Rapid B cell apoptosis induced by antigen receptor ligation does not require Fas (CD95/APO-1), the adaptor protein FADD/MORT1 or CrmA-sensitive caspases but is defective in both MRL-+/+ and MRL-lpr/lpr mice

Tsutomu Yoshida1, Tetsuya Higuchi1,2, Hiroyuki Hagiyama1,3, Andreas Strasser4, Kiyoshi Nishioka2 and Takeshi Tsubata1

1 Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510, Japan
2 Department of Dermatology and
3 First Department of Internal Medicine, Faculty of Medicine, Tokyo Medical and Dental University, Tokyo 113-8519, Japan
4 The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3050, Australia

Correspondence to: T. Tsubata


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Antigen receptor ligation-induced apoptosis is thought to play a role in self-tolerance by deleting autoreactive lymphocytes. Antigen receptor ligation-induced apoptosis of mature T cells and T cell lines requires autocrine or paracrine activation of Fas (CD95/APO-1). Whether B cell antigen receptor (BCR)-mediated apoptosis requires Fas or related molecules is unclear. Here we demonstrate that expression of either CrmA, the cowpox virus serpin, or an inhibitor of the adapter protein FADD/MORT1 blocks Fas-mediated apoptosis but has no effect on BCR ligation-induced apoptosis of the B cell line WEHI-231. In contrast, expression of Bcl-2 blocks BCR-mediated but not Fas-induced apoptosis in WEHI-231 cells. These results indicate that BCR ligation activates an apoptotic signaling pathway distinct from Fas-mediated apoptosis in WEHI-231 cells, and that BCR-mediated apoptosis of WEHI-231 cells does not require Fas or related molecules such as DR3, DR4 and DR5, as all of these death receptors require FADD/MORT1 and/or CrmA-sensitive caspases for induction of apoptosis. Moreover, extensive BCR ligation induces death of mature B cells from C57BL/6-lpr/lpr mice as efficiently as those from C57BL/6 mice, indicating that Fas is not essential for BCR-mediated apoptosis of mature B cells. In contrast, BCR ligation-induced apoptosis is reduced in mature B cells from MRL mice and this is not affected by the lpr mutation. Since MRL-lpr/lpr mice but not C57BL/6-lpr/lpr mice develop severe autoimmune disease, defects in BCR-mediated apoptosis in the MRL background, together with lpr mutation, may contribute to the development of severe autoimmune disease in MRL-lpr/lpr mice by allowing survival of self-reactive B cells.

Keywords: apoptosis, B lymphocyte, BCR, CrmA, MRL mice


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Studies on transgenic mice expressing autoantibodies have demonstrated that interaction with self-antigens can either inactivate or eliminate self-reactive B cells, allowing maintenance of self-tolerance (13). When challenged with membrane-bound autoantigens, self-reactive B cells are either rapidly eliminated, presumably by apoptosis, or alter the antigen specificity by receptor editing. In contrast, soluble autoantigens can cause functional inactivation (anergy) of self-reactive B cells. Anergized B cells also undergo apoptosis, albeit more slowly than apoptosis of B cells encountered with membrane-bound autoantigens (4). These findings suggest that strong B cell antigen receptor (BCR) ligation by membrane-bound antigens induces rapid apoptosis, whereas mild BCR ligation by soluble antigens causes apoptosis more slowly. BCR-mediated apoptosis has been demonstrated in vitro in transformed B cell lines, such as WEHI-231 (5,6), and in normal mature B cells (711). When BCR are extensively ligated, using immobilized anti-Ig antibody, most mature B cells undergo apoptosis within 48 h, whereas only a small fraction of mature B cells undergo apoptosis in 48 h by milder BCR ligation using soluble anti-Ig antibody (11) in agreement with the observations from studies on autoantibody-transgenic mice. BCR ligation-induced apoptosis is abnormally reduced in NZB and (NZBxNZW)F1 mice, which are prone to systemic autoimmune disease (7,10), and it is possible that this defect is involved in generation of self-reactive B cells and autoantibody production.

Fas is essential for TCR ligation-induced apoptosis of mature T cells and T cell lines both in vivo and in vitro(1219). Signaling through TCR up-regulates expression of both Fas and Fas ligand (FasL), and causes Fas-mediated apoptosis by an autocrine mechanism. Fas has been reported to be required for apoptosis of B cells anergized by interaction with soluble antigens (20). This suggests that mild BCR ligation induces Fas-mediated apoptosis at least in vivo, although it is not known whether this apoptosis is induced by an autocrine pathway. In contrast, Kozono et al. (21) have demonstrated that treatment with soluble anti-Ig antibody for 24 h induces apoptosis normally in lpr B cells, indicating that Fas is not essential for apoptosis induced by mild BCR ligation. However, this treatment induces apoptosis only in a small fraction of B cells (21), whereas most of the B cells exposed to soluble antigens in vivo are thought to slowly undergo apoptosis by a Fas-dependent mechanism (20). Therefore, significance of the Fas-independent pathway for apoptosis induced by mild BCR ligation is not yet clear. Moreover, it has also not yet been elucidated whether Fas is essential for B cell apoptosis induced by strong BCR ligation.

MRL-lpr/lpr mice carry a loss-of-function mutation of the Fas gene (lpr mutation) (22). These mice develop lymphadenopathy and a severe systemic lupus erythematosus (SLE)-like autoimmune disease, characterized by production of autoantibodies to nuclear antigens (23,24). Development of the severe autoimmune disease requires both the lpr mutation and unknown defects in the MRL background because the lpr mutation causes lymphadenopathy but not SLE-like autoimmunity on different genetic backgrounds such as C57BL/6. In mice carrying the lpr mutation, the defect in Fas-mediated apoptosis makes both antigen-stimulated T cells and B cells exposed to soluble self-antigens fail to die (12,14,19), and it is thought that these defects cause lymphadenopathy due to accumulation of activated T cells and autoantibody production by survival of self-reactive B cells (20). However, the defects of the MRL background involved in the development of severe autoimmune disease in MRL-lpr/lpr mice are not yet understood.

Fas requires both the adapter protein FADD (also called MORT1) and caspase-8 for transmission and execution of the apoptotic signal (2527). FADD and caspase-8 are also essential for apoptosis mediated by other members of the tumor necrosis factor (TNF) receptor family that have a death domain in the cytoplasmic region such as type 1 TNF receptor, DR4, DR5 and DR6 (2731). To assess whether Fas is involved in BCR-mediated apoptosis of human B lymphoma cells, Lens et al. (32) have blocked Fas signaling by expression of a dominant interfering mutant of FADD. However, FADD appears to play a role in cell proliferation as well as in apoptotic signals (33,34). To circumvent this potential complication, we introduced an expression construct for the cowpox virus serpin CrmA, a potent inhibitor of caspase-8 (35), into the mouse B cell line WEHI-231, and demonstrated that CrmA blocks Fas-mediated but not BCR-mediated apoptosis. We further demonstrated that extensive BCR cross-linking by immobilized anti-Ig antibody induced apoptosis in B cells from C57BL/6-lpr/lpr mice as efficiently as in B cells from C57BL/6 mice. These results clearly indicated that Fas is dispensable for rapid BCR-mediated apoptosis. Moreover, DR3, DR4, DR5 and DR6 also do not appear to be required for BCR-mediated apoptosis as those death receptors also depend on CrmA-sensitive caspases for apoptosis. In contrast, B cell apoptosis induced by immobilized anti-Ig antibodies was defective in both MRL-+/+ and MRL-lpr/lpr mice. The defect in BCR-mediated apoptosis of the MRL background, together with the lpr mutation, may play a role in the development of severe autoimmune disease in MRL-lpr/lpr mice.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice and cells
C57BL/6, C57BL/6-lpr/lpr, MRL-+/+ and MRL-lpr/lpr mice were purchased from Sankyo Labo Service (Tokyo, Japan). Small dense B cells were purified from mouse spleens as described previously (10). WEHI-231.5 is a subclone of WEHI-231 obtained in our laboratory (36).

Plasmids and transfection
For construction of the expression plasmid for the FLAG-tagged dominant negative form of FADD (FADD-DN), a BglII fragment containing cDNA for FLAG-tagged human FADD lacking the N-terminal 79 amino acids was isolated from pEF-FLAGFADD-DN79-pGKpuro (34). After blunting both ends, the fragment was subcloned into EcoRI–XhoI-opened blunt-ended pMKITNeo (a gift of Dr Maruyama), resulting in pMKITFADD-DN. The expression plasmid pMKITCrmA coding for FLAG-tagged CrmA was constructed by blunting the ends of a KpnI fragment of pEF-FLAGcrmA-pGKpuro (37) followed by subcloning into EcoRI–XhoI-opened blunt-ended pMKITNeo. Transfection of WEHI231.5 with the expression plasmids was done as described previously (36). For construction of an expression vector for human bcl-2 (pMIK-bcl2), pMIKneo (-XbaI) was generated by digestion of the pMIKneo vector (a gift of Dr. Maruyama) with XbaI, followed by self-ligation. The EcoRI fragment of the plasmid Bcl-2#58 (a gift of Dr Seto) (38) containing full-length human bcl-2 was inserted into the EcoRI-opened pMIKneo(-XbaI).

Cell culture
Plastic-immobilized anti-Ig antibody was prepared by incubating PBS containing 20 µg/ml of F(ab')2 fragments of affinity-purified goat anti-mouse IgM antibody (ICN, Aurora, OH) in wells of plastic dishes at 37°C overnight, followed by washing with PBS. Small dense B cells were cultured in RPMI 1640 medium supplemented with 10% FCS, 50 µM 2-mercaptoethanol and 2 mM L-glutamine with immobilized anti-Ig antibody, 1 µg/ml anti-mouse CD40 mAb HM40-3 (a gift of Dr Yagita) or both. Cells (1.0x104) of WEHI-231.5 and its transfectants were cultured in 100 µl of medium with 10 µg/ml of anti-mouse CD40 mAb HM40-3 for 24 h and then cultured with 10 µg/ml of anti-mouse Fas mAb Jo-2 (PharMingen, San Diego, CA). Alternatively, cells were cultured in 100 µL of medium with 10 µg/ml of F(ab')2 fragments of goat anti-mouse IgM antibody (ICN) for 24 or 48 h.

Cell stimulation and Western blotting
Small dense B cells were treated with 20 µg/ml of F(ab')2 fragments of goat anti-mouse IgM antibody (ICN) for at 37°C and lysed in SDS sample buffer. Cell lysates were separated by SDS–PAGE. Proteins were transferred to PVDF membrane (Amersham Pharmacia Biotech, Piscataway, NJ), and reacted with either peroxidase-conjugated anti-phosphotyrosine mAb 4G10 (Upstate Biotechnology, Lake Placid, NY), the combination of rabbit anti-phospho-p42/p44 MAP kinase antibody (New England Biolabs, Beverly, MA) and peroxidase- conjugated anti-rabbit IgG antibody (New England Biolabs), or the combination of rabbit anti-ERK2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and peroxidase-conjugated anti-rabbit IgG antibody (Amersham, Little Chalfont, UK). WEHI-231 cells and its transfectants were lysed in phospholysis buffer (39), and 50 µg of lysates was subjected to 13% SDS–PAGE. Proteins were transferred to PVDF membrane (Amersham Pharmacia Biotech) and incubated with 10 µg/ml of anti-FLAG-M2 mAb (Eastman Kodak, New Haven, Connecticut), followed by reaction with horseradish peroxidase-labeled anti-mouse IgG antibody (Southern Biotechnology Associates, Birmingham, AL). Proteins were detected using the ECL Western system (Amersham Pharmacia Biotech).

Flow cytometry
Small dense B cells were stained with FITC-labeled anti-mouse CD3 mAb (PharMingen) and phycoerythrin (PE)-labeled anti-mouse IgM antibody (Southern Biotechnology). WEHI-231 cells and its transfectants were incubated with biotin-labeled anti-mouse Fas mAb Jo-2 (PharMingen) in the presence of anti-Fc{gamma} receptor antibody 2.4G2, followed by incubation with FITC-labeled streptavidin (Dako, Carpinteria, CA). Alternatively, WEHI-231 cells and its transfectants were incubated with FITC-conjugated anti-mouse IgM antibody (Southern Biotechnology). For detection of cells containing hypodiploid nuclei, cells were incubated in PBS containing 0.15 % Triton X-100 and 5 µg/ml of RNase, followed by staining with 50 µg/ml of propidium iodide. For cell cycle analysis, cells were incubated with 20 µM of BrdU (Sigma, St Louis, MO) at 37°C for 10 min. Cells were collected, fixed with 70% ethanol and treated with 4 N HCl containing 0.5% Triton X-100 at room temperature for 20 min. After washing, cells were suspended in PBS containing 1% BSA and 10 µg/ml propidium iodide. Cells were analyzed by flow cytometry using a FACSCalibur (Becton Dickinson, San Jose, CA).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
BCR-mediated apoptosis of WEHI-231 cells requires neither FADD nor CrmA-sensitive caspases
To investigate whether Fas signaling is involved in BCR-mediated apoptosis of the mouse B cell line WEHI-231, we transfected WEHI-231.5 cells with expression vectors encoding either a dominant negative form of FADD (FADD-DN) or the cowpox virus serpin CrmA. Since both FADD-DN and CrmA are tagged with FLAG, expression of FADD-DN and CrmA was confirmed in transfectants by Western blot analysis using anti-FLAG mAb (Fig. 1A and BGo). WEHI-231.5 cells expressed a small amount of Fas on the surface and its expression was slightly increased by treatment with anti-CD40 mAb (Fig. 1CGo). Both the FADD-DN transfectants and CrmA transfectants showed the levels of Fas expression similar to the parent WEHI-231.5, regardless of the presence or absence of anti-CD40 mAb. Untreated WEHI-231.5 did not undergo apoptosis by treatment with anti-Fas mAb Jo-2 (data not shown). However, when pretreated with anti-CD40 mAb for 24 h, ~50% of the WEHI-231.5 cells died upon stimulation with anti-Fas mAb (Fig. 1DGo). This is consistent with previous studies which showed that CD40 signaling enhances Fas-mediated apoptosis of B cells (4043). In contrast, this treatment killed only a very small number of FADD-DN transfectants and CrmA transfectants. These results demonstrate that expression of either FADD-DN or CrmA blocks Fas-mediated apoptosis in WEHI-231 cells.



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Fig. 1. Expression of a dominant negative mutant of FADD (FADD-DN) or CrmA blocks Fas-mediated cell death in WEHI231.5 cells. (A) Expression of FADD-DN. Parental WEHI-231.5 cells and WEHI-231.5 FADD-DN transfectants (W/FADD-DN1 and W/FADD-DN-8) were lysed in phospholysis buffer. Cell lysates were subjected to Western blot analysis using anti-FLAG mAb. (B) Expression of CrmA. Parental WEHI-231.5 cells and WEHI-231.5 CrmA transfectants (W/CrmA-2 and W/CrmA-4) were lysed in phospholysis buffer. Cell lysates were subjected to Western blot analysis using anti-FLAG mAb. (C) Expression of Fas in WEHI-231.5 transfectants. Parental WEHI-231.5, W/FADD-DN1 and W/CrmA-2 were treated with anti-CD40 mAb HM40-3 for 24 h or left untreated. Cells were stained with biotinylated anti-mouse Fas mAb Jo-2 and FITC-labeled streptavidin, and analyzed by flow cytometry. Cells stained with FITC-labeled streptavidin alone were used as negative controls (filled histogram). (D) Fas-induced death of WEHI-231.5 transfectants. Parental WEHI-231.5 cells, WEHI-231.5 FADD-DN transfectants (W/FADD-DN1 and W/FADD-DN-8) and WEHI-231.5 CrmA transfectants (W/CrmA-2 and W/CrmA-4) were first treated with anti-mouse CD40 mAb HM40-3 for 24 h and then stimulated with anti-mouse Fas mAb Jo-2 for 24 h. Numbers of live and dead cells were counted by Trypan blue exclusion and percentages of dead cells were calculated. The data represent mean ± SD of triplicate cultures. Representative data of three experiments are shown.

 
Both WEHI-231.5 FADD-DN transfectants and WEHI-231.5 CrmA transfectants expressed similar levels of surface IgM to the parent WEHI-231.5 (Fig. 2AGo). Upon treatment with 10 µg/ml of polyclonal anti-IgM antibody for 48 h, both WEHI-231.5 FADD-DN transfectants and WEHI-231.5 CrmA transfectants died as efficiently as parental WEHI-231.5 cells (Fig. 2BGo). Moreover, these transfectants showed a similar increase in the percentage of cells with hypodiploid nuclei, characteristic for apoptosis, as did parental WEHI-231.5 cells (Fig. 2CGo). These results indicate that BCR ligation induces apoptosis in both WEHI-231.5 FADD-DN transfectants and WEHI-231.5 CrmA transfectants as efficiently as in parental WEHI-231.5 cells. Taken together, the adapter FADD and CrmA-sensitive caspases are essential for Fas-mediated but not BCR-mediated apoptosis in WEHI-231.



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Fig. 2. Expression of a dominant negative mutant of FADD (FADD-DN) or CrmA does not block BCR-mediated apoptosis of WEHI231.5 cells. (A) Expression of surface IgM. Parental WEHI-231.5 cells, W/FADD-DN1 cells and W/CrmA-2 cells were stained with FITC-conjugated anti-mouse IgM antibody, and analyzed by flow cytometry. Unstained cells were used as negative controls (filled histogram). (B) BCR-mediated death of WEHI-231.5 transfectants. Parental WEHI-231.5 cells, WEHI-231.5 FADD-DN transfectants (W/FADD-DN1 and W/FADD-DN-8) and WEHI-231.5 CrmA transfectants (W/CrmA-2 and W/CrmA-4) were cultured with 10 µg/ml F(ab')2 fragments of goat anti-mouse IgM antibody. After 48 h, numbers of live and dead cells were counted by Trypan blue exclusion and percentages of dead cells were calculated. The data represent mean ± SD of triplicate cultures. Representative data of three experiments are shown. (C) Measurement of cells with hypodiploid nuclei. Parental WEHI-231.5 cells, W/FADD-DN1 cells and W/CrmA-2 cells were cultured with 10 µg/ml of F(ab')2 fragments of goat anti-mouse IgM antibody. After 48 h, cells were suspended in PBS containing 0.15 % Triton-X 100, 5 µg/ml of RNase and 50 µg/ml propidium iodide, and were analyzed by flow cytometry. Percentages of cells with hypodiploid nuclei are indicated.

 
Bcl-2 and its homologues inhibit many cell death pathways, but are poor antidotes to apoptosis induced by `death receptors' such as Fas (37). We therefore studied the effects of Bcl-2 on apoptosis of WEHI-231 induced by either Fas or BCR ligation. When WEHI-231 transfected with human bcl-2 (WEHI-bcl2) and WEHI-231.5 were cultured with anti-IgM antibodies, cell death of WEHI-bcl2 was markedly reduced compared to that of WEHI-231.5 cells (Fig. 3AGo), although they expressed similar levels of BCR (data not shown). In contrast, treatment with the anti-Fas mAb Jo-2-induced cell death in WEHI-bcl2 cells as efficiently as in WEHI-231.5 cells (Fig. 3BGo). These results demonstrate that Bcl-2 can block BCR-mediated but not Fas-mediated apoptosis of WEHI-231 cells. This underscores that BCR ligation induces apoptosis in WEHI-231 cells by a signaling pathway that is distinct from that activated by Fas ligation.



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Fig. 3. Expression of Bcl-2 blocks BCR-mediated but not Fas-mediated apoptosis in WEHI-231 cells. WEHI-231.5 cells and WEHI-bcl2 cells were cultured with or without 10 µg/ml of F(ab')2 fragments of goat anti-mouse IgM antibody (A). Alternatively, these cells were first treated with anti-mouse CD40 mAb HM40-3 for 24 h and then stimulated with or without anti-mouse Fas mAb Jo-2 for 24 h (B). Numbers of live and dead cells were counted by Trypan blue exclusion and percentages of dead cells were calculated. The data represent mean ± SD of triplicate cultures.

 
BCR-mediated death is defective in MRL mice
Previously, Kozono et al. demonstrated that treatment with soluble anti-IgM antibody induces rapid apoptosis in a small fraction of B cells in a Fas-independent manner (21). However, rapid BCR-mediated apoptosis has been suggested to require extensive BCR ligation and is typically induced by using immobilized anti-Ig antibody that cross-link BCR extensively (711). To investigate whether Fas is required for apoptosis of normal B cells induced by extensive BCR ligation, we purified splenic B cells from 8- to 12-week-old C57BL/6 and C57BL/6-lpr/lpr mice, and cultured them in plastic wells containing immobilized anti-IgM antibody or in medium alone. After 24 or 48 h, we collected the cells, and measured the percentages of live and dead cells by Trypan blue exclusion. Treatment with anti-IgM antibody significantly increased the percentage of dead cells in both C57BL/6 and C57BL/6-lpr/lpr B cells at both 24 and 48 h, and no significant difference was observed between normal and lpr mutant cells (Fig. 4Go). This result demonstrates that Fas is not required for BCR ligation-induced apoptosis of normal splenic B cells.



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Fig. 4. Fas is not required for BCR-mediated death of spleen B cells. Small dense B cells from 8-week-old C57BL/6 (circles) and C57BL/6-lpr/lpr mice (triangles) were cultured in the presence (open symbols) or absence (closed symbols) of immobilized anti-mouse IgM antibody. After 24 or 48 h, cells were collected. Numbers of live and dead cells were counted by Trypan blue exclusion and percentages of dead cells were calculated. The data represent mean ± SD of triplicate cultures. Representative data of three experiments are shown.

 
Next, we tested the impact of BCR ligation on B cells from MRL-+/+ and MRL-lpr/lpr mice. As a control, we used spleen B cells of C57BL/6 mice. Although treatment with immobilized anti-IgM antibody for 24 h markedly enhanced the killing of C57BL/6 B cells, the same treatment increased the percentage of dead cells in both MRL-+/+ and MRL-lpr/lpr B cells only marginally (Fig. 5Go). Moreover, the percentage of dead cells was significantly reduced in MRL B cells comparing to C57BL/6 mice by prolonged treatment with anti-Ig for 48 h. This result indicated that BCR-mediated death is defective in both MRL-+/+ and MRL-lpr/lpr B cells, and suggested that the MRL background carries a defect in BCR-mediated apoptosis.



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Fig. 5. BCR-mediated apoptosis is defective in both MRL-+/+ and MRL-lpr/lpr mice. Small dense B cells from 12-week-old C57BL/6, MRL-+/+ (MRL/+) and MRL-lpr/lpr (MRL/lpr) mice were cultured for 24 (A) or 48 (B) h with or without immobilized anti-IgM antibody. Number of live and dead cells were counted by Trypan blue exclusion and percentages of dead cells were calculated. The data represent mean ± SD of triplicate cultures. Representative data of three experiments are shown.

 
Finally, we assessed whether MRL B cells have defects in expression or signaling function of BCR. However, surface expression of IgM on CD3 spleen cells of both MRL-+/+ and MRL-lpr/lpr mice was comparable to that of C57BL/6 and C57BL/6-lpr/lpr mice (Fig. 6AGo), indicating that BCR expression is not defective in MRL B cells. Moreover, treatment with anti-IgM antibody induces a similar level of phosphorylation of various cellular substrates including ERK in B cells of MRL-+/+ mice or C57BL/6 mice (Fig. 6B and CGo), indicating that MRL B cells do not carry a generalized defect in BCR signaling. To further assess whether MRL B cells carry a defect in stimulatory signaling by extensive BCR ligation, we tested cell cycle entry of B cells cultured with or without immobilized anti-IgM antibody. Since immobilized anti-IgM antibody induces death of almost all the C57BL/6 B cells, we blocked B cell apoptosis by treatment with anti-CD40 antibody. Treatment with anti-CD40 antibody alone induced cell cycle entry to the S phase only a small fraction of B cells (Fig. 7Go). However, treatment with immobilized anti-IgM together with anti-CD40 efficiently induced cell cycle entry in both MRL-+/+ and C57BL/6 B cells. This result indicated that treatment with anti-CD40 antibody alone is not sufficient for efficient cell cycle entry of B cells, and that extensive BCR ligation by immobilized anti-Ig antibody induces cell cycle entry of anti-CD40-stimulated B cells of both MRL and C57BL/6 mice. Therefore, the stimulatory effect of extensive BCR ligation on cell cycle entry was not defective in MRL B cells. MRL B cells appear to have a specific defect in an apoptotic signaling pathway activated by extensive BCR ligation.



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Fig. 6. Expression and signaling function of BCR in MRL B cells. (A) Surface expression of BCR. Total spleen cells from 8-week-old C57BL/6, C57BL/6-lpr/lpr, MRL-+/+ and MRL-lpr/lpr mice were stained with phycoerythrin-labeled anti-mouse IgM antibody and FITC-labeled anti-mouse CD3 mAb. Expression of IgM on CD3 cells was analyzed by flow cytometry. As controls, the same cells were stained with FITC-labeled anti-mouse CD3 mAb alone and analyzed in parallel (filled histogram). (B and C) Phosphorylation of cellular substrates by BCR ligation. Spleen B cells were purified from 8-week-old C57BL/6 and MRL-+/+ mice. Cells were treated with 20 µg/ml of F(ab')2 fragments of anti-mouse IgM antibody at 37°C for the indicated time and lysed in sample buffer. Cell lysates were separated by SDS-PAGE. (B) Proteins were analyzed by Western blotting using anti-phosphotyrosine mAb. (C) Proteins were analyzed by Western blotting using anti-phospho-MAP kinase antibody. The same blot was reprobed with anti-ERK2 antibody to document equal loading.

 


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Fig. 7. Treatment with immobilized anti-IgM antibody induces cell cycle entry of both MRL and C57BL/6 B cells in the presence of 1 µg/ml anti-CD40 mAb. Spleen B cells obtained from 8 wk-old C57BL/6, and MRL-+/+ mice were cultured with immobilized anti-IgM antibody, anti-CD40 or both for 30 (A and B) or 35 (B) h. Cells were then incubated with BrdU at 37°C for 10 min, collected and fixed with 70% ethanol. Cells were stained with FITC-labeled anti-BrdU antibody and propidium iodide (PI), and analyzed by flow cytometry. Please note that C57BL/6 B cells treated with immobilized anti-IgM were not analyzed because almost all the cells died by the treatment. (A) Cell cycle analysis by flow cytometry. Percentages of cells at the G0/G1, S and G2/M phases were indicated. (B) Percentages of cells in S phase. NT, not tested.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We demonstrate that WEHI-231 cells expressing either FADD-DN or CrmA undergo apoptosis in 48 h by BCR ligation but no longer die by Fas ligation, whereas a sizable fraction of WEHI-231 are killed by anti-Fas antibody. This indicates that expression of either FADD-DN or CrmA blocks Fas-mediated but not BCR-mediated apoptosis of WEHI-231 cells. Since FADD appears to play a role in cell proliferation as well as apoptotic signaling (33,34), FADD-DN might inhibit signaling for B cell growth and survival as well as Fas signaling. However, CrmA blocks Fas-induced apoptosis by inactivating caspase-8 and has no influence on cell proliferation (35,37,44). Since expression of CrmA also fails to block BCR-mediated apoptosis in WEHI-231, Fas signaling appears dispensable for this process. This is in agreement with the previous finding by Kozono et al. that mild BCR ligation induces apoptosis of B cells in 24 h in both C57BL/6 and C57BL/6-lpr/lpr mice, carrying a mutation in Fas, although this treatment kills only a small fraction of B cells (21). Apoptosis by extensive BCR ligation is more efficient than that induced by mild BCR ligation (711), suggesting that extensive BCR ligation generates a distinct apoptotic signaling from that induced by mild BCR ligation. However, our results demonstrate that extensive BCR ligation induces death of B cells of C57BL/6-lpr/lpr mice as efficiently as in normal C57BL/6 B cells. Fas is thus dispensable in BCR-mediated occurring rapidly within 48 h in both normal B cells and WEHI-231. Related death receptors also appear to be dispensable for BCR ligation-induced cell killing because apoptosis of BCR-ligated WEHI-231 is not inhibited by CrmA, which blocks pro-apoptotic activity of Fas-related death receptors.

In culture, extensive BCR cross-linking induces apoptosis within 48 h in normal B cells (10,11). In contrast, soluble anti-Ig antibodies that mildly ligate BCR do not induce apoptosis within 48 h in most B cells, but may induce apoptosis more slowly. Indeed, interaction with soluble antigens, which ligate BCR mildly, induces B cell apoptosis within mice over the course of several days (4). It is not possible to reproduce this process in culture because a substantial fraction of normal B cells die even in the absence of BCR ligation within 48 h. B cell apoptosis induced by soluble antigens within mice has been shown to require Fas (20), whereas our data presented here demonstrate that Fas is not essential for apoptosis induced by immobilized anti-Ig antibody. Extensive BCR ligation may thus be required for activation of Fas-independent apoptosis. This is in agreement with the finding that Fas is not essential for clonal deletion of self-reactive B cells by membrane-bound antigens that cross-link BCR extensively (45). Taken together, BCR ligation appears to activate both Fas-dependent and -independent pathways to apoptosis. The Fas-dependent apoptosis takes several days, whereas the Fas-independent apoptosis is rapid but requires strong BCR ligation. This is in contrast to TCR ligation-induced apoptosis of mature T cells, which requires Fas in culture and within mice (1219). In both T and B cells, antigen receptor signaling can activate Fas-dependent apoptosis by up-regulating expression of Fas and FasL (1219,46). However, mature B cells differ from mature T cells in that mature B cells but not mature T cells are capable of activating Fas-independent apoptosis by antigen receptor ligation.

Since BCR ligation induces rapid apoptosis of WEHI-231 cells independently of Fas signaling, BCR-mediated apoptosis of WEHI-231 may be a useful tool to analyze the Fas-independent pathway of BCR-mediated apoptosis. Recent studies have identified members of the TNF receptor family that can transmit apoptotic signals (e.g. DR3, DR4, DR5 and DR6) (29,31,4749). Since many of these receptors are expressed in lymphocytes, they might be involved in lymphocyte apoptosis (47). However, BCR-mediated apoptosis of WEHI-231 is not inhibited by expression of CrmA (Fig. 2Go), which blocks apoptosis mediated by Fas, DR3, DR4, DR5 and DR6, most likely by inhibiting caspase-8 (29,47). Since Fas-mediated apoptosis is blocked in WEHI-231 CrmA transfectants, expression of CrmA is sufficient for inhibiting apoptosis mediated by Fas, and probably DR3, DR4, DR5 and DR6. Therefore, BCR-mediated apoptosis of WEHI-231 does not involve members of the TNF receptor family that have a death domain.

We demonstrate here that Bcl-2 blocks BCR-mediated but not Fas-induced apoptosis in WEHI-231 cells. This finding is consistent with the notion that Fas-mediated apoptosis is distinct from Bcl-2-regulated apoptosis at least in lymphoid cells (37,50). Bcl-2-regulated apoptosis is accompanied by mitochondrial dysfunction, resulting in release of pro-apoptotic factors from mitochondria such as cytochrome c. Cytochrome c can interact with the adapter protein Apaf-1 and activate caspase-9, which then processes caspase-3. Bcl-2 and its homologs such as Bcl-xL prevent Apaf-1-mediated activation of caspase-9 (51,52). Since BCR-mediated apoptosis of WEHI-231 is blocked by Bcl-2 (Fig. 3Go) (53) or Bcl-xL (54), this process appear to involve Apaf-1-mediated caspase-9 activation and mitochondrial dysfunction (55). In contrast, Fas ligation activates caspase-8, which associates with Fas via the adapter molecule FADD and is capable of activating caspase-3 without inducing mitochondrial dysfunction in many cell types (type I cells). In other cell types (type II cells) such as primary hepatocytes, however, Fas has been reported to induce apoptosis via mitochondrial dysfunction (56). Since Bcl-2 is not able to inhibit Fas-mediated apoptosis in WEHI-231 cells, they are type I cells and Fas-mediated apoptosis of WEHI-231 is distinct from Bcl-2-regulated apoptosis such as that induced by BCR ligation. Taken together, Fas-mediated apoptosis is distinct from BCR-mediated apoptosis in WEHI-231.

The defect in Fas (lpr mutation) plays an important role in the development of the severe autoimmune disease in MRL-lpr/lpr mice because it occurs in MRL-lpr/lpr but not MRL-+/+ mice (23,57). However, lpr mice with other backgrounds, such as C57BL/6, do not develop severe autoimmune disease, indicating that the defects in the MRL background are also required for the development of the severe autoimmune disease in MRL-lpr/lpr mice. We demonstrate here that the MRL background carries a Fas- independent defect in BCR-mediated apoptosis. Since MRL B cells show normal expression of BCR, normal phosphorylation of cellular substrates upon BCR ligation (Fig. 6Go) and normal response to a proliferation-inducing signal generated by extensive BCR ligation (Fig. 7Go), they carry a restricted defect in apoptotic signaling but not a general defect in BCR signaling. This defect may be involved in development of autoimmune disease by allowing the survival of self-reactive B cells even in the presence of self-antigens. It is possible that the MRL background carries additional defects which promote development of autoimmune disease. As discussed above, apoptosis induced by antigen receptor ligation can involve both Fas-dependent and -independent pathways. The Fas-independent defect in BCR-mediated apoptosis in the MRL background may thus synergize with the defect in Fas (lpr mutation), resulting in severe defects in antigen-induced deletion of self-reactive B cells.

We and others have previously demonstrated that BCR-mediated apoptosis is defective in NZB and (NZBxNZW)F1 mice, which are also prone to systemic autoimmune disease, although these studies assessed only the rapid BCR ligation-induced apoptosis, which is independent of Fas (7,10,21). Moreover, autoimmune disease-prone BXSB mice (58) and patients with SLE (59, 60) overexpress CD40 ligand on lymphocytes. Since CD40 signaling blocks BCR-mediated apoptosis (811,61,62), BXSB mice and patients with SLE may also have a defect in BCR-mediated apoptosis. Since BCR-mediated apoptosis is reduced in multiple independent mouse lines prone to systemic autoimmune diseases and is thought to be abnormal in SLE patients, this defect may play an important role in the pathogenesis of systemic autoimmune diseases.


    Acknowledgments
 
We thank Drs K. Maruyama (Tokyo Medical and Dental University), H. Yagita (Juntendo University), M. Seto (Aich Cancer Center Research Institute) and D. C. S. Huang (WEHI) for reagents. This work was supported by grants from the Ministry of Education, Science, Sport and Culture, Ministry of Health and Welfare, the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Drug ADR Relief, R & D Promotion and Product Reviews of Japan, and Atsuko Ohuchi Memorial Research Fund (T. T), and the Leukemia Society of America, the Cancer Research Institute (New York) and the Dr Josef Steiner Cancer Research Foundation and the NHMRC (A. S.).


    Abbreviations
 
BCR B cell antigen receptor
FasL Fas ligand
SLE systemic lupus erythematosus
PE phycoerythrin
TNF tumor necrosis factor

    Notes
 
The first two authors contributed equally to this work.

Transmitting editor: S.-I. Nishikawa

Received 15 October 1999, accepted 4 January 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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