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International Immunology Advance Access originally published online on May 30, 2006
International Immunology 2006 18(7):1127-1137; doi:10.1093/intimm/dxl047
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Spontaneous B cell hyperactivity in autoimmune-prone MRL mice

Anastasia Nijnik1, Helen Ferry1, Graham Lewis1, Eleni Rapsomaniki1, Janson C. H. Leung1, Angelika Daser1, Teresa Lambe1, Christopher C. Goodnow2 and Richard J. Cornall1

1 Henry Wellcome Building of Molecular Physiology, Oxford University, Roosevelt Drive, Oxford, OX3 7BN, UK
2 Australian Cancer Research Foundation Genetics Laboratory, John Curtin School of Medical Research, Australian National University, Canberra, ACT 2601, Australia

Correspondence to: R. Cornall; E-mail: richard.cornall{at}ccmp.ox.ac.uk


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
The MRL-lpr/lpr mouse strain is a commonly used model of the human autoimmune disease systemic lupus erythematosus (SLE). Although much is known about the contribution of the lpr Fas mutation to B cell tolerance breakdown, the role of the genetic background of the MRL strain itself is less well explored. In this study, we use the MD4 anti-hen egg lysozyme Ig (IgHEL) transgenic system to explore B cell function in MRL+/+ and non-autoimmune mice. We demonstrate that MRL IgHEL B cells show spontaneous hyperactivity in the absence of self-antigen, which is associated with low total B cell numbers but an expansion of the marginal zone B cell population. However, B cell anergy is normal in the presence of soluble lysozyme [soluble hen egg lysozyme (sHEL)], and MRL IgHEL B cells undergo normal elimination in the presence of sHEL when competing with a polyclonal C57BL/6 B cell repertoire. We conclude that B cell hyperactivity may contribute to the autoimmune phenotype of MRL+/+ and MRL-lpr/lpr strains when it initiates antibody responses to rare or sequestered antigens that are below the threshold for tolerance induction, but that there is no B cell intrinsic defect in anergy in MRL mice.

Keywords: B lymphocytes, systemic lupus erythematosus (SLE), tolerance


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Systemic lupus erythematosus (SLE) is a common but clinically heterogeneous systemic autoimmune disease, which is characterised by auto-antibodies and immune complex formation leading to widespread organ damage. Because the causes of the disease are complex and polygenic, much work has been done to explore basic pathophysiology using mouse models, including the MRL-lpr/lpr mouse (1, 2). This strain develops SLE-like auto-antibody profiles, as well as splenomegaly, lymphadenopathy, fatal nephritis and vasculitis (1, 2). The genetic contributions to the autoimmunity in MRL-lpr/lpr mice consist of both the lpr mutation, which is a splicing error in Fas/CD95 gene (3, 4), and several as yet unidentified susceptibility genes in the MRL strain itself (5–7). The contribution of the MRL background to autoimmune susceptibility is demonstrated by finding that the lpr mutation results in a less severe phenotype when bred onto non-autoimmune strains (8, 9). Furthermore, in contrast to non-autoimmune prone strains, wild-type MRL-+/+ mice also develop an autoimmune disease but of a later onset and milder nature than MRL-lpr/lpr (2, 8). The polygenic nature of SLE in MRL suggests that, as in humans, susceptibility is determined by defects in multiple tolerance checkpoints. The aim of dissecting these mechanisms in MRL mice is to gain a better understanding of the aetiology of systemic autoimmune disorders in humans.

One way to reduce the inherent complexity of the B cell repertoire is to use Ig transgenic models. This approach has been used to track large populations of antigen-specific B cells in vivo and in this way define the checkpoints normally responsible for tolerance in non-autoimmune animals (10). These studies show that there is a spectrum of different checkpoints, which are triggered depending on the affinity of B cell receptor (BCR) and the concentration and avidity of self-antigen. This is illustrated in the hen egg lysozyme (HEL) transgenic model in which MD4 anti-hen egg lysozyme Ig (IgHEL) transgenic B cells develop in the presence of different forms of HEL expressed as a neo-self-antigen. Thus, self-reactive IgHEL B cells encountering systemic cell-surface membrane bound HEL are eliminated at the immature cell stage (11). In contrast, B cells that encounter soluble antigen [soluble hen egg lysozyme (sHEL)] above 10–20 ng ml–1 are functionally inactivated (anergic) (12) and in a mixed B cell repertoire have a shortened lifespan due to competitive exclusion from B cell follicles of secondary lymphoid organs and failure to bind adequate levels of the survival factor, B cell activating factor (BAFF) (13). B cells that encounter self-antigen at levels below 10 ng ml–1 are neither rendered anergic nor activated, indicating that they are functionally naive and in a state of immunological ignorance (14).

Transgenic systems have already been used successfully to dissect the role of lpr mutation in tolerance breakdown. Both B and T cell intrinsic effects of the mutation play a role in tolerance breakdown (15–17). lpr does not impede thymic negative selection of T lymphocytes (18–21) and does not interfere with central deletion of autoreactive B cells (22–24). Instead, it interferes with mechanisms of peripheral T cell tolerance (18, 21), prevents Fas-dependent elimination of anergic B cells by CD4 T cells (25) and results in provision of T cell help to autoreactive B cells (26). Thus, lpr mutation leads to an age-dependent B cell tolerance breakdown in MRL-lpr/lpr mice carrying Ig transgenes that recognise double-stranded DNA (dsDNA) (27, 28). To a lesser extent tolerance breakdown also occurs in C57BL/6 (B6)-lpr/lpr animals carrying the IgHEL transgene in the presence of sHEL (22). These findings emphasise the importance of strain-specific effects of background genes.

Despite the genetic evidence, little is known about the functional effects of individual MRL autoimmune susceptibility genes. In contrast to MRL-lpr/lpr, tolerance is intact in MRL-+/+ mice transgenic for VH3H9 heavy chain of an anti-dsDNA Ig, since no high-affinity anti-dsDNA antibodies are produced (28). However, anti-dsDNA VH3H9 transgenic B cells show less IgM down-modulation and developmental arrest in MRL+/+ than in non-autoimmune BALB/c strain (28) and, despite normal follicular exclusion, have a longer lifespan (28, 29). These findings suggest that there may be a partial breakdown in anergy in VH3H9 transgenic MRL mice, which is not quite sufficient to cause autoimmunity. However, the absence of an antigen-free control in the anti-dsDNA transgenic systems makes it impossible to distinguish between spontaneous and antigen-induced effects in these mice. Moreover, although there are physiological reasons to study a more polyclonal B cell repertoire, generated by the pairing of different light chains with the VH3H9 transgene and by receptor editing (30), this adds complexity to the model.

To investigate further the development of B cells in MRL mice, we introduced the IgHEL transgene and MHC H2b from B6 strain using a congenic strategy. This approach allowed us to track and compare IgHEL-specific B cells from the two strains and study their functional properties, independent of BCR specificity, affinity or MHC, and in the presence or absence of self-antigen. Our findings demonstrate spontaneous B cell hyperactivity, with increased plasma cell differentiation and antibody production, and enlarged marginal zones (MZs) in MRL IgHEL mice in the absence of cognate antigen. However, we detect no difference in B cell anergy or survival. This suggests that the autoimmunity in MRL may be due to B cell hyperactivity leading to auto-antibodies against antigens that normally fall below the threshold for anergy induction, including many intracellular proteins (14).


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
The IgHEL transgene, which encodes heavy and light chains of IgM and IgD antibody isotypes (12), and H2b MHC haplotype were both backcrossed from MD4 B6 line onto MRL strain for 10 generations before inter-crossing to generate H2b homozygous animals. ML5 B6 transgenic line expressing sHEL at 20 ng ml–1 in the serum has been previously described (12). Mice were matched for age and gender and were typically 8–12 weeks in age. All animals were maintained under specific pathogen-free conditions and the experiments were performed under UK Home Office Licence 30/1975.

Flow cytometry, antibody measurements, immunohistochemistry and chimeras
Four-colour FACS analysis was performed on a FACSCaliburTM flow cytometer with CellQuest software (Becton Dickinson). Serum anti-HEL IgMa was measured by ELISA and splenic anti-HEL IgMa-secreting plasma cells were enumerated by enzyme-linked immunospot (ELISPOT) assay (31). Radiation chimeras and immunohistochemistry were as previously described (31).

In vitro stimulation of plasma cell differentiation
Splenocytes and mesenteric lymph node (MLN) cells of MRL and B6 IgHEL mice were cultured at 1 x 106 cells ml–1 in RPMI1640 (Sigma), 10% FCS (First Link UK, Ltd), 2 mM L-glutamine (Sigma), 10 mM HEPES pH 7.4 (GIBCO), 100 µg ml–1 streptomycin, 100 U ml–1 penicillin (Sigma), 5 x 10–5 M 2-mercaptoethanol (GIBCO), with 10–0.1 µg ml–1 of LPS (Sigma), or with 10–0.1 µg ml–1 anti-mouse CD40 HM40-3 (BD PharMingen) and 5 ng ml–1 IL4 (Sigma), for 3 days at 37°C and 5% CO2. Anti-HEL IgMa-secreting plasma cells were enumerated by ELISPOT assay (31).

Single-cell PCR analysis of VHDHJH and V{kappa}J{kappa} rearrangements
Flow cytometric sorting of single cells for PCR analysis was performed using a DakoCytomation MoFlo with a Cyclone module. Single B220/syndecan-1/HSA+ plasma cells were sorted from MRL IgHEL, B6 IgHEL and B6 non-transgenic mice and were amplified in two rounds of PCR to detect VHDHJH and V{kappa}J{kappa} products, as described previously (32, 33). To control for any amplification bias toward the transgene, an experiment mixed eight transgenic plasma cell DNA samples 50:50 with eight non-transgenic plasma cell DNA samples. Half of each mixture was first-round amplified for VHDHJH products as normal, and the other half were amplified as normal but excluding the VHB (and cross-reacting VHC) primer sets, known to amplify VH2 family products (this family containing the IgHEL transgene). Second round PCR analysis was performed and products were analysed as normal. Where detected, endogenous VDJ products were amplified equally well in the presence and absence of amplified transgene products.

Adoptive cell transfers
Splenocytes from IgHEL transgenic MRL and B6 mice were labelled with 5 mM 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) using Vybrant Kit (Molecular Probes). A total of 5 x 106 or 2 x 107 of CD45.1 allotype marked spleen cells were injected into the tail vein of ML5 and non-transgenic B6 animals for follicular exclusion and B cell lifespan experiments, respectively. In follicular exclusion studies, spleens were collected 18 h later, frozen in CryoMcBED (Bright Instrument) and 7 µm acetone-fixed cryostat sections were stained with anti-B220–biotin (BD PharMingen) and streptavidin–AlexaFluor568 (Molecular Probes).

Statistical analysis
Statistical analysis were performed using GraphPad Prism version 4.00 (http://www.graphpad.com). Comparisons were by unpaired two-tailed t-test and data presented as mean with 95% confidence limits, unless otherwise stated.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Total B cell numbers are reduced in MRL mice
To study the effects of the genetic background of MRL strain on B cell function independently of strain-specific variations in B cell repertoire and MHC haplotype, anti-HEL Ig transgene (IgHEL) and MHC H2b haplotype were backcrossed from B6 MD4 line (12) onto MRL strain for 10 generations and fixed by inter-crossing. As well as controlling for direct effects of the MHC, the creation of MHC-identical strains allowed us to compare MHC expression between the strains and to use radiation chimeras and adoptive transfers to study B cell tolerance in B6 mice expressing HEL as a self-antigen.

All B cells in naive mice showed specificity for HEL, which permitted functional and phenotypic studies of large and homogenous populations of cells. B cell development in IgHEL transgenic MRL animals in the absence of HEL antigen was normal as indicated by B220, IgM and IgD expression (data not shown), however several differences between MRL and B6 IgHEL B cells were observed. In particular, there was a reduction in the immature and mature HEL-binding B cells in the bone marrow (P < 0.05 and P < 0.001, respectively), and in the mature B cells in the spleen (P < 0.01) of MRL mice (Fig. 1). There was also a reduction in the proportion of IgHEL B cells in the MLNs (P < 0.001, n ≥ 6); however, due to the increased overall lymph node cellularity (Table 1), this did not translate into a reduction in the absolute B cell number in the organ (Fig. 1). We also did not observe a reduction in B cells in the peritoneal cavity of MRL mice (Fig. 1), though difficulty in accurately collecting peritoneal cells often leads to a large variation in cell counts between animals. As the IgHEL transgene is not normally permissive for B1 cell development, no B1 cells were present in IgHEL mice of either strain. In contrast, the absolute numbers of T cells in secondary lymphoid organs of MRL mice were elevated (Table 1), which indicates that the reduction in B cells in MRL is not due to an overall reduction in the lymphocyte lineage. There was no obvious difference in other cell population in the forward-side scatter profiles of the lymphoid organs of MRL mice.


Figure 1
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  		Fig. 1 Absolute numbers of HEL-binding B cells in the bone marrow, spleen, MLNs and peritoneal cavity of MRL IgHEL and B6 IgHEL animals. Numbers of immature (IgMa+/IgDa–) and mature (IgMa+/IgDa+) B220+ lymphocytes in the bone marrow, and of HEL-binding B220+ lymphocytes in the spleen, MLNs and peritoneum of MRL and B6 IgHEL transgenics. Columns show arithmetic means and bars represent the 95% confidence limits, n ≥ 6; bone marrow counts per one tibia and femur.

 

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  		Table 1 Absolute cell counts and the numbers of CD4 and CD8 T cells in lymphoid organs of MRL IgHEL and B6 IgHEL mice

 
There was no difference in the expression of CD86 activation marker on HEL-binding B cells between the strains, but MHC class II levels on MRL IgHEL B cells were reduced (median fluorescence of splenic B220+HEL+ B cells stained for MHCII: B6 = 212 ± 19, MRL = 159 ±18, n ≥ 5, P < 0.01; data not shown). We also analysed the expression of CD44, CD69, CD62L, CD45RB and CD25 markers on CD4 T cells in MRL and B6 IgHEL mice and found no differences in the proportion of activated and antigen-experienced CD4 T cells between the strains (data not shown).

MZ B cells are increased in MRL mice
Previous reports have shown increased numbers of MZ B cells in MRL mice (28), and our findings confirm this (Fig. 2) and additionally show that the MZ expansion occurs in the absence of self-antigen. The absolute number of MZ CD21highCD23low B cells was elevated in MRL IgHEL mice (P < 0.005) (Fig. 2A), and MZ CD21highCD23low B cells represented 30 (±3)% of B220+ cells in the spleen of MRL IgHEL, as compared with 12 (±2)% in B6 IgHEL animals (Fig. 2B). Histochemistry of the spleen confirmed enlarged MRL IgHEL MZs, as shown by the increase in the size of B cell-rich B220+ (brown) areas outside of the MOMA-1+ (red) ring of metalophillic macrophages that surrounds splenic follicles (Fig. 2C) (34). Over 95% of MZ B cells of both strains were HEL binding (Fig. 2D) and, as expected, MZ B cells had a higher forward scatter and expressed increased levels of CD9 and CD86 compared with follicular B cell of the same mouse strain (Fig. 2D). This phenotype (34, 35) is associated with increased propensity of these cells to differentiate into plasma cells (35, 36).


Figure 2
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  		Fig. 2 MZ expansion in MRL mice. (A) Absolute numbers of B220+CD21highCD23low MZ B cells in the spleen of B6 and MRL IgHEL mice. Columns represent means and bars represent 95% confidence intervals, n ≥ 10. (B) CD21/CD23 flow cytometry profiles of the spleens of B6 and MRL IgHEL mice, gated on B220+ B cells. Percentage of MZ B cells as a fraction of total B cells is indicated. (C) Histochemistry of the spleen of B6 and MRL IgHEL mice. Spleen sections were stained for B220 (brown) and MOMA1 (red) to reveal B cells and metalophillic macrophages, respectively. Data representative of slides from three animals of each strain. (D) Histograms of forward scatter, and anti-HEL BCR, CD9, and CD86 expression on MZ B cells (black line) and follicular B cells (grey line) of B6 and MRL IgHEL mice. Histograms are representative of four mice of each strain.

 
Spontaneous B cell hyperactivity in MRL mice
We next looked at the level of antibody production in MRL IgHEL and B6 IgHEL mice. MRL IgHEL animals had increased numbers of anti-HEL plasma cells in the spleen (P < 0.05) (Fig. 3A) and a 5-fold increase in the serum anti-HEL IgMa levels (P < 0.005) (Fig. 3B), compared with B6 animals with the same IgHEL B cell repertoire. This shows that there is a large increase in plasma cell differentiation in IgHEL MRL, especially when the overall reduction in B cell number in MRL is taken into account.


Figure 3
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  		Fig. 3 Spontaneous B cell hyperactivity in MRL mice. (A) Anti-HEL IgMa-secreting splenic plasma cell numbers and (B) serum anti-HEL IgMa titres in B6 and MRL IgHEL mice, enumerated by ELISPOTs and ELISA, respectively. Columns represent means and bars represent 95% confidence intervals, n ≥ 10 and n ≥ 8, respectively.

 
To test for intrinsic B cell hyperactivity to co-stimulation, we next stimulated MRL and B6 IgHEL spleen and MLN cells with LPS or anti-CD40 and IL4 in vitro. This showed no increase in anti-HEL IgMa-secreting plasma cell differentiation in MRL in either tissue (Fig. 4), suggesting that increased plasma cell differentiation in MRL in vivo is not due to hypersensitivity to Toll-like receptor 4 (TLR4) stimulation or anti-CD40/IL4 T-dependent stimulation. However, this does not exclude the possibility that hypersensitivity to other co-stimuli or increased delivery of any of the co-stimulatory signals may contribute to increase plasma cell differentiation in MRL in vivo.


Figure 4
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  		Fig. 4 In vitro plasma cell differentiation in MRL and B6 IgHEL mice. Splenocytes and MLN cells were stimulated with different concentrations of LPS or anti-CD40 and IL4 in vitro for 3 days, and anti-HEL IgMa plasma cells in cultures were enumerated by ELISPOTs. Bars represent means and 95% confidence intervals, n = 3 for all groups.

 
Increased plasma cell differentiation in MRL is not associated with increased co-expression of endogenous BCRs
The recent description of mice expressing dual BCRs raised the possibility that the increase in IgHEL MRL plasma cells might not be spontaneous but instead due to the direct stimulation of co-expressed endogenous BCRs by self or foreign antigens (37, 38). To explore this, we performed single-cell PCR of rearranged Ig genes on flow-sorted syndecan-1+, HSA+ plasma cells from IgHEL MRL and IgHEL B6 transgenic mice. In a non-transgenic B6 control sorted using the same gate, the frequency of detection of plasma cells was comparable to previous studies (32, 33). Ten out of 16 (62.5%) sorted non-transgenic cells in the plasma cell gate generated a rearranged endogenous VDJ heavy-chain allele by PCR and 10/16 had an endogenous rearranged VJ kappa allele. In contrast, only 1/6 (17%) MRL and 1/6 (17%) B6 IgHEL plasma cells PCR positive for the IgHEL heavy-chain transgene also had a rearranged endogenous heavy chain by this assay and only 0/5 (0%) MRL and 1/7 (14%) of B6 IgHEL plasma cells amplified an endogenous VJ kappa light chain in addition to the transgenic kappa chain (Table 2). By mixing cells, we were able to show that PCR of the transgene did not suppress amplification of endogenous rearrangements in the non-transgenic cells. Therefore, the data suggest that the IgHEL transgene suppresses endogenous VDJ and VJ kappa rearrangements in the majority of plasma cells in both MRL and B6. Although this limited analysis cannot exclude the possibility that rare dual expressing cells differentiate preferentially in MRL, this seems an unlikely explanation given the large increase in plasma cell numbers in IgHEL MRL mice.


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  		Table 2 Single-cell PCR analysis of endogenous BCR V(D)J rearrangements in single syndecan-1+ HSA+ plasma cells, sorted from the spleen of B6 and MRL IgHEL mice

 
MRL B cells show no defect in anergy
To analyse tolerance induction in B cells from MRL mice, chimeras were generated by injecting the MHC-identical bone marrow from either MRL IgHEL or B6 IgHEL mice into irradiated B6 ML5 recipients expressing cognate antigen sHEL at 20 ng ml–1 in the serum (12) and into non-transgenic B6 control recipients. The B/T cell ratio in the spleen of non-transgenic recipients reconstituted with MRL IgHEL bone marrow was reduced compared with non-transgenic recipients reconstituted with B6 IgHEL bone marrow (MRL = 1.7 ± 0.2, B6 = 3.6 ± 0.6, P < 0.001) and a similar difference was seen in the MLNs (P < 0.001). However, the differences in the absolute numbers of B cells in the chimeras were not significant, perhaps due to large variation in reconstitution of the chimeras (Fig. 5A). This suggests that altered B/T ratio may be at least partly intrinsic to the MRL haematopoietic lineage. Furthermore, in the absence of sHEL, MRL MZ expansion persisted in the chimeras: in the non-transgenic recipients reconstituted with MRL IgHEL bone marrow, MZ B cells represented 21 (±6.6)% of splenic B cells, compared with 14 (±4.5)% in mice reconstituted with B6 IgHEL bone marrow (P < 0.05). Non-transgenic recipients reconstituted with MRL IgHEL bone marrow also had elevated anti-HEL splenic plasma cell numbers compared with mice reconstituted with B6 IgHEL bone marrow (Fig. 5B; P = 0.05 using a one-tailed t-test based on a prior hypothesis), but there was no difference in the level of anti-HEL IgMa antibody by ELISA. MHC class II expression was also reduced on naive MRL B cells compared with naive B6 B cells in the chimeras, with the median fluorescence of naive MRL IgHEL B cells being 117 ± 19, compared with 187 ± 32 for B6 IgHEL (P < 0.01). This suggests that several features of MRL strain are at least partly intrinsic to the MRL haematopoietic cell lineage.


Figure 5
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  		Fig. 5 Normal B cell anergy against soluble antigen in MRL mice. (A) Absolute numbers of HEL-binding B cells in the bone marrow and spleen of non-transgenic and sHEL transgenic irradiated recipients reconstituted with either MRL or B6 IgHEL bone marrow. Columns represent means and bars represent 95% confidence intervals, n ≥ 8. (B) Splenic anti-HEL IgMa-secreting plasma cell numbers and serum anti-HEL IgMa antibody levels in non-transgenic and sHEL transgenic irradiated recipients reconstituted with either MRL or B6 IgHEL bone marrow. Columns represent means and bars represent 95% confidence intervals, n ≥ 4. (C) Flow cytometry profiles of B220+ splenic B cells from non-transgenic and sHEL transgenic recipients reconstituted with either MRL or B6 IgHEL bone marrow, stained with antibodies to IgMa and IgDa. (D) Expression of B220, anti-HEL BCR, CD21 and MHC class II on splenic B cells in sHEL transgenic mice reconstituted with either B6 IgHEL (black line) or MRL IgHEL (dark grey line) bone marrow, and in non-transgenic controls reconstituted with B6 IgHEL bone marrow (light grey area). B220 histogram is gated on CD45.1+ IgDa+ cells; other histograms are gated on CD45.1+ B220+ cells. Data are representative of four mice in each group.

 
In the presence of sHEL antigen, MRL B cell anergy was established and maintained successfully. In sHEL-expressing B6 recipients, antigen-experienced MRL IgHEL B cells had anergic phenotype and exhibited the same degree of IgM and CD21 down-modulation as anergic B6 IgHEL B cells (Fig. 5C and D). Antigen exposure induced equivalent MZ depletion in both strains and also up-regulated MHC class II on MRL IgHEL B cells to the same level as on anergic B6 IgHEL B cells (Fig. 5D). Importantly, similar low numbers of anti-HEL plasma cells were present in the spleen of sHEL-expressing animals reconstituted with either MRL IgHEL or B6 IgHEL bone marrow and there was no detectable anti-HEL IgMa in the serum of either group (Fig. 5B). Thus, B cell anergy to soluble HEL is normal in MRL mice.

MRL B cells show no intrinsic defect in localisation to the T zone or competitive elimination
Previous findings showed normal follicular exclusion but increased survival of autoreactive B cells in MRL (29). Follicular exclusion of B cells in the presence of cognate antigen is a tolerance checkpoint that acts in the presence of competitor B cells in a polyclonal B cell repertoire (39, 40) and hence cannot be studied in IgHEL transgenic animals directly. Thus, in order to analyse MRL B cell follicular exclusion in our system, we performed adoptive transfers of CFSE-labelled CD45.1 allotype marked MRL IgHEL and B6 IgHEL splenocytes into MHC-identical ML5 mice expressing sHEL and into non-transgenic control recipients, as previously described (39). MRL cells were normally excluded from follicles at 18 h after transfer (Fig. 6A), consistent with previous reports (28). Competitive elimination of autoreactive MRL B cells was comparable to that of B6 B cells, as equivalent numbers of B cells remained in the spleen of ML5 recipient on day 5 after transfer (Fig. 6B). This suggests that the previously reported increased survival of autoreactive B cells in MRL mice (29) is not due to a B cell intrinsic defect.


Figure 6
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  		Fig. 6 Follicular exclusion and competitive survival of autoreactive MRL B cells. (A) Fluorescent microscopy of spleen sections from non-transgenic and sHEL transgenic mice transferred with CFSE-labelled B6 and MRL IgHEL splenocytes. Spleen sections collected at 18 h after transfer were stained for B220 (red) to reveal B cell follicles. (B) The ratio of the number of donor-derived IgHEL B cells (B220+ CD45.1+ CFSE+) and T cells (CD4+ CD45.1+ CFSE+), surviving at 5 days after transfer of B6 or MRL IgHEL splenocytes into B6 CD45.2+ non-transgenic or sHEL transgenic recipients.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Our data show that spontaneous B cell hyperactivity is a characteristic of MRL strain that predisposes to increased plasma cell differentiation and elevated serum anti-HEL antibody levels in IgHEL transgenic MRL mice in the absence of self-antigen. We show that B cell hyperactivity is independent of strain-specific variations in BCR specificity and MHC and is at least partially transferable with the bone marrow of MRL mice. Although we cannot exclude very low-affinity interactions between IgHEL B cells and endogenous antigens, there is no evidence that such interactions have any effect on plasma cell differentiation. Indeed, B6 IgHEL B cells remain functionally naive and do not differentiate into increased plasma cells even when they are exposed to sHEL at 2–10 ng ml–1 (14). We cannot exclude a strain-specific effect of rare co-expression of endogenous receptors on plasma cell differentiation, but given the rarity of dual expressors, this is unlikely to explain the very substantial increase of plasma cells in MRL IgHEL transgenic mice. These facts suggest that increased plasma cell differentiation in MRL may be largely B cell antigen independent and that this may contribute to increased autoimmune susceptibility in this strain.

The extent to which autoimmune disease is due to a failure of self versus non-self recognition or due to abnormal antigen-independent events, such as the polyclonal activation of lymphocytes from innate hyperactivity or exaggerated co-stimulation (41, 42), has been the subject of a long debate. Over the years, there has been excellent support for a failure of self versus non-self recognition, which has become the dominant paradigm; for example, in AIRE deficiency due to the failed central deletion of autoreactive lymphocytes in the absence of thymic self-antigen expression (43), or in disease due to increased peripheral survival of autoreactive lymphocytes in the absence of cell death factors, Fas or Bim (25, 44, 45). However, support for antigen-independent events has been lacking, largely because of the difficulty in distinguishing antigen-specific from antigen-independent effects in vivo. Our description of B cell hyperactivity in MRL is evidence that antigen-independent mechanisms may be important in this model of spontaneous SLE.

B cell hyperactivity, antigen independent or not, seems to be an important contributor to autoimmune disease in other polygenic murine models of SLE and possibly in human patients (46). For example, in the NZB x NZW/F1 mouse strain, polyclonal B cell activation precedes the development of an antigen-driven autoimmune disease (47) and B cell hyperactivity is linked to the Sle2 and Imh/Nba1/Lbw2 loci on chromosome 4 (48–51). In theory, B cell hyperactivity could arise from cell-intrinsic differences in BCR or co-stimulatory signalling or from differences in the availability and form of antigens and co-stimulatory signals. B cell hyperactivity arising through both mechanisms may cause or contribute to systemic autoimmunity (46). For example, spontaneous and targeted mutations that increase the strength of BCR signalling (CD22, SHP1, Lyn, CD19) lead to the production of auto-antibodies against intracellular antigens (52–54) and can also result in spontaneous B cell class switching to more pathogenic antibody isotypes (K. Silver, in preparation). Likewise, co-stimulation via TLR4 from LPS administration accelerates the disease in MRL and other autoimmune mouse strains (55, 56). Interestingly, our data shows that MRL are not hypersensitive to either LPS or anti-CD40/IL4 stimulation in vitro, but hypersensitivity to other stimuli or increased delivery of these and other stimulatory signals may contribute to plasma cell differentiation in MRL in vivo. Increased availability of self-antigens and co-stimulatory signals in MRL could arise from increased release of intracellular polyclonal activators, such as CpG (57) and heat shock proteins (58), from dying cells and from a decreased rate of their clearance. Indeed, MRL mice are defective in apoptotic cell clearance (59) and MRL C1q knockouts, which have a more severe defect in apoptotic cell clearance, develop more acute autoimmune symptoms (60). Furthermore, recently reported T cell hyperactivity (61) and defective T cell anergy (62) in the MRL strain might lead to increased T-dependent co-stimulation of MRL B cells, though in our model the effects on IgHEL B cells would be antigen independent.

It is likely that spontaneous hyperactivity will have the greatest chance of triggering autoimmunity against antigens that fall below the threshold required for antigen-specific B cell tolerance. This category of self-antigens includes many sequestered proteins, including intracellular antigenic targets in SLE and other systemic forms of autoimmunity (14, 63, 64). This principle is reinforced in the HEL transgenic model by finding that ubiquitous but highly sequestered intracellular HEL is either neutral or immunogenic to autoreactive B cells (31). Defects in apoptotic cell clearance that expose sequestered auto-antigens to ignorant B cells may have a particular role in making them immunogenic, as shown by the activation of anti-Sm B cells in Mer knockout mice (64). Thus, MRL B cell hyperactivity may act in synergy with the reported defect in apoptotic cell clearance in MRL mice (59) to target auto-antibody responses against intracellular antigens, typical of SLE.

Our description of increased MZs in MRL confirms a similar finding in MRL VH3H9 anti-dsDNA Ig transgenics (28), but extends this earlier observation by showing that this expansion is antigen independent and probably intrinsic to MRL bone marrow lineage. The role of BCR signalling in MZ development is controversial (65). In some mouse models, MZ expansion is associated with reduced strength of BCR stimulation or impaired BCR signalling, whereas increased BCR signalling favours follicular or B1 cell fates (66). In other models, interaction with self-antigen drives B cells toward MZ development (67). The absence of increased IgM modulation on naive IgHEL MRL B cells suggests that there is no substantial increase in proximal BCR activity and therefore the reason for the increased MZ is unexplained. In future it would be of interest to compare BCR-independent pathways essential for MZ development and retention, such as the Notch2 signalling pathway (68), and the integrin and chemokine receptor signalling pathways involving proteins pyk2, Dock2, and Lsc (66) between MRL and B6 strains.

The increased number of MZ B cell in MRL mice may play a role in the hyperactivity phenotype. MZ B cells are known to differentiate into plasma cells faster than follicular B cells (36) and to express higher levels of activation marker CD9 (35), which is also expressed on plasma cells (35). It is also known that autoreactive B cell clones are enriched in the MZ compartment (37, 69) and that MZ is the source of auto-antibody secreting plasma cells in a number of other mouse models of SLE, including anti-Sm heavy-chain Ig transgenic Mer knockout mice (64) and anti-dsDNA heavy-chain Ig transgenic mice treated with oestrogen (70). The ability of MZs to recognise particulate antigens (71) may make them responsive to SLE auto-antigens, as they are presented to the immune system on the surface of apoptotic blebs (72). In this way, the defective clearance of apoptotic cell debris (59) and enlarged MZs in MRL (28) may interact in predisposing this strain to autoimmunity.

Although B cell hyperactivity would predispose MRL mice to greater autoimmunity against rare antigens, there is no evidence from our chimeric experiments of a defect in B cell anergy against more abundant antigens. In the presence of sHEL on a B6 background, both MRL and B6 IgHEL B cells had similar IgM down-modulation and similar levels of other cell-surface developmental markers, and did not differentiate into plasma cells. This contrasts with the lesser degree of developmental arrest of anti-dsDNA B cells in MRL compared with BALB/c mice in the VH3H9 transgenic model (28), even though tolerance is preserved in both systems. The difference in B cell phenotype between the two transgenic systems may be due to the different nature of the antigens and BCRs, or due to trans effects of non-haematopoietic MRL environment that were undetectable in sHEL B6 recipients in this study.

Non-cell intrinsic effects of MRL environment may also be critical in determining strain-specific differences in the survival of autoreactive B cells. In non-autoimmune animals, such as BALB/c and B6, autoreactive anti-DNA and IgHEL B cells are excluded from follicles and have reduced lifespan in the presence of antigen and non-autoreactive competitor B cells (39, 40). This tolerance checkpoint operates due to increased requirement of anergic B cells for the pro-survival cytokine BAFF (13), and its importance is illustrated by the finding that increased expression of BAFF leads to autoimmunity in these mouse strains. For these reasons, it is striking that in MRL mice, anti-dsDNA anergic B cells survive for longer periods (29). Our experiments suggest that this enhanced survival may be due to trans effects of MRL environment, because we observed no intrinsic difference in the survival of B6 and MRL IgHEL B cells transferred into sHEL B6 recipients. This excludes possible inter-strain differences in the up-regulation of intracellular pro-apoptotic factor Bim as a cause of increased survival. However, lower B cell numbers in MRL mice, reported in this paper, may result in a reduced competition for the limiting amount of BAFF, and lead to increased survival of B cells with autoreactive specificities. Under these circumstances, the severity of disease in MRL-lpr/lpr mice compared with other lpr/lpr mutants could be due to increased survival of anergic B cells, as well as the deficiency in Fas-dependent T cell mediated killing. These factors will combine with B cell hyperactivity, and the reported defects in apoptotic cell clearance (59), negative regulation of T cell proliferation (61) and T cell anergy (62) in MRL mice to trigger systemic autoimmunity. Experiments to define the genetic and molecular mechanisms underlying B cell hyperactivity in MRL mice and humans may help to identify key pathways underlying disease and highlight new therapeutic targets.


   Acknowledgements
 
A.N. is supported by a Wellcome Trust Prize Studentship and R.J.C. is a Wellcome Trust Senior Clinical Fellow. We thank members of the Cornall laboratory for review of the manuscript, Nigel Rust for cell sorting and the staff of Biomedical Services Unit, Oxford for excellent animal husbandry. This work was supported by the Wellcome Trust.


   Abbreviations
 
B6, C57BL/6
BAFF, B cell activating factor
BCR, B cell receptor
CFSE, 5,6-carboxylfluorescein diacetate succinimidyl ester
dsDNA, double-stranded DNA
ELISPOT, enzyme-linked immunospot
HEL, hen egg lysozyme
IgHEL, MD4 anti-hen egg lysozyme Ig
MLN, mesenteric lymph node
MZ, marginal zone
sHEL, soluble hen egg lysozyme
SLE, systemic lupus erythematosus
TLR4, Toll-like receptor 4

   Notes
 
Transmitting editor: D. Tarlinton

Received 12 August 2005, accepted 26 April 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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