Prevention of B cell antigen receptor-induced apoptosis by ligation of CD40 occurs downstream of cell cycle regulation
Wendelina J. M. Mackus1,2,
Susanne M. A. Lens2,4,
René H. Medema3,4,
Mark J. Kwakkenbos2,
Ludo M. Evers1,
Marinus H. J. van Oers1,
René A. W. van Lier2 and
Eric Eldering2
1 Department of Hematology and 2 Department of Experimental Immunology, Academic Medical Center, PO Box 22660, 1100 DD Amsterdam, The Netherlands 3 Department of Hematology, University Medical Center, 3584 CX Utrecht, The Netherlands 4 Present address: Department of Molecular Biology H8, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
Correspondence to: E. Eldering; E-mail: e.eldering{at}amc.uva.nl
Transmitting editor: E. A. Clark
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Abstract
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Cross-linking of the B cell antigen receptor (BCR) on germinal center B cells can induce growth arrest and apoptosis, thereby eliminating potentially autoreactive B cells. Using the Burkitt lymphoma cell line Ramos as a model, we studied the commitment to apoptosis following growth arrest, as well as how triggering of CD40 or addition of tumor necrosis factor (TNF)-
can interfere to block cell death. Both BCR triggering and direct induction of growth arrest by sodium butyrate (n-But) caused hypophosphorylation of the retinoblastoma protein (pRb), followed by apoptosis. Interestingly, although CD40 ligation or TNF-
efficiently prevented BCR-induced and n-But-induced apoptosis, these co-stimuli did not inhibit, but rather augmented, growth arrest. Analysis of cell cycle regulators showed that each apoptotic and Th stimulus distinctly affected cyclins or cyclin-dependent kinase inhibitors, indicating that growth arrest can be uncoupled from apoptosis. BCR ligation and growth arrest activated the intrinsic or mitochondrial route of apoptosis. CD40 ligation and TNF-
prevented release of cytochrome c and activation of caspase-3, which could not be explained by effects on the expression of Bcl-2, Bcl-xL or Bax. Finally, the onset of BCR-induced apoptosis occurred after 1012 h and addition of CD40 mAb or TNF-
at that point still prevented further execution of apoptosis. We conclude that in mature B cells apoptosis is not an obligatory event following growth arrest. Instead, commitment to apoptosis can be rapidly controlled by T cells via CD40 ligand and TNF-
, downstream of the pRb-regulated restriction point of the cell cycle, but prior to mitochondrial cytochrome c release.
Keywords: B lymphocytes, CD40, cell cycle, cell death, TB cell collaboration
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Introduction
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Elimination of superfluous or harmful B cells is essential to ensure B cell homeostasis as well as the specificity of the immune response and to prevent autoimmunity. Potentially autoreactive B cell clones can be purged from the repertoire by the process of clonal deletion which occurs at various stages during B cell development in bone marrow and secondary lymphoid organs (1). Clonal deletion can be mimicked in vitro by ligation of the B cell antigen receptor (BCR) on either primary germinal center B cells or mature and immature B lymphoma cell lines. Triggering of the BCR on these cells induces programmed cell death (apoptosis), which is prevented via CD40 after interaction with CD40 ligand (CD154) present on activated CD4 Th cells (2,3). In addition, tumor necrosis factor (TNF)-
, IFN-
and IFN-ß have also been reported to inhibit BCR-induced apoptosis of Ramos cells (2,4,5). Relatively little is known about the early biochemical events leading to BCR-triggered apoptosis and how apoptosis is modulated by T cell signals such as ligation of CD40.
Upon induction of apoptosis a cascade of aspartate-specific cysteine proteases is activated. These caspases can be divided into initiator and executioner caspases, and their activation results in cleavage of a distinct set of substrates, which finally leads to regulated disintegration of the cell (6). BCR-induced apoptosis has been shown to involve the executioner caspases-3 and -7 (79), which are activated independently of death receptors and caspase-8 (10). In primary B cells (8) and in mature (11) as well as immature (12) B cell lines the involvement is suggested of the intrinsic apoptosis route of cytochrome c/Apaf-1/caspase-9, connecting mitochondria to executioner caspases (1315).
In addition to leading to apoptosis, cross-linking of BCR has been shown to lead to induction of growth arrest, which is thought to precede the activation of the cell death program (1619). Commitment to cell cycle progression takes place in mid-to-late G1 at the restriction point where phosphorylation of the retinoblastoma protein (pRb) leads to inactivation of its growth suppressive function, thereby driving cells into S phase (20,21). Phosphorylation of pRb is mediated by complexes of cyclins and cyclin-dependent kinase (cdk) (2224), which in turn can be inhibited by cdk inhibitors (CKI) such as p21 (25,26) and p27 (27). Growth arrest in G1 phase of the cell cycle can also be chemically induced by sodium butyrate (n-But), a multifunctional agent which efficiently inhibits cell proliferation and induces differentiation or apoptosis in numerous cell lines and cell types (2831). n-But reversibly inhibits histone deacetylase, which results in inhibition of transcription and an arrest in G1 via, among others, up-regulation of expression of p21 (29).
In the present study, we investigated the commitment to apoptosis following G1 arrest induced by BCR cross-linking or n-But treatment of the EpsteinBarr virus-negative Burkitt lymphoma cell line Ramos. Furthermore, we addressed how and at what point apoptosis was inhibited by co-stimulatory signals. We provide evidence that apoptosis can be uncoupled from growth arrest by ligation of CD40 or addition of TNF-
. This prevention of apoptosis is effectuated downstream of cell cycle regulatory events and upstream of cytochrome c release from mitochondria.
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Methods
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Antibodies and reagents
Anti-human IgM mAb (CLB/MH15) and anti-CD40 mAb (CLB-CD40/1, clone 14G7) were produced at the CLB Sanquin Blood Foundation (CLB, Amsterdam, The Netherlands). Human rTNF-
was purchased from R & D Systems (Abingdon, UK). The cell cycle inhibitor n-But and propidium iodide (PI) were purchased from Sigma (St Louis, MO). FITC-labeled Annexin V (APOPTEST-FITC) was purchased from ImmunoQuality Products (Groningen, The Netherlands).
The following antibodies were used in the immunoblot analyses. mAb to Bcl-2 (Dako, Glostrup, Denmark), cytochrome c (7H8.2C12), pRb (G3-245), Bcl-xL and p27Kip1 (p27) (Transduction Laboratories, Lexington, KY), tubulin (Sigma), and cyclin D3 (Ab-2) (Calbiochem Novabiochem, La Jolla, CA). Polyclonal antibodies to caspase-3 proform and Bax (PharMingen, San Diego, CA), caspase-3 cleaved forms (New England Biolabs, Beverly, MA), and p21WAF1/Cip1 (p21) (C-19) (Santa Cruz Biotechnologies, Santa Cruz, CA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse Ig was from the CLB. HRP-conjugated swine anti-rabbit Ig and HRP-conjugated goat anti-rabbit Ig were both from Dako.
Cell culture and cell stimulation
The Ramos.FSA cell line, which is highly sensitive to anti-Fas mAb, is a clone derived from the human Burkitt lymphoma cell line Ramos and was previously generated in our laboratory (10). Ramos.FSA cells, which we will refer to as Ramos for brevity, were cultured in IMDM (Gibco Life Technology, Paisley, UK) supplemented with 10% (v/v) heat-inactivated FCS (ICN, Meckenhein, Germany), 100 U/ml penicillin, 100 µg/ml streptomycin and L-glutamine (Gibco).
Cells were stimulated in 24-well plates or 25-mm2 culture flasks (0.251.0 x 106/ml) with the anti-IgM mAb (5 µg/ml) or with n-But (2.5 mM) for 24 h unless indicated otherwise. Anti-CD40 mAb (either ascites dilution 1:1000 or purified mAb (concentration) or rTNF-
(25 ng/ml) was added where indicated. In all experiments identical results were obtained when using culture supernatants of the anti-CD40 hybridoma as compared to anti-CD40 mAb ascites.
Detection of apoptotic cells
Phosphatidylserine (PS) exposure on apoptotic cells was measured as described previously (32). In brief, cells were harvested and washed in ice-cold HEPES buffer (10 mM HEPES, 150 mM KCl, 1 mM MgCl2 and 1.3 mM CaCl2, pH 7.4) supplemented with 1 mg/ml glucose and 0.5% (w/v) BSA. Cells were then incubated with FITC-labeled Annexin V (diluted 1:200 in HEPES buffer) for 15 min and washed again in HEPES buffer. Just before analysis of the samples by flow cytometry (FACSCalibur, Becton Dickinson, San Jose, CA), PI was added (final concentration 5 µg/ml) to distinguish necrotic cells (Annexin V/PI+) from early apoptotic cells (Annexin V+/PI) and late apoptotic cells (Annexin V+/PI+). Upon induction of apoptosis Annexin VFITC positivity of cells increased by at least 1 log scale. The samples were analyzed on a FACSCalibur (Becton Dickinson) equipped with CellQuest software. The cytometer was calibrated by eye with fluochrome beads supplied by the manufacturer.
Cell cycle analysis
DNA profiles were obtained by staining cells with PI. Cells (0.2 x 106/ml) were washed with ice-cold PBS and resuspended in 100 µl PBS containing PI (50 µg/ml), RNase A (100 µg/ml) (Sigma) and 0.1% (v/v) Tween 20%. After incubation on ice for 45 min, cells were analyzed on a FACSCalibur as described above. Gates were set to exclude cell aggregates, and particles of subcellular cells as defined by FSC and SSC scatter. The distribution of cells in the different phases of the cell cycle was analyzed by the Modfit Analysis program (Becton Dickinson).
Immunoblotting
After washing with ice-cold PBS, cells (5.0 x 106) were suspended in lysis buffer (1% NP-40, 0.01 M triethanolamineHCl, pH 7.8, 0.15 M NaCl, 5 mM EDTA, 0.02 mg/ml ovomucoid trypsin inhibitor, 1 mM PMSF, 0.02 mg/ml leupeptin and 25 µM phenylarsine oxide). After 515 min on ice, lysates were cleared by centrifugation at 13,000 g for 15 min. Protein contents were determined by the BioRad protein assay (BioRad, München, Germany) and equal protein loading was in all cases confirmed by Ponceau Red staining and was indicated by detection of tubulin. Depending on the protein detected, 30100 µg of protein per lane was subjected to SDSPAGE under reduced conditions as follows: pRb (6%), cyclin D3 (12%), Bcl-2, Bcl-xL, Bax, cytochrome c and caspase-3 (13%) or p21 and p27 (15%). After transfer to nitrocellulose or PVDF membranes (Hybond-C or Hybond-P; Amersham, Little Chalfont, UK), blots were blocked with 5% non-fat dry milk in TBS-T (10 mM Tris, 150 mM NaCl and 0.01% Tween 20, pH 8.0). Blots were probed with the indicated antibodies diluted in TBS-T containing 2.5% non-fat dry milk. Immunoreactive proteins were visualized using HRP-conjugated Ig (goat anti-mouse, goat anti-rat or swine anti-rat) and enhanced chemiluminescence (ECL; Amersham). For detection of caspase-3, blots were first probed with polyclonal antibody recognizing the cleaved forms of caspase-3 and subsequently the same blots were reprobed with the polyclonal antibody detecting the caspase-3 proform. Control lanes were cell lysates obtained from the following cell lines: in Fig. 3, non-stimulated Jurkat cells (cyclin D3), adranomycine-induced p21-over-expressing UTA6 cells (p21, p27); in Fig. 5, the non-stimulated EpsteinBarr virus-positive B cell line 9H9 expressing high levels of Bcl-2 and the non-stimulated Ramos subclone FR3 (10) expressing high levels of Bcl-xL.

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Fig. 3. Uncoupling of apoptosis and growth arrest by CD40 is regulated downstream of the cell cycle. Cells were stimulated as described in Fig. 2 and lysates were subjected to Western blotting. Apoptosis was detected by Annexin VFITC/PI staining via FACS analysis and percentage of apoptosis is indicated for each stimulus. Experiments were performed at least twice and a representative blot is shown. (A) mAb reactive with both hyperphosphorylated (pRbPPP) and hypophosphorylated (pRb) species of pRb were used (indicated with arrows). Anti-BCR, n-But and anti-CD40 (upper panel) or TNF- stimulation (lower panel) induced G1 arrest as detected by hypophosphorylated pRb. (B) mAb against cyclin D3 (upper panel) and p27 (lower panel) and polyclonal antibodies against p21 (middle panel) were used to detect expression of these cell cycle regulatory proteins after stimulation with anti-BCR, n-But and anti-CD40 stimulation. Control lanes are lysates of cells over-expressing either cyclin D3, p21 or p27. The internalized and degraded heavy chain (29 kDa) of the apoptosis-inducing anti-IgM mAb are indicated with an asterisk.
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Fig. 5. CD40 ligation does not change Bcl-2, Bcl-xL and Bax expression. Cells were stimulated with or without anti-CD40 mAb (1:1000 ascites dilution) for 24 h, and expression of (A) Bcl-2 (upper panel) and Bcl-xL (lower panel) was detected by Western blotting. mAb directed against Bcl-2 and Bcl-xL were used. Controls are the EpsteinBarr virus-positive B cell line 9H9 expressing high Bcl-2 protein levels (Bcl-2) and Ramos.FR3 (see Methods), which expresses higher levels of Bcl-xL. (B) Expression of Bax was detected by Western blotting using polyclonal antibodies in both the presence and absence of apoptotic stimuli in combination with anti-CD40 mAb. Cells were stimulated as described in Fig. 2.
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Preparation of cytosolic cell extracts for analysis of cytochrome c release
Cells (5.0 x 106/sample) were washed 3 times in ice-cold PBS and resuspended in 100 µl of extraction buffer (50 mM PIPESKOH, pH 7.4, 220 mM mannitol, 68 mM sucrose, 50 mM KCl, 5 mM EGTA, 2 mM MgCl2, 1 mM dithiothreitol, and protease inhibitors leupeptin, trypsin and PMSF; concentrations as described for the NP-40 lysis buffer). Washed cells were allowed to swell on ice for 3060 min. This was followed by incubation with digitonin (final concentration 200 µg/ml) for 15 min at 4°C. Lysates were centrifuged at 13,000 g for 15 min, and supernatants were harvested and stored at 20°C until use.
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Results
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Induction of apoptosis and growth arrest of Ramos cells by BCR cross-linking and n-But treatment
In cells stimulated by cross-linking the BCR with anti-IgM mAb for 24 h, apoptosis was induced as detected by Annexin VFITC/PI positivity (Fig. 1A) (2). This was accompanied by arrest of cells at G0/G1 phase of the cell cycle (Fig. 1B). A significant increase in the number of cells in the G0/G1 phase and a concomitant decrease in S phase was detected in BCR-cross-linked cells. The induction of growth arrest was also apparent by the decrease in the number of cells in G2/M phase, as these cells are the first cells entering G1 in the next round of cell division (Fig. 1B). When becoming apoptotic, Ramos cells do not clearly express <2N DNA content as detected by a sub-G1 peak (33).

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Fig. 1. Induction of growth arrest and apoptosis after BCR cross-linking or n-But treatment. Ramos cells were incubated with anti-IgM mAb (5 µg/ml) or n-But (2.5 mM) for 24 h, and apoptosis (Annexin VFITC/PI) (A) and cell cycle phase distribution of cells in G0/G1, S and G2/M (PI staining) (B) were detected. Data represent one out of at least five experiments.
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After direct induction of growth arrest by the cell cycle inhibitor n-But, induction of apoptosis was investigated. Upon treatment of cells for 24 h with 2.5 mM n-But, apoptosis was comparable to BCR-induced apoptosis (Fig. 1A). Furthermore, as expected, an extensive increase in the number of cells in G0/G1 phase was detected (Fig. 1B). When measured at later timepoints (4872 h), the percentages of apoptotic cells increased to
90% after stimulation by both BCR (10) and n-But (data not shown). Thus, induction of growth arrest either via BCR cross-linking or via n-But treatment resulted in apoptosis in Ramos cells.
Uncoupling of apoptosis and growth arrest by ligation of CD40 or stimulation with TNF-
BCR-mediated apoptosis can be inhibited by signals derived from Th cells such as CD40 ligand and TNF-
(24). It should be noted that Ramos cells do express TNF-RII but do not express TNF-RI as shown via FACS analysis with specific antibodies (2), therefore no apoptosis can be induced via recruitment of TRADD and FADD (34) by addition of TNF-
alone. After direct induction of G1 arrest by n-But, it was investigated if apoptosis could be rescued by CD40 ligation or TNF-RII triggering (Fig. 2A). In the case of BCR cross-linking, apoptosis was reduced to background levels by anti-CD40 mAb or TNF-
and apoptosis induced by 2.5 mM n-But could be partially inhibited to
60% of apoptosis in the absence of co-stimulation. To test whether apoptosis induced by n-But could be completely prevented by co-stimulation, experiments were performed with varying n-But concentrations. Complete prevention of apoptosis by CD40 ligation or TNF-
was observed at a concentration of 1.25 mM (Fig. 2B), indicating that apoptosis following direct induction of growth arrest can still be fully rescued by co-stimulation. Since the apoptotic response was subject to experimental variation at low n-But concentration plus anti-CD40 mAb or TNF-
, further tests were performed using 2.5 mM n-But.
The distribution of cells in the different phases of the cell cycle after CD40 ligation alone and in combination with apoptotic stimuli is shown in Fig. 2(C). CD40 ligation in combination with either BCR cross-linking or n-But treatment did not result in the rescue of G1 arrest. Instead, ligation of CD40 was able to induce a G1 arrest by itself and to enhance G1 arrest induced by n-But. The CD40-induced G1 arrest was transient, as cells subsequently continued to cycle (data not shown) (35). Thus, whereas apoptosis was rescued by CD40 ligation and TNF-
, the G1 arrest was sustained or even enhanced, suggesting that apoptosis can be uncoupled from growth arrest by co-stimulatory signals.
CD40 and TNF-
uncouple growth arrest and apoptosis downstream of the restriction point of the cell cycle
To further dissect the signaling pathway of growth arrest and apoptosis, and the possible uncoupling of these two processes by co-stimulatory signals, the involvement of regulatory cell cycle proteins was investigated. Since it is known that pRb in its hypophosphorylated form functions as a growth suppressor (36,37), the phosphorylation state of pRb was monitored. Apoptotic stimulation resulted in hypophosphorylation of pRb as two lower bands of the protein appeared on the blot (Fig. 3A, lanes 3 and 4) when compared to unstimulated cells (Fig. 3A, lane 1: medium), which is in accordance with induction of growth arrest. Furthermore, upon ligation of CD40 or TNF-
alone (Fig. 3A, lane 2) similar hypophosphorylated bands of pRb were observed. The effect on pRb hypophosphorylation of CD40 ligation or TNF-
stimulation in combination with apoptotic stimuli is shown in Fig. 3(A, lanes 5 and 6). Although apoptosis was prevented, hypophosphorylation of pRb was not inhibited, thereby confirming the data obtained by flow cytometry (see Fig. 2). Thus, the induction of growth arrest upon stimulation with either anti-IgM mAb, n-But or upon CD40 triggering or TNF-
stimulation is reflected by hypophosphorylated pRb. Furthermore, hypophosphorylation of pRb does not strictly correlate with apoptosis.
Next, expression of regulatory cell cycle proteins such as cyclins, cdk and CKI was investigated. These proteins are important in G1/S phase transition of cells through their ability to regulate pRb phosphorylation. No differences in expression of either cyclin E or the cdks 2, 4 and 6 were found (data not shown). However, stimuli-specific effects at the level of the cell cycle regulators cyclin D3, CKI p21 and CKI p27 were detected. Ramos cells do not detectably express cyclin D1 nor cyclin D2 [(38) and data not shown]. Expression of cyclin D3 was specifically down-regulated after BCR cross-linking (Fig. 3B, upper panel), whereas expression of p21 was up-regulated by n-But (Fig. 3B, middle panel) and expression of p27 was up-regulated by CD40 triggering (Fig. 3B, lower panel). Each of these events results in hypophosphorylation of pRb, explaining the observed growth arrest as induced by the three signals individually. Significantly, CD40 triggering was not dominant over the cell cycle effects induced by either BCR cross-linking or n-But. Expression of cyclin D3 remained down-regulated when CD40 was ligated in addition to BCR cross-linking (Fig. 3B, upper panel) and p21 was still up-regulated after n-But treatment (Fig. 3B, middle panel).
Collectively, these results indicate that the growth arrest was sustained or even enhanced by ligation of CD40 or triggering with TNF-
, whereas apoptosis was prevented. Furthermore, the uncoupling of apoptosis from growth arrest was apparently regulated by these signals downstream of the restriction point of the cell cycle.
CD40 and TNF-
block apoptosis upstream of cytochrome c release
Next, the apoptosis signaling pathway was investigated. Previously, we and others showed that the BCR activates the cell death program in a FADD-independent manner (8,10,39,40), demonstrating that an apoptosis route activated by a death receptor, which signals via caspase-8, is apparently not involved. Consequently, rescue of apoptosis mediated by CD40 signals and TNF-
may be at the level of the mitochondria. BCR cross-linking or n-But treatment induced the release of cytochrome c from mitochondria into the cytosol (Fig. 4A). This subsequently resulted in caspase-3 activation as evidenced by the p19 and p17 cleavage products of caspase-3 (Fig. 4B), and was followed by execution of apoptosis as detected by FACS analysis using FITC-labeled Annexin V and PI. Significantly, cytochrome c release as well as caspase-3 activation were inhibited when apoptosis was rescued by ligation of CD40 or by stimulation with TNF-
(Fig. 4A and B).

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Fig. 4. CD40 ligation and TNF- prevent apoptosis by blocking cytochrome c release. Cells were stimulated as described in Fig. 2 and (cytosolic) extracts were subjected to Western blotting. Apoptosis was detected by Annexin VFITC/PI staining via FACS analysis and the percentage of apoptosis is indicated for each stimulus which is the mean of two identical experiments. (A) mAb were used to detect the 15-kDa cytochrome c band. (B) Polyclonal antibodies against caspase-3 were used to detect the proform (p32) and cleaved forms of caspase-3 (p19, p17), which are indicated with arrows. Non-specific bands are indicated with an asterisk. (C) Blots were reprobed with antibodies directed against tubulin (55 kDa) as control for equal loading.
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Since mitochondrial homeostasis can be influenced by anti- and pro-apoptotic Bcl-2 family members (41) and since expression of these proteins can be under control of CD40 (42,43), the expression of Bcl-2, Bcl-xL or Bax was investigated via Western blotting (Fig. 5) and via RT-PCR approach (data not shown). In Ramos cells Bcl-2 is expressed below detectable limits in Western blots (Fig. 5A, upper panel) as well as in RT-PCR. Ligation of CD40 did not induce the expression of this anti-apoptotic protein. Bcl-xL was barely detectable in both Western blots and RT-PCR. After CD40 ligation no changes in protein expression of Bcl-xL could be observed (Fig. 5A, lower panel), whereas a 2.5-fold increase was detected by the more sensitive RT-PCR method. The expression of the pro-apoptotic protein Bax was monitored after stimulation of cells with anti-IgM mAb or n-But in combination with anti-CD40 mAb (Fig. 5B). Co-stimulation via CD40 did not influence Bax protein levels in anti-IgM mAb- or n-But-treated or untreated cells. Also, no changes in Bax levels were observed in the RT-PCR after CD40 co-stimulation. Thus, a complete inhibition of cytochrome c release from the mitochondria by CD40 is observed together with minimal changes in these Bcl-2 family members.
Rescue of apoptosis induced by BCR cross-linking or n-But is under rapid control of co-stimulatory signals
Since the above findings suggested that the uncoupling of growth arrest and apoptosis is regulated downstream of cell cycle regulation, it was considered that anti-apoptotic signaling by ligation of CD40 or stimulation with TNF-
might include a post-transcriptional mechanism which interferes with the apoptotic signal before it reaches the mitochondrion. Experiments in which transcription or translation was inhibited by actinomycin D or cycloheximide respectively were difficult to interpret because of their cytotoxic effect on Ramos cells (Mackus et al., unpublished observation). In an effort to further investigate how CD40 and TNF-
can interfere with the intrinsic apoptosis pathway, the inhibition of cell death was monitored over time. After ligation of the BCR, apoptosis started at
12 h (Fig. 6A). Subsequently, agonistic CD40 mAb was added either simultaneously or at the indicated timepoints after ligation of the BCR (Fig. 6B). CD40 mAb was able to rescue cells from BCR-mediated apoptosis completely, even when added 12 h after ligation of the BCR. Significantly, this is the timepoint at which cell death started to occur (Fig. 6A and B). When CD40 mAb was added later, it could no longer protect cells that were undergoing apoptosis. The same qualitative effect was observed for n-But-induced apoptosis (Fig. 6C). Although TNF-
(or anti-CD40; not shown) could not completely rescue cells from apoptosis induced by 2.5 mM n-But (see also Fig. 2B), the inhibitory effect on apoptosis was still apparent when TNF-
was added after 10 h. Again, when TNF-
was added later, protection of cells undergoing apoptosis was no longer observed. These results suggest that signaling via ligation of CD40 or stimulation with TNF-
may inhibit apoptosis via a direct post-transcriptional mechanism other than modulation of Bcl-2 family members.
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Discussion
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The present study addresses the connection between BCR-mediated apoptosis and cell cycle arrest. Specifically, a potential causal link between a G1 arrest and apoptosis in B cells was investigated, as well as the point in the signaling cascade at which co-stimulatory T cell signals can interfere with apoptosis. Earlier work had demonstrated that the Ramos B cell line responds to stimulation of CD95 and BCR by undergoing apoptosis, and that triggering of CD40 or co-stimulation with TNF-
differentially modulates the outcome, such that it enhances CD95-mediated but blocks BCR-mediated apoptosis (10). As such, the Ramos system is regarded as a generic model for selection of mature or germinal center B cells (4,10,17,42,44).
First, it was established that BCR triggering causes a G1 arrest and apoptosis, and that direct induction of growth arrest in G1 with n-But similarly causes apoptosis. The fact that cell cycle arrest itself induces apoptosis which is biochemically indistinguishable from BCR ligation already indicates that apoptosis follows upon cell cycle arrest. Also, timecourse analysis of n-But stimulations corroborated that G1 arrest occurs prior to PS exposure (unpublished data). The alternative possibility, that in fact two independent separate pathways operate in cell cycle arrest and apoptosis, would require experimental conditions whereby the cell cycle is unaffected during apoptosis induction via the BCR, which is indeed the opposite of what we have observed. Based on these considerations, we interpret our data to indicate that cell cycle arrest precedes apoptosis and that CD40 ligation can uncouple these two events downstream of the pRb restriction point of the cell cycle. Induction of apoptosis was determined by release of cytochrome c, by activation of caspase-3, and by high PS exposure on the outer cell membrane and later loss of membrane integrity (see Figs 1 and 4). In addition, activation of caspase-9, cleavage of PARP, DNA fragmentation and chromatin condensation were consistently observed upon both BCR triggering and induction of growth arrest via n-But (Eldering and Mackus, unpublished observations). These hallmarks of apoptosis exclude that the PS exposure was solely due to the activation or developmental status of the cells (45,46). Quantitative differences were observed in the numbers of cells in G0/G1 phase after BCR triggering and after n-But treatment. However, the analysis of the phosphorylation status of the pRb cell cycle regulator confirmed the qualitative assessment of a G1 arrest. Furthermore, an important finding was that, although signaling via CD40 or with TNF-
clearly blocked both BCR- and n-But-mediated apoptosis, these signals did not prevent the G1 arrest but rather augmented it. Indeed, anti-CD40 antibodies and TNF-
themselves induced a transient cell cycle block, as measured at the level of diploid DNA content and pRb phosphorylation status (see also below). An earlier report on the link between cell cycle and apoptosis induction in B lymphoma cells described an experimental system in which the phosphorylation status of pRb was uncoupled from cell cycle progression and apoptosis (47). Furthermore, a purported reciprocal relationship between pRb phosphorylation status and Bcl-2 activity complicates interpretation of the data (47). In contrast, our results with Ramos cells clearly demonstrated a relationship between the phosphorylation status of pRb and cell cycle progression. Therefore, it follows that in this model of mature B cells, G1 arrest does not necessarily result in apoptosis, since these processes can be uncoupled by ligation of CD40 or stimulation with TNF-
.
A more detailed analysis of cell cycle regulators indicated that the different apoptotic signals studied resulted in distinct responses, i.e. down-regulation of cyclin D3 after BCR cross-linking and up-regulation of p21 after n-But treatment. CD40 signaling resulted in up-regulation of p27 and thus represented a third manner in which a cell cycle arrest can be explained by ultimately regulating pRb phosphorylation. An important point from these analyses is that, although CD40 triggering overrules the apoptotic signals, this effect is exerted downstream of the restriction point of the cell cycle.
As mentioned above, we studied three signals that can influence pRb status via apparently different mechanisms. These findings differ from some reports on the immature murine B cell line WEHI-231, where CD40 either can directly oppose the G1 arrest by preventing p27 accumulation (4850) or can influence the NF-
B transcription factor (51,52), or can up-regulate specific anti-apoptotic members of the Bcl-2 family (43,53,54). Any actual discrepancy with the Ramos cells described here might result from differences in maturation status of the two B cell models. It is conceivable that at an immature stage of B cell development CD40 signals predominantly lead to effects at cell cycle level and/or transcriptional level, while at a more mature differentiation stage additional regulatory pathways are invoked. Indeed, a recent report also describes growth inhibitory effects of CD40 in mature, but not in immature, murine B cells (55). This observation is consistent with our findings in the human system, although it cannot formally be excluded that variable findings obtained with malignant cell lines are caused by aberrant signaling.
Recent research from numerous groups has culminated in the concept that apoptotic signaling can be divided into an extrinsic part and an intrinsic part. The former is generally assumed to convey death-receptor signals via FADD and the prime example is CD95. The second path involves various stress signals within the cell, which ultimately all result in release of cytochrome c [and other factors, for review, see (56)] from the mitochondria. Both pathways eventually converge at the level of the executioner caspases. Our findings regarding BCR- and n-But-mediated apoptosis fully agree with reports that BCR triggering can activate the intrinsic or mitochondria-dependent route of apoptosis (8,11,12). Furthermore, we show that CD40 and TNF-
co-stimulation blocked pro-apoptotic signals by preventing release of cytochrome c. Importantly, after BCR triggering an anti-apoptotic effect of CD40 could be detected up to the point of actual onset of apoptosis. This was also seen for the CD40- and TNF-
-mediated decrease in apoptosis after n-But treatment. These observations suggest a relatively rapid proteinprotein interaction as the basis for the block in apoptosis.
If such a direct anti-apoptotic effect of CD40 can intercept cell cycle signals before they trigger cytochrome c release from the mitochondria, this apparently complements or overrides the well-described transcriptional effect of CD40 signaling. Transcriptional regulation of anti-apoptotic mediators by CD40 signaling is mediated by the intensely studied NF-
B family of transcription factors (51,5759). In the mature B cell system studied here, we found that protein levels of obvious candidates to tip the apoptotic balance such as Bcl-2, Bcl-xL and Bax, were not significantly changed upon CD40 stimulation. Our data agree with reports that CD40 cross-linking does not alter expression of Bax in both murine (60) and human B cells (61). Of course, other Bcl-2 family members may be involved, e.g. Bfl-1/A1 is reported to be up-regulated in a CD40-dependent fashion in B cells (53,62). Furthermore, a direct effect of CD40 and TNF-
via TRAF-2, -3 or -5 recruitment (59,6365) and engagement of the IAP family of caspase inhibitors might be proposed (66,67), based on reported interactions in other systems. We are currently evaluating the relative contribution of transcriptional regulation of a comprehensive set of pro- and anti-apoptotic regulators by a multiplex RT-PCR approach. In addition, a functional assay to measure cytochrome c release from isolated mitochondria will be able to identify pro- and anti-apoptotic proteins in cytosolic preparations. These combined approaches will eventually allow complete evaluation of the relative contributions of transcriptional and post-transcriptional mechanisms. Regardless, the observed complete block in cytochrome c release after BCR triggering and CD40/TNF-
co-stimulation clearly implies a point at or upstream of the mitochondrial threshold for cytochrome c release as the major site for T cell prevention of apoptosis.
These considerations, combined with the earlier conclusion that CD40 or TNF-
co-stimulation predominantly interferes with apoptosis downstream of the cell cycle, can tentatively be interpreted in the context of germinal center selection and affinity maturation. BCR ligation alone would trigger apoptosis after a cell cycle arrest, but in the presence of appropriate co-stimulation, the G1 arrest would be maintained but apoptosis is stalled. The B cell is thus provided with an increased time frame to integrate diverse signals arriving from Th and follicular dendritic cells. This prolonged G0/G1 phase can be expected to resolve itself after a finite timespan depending on, for instance, protein turnover or additional signals from neighboring cells.
We conclude that in Ramos B cells, a BCR-triggered G1 arrest is not obligatory followed by apoptosis, because the two processes can be uncoupled by co-stimulatory T cell signals. The block in apoptosis mediated by anti-CD40 mAb or TNF-
occurs downstream of the pRb-regulated restriction point of the cell cycle, but takes place prior to cytochrome c release from mitochondria. These observations and their interpretation provide a framework for further understanding of B cell selection.
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Acknowledgements
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The authors would like to thank Bianca den Drijver and Saskia Smulders for technical assistance. This work was supported by grant 99-1998 (to W. J. M. M.) and grant 99-1996 (to E. E.) from the Dutch Cancer Society
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Abbreviations
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BCRB cell antigen receptor
cdkcyclin-dependent kinase
CKIcyclin-dependent kinase inhibitor
HRPhorseradish peroxidase
n-Butsodium butyrate
PIpropidium iodide
pRbretinoblastoma protein
PSphosphatidylserine
TNFtumor necrosis factor
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