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*
Institut National de la Santé et de la Recherche Médicale Unité 404, "Immunité et Vaccination," Lyon, France; and
Tumor Immunology Program, German Cancer Research Center, Heidelberg, Germany
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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m). In type II
cells, DISC formation is strongly reduced and activation of caspase-8
mainly occurs downstream of the mitochondria, consecutive to m
disruption. Although mitochondria are activated in both cell types,
only type II cells depend on the proapoptotic function of the
mitochondria to execute the death program initiated by ligation of
Fas. Another DED-containing protein called FLICE inhibitory protein (FLIP) has also been reported to bind to the Fas/FADD complex under certain circumstances. FLIP was initially described as a virus-encoded apoptosis inhibitory protein, but its cellular homologue has been independently discovered by several groups and named c-FLIP/CASH/Casper/CLARP/FLAME/I-FLICE/MRIT/Usurpin (12, 13, 14, 15, 16, 17, 18, 19). c-FLIP contains two N-terminal DED motifs and can be expressed either as a long (c-FLIPL) or as a short (c-FLIPS) protein form through alternative splicing. The C-terminal region of c-FLIPL presents homology with the proteolytic domain of caspase-8 but lacks some of the critical residues required for its catalytic activity. This caspase-like domain is absent in c-FLIPS. The function of cFLIP is not entirely clear yet because its overexpression in mammalian cell lines has been shown to either promote (14, 15, 18) or prevent (12, 16, 17, 19) apoptosis.
The Fas/Fas ligand (L) system plays a critical role in the homeostatic regulation of activated peripheral T cells and as an effector of cytotoxic lymphocytes activity (see Ref. 20 for review). In B cells, Fas is instrumental in the maintenance of peripheral B cell tolerance and prevention of misguided T cell help (21, 22). We and others have shown that CD40 stimulation is required to induce expression of Fas on virgin B cells and to render them sensitive to Fas-induced apoptosis (23, 24, 25). In the current study, we have addressed two issues. First, what is the contribution of the mitochondria to transduction of the Fas death signal in CD40L-activated virgin human B cells? Second, what is the potential role of c-FLIP during Fas-induced apoptosis of activated B cells?
We report here that the Fas death program in B cells bypasses the mitochondrial Apaf-1 pathway because we have observed that blocking cytochrome c/Apaf-1/procaspase-9 complex formation or activation of caspase-9 does not prevent induction of apoptosis upon Fas triggering. Our results are compatible with the conclusion that caspase-8 is activated at the level of the DISC in Fas-sensitive B cells. FADD and the zymogen form of caspase-8 are constitutively expressed at high levels in virgin B cells, whereas expression of cFLIP is marginal. In contrast, c-FLIP, but neither FADD nor procaspase-8, is strongly up-regulated upon B cell activation through CD40 or the B cell receptor (BCR). Unexpectedly, we have found that Fas ligation on sensitive B cells not only allows for the recruitment but also for the processing of c-FLIPL at the receptor level. We propose that c-FLIP could be instrumental in transient protection of CD40L-activated B cells from Fas-induced apoptosis. The implications of these findings on B cell physiology are discussed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The trimeric human CD40L/leucine zipper fusion protein was
kindly provided by Dr. R. Armitage (Immunex, Seattle, WA) and was used
at 500 ng/ml throughout the study. Soluble recombinant human FLAG-Fas
ligand (Alexis, San Diego, CA) was used at 50 ng/ml and was aggregated
by using the anti-FLAG mAb M2 (Sigma, St. Louis, MO) at 1
µg/ml. Three different anti-Fas/CD95 Abs were used. The agonistic
mAb 7C11 (IgM) was used for biological assays and was purchased from
Immunotech (Marseille, France). The anti-APO-1 mAb
(26) was used for immunoprecipitation of the DISC. A
rabbit polyclonal Ab (Santa Cruz Biotechnology, Santa Cruz, CA) was
used for revelation of Fas in the immunoprecipitates. Purified mouse
myeloma proteins used as isotypic controls for mAb 7C11 (IgM) and
anti-APO-1 (IgG3-
) were obtained from Sigma. mAbs 7C11 and
anti-APO-1 were used at 200 ng/ml and 3 µg/ml, respectively.
The C15 mAb recognizing the p18 subunit of caspase-8 and the
anti-c-FLIP mAb NF6 have been described previously (27, 28). The anti-FADD and ß-actin mAbs were purchased from
Transduction Laboratories (Lexington, KY) and Sigma, respectively. The
caspase-8 colorimetric substrate Ac-IETD-pNA was purchased from Bachem
(Bubendorf, Switzerland). HRP-conjugated sheep anti-mouse Abs
(Amersham Life Science, Little Chalfont, U.K.) were used for
development of the immunoblots performed on cell lysates with the
anti-caspase-8, c-FLIP, and FADD mAbs. For DISC analysis by Western
blotting, isotype-specific HRP-conjugated secondary Abs (Southern
Biotechnology Associates, Birmingham, AL) were used: goat
anti-mouse IgG1 (for the c-FLIP and FADD mAbs) and goat
anti-mouse IgG2b (for the anti-caspase-8 mAb). HRP-conjugated
donkey anti-rabbit Abs were used for the Western blot analysis of
Fas in the immunoprecipitates. Oligomycin was purchased from Sigma and
was used at a final concentration of 2.5 µM throughout the study. The
caspase inhibitors (z-VAD-fmk, z-IETD-fmk, and z-LEHD-fmk) as well as
the control peptide z-FA fmk were purchased from Calbiochem
(Nottingham, U.K.) and used at the final concentration of 50
µM.
Cells
Purified tonsillar B cells were isolated as previously described (29). A modified version of the protocol originally described by Feuillard et al. (30) was used to enrich virgin B cells from the tonsillar B cell suspension by negative selection. Briefly, germinal center (GC) and memory B cells were depleted after two successive rounds of rosetting performed with SRBC coated with anti-CD38 and anti-CD80 mAbs, respectively. The purity of the negatively selected virgin B cell populations ranged between 70 and 85% due to the heterogeneous distribution of CD80 on memory B cells.
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. Fas-sensitive B blasts were obtained from virgin B cells after 48 h of stimulation by soluble trimeric CD40L, except where indicated. For primary cultures, B cells were seeded at the density of 1 x 107 cells/well in six-well plates. For secondary cultures, viable B blasts recovered by density gradient centrifugation were seeded at 5 x 106 cells/well in 12-well plates in the presence of the anti-Fas mAb 7C11 or its isotypic control. In certain experiments, the anti-Fas mAb was substituted by the optimal concentration (50 ng/ml) of the soluble recombinant FLAG-FasL combined with an anti-FLAG mAb as a cross-linker. Control cultures were only treated with the anti-FLAG mAb.
Western blot and immunoprecipitation
For the Western blot analysis, cells (5 x 106 per sample) were washed twice with cold PBS, resuspended in 100 µl of lysis buffer (10 mM Tris, pH 7.6, 150 mM NaCl, 1% Triton X-100, 10 mM EDTA) supplemented with a protease inhibitor cocktail (Boehringer Mannheim, Indianapolis, IN) and incubated for 15 min at 4°C. The cell-free supernatants were recovered by centrifugation of the suspension at 10,000 x g for 15 min at 4°C. The protein concentration of the extracts was determined by the Lowry method (Bio-Rad, Richmond, CA). For each sample, 30 µg of protein were loaded on the gel, then separated by 12% SDS-PAGE and transferred to Hybond nitrocellulose membrane (Amersham Life Science). Following transfer, the immunoblots were blocked by incubating with 5% nonfat dry milk in TBS and 0.1% Tween. The blots were next probed overnight with the appropriate dilution of the primary Abs (anti-caspase-8, c-FLIP, FADD or ß actin) at 4°C and revealed with the HRP-conjugated sheep anti-mouse polyclonal Ab (Amersham) for 1 h at room temperature. After washing with PBS/Tween, the blots were developed using the ECL chemiluminescence method (Pierce, Rockford, IL) according to the manufacturer’s protocol.
Immunoprecipitation of the Fas/CD95 DISC was conducted as described previously (7). Briefly, 1 x 107 B blasts were treated with the anti-APO-1 mAb (3 µg/ml) at 37°C for different time intervals and lysed in lysis buffer (30 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM PMSF, 1% Triton X-100, and 10% glycerol) for 15 min at 4°C. For the immunoprecipitation control, 1 x 107 freshly isolated B blasts were first lysed and then supplemented with the anti-APO-1 mAb. The Fas DISC was then precipitated overnight at 4°C with protein A-Sepharose (Sigma). The Sepharose beads were spun down, washed, resuspended in SDS-gel sample buffer, and boiled at 95°C for 3 min. Immunoprecipitates were separated by 12% SDS-PAGE and immunoblotted with the anti-Fas, FADD, caspase-8, and c-FLIP Abs.
Measurement of caspase-8 enzymatic activity
B blasts were seeded at 5 x 106 cells/well in 6-well plates in the presence of the anti-Fas mAb 7C11 or its isotypic control. Cells were harvested at different time points, washed with ice-cold PBS, and lysed in lysis buffer (HEPES 100 mM, sucrose 250 mM, EDTA 2 mM, CHAPS 0, 1%, DTT 5 mM, aprotinin 10 mg/ml, leupeptin 5 mg/ml, PMSF 1 mM). The lysates were then incubated with a 200 µM concentration of the colorimetric substrate IETD-pNA (Bachem) for 6 h at 37°C. The samples were read on a spectrophotometer set at 405 nm emission wavelength.
Measurement of caspase-3 cleavage
Percentages of cells with active caspase-3 were estimated by flow cytometry staining using a PE-coupled rabbit Ab recognizing the active cleavage product of caspase-3 (PharMingen/Becton Dickinson, Franklin Lakes, NJ). Cells were permeabilized with PermeaFix (Ortho Diagnostics, Raritan, NJ) before labeling. An unrelated isotype-matched PE-conjugated mAb was used as a negative control.
Assays for apoptosis
Quantitation of the proportion of apoptotic cells was made with:
1) the 3,3' dihexyloxacarbocyanine iodide (DiOC6)
dye (Molecular Probes, Eugene, OR), which reveals disruption of the
mitochondrial transmembrane potential (m) as described by Zamzami
et al. (31). In this assay, apoptotic cells are identified
by their decreased m
(DiOC6low); and 2) biotinylated
annexin V (Boehringer Mannheim), which detects the translocation of
phosphatidyl serines (PS) from the inner side to the outer leaflet of
the plasma membrane on apoptotic cells. Staining was revealed with
FITC-conjugated avidin (Immunotech, Marseille, France) used at 2.5
µg/ml. Immunofluorescence stainings were analyzed on a FACScan flow
cytometer using the LYSIS II software (Becton Dickinson). For cultures
conducted with the agonistic anti-Fas mAb 7C11, percentages of
specific cell death were calculated as follows: 100 x (apoptosis
with mAb 7C11 (%) - apoptosis with the isotype-matched control
mAb (%))/(100% - apoptosis with the isotype-matched control mAb
(%)). For cultures conducted with the FLAG-FasL, calculation of the
percentages of specific cell death were calculated as follows: 100
x (apoptosis with the soluble FasL aggregated by the anti-FLAG mAb
(%) - apoptosis with the anti-FLAG mAb alone (%))/(100% -
apoptosis with the anti-FLAG mAb alone (%)). Depending on the
read-out system used, cells were scored as apoptotic if: 1) they
exhibited disruption of m
(DiOC6low), 2) they externalized
PS (annexin V-positive), and 3) they expressed activated caspase-3.
Blockade of the mitochondrial Apaf-1 pathway
The experimental approach used relies on dATP depletion to prevent formation of the ATP-dependent cytochrome c/Apaf-1/pro-caspase-9 complex. For this purpose, B blasts were first preincubated for 1 h 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, to prevent production of ATP from both glycolysis and oxidative phosphorylation, as described previously (32). The cells were then cultured with either the anti-Fas mAb 7C11 or its isotypic control in the same medium and processed for Western blotting or measurement of apoptosis.
Measurement of intracellular ATP
The cellular ATP content was measured using a bioluminescence kit (Roche Molecular Biochemicals, Mannheim, Germany), based on the luciferin/luciferase assay, according to the manufacturer’s protocol. Briefly, 106 cells were cultured in complete RPMI medium or in glucose-free medium supplemented with oligomycin and treated with either the anti-Fas mAb 7C11 or its isotypic control. After the indicated times, cells were washed with PBS, lysed in the appropriate buffer, and incubated for 10 min at 4°C. After removal of cellular debris, ATP content was measured with a luminometer. ATP values in the samples were calculated using a calibration curve established with graded concentrations of free ATP. For each time point, the ATP content of cells cultured in complete medium without oligomycin and supplemented with the control mAb was considered as the reference value. Results are expressed as percent of the ATP content of the control sample.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because CD40 ligation induces susceptibility of mature B cells to
Fas-mediated apoptosis (23, 24, 25), CD40L-activated B cell
blasts were used as a model for Fas-sensitive B cells. To gain insight
into the molecular mechanisms responsible for execution of the Fas
death program, we first examined whether engagement of Fas on sensitive
B blasts was followed by cleavage of caspase-8. All experiments were
conducted with enriched virgin B cells, i.e., depleted of GC and memory
B cells, because we had found caspase-8 to be constitutively activated
in memory B cells ex vivo and spontaneously cleaved in cultured GC B
cells (data not shown). Consequently, enriched virgin B cells were
stimulated for 48 h with soluble trimeric CD40L and next
recultured with the anti-Fas mAb 7C11 or its isotypic control. Cell
lysates were prepared at different time points of the secondary culture
and probed with the anti-FADD and caspase-8 mAbs or tested for
caspase-8 enzymatic activity using the caspase-8-specific colorimetric
substrate IETD-pNA. As illustrated by Fig. 1
A, the zymogen but not the
activated form of caspase-8 is constitutively expressed in the blasts
recovered from the primary culture (first lane on the left). The 18-kDa
active cleavage product of caspase-8 is not detected in any of the
samples that received the control mAb. By contrast, the processed form
of caspase-8 appears in the anti-Fas-treated samples after 4 and
7 h of stimulation. FADD is also constitutively expressed in
virgin B blasts, and its levels of expression remain constant
whatever the culture conditions and the time point considered. Data
acquired from the enzymatic activity assay (Fig. 2B) are in agreement with the
Western blot results because a significant rise in caspase-8 enzymatic
activity is observed in anti-Fas-treated cells as compared with
cultures receiving the isotype-matched control mAb. In fact, the
appearance of caspase-8 enzymatic activity coincides with the release
of its 18-kDa active cleavage product: it is first detectable by 4
h of stimulation and is expressed until 12 h of culture.
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To explore the role of the mitochondria in execution of the Fas
apoptotic program in activated human B cells, experiments were first
conducted with various caspase inhibitors. The caspase-8-specific
blocking peptide z-IETD was used to determine whether activation of
caspase-8 in CD40L-activated B cells is a pre- or a postmitochondrial
event. We reasoned that, in the former case, blocking caspase-8
activation should prevent the Fas-induced disruption of the
mitochondrial transmembrane potential (m). The caspase-9-specific
blocking peptide z-LEHD was used to determine the implication of
caspase-9 and thus participation of the so-called "apoptosome"
(33) in Fas-induced killing of CD40L-activated B cells.
The cathepsin B inhibitor z-FA and the broad-range caspase inhibitory
peptide z-VAD were used as negative and positive controls,
respectively. The fluoromethylketone derivatives of the four peptides
mentioned above were thus tested for their capacity to inhibit the
Fas-induced disruption of the mitochondrial potential and PS exposure
in CD40L-induced virgin B blasts. Finally, to exclude any artifactual
finding resulting from the use of an agonistic Abs to ligate Fas, these
experiments were conducted both with the anti-Fas mAb 7C11 (or its
isotypic control) and with a recombinant human FLAG-FasL molecule
aggregated with and anti-FLAG mAb as reported elsewhere
(34). Fig. 2
shows that similar results are obtained when
Fas is engaged by the anti-Fas Ab or by the aggregated soluble
FasL, therefore indicating that, in our hands, the anti-Fas mAb
7C11 reliably mimics the effect of the physiological ligand.
Furthermore, as compared with control cultures conducted in the
presence of z-FA, both z-VAD and z-IETD reduced PS externalization and
prevented loss of mitochondrial transmembrane potential consecutive to
Fas ligation. In contrast, z-LEHD only marginally affected both these
parameters. These results have two implications. First, they indicate
that caspase-8 participates in transducing the death signal from Fas to
the mitochondria. Hence, most of caspase-8 activity promoted by Fas
must result from cleavage of its zymogen form upstream of the
mitochondria. Second, they suggest that caspase-9 activity is not
mandatory for induction of the mitochondrial and membrane alterations
in response to Fas ligation on CD40L-activated B cells. To explore
further the involvement of the mitochondria in activation of caspase-8
in sensitive B cells, we decided to impair the proapoptotic
mitochondrial function by preventing dATP-dependent formation of the
apoptogenic cytochrome c/Apaf-1/pro-caspase-9 complex. For
this purpose, CD40L-activated B cell blasts were treated with
oligomycin in glucose-free medium, a procedure known to prevent
production of dATP from both glycolysis and oxidative phosphorylation.
PS externalization, m, and the cleavage of caspase-8 were
compared in oligomycin-treated and untreated blasts following
triggering of Fas. Although the use of oligomycin in glucose-free
medium severely reduced (by 95% or more) the intracellular ATP levels
(Fig. 3A), it blocked neither
the mitochondrial (m) nor the membrane (PS exposure)
manifestations of apoptosis consecutive to Fas ligation on sensitive B
blasts (Fig. 3B). Instead, we consistently observed that
dATP depletion enhanced both Fas-induced disruption of the
mitochondrial potential and PS externalization. As illustrated by Fig. 3C, dATP depletion also failed to abrogate the proteolytic
cleavage of caspase-8, because both its intermediate (p43/p41) and
active (p18) cleavage products were produced in oligomycin-treated
blasts. Altogether, these data support the notion that the
mitochondrial Apaf-1 pathway is not responsible for activation of
caspase-8 and execution of the apoptotic program promoted by Fas
ligation on CD40L-induced B blasts.
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To monitor the constitutive and activation-induced expression of
cFLIP in virgin B cells, Western blot analysis of c-FLIP was conducted
in lysates of isolated virgin B cells before and after 24 h of
stimulation with CD40L, anti-Ig Abs, or the combination of both
reagents. Fig. 4A shows that
cFLIP is virtually undetectable in freshly isolated virgin B cells.
However, both the long (c-FLIPL) and the short
(c-FLIPS) isoforms of c-FLIP are induced in B
cells upon separate or concomitant triggering of CD40 and the BCR. In
contrast, the levels of expression of FADD and procaspase-8 remained
unchanged after in vitro B cell activation. Because Fas triggering on
model cell lines induces cleavage of c-FLIPL into
a 43-kDa product at the level of the DISC (12, 28), we
next investigated the impact of Fas ligation on the processing of
c-FLIP in sensitive B blasts. CD40L-activated B cells were thus
recultured for 4 h with the anti-Fas mAb or its isotypic
control and tested for the expression of c-FLIP at the end of the
secondary culture by Western blot analysis. Fig. 4B shows
that Fas triggering promotes processing of
c-FLIPL in sensitive B blasts because its
p43-cleaved form was produced in the anti-Fas-treated samples but
not in control cultures. To investigate the kinetics of expression of
c-FLIP following CD40 ligation, cell lysates were prepared from
virgin B cells stimulated for 12, 24, 48, and 72 h with soluble
trimeric CD40L and successively probed with the anti-c-FLIP and ß
actin Abs. As shown in Fig. 5, A and B, expression of both c-FLIP isoforms
peaked after 24 h of CD40 activation and decreased thereafter to
reach its lowest level after 72 h of stimulation. We next
determined whether the levels of expression of c-FLIP had any impact on
the susceptibility of activated B cells to Fas-induced apoptosis. For
this purpose, B blasts recovered after 24 h
(c-FLIPhigh) and 72 h
(c-FLIPlow) of CD40L stimulation were
compared for: 1) their kinetics of entry into apoptosis upon Fas
ligation, and 2) the amplitude of their apoptotic response to the
anti-Fas mAb 7C11. Both m and the proportions of cells with
active caspase-3 were estimated at different time points of the
secondary cultures. As illustrated by Fig. 5, C and
D, both read-outs concurred to indicate that the apoptotic
response of 72-h blasts develop more rapidly and is of higher magnitude
than that of the 24-h blasts.
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, FADD
as well as the p43-cleavage products of c-FLIPL
and caspase-8 were coprecipitated with Fas in cells that had been
stimulated with the anti-Fas mAb APO-1. No signal was obtained with
the FADD, c-FLIP, and caspase-8 Abs in freshly isolated B blasts,
despite the efficient precipitation of Fas (left lane on the blot).
This observation testifies that association of these three proteins
with Fas is consecutive to ligation of the receptor on sensitive B
blasts. No significant DISC formation was found after short exposure
(10 min) of B blasts to the agonistic anti-Fas mAb APO-1 (data not
shown). Interestingly, c-FLIP was already associated with Fas and
cleaved after 3 h of anti-Fas stimulation, whereas the
processed form of caspase-8 is only found in the immunoprecipitate
after 4 h of anti-Fas stimulation, suggesting that recruitment
of c-FLIP to the DISC may precede that of caspase-8. Therefore, both
c-FLIP and caspase-8 associate with the Fas DISC following ligation of
Fas on sensitive B cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In our hands, the expression of both c-FLIP isoforms is strongly up-regulated when virgin B cells are activated by separate or concomitant ligation of the BCR and CD40. Although we did not observe complete disappearance of c-FLIP upon prolonged CD40 stimulation as reported by Van Parijs et al. (47) for murine B cells, we did find that its level of expression significantly decreases between 24 and 72 h of CD40 stimulation. It has also been documented that the levels of c-FLIP transcript in mouse splenic B cells treated with anti-Ig Abs are enhanced during the first 24 h of stimulation but return to baseline levels thereafter (48). Altogether, these findings support the notion that c-FLIP expression in activated B lymphocytes is transient. As previously observed for Fas-sensitive T cells after prolonged culturing with PHA and IL-2 (28), we found c-FLIP to be recruited and cleaved at the level of the DISC in response to Fas triggering of sensitive B cells. One possible explanation for this finding would be that association of c-FLIP with the DISC is required for transduction of the Fas death signal. This hypothesis concurs with several studies showing that c-FLIP can behave as a proapoptotic molecule when it is overexpressed by transfection in mammalian cell lines (14, 15, 18). Although the latter possibility cannot be definitely excluded, it is not compatible with the wealth of data that document the inhibitory function of c-FLIP toward death receptor-mediated apoptosis. Indeed, c-FLIP expression has been correlated with interruption of the Fas death signal in melanoma cell lines (12), Con A-activated Jurkat cells (49), T cells from IL-2 knockout mice (50), and in vitro-differentiated macrophages (51). Moreover, enforced expression of c-FLIP has been shown to protect B and T lymphocytes as well as tumor cells from death receptor-induced apoptosis in vivo (47, 52, 53). The pull-down system we have used for analysis of c-FLIP expression does not allow us to determine whether c-FLIP and caspase-8 are part of the same DISC or whether two separate types of DISC, containing either caspase-8 or c-FLIP, are formed within the CD40-activated B cell population in response to Fas ligation. However, experiments conducted on stable c-FLIP transfectants have demonstrated that c-FLIP, caspase-8, and Fas can be associated within the same complex following engagement of Fas (28). Finally, the inverse correlation we found between the levels of expression of c-FLIP and the degree of susceptibility of CD40L-activated B blasts to Fas-induced apoptosis further supports the notion that c-FLIP behaves as a negative regulator of the Fas signaling pathway in activated human B cells.
One is then left with the open question: why should a molecule capable
of antagonizing the Fas death signal associate with the Fas receptor in
sensitive cells? Our interpretation of this finding is that c-FLIP is
instrumental in delaying the onset of apoptosis in cells that are
doomed for death. As a matter of fact, as opposed to model
lymphoblastoid or lymphoma cell lines in which cleavage of caspase-8 is
detected within min of Fas triggering, activation of caspase-8 in
activated virgin B cells does not occur until 4 h of Fas
stimulation. The results presented herein are thus compatible with the
hypothesis that c-FLIP could serve to attenuate the efficiency of the
Fas signal in activated B lymphocytes. The transient expression of
c-FLIP in activated B cells could offer an ultimate opportunity for B
cells that have been signaled through Fas to be rescued by Ag or
trophic factors. Alternatively, c-FLIP could be instrumental in
suspending death in Fas-targeted B cells long enough to allow them to
release chemoattractants able to mobilize phagocytic cells. Indeed, a
defective clearance of apoptotic cells could favor inflammation or
autoimmune reactions due to the release by dying cells of cryptic or
potential self Ag. In keeping with this, it has been described that two
chemokines (IL-8 and MIP1
) known to promote recruitment of
phagocytes are produced by apoptotic cells in response to Fas or TNF
ligation (54) or treatment with chemotherapeutic drugs
(55).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Thierry Defrance, Institut National de la Santé et de la Recherche Médicale Unité 404, Avenue Tony Garnier, 69365, Lyon, Cedex 07, France.
3 Abbreviations used in this paper: DD, death domain; DED, death effector domain; FADD, Fas-associated DD-containing protein; FLICE, FADD-like IL-1ß-converting enzyme; FLIP, FLICE-like inhibitory protein, GC, germinal center; PS, phosphatidyl serines; DISC, death-inducing signaling complex; L, ligand; BCR, B cell receptor; GC, germinal center.
Received for publication February 16, 2000. Accepted for publication June 26, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ligation of Fas antigen. J. Immunol. 155:4147.[Abstract]
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C. Kern, J.-F. Cornuel, C. Billard, R. Tang, D. Rouillard, V. Stenou, T. Defrance, F. Ajchenbaum-Cymbalista, P.-Y. Simonin, S. Feldblum, et al. Involvement of BAFF and APRIL in the resistance to apoptosis of B-CLL through an autocrine pathway Blood, January 15, 2004; 103(2): 679 - 688. [Abstract] [Full Text] [PDF]
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P. K. A. Mongini, A. E. Jackson, S. Tolani, R. J. Fattah, and J. K. Inman Role of Complement-Binding CD21/CD19/CD81 in Enhancing Human B Cell Protection from Fas-Mediated Apoptosis J. Immunol., November 15, 2003; 171(10): 5244 - 5254. [Abstract] [Full Text] [PDF]
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F. Zazzeroni, S. Papa, A. Algeciras-Schimnich, K. Alvarez, T. Melis, C. Bubici, N. Majewski, N. Hay, E. De Smaele, M. E. Peter, et al. Gadd45{beta} mediates the protective effects of CD40 costimulation against Fas-induced apoptosis Blood, November 1, 2003; 102(9): 3270 - 3279. [Abstract] [Full Text] [PDF]
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I. Schmitz, A. Krueger, S. Baumann, H. Schulze-Bergkamen, P. H. Krammer, and S. Kirchhoff An IL-2-Dependent Switch Between CD95 Signaling Pathways Sensitizes Primary Human T Cells Toward CD95-Mediated Activation-Induced Cell Death J. Immunol., September 15, 2003; 171(6): 2930 - 2936. [Abstract] [Full Text] [PDF]
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M. Fukui, R. Imamura, M. Umemura, T. Kawabe, and T. Suda Pathogen-Associated Molecular Patterns Sensitize Macrophages to Fas Ligand-Induced Apoptosis and IL-1{beta} Release J. Immunol., August 15, 2003; 171(4): 1868 - 1874. [Abstract] [Full Text] [PDF]
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 E. M. Maggio, A. van den Berg, D. de Jong, A. Diepstra, and S. Poppema Low Frequency of FAS Mutations in Reed-Sternberg Cells of Hodgkin's Lymphoma Am. J. Pathol., January 1, 2003; 162(1): 29 - 35. [Abstract] [Full Text] [PDF]
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 L Leoncini, S Lazzi, C Bellan, and P Tosi Cell kinetics and cell cycle regulation in lymphomas J. Clin. Pathol., September 1, 2002; 55(9): 648 - 655. [Abstract] [Full Text] [PDF]
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R. K. Thomas, A. Kallenborn, C. Wickenhauser, J. L. Schultze, A. Draube, M. Vockerodt, D. Re, V. Diehl, and J. Wolf Constitutive Expression of c-FLIP in Hodgkin and Reed-Sternberg Cells Am. J. Pathol., April 1, 2002; 160(4): 1521 - 1528. [Abstract] [Full Text] [PDF]
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P. Chu, D. Deforce, I. M. Pedersen, Y. Kim, S. Kitada, J. C. Reed, and T. J. Kipps Latent sensitivity to Fas-mediated apoptosis after CD40 ligation may explain activity of CD154 gene therapy in chronic lymphocytic leukemia PNAS, March 19, 2002; 99(6): 3854 - 3859. [Abstract] [Full Text] [PDF]
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N. A. Maianski, F. P. J. Mul, J. D. van Buul, D. Roos, and T. W. Kuijpers Granulocyte colony-stimulating factor inhibits the mitochondria-dependent activation of caspase-3 in neutrophils Blood, January 15, 2002; 99(2): 672 - 679. [Abstract] [Full Text] [PDF]
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 A. Krueger, S. Baumann, P. H. Krammer, and S. Kirchhoff FLICE-Inhibitory Proteins: Regulators of Death Receptor-Mediated Apoptosis Mol. Cell. Biol., December 15, 2001; 21(24): 8247 - 8254. [Full Text] [PDF]
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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]
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A. L. Snow, L. J. Chen, R. R. Nepomuceno, S. M. Krams, C. O. Esquivel, and O. M. Martinez Resistance to Fas-Mediated Apoptosis in EBV-Infected B Cell Lymphomas Is Due to Defects in the Proximal Fas Signaling Pathway J. Immunol., November 1, 2001; 167(9): 5404 - 5411. [Abstract] [Full Text] [PDF]
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M. van Eijk, J. P. Medema, and C. de Groot Cutting Edge: Cellular Fas-Associated Death Domain-Like IL-1-Converting Enzyme-Inhibitory Protein Protects Germinal Center B Cells from Apoptosis During Germinal Center Reactions J. Immunol., June 1, 2001; 166(11): 6473 - 6476. [Abstract] [Full Text] [PDF]
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 A. Hennino, M. Berard, P. H. Krammer, and T. Defrance Flice-Inhibitory Protein Is a Key Regulator of Germinal Center B Cell Apoptosis J. Exp. Med., February 19, 2001; 193(4): 447 - 458. [Abstract] [Full Text] [PDF]
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 A. Krueger, I. Schmitz, S. Baumann, P. H. Krammer, and S. Kirchhoff Cellular FLICE-inhibitory Protein Splice Variants Inhibit Different Steps of Caspase-8 Activation at the CD95 Death-inducing Signaling Complex J. Biol. Chem., June 1, 2001; 276(23): 20633 - 20640. [Abstract] [Full Text] [PDF]
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