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Evidence that Yaa-induced loss of marginal zone B cells is a result of dendritic cell-mediated enhanced activation
Journal of Autoimmunity 34 (2010) 349e355
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Journal of Autoimmunity journal homepage: www.elsevier.com/locate/jautimm
Evidence that Yaa-induced loss of marginal zone B cells is a result of dendritic cell-mediated enhanced activation Marie-Laure Santiago-Raber a, Hirofumi Amano b, Eri Amano b, Liliane Fossati-Jimack c, Lee Kim Swee d, Antonio Rolink d, Shozo Izui a, * a
Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Geneva 1211, Switzerland Department of Internal Medicine, Juntendo University School of Medicine, Tokyo, Japan Rheumatology Section, Hammersmith Campus, Imperial College London, London, UK d Department of Immunology, University of Basel, Basel, Switzerland b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 December 2009 Received in revised form 5 January 2010 Accepted 7 January 2010
The development of systemic lupus is accelerated by the Yaa (Y-linked autoimmune acceleration) mutation, which is the consequence of a translocation of the telomeric end containing the Tlr7 gene from the X chromosome onto the Y chromosome. However, the loss of marginal zone (MZ) B cells, one of the Yaa-linked cellular abnormalities, has previously been shown to be unrelated to the Tlr7 gene duplication, and the present study therefore aimed to investigate the mechanism responsible for MZ B-cell loss. Analyses of Yaa and non-Yaa C57BL/6 male mice expressing an MD4 anti-HEL IgM transgene or those deficient in fms-like tyrosine kinase 3 ligand (FL) revealed that the proportion of MZ B cells in these Yaa mice was comparable to that of the respective non-Yaa control mice. Notably, the activation of MZ B cells was compromised in both of these transgenic model systems, due to the absence of cognate antigens or the impaired development of dendritic cells, respectively. These results contrasted with the loss of MZ B cells in non-Yaa mice treated with FL and the lack of accumulation of MZ B cells in Yaa mice treated with a B-cell survival factor, BAFF. Taken together, our results suggest that the persistent and enhanced activation of Yaa-bearing hyperactive MZ B cells by dendritic cells is responsible for the loss of this B-cell subset in Yaa mice. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Systemic lupus erythematosus Marginal zone B cell Dendritic cell The Yaa mutation
1. Introduction The BXSB strain of mice spontaneously develops an autoimmune syndrome with features of systemic lupus erythematosus (SLE) that affects males much earlier than females [1]. The accelerated development of SLE in BXSB male mice results from the genetic abnormality, Yaa (Y-linked autoimmune acceleration), present on the BXSB Y chromosome [2]. Studies of Yaa and non-Yaa double bone marrow chimeric mice showed that anti-DNA autoantibodies are selectively produced by B cells bearing the Yaa mutation, and that T cells from both Yaa and non-Yaa origin efficiently promote anti-DNA autoantibody responses [3,4]. Based on these results, it has been speculated that the Yaa defect may decrease the threshold for BCR-mediated signaling, thereby triggering and excessively stimulating autoreactive B cells. More recently, the Yaa mutation was identified to be a consequence of
* Corresponding author. Tel.: þ41 22 379 5741; fax: þ41 22 379 5746. E-mail address: [email protected] (S. Izui). 0896-8411/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaut.2010.01.001
a translocation of the telomeric end containing the gene encoding TLR7 from the X chromosome onto the Y chromosome [5,6]. Since the synergistic engagement of TLR7 and BCR in response to nuclear antigens could induce the activation of autoreactive B cells in SLE [7,8], the Tlr7 gene duplication has been proposed to be the etiologic basis for the Yaa-mediated enhancement of disease [5,6,9,10]. Newly generated B cells in the bone marrow emigrate to the spleen, where they further differentiate into follicular or marginal zone (MZ) B cells. Follicular B cells are IgMintIgDhiCD21intCD23hi, and MZ B cells located at the junction of white and red pulps are IgMhiIgDloCD21hiCD23neg/lo [11e13]. While follicular B cells respond to T-dependent antigens, MZ B cells play a critical role in host defense against T-independent blood-borne pathogens [14,15]. Significantly, mice bearing the Yaa mutation display a marked reduction of the MZ B-cell subset, which results from a defect intrinsic to Yaa-bearing B cells, independently of the development of SLE [16]. Although the Tlr7 gene duplication was proposed as a cause of this cellular defect [9], we observed that the extent of the MZ B-cell loss in C57BL/6 mice bearing the Yaa mutation (B6.Yaa) was essentially unchanged by the introduction of the Tlr7 null mutation on the X chromosome which
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left only a single “Yaa-copy” of the Tlr7 gene expressed [17]. These results indicated that the loss of MZ B cells occurring in Yaa-bearing mice cannot be explained by the Tlr7 gene duplication alone and suggested a critical contribution of other duplicated genes in the translocated X chromosomal end. Since the loss of MZ B cells in Yaa mice can be related to the hyperactive phenotype of Yaa-bearing B cells, which contributed to the accelerated development of murine SLE, it remains important to define the mechanism responsible for this Yaa-linked MZ B-cell loss. Studies on several different genetically manipulated mice revealed that the reduction of MZ B cells could be related to hypersensitive BCR signaling [18e21]. It has been hypothesized that the strength of the signal elicited via the BCR may regulate the lineage commitment of mature B cells into follicular vs. MZ B cells, with hypersensitive BCR signaling favoring an accelerated maturation towards follicular B cells [22]. Thus, a hyperactive phenotype of Yaa-bearing B cells could contribute to the enhanced maturation towards follicular B cells and the decrease in MZ B cells [16]. An alternative possibility could be that the reduction of Yaa-bearing MZ B cells is due to their constant and enhanced activation followed by subsequent emigration from the MZ into the red pulp or B-cell follicles [23e26]. In the present study, we provide evidence that enhanced activation by dendritic cells (DC) is likely to be responsible for the loss of MZ B cells in B6.Yaa mice.
(Serotec, Kidlington, England) mAb in the presence of 2.4G2 antiFcgRII/III mAb. 2.4. Treatment with recombinant FL or BAFF in vivo and in vitro
2. Materials and methods
Mice were subcutaneously injected daily for 9 days with 1 mg of mouse serum albumin (Sigma-Aldrich) plus 10 mg of human recombinant FL (a kind gift of Amgen Inc., Thousand Oaks, CA) or with mouse serum albumin alone. One day after the last injection of FL, the proportion of MZ B cells in spleens was analyzed by flow cytometric and immunohistochemical analysis. The in vivo effect of BAFF was determined in mice injected i.v. on days 0 and 14 with 100 mg of human recombinant BAFF, prepared as described previously [31]. Seven days after the second injection of BAFF, the size of MZ B-cell compartments was assessed. To determine the survival of splenic B cells in vitro, B cells were purified from spleen cells using mouse B-cell enrichment kit (StemCell Technologies, Grenoble, France). The purity of B cells, as documented by flow cytometric analysis, was superior to 95%. Purified B cells were cultured in IMDM containing 5 105 2-ME, 0.03% primatone (Quest International, Naarden, The Netherlands) and 5% FCS at 5 105 cells/ml in the presence or absence of 500 ng/ml of recombinant BAFF. At various time points viable cells were counted by the trypan blue exclusion test. Serum levels of BAFF were quantified by using BAFF, Soluble (mouse) ELISA kit (Apotech Corporation, Epalinges, Switzerland).
2.1. Mice
2.5. Quantitative RT-PCR
B6 mice expressing an MD4 anti-hen egg lysozyme (HEL) IgM transgene [27] were created by backcross procedures at the 8th generation. Mice deficient in C3 [28], kindly provided by Dr M. Carroll, Harvard Medical School, Boston, were backcrossed for 7 generations with B6 mice. B6 mice deficient in fms-like tyrosine kinase 3 ligand (FL), generated by gene targeting in B6-derived ES cells [29], were purchased from Taconic Farms, Inc., Germantown, NY. The Yaa mutation was introduced into these different transgenic mice by crossing with B6.Yaa mice [30]. Animal studies described in the present study have been approved by the Ethics Committee for Animal Experimentation of the Faculty of Medicine, University of Geneva.
RNA from spleen cells was purified with TRIzol reagent (Invitrogen AG, Basel, Switzerland). The abundance of Flt3l mRNA was quantified by real-time RT-PCR with cDNA prepared from RNA. Flt3l cDNA was amplified using a forward primer (50 -TTCAGCCA CAGTCCCATCTC-30 ) and reverse primer (50 -CCTGCCACAGTCTTC AGTTG-30 ). PCR was performed using the iCycler iQ Real-Time PCR Detection System (Bio-Rad, Philadelphia, PA) and iQ SYBR green Supermix (Bio-Rad). Results were quantified relative to a standard curve generated with serial dilutions of a reference cDNA preparation from spleen cells and normalized using TATA-binding protein mRNA. 2.6. Statistical analysis
2.2. Flow cytometric analysis Flow cytometry was performed using three- or four-color staining of spleen cells, and analyzed with a FACSCalibur (Becton Dickinson, Mountain View, CA). The following antibodies were used: anti-CD21 (7G6), anti-CD23 (B3B4), anti-B220 (RA3-6B2), anti-CD11c (N418), anti-MHC class II I-A (Y3P) and anti-BAFFreceptor (BAFF-R; 9B9) [31]. For the analysis of splenic DC, spleen cells were prepared according to the method described by Kamath et al. [32]. Briefly, spleen fragments were digested with a mixture of 1 mg/ml Liberase (Roche Diagnostics, Mannheim, Germany) and 1 mg/ml DNAse I (SigmaeAldrich, St Louis, MO) for 20 min at room temperature and treated with 10 mM EDTA to disrupt T cell-DC complexes. Cells were then filtered with a cell strainer (70 mm), resuspended and stained in PBS containing 1% BSA, 2 mM EDTA and a saturating concentration of 2.4G2 anti-FcgRII/III mAb. 2.3. Immunohistochemistry Spleens were embedded in Tissue-Tek O.C.T. compound (Miles, Elkhart, IN) and snap-frozen in liquid nitrogen. Four mm frozen sections were stained with PE-labeled anti-IgM (1B4B1; Southern Biotechnology, Birmingham, AL) and FITC-labeled MOMA-1
Statistical analyses were performed using the ManneWhitney U-test. Probability values >5% were considered insignificant. 3. Results 3.1. No reduction of MZ B cells in MD4 anti-HEL IgM transgenic B6.Yaa male mice We have previously shown that the proportion of MZ B cells in lupus-prone BXSB Yaa males expressing an Sp6 anti-TNP/DNA transgenic IgM was substantially increased, as compared with nontransgenic littermates, reaching a level nearly identical to that of non-Yaa BXSB transgenic males [16]. The increase in the size of the MZ B-cell compartment in the Sp6 transgenic BXSB mice could be related to the findings that autoreactive B cells tend to be accumulated in the MZ [33,34]. However, larger MZ B-cell compartments have also been reported in non-autoreactive IgM transgenic mice such as MD4 anti-HEL transgenic mice [27]. Therefore, we determined whether B cells expressing an MD4 anti-HEL IgM transgene similarly accumulated in the MZ of B6.Yaa male mice. As was the case of Sp6 transgenic mice, the proportion of MZ B cells in MD4 transgenic B6.Yaa males (means SD: 15.6 2.4%) was as
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we determined whether the MZ B-cell compartment of B6.Yaa mice can be restored by the absence of C3, as a result of down-modulated activation of MZ B cells in response to T-independent blood-borne antigens. Percentages of MZ B cells were significantly increased in both C3-deficient Yaa and non-Yaa B6 mice as compared to C3-sufficient counterparts (P < 0.005; Fig. 2). Notably, the proportion of MZ B cells in C3/ B6.Yaa mice (6.0 2.4%) was comparable to that in C3-sufficient non-Yaa B6 mice (6.2 0.6%), but still lower than that of C3/ non-Yaa mice (9.7 2.6%). These data suggested that the loss of MZ B cells in B6.Yaa mice may be in part a consequence of an enhanced complement-dependent activation of MZ B cells. 3.3. Rescue of MZ B-cell loss in B6.Yaa male mice deficient in FL, and loss of MZ B cells in B6 male mice treated with recombinant FL Studies have shown that blood-borne antigens are transported into the MZ by DC and that interaction of antigen-bearing DC with
Fig. 1. No reduction of MZ B cells in MD4 anti-HEL IgM transgenic Yaa male mice. CD21hiCD23neg/lo MZ B cells in spleen cells of 2 mo-old MD4 transgenic B6 Yaa and non-Yaa male mice and of their non-transgenic littermates were stained with a combination of anti-B220, anti-CD21 and anti-CD23 mAb, and gated for B220þ cells. Representative results from 5 to 6 mice in each group of mice are shown, and mean percentages of MZ B cells among B220þ cells are indicated (upper panel). Percentages of MZ B cells among B220þ cells of individual B6 mice of different genotypes are shown, and mean values are indicated by horizontal lines (lower panel).
large as that of MD4 transgenic non-Yaa males (17.5 4.4%; Fig. 1). These results contrasted with marked differences in MZ B cells between non-transgenic Yaa and non-Yaa male littermates (2.5 0.5% vs. 5.8 0.9%, P < 0.01). Since anti-HEL transgenic B cells were not expected to be spontaneously activated because of the absence of cognate antigens, these data suggested that lack of reduction of Sp6 and MD4 transgenic B cells in the MZ of Yaabearing mice could be due to a limited antigen-specific activation of these B cells, thereby prevented their depletion from the MZ. 3.2. Partial recovery of MZ B-cell compartment in C3-deficient B6.Yaa male mice MZ B cells are known to play the major role in immune responses against T-independent blood-borne pathogens [14]. Therefore, we hypothesized that the Yaa-linked loss of MZ B cells could be a result of their excessive activation in response to T-independent antigens. Since complement plays a substantial role in the development of T-independent antibody responses [35,36],
Fig. 2. Partial recovery of MZ B-cell compartment in C3-deficient B6.Yaa male mice. Representative results for CD21hiCD23neg/lo MZ B cells in spleen cells of 2 mo-old C3/ Yaa and non-Yaa B6 male mice and of their control littermates (5e8 mice in each group) with mean percentages of MZ B cells among B220þ cells are shown (upper panel). Percentages of MZ B cells among B220þ cells of individual B6 mice of different genotypes are shown (lower panel).
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MZ B cells can initiate T-independent immune responses [14,23]. Therefore, we investigated the possible role of DC in the activationinduced loss of MZ B cells in B6.Yaa mice. We introduced the Flt3l null mutation into B6.Yaa mice, thereby impairing the development of DC, and then assessed the size of the MZ B-cell compartment. As expected, percentages of CD11cþ MHC class II I-Aþ DC in spleens were markedly diminished in 2 mo-old FL-deficient B6.Yaa mice (means of 5 mice SD: 1.4 1.0%), as compared with FL-sufficient control mice (4.3 0.7%, P < 0.01). This decrease of DC in spleens
was accompanied by a marked increase in the proportion of MZ B cells in FL/ B6.Yaa mice (FL/ Yaa: 12.7 2.1%, FLþ/þ Yaa: 2.6 2.1%, P < 0.005; Fig. 3A). More significantly, percentages of MZ B cells in FL/ B6.Yaa male mice were almost comparable to those in FL/ B6 non-Yaa male mice (14.1 1.5%). In agreement with the flow cytometric analysis, immunohistological examination of spleens showed that the loss of a characteristic rim of IgMþ MZ B cells at the periphery of the follicles and separated by MOMA-1þ macrophages in B6.Yaa mice was rescued in the spleens of FL/
Fig. 3. Rescue of MZ B-cell loss in B6.Yaa male mice deficient in FL, and loss of MZ B cells in B6 mice treated with FL. A. Representative results for CD21hiCD23neg/lo MZ B cells in spleen cells of FL-deficient (FL/) Yaa and non-Yaa B6 male mice and of their control (FLþ/þ) littermates (5e8 mice in each group) with mean percentages of MZ B cells among B220þ cells are shown (upper panel). Percentages of MZ B cells in B220þ cells of individual B6 mice of different genotypes are shown (lower panel). B. Spleen sections from FL-deficient (FL/) Yaa and non-Yaa B6 male mice and of their control littermates were stained with PE-labeled anti-IgM (red) and FITC-labeled anti-MOMA-1 (green). Representative results obtained from 4 mice in each group are shown. C.10 mg of human recombinant FL were daily injected into 2 mo-old B6 male mice for 9 days, and percentages of CD11cþB220 myeloid and CD11cþB220þ plasmacytoid DC in spleen cells were analyzed on day 10. Representative results from FL-treated and control mice (5 mice in each group) with mean percentages of DC in spleen cells are shown. D. Representative results for CD21hiCD23neg/lo MZ B cells in spleen cells of FL-treated and control B6 male mice (5 mice in each group) with mean percentages of MZ B cells among B220þ cells are shown (upper panel). Percentages of MZ B cells in B220þ cells of individual B6 mice are shown (lower panel).
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B6.Yaa males (Fig. 3B). Notably, the size of this B cell population was enlarged in FL/ B6.Yaa males and comparable to that in FL/ B6 non-Yaa males. These results indicated that the impaired development of DC prevented the loss of MZ B cells in B6.Yaa mice. To further define the role of DC in the reduction of MZ B cells in B6.Yaa mice, we determined whether increased production of DC following injections of FL could induce a depletion of MZ B cells in B6 male mice. As reported previously [37], mice daily treated with human recombinant FL for 9 days displayed higher percentages of DC in spleens (means of 5 mice SD: CD11cþB220 myeloid DC, 17.5 1.6% and CD11cþB220þ plasmacytoid DC, 8.0 1.9%) than control mice (myeloid DC, 2.4 0.2% and plasmacytoid DC, 2.2 0.1%, P < 0.01; Fig. 3C). In FL-treated B6 mice, the proportions of MZ B cells were markedly diminished (1.9 0.2%) as compared with those in control mice (6.3 0.8%, P < 0.01; Fig. 3D). The loss of MZ B cells in FL-treated B6 mice was confirmed by immunohistological analysis (data not shown). These data collectively suggested that DC-mediated enhanced activation of MZ B cells could be responsible for their depletion from the MZ compartment in B6.Yaa mice. Results obtained with FL-deficient or FL-injected B6 mice prompted us to investigate whether B6.Yaa mice displayed increased production of DC or FL as compared with wild-type B6 mice. However, no significant differences in percentages of myeloid and plasmacytoid DC in spleens were observed between 2 mo-old Yaa and non-Yaa B6 male mice (data not shown). In addition, quantitative RT-PCR failed to show any measurable differences in Flt3l mRNA abundance in spleen cells from 2 mo-old Yaa and nonYaa B6 male mice (means of 3 mice SD: Yaa, 0.96 0.13; non-Yaa, 0.83 0.14). 3.4. Lack of accumulation of MZ B cells in BAFF-injected B6.Yaa male mice BAFF is a potent survival factor for MZ B cells [38,39]. If the loss of MZ B cells was indeed a consequence of their persistently increased activation and subsequent depletion, we should expect that an increase in the number of MZ B cells in B6.Yaa male mice following treatment with BAFF is limited as compared with that in B6 male mice. To address this question, 2 mo-old Yaa and non-Yaa B6 male mice were treated i.v. with 100 mg of human recombinant BAFF on days 0 and 14, and on day 21 the size of MZ B-cell compartments was assessed by flow cytometric analysis. As expected, percentages of MZ B cells in splenic B cells of B6 males treated with BAFF (means of 4 mice SD: 16.0 3.0%) were more than 2-fold higher than those in control B6 males (6.9 1.0%, P < 0.05; Fig. 4A). In contrast, BAFF-treated B6.Yaa males failed to display an increase in MZ B cells (BAFF-treated: 3.4 0.6%; PBS-treated: 3.3 0.1%). Concordantly, immunohistochemical analysis of spleens showed an increased accumulation of MZ B cells in BAFF-treated B6 mice, but not in BAFFtreated B6.Yaa mice (data not shown). To exclude the possibility that the lack of accumulation of MZ B cells in BAFF-treated B6.Yaa male mice was due to the failure of Yaabearing B cells to respond to stimulation by BAFF, we determined the functional expression of BAFF-R, which plays a critical role in the BAFF-dependent survival of mature B cells [31]. When splenic B cells from 2 mo-old B6 male mice were cultured with human recombinant BAFF, marked and comparable increases in life-span of B cells from both Yaa and non-Yaa B6 mice were observed (Fig. 4B). Indeed, more than 60% of B cells were still alive in a 6-day culture in the presence of recombinant BAFF, while only w10% of B cells were alive without BAFF. In addition, flow cytometric analysis confirmed the comparable expression of BAFF-R on MZ B cells in B6 and B6.Yaa mice (mean fluorescence intensity of 3 mice SD: B6, 53.0 3.5; B6.Yaa, 49.7 2.6). Moreover, we also ruled out the possibility that
Fig. 4. Lack of accumulation of MZ B cells in BAFF-injected B6.Yaa male mice. A. 100 mg of human recombinant BAFF were injected into 2 mo-old Yaa and non-Yaa B6 male mice on days 0 and 14, and percentages of MZ B cells among B220þ cells of spleen were analyzed on day 21. Representative results from BAFF-treated and control mice (4 mice in each group) with mean percentages of MZ B cells among B220þ cells are shown (upper panel). Percentages of MZ B cells among B220þ cells of individual B6 mice are shown (lower panel). B. B cells purified from spleens of 2 mo-old Yaa (B) and non-Yaa (C) B6 male mice were cultured in the absence (dotted lines) or presence (solid lines) of 500 ng/ml of recombinant BAFF, and viable cells were recorded every 2 days. Results are expressed as mean percentages (SD) of 3 mice.
the loss of MZ B cells in B6.Yaa mice was due to a lower production of BAFF by DC and macrophages, since serum levels of BAFF in 2 mo-old B6.Yaa males (means of 12 mice SEM, 11.1 2.2 ng/ml) were not lower than those in B6 males (5.1 1.1 ng/ml).
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4. Discussion The present study was designed to investigate the mechanism responsible for the loss of MZ B cells due to the presence of the Yaa mutation in mice. Analysis of Yaa and non-Yaa B6 male mice expressing the MD4 anti-HEL IgM transgene and of those deficient in FL revealed that the proportion of MZ B cells in Yaa male mice was comparable to that of the respective non-Yaa control male mice. These results, obtained under conditions in which MZ B cells were poorly activated, collectively suggest that the loss of MZ B cells in Yaa male mice likely results from the persistently elevated activation of this subset of B cells by DC. We have previously shown that lupus-prone BXSB Yaa male mice expressing the Sp6 anti-TNP/DNA IgM developed a MZ B-cell compartment almost comparable to that of their non-Yaa counterparts [16]. It was speculated that self-antigens may trigger Sp6 antiTNP/DNA B cells, thereby promoting the differentiation towards and/or accumulation of MZ B cells in Yaa-bearing mice. However, the finding that the proportion of MZ B cells in MD4 anti-HEL transgenic mice was also not reduced in the presence of the Yaa mutation argues against this possibility. Instead, these results rather suggest that the lack of activation of anti-HEL transgenic B cells, owed to the absence of cognate antigens, may be responsible for their accumulation in the MZ. This concept of an enlarged MZ B-cell compartment due to a lack of B-cell activation is compatible with previous findings of larger MZ B-cell compartments in different IgM transgenic lines which were derived from diverse sources, independently of the autoreactivity of their BCR [40]. The analysis of MD4 anti-HEL and other IgM transgenic mice supports the idea that, since the Yaa mutation could act as a positive BCR regulator [41], the loss of MZ B cells in Yaa-bearing mice is a result of the constantly elevated antigen-specific activation of this subset of B cells. This conclusion was further supported by a number of other findings. First, we observed complete recovery of MZ B cells in B6.Yaa male mice deficient in FL, in which the DC-mediated activation of MZ B cells was markedly compromised because of reduced development of DC [29]. The importance of DC in the activation-induced loss of MZ B cells was further supported by the fact that treatment with recombinant FL resulted in a marked depletion of MZ B cells in non-Yaa B6 mice. Notably, we observed no significant constitutive increases in the number of DC and in the abundance of Flt3l mRNA in spleen of B6.Yaa male mice, indicating that the Yaa-linked loss of MZ B cells was not directly related to an increased production of DC. Moreover, we observed that treatment with the potent B-cell survival factor BAFF led to increases in the proportion of MZ B cells in B6 male mice, but not in B6.Yaa male mice, despite the fact that the expression of BAFF-R on MZ B cells was not defective in B6.Yaa mice. It is likely that injection of BAFF promoted the survival of MZ B cells in Yaa mice, but their enhanced activation led to their constant emigration from the MZ, resulting eventually in unchanged depletion of this B-cell subset. Notably, our interpretation is in agreement with recent findings that the size of MZ B-cell compartment was completely restored in IL-21-deficient BXSB Yaa mice, in which the activation of B cells was strongly suppressed as judged by a marked reduction of serum levels of autoantibodies [42]. It has been established that MZ B cells are critically involved in T-independent antibody responses against blood-borne antigens [14,23], in which complement plays a considerable role [35,36]. However, in contrast to results obtained with FL-deficient mice, analysis of C3-deficient Yaa male mice revealed a less complete restoration of MZ B cells. As T-independent antibody responses are not totally dependent on complement, as documented in C3- or CD21-deficient mice [35,36], it is possible that the interaction of CD21 with C3-opsonized antigens might be only partially
responsible for the activation of MZ B cells induced following the interaction with DC carrying blood-borne antigens. Our present studies provide evidence that the Yaa-linked loss of MZ B cells is a consequence of their excessive activation, due to the hyperactive phenotype conferred by the Yaa mutation, in response to environmental antigens presented by DC. This conclusion is compatible with the loss of MZ B cells in several mutant mice, in which B cells become hypersensitive to BCR signaling [18e21]. Our previous analysis revealed that the loss of the MZ B cells in B6.Yaa male mice cannot be explained by the Tlr7 gene duplication alone [17]. This suggests that gene(s) other than Tlr7 present in the translocated X chromosome additionally contributes to the hyperactive phenotype of Yaa-bearing B cells, thereby promoting the activation of autoreactive B cells. An interesting candidate is the Tlr8 gene, located next to Tlr7, since murine TLR8 has been shown to be activated by a combination of imidazoquinoline and polyT oligodeoxynucleotide [43], while it is inactive in response to any known human TLR7/8 agonist alone [44e46]. Although we have recently observed that B6.Yaa mice bearing the Tlr8 null mutation on the X chromosome still displayed a loss of MZ B cells, we cannot exclude the possibility that the duplication of the Tlr7 and Tlr8 genes in concert contributes to the loss of MZ B cells. Clearly, further studies on the Yaa-linked hyperactive phenotype of B cells and the loss of MZ B cells will help to determine additional target molecules central to the development of SLE, thereby facilitating the design of novel therapeutic strategies in human SLE.
Acknowledgments We thank Dr Thomas Moll for critically reading the manuscript and Ms M. Alvarez, Ms C. Manzin, Mr G. Brighouse and Mr G. Celetta for their excellent technical help. This work was supported by grants from the Swiss National Foundation for Scientific Research and from the Alliance for Lupus Research.
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Evidence that Yaa-induced loss of marginal zone B cells is a result of dendritic cell-mediated enhanced activation Marie-Laure Santiago-Raber a, Hirofumi Amano b, Eri Amano b, Liliane Fossati-Jimack c, Lee Kim Swee d, Antonio Rolink d, Shozo Izui a, * a
Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Geneva 1211, Switzerland Department of Internal Medicine, Juntendo University School of Medicine, Tokyo, Japan Rheumatology Section, Hammersmith Campus, Imperial College London, London, UK d Department of Immunology, University of Basel, Basel, Switzerland b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 December 2009 Received in revised form 5 January 2010 Accepted 7 January 2010
The development of systemic lupus is accelerated by the Yaa (Y-linked autoimmune acceleration) mutation, which is the consequence of a translocation of the telomeric end containing the Tlr7 gene from the X chromosome onto the Y chromosome. However, the loss of marginal zone (MZ) B cells, one of the Yaa-linked cellular abnormalities, has previously been shown to be unrelated to the Tlr7 gene duplication, and the present study therefore aimed to investigate the mechanism responsible for MZ B-cell loss. Analyses of Yaa and non-Yaa C57BL/6 male mice expressing an MD4 anti-HEL IgM transgene or those deficient in fms-like tyrosine kinase 3 ligand (FL) revealed that the proportion of MZ B cells in these Yaa mice was comparable to that of the respective non-Yaa control mice. Notably, the activation of MZ B cells was compromised in both of these transgenic model systems, due to the absence of cognate antigens or the impaired development of dendritic cells, respectively. These results contrasted with the loss of MZ B cells in non-Yaa mice treated with FL and the lack of accumulation of MZ B cells in Yaa mice treated with a B-cell survival factor, BAFF. Taken together, our results suggest that the persistent and enhanced activation of Yaa-bearing hyperactive MZ B cells by dendritic cells is responsible for the loss of this B-cell subset in Yaa mice. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Systemic lupus erythematosus Marginal zone B cell Dendritic cell The Yaa mutation
1. Introduction The BXSB strain of mice spontaneously develops an autoimmune syndrome with features of systemic lupus erythematosus (SLE) that affects males much earlier than females [1]. The accelerated development of SLE in BXSB male mice results from the genetic abnormality, Yaa (Y-linked autoimmune acceleration), present on the BXSB Y chromosome [2]. Studies of Yaa and non-Yaa double bone marrow chimeric mice showed that anti-DNA autoantibodies are selectively produced by B cells bearing the Yaa mutation, and that T cells from both Yaa and non-Yaa origin efficiently promote anti-DNA autoantibody responses [3,4]. Based on these results, it has been speculated that the Yaa defect may decrease the threshold for BCR-mediated signaling, thereby triggering and excessively stimulating autoreactive B cells. More recently, the Yaa mutation was identified to be a consequence of
* Corresponding author. Tel.: þ41 22 379 5741; fax: þ41 22 379 5746. E-mail address: [email protected] (S. Izui). 0896-8411/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaut.2010.01.001
a translocation of the telomeric end containing the gene encoding TLR7 from the X chromosome onto the Y chromosome [5,6]. Since the synergistic engagement of TLR7 and BCR in response to nuclear antigens could induce the activation of autoreactive B cells in SLE [7,8], the Tlr7 gene duplication has been proposed to be the etiologic basis for the Yaa-mediated enhancement of disease [5,6,9,10]. Newly generated B cells in the bone marrow emigrate to the spleen, where they further differentiate into follicular or marginal zone (MZ) B cells. Follicular B cells are IgMintIgDhiCD21intCD23hi, and MZ B cells located at the junction of white and red pulps are IgMhiIgDloCD21hiCD23neg/lo [11e13]. While follicular B cells respond to T-dependent antigens, MZ B cells play a critical role in host defense against T-independent blood-borne pathogens [14,15]. Significantly, mice bearing the Yaa mutation display a marked reduction of the MZ B-cell subset, which results from a defect intrinsic to Yaa-bearing B cells, independently of the development of SLE [16]. Although the Tlr7 gene duplication was proposed as a cause of this cellular defect [9], we observed that the extent of the MZ B-cell loss in C57BL/6 mice bearing the Yaa mutation (B6.Yaa) was essentially unchanged by the introduction of the Tlr7 null mutation on the X chromosome which
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left only a single “Yaa-copy” of the Tlr7 gene expressed [17]. These results indicated that the loss of MZ B cells occurring in Yaa-bearing mice cannot be explained by the Tlr7 gene duplication alone and suggested a critical contribution of other duplicated genes in the translocated X chromosomal end. Since the loss of MZ B cells in Yaa mice can be related to the hyperactive phenotype of Yaa-bearing B cells, which contributed to the accelerated development of murine SLE, it remains important to define the mechanism responsible for this Yaa-linked MZ B-cell loss. Studies on several different genetically manipulated mice revealed that the reduction of MZ B cells could be related to hypersensitive BCR signaling [18e21]. It has been hypothesized that the strength of the signal elicited via the BCR may regulate the lineage commitment of mature B cells into follicular vs. MZ B cells, with hypersensitive BCR signaling favoring an accelerated maturation towards follicular B cells [22]. Thus, a hyperactive phenotype of Yaa-bearing B cells could contribute to the enhanced maturation towards follicular B cells and the decrease in MZ B cells [16]. An alternative possibility could be that the reduction of Yaa-bearing MZ B cells is due to their constant and enhanced activation followed by subsequent emigration from the MZ into the red pulp or B-cell follicles [23e26]. In the present study, we provide evidence that enhanced activation by dendritic cells (DC) is likely to be responsible for the loss of MZ B cells in B6.Yaa mice.
(Serotec, Kidlington, England) mAb in the presence of 2.4G2 antiFcgRII/III mAb. 2.4. Treatment with recombinant FL or BAFF in vivo and in vitro
2. Materials and methods
Mice were subcutaneously injected daily for 9 days with 1 mg of mouse serum albumin (Sigma-Aldrich) plus 10 mg of human recombinant FL (a kind gift of Amgen Inc., Thousand Oaks, CA) or with mouse serum albumin alone. One day after the last injection of FL, the proportion of MZ B cells in spleens was analyzed by flow cytometric and immunohistochemical analysis. The in vivo effect of BAFF was determined in mice injected i.v. on days 0 and 14 with 100 mg of human recombinant BAFF, prepared as described previously [31]. Seven days after the second injection of BAFF, the size of MZ B-cell compartments was assessed. To determine the survival of splenic B cells in vitro, B cells were purified from spleen cells using mouse B-cell enrichment kit (StemCell Technologies, Grenoble, France). The purity of B cells, as documented by flow cytometric analysis, was superior to 95%. Purified B cells were cultured in IMDM containing 5 105 2-ME, 0.03% primatone (Quest International, Naarden, The Netherlands) and 5% FCS at 5 105 cells/ml in the presence or absence of 500 ng/ml of recombinant BAFF. At various time points viable cells were counted by the trypan blue exclusion test. Serum levels of BAFF were quantified by using BAFF, Soluble (mouse) ELISA kit (Apotech Corporation, Epalinges, Switzerland).
2.1. Mice
2.5. Quantitative RT-PCR
B6 mice expressing an MD4 anti-hen egg lysozyme (HEL) IgM transgene [27] were created by backcross procedures at the 8th generation. Mice deficient in C3 [28], kindly provided by Dr M. Carroll, Harvard Medical School, Boston, were backcrossed for 7 generations with B6 mice. B6 mice deficient in fms-like tyrosine kinase 3 ligand (FL), generated by gene targeting in B6-derived ES cells [29], were purchased from Taconic Farms, Inc., Germantown, NY. The Yaa mutation was introduced into these different transgenic mice by crossing with B6.Yaa mice [30]. Animal studies described in the present study have been approved by the Ethics Committee for Animal Experimentation of the Faculty of Medicine, University of Geneva.
RNA from spleen cells was purified with TRIzol reagent (Invitrogen AG, Basel, Switzerland). The abundance of Flt3l mRNA was quantified by real-time RT-PCR with cDNA prepared from RNA. Flt3l cDNA was amplified using a forward primer (50 -TTCAGCCA CAGTCCCATCTC-30 ) and reverse primer (50 -CCTGCCACAGTCTTC AGTTG-30 ). PCR was performed using the iCycler iQ Real-Time PCR Detection System (Bio-Rad, Philadelphia, PA) and iQ SYBR green Supermix (Bio-Rad). Results were quantified relative to a standard curve generated with serial dilutions of a reference cDNA preparation from spleen cells and normalized using TATA-binding protein mRNA. 2.6. Statistical analysis
2.2. Flow cytometric analysis Flow cytometry was performed using three- or four-color staining of spleen cells, and analyzed with a FACSCalibur (Becton Dickinson, Mountain View, CA). The following antibodies were used: anti-CD21 (7G6), anti-CD23 (B3B4), anti-B220 (RA3-6B2), anti-CD11c (N418), anti-MHC class II I-A (Y3P) and anti-BAFFreceptor (BAFF-R; 9B9) [31]. For the analysis of splenic DC, spleen cells were prepared according to the method described by Kamath et al. [32]. Briefly, spleen fragments were digested with a mixture of 1 mg/ml Liberase (Roche Diagnostics, Mannheim, Germany) and 1 mg/ml DNAse I (SigmaeAldrich, St Louis, MO) for 20 min at room temperature and treated with 10 mM EDTA to disrupt T cell-DC complexes. Cells were then filtered with a cell strainer (70 mm), resuspended and stained in PBS containing 1% BSA, 2 mM EDTA and a saturating concentration of 2.4G2 anti-FcgRII/III mAb. 2.3. Immunohistochemistry Spleens were embedded in Tissue-Tek O.C.T. compound (Miles, Elkhart, IN) and snap-frozen in liquid nitrogen. Four mm frozen sections were stained with PE-labeled anti-IgM (1B4B1; Southern Biotechnology, Birmingham, AL) and FITC-labeled MOMA-1
Statistical analyses were performed using the ManneWhitney U-test. Probability values >5% were considered insignificant. 3. Results 3.1. No reduction of MZ B cells in MD4 anti-HEL IgM transgenic B6.Yaa male mice We have previously shown that the proportion of MZ B cells in lupus-prone BXSB Yaa males expressing an Sp6 anti-TNP/DNA transgenic IgM was substantially increased, as compared with nontransgenic littermates, reaching a level nearly identical to that of non-Yaa BXSB transgenic males [16]. The increase in the size of the MZ B-cell compartment in the Sp6 transgenic BXSB mice could be related to the findings that autoreactive B cells tend to be accumulated in the MZ [33,34]. However, larger MZ B-cell compartments have also been reported in non-autoreactive IgM transgenic mice such as MD4 anti-HEL transgenic mice [27]. Therefore, we determined whether B cells expressing an MD4 anti-HEL IgM transgene similarly accumulated in the MZ of B6.Yaa male mice. As was the case of Sp6 transgenic mice, the proportion of MZ B cells in MD4 transgenic B6.Yaa males (means SD: 15.6 2.4%) was as
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we determined whether the MZ B-cell compartment of B6.Yaa mice can be restored by the absence of C3, as a result of down-modulated activation of MZ B cells in response to T-independent blood-borne antigens. Percentages of MZ B cells were significantly increased in both C3-deficient Yaa and non-Yaa B6 mice as compared to C3-sufficient counterparts (P < 0.005; Fig. 2). Notably, the proportion of MZ B cells in C3/ B6.Yaa mice (6.0 2.4%) was comparable to that in C3-sufficient non-Yaa B6 mice (6.2 0.6%), but still lower than that of C3/ non-Yaa mice (9.7 2.6%). These data suggested that the loss of MZ B cells in B6.Yaa mice may be in part a consequence of an enhanced complement-dependent activation of MZ B cells. 3.3. Rescue of MZ B-cell loss in B6.Yaa male mice deficient in FL, and loss of MZ B cells in B6 male mice treated with recombinant FL Studies have shown that blood-borne antigens are transported into the MZ by DC and that interaction of antigen-bearing DC with
Fig. 1. No reduction of MZ B cells in MD4 anti-HEL IgM transgenic Yaa male mice. CD21hiCD23neg/lo MZ B cells in spleen cells of 2 mo-old MD4 transgenic B6 Yaa and non-Yaa male mice and of their non-transgenic littermates were stained with a combination of anti-B220, anti-CD21 and anti-CD23 mAb, and gated for B220þ cells. Representative results from 5 to 6 mice in each group of mice are shown, and mean percentages of MZ B cells among B220þ cells are indicated (upper panel). Percentages of MZ B cells among B220þ cells of individual B6 mice of different genotypes are shown, and mean values are indicated by horizontal lines (lower panel).
large as that of MD4 transgenic non-Yaa males (17.5 4.4%; Fig. 1). These results contrasted with marked differences in MZ B cells between non-transgenic Yaa and non-Yaa male littermates (2.5 0.5% vs. 5.8 0.9%, P < 0.01). Since anti-HEL transgenic B cells were not expected to be spontaneously activated because of the absence of cognate antigens, these data suggested that lack of reduction of Sp6 and MD4 transgenic B cells in the MZ of Yaabearing mice could be due to a limited antigen-specific activation of these B cells, thereby prevented their depletion from the MZ. 3.2. Partial recovery of MZ B-cell compartment in C3-deficient B6.Yaa male mice MZ B cells are known to play the major role in immune responses against T-independent blood-borne pathogens [14]. Therefore, we hypothesized that the Yaa-linked loss of MZ B cells could be a result of their excessive activation in response to T-independent antigens. Since complement plays a substantial role in the development of T-independent antibody responses [35,36],
Fig. 2. Partial recovery of MZ B-cell compartment in C3-deficient B6.Yaa male mice. Representative results for CD21hiCD23neg/lo MZ B cells in spleen cells of 2 mo-old C3/ Yaa and non-Yaa B6 male mice and of their control littermates (5e8 mice in each group) with mean percentages of MZ B cells among B220þ cells are shown (upper panel). Percentages of MZ B cells among B220þ cells of individual B6 mice of different genotypes are shown (lower panel).
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MZ B cells can initiate T-independent immune responses [14,23]. Therefore, we investigated the possible role of DC in the activationinduced loss of MZ B cells in B6.Yaa mice. We introduced the Flt3l null mutation into B6.Yaa mice, thereby impairing the development of DC, and then assessed the size of the MZ B-cell compartment. As expected, percentages of CD11cþ MHC class II I-Aþ DC in spleens were markedly diminished in 2 mo-old FL-deficient B6.Yaa mice (means of 5 mice SD: 1.4 1.0%), as compared with FL-sufficient control mice (4.3 0.7%, P < 0.01). This decrease of DC in spleens
was accompanied by a marked increase in the proportion of MZ B cells in FL/ B6.Yaa mice (FL/ Yaa: 12.7 2.1%, FLþ/þ Yaa: 2.6 2.1%, P < 0.005; Fig. 3A). More significantly, percentages of MZ B cells in FL/ B6.Yaa male mice were almost comparable to those in FL/ B6 non-Yaa male mice (14.1 1.5%). In agreement with the flow cytometric analysis, immunohistological examination of spleens showed that the loss of a characteristic rim of IgMþ MZ B cells at the periphery of the follicles and separated by MOMA-1þ macrophages in B6.Yaa mice was rescued in the spleens of FL/
Fig. 3. Rescue of MZ B-cell loss in B6.Yaa male mice deficient in FL, and loss of MZ B cells in B6 mice treated with FL. A. Representative results for CD21hiCD23neg/lo MZ B cells in spleen cells of FL-deficient (FL/) Yaa and non-Yaa B6 male mice and of their control (FLþ/þ) littermates (5e8 mice in each group) with mean percentages of MZ B cells among B220þ cells are shown (upper panel). Percentages of MZ B cells in B220þ cells of individual B6 mice of different genotypes are shown (lower panel). B. Spleen sections from FL-deficient (FL/) Yaa and non-Yaa B6 male mice and of their control littermates were stained with PE-labeled anti-IgM (red) and FITC-labeled anti-MOMA-1 (green). Representative results obtained from 4 mice in each group are shown. C.10 mg of human recombinant FL were daily injected into 2 mo-old B6 male mice for 9 days, and percentages of CD11cþB220 myeloid and CD11cþB220þ plasmacytoid DC in spleen cells were analyzed on day 10. Representative results from FL-treated and control mice (5 mice in each group) with mean percentages of DC in spleen cells are shown. D. Representative results for CD21hiCD23neg/lo MZ B cells in spleen cells of FL-treated and control B6 male mice (5 mice in each group) with mean percentages of MZ B cells among B220þ cells are shown (upper panel). Percentages of MZ B cells in B220þ cells of individual B6 mice are shown (lower panel).
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B6.Yaa males (Fig. 3B). Notably, the size of this B cell population was enlarged in FL/ B6.Yaa males and comparable to that in FL/ B6 non-Yaa males. These results indicated that the impaired development of DC prevented the loss of MZ B cells in B6.Yaa mice. To further define the role of DC in the reduction of MZ B cells in B6.Yaa mice, we determined whether increased production of DC following injections of FL could induce a depletion of MZ B cells in B6 male mice. As reported previously [37], mice daily treated with human recombinant FL for 9 days displayed higher percentages of DC in spleens (means of 5 mice SD: CD11cþB220 myeloid DC, 17.5 1.6% and CD11cþB220þ plasmacytoid DC, 8.0 1.9%) than control mice (myeloid DC, 2.4 0.2% and plasmacytoid DC, 2.2 0.1%, P < 0.01; Fig. 3C). In FL-treated B6 mice, the proportions of MZ B cells were markedly diminished (1.9 0.2%) as compared with those in control mice (6.3 0.8%, P < 0.01; Fig. 3D). The loss of MZ B cells in FL-treated B6 mice was confirmed by immunohistological analysis (data not shown). These data collectively suggested that DC-mediated enhanced activation of MZ B cells could be responsible for their depletion from the MZ compartment in B6.Yaa mice. Results obtained with FL-deficient or FL-injected B6 mice prompted us to investigate whether B6.Yaa mice displayed increased production of DC or FL as compared with wild-type B6 mice. However, no significant differences in percentages of myeloid and plasmacytoid DC in spleens were observed between 2 mo-old Yaa and non-Yaa B6 male mice (data not shown). In addition, quantitative RT-PCR failed to show any measurable differences in Flt3l mRNA abundance in spleen cells from 2 mo-old Yaa and nonYaa B6 male mice (means of 3 mice SD: Yaa, 0.96 0.13; non-Yaa, 0.83 0.14). 3.4. Lack of accumulation of MZ B cells in BAFF-injected B6.Yaa male mice BAFF is a potent survival factor for MZ B cells [38,39]. If the loss of MZ B cells was indeed a consequence of their persistently increased activation and subsequent depletion, we should expect that an increase in the number of MZ B cells in B6.Yaa male mice following treatment with BAFF is limited as compared with that in B6 male mice. To address this question, 2 mo-old Yaa and non-Yaa B6 male mice were treated i.v. with 100 mg of human recombinant BAFF on days 0 and 14, and on day 21 the size of MZ B-cell compartments was assessed by flow cytometric analysis. As expected, percentages of MZ B cells in splenic B cells of B6 males treated with BAFF (means of 4 mice SD: 16.0 3.0%) were more than 2-fold higher than those in control B6 males (6.9 1.0%, P < 0.05; Fig. 4A). In contrast, BAFF-treated B6.Yaa males failed to display an increase in MZ B cells (BAFF-treated: 3.4 0.6%; PBS-treated: 3.3 0.1%). Concordantly, immunohistochemical analysis of spleens showed an increased accumulation of MZ B cells in BAFF-treated B6 mice, but not in BAFFtreated B6.Yaa mice (data not shown). To exclude the possibility that the lack of accumulation of MZ B cells in BAFF-treated B6.Yaa male mice was due to the failure of Yaabearing B cells to respond to stimulation by BAFF, we determined the functional expression of BAFF-R, which plays a critical role in the BAFF-dependent survival of mature B cells [31]. When splenic B cells from 2 mo-old B6 male mice were cultured with human recombinant BAFF, marked and comparable increases in life-span of B cells from both Yaa and non-Yaa B6 mice were observed (Fig. 4B). Indeed, more than 60% of B cells were still alive in a 6-day culture in the presence of recombinant BAFF, while only w10% of B cells were alive without BAFF. In addition, flow cytometric analysis confirmed the comparable expression of BAFF-R on MZ B cells in B6 and B6.Yaa mice (mean fluorescence intensity of 3 mice SD: B6, 53.0 3.5; B6.Yaa, 49.7 2.6). Moreover, we also ruled out the possibility that
Fig. 4. Lack of accumulation of MZ B cells in BAFF-injected B6.Yaa male mice. A. 100 mg of human recombinant BAFF were injected into 2 mo-old Yaa and non-Yaa B6 male mice on days 0 and 14, and percentages of MZ B cells among B220þ cells of spleen were analyzed on day 21. Representative results from BAFF-treated and control mice (4 mice in each group) with mean percentages of MZ B cells among B220þ cells are shown (upper panel). Percentages of MZ B cells among B220þ cells of individual B6 mice are shown (lower panel). B. B cells purified from spleens of 2 mo-old Yaa (B) and non-Yaa (C) B6 male mice were cultured in the absence (dotted lines) or presence (solid lines) of 500 ng/ml of recombinant BAFF, and viable cells were recorded every 2 days. Results are expressed as mean percentages (SD) of 3 mice.
the loss of MZ B cells in B6.Yaa mice was due to a lower production of BAFF by DC and macrophages, since serum levels of BAFF in 2 mo-old B6.Yaa males (means of 12 mice SEM, 11.1 2.2 ng/ml) were not lower than those in B6 males (5.1 1.1 ng/ml).
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4. Discussion The present study was designed to investigate the mechanism responsible for the loss of MZ B cells due to the presence of the Yaa mutation in mice. Analysis of Yaa and non-Yaa B6 male mice expressing the MD4 anti-HEL IgM transgene and of those deficient in FL revealed that the proportion of MZ B cells in Yaa male mice was comparable to that of the respective non-Yaa control male mice. These results, obtained under conditions in which MZ B cells were poorly activated, collectively suggest that the loss of MZ B cells in Yaa male mice likely results from the persistently elevated activation of this subset of B cells by DC. We have previously shown that lupus-prone BXSB Yaa male mice expressing the Sp6 anti-TNP/DNA IgM developed a MZ B-cell compartment almost comparable to that of their non-Yaa counterparts [16]. It was speculated that self-antigens may trigger Sp6 antiTNP/DNA B cells, thereby promoting the differentiation towards and/or accumulation of MZ B cells in Yaa-bearing mice. However, the finding that the proportion of MZ B cells in MD4 anti-HEL transgenic mice was also not reduced in the presence of the Yaa mutation argues against this possibility. Instead, these results rather suggest that the lack of activation of anti-HEL transgenic B cells, owed to the absence of cognate antigens, may be responsible for their accumulation in the MZ. This concept of an enlarged MZ B-cell compartment due to a lack of B-cell activation is compatible with previous findings of larger MZ B-cell compartments in different IgM transgenic lines which were derived from diverse sources, independently of the autoreactivity of their BCR [40]. The analysis of MD4 anti-HEL and other IgM transgenic mice supports the idea that, since the Yaa mutation could act as a positive BCR regulator [41], the loss of MZ B cells in Yaa-bearing mice is a result of the constantly elevated antigen-specific activation of this subset of B cells. This conclusion was further supported by a number of other findings. First, we observed complete recovery of MZ B cells in B6.Yaa male mice deficient in FL, in which the DC-mediated activation of MZ B cells was markedly compromised because of reduced development of DC [29]. The importance of DC in the activation-induced loss of MZ B cells was further supported by the fact that treatment with recombinant FL resulted in a marked depletion of MZ B cells in non-Yaa B6 mice. Notably, we observed no significant constitutive increases in the number of DC and in the abundance of Flt3l mRNA in spleen of B6.Yaa male mice, indicating that the Yaa-linked loss of MZ B cells was not directly related to an increased production of DC. Moreover, we observed that treatment with the potent B-cell survival factor BAFF led to increases in the proportion of MZ B cells in B6 male mice, but not in B6.Yaa male mice, despite the fact that the expression of BAFF-R on MZ B cells was not defective in B6.Yaa mice. It is likely that injection of BAFF promoted the survival of MZ B cells in Yaa mice, but their enhanced activation led to their constant emigration from the MZ, resulting eventually in unchanged depletion of this B-cell subset. Notably, our interpretation is in agreement with recent findings that the size of MZ B-cell compartment was completely restored in IL-21-deficient BXSB Yaa mice, in which the activation of B cells was strongly suppressed as judged by a marked reduction of serum levels of autoantibodies [42]. It has been established that MZ B cells are critically involved in T-independent antibody responses against blood-borne antigens [14,23], in which complement plays a considerable role [35,36]. However, in contrast to results obtained with FL-deficient mice, analysis of C3-deficient Yaa male mice revealed a less complete restoration of MZ B cells. As T-independent antibody responses are not totally dependent on complement, as documented in C3- or CD21-deficient mice [35,36], it is possible that the interaction of CD21 with C3-opsonized antigens might be only partially
responsible for the activation of MZ B cells induced following the interaction with DC carrying blood-borne antigens. Our present studies provide evidence that the Yaa-linked loss of MZ B cells is a consequence of their excessive activation, due to the hyperactive phenotype conferred by the Yaa mutation, in response to environmental antigens presented by DC. This conclusion is compatible with the loss of MZ B cells in several mutant mice, in which B cells become hypersensitive to BCR signaling [18e21]. Our previous analysis revealed that the loss of the MZ B cells in B6.Yaa male mice cannot be explained by the Tlr7 gene duplication alone [17]. This suggests that gene(s) other than Tlr7 present in the translocated X chromosome additionally contributes to the hyperactive phenotype of Yaa-bearing B cells, thereby promoting the activation of autoreactive B cells. An interesting candidate is the Tlr8 gene, located next to Tlr7, since murine TLR8 has been shown to be activated by a combination of imidazoquinoline and polyT oligodeoxynucleotide [43], while it is inactive in response to any known human TLR7/8 agonist alone [44e46]. Although we have recently observed that B6.Yaa mice bearing the Tlr8 null mutation on the X chromosome still displayed a loss of MZ B cells, we cannot exclude the possibility that the duplication of the Tlr7 and Tlr8 genes in concert contributes to the loss of MZ B cells. Clearly, further studies on the Yaa-linked hyperactive phenotype of B cells and the loss of MZ B cells will help to determine additional target molecules central to the development of SLE, thereby facilitating the design of novel therapeutic strategies in human SLE.
Acknowledgments We thank Dr Thomas Moll for critically reading the manuscript and Ms M. Alvarez, Ms C. Manzin, Mr G. Brighouse and Mr G. Celetta for their excellent technical help. This work was supported by grants from the Swiss National Foundation for Scientific Research and from the Alliance for Lupus Research.
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