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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Objective

To determine whether overexpression of BAFF can promote systemic lupus erythematosus (SLE)–like autoimmunity in mice that are otherwise autoimmune-resistant.

Methods

We used class II major histocompatibility complex (MHC)–deficient C57BL/6 (B6) mice as a model of resistance to SLE and Sles1-bearing B6 mice as a model of resistance to the autoantibody-promoting capacity of the Sle1 region. We generated BAFF-transgenic (Tg) counterparts to these respective mice and evaluated lymphocyte phenotype, serologic autoimmunity, renal immunopathology, and clinical disease in the BAFF-Tg and non-Tg mouse sets.

Results

Although constitutive BAFF overexpression did not lead to B cell expansion in class II MHC–deficient B6 mice, it did lead to increased serum IgG autoantibody levels. Nevertheless, renal immunopathology was limited, and clinical disease did not develop. In B6 and B6.Sle1 mice, constitutive BAFF overexpression led to increased numbers of B cells and CD4+ memory cells, as well as increased serum IgG and IgA autoantibody levels. Renal immunopathology was modestly greater in BAFF-Tg mice than in their non-Tg counterparts, but again, clinical disease did not develop. Introduction of the Sles1 region into B6.Sle1.Baff mice abrogated the BAFF-driven increase in CD4+ memory cells and the Sle1-driven, but not the BAFF-driven, increase in serum IgG antichromatin levels. Renal immunopathology was substantially ameliorated.

Conclusion

Although constitutive BAFF overexpression in otherwise autoimmune-resistant mice led to humoral autoimmunity, meaningful renal immunopathology and clinical disease did not develop. This raises the possibility that BAFF overexpression, even when present, may not necessarily drive disease in some SLE patients. This may help explain the heterogeneity of the clinical response to BAFF antagonists in human SLE.

B cell–activating factor belonging to the tumor necrosis factor family (BAFF; also known as B lymphocyte stimulator [BLyS]) is a 285–amino acid type II transmembrane protein member of the tumor necrosis factor ligand superfamily (1, 2). Constitutive overexpression of BAFF in BAFF-transgenic (Tg) mice that, otherwise, are not autoimmune-prone leads to systemic lupus erythematosus (SLE)–like features (3–5).

In humans, circulating levels of BAFF are elevated in many systemic rheumatic disorders, including SLE (6, 7). Importantly, overexpression of BAFF is not immutably associated with autoimmune disease. For example, patients with various hematologic malignancies or immunodeficiency disorders harbor elevated circulating levels of BAFF (8–11). Thus, BAFF may be incapable of driving clinically important autoimmunity under certain in vivo conditions. To test this possibility, we turned to 2 murine models in which resistance to autoimmunity has been conferred through discrete genetic manipulations.

The first model uses C57BL/6 (B6)–congenic mice that are genetically deficient in the I-Aβ chain (B6.Aβ−/− mice) and, therefore, do not express class II major histocompatibility complex (MHC). Circulating autoantibody levels in SLE-prone mice are markedly reduced in the absence of class II MHC (12, 13), highlighting the vital role of class II MHC molecules in the development of humoral autoimmunity.

The second model uses B6-congenic mice homozygous for the Sle1 region (B6.Sle1 mice) (14). These mice develop elevated titers of circulating IgG antichromatin autoantibodies but rarely develop renal disease (15, 16). Full-blown disease develops when other genetic “insults” are superimposed, such as the Sle2 or Sle3 regions (17, 18). Importantly, there are genetic modifiers, especially the Sles1 region, which abrogate the ability of B6.Sle1 mice to generate SLE-associated autoantibodies (19). Circulating autoantibody levels in B6.Sle1.Sles1 mice are identical to those in B6 wild-type mice, and B6.Sle1.Sles1 mice do not develop end-organ (kidney) disease, even when the Sle2 or Sle3 region is introgressed.

In this study, we demonstrated that constitutive overexpression of BAFF in BAFF-Tg B6.Aβ−/− mice overcomes the resistance to autoimmunity associated with class II MHC deficiency and promotes humoral autoimmunity. Nevertheless, the renal immunopathology is very modest, and clinical disease does not develop. BAFF-Tg B6.Sle1.Sles1 mice also develop humoral autoimmunity, but the constitutive overexpression of BAFF is incapable of reversing the Sles1-mediated inhibition of Sle1-dependent autoimmunity, and renal immunopathology is very limited. Thus, the consequences of BAFF overexpression on in vivo autoimmunity may be highly dependent upon the genetic background of the host, raising the possibility that BAFF overexpression, even when present, may not necessarily be driving disease in some SLE patients. Accordingly, the clinical effects of therapeutic BAFF neutralization may be widely disparate across the heterogeneous population of patients with SLE.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Mice.

B6.Sle1, B6.Sles1, B6.Sle1.Sles1, B6.Aβ−/−, and B6 mice hemizygous for a murine BAFF transgene (B6.Baff mice) have been previously described (13, 14, 19). B6.Sle1.Baff, B6.Sle1.Sles1.Baff, and B6.Aβ−/−.Baff mice were generated by crossing the relevant parents and screening by polymerase chain reaction (PCR) analysis and/or flow cytometry (20, 21). Mice of either sex were assessed.

All mice were maintained at the University of Southern California. The experiments were approved by the Institutional Animal Care and Use Committee.

Cell surface staining.

Murine spleen mononuclear cells were stained with fluorochrome-conjugated monoclonal antibody specific for murine CD3, CD4, CD8, CD44, CD62L, CD45R (B220), CD19, CD21, or CD23 (from either BD PharMingen or eBioscience) and then analyzed by flow cytometry (22).

Serum Ig and spleen Ig-secreting cells.

Sera were analyzed for levels of total IgM and total IgG by enzyme-linked immunosorbent assay (ELISA) (21). Levels of Ig-secreting cells in the spleen were determined by the reverse hemolytic plaque assay (23).

Serum autoantibodies.

Serum levels of IgM, IgA, IgG, and IgG subclass autoantibodies to chromatin were determined by ELISA (15). Pooled sera from B6.Sle1.Sle2.Sle3 mice were used to generate a standard curve. Values are presented in arbitrary units, with 100 units/ml defined as the activity of a 1:100 dilution of the pooled B6.Sle1.Sle2.Sle3 mouse sera. Since the Ig representing 1 unit differs across the individual Ig classes/subclasses, the units/ml values are comparable only within a given Ig class/subclass.

Kidney immunofluorescence (IF).

Sections of snap-frozen kidneys were stained for IgG, IgA, or C3 using fluorescein isothiocyanate–conjugated goat antibodies against the respective targets (from either SouthernBiotech or MP Biomedicals). From each mouse cohort, 4 kidneys were randomly chosen, and 14–25 glomeruli from each kidney were scored qualitatively for IgG, IgA, or C3 deposition, using a 0–3 scale, where 0 = negative, 1 = weak, 2 = moderate, and 3 = strong fluorescence.

Kidney histology.

Sections of formalin-fixed kidneys were stained with hematoxylin and eosin and were assessed for glomerular hypercellularity, mesangial matrix expansion, interstitial cellular infiltration, and tubular atrophy or dilation.

Statistical analysis.

All analyses were performed using SigmaPlot software (SPSS). P values less than or equal to 0.05 were considered significant. When needed, raw results were log-transformed to achieve normality. Parametric testing between 2 groups was performed with the t-test, and parametric testing among 3 groups was performed with one-way analysis of variance (ANOVA). When log-transformation failed to generate normally distributed data or the equal variance test was not satisfied, nonparametric testing was performed with the Mann-Whitney rank sum test between 2 groups and with Kruskal-Wallis one-way ANOVA on ranks among 3 groups. Since the IF scores are ordinal, nonparametric testing was performed.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Divergent effects of constitutive BAFF overexpression on spleen B cells in B6.Aβ+/− and B6.Aβ−/− mice.

The spleen lymphocyte phenotype was assessed in 4-month-old B6.Aβ+/−, B6.Aβ+/−.Baff, B6.Aβ−/−, and B6.Aβ−/−.Baff littermate mice (Figure 1). As expected, the phenotype of class II MHC–deficient B6.Aβ−/− mice differed from that of their class II MHC–sufficient B6.Aβ+/− littermates. Total spleen mononuclear cells were modestly increased (P = 0.030), CD3+ cells were decreased, although not significantly (P = 0.234), CD4+ cells were markedly decreased (P < 0.001), CD8+ cells were increased, although not significantly (P = 0.224), and total B220+ cells and marginal zone B cells (B220+CD21+CD23low) were greatly increased (P = 0.027 and P < 0.001, respectively) in B6.Aβ−/− mice as compared with B6.Aβ+/− mice.

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Figure 1. Spleen T cells and B cells in B6.Aβ+/−, B6.Aβ+/−.Baff, B6.Aβ−/−, and B6.Aβ−/−.Baff mice. Spleen levels of total mononuclear cells (A), CD3+ cells (B), CD4+ cells (C), CD8+ cells (D), total B cells (B220+) (E), and marginal zone B cells (B220+CD21+CD23low) (F) in the indicated littermate mice (4 months old) are plotted. Each symbol represents an individual mouse. The composite results are shown as box plots. The lines inside the boxes indicate the medians; the outer borders of the boxes indicate the 25th and 75th percentiles; and the bars extending from the boxes indicate the 10th and 90th percentiles.

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Constitutive overexpression of BAFF had disparate effects on lymphocyte phenotypes in B6.Aβ+/− and B6.Aβ−/− littermates. In comparison to non-Tg B6.Aβ+/− mice, total spleen mononuclear cells in B6.Aβ+/−.Baff mice were greatly increased (P = 0.002), CD3+ cells were modestly increased (P = 0.058), CD4+ cells and CD8+ cells were increased, though not significantly (P = 0.109 and P = 0.587, respectively), and total B220+ cells and marginal zone B cells were markedly increased (P < 0.001 for each comparison).

The phenotypic consequences of constitutive BAFF overexpression in B6.Aβ−/− mice were strikingly different. In comparison to non-Tg B6.Aβ−/− mice, no significant increases (or decreases) in total spleen mononuclear cells, CD3+ cells, CD4+ cells, CD8+ cells, total B220+ cells, or marginal zone B cells were appreciated in B6.Aβ−/−.Baff mice (P ≥ 0.290 for each comparison). Thus, although constitutive overexpression of BAFF leads to marked expansion of spleen B cells in a class II MHC–sufficient host, it fails to do so in a class II MHC–deficient host.

BAFF-driven serologic autoimmunity in the absence of class II MHC.

BAFF-driven B cell expansion in B6.Aβ+/−.Baff mice was paralleled by large increases in spleen levels of Ig-secreting cells and in serum levels of total IgG (P < 0.001 and P = 0.008, respectively; Figures 2A and B). Similar increases in spleen Ig-secreting cells (P = 0.009) and serum total IgG levels (P = 0.001) were also observed in B6.Aβ−/−.Baff mice despite the absence of BAFF-driven B cell expansion. Of note, levels of spleen Ig-secreting cells were greater in B6.Aβ−/− mice than in B6.Aβ+/− mice (P < 0.001), despite serum total IgG levels being considerably greater in the latter than in the former (P < 0.001). This likely reflects impaired Ig class switching in B6.Aβ−/− mice consequent to the marked reduction in CD4+ cells in B6.Aβ−/− mice. Consistent with this notion, serum total IgM levels were greater in B6.Aβ−/− mice than in B6.Aβ+/− mice (P = 0.031) (Figure 2C). As expected, serum total IgM levels in B6.Aβ+/−.Baff and B6.Aβ−/−.Baff mice were each substantially greater (P < 0.001 and P = 0.003, respectively) than in their corresponding non-Tg littermates.

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Figure 2. Spleen Ig-secreting cells and serum levels of total IgG, total IgM, and antichromatin antibody of different Ig classes and subclasses in B6.Aβ+/−, B6.Aβ+/−.Baff, B6.Aβ−/−, and B6.Aβ−/−.Baff mice. Spleen levels of Ig-secreting cells (A) and serum levels of total IgG (B), total IgM (C), IgG antichromatin (D), IgM antichromatin (E), IgA antichromatin (F), IgG1 antichromatin (G), IgG2b antichromatin (H), and IgG3 antichromatin (I) in the indicated littermate mice are plotted. Each symbol represents an individual mouse. The composite results are shown as box plots. The lines inside the boxes indicate the medians; the outer borders of the boxes indicate the 25th and 75th percentiles; and the bars extending from the boxes indicate the 10th and 90th percentiles.

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Serum IgG antichromatin antibody levels were also increased in both B6.Aβ+/−.Baff and B6.Aβ−/−.Baff mice relative to their non-Tg counterparts (P = 0.003 and P = 0.002, respectively) (Figure 2D). This largely held true across all the tested IgG isotypes (P = 0.068 and P = 0.089, respectively, for IgG1, P = 0.009 and P = 0.028, respectively, for IgG2b, and P = 0.005 and P = 0.212, respectively, for IgG3) (Figures 2G–I). In contrast, neither serum IgM antichromatin antibody levels (Figure 2E) nor serum IgA antichromatin levels (Figure 2F) were significantly greater in B6.Aβ+/−.Baff and B6.Aβ−/−.Baff mice relative to their non-Tg counterparts (P = 0.224 and P = 0.577, respectively, for IgM antichromatin and P = 0.246 and P = 0.293, respectively, for IgA antichromatin).

Limited BAFF-driven renal immunopathology in the absence of class II MHC.

To increase the likelihood of detecting renal immunopathology, we studied 12-month-old mice (Figure 3). Glomerular deposition of IgG, C3, and IgA in B6.Baff mice was not as strong as that previously observed by other investigators (24), perhaps due to subtle genetic differences among the various B6.Baff mouse lines and/or differences in environmental factors. Regardless, no significant differences in IF scores for glomerular IgG deposition were appreciated between B6 and B6.Baff mice (P = 0.185) or between B6.Aβ−/− and B6.Aβ−/−.Baff mice (P = 0.129). The IF score for glomerular C3 deposition was actually lower in B6.Baff mice than in B6 mice (P = 0.005), but it was greater in B6.Aβ−/−.Baff mice than in B6.Aβ−/− mice (P < 0.001). The IF scores for glomerular IgA deposition were greater in B6.Baff and B6.Aβ−/−.Baff mice than in B6 and B6.Aβ−/− mice (P = 0.005 and P = 0.006, respectively). Importantly, mesangial hypercellularity was noted only in B6.Baff mice, not B6.Aβ−/−.Baff (or B6 or B6.Aβ−/−) mice. Consistent with the limited renal immunopathology, BAFF overexpression did not promote overt clinical kidney disease (severe proteinuria and/or increased mortality rates) in any of these mice.

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Figure 3. Renal immunofluorescence and histologic assessments in B6, B6.Baff, B6.Aβ−/−, and B6.Aβ−/−.Baff mice. Kidney sections from the indicated mice were stained for IgG (A–D), C3 (E–H), and IgA (I–L) immunofluorescence or were stained with hematoxylin and eosin (H&E) (M–P) for standard histologic evaluation. Representative sections are illustrated. The yellow arrows in E and H point to areas of true glomerular C3 deposition. The blue arrows in F and G point to areas of nonspecific staining. The black arrow in N points to mesangial hypercellularity. (Original magnification × 400 in A–L; × 200 in M–P.)

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Divergent effects of constitutive BAFF overexpression on spleen T cells in B6.Sle1 and B6.Sle1.Sles1 mice.

Since deficiency of class II MHC leads to profound disturbances in T cell physiology, it remained uncertain whether constitutive BAFF overexpression could overcome an autoimmune-resistant state and promote autoimmunity under less severe in vivo conditions. Accordingly, we turned our investigations to B6.Sle1 and B6.Sle1.Sles1 mice, in which expression of class II MHC is intact.

The lymphocyte phenotype of B6.Sle1 mice (6 months of age) differed from that of age-matched B6 mice. The levels of total spleen mononuclear cells, CD3+ cells, and CD4+ cells were each modestly increased (P = 0.030, P = 0.021, and P = 0.038, respectively) (Figures 4A–C), whereas CD8+ cells were not (P = 0.477) (data not shown). The increases in CD4+ cells included both memory (CD44+CD62L–) (Figure 4D) and naive (CD44–CD62L+) (data not shown) cells (P = 0.061 and P = 0.013, respectively). Total B220+ cell and marginal zone B cell levels were increased to an even greater extent (P = 0.005 and P = 0.002, respectively) (Figures 4E and F). In toto, these results are consistent with previously reported findings (17, 25, 26).

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Figure 4. Spleen T cells and B cells in B6, B6.Baff, B6.Sle1, B6.Sle1.Baff, B6.Sle1.Sles1, and B6.Sle1.Sles1.Baff mice. Spleens from 2 separate sets of the indicated mice at age 6 months (A–F) and at age 12 months (G–L) were analyzed for levels of total mononuclear cells (A and G), CD3+ cells (B and H), CD4+ cells (C and I), CD4+ memory cells (CD4+CD44+CD62L–) (D and J), total B cells (B220+ or CD19+) (E and K), and marginal zone B cells (B220+CD21+CD23low) (F and L). Each symbol represents an individual mouse. The composite results are shown as box plots. The lines inside the boxes indicate the medians; the outer borders of the boxes indicate the 25th and 75th percentiles; and the bars extending from the boxes indicate the 10th and 90th percentiles.

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The effects of introducing a BAFF transgene were similar in B6 and B6.Sle1 mice (Figures 4A–F). In each case, numbers of total spleen mononuclear cells, total B cells, and marginal zone B cells were greatly increased (P < 0.001 for each comparison). Although expansions in CD3+ cells (P = 0.601 and P = 0.102, respectively), CD8+ cells (P = 0.059 and P = 0.213, respectively), and CD4+ naive cells (P = 0.092 and P = 0.963, respectively) were limited at most, CD4+ memory cells were substantially expanded (P = 0.002 and P = 0.009, respectively).

A second set of mice (12 months of age) was studied to assess the effect of the Sles1 region on the lymphocyte phenotype. Although spleen levels of total mononuclear cells, CD3+ cells, CD4+ cells, CD4+ memory cells, B (CD19+) cells, and marginal zone B cells were similar in B6.Sle1 and B6.Sle1.Sles1 mice (P ≥ 0.094 for each comparison), the phenotypes of B6.Sle1.Baff and B6.Sle1.Sles1.Baff mice were strikingly different (Figures 4G–L). Whereas B6.Sle1.Baff mice harbored greater numbers of total spleen mononuclear cells (P < 0.001), CD4+ cells (P = 0.034), and CD4+ memory cells (P = 0.003) than did B6.Sle1 mice, none of these cell populations were significantly greater in B6.Sle1.Sles1.Baff than in B6.Sle1.Sles1 mice (P ≥ 0.113 for each comparison). Nonetheless, B6.Sle1.Sles1.Baff mice did harbor greater numbers of CD19+ cells (P < 0.001) and marginal zone B cells (P = 0.019) than did B6.Sle1.Sles1 mice, similar to the expansions of these populations in B6.Sle1.Baff mice relative to B6.Sle1 mice (P < 0.001 for each comparison).

The disparate effects of constitutive BAFF overexpression in B6.Sle1.Baff mice as compared with B6.Sle1.Sles1.Baff mice resulted in the latter group having a T cell phenotype more similar to that of B6.Sle1 mice than to that of B6.Sle1.Baff mice but a B cell phenotype more similar to that of B6.Sle1.Baff mice than to that of B6.Sle1 mice. Total spleen mononuclear cell levels in B6.Sle1.Sles1.Baff mice were modestly lower than those in B6.Sle1.Baff mice (P = 0.021) but were modestly greater than those in B6.Sle1 mice (P = 0.013). These changes can be completely explained by selective effects on CD3+, total CD4+, and CD4+ memory cells. CD3+ cell numbers in B6.Sle1.Sles1.Baff mice were substantially reduced in comparison to those in B6.Sle1.Baff mice (P = 0.006) and in B6.Sle1 mice (P = 0.012). Levels of total CD4+ cells and CD4+ memory cells in B6.Sle1.Sles1.Baff mice were also greatly reduced in comparison to those in B6.Sle1.Baff mice (P = 0.002 and P < 0.001, respectively) and were modestly, albeit not significantly, reduced in comparison to those in B6.Sle1 mice (P = 0.111 and P = 0.060, respectively). In sharp contrast, levels of total B cells (CD19+) and marginal zone B cells in B6.Sle1.Sles1.Baff mice were identical to those in B6.Sle1.Baff mice (P = 0.799 and P = 0.280, respectively) and were markedly greater than those in B6.Sle1 mice (P < 0.001 for each comparison).

BAFF-driven serologic autoimmunity in B6.Sle1 and B6.Sle1.Sles1 mice.

Consistent with previous reports (15, 16, 19), B6.Sle1 mice in the present study developed considerable serum levels of IgG antichromatin, whereas the levels in B6, B6.Sles1, or B6.Sle1.Sles1 mice were, with the exception of a single mouse, uniformly very low (P < 0.001 for the comparison of B6.Sle1 mice with the other non-Tg mice) (Figure 5A). Constitutive overexpression of BAFF led to development of serum IgG antichromatin antibodies in B6.Baff mice (P < 0.001 versus B6 mice), which was qualitatively reflected across all IgG subclasses tested (P = 0.017 for IgG1, P = 0.002 for IgG2b, and P < 0.001 for IgG3) (Figures 5B–D). Of note, constitutive overexpression of BAFF had no incremental effect in B6.Sle1.Baff mice on the already-elevated serum IgG antichromatin antibody levels present in B6.Sle1 mice (P = 0.443), suggesting that the autoimmune process that leads to such autoantibodies was already maximal in Sle1-bearing hosts. Importantly, serum IgG antichromatin levels in B6.Sle1.Sles1.Baff mice were neither reduced to the levels in B6 mice (P < 0.001) nor elevated to the levels in B6.Sle1.Baff mice (P = 0.003). Rather, serum IgG antichromatin levels in B6.Sle1.Sles1.Baff mice were identical with those in B6.Baff mice (P = 0.896).

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Figure 5. Serum IgG and IgA antichromatin antibody levels in B6, B6.Baff, B6.Sle1, B6.Sle1.Baff, B6.Sles1, B6.Sle1.Sles1, and B6.Sle1.Sles1.Baff mice. Serum levels of IgG (A), IgG1 (B), IgG2b (C), IgG3 (D), and IgA (E) antichromatin in the indicated mice are plotted. Each symbol represents an individual mouse. The composite results are shown as box plots. The lines inside the boxes indicate the medians; the outer borders of the boxes indicate the 25th and 75th percentiles; and the bars extending from the boxes indicate the 10th and 90th percentiles.

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Although, as expected, serum IgG antichromatin antibody levels in B6.Sle1 mice were considerably greater than those in B6 mice, serum IgA antichromatin levels were not (P = 0.943) (Figure 5E). Nonetheless, serum IgA antichromatin levels were significantly increased in B6.Baff, B6.Sle1.Baff, and B6.Sle1.Sles1.Baff mice in comparison to the corresponding non-Tg mice (P = 0.006, P < 0.001, and P = 0.003, respectively). This is consistent with the increased serum IgA levels previously reported in BAFF-Tg mice (4, 27).

Limited BAFF-driven renal immunopathology in Sles1-bearing hosts.

Glomerular deposition of IgG, C3, and IgA was assessed in 12-month-old B6.Sle1, B6.Sle1.Baff, B6.Sle1.Sles1, and B6.Sle1.Sles1.Baff mice (Figures 6A–L). As assessed by IF scores, glomerular IgG deposition and glomerular IgA deposition were each greater in B6.Sle1.Baff mice than in B6.Sle1 mice (P < 0.001 for each comparison), whereas glomerular C3 deposition was not (P = 0.123). Of note, levels of IgG and IgA deposition in B6.Sle1.Sles1 mice were each similar to those in B6.Sle1 mice (P = 0.424 and P = 0.062, respectively), whereas C3 deposition was markedly reduced (P < 0.001). Importantly, glomerular deposition of IgG, C3, and IgA was greater in B6.Sle1.Sles1.Baff mice than in B6.Sle1.Sles1 mice (P < 0.001, P = 0.039, and P < 0.001, respectively).

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Figure 6. Renal immunofluorescence and histologic assessments in B6.Sle1 (Sle1), B6.Sle1.Baff (Sle1.Baff), B6.Sle1.Sles1 (Sle1.Sles1), and B6.Sle1.Sles1.Baff (Sle1.Sles1.Baff) mice. Kidney sections from the indicated mice were stained for IgG (A–D), C3 (E–H), and IgA (I–L) immunofluorescence or were stained with hematoxylin and eosin (H&E) (M–P) for standard histologic evaluation. Representative sections are illustrated. The yellow arrows in E and F point to areas of true glomerular C3 deposition. The blue arrow in G points to an area of nonspecific staining. The black arrow in N points to mesangial hypercellularity. (Original magnification × 400 in A–L; × 200 in M–P.)

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We previously reported that B6.Sle1.Baff mice developed glomerular disease by 3 months of age (28). Similar renal disease (predominantly mesangial hypercellularity) was noted in 12-month-old B6.Sle1.Baff mice but was not appreciated in B6.Sle1, B6.Sle1.Sles1, or B6.Sle1.Sles1.Baff mice (Figures 6M–P). None of these mice developed overt clinical disease by 12 months of age (data not shown).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Constitutive overexpression of BAFF in non–autoimmune-prone hosts is known to lead to B cell expansion, high titers of circulating IgG autoantibodies, and in some cases, Ig deposition in the kidneys (3–5). We report here that constitutive BAFF overexpression can overcome some, but not all, of a host's genetic resistance to SLE-like autoimmunity.

The first model we studied took advantage of the strong dependence on class II MHC expression that development of circulating autoantibodies and disease has in SLE-prone mice (12, 13), providing prima facie evidence that a class II MHC–deficient state creates an autoimmune-resistant environment. In comparison to their class II MHC–sufficient B6.Aβ+/− littermates, class II MHC–deficient B6.Aβ−/− mice harbored increased spleen B cells and Ig-secreting cells along with the expected marked decrease in CD4+ cells (Figures 1 and 2). Consistent with this decrease in CD4+ cells, total serum IgG levels were substantially lower in B6.Aβ−/− mice than in their B6.Aβ+/− littermates. Not surprisingly, serum levels of SLE-associated IgG autoantibodies were low in these non–autoimmune-prone B6.Aβ−/− and B6.Aβ+/− mice.

Introduction of a BAFF transgene into B6.Aβ+/− mice led, as expected, to increased numbers of spleen B cells and Ig-secreting cells and to increased levels of serum total IgG and IgG antichromatin antibody, although the increase in serum levels of neither IgM nor IgA antichromatin antibodies achieved statistical significance. Furthermore, although spleen B cell numbers did not increase in B6.Aβ−/−.Baff mice, spleen levels of Ig-secreting cells and serum levels of total IgG and IgG autoantibodies did, despite the paucity of CD4+ cells and the complete absence of class II MHC. Whether the inability of class II MHC–deficient B cells to expand in response to BAFF overexpression reflects an inherent defect of such B cells or whether it is consequent to the marked reduction in CD4+ cells remains to be determined. Definitive studies will require the generation of mice in which class II MHC deficiency is limited solely to B cells.

Regardless, the increased levels of IgG autoantibodies in B6.Aβ−/−.Baff mice proves that the effects of constitutive overexpression of BAFF on humoral autoimmunity do not require expression of H-2b, a haplotype that permits the development of SLE-associated IgG autoantibodies in BALB/c and C3H mice (29). Moreover, the divergent effects of BAFF overexpression on B cells and IgG autoantibody levels in B6.Aβ−/− mice highlight the in vivo separability of B cell numbers from B cell–derived products. Along this vein, we have previously documented serologic autoimmunity in BAFF-deficient NZM2328 mice despite their lifelong profound reductions in B cells (30).

The absence of BAFF-driven B cell expansion in B6.Aβ−/− mice sharply contrasts with the BAFF-driven B cell expansion observed in wild-type B6 mice or B6 mice completely devoid of T cells (24). It is possible, but unlikely, that class II MHC–deficient B cells inherently have impaired proliferative capacity in response to BAFF overexpression, since BAFF overexpression leads to increased numbers of Ig-secreting cells and increased production of IgG autoantibodies by such class II MHC–deficient cells. An alternative, non–mutually exclusive, explanation invokes CD4+ Treg cells, which are increased in BAFF-Tg mice (24, 31). CD4+FoxP3+ Treg cells in class II MHC–deficient hosts differ phenotypically from those in class II MHC–intact hosts (32), so their respective inhibitory effects on BAFF-driven B cell expansion may also differ. A third, non–mutually exclusive, explanation invokes CD8+ cells. In contrast to class II MHC–intact hosts in which most FoxP3+ cells are CD4+, the FoxP3+ cells in class II MHC–deficient hosts are evenly distributed between CD4+ and CD8+ subsets (32). Thus, the regulatory role of CD8+ Treg cells in class II MHC–deficient hosts may be considerable, and such cells may modulate what otherwise would be unfettered BAFF-driven expansion of B cells. Additional experimentation, including the genetic depletion of CD8+ cells, will be required to definitively determine the mechanisms underlying the absence of BAFF-driven B cell expansion in B6.Aβ−/− mice.

The increase in circulating IgG autoantibodies in B6.Aβ−/−.Baff mice was associated with no incremental glomerular deposition of IgG and only very modest incremental glomerular deposition of C3. Importantly, no changes in the renal histopathologic appearance were noted in B6.Aβ−/−.Baff mice (Figure 3). These findings must be reconciled with a previous report of substantial renal immunopathologic changes in BAFF-Tg, T cell–deficient B6 mice (24). It may be that CD4+ and/or CD8+ Treg cells that are present in class II MHC–deficient hosts but absent from T cell–deficient hosts play a protective immunomodulatory role. Alternatively, subtle differences in genetic backgrounds and/or local environments may have affected the immunopathologic outcomes. Further investigation in this area is warranted.

The second model we studied took advantage of the ability of the Sle1 region to confer development of IgG antichromatin autoantibodies upon B6 mice and the ability of the Sles1 region to abrogate such autoimmunity (15, 16, 19). Accordingly, a Sles1-bearing host is resistant to Sle1-dependent autoimmunity (although not necessarily resistant to Sle1-independent autoimmunity).

In Sle1-expressing mice, functional changes are independently expressed in both B cells and T cells (26, 33). The Sle1 locus is a complex of multiple susceptibility alleles (25), including Cr2 (34), 4 Slam family genes (Slam, Ly108, Cd84, and Cd48) (35), and Fcgr2b (36). Both Sle1 (NZW) alleles of Cr2 and Fcgr2b alter germinal center B cell function (34, 36), and the Sle1 (NZW) allele of Ly108 impedes negative selection of transitional B cells (37). Importantly, both B cells and T cells (including CD4+ memory cells) were increased in B6.Sle1 mice (Figure 4), and introduction of a BAFF transgene led to further expansions in these cells. Determination of how BAFF overexpression interacts with the individual susceptibility alleles within the Sle1 locus will require breeding of the BAFF transgene into the respective individual subcongenic strains. Regardless, expansion of CD4+ memory cells may be especially important, since expansion of this population in SLE-prone NZM2328 mice closely reflects the onset and severity of clinical disease (22, 38).

One remarkable finding was that BAFF-driven expansion of T cells was abrogated in B6.Sle1.Sles1.Baff mice, whereas BAFF-driven expansion of B cells was unaffected. Despite this lack of effect on BAFF-driven B cell expansion, the IgG autoantibody profile of B6.Sle1.Sles1.Baff mice resembled that of B6.Baff mice rather than B6.Sle1.Baff mice (Figure 5), raising the possibility that the Sle1-associated component of the IgG autoimmune response is largely driven by T cells. Indeed, the Sle1a and Sle1c subregions can promote the development of chromatin-specific autoreactive CD4+ cells (39). Since the Sles1 locus maps to a small region that contains the class II MHC genes and abrogates Sle1-driven T cell activation while leaving Sle1-driven ERK phosphorylation in B cells intact (40), it may inhibit autoreactive CD4+ cells in B6.Sle1.Sles1.Baff mice, and such inhibition may be resistant to BAFF overexpression. Nonetheless, the Sles1 region may also importantly affect Sle1-driven B cell activation (as opposed to B cell expansion) and/or autoantibody affinity maturation and thereby contribute to inhibition of Sle1-associated autoimmunity through T cell–independent means. Formal testing of these possibilities is needed, and B6.Sles1.Baff mice could be especially informative in establishing the effects of the Sles1 region on the biologic consequences of constitutive BAFF overexpression.

In any case, BAFF overexpression appears to be completely capable of promoting Sle1-independent IgG (and IgA) autoantibody production. The inability of a Sles1-bearing host to overcome the autoantibody-promoting effects of BAFF overexpression is reminiscent of the inability of a Sles1-bearing host to overcome the autoantibody-promoting effects of the Sle2 or Sle3 regions (19). Regardless, and consistent with their elevated circulating levels of IgG autoantibody, glomerular deposition of IgG and C3 in B6.Sle1.Sles1.Baff mice was greater than that in B6.Sle1.Sles1 mice. In contrast, no renal histopathology was noted in B6.Sle1.Sles1.Baff mice (Figure 6), and clinical disease did not develop.

Our collective findings in 2 disparate models of autoimmune resistance demonstrated a discordance among glomerular IgG deposition, glomerular C3 deposition, glomerular IgA deposition, and renal histopathology. Glomerular deposition of IgG and IgA in B6.Sle1.Baff mice was greater than that in B6.Sle1 mice, but glomerular deposition of C3 was similar. Conversely, glomerular C3 and IgA deposition in B6.Aβ−/−.Baff mice was greater than that in B6.Aβ−/− mice, but glomerular IgG deposition was similar. Furthermore, no renal histopathology developed in B6.Sle1.Sles1.Baff mice despite considerable glomerular deposition of IgG, C3, and IgA.

Indeed, the limited renal immunopathology and absence of clinical disease in 12-month-old B6.Baff mice indicate that longstanding overexpression of BAFF requires additional factors to promote bona fide disease. The similar renal immunopathology in the 12-month-old B6.Sle1.Baff mice in the present study and in the 3-month-old B6.Sle1.Baff mice previously described (28) suggests that the immunopathology promoted by BAFF overexpression reaches a plateau. Only in the presence of other genetic and/or environmental factors might this finite BAFF-driven immunopathology progress to clinical disease.

The differential ability of BAFF overexpression to promote or permit different facets of autoimmunity may have important ramifications for human disease. Although BAFF antagonists have been highly effective in treating murine SLE (5, 41–43), results in treating human SLE have been less impressive (44–46). It may be that in some patients, only certain autoimmune elements of SLE are promoted or permitted by BAFF. If clinical disease in any given patient is largely BAFF-independent, then that patient would not be expected to respond to a BAFF antagonist. Future studies will be needed to distinguish patients whose disease is largely BAFF-driven from those whose disease is not.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Stohl had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Stohl.

Acquisition of data. Stohl, Jacob, Guo, Morel.

Analysis and interpretation of data. Stohl, Guo.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES
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