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. 2002 Jun;128(3):436–443. doi: 10.1046/j.1365-2249.2002.01878.x

The molecular mechanism in activation-induced cell death of an Ag-reactive B cell clone

T HAMANO 1, T IWASAKI 1, A OGATA 1, N HASHIMOTO 1, E KAKISHITA 1
PMCID: PMC1906264  PMID: 12067298

Abstract

TPA-1 is a subclone of B cell hybridomas established by somatic hybridization using B cells of A/J mice immunized with TNP-LPS, and expresses a receptor for TNP on the cell membrane. The present study showed that TPA-1 was induced to apoptotic cell death upon treatment with TNP-BSA. Therefore, TPA-1 is considered to provide a good model for the study on activation-induced cell death of mature B cells induced by soluble antigen. TNP-BSA treatment caused the generation of a large amount of intracellular reactive oxygen species (ROS) of TPA-1, and the addition of the monovalent thiol-reactive compound: monochlorobimane (MCB) rescued it from apoptosis as well as the antioxidant reagent: N-acetyl-l-cysteine. Furthermore, MCB markedly inhibited the generation of ROS and prevented the disruption of mitochondrial membrane potential that was induced by TNP-BSA treatment. In addition, it counteracted the effect of TNP-BSA on the expression of the Bcl-2 family, resulting in down-regulation of Bax and Bad and up-regulation of Bcl-XL. Taken together, these results suggest strongly that oxidative stress of mitochondria may be involved directly in apoptotic cell death by engagement of antigen receptors on mature B cells with soluble antigen.

Keywords: activation-induced cell death, B cell clone, Bcl-2 family, mitochondria, reactive oxygen species

INTRODUCTION

Apoptosis is physiological cell death occurring commonly during lymphoid development, which is morphologically distinguishable from necrosis [1,2]. It is well known that a large pool of newly formed B cells is produced which expresses a broad array of surface IgM receptors, including self-antigens [3]. Elimination of self-reactive B cells appears to occur at the immature stage of B cell development, and depends on the interaction of the surface IgM receptor with self-antigens; low affinity interactions with the IgM receptor are sufficient to induce growth inhibition, whereas high affinity interactions are required to induce clonal expansion of mature B cells [4]. Cross-linking of surface IgM with anti-IgM antibodies leads the immature B cell line: WEHI-231 to undergo growth arrest and apoptotic cell death [5,6]. Therefore, it has been extensively used as a model system for the study on the mechanism by which engagement of surface IgM results in activation-induced cell death (AICD). Recent findings show that mature B cells can also be induced to apoptosis upon stimulatory conditions such as extensive surface IgM ligation with immobilizing antibodies [7–9] and MHC class II cross-linking [10]. Antigen-specific B cells in the germinal centre are killed rapidly by apoptosis in situ when they encounter soluble antigen [11]. These findings suggest that apoptosis may affect lymphoid cells throughout their ontogeny, and contribute to the maintenance of peripheral tolerance as well as more generally to the regulation of immune responses. However, the signalling mechanism in AICD of mature B cell, but not immature B cells, is not understood fully.

Recent evidence indicates that mitochondria is directly involved in apoptotic cell death [12,13]. In brief, early after induction of apoptosis, mitochondrial permeability transition (PT) and loss of mitochondrial membrane potential (ΔΨm) can be observed, resulting in releasing the pro-apoptotic protein cytochrome C and apoptosis-inducing factor (AIF) from the mitochondrial intermembrane space to the cytosol. Some caspases and AIF are themselves able to induce mitochondrial PT and thus amplify mitochondria activation [14,15]. The integrity of mitochondria is regulated by proto-oncogenes of the Bcl-2 family. Namely, anti-apoptotic family members such as Bcl-2 and Bcl-XL protect mitochondria against various apoptotic conditions, while pro-apoptotic family members such as Bax, Bad, Bak, Bid, Bik and Bim induce the release of cytochrome C from mitochondria and the loss of ΔΨm[13]. In addition, Bcl-2-like proteins inhibit caspase adaptors, and pro-apoptotic molecules that share only a single Bcl-2 homology domain, BH3, bind to the anti-apoptotic Bcl-2 family member and promote apoptosis by blocking its function. Recently, it has been reported that engagement of B cell receptors (BCR) on CD40L-activated B cells induces breakdown of ΔΨm, which leads to processing of caspase 9 and subsequent activation of downstream effector caspases [16]. Intracellular reactive oxygen species (ROS) have been found to mediate apoptosis in several model systems including excitotoxic neural cell death [17], PMA-induced death of neutrophils [18], glucocorticoid-mediated cell death of thymocytes [19] and HIV-induced death of T cells [20], as well as in the case of the WEHI-231 cell line treated with anti-IgM [21]. Because the intracellular reduction/oxidation (REDOX) system can regulate mitochondrial PT [22], it is possible that ROS are involved in apoptosis by mediating mitochondrial PT. However, little is known about the molecular mechanisms of signal transduction pathways linking BCR to sites of intracellular ROS generation. Both the requirement of intracellular ROS for activation of downstream target proteins and the mechanisms of ROS generation are subjects of ongoing research.

In this paper, we established a model system for the study of AICD of mature B cells by using the TPA-1 cell line, an antigen-reactive B cell clone established by somatic hybridization, and investigated the molecular mechanism in the process of AICD. We describe here that the generation of intracellular ROS produced by ligation of B cell receptor, resulting in the disturbance of the intracellular REDOX system, may be directly involved in the induction of apoptotic cell death of B cells activated with soluble antigen.

MATERIALS AND METHODS

Antigen-reactive B cell clone

TPA-1 is a subclone of B cell hybridomas established by somatic hybridization between B cells in the spleen of A/J mice immunized with TNP-LPS and a HAT medium-sensitive mutant of the B lymphoma line, as described previously [23]. It expresses a receptor for TNP (TNP-R) on the cell membrane, as well as MHC class II, CD25, CD40, CD45R and CD71 [24]. The cells were grown continuously in RPMI 1640 medium supplemented with 5% FCS (MA Bioproducts, Walkersville, MD, USA), 2 mml-glutamine, 5 × 10–4m 2-ME, 100 μg/ml streptomycin, 100 μg/ml penicillin and 0·25μg/ml amphotericin B. Usually, TPA-1 (5 × 104 cells/ml) was treated with TNP-BSA (20μg/ml) for various periods in complete RPMI-1640 medium and was analysed for the study of apoptotic changes.

Reagents

BSA and N-acetyl-l-cysteine (NAC) were purchased from Sigma Chemical Co., St Louis, MO, USA; 2,4,6-trinitrobenzenesulphonic acid from Wako Pure Chemical Inc., Osaka, Japan; TNP-alanine (TNP-ALA) from Research Organics Inc., Cleveland, OH, USA; 3,3′-dihexyloxacarbocyanine iodide (DiOC6), 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) and monochlorobimane (MCB) from Molecular Probes, Eugene, OR, USA; each affinity-purified rabbit antibody to Bax, Bad and Bcl-XL from Santa Cruz Biotech, Santa Cruz, CA, USA; and the caspase inhibitor z-VAD-fmk from Calbiochem, La Jolla, CA, USA. TNP-BSA was prepared as previously described [24] and throughout used in this experiment as soluble antigen.

Flow cytometry (FCM) analysis for a quantitative assay of apoptotic cell death

For a quantitative assay of apoptotic cell death, TPA-1 was incubated with TNP-BSA (20μg/ml) for 72 h and suspended in hypotonic DNA staining buffer containing 50μg/ml propidium iodide (PI), 0·1% sodium citrate and 0·1% Triton X-100. It was then subjected to FCM analysis as reported previously [25], and apoptotic cell nuclei containing hypodiploid DNA were enumerated as a percentage of the total population.

Assessment of intracellular ROS levels and changes in ΔΨm

The level of intracellular ROS was determined by analysis with a FACScan using H2DCFDA as a fluorescent probe [26]. In brief, cells (5 × 104/ml) were treated for 24 h under various conditions, and were loaded with 5 μm H2DCFDA for 30 min at 37°C, then the fluorescence intensity of 2 × 104 cells from each sample was measured by FCM. To evaluate ΔΨm, cells were incubated with 50 nm DiOC6 for 30 min at 37°C and were analysed by FCM [26].

Conforcal laser scanning microscopy (CLSM)

Analysis with CLSM was performed to evaluate changes in each expression of Bax, Bad and Bcl-XL at a single cell level. Briefly, after incubation with TNP-BSA (20μg/ml) for 24 h, cells were fixed with 0·5% paraformaldehyde in PBS at 4°C overnight, and were permeabilized with 0·1% saponin in PBS. Then, cells were reacted with each affinity-purified rabbit antibody to Bax, Bad and Bcl-XL for 3h at 4°C, followed by incubation with FITC-F(ab′)2 fragments of affinity-purified goat antirabbit IgG (Cappel Laboratories, Cochranville, PA, USA) for 1h at 4°C. These cells were analysed with a Leica CLSM (Deerfield, IL, USA). As a control, cells were reacted with affinity-purified rabbit normal IgG, followed by incubation with FITC-F(ab′)2 fragments of affinity-purified goat antirabbit IgG.

Assay of the caspase 3 activity

The activation of caspase 3 upon TNP-BSA treatment was determined in cellular extracts with a caspase 3 assay using Ac-DEVD-AFC as a substrate. Briefly, TPA-1 was treated with TNP-BSA (20 μg/ml) for 48 h, washed three times with PBS, and lysed in cell lysis buffer (10 mm HEPES/KOH, 2 mm EDTA, 5 mm DTT, 1 mm PMSF, 10μg/ml pepstatin A, 20μg/ml leupeptin and 10μg/ml aprotinin). Cell extracts were then centrifuged at 13 000 r.p.m. for 10 min, and supernatants were transferred to new tubes. A total of 100μg protein was incubated with 50μm Ac-DEVD-AFC in a 96-well plate at 37°C for 60 min. The release of fluorogenic AFC was measured by a spectrofluorometer using an exitation wavelength of 400 nm and an emission wavelength of 505 nm.

RESULTS

Effects of TNP-BSA treatment on TPA-1

The cell growth of TPA-1 is markedly inhibited in the presence of 20μg/ml of TNP-BSA (data not shown). Therefore, we attempted to investigate the mechanism by which TNP-BSA treatment inhibits its cell growth and performed a quantitative assay of apoptotic cell death by FCM with PI staining. As shown in Fig. 1, a large number of the nuclei of cells treated with TNP-BSA for 72h were hypodiploid and apoptotic: a level of apoptotic cell death reached the maximum after 72 h, although it was slight after 24h and modest after 48 h, respectively (data not shown). However, its cell death was almost completely rescued by the addition of TNP-ALA (5μg/ml), a non-antigenic TNP-conjugate, indicating that engagement of surface TNP-R on TPA-1 directly mediates its apoptotic cell death. Recent work has shown that anti-oxidants inhibit apoptosis in a variety of cells, demonstrating that oxidative stress may be involved during apoptotic cell death. NAC is an antioxidant with direct radical scavenging capabilities that can also serve as a precursor for glutathione [27]. To investigate whether oxidative stress was required for the induction of apoptotic cell death, we examined the effect of NAC on the induction of apoptotic cell death of TPA-1. As shown in Fig. 1, the addition of NAC blocked its apoptosis in a dose-dependent fashion. The monovalent substitution of thiols: MCB has been described to function as a superoxide dismutase mimetic, prevent opening of the PT pore and exhibit an efficient inhibitory effect on some apoptotic cascades [28]. Therefore, we attempted to examine the effect of MCB on the induction of apoptosis of TPA-1, and found that the addition of MCB (2 μm and 5 μm) rescued TPA-1 from apoptosis as well as NAC (Fig. 1), although it modestly rescued at the dose of 1μm and was toxic to cells at the dose of 10μm (data not shown).

Fig. 1.

Fig. 1

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Induction of apoptotic cell death by TNP-BSA treatment. TPA-1 was treated with TNP-BSA (20 μg/ml) for 72 h in the presence or absence of TNP-ALA, NAC and MCB. A quantitative assay of apoptotic cell death was performed by FCM with PI staining. Data are representative of five different experiments.

The generation of intracellular ROS upon treatment with TNP-BSA

It has been reported recently that intracellular ROS mediate apoptosis in a number of cell types. Therefore, a level of intracellular ROS upon treatment with TNP-BSA was measured by FCM using H2DCFDA as an indicator. As shown in Fig. 2, TNP-BSA treatment caused the generation of intracellular ROS in a dose-dependent fashion, and it showed a sixfold increase at a dose of 20μg/ml compared with a medium control, but its level was diminished in the presence of TNP-ALA. The result indicates that engagement of TNP-R directly causes the generation of a high level of intracellular ROS. More importantly, MCB blocked the generation of intracellular ROS in a dose-dependent fashion, although the caspase inhibitor z-VAD-fmk did not show any effect on its generation (Fig. 2). The results suggest strongly that a high level of intracellular ROS may be a critical event during the apoptotic process.

Fig. 2.

Fig. 2

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Measurement of a level of intracellular ROS by FCM. TPA-1 was treated with various concentrations of TNP-BSA for 24 h in the presence or absence of TNP-ALA, MCB and z-VAD-fmk. A level of intracellular ROS was measured by FCM with H2DCFDA (5 μm) as an indicator. Data are shown as the mean ± s.e.m. of five different experiments.

Reduction in ΔΨm upon treatment with TNP-BSA

It has been reported that cells rapidly lose ΔΨm during apoptosis, and this process can be measured by FCM with DiOC6, which accumulates in mitochondria that has an intact ΔΨm [26]. As in Fig. 3, TNP-BSA treatment diminished a level of its incorporation in a dose-dependent fashion, with approximately 75% reduction observed at 20μ/ml of TNP-BSA compared with a medium control. The result suggests that TNP-BSA treatment results in a remarkable reduction in ΔΨm of TPA-1. In contrast, the addition MCB prevented the ΔΨm disruption that was induced by TNP-BSA in a dose-dependent fashion, although z-VAD-fmk did not show any effect on the ΔΨm disruption under the same conditions (Fig. 3). Collectively, these results suggest strongly that the thiol oxidation in mitochondria may be a critical event during the apoptotic process. Recently, Mn (III) tetrakis porphyrin, a superoxide dismutase mimetic, has been reported to protect superantigen-activated T cells from ROS generation and loss of ΔΨm resulting in rescue from AICD [29]. Our present finding is consistent with the previous report, supporting the idea that the disruption of ΔΨm may be critical for the induction of apoptosis.

Fig. 3.

Fig. 3

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Measurement of a level of Δÿm by FCM. TPA-1 was treated with various concentrations of TNP-BSA for 24h in the presence or absence of TNP-ALA, MCB and z-VAD-fmk. A level of ΔΨm was determined by FCM with DiOC6 (50 nm) as an indicator. Data are shown as the mean ± s.e.m. of five different experiments.

Changes in each expression of Bax, Bad and Bcl-XL in TPA-1

An increasing number of genes that belong to the Bcl-2 family have been discovered that are involved in regulating apoptosis [30]. Subsequent studies have demonstrated that Bcl-XL inhibits apoptosis in the response to a variety of different stimuli as well as Bcl-2, including irradiation, chemotherapeutic agents, viral infection, growth factor withdrawal and glucocorticoids. In contrast, several additional Bcl-2 related proteins, including Bax, Bad, Bak, Bid and Bim have been shown to promote apoptosis [30]. Therefore, we attempted to determine changes in each expression of Bax, Bad and Bcl-XL in TPA-1 upon treatment with TNP-BSA by using a CLSM at a single cell level. As shown in Fig. 4, TNP-BSA treatment caused up-regulation of each expression of Bax and Bad, but down-regulation of Bcl-XL, while the addition of MCB (2μm) counteracted its effect resulting in down-regulation of Bax and Bad, but up-regulation of Bcl-XL. The results suggest that intracellular ROS may regulate each expression of Bax, Bad and Bcl-XL.

Fig. 4.

Fig. 4

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Changes in each expression of Bax, Bad and Bcl-XL. TPA-1 was treated with TNP-BSA (20μg/ml) for 24h in the presence or absence of MCB (2μm). Changes in each expression of Bax, Bad and Bcl-XL were determined by CLSM at a single cell level. Data are representative of three different experiments.

Caspase dependence of apoptosis

Apoptosis triggered by engagement of B cell receptors with anti-IgM antibodies has been shown to be dependent on the activation of caspases [16,31,32]. To examine whether apoptosis of TPA-1 induced by TNP-BSA treatment requires the activation of caspases, the broad caspase inhibitor z-VAD-fmk was added to the culture. As shown in Fig. 5, z-VAD-fmk prevented its apoptotic cell death in a dose-dependent fashion. Next, the activation of caspase 3 was determined by using a DEVD peptide, a substrate relatively selective for caspase 3. TNP-BSA treatment for 48h caused a remarkable increase in the activation of caspase 3, while its activation was decreased significantly in the presence of MCB (Fig. 6). Taken together, these findings suggest that apoptotic cell death of TPA-1 induced by treatment with TNP-BSA is caspase-dependent, and intracellular ROS may play an important upstream role in activating caspase 3.

Fig. 5.

Fig. 5

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Effects of z-VAD-fmk on apoptotic cell death. TPA-1 was treated with TNP-BSA (20 μg/ml) for 72 h in the presence of various concentrations of z-VAD-fmk and a comparable dose of DMSO. A quantitative assay of apoptotic cell death was performed by FCM with PI staining. Data are representative of five different experiments.

Fig. 6.

Fig. 6

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Assay of the caspase 3 activity. TPA-1 was treated with TNP-BSA (20 μg/ml) for 48 h in the presence or absence of TNP-ALA, MCB and z-VAD-fmk. The activation of caspase 3 was determined by detection of cleavage Ac-DEVD-AFC. Data are shown as the mean ± s.e.m. of five different experiments.

DISCUSSION

The immature B lymphoma cell line WEHI-231 has mainly been used for a model study on antigen-induced apoptosis of B cells upon stimulation with anti-IgM antibodies [5,6]. However, it has been reported recently that mature B cells are also triggered to apoptosis after cross-linking of surface IgM receptors using plastic-immobilized antibodies or biotinylated antibodies with avidin [7,8,16,31]. Accumulating evidence suggests that AICD may affect B cell lineages throughout its ontogeny and contribute to the regulation of immune responses. In the present study, we investigated the molecular mechanism by which mature B cells result in apoptotic cell death through cross-linking of surface IgM receptors with soluble antigen, but not anti-IgM antibodies, by using TPA-1 as a model for antigen-reactive B cells. TPA-1 is a subclone of B cell hybridomas established between B cells in the spleen of A/J mice immunized with TNP-LPS and a HAT medium-sensitive B lymphoma mutant line, as described previously [24]. It expresses characteristic surface markers of mature B cells, such as IgM, MHC class II, CD25, CD40, CD45R and CD71. It also exhibits TNP-R on the cell membrane derived from TNP-reactive B cells of mice immunized with TNP-LPS used for cell fusion [24]. The obvious advantage of using this kind of a B cell hybridoma is that it may possess functional genes from normal B cells used for cell fusion, in addition to its monoclonal origin. Thus, it is capable of providing a good model system for the study on immune responses of mature B cells as previously reported [23,24,33]. In this paper, TPA-1 was shown to be induced to apoptotic cell death upon treatment with TNP-BSA, and its apoptosis was initiated directly by cross-linking of surface TNP-R with TNP-BSA, as the addition of a non-antigenic reagent, TNP-ALA, blocked its apoptosis almost completely. Recently, CD40 signalling has been reported to block apoptosis in B cells after BCR cross-linking [34,35]. In this study, the addition of anti-CD40 antibodies significantly rescued TPA-1 from apoptosis induced by TNP-BSA treatment (data not shown). Taken together, the result is consistent with the previous report demonstrating that antigen-specific B cells with high affinity are rapidly killed by apoptosis in situ when they encounter soluble antigen in germinal centres [11].

It has been shown that intracellular ROS may serve as a second messenger linking CD40 engagement on B cells to downstream activation events [36], and act in signal transduction pathways linking TNF family receptors on T cells to the activation of NF-κB [37]. However, recent findings have demonstrated that a high level of intracellular ROS promotes cytotoxicity via mechanisms very similar to the alterations observed in apoptotic cells, including plasma and nuclear membrane blebbing, elevations in the intracellular Ca2+ concentration, and protease activation [38]. Oxidative signals for various types of cells can originate both extracellularly and intracellularly. Intracellular sources of ROS include mitochondrial oxidation, the microsomal cytochrome P450 system and plasma membrane-NAD (P) h oxidases. ROS within mitochondria are of particular importance during the apoptotic process, since cellular activation requires increased oxidative phosphorylation. The electron transport chain may become uncoupled, resulting in the direct reduction of O2 by electrons that is normally directed towards oxidative phosphorylation. ROS are produced by the increased rates of respiration within mitochondria of the activated cells, because it is known to be a byproduct of the mitochondrial electron transport chain. In the present study, we found that TNP-BSA treatment caused the generation of a large amount of intracellular ROS through engagement of TNP-R. Our findings provide evidence that the ΔΨm disruption through the generation of intracellular ROS constitutes an important pathway in BCR-induced apoptosis of mature B cells. To date, there is no direct evidence indicating that the activation of caspases requires intracellular ROS. Instead, caspases may function as negative regulators of the ROS production, because the activation of caspases may counteract its production [39,40]. However, our observation that the caspase inhibitor z-VAD-fmk could not prevent the intracellular ROS generation indicates that its generation is upstream of the caspase activation in BCR-induced apoptotic cell death of B cells; this is consistent with recent reports demonstrating that the intracellular ROS generation is early event, whereas the cleavage of poly (ADP-ribose) polymerase is a late event [41,42]. Remarkably, MCB blocked its generation and prevented the loss of ΔΨm that was induced by TNP-BSA treatment, resulting in rescue of TPA-1 from apoptotic cell death. These results are consistent with previous report that MCB is an efficient inhibitor of the apoptotic cascade in thymocytes induced by treatment with diamide, glucocorticoid, irradiation and topoisomerase inhibition [28]. While, it has been speculated that AICD of T cells results from stimulation of certain death gene programs [43]. Therefore, it may be the possibility that a high level of intracellular ROS results in the activation of some genes responsible for apoptosis, conceivably through an oxidative stress-responsive nuclear transcription factor such as NF-κB.

The Bcl-2 family members act on mitochondria to regulate apoptosis presumably by influencing PT, and mitochondria undergoing PT can release several factors including a protease and a protease activator: cytochrome C [13,22]. In the present study, TNP-BSA treatment resulted in up-regulation of each expression of the pro-apoptotic Bax and Bad, but down-regulation of anti-apoptotic Bcl-XL in TPA-1. While, the superoxide dismutase mimetic MCB counteracted these effects, consisting with evidence that it rescued TPA-1 from apoptosis. In many experimental systems, the intracellular REDOX potential, which is largely determined by the ratio of oxidized and reduced glutathione, regulates the propensity of cells to undergo apoptosis [44,45]. Also, there is an oxidative shift in the cellular REDOX state which thereby modifies the nature of the stimulatory signal and which results in cell death as opposed to proliferation [46]. Taken together, the present study suggests strongly that the generation of a large amount of intracellular ROS may be involved directly in the disturbance of intracellular REDOX potentials, resulting in AICD of mature B cells stimulated with soluble antigen.

Acknowledgments

We are grateful to Mr K. Kaji, Mr H. Kubo, Mr M. Yagi, Mr K. Hamada and Ms H. Seki of Common Research Laboratory of Hyogo College of Medicine for their help in performing the present experiment.

This study was supported in part by the grant from the Osaka Medical Research Foundation for Incurable Diseases.

REFERENCES

  • 1.Steller H. Mechanisms and genes of cellular suicide. Science. 1995;267:1445–9. doi: 10.1126/science.7878463. [DOI] [PubMed] [Google Scholar]
  • 2.Jacobson MD, Wei M, Raff MC. Programmed cell death in animal development. Cell. 1997;88:347–54. doi: 10.1016/s0092-8674(00)81873-5. [DOI] [PubMed] [Google Scholar]
  • 3.Nossal GJV. Negative selection of lymphocyte. Cell. 1994;76:229–39. doi: 10.1016/0092-8674(94)90331-x. [DOI] [PubMed] [Google Scholar]
  • 4.Hartley SB, Cooke MP, Fulcher DA, et al. Elimination of self-reactive B lymphocytes proceeds in two stages: arrested development and cell death. Cell. 1993;72:325–35. doi: 10.1016/0092-8674(93)90111-3. [DOI] [PubMed] [Google Scholar]
  • 5.Harsbold J, Klaus GG. Anti-immunoglobulin antibodies induce apoptosis in immature B cell lymphomas. Eur J Immunol. 1990;20:1685–90. doi: 10.1002/eji.1830200810. [DOI] [PubMed] [Google Scholar]
  • 6.Hibner U, Benhamous LE, Haury M, Cazenave PA, Sarthou P. Signaling of programmed cell death induction in WEHI-231 B lymphoma cells. Eur J Immunol. 1993;23:2821–5. doi: 10.1002/eji.1830231115. [DOI] [PubMed] [Google Scholar]
  • 7.Parry SL, Holman M, Hasbold J, Klaus GGB. Plastic-immobilized anti-μ or anti-δ antibodies induce apoptosis in mature murine B lymphocytes. Eur J Immunol. 1994;24:974–9. doi: 10.1002/eji.1830240429. [DOI] [PubMed] [Google Scholar]
  • 8.Parry SL, Hasbold J, Holman M, Klaus GGB. Hypercross-linking surface IgM and IgD receptors on mature B cells induces apoptosis that is reversed by costimulation with IL-4 and anti-CD40. J Immunol. 1994;152:2821–9. [PubMed] [Google Scholar]
  • 9.Chaouchi N, Vazquez A, Galanaud P, Leprince C. B cell antigen receptor-mediated apoptosis. Importance of accessory molecules CD19 and CD22, and of surface IgM cross-linking. J Immunol. 1995;154:3096–104. [PubMed] [Google Scholar]
  • 10.Newell MK, Vanderwall J, Beard S, Freed JH. Ligation of major histcompatibility complex class II molecules mediates apoptotic cell death in resting B lymphocytes. Proc Natl Acad Sci USA. 1993;90:10459–10463. doi: 10.1073/pnas.90.22.10459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Pulendran B, Kannourakis G, Nouri S, Smith KGS, Nossal GJV. Soluble antigen can cause enhanced apoptosis of germinal-centre B cells. Nature. 1995;375:331–4. doi: 10.1038/375331a0. [DOI] [PubMed] [Google Scholar]
  • 12.Kroemer GN, Zamzami N, Susin SA. Mitochondrial control of apoptosis. Immunol Today. 1997;18:44–51. doi: 10.1016/s0167-5699(97)80014-x. [DOI] [PubMed] [Google Scholar]
  • 13.Green DR, Reed JC. Mitochondria and apoptosis. Science. 1998;281:1309–12. doi: 10.1126/science.281.5381.1309. [DOI] [PubMed] [Google Scholar]
  • 14.Liu X, Kim CN, Yang J, Jemmerson R, Wang X. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome C. Cell. 1996;86:147–57. doi: 10.1016/s0092-8674(00)80085-9. [DOI] [PubMed] [Google Scholar]
  • 15.Susin SA, Lorenzo HK, Zamzami N, et al. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature. 1999;397:441–6. doi: 10.1038/17135. [DOI] [PubMed] [Google Scholar]
  • 16.Berard M, Mondiere P, Casamayor-Palleja M, et al. Mitochondria connects the antigen receptor to effector caspases during B cell receptor-induced apoptosis in normal human B cells. J Immunol. 1999;163:4655–62. [PubMed] [Google Scholar]
  • 17.Patel M, Day BJ, Crapo JD, Fridovich I, McNamara JO. Requirement for superoxide in excitotoxic cell death. Neuron. 1996;16:345–55. doi: 10.1016/s0896-6273(00)80052-5. [DOI] [PubMed] [Google Scholar]
  • 18.Fadeel B, Ahlin A, Henter JI, Orrenius S, Mampton MB. Involvement of caspases in neutrophil apoptosis: regulation by reactive oxygen species. Blood. 1995;92:4808–18. [PubMed] [Google Scholar]
  • 19.Buttke TM, Sandstrom PA. Redox regulation of programmed cell death in lymphocytes. Free Radic Res. 1995;22:389–97. doi: 10.3109/10715769509147548. [DOI] [PubMed] [Google Scholar]
  • 20.Dobmeyer TS, Findhammer S, Dobmeyer JM, et al. Ex vivo induction of apoptosis in lymphocytes is mediated by oxidative stress: role for lymphocyte loss in HIV infection. Free Radic Biol Med. 1997;22:775–85. doi: 10.1016/s0891-5849(96)00403-0. [DOI] [PubMed] [Google Scholar]
  • 21.Fang W, Rivard JJ, Ganser JA, et al. Bcl-XL rescues WEHI 231 B lymphocytes from oxidant-mediated death following diverse apoptotic stimuli. J Immunol. 1995;155:66–75. [PubMed] [Google Scholar]
  • 22.Kroemer G, Reed JC. Mitochondrial control of cell death. Nature Med. 2000;6:513–9. doi: 10.1038/74994. [DOI] [PubMed] [Google Scholar]
  • 23.Hamano T, Kim KJ, Leiserson WM, Asofsky R. Establishment of B cell hybridomas with B cell surface antigens. J Immunol. 1982;29:1403–6. [PubMed] [Google Scholar]
  • 24.Hamano T, Murata Y, Yamasaki T, Yasuda Y, Iwasaki T, Nagai K. Establishment of an antigen-specific B cell clone by somatic hybridization. J Immunol. 1987;139:2556–61. [PubMed] [Google Scholar]
  • 25.Nicoletti I, Migliorati G, Pagliaci MC, Grignani F, Riccardi CA. A rapid and simple methods for measuring thymocytes apoptosis by propidium iodide staining and flow cytometry. J Immunol Meth. 1991;139:271–9. doi: 10.1016/0022-1759(91)90198-o. [DOI] [PubMed] [Google Scholar]
  • 26.Zamzami N, Marchetti P, Castedo M, et al. Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J Exp Med. 1995;181:1661–71. doi: 10.1084/jem.181.5.1661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Olsson B, Johansson M, Gabrielsson J, Bolme P. Pharmacokinetics and bioavailability of reduced and oxidized N-acetyl-cysteine. Eur J Clin Pharmacol. 1988;34:77–82. doi: 10.1007/BF01061422. [DOI] [PubMed] [Google Scholar]
  • 28.Marchetti P, Decausin D, Macho A, et al. Redox regulation of apoptosis: impact of thiol oxidation status on mitochondrial function. Eur J Immunol. 1997;27:289–96. doi: 10.1002/eji.1830270142. [DOI] [PubMed] [Google Scholar]
  • 29.Hildeman D, Mitchell T, Teague TK, et al. Reactive oxygen species regulate activation-induced T cell apoptosis. Immunity. 1999;10:735–44. doi: 10.1016/s1074-7613(00)80072-2. [DOI] [PubMed] [Google Scholar]
  • 30.Reed JC. Double identity for proteins of the Bcl-2 family. Nature. 1997;387:773–6. doi: 10.1038/42867. [DOI] [PubMed] [Google Scholar]
  • 31.Chen W, Wang HG, Srinivasula SM, Alnemri ES, Cooper NR. B cell apoptosis triggered by antigen receptor ligation proceeds via a novel caspase-dependent pathway. J Immunol. 1999;163:2483–91. [PubMed] [Google Scholar]
  • 32.Bras A, Ruiz-Vela A, Buitrago GD, Martinez AC. Caspase activation by BCR cross-linking in immature B cells. differential effects on growth arrest and apoptosis. FASEB J. 1999;13:931–44. doi: 10.1096/fasebj.13.8.931. [DOI] [PubMed] [Google Scholar]
  • 33.Hamano T, Asofsky R. Functional studies on B cell hybridomas with B cell surface antigens. I. Effects of anti-immunoglobulin antibodies on proliferation and differentiation. J Immunol. 1983;130:2027–32. [PubMed] [Google Scholar]
  • 34.Lee JR, Koretzky GA. Production of reactive oxygen intermediates followingCD40 ligation correlates with c-Jun N-terminal kinase activation and IL-6 secretion in murine B lymphocytes. Eur J Immunol. 1998;28:4188–97. doi: 10.1002/(SICI)1521-4141(199812)28:12<4188::AID-IMMU4188>3.0.CO;2-B. [DOI] [PubMed] [Google Scholar]
  • 35.Tsubata T, Wu J, Honjo T. B-cell apoptosis induced by antigen receptor crosslinking is blocked by a T-cell signal through CD40. Nature. 1993;364:645–8. doi: 10.1038/364645a0. [DOI] [PubMed] [Google Scholar]
  • 36.Gordon J, Pound JD. Fortifying B cells with CD154: an engaging tale of many hues. Immunology. 2000;100:269–80. doi: 10.1046/j.1365-2567.2000.00074.x. 10.1046/j.1365-2567.2000.00074.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Schreck R, Rieber P, Baeuerle PA. Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappa B transcription factor and HIV-1. EMBO J. 1991;10:2247–58. doi: 10.1002/j.1460-2075.1991.tb07761.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Buttke TM, Sandstrom PA. Oxidative stress as a mediator of apoptosis. Immunol Today. 1994;15:7–10. doi: 10.1016/0167-5699(94)90018-3. [DOI] [PubMed] [Google Scholar]
  • 39.Tan S, Sagara Y, Liu Y, Maher P, Schubert D. The regulation of reactive oxygen species production during programmed cell death. J Cell Biol. 1998;141:1423–32. doi: 10.1083/jcb.141.6.1423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Vercammer D, Beyaert R, Denecker D, et al. Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis factor. J Exp Med. 1998;187:1477–85. doi: 10.1084/jem.187.9.1477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Li PF, Dietz R, von Hardorf R. p53 regulates mitochondrial membrane potential through reactive oxygen species and induces cytochrome c-independent apoptosis blocked by Bcl-2. EMBO J. 1999;18:6027–36. doi: 10.1093/emboj/18.21.6027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Chandel NS, Schumacker PT, Arch H. Reactive oxygen species are downstream products of TRAF-mediated signal transduction. J Biol Chem. 2001;276:42728–36. doi: 10.1074/jbc.M103074200. [DOI] [PubMed] [Google Scholar]
  • 43.Shi Y, Glynn JM, Guilbert LJ, et al. Role for c-myc in activation-induced apoptotic cell death in T cell hybridomas. Science. 1992;257:212–4. doi: 10.1126/science.1378649. [DOI] [PubMed] [Google Scholar]
  • 44.Polyak K, Xia Y, Zweier JL, Kinzler KW, Vogelstein B. A model for p53-induced apoptosis. Nature. 1997;389:300–5. doi: 10.1038/38525. [DOI] [PubMed] [Google Scholar]
  • 45.Hall AG. The role of glutathione in the regulation of apoptosis. Eur J Clin Invest. 1999;29:238–45. doi: 10.1046/j.1365-2362.1999.00447.x. [DOI] [PubMed] [Google Scholar]
  • 46.Voehringer D, Hirschberg DL, Xiao J, et al. Gene microarray identification of redox and mitochondrial elements that control resistance or sensitivity to apoptosis. Proc Natl Acad Sci USA. 2000;97:2680–5. doi: 10.1073/pnas.97.6.2680. [DOI] [PMC free article] [PubMed] [Google Scholar]

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