| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Institut National de la Santé et de la Recherche Médicale U404, Immunité et Vaccination, Lyon, France
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The apoptotic process can be induced by direct cellular damages such as those caused by irradiation, but also by triggering surface receptors. Some of these, such as the death domain (DD)3-containing receptors, mainly function as apoptosis-inducing molecules (6). Others, like the Ag receptors and MHC class II molecules, only behave as such under certain strictly defined circumstances. Although the downstream events connected to the DD-containing receptors are becoming better understood, the intracellular death cascade linked to Ag receptors remains relatively obscure.
The efficiency of the DD-containing receptors at transducing death signals is partly due to their ability to rapidly form a multimolecular complex (death-inducing signaling complex or DISC) connected to their intracytoplasmic tail (7). This complex is constituted within seconds of ligand binding and induces death of the target cells within several hours. Most of the damages inflicted to the cell by the DD-containing receptors are due to the activation of a peculiar type of cysteine proteases, best known as caspases. These proteolytic enzymes act in a stepwise fashion and can behave as substrates for each other (8, 9). The initiator caspases (caspase-8 or -10) are recruited and activated within the DISC (10, 11). They in turn activate downstream effector caspases (6) that are responsible for the characteristic cellular alterations associated with apoptosis, such as phosphatidylserine (PS) exposure or DNA damage.
Most of our current knowledge of the apoptosis effectors recruited by the BCR comes from experiments conducted with transformed immature (12) or mature B cell lines (13, 14). These studies have established that, as for most apoptotic pathways described to date, the death signal transmitted through the BCR induces activation of effector caspases (caspase-3 in particular). Nevertheless, the cytoplasmic mediators that allow the BCR to switch from a positive to a negative regulatory function and that connect the Ag receptor to the executioners of apoptosis are presently unknown.
It has been proposed that BCR-induced death could be instrumental in censoring self-reactive mutants generated through V gene hypermutation in the germinal center (GC) (15, 16, 17). We have previously shown that susceptibility of mature B cells to BCR-induced apoptosis is not a specific feature of GC B cells, but that human memory B cells can also be sensitized by CD40L or anti-Ig Abs to undergo apoptosis upon subsequent challenge of the Ag receptor (18). We have thus hypothesized that all mature B cells become sensitive to BCR-induced killing provided that they have received the adequate activation stimulus.
We have decided to explore two aspects of the death process connected to the BCR in normal human B cells: 1) the parameters that regulate the acquisition of their susceptibility to BCR-induced killing; 2) the molecular mechanisms responsible for execution of the apoptotic program. Although both GC and memory B cells are sensitive to BCR-induced killing, CD40L-stimulated memory B cells were chosen as the experimental model because of their lower tendency to enter spontaneous apoptosis in culture.
We report in this work that susceptibility of activated memory B cells to BCR-induced apoptosis relies on their entry in the S phase of the cell cycle. Execution of the BCR-induced apoptotic program is then divided into two phases: 1) an early caspase-independent step that leads to alteration of the mitochondrial permeability; 2) a later caspase-dependent step responsible for the ultimate manifestations of BCR-induced apoptosis. Our results demonstrate that the disruption of the mitochondrial function during the first phase initiates a downstream effector caspase cascade that involves activation of caspases-9 and -3.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The trimeric human CD40L/leucine zipper fusion protein was kindly provided by Dr. R. Armitage (Immunex, Seattle, WA) and was used at 1 µg/ml throughout the study. F(ab')2 fragments of rabbit Abs anti-human IgM/G/A (H + L) were purchased from Jackson ImmunoResearch (West Grove, PA) and used at 5 µg/ml. Rabbit anti-human IgM Abs coupled to polyacrylamide beads were purchased from Bio-Rad (Richmond, CA) and used at 20 µg/ml. The agonistic anti-CD95/Fas mAb 7C11 (IgM) was purchased from Immunotech (Marseilles, France). Purified mouse IgM myeloma protein used as the isotypic control for mAb 7C11 was obtained from Sigma (St. Louis, MO). Both the 7C11 mAb and its isotypic control were used at 100 ng/ml. The mouse mAb directed against poly(ADP-ribose) polymerase (PARP) and the polyclonal rabbit Ab against caspase-3 were purchased from Biomol (Plymouth Meeting, PA) and PharMingen (San Diego, CA), respectively. The broad range caspase inhibitor Z-VAD-fmk was purchased from Bachem (Bubendorf, Switzerland). The caspase-8-specific inhibitor Z-IETD-fmk and the caspase-9-specific inhibitor Z-LEHD-fmk were purchased from Calbiochem (Nottingham, U.K.). Z-VAD-fmk, Z-LEHD-fmk, and Z-IETD-fmk peptides being dissolved in DMSO, control cultures were performed in the presence of the appropriate dilution of DMSO. The cell cycle blockers aphidicolin, deferoxamine, and mimosine were purchased from Sigma and used at 1 µg/ml, 300 µM, and 600 µM, respectively.
Cells
Purified tonsillar B cells were isolated as previously described (19). IgD-positive and IgD-negative B cells were separated using anti-IgD-coated SRBC, as described in detail by Feuillard et al. (20). Memory B cells were isolated from the IgD- population, as previously described (2). Experiments were conducted either on isolated memory B cells or on the Burkitt lymphoma cell line BL60. The choice of memory B cells to study the intracellular apoptotic pathway connected to the BCR in normal mature B cells was motivated by the fact that this subset is highly sensitive to BCR-induced apoptosis, but less prone to undergo spontaneous apoptosis than its GC counterpart.
Cultures
All cultures were made in RPMI 1640 supplemented with 10% selected heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2% HEPES (all from Life Technologies, Grand Island, NY), except where indicated. Tonsillar B cell cultures were performed in two steps. First, B cell blasts were generated by stimulating B cells for 48 h with soluble trimeric CD40L at the density of 1 x 107 cells/well in six-well plates. Second, viable B cell blasts were recovered by density-gradient centrifugation and seeded at 1 x 105 cells/well in 96-well round-bottom microtiter plates in a final volume of 0.1 ml for the indicated times. We had observed that persistent CD40 stimulation not only failed to inhibit anti-Ig-induced apoptosis in B blasts, but rather decreased their spontaneous apoptosis (data not shown). Therefore, all secondary cultures of B blasts were established in the presence of CD40L, with or without addition of F(ab')2 fragments of rabbit anti-human Igs Abs. For the experiments conducted on the IgM-expressing Burkitt lymphoma cell line BL60, cells were seeded at 1 x 105 cells/well and cultured with or without immobilized rabbit anti-human IgM Abs. Percentages of specific cell death were calculated as follows: 100 x (apoptosis with anti-Ig Abs (%) - spontaneous apoptosis (%))/(100% - spontaneous apoptosis (%)). Intracellular ATP was depleted by incubating BL60 cells in glucose-free RPMI 1640 medium (Sigma) supplemented with 10% selected heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2.5 µM oligomycin, an inhibitor of F0F1-ATPases (21), to prevent production of ATP from both glycolysis and oxidative phosphorylation.
Measurement of apoptosis
Quantitation of apoptotic cells was made with: 1) The 3,3'
dihexyloxacarbocyanine iodide (DiOC6)
fluorochrome (Molecular Probes, Eugene, OR), which reveals disruption
of the mitochondrial transmembrane potential (
m), as described by
Zamzami et al. (22). In this assay, apoptotic cells are
identified by their decreased m
(DiOC6low). 2) Biotinylated
annexin V (Boehringer Mannheim, Mannheim, Germany) used according to
the manufacturer’s protocol to detect the translocation of PS from the
inner side to the outer leaflet of the plasma membrane on apoptotic
cells. The annexin V staining was revealed with FITC-conjugated avidin
(Immunotech) used at 2.5 µg/ml.
Immunofluorescence stainings were analyzed on a FACScan flow cytometer using the Lysis II software (Becton Dickinson, San Jose, CA).
Western blot analysis
Briefly, 5 x 106 cells were pelleted, washed twice with PBS, and resuspended in 100 µl of lysis buffer (62.5 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 2% 2-ME, supplemented with a protease inhibitor mixture (Boehringer Mannheim)). Samples were run on 8% (PARP) or 15% (caspase-3) SDS polyacrylamide gels. Following transfer to nitrocellulose membrane, the immunoblots were blocked by incubating with 0.5% BSA, Tris-buffered saline, and 0.1% Tween 20 (TBS-T), and probed overnight with the anti-PARP mAb or the anti-caspase-3 polyclonal Ab at a dilution of 1/2000 prepared in 0.5% BSA TBS-T. The immunoblots were then probed with HRP-conjugated goat anti-mouse Igs Abs (PARP) or goat anti-rabbit Ig Abs (caspase-3) (both from Amersham Life Science, Little Chalfont, U.K.) and developed using the ECL system (Pierce, Rockford, IL).
Measurement of caspase-3 enzymatic activity
The cell-permeable fluorigenic peptidic substrate PhiPhiLux-G2D2 (OncoImmunin, College Park, MD) containing the cleavage site DEVD was used to monitor caspase-3-like activity in intact cells. Cells were incubated with the substrate solution for 1 h at 37°C in the dark, according to the manufacturer’s instructions. The fluorescence emission revealing the release of the fluorescent product was analyzed on a flow cytometer in the FL2 channel.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We have previously demonstrated that both GC (19) and memory B cells (18) acquire susceptibility to BCR-induced apoptosis upon primary stimulation via CD40. We thus postulated that activation stimuli induce a rewiring of the Ag receptor to an apoptotic pathway in mature B cells. This led us to propose that BCR-induced apoptosis in the mature B cell lineage fulfills a function similar to that ascribed to AICD in the T cell compartment.
It has been demonstrated that the susceptibility of mature T cells to
Ag receptor-induced apoptosis is acquired upon their entry into the S
phase of the cell cycle (23). CD40L-activated and resting
memory B cells, which are respectively sensitive and resistant toward
BCR-induced apoptosis, also differ by their cycling status
(18). We thus examined whether the acquisition of
susceptibility toward BCR-induced killing in normal mature B cells
could also rely on their entry in a particular phase of the cell cycle.
To address this issue, different cell cycle blocking agents were tested
for their capacity to inhibit the proapoptotic effect of anti-Ig
Abs on activated mature B cells. Mimosine and deferoxamine were used to
arrest cells in the G1 phase of the cycle, while
aphidicolin was used to arrest cells in the S phase of the cell cycle.
Isolated memory B cells were first stimulated for 48 h with
CD40L in the presence or absence of either aphidicolin, deferoxamine,
or mimosine. For secondary cultures, viable B blasts were restimulated
with CD40L, in the presence or absence of F(ab')2
fragments of rabbit anti-human Ig Abs for 24, 48, and 72 h.
When added, the cell cycle blockers were present throughout the primary
and the secondary cultures. Estimation of the mitochondrial
transmembrane potential with the fluorescent dye
DiOC6 was used to monitor the proportions of
apoptotic cells at the end of the secondary cultures. None of the cell
cycle blockers used induced any significant rise in the proportions of
apoptotic cells per se. As illustrated by Fig. 1
, aphidicolin does not inhibit the
anti-Ig-driven apoptosis of CD40L-activated blasts, whatever the
time point considered. By contrast, blockade of the cells in the
G1 phase of the cycle with deferoxamine or
mimosine strongly impairs their capacity to undergo apoptosis following
triggering of the BCR. Mimosine, for example, caused more than a 50%
reduction in the levels of specific apoptosis at 72 h. Therefore,
the susceptibility of mature B cells to Ag receptor-induced death is
acquired as they enter the S phase of the cell cycle. This finding
suggests that the cycling potential of mature B cells is one of the
parameters that regulates their threshold of sensitivity to BCR-induced
apoptosis.
|
We next studied the signaling elements involved during the
effector phase of BCR-induced apoptosis in activated memory B cells. To
explore the caspase dependency of this apoptotic pathway, we compared
the impact of the broad range caspase inhibitor Z-VAD-fmk on the
apoptosis of CD40L-activated memory B cells driven either by
anti-Ig or anti-Fas Abs. Two types of assays were used to
evaluate the levels of apoptosis: first, estimation of the disruption
of the mitochondrial transmembrane potential revealed by the
DiOC6 fluorochrome; second, examination of the
membrane PS exposure, revealed by the binding of annexin V. As shown in
Fig. 2A, Z-VAD-fmk inhibits in
a dose-dependent fashion the PS externalization subsequent to
anti-Ig treatment, whereas it only marginally affects the loss of
mitochondrial transmembrane potential. On average, a 200 µM
concentration of Z-VAD-fmk causes a 74% reduction of the specific
apoptosis levels as estimated by annexin V binding, whereas it only
induces a 19% reduction in the levels of specific apoptosis as
determined by DiOC6 staining. Because this slight
inhibition of loss of m could only be detected at the latest
culture point (72 h), it is evocative of a secondary positive feedback
mechanism whereby activated caspases would amplify the mitochondrial
damages. These observations suggest that alteration of the plasma
membrane integrity subsequent to BCR ligation is dependent on
activation of caspases, while disruption of the mitochondrial potential
is not. In striking contrast, the loss of m and PS
externalization induced by the anti-Fas mAb 7C11 are both
caspase-dependent events, because they are inhibited by Z-VAD-fmk (Fig. 2B). Besides, a concentration of Z-VAD 20-fold lower than
that required to block the BCR-induced membrane alterations is
sufficient for complete inhibition of anti-Fas-induced PS exposure.
Altogether, these findings suggest that in activated memory B cells,
the Fas death pathway is more critically dependent on caspase
activation than the BCR death pathway. Nevertheless, to confirm the
involvement of caspases in the BCR apoptotic pathway, studies were then
undertaken to directly assess the caspase activity following engagement
of the BCR on CD40L-induced memory B blasts. There are at least 10
caspases identified to date, but the effector caspase-3 has been shown
to be activated in response to multiple apoptotic stimuli (6, 9). Moreover, the involvement of caspase-3 in the BCR-induced
apoptotic pathway has been documented in several B cell lymphoma models
(13, 14). Therefore, we examined whether engagement of the
BCR on CD40L-activated memory B cells could induce the cleavage of the
typical caspase-3 substrate PARP. For this purpose, CD40L-stimulated
memory B blasts were recultured with CD40L in the presence or absence
of anti-Ig Abs, with the caspase inhibitor Z-VAD-fmk (200 µM) or
the appropriate dilution of DMSO as a negative control. Analysis of the
PARP protein was performed by Western blot in cell lysates prepared
from cells recovered 72 h after the onset of the secondary
cultures. As shown in Fig. 3, the
full-length 116-kDa form of PARP is present in all four samples tested.
Spontaneous cleavage of PARP occurs to some extent in untreated blasts
(receiving neither anti-Ig Abs nor Z-VAD-fmk), as visualized by a
faint 85-kDa band on the gel. Nevertheless, the 85-kDa cleavage
fragment of PARP is only clearly detectable in anti-Ig-treated B
cells. This assertion is further supported by the densitometry analysis
of the 116- and 85-kDa bands (lower panel), which
indicates that the ratio of the cleaved form vs the uncleaved form of
PARP is at least 3 times higher in anti-Ig-treated blasts than in
untreated blasts. We next used the cell-permeable fluorigenic substrate
PhiPhilux-G2D2 to confirm
the involvement of a caspase-3-like activity in BCR-induced apoptosis.
CD40L-activated memory B blasts were recultured with CD40L, in the
presence or absence of anti-Ig Abs, with or without Z-VAD-fmk.
Memory B blasts treated with the agonistic anti-Fas mAb 7C11 were
used as positive control because caspase-3 has been shown to be
involved in the Fas apoptotic pathway in both mature human B and T cell
lines (13, 24). The capacity of B blasts to cleave the
PhiPhilux-G2D2 substrate
was estimated 60 h after the onset of the secondary cultures. The
representative experiment shown in Fig. 4A indicates that ligation of
Fas on memory B blasts induces an increase in the proportion of cells,
producing a significant fluorescence emission upon addition of the
substrate (64% in anti-Fas-treated cultures vs 19% in control
cultures). Ligation of the BCR (Fig. 4B) also induces a rise
in the percentage of blasts able to cleave the PhiPhiLux
G2D2 substrate (42% in anti-Ig-stimulated cultures vs
20% in untreated cultures). Addition of Z-VAD-fmk at the onset of the
secondary culture completely suppresses the anti-Ig-induced
caspase-3-like activity (Fig. 4C). These results provide
direct evidence for an activation of a caspase-3-like protease during
BCR-induced apoptosis in activated memory B cells. Because other
caspases such as caspase-7 could also be responsible for the
BCR-induced cleavage of PARP and of the fluorigenic substrate PhiPhiLux
G2D2, we tested by Western
blotting whether anti-Ig Abs promote the release of the active
cleavage product of caspase-3. As shown in Fig. 5, in memory B blasts, anti-Ig Abs
promote a specific rise in the expression of the 17-kDa active cleavage
product of caspase-3. Altogether, these findings indicate that the BCR
apoptotic pathway in activated memory B cells involves the late
activation of caspase-3 (Fig. 4D).
|
|
|
|
The observation that PS exposure but not perturbation of m
is blocked by Z-VAD-fmk in anti-Ig-treated blasts suggested that
the alteration of the plasma membrane induced by BCR triggering on
activated B cells is a caspase-dependent process, while the
premitochondrial phase is not. Because we found chemical agents known
to block the mitochondrial function to be toxic for normal B cells, we
used a cell line model to determine whether caspase activation is
consecutive to the BCR-induced mitochondrial alterations. Because
Burkitt lymphoma (BL) cell lines have long been recognized to be
sensitive to BCR-induced apoptosis (25), experiments were
thus undertaken with BL60 cells to address this issue. To test the
validity of the Burkitt lymphoma model, we first determined the effect
of the broad spectrum caspase inhibitor Z-VAD-fmk on the
anti-Ig-induced death of BL60 cells. For this purpose, both
DiOC6 staining and annexin V binding were
performed on BL60 cells cultured with or without immobilized
anti-IgM Abs, in the presence or absence of Z-VAD-fmk. As shown in
Table I, BCR-induced death of BL60 cells
is rapid and massive, because the rates of specific apoptosis were 85
and 77% on the basis of DiOC6 staining and
annexin V binding, respectively, after 24 h of culture with
anti-IgM Abs. The caspase inhibitor Z-VAD-fmk marginally affected
the disruption of the mitochondrial potential (80% vs 85% specific
apoptosis in control cultures), but dramatically reduced PS
externalization (26% vs 77% specific apoptosis in control cultures)
induced by anti-IgM Abs. This finding indicates that the
mitochondrial and membrane alterations induced by anti-Ig Abs
follow the same scheme in primary activated memory B blasts and in BL60
cells, that is the loss of m is caspase independent, while
alterations of the plasma membrane integrity require activation of
caspases. We next investigated whether caspase activation during
BCR-induced death is under the control of the mitochondria or regulated
by a separate pathway independent from the mitochondria. To
discriminate between these two possibilities, we decided to block the
dATP-dependent association of cytochrome c, Apaf-1 (the
mammalian homologue of CED-4), and procaspase-9. This multiprotein
complex promotes processing of caspase-9, which in turn cleaves and
activates caspase-3 (26). ATP levels were thus
artificially reduced in the BL60 cell line by culturing the cells in a
glucose-free medium in the presence of 2.5 µM oligomycin, as
previously described (27, 28). This procedure prevents
production of dATP from both glycolysis and oxidative phosphorylation.
The anti-Ig-mediated PS exposure was first estimated on BL60 cells
incubated or not in glucose-free medium, supplemented or not with
oligomycin. As shown in Fig. 6A, neither glucose
deprivation nor the addition of oligomycin significantly impaired the
BCR-induced apoptosis of BL60 cells, when done separately. By contrast,
a strong reduction of the anti-Ig-induced PS externalization was
observed when cultures were made in glucose-free medium in the presence
of oligomycin (22% vs 65% specific apoptosis in control cultures). We
next investigated whether dATP depletion and subsequent blockade of the
proapoptotic function of the mitochondria have an impact on the
activation of caspase-3 consecutive to the engagement of the BCR. PARP
cleavage was therefore examined in BL60 cells cultured in glucose-free
medium, with or without anti-IgM Abs, in the presence or absence of
oligomycin. As expected, in the absence of oligomycin, ligation of the
BCR on BL60 induces the appearance of the 85-kDa cleavage product of
PARP. Addition of oligomycin completely prevented the
anti-Ig-induced degradation of PARP, thus indicating that caspase-3
activation during BCR-mediated apoptosis occurs downstream of the
mitochondria. Because these data were evocative of a possible role for
the cytochrome c/Apaf-1/procaspase-9 complex in the
processing of effector caspases-3 induced by anti-Ig Abs, we next
investigated whether caspase-9 intervenes in the apoptotic process
elicited by the BCR in CD40L-activated memory B cells. For this
purpose, memory B blasts were recultured with or without anti-Ig
Abs, in the presence or absence of serial dilutions of the caspase-9
inhibitory peptide Z-LEHD-fmk. Specific apoptosis was estimated after
72 h of culture by evaluation of PS exposure. As shown in Fig. 7, the caspase-9 inhibitory peptide
Z-LEHD-fmk blocked the proapoptotic effect exerted by anti-Ig Abs
on CD40L-activated memory B cells, in a dose-dependent manner, reaching
complete suppression at a 50 µM concentration. To investigate further
the caspases involved in the BCR death pathway, we next compared the
impact of the caspase-8 inhibitory peptide Z-IETD-fmk on the death of
activated memory B cells promoted either by anti-Fas or anti-Ig
Abs. The results of a representative experiment illustrated by Fig. 8 show that the broad range caspase
inhibitor Z-VAD-fmk strongly reduces apoptosis caused by BCR or Fas
engagement. In contrast, while Z-IETD-fmk brought about the same level
of inhibition of apoptosis than Z-VAD-fmk in anti-Fas-treated
cells, it only marginally affected the BCR-induced PS exposure.
Z-IETD-fmk was still unable to inhibit BCR-induced death even when its
concentration was increased up to 200 µM (data not shown). This
supports the conclusion that caspase-8 activation is not primarily
involved in the BCR-induced caspases cascade, leading to death in
CD40L-induced memory blasts. Altogether, these findings are compatible
with the notion that triggering the BCR on activated B lymphocytes
induces a mitochondrial damage, which in turn causes the activation of
caspases-9 and -3.
|
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The data shown in this work are in agreement with this hypothesis because, as previously described for T cell AICD (23), B cells acquire susceptibility to BCR-induced death upon entry into the S phase of the cell cycle. Because proliferating cells are the preferential targets of the negative regulatory control exerted by the BCR, it can thus be speculated that the sensitivity of mature B cell subsets to AICD should be closely correlated with their ability to cycle in response to primary activation stimuli. In return, the negative feedback control exerted by Ag on B cell responses should not cause massive or premature deletion of the activated B cell pool in the course of the humoral response. Both CD40L and IL-4 have been described to protect Burkitt lymphoma and immature B cell lines from BCR-induced killing (31, 32). This suggests that the proapoptotic function of the BCR on activated B cells could be balanced by T cells. Both Ag and T cell influences would thus contribute to maintain homeostasis of the B cell compartment by regulating the size of the activated B cell pool during the expansion phase of the humoral response. However, neither CD40L nor various T cell-derived mediators (including IL-4) were able to counteract the death-promoting effect of surrogate Ag on activated memory B cells (data not shown). Therefore, the nature of the putative factors conferring resistance to AICD on activated memory B cells still remains elusive.
We next explored the molecular effector mechanisms involved in the death pathway connected to the BCR in activated memory B cells. One of the first transductional events associated with triggering of DD-containing receptors is the recruitment and activation of an initiator caspase (caspase-8 or -10) that is responsible for the subsequent cascade of damages that occurs during the apoptotic cell death (10, 11). Two features of the BCR-induced apoptosis described herein argue against the possibility that B cell AICD could involve the interaction between a DD-containing receptor and its ligand, as it is the case for T cell AICD. First, a caspase-8 inhibitory peptide does not counteract the proapoptotic effect of anti-Ig Abs in CD40L-induced memory blasts. Second, transduction of the death signal from the Ag receptor to the mitochondria does not rely on caspase processing. The ability of Z-VAD-fmk to prevent the membrane but not the mitochondrial alterations resulting from BCR ligation on activated memory B cells strongly argues for a partitioning of the BCR-induced death pathway into a caspase-independent phase and a caspase-dependent one, intervening upstream and downstream of the mitochondria, respectively. We extended this observation by showing that BCR-induced mitochondrial dysfunctioning is involved in controlling caspase activation during this process. This assertion is supported by two lines of evidence. First, dATP depletion, which prevents formation of the cytochrome c/Apaf-1/procaspase-9 complex, inhibits both the BCR-induced PS exposure and PARP cleavage in BL60 cells. Second, the inhibitory effect exerted by the Z-LEHD-fmk peptide on the BCR death pathway suggests that caspase-9 participates in the death effector cascade during BCR-induced apoptosis of CD40L-activated memory B cells. These results favor the hypothesis that propagation of the death signal from the BCR involves the mitochondria-dependent cleavage of procaspase-9 that in turn activates other downstream apoptosis effectors.
Two independent sets of data establish that caspase-3 is
activated during BCR-induced apoptosis in activated memory B cells: 1)
the caspase substrates PARP and PhiPhiLux
G2D2 are cleaved in
anti-Ig-treated cells; 2) anti-Ig Abs enhance the release of
the active cleavage product of caspase-3 in CD40L-activated memory B
cells. We found activation of caspase-3 to be delayed during
BCR-induced apoptosis with respect to its kinetics of activation during
Fas-mediated killing (data not shown). However, our observation that
mitochondrial damages precede caspase activation and PS exposure
following BCR ligation suggests that anti-Ig-treated cells are
already committed to death before caspase processing occurs. Therefore,
the late and moderate activation of caspases during BCR-induced
apoptosis should not be regarded as an indication of the poor
efficiency of the death signaling pathway triggered by the Ag receptor
in B cells. Interestingly, Graves and colleagues (33)
recently provided evidence that protein neosynthesis is required for
BCR-induced apoptosis to occur in a B lymphoma cell line. We have shown
in this work that the postmitochondrial phase of BCR-induced death
essentially relies on processing of preformed caspases. It is thus
tempting to speculate that de novo synthesis of a protein component is
more likely to be involved in transduction of the apoptotic signal from
the BCR to the mitochondria. However, because Z-VAD-fmk does not
prevent the first cleavage step of caspase-3 (which produces the
intermediate p20/p12 inactive heterodimer) during BCR-mediated
apoptosis of Ramos cells (13), we cannot formally exclude
that proteases other than caspases participate in the BCR-mediated
activation of caspase-3 in activated memory B cells. A hypothetical
model for the transduction of the apoptotic signal through the BCR,
based on our present experimental findings and on the published
literature, is depicted in Fig. 9.
|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Thierry Defrance, Institut National de la Santé et de la Recherche Médicale U404, Avenue Tony Garnier, 69365, Lyon, Cedex 07, France. E-mail address:
3 Abbreviations used in this paper: DD, death domain; AICD, activation-induced cell death; BCR, B cell receptor; BL, Burkitt lymphoma; DiOC6, 3,3' dihexyloxacarbocyanine iodide; DISC, death-inducing signaling complex; GC, germinal center; PARP, poly(ADP-ribose) polymerase; PS, phosphatidylserine; CD40L, CD40 ligand; Z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; Z-IETD-fmk, benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone; Z-LEHD-fmk, benzyloxycarbonyl-Leu-Glu-Mis-Asp-fluoramethylketone.
Received for publication June 7, 1999. Accepted for publication August 10, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  M. Poggianella, M. Bestagno, and O. R. Burrone The Extracellular Membrane-Proximal Domain of Human Membrane IgE Controls Apoptotic Signaling of the B Cell Receptor in the Mature B Cell Line A20 J. Immunol., September 15, 2006; 177(6): 3597 - 3605. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Hausl, R. U. Ahmad, M. Sasgary, C. B. Doering, P. Lollar, G. Richter, H. P. Schwarz, P. L. Turecek, and B. M. Reipert High-dose factor VIII inhibits factor VIII-specific memory B cells in hemophilia A with factor VIII inhibitors Blood, November 15, 2005; 106(10): 3415 - 3422. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Xing, H. Igarashi, X. Wang, and N. Sakaguchi Protein phosphatase subunit G5PR is needed for inhibition of B cell receptor-induced apoptosis J. Exp. Med., September 6, 2005; 202(5): 707 - 719. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Daniel, M. B. Arvelo, V. I. Patel, C. R. Longo, G. Shrikhande, T. Shukri, J. Mahiou, D. W. Sun, C. Mottley, S. T. Grey, et al. A20 protects endothelial cells from TNF-, Fas-, and NK-mediated cell death by inhibiting caspase 8 activation Blood, October 15, 2004; 104(8): 2376 - 2384. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Varambally, Y. Bar-Dayan, J. Bayry, S. Lacroix-Desmazes, M. Horn, M. Sorel, Y. Bar-Dayan, G. Ruberti, M. D. Kazatchkine, and S. V. Kaveri Natural human polyreactive IgM induce apoptosis of lymphoid cell lines and human peripheral blood mononuclear cells Int. Immunol., March 1, 2004; 16(3): 517 - 524. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Mouhamad, L. Besnault, M. T. Auffredou, C. Leprince, M. F. Bourgeade, G. Leca, and A. Vazquez B Cell Receptor-Mediated Apoptosis of Human Lymphocytes Is Associated with a New Regulatory Pathway of Bim Isoform Expression J. Immunol., February 15, 2004; 172(4): 2084 - 2091. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.D. Garlichs, S. Kozina, S. Fateh-Moghadam, B. Tomandl, C. Stumpf, S. Eskafi, D. Raaz, A. Schmeisser, A. Yilmaz, J. Ludwig, et al. Upregulation of CD40-CD40 Ligand (CD154) in Patients With Acute Cerebral Ischemia Stroke, June 1, 2003; 34(6): 1412 - 1418. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. S. Goodyear and G. J. Silverman Death by a B Cell Superantigen: In Vivo VH-targeted Apoptotic Supraclonal B Cell Deletion by a Staphylococcal Toxin J. Exp. Med., May 5, 2003; 197(9): 1125 - 1139. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B.-J. Kroesen, S. Jacobs, B. J. Pettus, H. Sietsma, J. W. Kok, Y. A. Hannun, and L. F. M. H. de Leij BcR-induced Apoptosis Involves Differential Regulation of C16 and C24-Ceramide Formation and Sphingolipid-dependent Activation of the Proteasome J. Biol. Chem., April 18, 2003; 278(17): 14723 - 14731. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. E. Muscarella and S. E. Bloom Cross-linking of Surface IgM in the Burkitt's Lymphoma Cell Line ST486 Provides Protection against Arsenite- and Stress-induced Apoptosis That Is Mediated by ERK and Phosphoinositide 3-Kinase Signaling Pathways J. Biol. Chem., January 31, 2003; 278(6): 4358 - 4367. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Hase, Y. Kanno, H. Kojima, C. Morimoto, K. Okumura, and T. Kobata CD27 and CD40 Inhibit p53-independent Mitochondrial Pathways in Apoptosis of B Cells Induced by B Cell Receptor Ligation J. Biol. Chem., November 27, 2002; 277(49): 46950 - 46958. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. J. M. Mackus, S. M. A. Lens, R. H. Medema, M. J. Kwakkenbos, L. M. Evers, M. H. J. v. Oers, R. A. W. v. Lier, and E. Eldering Prevention of B cell antigen receptor-induced apoptosis by ligation of CD40 occurs downstream of cell cycle regulation Int. Immunol., September 1, 2002; 14(9): 973 - 982. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Herold, A. W. Kuss, C. Kraus, and I. Berberich Mitochondria-Dependent Caspase-9 Activation Is Necessary for Antigen Receptor-Mediated Effector Caspase Activation and Apoptosis in WEHI 231 Lymphoma Cells J. Immunol., April 15, 2002; 168(8): 3902 - 3909. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Owyang, J. R. Tumang, B. R. Schram, C. Y. Hsia, T. W. Behrens, T. L. Rothstein, and H.-C. Liou c-Rel Is Required for the Protection of B Cells from Antigen Receptor-Mediated, But Not Fas-Mediated, Apoptosis J. Immunol., November 1, 2001; 167(9): 4948 - 4956. [Abstract] [Full Text] [PDF]
|
|
