An Ig µ-heavy chain transgene inhibits systemic lupus erythematosus immunopathology in autoimmune (NZB x NZW)F1 mice
Ute Wellmann,
Miriam Letz,
Andrea Schneider,
Kerstin Amann1 and
Thomas H. Winkler
Department of Genetics, Hematopoiesis Unit, Nikolaus Fiebiger Center for Molecular Medicine and
1 Institute for Pathology, University of Erlangen-Nürnberg, 91054 Erlangen, Germany
Correspondence to:
T. Winkler
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Abstract
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Intrinsic defects in the B lymphoid lineage are involved in predisposition for systemic lupus erythematosus in (NZB x NZW)F1 (NZB/W) mice. In addition, a contribution of CD4+ T cells has been shown to be crucial for the development of fatal glomerulonephritis. To further dissect the role of B and T cells in lupus immunopathology we used Ig µ-heavy chain (µHC) transgenic (Tg) NZB/W mice that we recently established to study mechanisms of B cell tolerance. The Tg NZB/W mice have a very restricted B cell repertoire and only a very minor population of B cells having endogenously rearranged µHC Ig loci are able to undergo isotype switch. Here we analyzed the influence of the restricted B cell repertoire on the development of IgG anti-DNA antibodies and glomerulonephritis as well as the hyperactivation of Th cells. IgG anti-DNA antibodies developed delayed but consistently in the Tg NZB/W mice, suggesting that a strong selective mechanism for the development of these autoantibodies is operative. Despite significant autoantibody titers in Tg NZB/W mice, very little immune deposits in the glomeruli and no evidence for renal inflammation were found. The Tg mice have a significantly prolonged survival time and most of the Tg mice lived much longer than 1 year. Interestingly, the generalized T cell activation that normally correlates and coincides with the progression of the disease in NZB/W mice is strongly reduced in older Tg animals. The absence of IgG3 anti-DNA antibodies and the strong reduction of IgG2a anti-DNA antibodies in the Tg mice suggests that particularly the activation of Th1 cells is inhibited. This result shows that a significant restriction in the B cell repertoire prevents hyperactivation of Th cells and supports the model that T cell hyperactivation in NZB/W mice is secondary to specific interactions with a subpopulation of presumably autoreactive B lymphocytes.
Keywords: anti-DNA antibodies, glomerulonephritis, T cell activation
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Introduction
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Female (NZBxNZW)F1 (NZB/W) mice develop an autoimmune disease that is very similar to human systemic lupus erythematosus (SLE), characterized by the production of autoantibodies against nuclear antigens and the development of fatal lupus nephritis (1). The immunopathogenesis in the NZB/W model is complex, and the primary immunological defects responsible for initiation and progression of the disease are not fully understood. B cells in NZB/W mice show a generalized hyperactivity (2) and this abnormality is intrinsic to the B cell lineage (3). This B cell hyperactivity is observed already early in life in NZB/W mice (4). High-affinity IgG anti-DNA autoantibodies spontaneously develop in female NZB/W mice at the age of 56 months (1). Several studies have shown that these autoantibodies arise from an antigen-driven expansion of few B cell clones that is most likely dependent on T cell help (57). The high-affinity IgG anti-DNA antibodies appear to play a major pathogenic role in the development of lupus glomerulonephritis (8). However, not all IgG anti-DNA autoantibodies are pathogenic. The exact mechanisms for the deposition of anti-DNA autoantibodies in the glomeruli as well as for the induction of an inflammatory response are not known (9).
The mechanisms for the activation and expansion of highly selected B cell clones remain unclear. It is possible that genetically determined hyperactivity and/or defects in central tolerance mechanisms in both the B and T cell lineage favor an autoantigen-driven immune response that is, in addition, controlled by stochastic events. A loss of T cell tolerance to chromatin has been proposed to be the primary event in the sequence of events that are responsible for the development of pathogenic anti-DNA autoantibodies and lupus nephritis (10). Finally, it remains a possibility that polyclonal and autoantigen-specific activation of T cells is secondary to an abnormality intrinsic to the B cell lineage.
We have recently established NZB/W mice that are transgenic (Tg) for an unmutated Ig µ-heavy chain (µHC) originating from an IgM anti-DNA hybridoma to study central tolerance mechanisms of B cells towards DNA (11). In this former study we showed that the introduction of the µHC transgene leads to a restricted B cell repertoire in which the majority of B cells express the Tg HC and only a minority of the B cells expresses endogenous encoded Ig. Only the minor fraction of B cells with endogenously rearranged Ig HC genes is able to undergo isotype switching and therefore can form IgG autoantibodies, whereas the majority expressing the Tg HC is not. These Tg NZB/W mice therefore allow us to study the influence of a significantly restricted B cell compartment on the development of autoimmune disease.
Here we demonstrate that the Tg NZB/W mice do not develop fatal glomerulonephritis despite the production of IgG anti-DNA antibodies. Furthermore, the small and restricted B cell repertoire does not support the accumulation of activated T cells that normally is observed in aged NZB/W mice. The Tg mice represent a model to show that a restricted B cell repertoire is sufficient to inhibit SLE immunopathology.
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Methods
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Mice
3-32 IgM HC Tg mice were generated as described (11) in the NZB background. NZB and NZW mice were initially obtained from IFFA CREDO (L'Arbresle, France) and bred in our facility. To obtain NZB/W Tg and wild-type offspring, NZW female mice were mated with Tg NZB males. The mice have been bred in the animal facilities of the Fiebiger Center for Molecular Medicine under specific pathogen-free conditions. Only female mice were used in the present study.
Statistical analysis
The Kaplan-Meier survival plot was calculated using GB-STAT software (Dynamic Microsystems, Silver Spring, MD). When a spontaneous death was recorded, the timepoints were entered as uncensored data. Mice which were sacrificed for analysis or were still alive at the time of the analysis were entered as censored data. The probability of equality of survival was calculated by log-rank test. The Tg versus wild-type NZB/W mice were tested for differences in the serum data by the MannWhitney U-test using the above- mentioned software. Differences were considered as significant for P < 0.05.
ELISA assays
The concentrations of IgG antibodies in the sera were determined by a sandwich ELISA assay. Goat anti-mouse IgG, Fc
-specific antiserum was coated, then serial dilutions of sera were incubated and bound IgG was detected using horseradish peroxidase-conjugated goat anti-mouse IgG antisera (all antisera were obtained from Dianova, Hamburg, Germany). The IgG concentrations were calculated from a standard curve using calibrated mouse serum (The Binding Site, Birmingham, UK). Total levels of IgG subclasses with a
-light chain in the sera were determined by coating rat anti-mouse
(clone 178.1) and using rat anti-mouse IgG subclass-specific secondary antibodies (A85-1, anti-IgG1; R19-15, anti-IgG2a; R12-13, anti-IgG2b; R40-82: anti-IgG3; all from PharMingen, Heidelberg, Germany).
To determine the relative IgG anti-double-stranded (ds) DNA activity in sera, microtiter plates were incubated with 20 µg/ml poly-L-lysine and then with 20 µg/ml native calf thymus DNA as described (12). Serial dilutions of sera were incubated and bound IgG was detected with a horseradish peroxidase-conjugated goat anti-mouse IgG antiserum. The relative IgG anti-dsDNA binding activity of the sera was calculated from a standard curve obtained with a serum pool of 4- to 5-month-old MRL/lpr mice and is presented as percentage of the binding activity of the serum pool. To assay for the IgG subclass anti-dsDNA activity, the ELISA was modified by using IgG subclass-specific antibodies for detection of IgG bound to DNA.
For the anti-histone ELISA a mixture of histones H1, H2A, H2B, H3 and H4 (Roche, Mannheim, Germany) was coated in a final concentration of 10 µg/ml in PBS, then serial dilutions of sera were incubated and bound IgG was detected with a horseradish peroxidase-conjugated goat anti-mouse IgG antiserum. The relative IgG anti-histone binding activity of the sera was calculated from a standard curve obtained with the MRL/lpr serum pool.
Evaluation of kidney disease
Mice were evaluated for albuminuria using Albustix (Bayer Diagnostics, Munich, Germany). Mice with albumin
300 mg/dl (3+) were considered to be positive for albuminuria.
For histological analysis, mice were killed and the kidneys were dissected in 1-mm thick slices perpendicular to the longitudinal axis. Using area-weighted sampling, five small pieces (2x2x1 mm) were taken for embedding in epon araldite. The remaining tissue slices were embedded in paraffin, and 2-µm thick sections were cut and stained with hematoxylin & eosin (HE), PAS and a fibrous tissue stain (Sirius red).
IgG Ig deposits were analyzed on cryosections; 5-µm thick cryosections of kidneys were fixed in acetone for 10 min at 20°C and IgG was detected with Cy-3-conjugated goat anti-mouse IgG, Fc
-specific antiserum (Dianova, Hamburg, Germany).
Flow cytometry analysis
Lymph node and spleen cells (25x105) were surface stained according to standard protocols using the following antibodies: FITC-conjugated anti-CD4 (GK1.5), phycoerythrin-conjugated anti-CD4 (GK1.5), biotinylated anti-CD69 (H1.2F3), biotinylated anti-CD25 (7D4), biotinylated anti-CD62 ligand (CD62L) (MEL-14) and FITC-conjugated anti-CD8 (53-6.7). All antibodies were obtained from PharMingen. Biotinylated antibodies were visualized with phycoerythrin-conjugated streptavidin (Molecular Probes, Leiden, Netherlands). All samples were analyzed on a FACSCalibur flow cytometer calibrated with FACSComp software with CaliBRITE beads (Becton Dickinson, Heidelberg, Germany). Collected events were gated for total lymphocytes defined by forward and side scatter characteristics using CellQuest software. A logarithmic scale was used for the axis of the dot-plots, which give the relative fluorescence intensities of events.
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Results
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Ig Tg NZB/W mice have a restricted B cell repertoire
We previously used 3-32 µHC Tg mice as a model to study tolerance mechanisms of B lymphocytes towards DNA (11). Lupus prone NZB/W mice showed intact central tolerance mechanisms compared to normal C57Bl/6 mice. B cells were reduced in numbers ~3-fold and light chain usage was strongly biased in Tg mice. The Tg and the endogenously encoded IgM can be distinguished by means of µ-allotype-specific reagents. B cells expressing endogenously encoded Ig accounted for only 5% of all B cells in 3-month-old Tg mice. Cells expressing the endogenous as well as the Tg allotype made up <1% of all B cells (11). Therefore young Tg NZB/W mice showed a 40- to 60-fold reduction in numbers of B cells expressing endogenously encoded IgM compared to wild-type NZB/W littermates. In older Tg mice (915 months) B cells expressing endogenously encoded IgM made up 30 ± 9% of the B cells in the spleen (mean ± SD from eight mice analyzed, data not shown), consistent with previous observations that endogenous Ig-expressing B cells accumulate in Ig-Tg mice (13). In summary, 3-32 µHC Tg NZB/W mice had a smaller and highly restricted B cell repertoire when compared to wild-type littermates. B cells expressing endogenously encoded Ig that would be able to undergo isotype switch and to differentiate into IgG- secreting plasma cells were strongly reduced in numbers.
The HC transgene prolongs the life of NZB/W mice
In the course of our analyses of Tg mice we noticed that not a single female Tg NZB/W mouse out of 25 mice with an average age of 48 weeks developed albuminuria. Wild-type female littermates developed significant albuminuria at the age of 58 months, as expected. We determined the survival time of Tg and wild-type NZB/W female mice. The KaplanMeier analysis for cumulative survival revealed 50% mortality for wild-type mice at the age of 78 months and Tg mice at 2224 months (Fig. 1
). The difference is highly significant (log-rank test for equality of survival: P < 106). At 12 months of age all female wild-type NZB/W mice had died or were severely sick and had been killed. In contrast, >90% of Tg mice were still alive and remained healthy without significant albuminuria at that timepoint.

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Fig. 1. KaplanMeier survival analysis of NZB/W mice. The plot shows the probability of cumulative survival of the mice as a function of age in weeks. NZB/W wild-type female mice (, 15 spontaneous deaths and 42 censored events) are compared with female Tg mice ( , nine spontaneous deaths and 62 censored events).
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The HC transgene delays development of serum IgG autoantibodies
Because anti-DNA antibodies have been implicated in the pathogenesis of glomerulonephritis in NZB/W mice we examined the IgG concentrations and IgG anti-DNA levels in the sera of Tg and wild-type NZB/W mice. As expected, wild-type NZB/W mice showed significant IgG-hypergammaglobulinemia with serum IgG concentrations of 16 ± 9 mg/ml at the age of 612 months (mean ± SD from 13 sera analyzed), whereas sera of 1-year-old BALB/c mice contained 6 ± 3 mg/ml IgG. In Tg NZB/W mice >6 months, serum IgG concentrations were found to be 8 ± 4 mg/ml (mean ± SD from 25 sera analyzed). This shows that endogenous Ig-expressing B cells in Tg mice mature and differentiate into IgG-secreting plasma cells.
Titers of IgG anti-DNA autoantibodies were evaluated in sera from mice with ages ranging from 3 to 15 months. Results are shown in Fig. 2(A)
. Female wild-type NZB/W mice started to produce elevated IgG anti-DNA antibody levels at the age of 46 months, whereas in Tg mice of the same age IgG anti-DNA antibodies could not be detected. All wild-type NZB/W mice developed elevated IgG anti-DNA antibodies at the age of 69 months. Fourteen out of 16 (88%) Tg mice at the age of 912 months and all Tg mice >1 year had elevated IgG anti-DNA titers. In summary, Tg NZB/W mice developed IgG anti-DNA antibodies, but on average 34 months later in life than wild-type NZB/W mice. The levels of IgG anti-DNA antibodies were lower in Tg mice.

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Fig. 2. Analysis of IgG autoantibody activity in sera of wild-type and Tg NZB/W mice. (A) IgG anti-dsDNA activities were determined by ELISA. Results are expressed in percentage binding as calculated from a standard curve obtained with a serum pool of 4- to 5-month- old MRL/lpr mice. Each point represents the data of a single NZB/W mouse ( , wild-type mice; , Tg mice). The age in months and the number of the analyzed mice is given below the plot. Horizontal lines indicate the mean values of IgG anti-DNA activity. Values >5% of the anti-DNA activity were considered positive. (B) IgG anti-histone activity of wild-type and Tg NZB/W mice relative to the MRL/lpr serum pool. Values >2% of the anti-histone activity were considered positive.
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The sera of the NZB/W mice were also tested for the presence of IgG anti-histone autoantibodies. Figure 2 (B)
shows the binding of the sera to histones mixed in the same ratio as found for the histones in nucleosomes. Elevated IgG anti-histone antibodies were detected in all wild-type NZB/W female mice >6 months. In Tg mice, the development of IgG anti-histone antibodies was delayed, similar to anti-dsDNA antibodies. In addition, anti-histone antibodies were observed with a lower penetrance in Tg compared to wild-type NZB/W mice.
Reduced production of IgG2a and IgG3 anti-DNA autoantibodies in Tg NZB/W mice
In NZB/W mice, anti-dsDNA antibodies are predominantly of the IgG2a and IgG3 isotype (1). To determine whether the IgG subclass pattern of anti-DNA antibodies was altered in the Tg mice, sera of 9- to 15-month-old Tg and wild-type NZB/W mice were examined. Whereas the IgG1 and IgG2b anti-dsDNA levels were in a similar range in Tg and wild-type NZB/W mice, IgG2a anti-dsDNA levels were significantly reduced in Tg mice (Fig. 3A
, P, < 0.001). IgG3 anti-dsDNA antibodies present in most of the 9- to 12-month-old wild-type NZB/W mice were barely detectable in the sera from 9- to 15-month-old Tg NZB/W mice.
We also determined the total IgG subclass levels in the sera of Tg and wild-type NZB/W mice. The concentrations of IgG2a and IgG3 were significantly reduced (P < 103 for both subclasses) in Tg mice compared to wild-type littermates (Fig. 2B
). In contrast, the IgG2b levels were not significantly different (see Fig. 5B
), and the IgG1 levels were less reduced than the levels of IgG2a and IgG3 (3- versus 11- and 14-fold reduction).

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Fig. 5. Representative flow cytometric analysis of the splenic T cell compartment of a Tg (left panels) versus a wild-type (right panels) 10-month-old NZB/W mouse. The dot-plots are gated on live lymphocytes by forward and side scatter, and show the surface expression of CD4 versus CD69 (top), CD25 (second row), CD62L (third row) and CD8 (bottom). The numbers give the percentages of gated lymphocytes in the quadrants.
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The HC transgene prevents severe glomerulonephritis
Renal disease in the glomeruli, the tubules and the vessels was analyzed on paraffin sections of total kidney specimens from wild-type and Tg NZB/W mice using light microscopy at various magnifications and a semiquantitative scoring system. Results are listed in Table 1
and representative sections are shown in Fig. 4
. The wild-type NZB/W mice developed lesions typical of severe lupus glomerulonephritis with glomerular enlargement due to extracellular matrix expansion, hypercellularity with intra- and extracapillary proliferation and influx of mononuclear cells, segmental tuft adhesion and focal crescent formation. Peripheral capillary walls showed thickening of the basement membrane as a sign of subendothelial immune complex deposition. In addition, in the animal with the most advanced glomerular changes, marked tubulointerstitial changes with tubular dilatation and atrophy, tubular casts and interstitial inflammation were seen. Vascular changes, i.e. wall thickening or vasculitis, were not observed.

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Fig. 4. Representative light microscopy and immunofluorescence photographs of renal tissue sections from wild-type and Tg NZB/W mice. (A) Glomerular morphology of a 13-month-old Tg NZB/W mouse. PAS-stained paraffin sections show only a slight increase in mesangial matrix but no hypercellularity. (B) Glomerular morphology of an 11-month-old wild-type NZB/W mouse. PAS-stained paraffin sections revealed enlargement of the glomeruli due to mesangial matrix expansion, hypercellularity with intracapillary proliferation and influx of mononuclear cells. (C) Direct immunofluorescence on a glomerulus of a 13-month-old Tg NZB/W mouse. Cryosections stained with Cy-3-conjugated anti-mouse IgG antiserum revealed only weak mesangial and pseudolinear IgG deposits. (D) Direct immunofluorescence on a glomerulus of an 11-month-old wild-type NZB/W mouse showing intense granular IgG staining of peripheral capillary walls and of some mesangial areas.
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In contrast to the findings in wild-type mice, histological examination of kidney sections of Tg mice at the age of 915 months revealed minor lesions with only slight to moderate mesangial matrix expansion and a slight increase in peripheral basement membrane thickness. Glomerular hypercellularity, tuft adhesions or crescent formation respectively were not seen. Tubulointerstitial or vascular abnormalities were not observed.
Immunofluorescence staining of kidney sections showed intense granular IgG deposits along the capillary walls (subendothelial and subepithelial) as well as in some mesangial areas in wild-type NZB/W mice with proteinuria (Fig. 4
). Kidney sections from Tg mice revealed no or only minimal glomerular IgG deposits with a weak mesangial and pseudolinear pattern, respectively (Fig. 4
).
Activated and memory T cells do not accumulate in HC Tg mice
As an expansion of activated CD4+ T cells with increased age in NZB/W mice has been documented (14), we analyzed whether the restricted B cell repertoire in Tg mice might have an influence on activation of CD4+ T cells in aged mice. CD69 and CD25 expression is up-regulated in activated T cells, and CD62L expression is down-regulated in activated and memory T cells as compared to naive T cells (15). We analyzed the expression levels of these markers on spleen and lymph node cells from NZB/W female mice. Figure 5
shows a representative flow cytometric analysis of the T cell compartment of 10-month-old wild-type and Tg NZB/W mice, and the results for all analyzed mice are summarized in Table 2
. Among CD4+ T cells in the spleen the wild-type NZB/W mice had significantly higher percentages of CD69+ and CD25+ activated cells. Also, the frequency of CD62Llow activated/memory T cells was higher in wild-type NZB/W mice as compared to Tg NZB/W mice (Table 2
). This relative expansion of activated/memory CD4+ T cells in wild-type mice is particularly striking in aged mice and coincides with an elevated CD4:CD8 ratio (Table 2
). This effect was not specific for the spleen, as the above accumulation was also significant in the lymph nodes of aged mice (Table 2
). The cumulative data suggest that the B cells in Tg NZB/W mice do not promote the accumulation of activated and memory T cells in contrast to the B cells in wild-type NZB/W mice.
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Discussion
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Although the susceptibility to lupus-like disease in NZB/W mice is a complex genetic trait (16) and multiple immunological abnormalities are operative (1,17), our data presented here clearly indicate that a restriction of the B cell repertoire is sufficient to significantly protect against severe lupus nephritis. Autoantibodies of the IgG class and particularly IgG anti-DNA antibodies are believed to play a major role in the pathogenesis of lupus nephritis (18,19). In the Tg mice described here, the development of significant IgG anti-DNA antibody titer is delayed when compared to wild-type NZB/W mice and the average titers of Tg NZB/W mice are slightly lower. Considering that IgG anti-DNA autoantibodies can only be derived from B cells that express endogenously encoded Ig and that these B cells make up only a very minor part of the B cell repertoire, it is striking that these antibodies develop at all to significant levels and reproducibly after ~1012 months. This finding clearly illustrates the strong selective pressure for the expansion of B cell clones expressing anti-DNA receptors that most likely is driven by autoantigen (5,20,21).
Despite the presence of IgG anti-DNA antibodies in the sera of Tg animals we found very little evidence for deposition of IgG in the glomeruli of the mice and accordingly only very minute pathological changes were observed. The protection from glomerulonephritis of the Tg mice correlated with a substantial change of the IgG subclasses of anti-dsDNA antibodies. The absence of IgG3 anti-DNA antibodies as well as the strong reduction of IgG2a anti-DNA antibodies in the Tg NZB/W mice might explain the protection from glomerulonephritis as several studies have shown the particular importance of these isotypes for pathogenicity (2224). Very similar to our results obtained with a µ-HC transgene in the NZB/W background, an IL-4 transgene in the (NZWxC57Bl/6.Yaa) background shifted the IgG subclass pattern of anti-DNA antibodies to predominantly IgG1 and prevented glomerulonephritis (24).
One other possible explanation for the absence of glomerular deposits might be that the anti-DNA repertoire in the Tg mice is very restricted. It has been documented extensively that only a subset of anti-DNA autoantibodies has nephritogenic properties (9,25,26). The strong restriction of the B cell repertoire in the Tg mice might not permit the development of antibodies that have the right properties to deposit in the glomeruli. It is unclear at the moment what molecular properties distinguish pathogenic from non- pathogenic anti-DNA autoantibodies and probably there are several mechanisms for the binding as well as the induction of tissue damage by anti-DNA antibodies (reviewed in 27). In addition, other autoantibody specificities including anti-nucleosome (28), anti-retroviral gp70 (29) and antibodies with cryoglobulin activity (30) have been shown to be involved in the induction of lupus nephritis. Further analysis of the fine specificity of the IgG anti-DNA antibodies in the Tg NZB/W mice and the comparison with antibodies from wild-type NZB/W mice should help in understanding the immunopathogenesis of lupus nephritis.
The role of T cells in the pathogenesis of SLE in NZB/W mice has been underlined in several studies (31,32). An expansion of activated Th cells is characteristic for lupus-prone New Zealand mice (14,33,34) as well as MRL/lpr mice (15). It is unclear whether hyperactivation of Th cells is a primary and intrinsic abnormality in NZB/W mice or whether it might be secondary to the B cell hyperactivation. Polyclonal B cell activation itself is clearly an intrinsic B cell defect in NZB/W mice, as several studies have shown (3,3537). Our data presented here demonstrate that spontaneous T cell activation is dependent on the B cell repertoire as the populations of activated T cells are markedly reduced in the HC Tg mice. Furthermore, the down-regulation of IgG2a and IgG3 anti-DNA as well as total serum antibodies in the Tg mice suggests that particularly the activation of Th1 cells is specifically inhibited.
Thus, our data support the idea that B cells might play a regulatory role for the expansion of activated CD4+ T cells that might include T cells specific for nuclear autoantigens like histones (10) in the NZB/W model. A series of elegant experiments recently demonstrated such a key regulatory role of B cells for fas-deficient and fas-intact MRL mice (3841). The similarity to the findings presented here is striking, as the genetic loci influencing the lupus-like disease in MRL/lpr mice (42) and New Zealand strains of mice (16,43) are distinct and little complementation between disease-prone genetic backgrounds is observed (1). Thus, it appears that B cells have a central role in the pathogenesis of genetically different mouse models for SLE.
Our observations that in HC Tg NZB/W mice T cell hyperactivation is reduced and that the mice are protected from severe nephritis is supportive for the model of lupus pathogenesis recently proposed by Chan et al. (44). An amplification loop consisting of B cells and Th cells as a primary event in lupus pathogenesis was proposed. In that loop autoreactive B cells function as antigen-presenting cells and promote T cell activation. Activated T cells provide further help for B cells, and each cycle might further amplify autoreactive B and T cell clones and at the same time further diversifies autoreactive specificities through epitope spreading (45). By this mechanisms the pathogenic potential of autoantibody specificities would be increased. In the context of this model the restriction of the B cell repertoire in the Tg NZB/W mice described here apparently is sufficient to inhibit such an amplification loop although significant IgG anti-DNA antibodies are induced.
Interestingly, substantial but incomplete depletion of CD4+ T cells by thymectomy and a short anti-CD4 treatment does not prevent severe autoimmune disease in NZB/W mice (46), whereas chronic anti-CD4 treatment does (31). A small subpopulation of CD4+ cells refractory to depletion is apparently sufficient to promote full expression of the fatal lupus-like disease in the NZB/W model, whereas a restriction of the B cells repertoire, as described here, does efficiently suppress autoimmune disease. These findings further warrant the evaluation of novel therapeutic approaches for SLE aiming at the depletion or suppression of B cells (39).
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Acknowledgments
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This work was supported by the Deutsche Forschungsgemeinschaft (SFB 263 and SFB 423). We would like to thank Jeanette Striepe for animal husbandry and Rosa Rückerl for technical assistance.
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Abbreviations
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µHC µ-heavy chain |
ds double-stranded |
NZB/W (NZBxNZW)F1 |
SLE systemic lupus erythematosus |
Tg transgenic |
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Notes
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Transmitting editor: S. Izui
Received 4 May 2001,
accepted 24 August 2001.
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References
|
---|
-
Theofilopoulos, A. N. and Dixon, F. J. 1985. Murine models of systemic lupus erythematosus. Adv. Immunol. 37:269.[ISI][Medline]
-
Izui, S., McConahey, P. J. and Dixon, F. J. 1978. Increased spontaneous polyclonal activation of B lymphocytes in mice with spontaneous autoimmune disease. J. Immunol. 121:2213.[Abstract]
-
Reininger, L., Winkler, T. H., Kalberer, C. P., Jourdan, M., Melchers, F. and Rolink, A. G. 1996. Intrinsic B cell defects in NZB and NZW mice contribute to systemic lupus erythematosus in (NZBxNZW)F1 mice. J. exp. Med. 184:853.[Abstract]
-
Moutsopoulos, H. M., Boehm-Truitt, M., Kassan, S. S. and Chused, T. M. 1977. Demonstration of activation of B lymphocytes in New Zealand black mice at birth by an immunoradiometric assay for murine IgM. J. Immunol. 119:1639.[Abstract]
-
Shlomchik, M., Mascelli, M., Shan, H., Radic, M. Z., Pisetsky, D., Mershak-Rothstein, A. and Weigert, M. 1990. Anti-DNA antibodies from autoimmune mice arise by clonal expansion and somatic mutation. J. Exp. Med. 171:265.[Abstract]
-
Tillman, D. M., Jou, N. T., Hill, R. J. and Marion, T. N. 1992. Both IgM and IgG anti-DNA antibodies are the products of clonally selective B cell stimulation in (NZBxNZW)F1 mice. J. Exp. Med. 176:761.[Abstract]
-
Winkler, T. H., Fehr, H. and Kalden, J. R. 1992. Analysis of immunoglobulin variable region genes from human IgG anti-DNA hybridomas. Eur. J. Immunol. 22:1719.[ISI][Medline]
-
Winfield, J. B., Faiferman, I. and Koffler, D. 1977. Avidity of anti-DNA antibodies in serum and IgG glomerular eluates from patients with systemic lupus erythematosus. J. Clin. Invest. 59:90.[ISI][Medline]
-
Ohnishi, K., Ebling, F., Mitchell, B., Singh, R., Hahn, B. and Tsao, B. 1994. Comparison of pathogenic and non-pathogenic murine antibodies to DNA: antigen binding and structural characteristics. Int. Immunol. 6:817.[Abstract]
-
Mohan, C., Adams, S., Stanik, V. and Datta, S. K. 1993. Nucleosome: a major immunogen for pathogenic autoantibody-inducing T cells of lupus. J. Exp. Med. 177:1367.[Abstract]
-
Wellmann, U., Werner, A. and Winkler, T. H. 2001. Altered selection processes of B lymphocytes in autoimmune NZB/W mice, despite intact central tolerance against DNA. Eur. J. Immunol. 31:2800.[ISI][Medline]
-
Winkler, T. H., Henschel, T. A., Kalies, I., Baenkler, H. W., Skvaril, F. and Kalden, J. R. 1988. Constant isotype pattern of anti-dsDNA antibodies in patients with systemic lupus erythematosus. Clin. Exp. Immunol. 72:434.[ISI][Medline]
-
Grandien, A., Coutinho, A. and Anderson, J. 1990. Selective peripheral expansion and activation of B cells expressing endogenous immunoglobulin in mu-transgenic mice. Eur. J. Immunol. 20:991.[ISI][Medline]
-
Rozzo, S. J., Drake, C. G., Chiang, B. L., Gershwin, M. E. and Kotzin, B. L. 1994. Evidence for polyclonal T cell activation in murine models of systemic lupus erythematosus. J. Immunol. 153:1340.[Abstract/Free Full Text]
-
Giese, T. and Davidson, W. F. 1992. Evidence for early onset, polyclonal activation of T cell subsets in mice homozygous for lpr. J. Immunol. 149:3097.[Abstract/Free Full Text]
-
Morel, L. and Wakeland, E. K. 1998. Susceptibility to lupus nephritis in the NZB/W model system. Curr. Opin. Immunol. 10:718.[ISI][Medline]
-
Kotzin, B. L. 1996. Systemic lupus erythematosus. Cell 85:303.[ISI][Medline]
-
Lefkowith, J. and Gilkeson, G. 1996. Nephritogenic autoantibodies in lupus: current concepts and continuing controversies. Arthritis Rheum. 39:894.[ISI][Medline]
-
Madaio, M. and Shlomchik, M. 1996. Emerging concepts regarding B cells and autoantibodies in murine lupus nephritis. B cells have multiple roles; all autoantibodies are not equal. J. Am. Soc. Nephrol. 7:387.[Abstract]
-
Marion, T. N., Tillman, D. M. and Jou, N. T. 1990. Interclonal and intraclonal diversity among anti-DNA antibodies from an (NZBxNZW)F1 mouse. J. Immunol. 145:2322.[Abstract/Free Full Text]
-
Friedmann, D., Yachimovich, N., Mostoslavsky, G., Pewzner-Jung, Y., Ben-Yehuda, A., Rajewsky, K. and Eilat, D. 1999. Production of high affinity autoantibodies in autoimmune New Zealand Black/New Zealand white F1 mice targeted with an anti-DNA heavy chain. J. Immunol. 162:4406.[Abstract/Free Full Text]
-
Takahashi, S., Nose, M., Sasaki, J., Yamamoto, T. and Kyogoku, M. 1991. IgG3 production in MRL/lpr mice is responsible for development of lupus nephritis. J. Immunol. 147:515.[Abstract/Free Full Text]
-
Takahashi, S., Fossati, L., Iwamoto, M., Merino, R., Motta, R., Kobayakawa, T. and Izui, S. 1996. Imbalance towards Th1 predominance is associated with acceleration of lupus-like autoimmune syndrome in MRL mice. J. Clin. Invest. 97:1597.[Abstract/Free Full Text]
-
Santiago, M. L., Fossati, L., Jacquet, C., Muller, W., Izui, S. and Reininger, L. 1997. Interleukin-4 protects against a genetically linked lupus-like autoimmune syndrome. J. Exp. Med. 185:65.[Abstract/Free Full Text]
-
Raz, E., Brezis, M., Rosenmann, E. and Eilat, D. 1989. Anti-DNA antibodies bind directly to renal antigens and induce kidney dysfunction in the isolated perfused rat kidney. J. Immunol. 142:3076.[Abstract/Free Full Text]
-
Vlahakos, D., Foster, M., Adams, S., Katz, M., Ucci, A., Barrett, K., Datta, S. and Madaio, M. 1992. Anti-DNA antibodies form immune deposits at distinct glomerular and vascular sites. Kidney Int. 41:1690.[ISI][Medline]
-
Hahn, B. H. 1998. Antibodies to DNA. N. Engl. J. Med. 338:1359.[Free Full Text]
-
Burlingame, R. W., Boey, M. L., Starkebaum, G. and Rubin, R. L. 1994. The central role of chromatin in autoimmune responses to histones and DNA in systemic lupus erythematosus. J. Clin. Invest. 94:184.[ISI][Medline]
-
Izui, S., McConahey, P. J., Clark, J. P., Hang, L. M., Hara, I. and Dixon, F. J. 1981. Retroviral gp70 immune complexes in NZBxNZW F2 mice with murine lupus nephritis. J. Exp. Med. 154:517.[Abstract]
-
Reininger, L., Berney, T., Shibata, T., Spertini, F., Merino, R. and Izui, S. 1990. Cryoglobulinemia induced by a murine IgG3 rheumatoid factor: skin vasculitis and glomerulonephritis arise from distinct pathogenic mechanisms. Proc. Natl Acad. Sci. USA 87:10038.[Abstract]
-
Wofsy, D. and Seaman, W. E. 1985. Successful treatment of autoimmunity in NZB/NZW F1 mice with monoclonal antibody to L3T3. J. Exp. Med. 161:378.[Abstract]
-
Early, G., Zhao, W. and Burns, C. 1996. Anti-CD40 ligand antibody treatment prevents the development of lupus-like nephritis in a subset of New Zealand blackxNew Zealand white mice. Response correlates with the absence of an anti-antibody response. J. Immunol. 157:3159.[Abstract]
-
Ishikawa, S., Akakura, S., Abe, M., Terashima, K., Chijiiwa, K., Nishimura, H., Hirose, S. and Shirai, T. 1998. A subset of CD4+ T cells expressing early activation antigen CD69 in murine lupus: possible abnormal regulatory role for cytokine imbalance. J. Immunol. 161:1267.[Abstract/Free Full Text]
-
Mohan, C., Yu, Y., Morel, L., Yang, P. and Wakeland, E. K. 1999. Genetic dissection of Sle pathogenesis: Sle3 on murine chromosome 7 impacts T cell activation, differentiation, and cell death. J. Immunol. 162:6492.[Abstract/Free Full Text]
-
Reininger, L., Radaszkiewicz, T., Kosco, M., Melchers, F. and Rolink, A. G. 1992. Development of autoimmune disease in SCID mice populated with long-term `in vitro' proliferating (NZBxNZW)F1 pre-B cells. J. Exp. Med. 176:1343.[Abstract]
-
Merino, R., Iwamoto, M., Fossati, L. and Izui, S. 1993. Polyclonal B cell activation arises from different mechanisms in lupus-prone (NZBxNZW)F1 and MRL/MpJ-lpr/lpr mice. J. Immunol. 151:6509.[Abstract/Free Full Text]
-
Sobel, E. S., Mohan, C., Morel, L., Schiffenbauer, J. and Wakeland, E. K. 1999. Genetic dissection of SLE pathogenesis: adoptive transfer of Sle1 mediates the loss of tolerance by bone marrow-derived B cells. J. Immunol. 162:2415.[Abstract/Free Full Text]
-
Shlomchik, M. J., Madaio, M. P., Ni, D., Trounstein, M. and Huszar, D. 1994. The role of B cells in lpr/lpr-induced autoimmunity. J. Exp. Med. 180:1295.[Abstract]
-
Chan, O. and Shlomchik, M. J. 1998. A new role for B cells in systemic autoimmunity: B cells promote spontaneous T cell activation in MRL-lpr/lpr mice. J. Immunol. 160:51.[Abstract/Free Full Text]
-
Chan, O. T., Hannum, L. G., Haberman, A. M., Madaio, M. P. and Shlomchik, M. J. 1999. A novel mouse with B cells but lacking serum antibody reveals an antibody-independent role for B cells in murine lupus. J. Exp. Med. 189:1639.[Abstract/Free Full Text]
-
Chan, O. T., Madaio, M. P. and Shlomchik, M. J. 1999. B cells are required for lupus nephritis in the polygenic, Fas-intact MRL model of systemic autoimmunity. J. Immunol. 163:3592.[Abstract/Free Full Text]
-
Watson, M. L., Rao, J. K., Gilkeson, G. S., Ruiz, P., Eicher, E. M., Pisetsky, D. S., Matsuzawa, A., Rochelle, J. M. and Seldin, M. F. 1992. Genetic analysis of MRL-lpr mice: relationship of the Fas apoptosis gene to disease manifestations and renal disease-modifying loci. J. Exp. Med. 176:1645.[Abstract]
-
Vyse, T. J. and Kotzin, B. L. 1998. Genetic susceptibility to systemic lupus erythematosus. Annu. Rev. Immunol. 16:261.[ISI][Medline]
-
Chan, O. T., Madaio, M. P. and Shlomchik, M. J. 1999. The central and multiple roles of B cells in lupus pathogenesis. Immunol. Rev. 169:107.[ISI][Medline]
-
Mamula, M. J., Lin, R. H., Janeway, C. A., Jr and Hardin, J. A. 1992. Breaking T cell tolerance with foreign and self co-immunogens. A study of autoimmune B and T cell epitopes of cytochrome c. J. Immunol. 149:789.[Abstract/Free Full Text]
-
Connolly, K., Roubinian, J. R. and Wofsy, D. 1992. Development of murine lupus in CD4-depleted NZB/NZW mice. Sustained inhibition of residual CD4+ T cells is required to suppress autoimmunity. J. Immunol. 149:3083.[Abstract/Free Full Text]