived from the donor partner tended to be more pronounced when paired with B6.B6.Thy1a than with B6.Sle1.Thy1a (i.e., group 3 vs group 4).



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  		FIGURE 3. Representative allele-specific flow cytometry contour plots of mixed chimeras showed that the presence of Sle3/5 could cause an increase in CD4:CD8 ratio and an increase in expression of the activation marker CD69 by T cells of non-Sle3/5 donor origin. Spleen cells were stained with combinations of either anti-CD90.1 or anti-CD90.2 along with Abs to CD4, CD8, and CD69. Following gating on CD90+ events, contour plots are shown for CD4 vs CD8 and CD4 vs CD69. For the CD4 vs CD69 plots, values shown in the top right quadrant are the percentage of gated cells. For CD4 vs CD8 plots, this value is the CD4:CD8 ratio.

 
As mentioned, it was surprising that cells derived from the B6.Sle1 donor had a lower CD4:CD8 ratio when paired with B6.Sle3/5. It was possible that these observed differences could be due to differences in the relative composition of the chimeras. Indeed, the contribution of B6.Sle3/5 to mixed chimeras when paired with B6.Sle1 was lower than when paired with B6. When the CD4:CD8 ratio was plotted as a function of composition, there was little to no difference in the regression slope between B6+B6.Sle3/5 and B6.Sle1+B6.Sle3/5 chimeras (0.0090 vs 0.0083, not significant) (Fig. 2C), suggesting that the difference was due to composition rather than to an intrinsic difference in B6.Sle1-derived T cells. In contrast, when B6 and B6.Sle1.Sle3/5 bone marrow were mixed, the CD4:CD8 ratio was much higher at equivalent compositions (Fig. 2C). This impression was confirmed in mixed chimeras in which B6.Sle1 and B6.Sle1.Sle3/5 bone marrow were coinfused (Fig. 2B). Taken together, the extrinsic effect of an intracellular integration of Sle1 and Sle3/5 increased the CD4:CD8 ratio in an additive fashion, whereas expression in different cell compartments did not.

T cell activation marker expression in mixed chimeras

In murine lupus, CD69 expression has been used as a marker of chronic activation of T cells in vivo as well as a marker of very early activation of T cells in vitro (22, 23). CD134 (also known as OX40), which is another T cell activation marker, functions as a costimulatory molecule for CD4+ T cells and contributes to humoral immune responses (24). To assess the effect of interactions of Sle1 and Sle3/5 on the activation status of CD4+ T cells, expression levels of CD69 and CD134 were evaluated by flow cytometric analysis. As shown in Fig. 4A, the percentage of CD69+ cells among B6.Sle1.Thy1a donor-derived CD4+ T cells was higher in B6.Sle1.Thy1a +B6.Sle3/5 (group 4) and B6.Sle1.Thy1a +B6.Sle1.Sle3/5 (group 5) chimeras than in group 2 B6.Sle1.Thy1a +B6 chimeras (33 vs 14%, p = 0.007 and 38 vs 14%, p = 0.00002, respectively). There was no significant difference between B6.Sle1.Thy1a +B6.Sle3/5 (group 4) and B6.Sle1.Thy1a +B6.Sle1.Sle3/5 (group 6) chimeras with respect to this phenotype. To evaluate the influence of composition, the percentage of CD69+CD4+ T cells was plotted as a function of the percentage of contribution by the Sle3/5 or Sle1.Sle3/5 donor (Fig. 4B). Although there was some difference in calculated slope based on the donor composition, the differences were not statistically significant. Overall, linear regression showed a positive correlation with contribution by either the Sle3/5 or the Sle1.Sle3/5 donor (slope = 0.24 ± 0.06; p < 0.001). This suggests that the intracellular integration of the Sle1- and Sle3/5-mediated pathways did not further potentiate the transactivation of other T cells not expressing either of these loci. In this limited data set, Sle1 alone (group 2) did not cause an elevation in CD69 expression by CD4+ T cells, a phenotype which has had limited penetrance in mixed chimeras (16). However, there was some enhancement when B6.Sle1-derived T cells were in the presence of cells of B6.Sle1.Sle3/5 origin (group 6).



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  		FIGURE 4. Allele-specific evaluation of T cell activation in mixed chimeras. A, The percentage CD4+ cells expressing CD69 was higher in B6.Sle1.Thy1a donor-derived T cells in the presence of B6.Sle3/5 (group 4) or B6.Sle1.Sle3/5 (group 5) donor cells than in the presence of B6 (group 2) donor cells (33.2 ± 10.5% vs 13.9 ± 6.3%, p = 0.007; 38.0 ± 26.8% vs 13.9 ± 6.3%, p = 0.00002). B, The B6.Sle3/5 or B6.Sle1.Sle3/5 donor cell contribution correlated with increased expression of CD69 by B6-derived CD4+ T cells. Linear regression analysis showed no difference between the effects of B6.Sle3/5 and B6.Sle1.Sle3/5 donors. C, Enhanced expression of CD134 in B6.Sle1.Sle3/5-contraining mixed chimeras. In the presence of B6.Sle1.Sle3/5- but not B6.Sle3/5-derived donor cells, CD134 expression by CD4+ T cells was in B6-derived CD4+ T cells (mean, 42.5 ± 26.5 vs 24.2 ± 16.4), but this did not reach statistical significance. However, CD134 expression appeared to be further enhanced by expression of the Sle1 susceptibility locus in the presence of B6.Sle1.Sle3/5-derived bone marrow (64.7 ± 14.5% vs 22.5 ± 7.1%), and this was statistically significant (p < 0.0001).

 
As shown in Fig. 4C, the percentage of CD134+ cells among B6.Sle1.Thy1a donor-derived CD4+ T cells was higher in B6.Sle1.Thy1a +B6.Sle1.Sle3/5 chimeras than in B6.Sle1.Thy1a +B6 and B6.Sle1.Thy1a +B6.Sle3/5 chimeras (65 vs 28%, p < 0.0001; 65 vs 23%, p < 0.0001). With respect to this phenotype, there was no significant difference between B6.Sle1.Thy1a +B6 and B6.Sle1.Thy1a +B6.Sle3/5 chimeras. The percentage among B6.Thy1a donor T cells appeared to be higher in B6.Thy1a +B6.Sle1.Sle3/5 chimeras than in B6.Thy1a +B6 and B6.Thy1a +B6.Sle3/5 chimeras, but the increase did not achieve statistical significance because of the broad distribution.

Alteration of B cell subsets in mixed chimeras

Perturbations in splenic B cell subsets have been described in (NZB x NZW)F1 mice and the parental strains (25, 26, 27). Although the increases in the marginal zone population has been linked most tightly with New Zealand Black-derived loci, differences have also been described between C57BL/6 (B6) and NZW mice (25), and our group has observed differences in transitional cell populations in B6.Sle1.Sle3/5 bicongenic mice (our unpublished observations). It was therefore of interest to phenotype splenic B cell subsets in mixed chimeras from mice receiving a combination of B6.Igha and B6.Sle1.Sle3/5 bone marrow (group 5). Spleen cells were stained for a combination with allele-specific IgM and a combination of B220, CD21, and CD23. As shown in Fig. 5, cells of B6.Sle1.Sle3/5 origin tended to accumulate at the transition between T1 and T2 cells, when compared with the normal B6 donor. These differences were statistically significant.



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  		FIGURE 5. In B6.Igha +B6.Sle1.Sle3/5 (group 5) mixed chimeras, B cells of B6.Sle1.Sle3/5 origin tended to accumulate at the T1/T2 transition. Freshly isolated splenic B cells were stained with combinations of anti-B220, anti-CD23, anti-CD21, and either anti-IgMa or anti-IgMb. After gating on B220+IgMa+ or B220+IgMb+ events, and displaying CD21 vs CD23, T1 cells were identified as CD21lowCD23low, T2 cells as CD21highCD23+, marginal zone cells (MZ) as CD21highCD23low, and follicular B cells (FC) as CD21+CD23+. By paired and unpaired t tests, the differences in the T1 and T2 subsets by donor origin were statistically significant (p < 0.001).

 
An increase in the percentage of lymphoid DCs in spleens of mixed chimeras

DCs are a candidate population of cells functionally expressing Sle3/5 (16). We found a significant increase in splenic lymphoid DCs, which were defined by the CD11b–CD11c+ I-Ab+ phenotype, in B6+B6.Sle1.Sle3/5 chimeras. Although previous studies have shown that this lymphoid DC phenotype defined by CD8 positivity or CD11b negativity does not truly reflect lymphoid origin, the functional properties have been reported to be different between myeloid DCs, which are defined by CD8 negativity or CD11b positivity (28, 29, 30). The percentage of lymphoid DCs was higher in spleens of B6+B6.Sle1.Sle3/5 chimeras than in those of B6+B6.Sle3/5 and B6+B6 chimeras (3.7 vs 2.3%, p < 0.05; 3.7 vs 1.8%, p < 0.01) (Fig. 6). This percentage also tended to be higher than that seen in B6.Sle1+B6.Sle3/5 chimeras, although the difference was not statistically significant (3.7 vs 3.1%, not significant). When compared with B6+B6.Sle3/5 or B6+B6 chimeras, B6.Sle1+B6.Sle3/5 chimeras exhibited a modest increase, again without achieving statistical significance. The myeloid DC compartment, defined as CD11b+CD11c+I-Ab+, showed a small increase in population in B6+B6.Sle1.Sle3/5 chimeras that did not reach statistical significance (data not shown). Taken together, an intracellular integration of Sle1 and Sle3/5 was required for a significant increase in lymphoid DCs. It remains unclear whether the interaction of Sle1 and Sle3/5 was extrinsic or intrinsic for DCs because allotype-specific analysis of the DC phenotype could not be performed in B6.Sle1+B6.Sle3/5 chimeras. Efforts are underway to prepare these mixed chimeras with the a allele and b allele of CD45.



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  		FIGURE 6. Splenic lymphoid DCs in mixed chimeras. The lymphoid DC phenotype was defined as I-Ab+ cells that also coexpressed CD11c but not CD11b. The percentage of lymphoid DCs was highest in the spleens of mice containing B6.Sle1.Sle3/5 bone marrow (p < 0.05 by ANOVA).

 
Autoantibody profiles in mixed chimeras

We have previously reported that B6.Sle1 mice produced IgG anti-H2A/H2B/dsDNA with minimal epitope spreading, whereas B6.Sle3/5 mice exhibited low-penetrant antinuclear Ab production with significant epitope spreading (3, 4). B6.Sle1.Sle3/5 have been shown to produce a high titer and a broad spectrum of antinuclear Abs (6, 7). To assess the effects of interaction between Sle1 and Sle3/5, serum autoantibodies in mixed chimeras were evaluated (Fig. 7).



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  		FIGURE 7. Allotype-specific serum IgG2a autoantibody profiles in mixed chimeras are shown for anti-H2A/H2B/dsDNA (A), anti-chromatin (B), and anti-dsDNA (C). High titers of IgG2a anti-dsDNA and anti-glomerular binding activity Abs were seen only in the mice containing B6.Sle1.Sle3/5-derived B cells, and the titers were predominantly derived from the B6.Sle1.Sle3/5 donor.

 
In mice receiving a combination of B6 and B6.Sle1 bone marrow, there was a modest increase in titers of IgG2a anti-H2A/H2B/dsDNA derived from the B6.Sle1 donor, as anticipated, although the titers were lower than we had seen in mice receiving lethal irradiation (Fig. 7A). Whether this was due to the difference in conditioning regimen or the change to the Ig H chain a allotype is unclear. Likewise, there was a subset of mice receiving B6 and B6.Sle3/5 bone marrow that developed anti-H2A/H2B/dsDNA Abs, but these were derived from both donors, also as previously reported (16). Coinfusion of B6.Sle1 and B6.Sle3/5 bone marrow resulted in a higher prevalence of autoantibodies, and these were derived nearly equally from both donors, suggesting the ability of Sle1 and Sle3/5 to function when expressed by different cell populations. This was also seen for anti-dsDNA (Fig. 7C) and was even more obvious in the anti-chromatin specificity (Fig. 7B). Despite this effect, mice receiving a combination of B6 and B6.Sle1.Sle3/5 bone marrow had significantly higher titers of anti-H2A/H2B/dsDNA and anti-dsDNA, although this effect did not hold for anti-chromatin. More importantly, however, was the fact that the autoantibodies for all of the specificities were disproportionately derived from the B6.Sle1.Sle3/5 donor, demonstrating an effect of an intracellular epistatic interaction between Sle1 and Sle3/5.

Epitope spreading in mixed chimeras

To evaluate further the ability of interactions between Sle1 and Sle3/5 to mediate epitope spreading, we examined the donorspecific profile of autoantibodies produced by individual chimeras. The influence of coinfusion of either Sle1-, Sle3/5-, or Sle1.Sle3/5-derived bone marrow on autoantibody production on B6-derived B cells is shown in Fig. 8A. In the presence of Sle3/5 or Sle1.Sle3/5-derived cells, B6-derived B cells could be induced to break tolerance. However, titers were low. We have previously shown that epitope spreading could extend to the anti-dsDNA specificity, albeit with low penetrance. In the case of this more limited sampling, little to no anti-dsDNA of B6 origin was seen when the infusion partner was B6.Sle3/5. Interestingly, two mice were induced to make limited anti-dsDNA when B6.Sle1.Sle3/5 was coinfused. These results contrasted with the outcome of B cells of B6.Sle1 origin (Fig. 8B). Coinfusion of B6 bone marrow resulted in IgG2a anti-H2A/H2B/dsDNA Abs, as expected. Again, the titers were limited. Two mice developed appreciable titers of anti-chromatin, but epitope spreading did not extend to anti-dsDNA. In the presence of B6.Sle3/5-derived bone marrow, there was marked potentiation of the anti-chromatin response and relatively little response solely to the anti-H2A/H2B/dsDNA inner nucleosome core. Epitope spreading to anti-dsDNA was minimal, however. The influence of B6.Sle1 on B6.Sle3/5-derived B cells was more limited (Fig. 8C), again as expected. There might have been a slightly increased tendency for B6.Sle3/5-derived B cells to develop anti-dsDNA Abs. If so, this might reflect the influence of B6.Sle1-derived T cells, as we have previously shown that Sle1 is functionally expressed on this population. These results contrasted markedly with the response of B6.Sle1.Sle3/5-derived B cells, in which Abs to all three specificities were seen at high concentrations (Fig. 8D).



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  		FIGURE 8. Epitope spreading of autoantibody specificities in mixed chimeras. B6.Sle1.Sle3/5-derived B cells showed evidence of intracellular epistatic interactions between Sle1 and Sle3/5. The effects of coinfusion of bone marrow from B6.Sle1, B6.Sle3/5, or B6.Sle1.Sle3/5 were assessed by IgG2a allotype-specific ELISA. The titers for each sample were normalized to the highest titer obtained for that specificity, as the original titers are in arbitrary units. A, The response of B6-derived B cells. B, The response of B6.Sle1-derived B cells. C, The response of B6.Sle3/5-derived B cells. D, The response of B6.Sle1.Sle3/5-derived B cells.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Epistatic interactions have been thought to play a critical role in the development of polygenic diseases such as lupus. Most previous studies on the epistatic effects of lupus genetic regions have been based on linkage analysis or multiple congenics (6, 7, 9, 10, 31). However, these approaches cannot address whether each epistatic interaction is based on an intracellular or extracellular pathway because any cell can potentially express each gene from each locus. These limitations can be overcome by using mixed chimeras reconstituted with hemopoietic cells from combinations of congenic strains expressing appropriate allelic markers. In this study, we have used such mixed chimeras to assess the interactions of Sle1 and Sle3/5, a synergistic combination that had been demonstrated by the development of aggressive autoimmunity in B6.Sle1.Sle3/5 mice. Based on our earlier observations that Sle1 was functionally expressed by T and B cells (14), and that Sle3 (and probably Sle5) was functionally expressed by non-B cell APCs (16), we had predicted that the coinfusion of Sle3/5-derived bone marrow cells would be sufficient to drive Sle1-derived B cells to demonstrate the full phenotypic expression seen in unmanipulated B6.Sle1.Sle3/5 mice. To our surprise, the results were more complicated.

The first phenotype we examined was splenomegaly. Splenomegaly has been associated with the progress and severity of mouse lupus (6, 7). Significantly enlarged spleens in B6+B6.Sle1.Sle3/5 (group 4) but not B6.Sle1+B6.Sle3/5 (group 5) mixed chimeras confirmed the impression that interactions between Sle1 and Sle3/5 were further enhanced when coexpressed by the same cell (Fig. 1). This impression was reinforced by evaluation of T cell phenotypes. In this model, coinfusion of Sle3/5-expressing bone marrow caused an increase in the CD4:CD8 ratio of B6-derived T cells, as expected (Figs. 1 and 2). However, this effect was not seen when paired with Sle1-expressing T cells. If anything, the CD4:CD8 ratio was decreased, an effect that was overcome when the paired bone marrow was of B6.Sle1.Sle3/5 origin (group 6). In addition, expression of CD134 (Fig. 4C) but not CD69 (Fig. 4A) was enhanced when the infusion partner was of B6.Sle1.Sle3 origin. In as much as enhanced expression of CD69 by CD4+ T cells is seen as an intrinsic property of Sle1-derived T cells, the latter finding is perhaps not surprising. In contrast, it has been reported that CD134 expression by T cells is augmented by CD28 costimulation in vitro, suggesting a contribution of APCs to up-regulation of CD134 in vivo (32). One potential explanation for our results regarding changes in T cell phenotype is that Sle1 and Sle3/5 are also functionally integrated within the DC population. Indeed, studies of DCs derived from group 5 mice showed an expansion of the lymphoid DCs (Fig. 6). Moreover, as shown in Fig. 2C, at limiting doses, Sle1.Sle3/5-derived bone marrow was more potent than the expression with Sle3/5 alone.

Because of these findings, it was of particular interest to evaluate B cell phenotypes in the mixed chimeras. If Sle3/5 were functioning purely through its effects on DCs, it would be expected that B6.Sle1-derived B cells would be induced to undergo epitope spreading and produce high titers of IgG anti-dsDNA characteristic of B6.Sle1.Sle3/5 bicongenics. Clearly, this was not the case, as B6.Sle1-derived B cells secreted high levels of anti-chromatin but not anti-dsDNA (Fig. 8). Although it might be argued that this was a result of an inadequate number of B6.Sle3/5-derived DCs cells because of dilution in mixed chimeras, these DCs caused a partial break in tolerance when partnered with B6-derived B cells, and the B6.Sle1.Sle3/5-derived B cells produced high titers of anti-dsDNA even when they were the minor donor partner (Fig. 2C). Taken together, we conclude that these loci were coexpressed by B cells and possibly by DCs and that an intracellular integration was required for full development of the lupus phenotype expressed in B6.Sle1.Sle3/5 mice. To our knowledge, this study is the first to present evidence for a functional interaction between two different lupus susceptibility loci within the same B cell to show that an intracellular integration of these loci is required for full phenotypic expression. This approach provided a novel insight into genetic pathways underlying autoimmunity and can be used as a model for better understanding of epistatic interactions of two susceptibility loci in polygenic diseases.

It remains possible that a high concentration of B6.Sle3/5-derived DCs could cause a full break in tolerance in Sle1-derived B cells. For the present studies, we used C57BL/6 (B6) mice deficient in Rag-1 expression as hosts, and the mice were lightly irradiated. This step reduced radiation-induced damage to the kidneys but likely permitted persistence of host-derived DCs. This was confirmed in a separate set of experiments using the pan-leukocyte marker CD45 (data not shown). We plan to pursue further the potential of Sle3/5 DCs by using B6.Sle3/5 mice deficient in Rag-1 expression as a host. It must be pointed out, however, that it was the fortuitous use of the present system that permitted us to see definitive evidence that Sle3/5 is functionally expressed by B cells. B cells coexpressing Sle1 and Sle3/5 might also have affected the CD4:CD8 ratio and CD134 expression on T cells due to the B cell altered APC function. Conversely, several groups have reported that normal DC development and function may depend upon lymphocytes (33, 34), making it possible that some of the phenotypes are interdependent. At present, we cannot determine whether the effect of an intracellular integration of Sle1 and Sle3/5 on the percentage of splenic lymphoid DCs was fully or partially intrinsic, and the B6.Sle3/5 Rag-deficient mice will help in this regard as well.

There are at least two possibilities with respect to a significant increase in the percentage of splenic lymphoid DC phenotype in B6+B6.Sle1.Sle3/5 (group 5) chimeras. One is that coexpression of the two loci by DCs resulted in preferential differentiation into this phenotype. Another possibility is that an intracellular integration of the two loci produced extrinsic signals to affect DC development. Treatment with Flt3 ligand (Flt3L) has been shown to preferentially induce the lymphoid DC phenotype (35). Interestingly, the chromosomal location of Flt3L gene overlaps with the Sle3/5 region, making this cytokine a candidate gene encoded by Sle3/5 (36). In this case, Sle1 might accelerate production of Flt3L or activate this cytokine through coexpression with Sle3/5 on the same cell. Fiz1 is a novel zinc finger protein interacting with Flt3 in intracellular signaling (37). The Fiz1 gene is also located on mouse chromosome 7, suggesting a possibility that this gene may be another candidate, which is encoded by Sle3/5 and expressed possibly on DCs (38).

It is of considerable interest that both extrinsic and intrinsic effects of Sle3/5 contributed to epitope spreading of autoantibodies. The contribution seems to differ by the autoantibody specificity. The extrinsic effect of Sle3/5 efficiently broke tolerance of chromatin-reactive B cells but was much less effective with regard to dsDNA-reactive B cells. The intracellular integration of Sle1 and Sle3/5 greatly facilitated the break in tolerance of dsDNA-reactive B cells, a more pathogenic autoantibody. Recently, it was shown that Sle3/5 impacts IgH CDR3 sequences, somatic mutations, and receptor editing, although it remains uncertain whether these effects are extrinsic and/or intrinsic (39). Cultured B6.Sle3/5 bone marrow-derived DCs have been found to display high gene expression of BAFF/BLyS (40), which has been shown to rescue autoreactive B cells from apoptosis (41, 42). This cytokine might be one contributor to the extrinsic effect of Sle3/5 on autoreactive B cells. If so, Sle3/5 may encode a molecule involved in the regulation of BAFF/Blys production but would not be BAFF/BlyS itself because this cytokine gene is located on a chromosomal interval different from Sle3/5 genetic region.

As mentioned earlier, CD134 expression profiles were not concordant with CD69 profiles among mixed chimeras. B6.Sle1.Sle3/5 but not B6.Sle3/5 donor cells increased CD134 expression markedly on B6.Sle1-derived donor CD4+ T cells and modestly on B6-derived donor CD4+ T cells, whereas both B6.Sle3/5 and B6.Sle1.Sle3/5 donor cells equally increased CD69 expression on B6.Sle1 but not B6 donor CD4+ T cells. Why B6.Sle1 donor T cells were more sensitive to the Sle3/5 effects on CD69 expression than B6 donor T cells remains unclear. Previous studies showing association of increased CD69 expression with lupus have suggested a contribution of intrinsic dysregulation of CD69 induction or apoptosis resistance (22, 43). Sle1 expression on T cells might alter regulation of CD69 expression or sensitivity to activation-induced cell death. CD134 signaling up-regulates CXCR5 expression on T cells, directing them to B cell follicles (24). Although the functional relevance of CD134 in lupus has not been clearly established, several studies have presented evidence that this costimulatory molecule is critical for a break in self-tolerance (44, 45, 46). Therefore, it is of interest that the increase in CD134 expression on CD4+ T cells correlated with full phenotypic expression of lupus and required coexpression of Sle1 and Sle3/5 probably on the same APCs.

Our ultimate goal is to identify lupus susceptibility locus-encoded genes responsible for the phenotypic expression and to establish a receipt for lupus induction. Further characterization of functional expression patterns of isolated loci along with their finer mapping should continue to complement each other. In addition, molecular characterization by genomics and proteomics is expected to be helpful to dissect lupus pathogenesis (8, 47). For example, genes and proteins differentially expressed in the spleen of B6.Sle1+B6.Sle3/5 (group 4) vs B6+B6.Sle1.Sle3/5 (group 5) mixed chimeras may reflect target molecules of epistatic interactions based on an intracellular integration of these two loci, which play a critical role in full development of the disease. Our efforts are underway to thoroughly dissect and reconstitute lupus pathogenesis.


   Acknowledgments
 
We thank Raquel Baert, Aimee Young, Deyadira Baez-Sierra, and Carla Moodie for expert technical assistance.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
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 Grants P01 AI39824, R01 AR44894, and R01 AI043454. Back

2 Current address: Department of Allergy and Immunology, Saitama Medical School, 38 Morohongo, Moroyama, Iruma-gun, Satama 350-0495, Japan. Back

3 Address correspondence and reprint requests to Dr. Eric S. Sobel, University of Florida Department of Medicine, Box 100221 J. Hillis Miller Health Center, Gainesville, FL 32610-0221. E-mail address: sobeles{at}medicine.ufl.edu Back

4 Abbreviations used in this paper: SLE, systemic lupus erythematosus; DC, dendritic cell; NZW, New Zealand White; Flt3L, Flt3 ligand. Back

Received for publication January 12, 2005. Accepted for publication May 10, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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