BAFF overexpression and accelerated glomerular disease in mice with an incomplete genetic predisposition to systemic lupus erythematosus

Authors


Abstract

Objective

To determine whether overexpression of BAFF can accelerate the development of systemic lupus erythematosus–associated end-organ disease in hosts with an underlying autoimmune diathesis.

Methods

We introduced a BAFF transgene (Tg) into autoimmune-prone B6.Sle1 and B6.Nba2 mice and evaluated these mice for serologic autoimmunity and renal pathology.

Results

B6.Sle1.BAFF and B6.Nba2.BAFF mice, but not non-Tg littermates, frequently developed severe glomerular pathology by 3 months of age. Age-matched B6.BAFF mice, despite renal Ig deposits and increases in B cells and Ig production similar to those in B6.Sle1.BAFF and B6.Nba2.BAFF mice, did not develop glomerular pathology. In B6.Sle1.BAFF and B6.Nba2.BAFF mice, severity of glomerular disease did not obligately correlate with circulating levels of IgG antichromatin and/or anti–double-stranded DNA antibodies or with amounts of these autoantibodies deposited in the kidneys. Even in mice with severe glomerular disease, renal tubulointerstitial infiltrates were very limited, and increased proteinuria was not detected.

Conclusion

BAFF-driven effects on glomerular pathology may be mediated, at least in part, by autoantibodies with specificities other than chromatin and/or by autoantibody-independent means. There is an uncoupling of BAFF-driven precocious glomerular pathology from concomitant development of clinically apparent renal disease, strongly suggesting that BAFF overexpression works in concert with other factors to promote overt renal disease.

Renal involvement in patients with systemic lupus erythematosus (SLE) is clinically apparent in ∼50% of cases and leads to considerable morbidity. The vast majority of SLE patients with renal involvement develop increased proteinuria, sometimes to degrees great enough to adversely affect systemic fluid balance and hemodynamics (nephrotic syndrome). Glomerular disease is widely believed to be the major contributor to this increased proteinuria. Indeed, the World Health Organization (WHO) classification of lupus nephritis focuses almost entirely on glomerular lesions, with little attention to tubular, interstitial, or vascular lesions. Accordingly, factors which promote glomerular disease in SLE may be vital to the pathogenesis of lupus nephritis.

One such candidate factor is B cell–activating factor belonging to the tumor necrosis factor family (BAFF). BAFF, also known as B lymphocyte stimulator (BLyS), TALL-1, THANK, tumor necrosis factor superfamily member 13B, and zTNF4, is a 285–amino acid type II transmembrane protein member of the tumor necrosis factor ligand superfamily (1–6). It is expressed by myeloid lineage cells (1–3, 5, 7, 8), bone marrow–derived radiation-resistant stromal cells (9), and T cells (10), and it is cleaved at the cell surface by a furin protease to release a soluble, biologically active 17-kd molecule (1, 7).

BAFF plays a vital role in B cell survival (11–17). BAFF-deficient mice display substantial (albeit incomplete) reductions in mature B cells and in baseline serum Ig levels and Ig responses to T cell–dependent and T cell–independent antigens (18–20). In contrast, constitutive overexpression of BAFF in BAFF-transgenic (Tg) mice leads to SLE-like features (elevated circulating titers of multiple autoantibodies and renal Ig deposits) (6, 21, 22). By 8 months of age (but not 5 months of age), BAFF-Tg mice may manifest increased proteinuria (22).

BAFF overexpression is associated with SLE in humans as well. Cross-sectional and longitudinal studies have documented increased circulating levels of BAFF in as many as 50% of SLE patients. At the population level, circulating levels of BAFF correlate with circulating levels of anti–double-stranded DNA (anti-dsDNA) antibodies (23–25) and with clinical disease activity (26).

Importantly, elevated circulating levels of BAFF are not specific to SLE. Many patients with rheumatoid arthritis and Sjögren's syndrome also harbor elevated circulating levels of BAFF (23, 24, 27, 28). Indeed, elevated circulating levels of BAFF do not necessarily result in autoimmune disease. For example, circulating BAFF levels are elevated in a large percentage of human immunodeficiency virus (HIV)–infected individuals (29, 30), but these subjects do not manifest features of SLE or related autoimmune disorders.

The common uncoupling of BAFF overexpression from the development of SLE-like illness suggests that BAFF overexpression may remain clinically silent in the absence of an independent underlying SLE diathesis. The prediction is that the greater the underlying SLE diathesis, the greater the effects of BAFF overexpression on the phenotypic expression of disease. To test this hypothesis, we utilized 2 distinct mouse lines congenic with C57BL/6 (B6) mice. B6 mice homozygous for the NZW mouse–derived Sle1 region (B6.Sle1 mice) (31) or for the NZB mouse–derived Nba2 region (B6.Nba2 mice) (32) each have an incomplete genetic predisposition to SLE, in that they spontaneously develop elevated circulating titers of IgG antichromatin autoantibodies but rarely develop renal disease (32–34). Full-blown disease is realized in these mice when other genetic insults are superimposed, such as expression of the Sle2 or Sle3 regions in the case of B6.Sle1 mice or by crossing with NZW mice in the case of either B6.Sle1 or B6.Nba2 mice (32, 35–37).

In this study, we demonstrate that the introduction of a BAFF Tg into B6.Sle1 or B6.Nba2 mice often led to the precocious development of severe glomerular pathology by 3 months of age. BAFF-Tg mice that bore neither the Sle1 nor the Nba2 region did not develop glomerular pathology at this young age. Glomerular lesions in this model did not immutably correlate with circulating levels of IgG autoantibodies against chromatin and/or dsDNA or with the amounts of these autoantibodies deposited in the kidneys, which raises the possibility that the BAFF-driven effects are mediated, at least in part, via nonchromatin autoantibodies and/or via autoantibody-independent means. Of note, little renal tubulointerstitial infiltration and no increased proteinuria were observed even among mice with severe glomerular disease. This points to an uncoupling of BAFF-driven precocious glomerular pathology from the concomitant development of clinically apparent renal disease and implies that factors in addition to BAFF contribute vitally to lupus nephritis in the BAFF-Tg model of SLE.

MATERIALS AND METHODS

Mice.

All mice were maintained at the University of Southern California (USC), and the experiments were approved by the Institutional Animal Care and Use Committee. Mice transgenic for murine BAFF (21) that had been backcrossed to B6 mice for >9 generations (B6.BAFF mice) were maintained as hemizygotes for the BAFF Tg. B6.Sle1.BAFF and B6.Nba2.BAFF mice were generated by crossing B6.Sle1 and B6.Nba2 mice (31, 32, 38), respectively, with B6.BAFF mice and screening the F1 progeny (obligate heterozygotes for the Sle1 and Nba2 regions, respectively) for the BAFF Tg by polymerase chain reaction (PCR) (see below). F1 mice bearing the BAFF Tg were backcrossed to B6.Sle1 and B6.Nba2 mice, and the resulting pups were screened for homozygosity for the Sle1 and Nba2 regions, respectively, by PCR (38) and for the BAFF Tg.

Parental B6.Sle1.BAFF and B6.Nba2.BAFF mice were mated with B6.Sle1 and B6.Nba2 mice, respectively, to yield experimental BAFF-Tg mice and control non-Tg littermates of either sex. As additional controls, B6.BAFF mice were mated with B6 mice to yield BAFF-Tg and non-Tg mice of either sex that bore neither the Sle1 nor the Nba2 region. No significant differences in the measured parameters were identified between male and female mice of any strain; therefore, the results for males and females were pooled.

Detection of the BAFF Tg.

Genomic DNA extracted from mouse tail clippings was PCR-amplified for 29 cycles each at 95°C for 1 minute, 64°C for 1 minute, and 72°C for 1 minute. The primer sequences used were 5′-GCAGTTTCACAGCGATGTCCT-3′ and 5′-GTCTCCGTTGCGTGAAATCTG-3′. The PCR products were subjected to electrophoresis in 1.5% agarose gels containing ethidium bromide, and bands were visualized under ultraviolet light. The band size for the BAFF Tg was ∼700 bp.

Cell surface staining.

Murine spleen mononuclear cells were stained singly or in combination with fluorescein isothiocyanate–conjugated, phycoerythrin-conjugated, or Cy-Chrome–conjugated monoclonal antibodies specific for murine CD3, CD4, CD8, CD44, CD62L, CD45R (B220), CD21, or CD23 (BD PharMingen, San Diego, CA) and then analyzed by flow cytometry (39).

Serum immunoglobulins and spleen Ig-secreting cell determinations.

Sera were assayed for levels of total IgG and total IgM by enzyme-linked immunosorbent assay (ELISA) (39). Spleen cells were assayed for numbers of total Ig-secreting cells by the reverse hemolytic plaque assay (40, 41). Each plaque-forming cell was taken as an Ig-secreting cell.

Serum autoantibody determinations.

Serum levels of IgG autoantibodies to chromatin and dsDNA were determined by ELISA (38). Quantitative values were obtained by use of standard curves obtained with monoclonal antibodies to the appropriate nuclear antigen (42). Samples from (NZB × NZW)F1 (NZB/NZW) and B6 mice were run in every assay as positive and negative controls, respectively.

Serum BAFF determination.

Serum BAFF levels were determined by a sandwich ELISA. Quantitative values were calculated from a standard curve of known concentrations of recombinant soluble murine BAFF (Biogen Idec, Cambridge, MA). The lower level of detection is 0.01 μg/ml.

Assessment of proteinuria.

Reagent strips for urinary protein (Albustix; Bayer, Elkhart, IN) were dipped in mouse urine. Results were assigned a score of 0–4+ by visual color comparison to the standard color key that was supplied.

Kidney histology.

Individual sections of formalin-fixed kidneys were stained with hematoxylin and eosin and with Masson's trichrome and were examined by light microscopy by one of us (MNK), who was blinded to the genotype of the mouse. Each case was assessed for the presence of glomerulonephritis (GN) using a modification of the WHO classification for lupus nephritis. Class I was assigned for normal histologic features by light microscopy. Class II was assigned for increases in mesangial matrix and/or cells. Class III was assigned for focal proliferative GN (<50% of glomeruli showing endocapillary proliferative changes, with or without crescents). Class IV was assigned for diffuse proliferative GN (>50% of glomeruli showing endocapillary proliferative changes, with or without crescents). The presence of mesangial and capillary wall deposits in the trichrome-stained sections was also noted.

Kidney immunofluorescence.

Five-micron sections of snap-frozen kidneys were cut and stained for total IgG deposition using fluorescein isothiocyanate–conjugated rabbit anti-mouse IgG antibodies (MP Biomedicals, Irvine, CA). Two of us (TNJ and BLK), who were blinded to the genotype of the mouse, scored the degree of staining using a 0–4 scale, where 0 = no detectable staining, 0.5 = trace staining in the mesangium only, 1 = staining in the mesangium only (<50% of glomeruli), 2 = staining in the mesangium only (>50% of glomeruli), 3 = strong staining in the mesangium (>50% of glomeruli) with occasional staining of capillary loops, and 4 = strong staining in the mesangium (>50% of glomeruli) with widespread staining of capillary loops.

Ig elution from kidneys.

One-half of a snap-frozen kidney from each mouse was thawed and minced with a clean razor blade. Kidneys from mice of a given cohort were pooled, and Ig was eluted (43). The eluates were dialyzed at 4°C against distilled water for 24 hours and then against phosphate buffered saline for an additional 24 hours. The total eluted IgG in each group was ∼2–5 μg, as determined by ELISA. The antigenic specificities of the eluted antibodies were measured by antigen-specific ELISAs, using eluates adjusted to a total IgG concentration of 500 ng/ml (44, 45).

Statistical analysis.

All analyses were performed using SigmaStat software (SPSS, Chicago, IL). Raw results were log-transformed to achieve normality. Parametric testing between 2 groups was performed by the t-test, and parametric testing among 3 groups was performed by one-way analysis of variance (ANOVA). When log-transformation failed to generate normally distributed data or when the equal variance test was not satisfied, nonparametric testing between 2 groups was performed with the Mann-Whitney rank sum test and among 3 groups by Kruskal-Wallis one-way ANOVA. Correlations were determined by Pearson's product-moment correlation for interval data and by Spearman's rank order correlation for ordinal data or for interval data that did not follow a normal distribution.

RESULTS

Numbers of splenic T cells and B cells and global Ig production in 3-month-old BAFF-Tg and non-Tg B6, B6.Sle1, and B6.Nba2 mice.

The numbers of splenic CD3+, CD4+, CD8+, and total B (B220+) cells were each modestly greater (geometric mean 31–68%) in non-Tg 3-month-old B6.Sle1 and B6.Nba2 mice than in age-matched non-Tg B6 mice (P ≤ 0.032 for each comparison) (Figures 1A–D), although the numbers of splenic marginal zone B cells were essentially the same (Figure 1E).

Figure 1.

Numbers of splenic T cells and B cells in 3-month-old BAFF-transgenic (Tg) and non-Tg B6.Sle1, B6.Nba2, and B6 mice. A–E, Splenic CD3+, CD4+, CD8+, total B (B220+), and marginal zone (MZ) B (B220+,CD21+,CD23low) cells were enumerated in the 3 groups of mice, which were categorized according to the expression (yes/no) of the BAFF Tg. Each symbol represents an individual mouse. The composite results for male plus female mice of each group are shown as box plots. The outer borders of the boxes indicate the 25th and 75th percentiles. Lines inside the boxes indicate the median. Bars extending from the boxes indicate the 10th and 90th percentiles.

Introduction of the BAFF Tg had similar effects on the numbers of CD3+, CD4+, CD8+, total B (B220+), and marginal zone B cells in B6, B6.Sle1, and B6.Nba2 mice. Splenic CD3+, CD4+, and CD8+ cell numbers in B6.Sle1.BAFF and B6.Nba2.BAFF mice remained modestly greater (geometric mean 58–116%) than those in B6.BAFF mice (P ≤ 0.004 for each comparison), with CD4+ and CD8+ cells displaying a more activated phenotype (greater percentages of CD44+,62L– cells and/or lesser percentages of CD44–,62L+ cells) in all BAFF-Tg mice than in their non-Tg littermates (data not shown). The numbers of splenic total B cells and marginal zone B cells in BAFF-Tg mice were each considerably greater (geometric mean 168–188% and 336–713%, respectively) than those in the corresponding non-Tg littermates (P < 0.001 for each comparison) (Figures 1D–E).

The numbers of splenic Ig-secreting cells were similar among non-Tg B6.Sle1, B6.Nba2, and B6 mice (Figure 2A). Serum total IgM levels in non-Tg B6.Sle1 and B6.Nba2 mice were lower (geometric mean 41–72%) than in non-Tg B6 mice (P < 0.001) (Figure 2B). Serum total IgG levels in non-Tg B6.Sle1 and B6.Nba2 mice were greater (geometric mean 29–96%) than in non-Tg B6 mice (P < 0.001) (Figure 2C). These serologic results paralleled those previously observed for corresponding mice tested at 8–9 months of age (38).

Figure 2.

Total Ig and autoantibody levels in 3-month-old BAFF-transgenic (Tg) and non-Tg B6.Sle1, B6.Nba2, and B6 mice. A–E, The numbers of splenic Ig-secreting cells, and serum levels of total IgM, total IgG, IgG antichromatin, and IgG anti–double-stranded DNA (anti-dsDNA) were measured in the 3 groups of mice, which were categorized according to the expression (yes/no) of the BAFF Tg. Each symbol represents an individual mouse. The composite results for male plus female mice of each group are shown as box plots. The outer borders of the boxes indicate the 25th and 75th percentiles. Lines inside the boxes indicate the median. Bars extending from the boxes indicate the 10th and 90th percentiles.

Introduction of the BAFF Tg resulted in increased numbers of splenic Ig-secreting cells (geometric mean 4.6–14.0-fold), increased levels of serum total IgM (geometric mean 2.4–8.5-fold), and increased levels of serum total IgG (geometric mean 1.8–3.5-fold) in B6, B6.Sle1, and B6.Nba2 mice (P ≤ 0.010 for each comparison).

Serum levels of BAFF were measured in all but 3 of the mice. There was marked variability in serum BAFF levels among the BAFF-Tg mice, with some mice harboring vastly elevated levels and others harboring levels that were not much greater than those in their non-Tg littermates (Figure 3). This variability among BAFF-Tg mice has been observed both in the colony at USC and in the colony at Biogen Idec. The basis for this variability is not known, but it likely reflects the inherent biologic properties of BAFF-Tg mice, rather than some unappreciated unique local environmental issue. In any case, the great variability did permit us to assess the in vivo consequences of differences in serum BAFF levels across a very wide range.

Figure 3.

Association between serum BAFF levels and renal pathology in 3-month-old BAFF-transgenic (Tg) and non-Tg B6.Sle1, B6.Nba2, and B6 mice. Serum BAFF levels from individual BAFF-Tg (circles) and non-Tg (squares) mice in the 3 groups are plotted against the corresponding class of renal pathology, based on the World Health Organization classification of histologic features.

When results were pooled from BAFF-Tg mice and their non-Tg littermates within a given cohort, splenic B cell numbers and all the tested parameters of global Ig production, including splenic Ig-secreting cell numbers, serum total IgM levels, and serum total IgG levels, uniformly correlated strongly with serum BAFF levels (Table 1). Among BAFF-Tg mice within a given cohort (excluding non-Tg littermates), serum BAFF levels did not necessarily correlate with each of these B cell parameters, indicating that factors in addition to circulating BAFF levels are critical determinants of B cell numbers and global Ig production.

Table 1. Correlations between serum BAFF levels and B cell/histology parameters*
Serum BAFF versusBAFF-transgenic mice and their nontransgenic littermatesBAFF-transgenic mice only
B6.Sle1B6.Nba2B6B6.Sle1B6.Nba2B6
rPrPrPrPrPrP
  • *

    MZ = marginal zone; anti-dsDNA = anti–double-stranded DNA; NA = not applicable.

Splenic B220+ cells0.732<0.0010.747<0.0010.4710.0100.5730.0480.1030.7150.0820.797
Splenic MZ B cells0.600<0.0010.792<0.0010.5070.0050.4900.1000.1100.709−0.0360.903
Splenic Ig-secreting cells0.686<0.0010.740<0.0010.757<0.0010.832<0.0010.5030.0640.7330.020
Serum IgM0.5310.0050.732<0.0010.626<0.0010.909<0.0010.785<0.0010.7550.006
Serum IgG0.618<0.0010.781<0.0010.5270.0030.825<0.0010.930<0.0010.6270.035
Serum IgG antichromatin−0.1830.3590.2870.130NANA0.1850.5570.0550.844NANA
Serum IgG anti-dsDNA0.0980.6220.676<0.001NANA0.3400.2630.947<0.001NANA
Renal histology0.5530.0050.5660.004NANA0.5500.0580.0940.749NANA

Disparate effects of constitutive overexpression of BAFF on circulating autoantibody levels among B6, B6.Sle1, and B6.Nba2 mice.

By 8–9 months of age, large percentages of B6.Sle1 and B6.Nba2 mice harbor detectable circulating IgG antichromatin autoantibodies (32–34, 38). By 3 months of age, there was already a significant difference in serum levels of IgG antichromatin in the B6 mice compared with the B6.Sle1 and the B6.Nba2 mice (P < 0.001) (Figure 2D). Serum levels of IgG anti-dsDNA in B6.Nba2 mice were significantly greater than those in either the B6 or the B6.Sle1 mice (P < 0.001) (Figure 2E). Of note, 3-month-old B6.BAFF mice harbored no detectable circulating IgG antichromatin or anti-dsDNA antibodies (Figures 2D and E).

Unexpectedly, the increases in serum levels of total IgG observed among B6.Sle1.BAFF mice in comparison to their non-Tg littermates were not paralleled by increases in serum levels of IgG antichromatin or anti-dsDNA autoantibodies (Figures 2D and E). We observed no significant correlations between serum levels of IgG antichromatin or anti-dsDNA antibodies and serum levels of BAFF among all the Sle1-bearing mice or among just the B6.Sle1.BAFF mice (Table 1). In contrast, serum levels of IgG anti-dsDNA antibodies were significantly greater in B6.Nba2.BAFF mice than in their non-Tg littermates (P < 0.001) (Figure 2E), and these levels correlated significantly with serum BAFF levels regardless of whether all the Nba2-bearing mice or just the B6.Nba2.BAFF mice were considered (Table 1). However, serum IgG antichromatin levels in B6.Nba2.BAFF mice were not significantly greater than those in their non-Tg littermates (Figure 2D), and no significant correlations were appreciated between serum levels of IgG antichromatin antibodies and BAFF among all the Nba2-bearing mice or among just the B6.Nba2.BAFF mice (Table 1). The differences in autoantibody profiles between Sle1-bearing and Nba2-bearing mice are consistent with previous observations (38) and reinforce the assertion that these mice are genetically distinct.

Constitutive overexpression of BAFF and precocious glomerular pathology in B6.Sle1 and B6.Nba2 mice, but not B6 mice.

The variable effects on serum IgG autoantibody levels notwithstanding, introduction of the BAFF Tg into B6.Sle1 or B6.Nba2 mice had a dramatic effect on the development of glomerular disease. Kidney sections from 12 B6.Sle1.BAFF mice, 12 B6.Sle1 non-Tg littermates, 12 B6.Nba2.BAFF mice, 12 B6.Nba2 non-Tg littermates, 11 B6.BAFF (without the Sle1 or Nba2 regions), and 13 B6 non-Tg littermates were analyzed. Despite their young age (3 months), 4 B6.Sle1.BAFF mice and 5 B6.Nba2.BAFF mice already had class III (focal proliferative) GN, and 1 B6.Sle1.BAFF mouse and 5 B6.Nba2.BAFF mice already had class IV (diffuse proliferative) GN (Figure 3).

Massive mesangial and subendothelial deposits, with or without glomerular hypercellularity, were frequently observed in kidneys from B6.Sle1.BAFF and B6.Nba2.BAFF mice (Figure 4). In contrast, such histologic changes were rare and, when present, were very mild in B6.Sle1 and B6.Nba2 non-Tg mice. Only 1 B6.Sle1 and 2 B6.Nba2 non-Tg mice displayed class III GN, and no B6.Sle1 or B6.Nba2 mice displayed class IV GN. Among the 24 Sle1-bearing mice and the 24 Nba2-bearing mice, the severity of GN correlated strongly with serum levels of BAFF (Table 1). Among the limited numbers of B6.Sle1.BAFF and B6.Nba2.BAFF mice analyzed (without consideration of their non-Tg littermates), a trend between the severity of GN and the serum levels of BAFF was still appreciated in B6.Sle1.BAFF mice but not in B6.Nba2.BAFF mice. Among B6.Sle1 and B6.Sle1.BAFF mice, the severity of GN correlated with neither the serum levels of IgG antichromatin antibodies (r = –0.055, P = 0.793) nor the serum levels of IgG anti-dsDNA antibodies (r = 0.221, P = 0.294). Among B6.Nba2 and B6.Nba2.BAFF mice, the severity of GN correlated with serum levels of IgG anti-dsDNA antibodies (r = 0.645, P < 0.001) but not with serum levels of IgG antichromatin antibodies (r = 0.248, P = 0.240).

Figure 4.

Renal histologic features in 3-month-old BAFF-transgenic (Tg) and non-Tg B6.Sle1, B6.Nba2, and B6 mice. Kidney sections were stained with hematoxylin and eosin (H&E) or Masson's trichrome. Sections are from 2 representative mice from each of the indicated groups. Large arrows indicate abnormal mesangial deposits, small arrows indicate abnormal subendothelial deposits, and arrowhead indicates an area of glomerular hypercellularity. These abnormalities were observed almost exclusively among B6.Sle1.BAFF and B6.Nba2.BAFF mice and not among B6, B6.BAFF, B6.Sle1, or B6.Nba2 mice. (Original magnification × 40.)

Importantly, all B6 and B6.BAFF mice displayed normal histologic features (WHO class I) or showed only mild, focal mesangial hypertrophy (class IIb) without any relationship to serum BAFF levels (Figure 3). Thus, at 3 months of age, the only BAFF-Tg mice to manifest substantial glomerular pathology were those that bore the Sle1 or Nba2 regions.

Of note, histologic changes in the kidneys did not faithfully parallel renal IgG deposition. IgG deposits were not detected in the kidneys of B6 mice (median immunofluorescence score 0.0 [n = 6]) but were present to a modest degree in the kidneys of B6.Sle1 (median immunofluorescence score 0.75 [n = 6]) and B6.Nba2 (median immunofluorescence score 0.75 [n = 5]) mice (P = 0.007). Importantly, renal IgG deposits were pronounced not only in the kidneys of B6.Sle1.BAFF (n = 6) and B6.Nba2.BAFF mice (n = 5), but also in the kidneys of B6.BAFF mice (n = 6) (Figure 5). Despite the lack of a statistically significant difference in the median immunofluorescence scores (2.75, 3.0, and 2.0, respectively) among these groups of mice, the B6.Sle1.BAFF and B6.Nba2.BAFF mice displayed considerable glomerular pathology, whereas the B6.BAFF mice displayed none (Figures 3 and 4).

Figure 5.

Immunofluorescence analysis of IgG deposits in kidneys from 3-month-old BAFF-transgenic (Tg) and non-Tg B6.Sle1, B6.Nba2, and B6 mice. Kidney sections were stained for IgG deposition using fluorescein isothiocyanate–conjugated rabbit anti-mouse IgG antibodies. Sections are from a representative mouse from each of the indicated groups. (Original magnification × 40.)

This discordance between renal Ig deposits and pathology cannot be reconciled solely on the basis of differences in specificities of the deposited Ig (Figure 6). Although eluates from B6.Nba2.BAFF kidneys contained more IgG autoantibodies against chromatin, dsDNA, laminin, and α-actinin than did eluates from the kidneys of B6.Nba2 non-Tg littermates, a similar pattern was not observed for eluates from kidneys of B6.Sle1.BAFF mice and their non-Tg littermates. A greater amount of IgG anti–α-actinin antibodies was eluted from kidneys of B6.Sle1.BAFF mice than from kidneys of B6.Sle1 non-Tg littermates, but no differences were appreciated in the amounts of IgG antichromatin or anti-dsDNA antibodies eluted from the kidneys of these groups of mice, and a considerably lesser amount of IgG antilaminin antibodies was eluted from kidneys of B6.Sle1.BAFF mice than from kidneys of B6.Sle1 non-Tg littermates. Similar amounts of IgG anti–type IV collagen antibodies were eluted from the kidneys of each mouse cohort.

Figure 6.

IgG autoantibodies in eluates of kidneys from 3-month-old BAFF-transgenic (Tg) and non-Tg B6.Sle1, B6.Nba2, and B6 mice. Eluates from pooled kidneys from B6, B6.BAFF, B6.Sle1, B6.Sle1.BAFF, B6.Nba2, and B6.Nba2.BAFF mice were assayed for IgG antibodies against chromatin (chrom), double-stranded DNA (dsDNA), laminin, α-actinin, and type IV collagen. Values are the mean and 1 SD optical density (OD) at 405 nm.

Absence of increased proteinuria in B6.Sle1.BAFF and B6.Nba2.BAFF mice despite severe glomerular disease.

None of the BAFF-Tg mice (including those with class III or class IV GN) had increased proteinuria (≥3+ by dipstick) relative to that in their non-Tg littermates (data not shown). Increased proteinuria has also not been observed in the limited numbers of 6-month-old B6.Sle1.BAFF and B6.Nba2.BAFF mice examined to date. Moreover, routine histologic and immunohistologic analyses revealed little tubulointerstitial inflammatory cell infiltrates even in kidneys with severe glomerular disease (Figure 4).

DISCUSSION

Using 2 distinct mouse lines congenic with B6 (B6.Sle1 and B6.Nba2) that each bears a genetic diathesis to SLE but rarely develops end-organ disease (32–34), we have demonstrated that constitutive overexpression of BAFF frequently leads to the development of severe glomerular pathology by as early as 3 months of age (Figure 3). When all Sle1-bearing mice or Nba2-bearing mice were considered, precocious glomerular pathology correlated strongly with serum levels of BAFF (Table 1). The greater prevalence of class IV GN among B6.Nba2.BAFF mice than among B6.Sle1.BAFF mice may reflect, in part, the greater serum BAFF levels in the former than in the latter group. Of note, a trend between serum BAFF levels and severity of GN was noted among B6.Sle1.BAFF mice but not among B6.Nba2.BAFF mice. This demonstrates that circulating BAFF levels, although certainly critical, are not obligately the dominant factor in determining the degree of glomerular pathology.

Importantly, similar constitutive overexpression of BAFF in B6 mice without the autoimmune proclivity imposed by the Sle1 or Nba2 regions does not lead to detectable changes in renal histology at this age, despite readily detectable IgG deposits in the kidneys (Figures 4 and 5). This indicates that BAFF overexpression per se is insufficient for the development of precocious glomerular disease and indicates that renal IgG deposits are not synonymous with histologic changes. BAFF-driven glomerular pathology fails to develop in age-matched B6 mice, despite similar BAFF-driven changes in T cells, B cells, and global Ig production among B6, B6.Sle1, and B6.Nba2 mice (Figures 1 and 2).

Regardless of whether all Sle1-bearing or Nba2-bearing mice were considered or whether just the respective BAFF-Tg mice were considered, serum BAFF levels correlated strongly with serum levels of total IgG (and total IgM) but not with serum levels of IgG antichromatin antibodies (Table 1). For the Sle1-bearing mice, the lack of correlation extended to serum levels of IgG anti-dsDNA antibodies as well. In humans with SLE or rheumatoid arthritis, circulating BAFF levels correlate significantly with anti-dsDNA antibodies and rheumatoid factor antibodies, respectively (23, 24). Nevertheless, the absence of a positive correlation between circulating levels of BAFF and some autoantibody has precedent. Despite one group of investigators documenting a correlation between circulating levels of BAFF and anti-SSA/Ro antibodies in patients with Sjögren's syndrome (28), no such correlation was seen by another group of investigators (27). Since BAFF can form heterotrimers with APRIL (46), it is possible that the lack of correlation between serum levels of BAFF and IgG autoantibodies may be due to differential biologic effects of BAFF homotrimers compared with those of BAFF/APRIL heterotrimers. Although we did not detect circulating APRIL in our mice (Stohl W, et al: unpublished observations), our ELISA may not be able to recognize APRIL complexed to BAFF. Thus, the relationship between circulating autoantibody levels and circulating BAFF levels is not straightforward, and further investigation will be necessary to clarify this.

In addition to serum levels of IgG antichromatin and IgG anti-dsDNA antibodies not immutably correlating with serum BAFF levels, serum levels of each of these autoantibodies did not correlate with the severity of glomerular disease among B6.Sle1 and B6.Sle1.BAFF mice, and serum levels of IgG antichromatin antibodies did not correlate with the severity of glomerular disease among B6.Nba2 and B6.Nba2.BAFF mice. This may indicate that IgG antichromatin or anti-dsDNA antibodies are not the pathogenic autoantibodies in these mice. The specificity of the true pathogenic autoantibodies may be something other than chromatin or dsDNA and, thus, remained unmeasured.

There is precedent for this notion. Although severe renal disease develops in association with substantial Ig deposition in the kidneys of both NZM 2328 and congenic NZM.C57Lc4 mice, the prevalence of detectable circulating IgG anti-dsDNA or IgG antihistone/DNA antibodies is high only in NZM 2328 mice but is very low in NZM.C57Lc4 mice (47). Moreover, IgG anti-dsDNA antibodies are plentiful in eluates of diseased kidneys from NZM 2328 mice but are virtually absent from eluates of diseased kidneys from NZM.C57Lc4 mice. However, multiple antibodies with reactivities to autoantigens in kidney and liver extracts are readily detectable in both, which raises the possibility that IgG autoantibodies with nonchromatin specificities are pathogenic.

While the amounts of IgG antichromatin, anti-dsDNA, antilaminin, and anti–α-actinin antibodies deposited in the kidneys of B6.Nba2.BAFF mice were uniformly greater than those in the kidneys of B6.Nba2 non-Tg littermates, the amounts of 3 of these autoantibodies deposited in the kidneys of B6.Sle1.BAFF mice were either equal to or were actually less than the amounts deposited in the kidneys of B6.Sle1 non-Tg littermates (Figure 6). An important caveat in regard to these experiments is that the antigenic specificities of these antibodies in vivo may not necessarily be the same as those measured in the in vitro assays. Nevertheless, the amounts of IgG antichromatin, anti-dsDNA, antilaminin, and anti–α-actinin antibodies deposited in the disease-free kidneys of B6.BAFF mice were very similar to the amounts deposited in the diseased kidneys of B6.Sle1.BAFF mice, so it is doubtful that any of these autoantibodies is truly pathogenic in Sle1-bearing or Nba2-bearing mice. This may explain why B6.Sle1 and B6.Nba2 mice do not develop renal disease despite having high titers of circulating antichromatin and anti-dsDNA antibodies. Functional testing of the isolated autoantibodies from B6.Sle1 and B6.Nba2 mice will be required to validate this thesis.

It is plausible that the pathogenesis of BAFF-driven glomerular disease includes autoantibody-insensitive and/or autoantibody-independent mechanisms in addition to autoantibody-dependent mechanisms. There are precedents for this from other models.

First, STAT-4–deficient NZM 2328 mice develop accelerated renal disease and mortality despite a modest decrease in circulating anti-dsDNA levels, whereas congenic STAT-6–deficient NZM 2328 mice develop markedly attenuated renal disease and improved survival despite serum anti-dsDNA levels equal to or greater than those of wild-type NZM 2328 mice (48).

Second, in related NZM 2410 mice, treatment with anti–interleukin-4 antibodies or the genetic ablation of STAT-6 was shown to ameliorate renal disease and enhance survival, whereas the genetic ablation of STAT-4 hastened renal disease and premature death. Despite the profound effects on morbidity and mortality, none of these interventions had appreciable effects on circulating levels of anti-dsDNA antibodies (49).

Third, treatment of NZB/NZW mice with one BAFF antagonist (TACI-Ig) had a dramatic salutary effect on renal disease and survival without measurable effects on circulating anti-dsDNA antibody levels (6) (although treatment of such mice with another BAFF antagonist [BAFF-R-Ig] did reduce circulating levels of anti-dsDNA antibodies in parallel with clinical improvement [50]).

Fourth, although B cell–deficient MRL-lpr/lpr mice do not develop nephritis or vasculitis (51), MRL-lpr/lpr mice bearing B cells that are incapable of secreting Ig (and, hence, harbor no circulating autoantibodies) do develop such disease (albeit in a milder form than that developed by wild-type MRL-lpr/lpr mice) (52). Thus, B cells clearly effect a disease-promoting function that is independent of their ability to produce autoantibodies but may be dependent upon the ability of B cells to promote the activation of autoreactive T cells (52, 53). By extension, the effects of BAFF overexpression on the precocious development of glomerular disease in B6.Sle1 and B6.Nba2 mice may relate more to the effects of BAFF on B cell numbers (Figure 1D) than to its effects on circulating autoantibody levels. Although Ig-secreting cells are present in the kidneys of diseased NZB/NZW mice (54), it remains unknown whether the local antibody production per se is critical to pathogenesis. Experiments are presently being designed to assess the effects of BAFF overexpression on the development of disease in mice bearing B cells incapable of secreting any Ig.

The question remains why, despite similar elevations in circulating BAFF levels, some hosts develop SLE-like disease but others do not. For example, some HIV-infected subjects harbor exceptionally high circulating levels of BAFF (29, 30), yet they do not develop features of SLE.

We postulate that BAFF overexpression amplifies an underlying latent diathesis to SLE, rather than creating an SLE-like disease de novo. In hosts with a relatively “high” latent SLE diathesis, such as B6.Sle1 and B6.Nba2 mice, the development of end-organ (glomerular) pathology requires only short-term overexpression of BAFF. Pathologic changes in hosts with an “intermediate” latent SLE diathesis would require longer-term BAFF overexpression, and in hosts with a “low” latent SLE diathesis, even long-term BAFF overexpression might be insufficient to induce pathology.

We suggest that wild-type B6 mice have an underlying “intermediate” latent SLE diathesis. In a large cohort of (B6 × 129)F2 mice, a B6-derived locus on chromosome 3 was shown to augment circulating levels of IgG antinuclear and antichromatin antibodies (55). Indeed, B6 mice deficient in Fcγ receptor IIB (FcγRIIB) spontaneously develop circulating antichromatin and anti-dsDNA autoantibodies along with immune complex GN and pulmonary vasculitis, whereas FcγRIIB-deficient BALB/c mice do not (56). Moreover, B6 mice deficient in programmed death 1 (PD-1) develop a clinical phenotype highlighted by arthritis and GN (57), whereas BALB/c mice deficient in PD-1 develop no signs of arthritis or GN but do develop a lethal dilated cardiomyopathy (58). Thus, discrete immune disturbances elicit SLE-like features in B6 mice, but the identical immune disturbances do not do so in BALB/c mice. Accordingly, it is not surprising that BAFF-Tg mice bearing a B6 or B6-mixed genetic background can develop SLE-like features with advancing age (6, 21, 22).

A final issue is the absence of severe proteinuria (≥3+ by dipstick) even in mice with class III or class IV GN. The discordance between proteinuria and glomerular pathology at 3 months of age strongly suggests that proteinuria may not be an adequate marker for (early) lupus nephritis, even when it is severe. Given the paucity of tubulointerstitial infiltrates even in mice with class IV GN (Figure 4), it may be that the combination of glomerular disease along with tubulointerstitial infiltrates is necessary for the development of severe proteinuria. We have not observed increased proteinuria in the limited number of 6-month-old B6.Sle1.BAFF and B6.Nba2.BAFF mice studied to date (data not shown), which strongly suggests that BAFF overexpression works in concert with other factors to promote the development of clinically apparent renal disease. Expression of these unidentified contributory factors likely requires neither the Sle1 nor the Nba2 region, since BAFF-Tg mice bearing neither region develop proteinuria by 8 months of age (22). Studies have been initiated to elucidate the nature of these factors.

Acknowledgements

The authors thank Hal Soucier for performing the flow cytometry and the Histology Service of the University of Southern California, Norris Comprehensive Cancer Center, for processing the tissue samples.

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