From the Laboratory of Immunology, INSERM U80 UCBL,
Hôpital E. Herriot, 69437 Lyon, France, the ¶ Laboratory
of Biochemistry and Pharmacology, INSERM U352, INSA-Lyon, 69621 Villeurbanne Cedex, France, and
Schering-Plough, Laboratory
for Immunological Research, 69571 Dardilly, France
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ABSTRACT |
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We recently demonstrated that the engagement of
HLA class I 1 domain induced Fas-independent apoptosis in human
T and B lymphocytes. We analyzed the signaling pathway involved in HLA
class I-mediated apoptosis in comparison with Fas (APO-1,
CD95)-dependent apoptosis. The mouse mAb90 or the rat
YTH862 monoclonal antibodies which bind the human HLA class I
1
domain induced the production of ceramide which was blocked by addition
of the phosphatidylcholine-dependent phospholipase C
inhibitor, D609. Furthermore, HLA class I-mediated apoptosis involved
at least two different caspases, an interleukin-1 converting
enzyme-like protease and another protease inhibited by the CPP32-like
protease inhibitor Ac-DEVD-CHO. Despite similarity between Fas and HLA
class I signaling pathways, we failed to demonstrate any physical
association between these two molecules. We also report that the
pan-caspase inhibitory peptide zVAD-fmk, but not Ac-DEVD-CHO and
Ac-YVAD-CHO, inhibited decrease of mitochondrial transmembrane
potential and generation of ceramide induced by anti-HLA class I and
anti-Fas monoclonal antibodies, whereas all three peptides
efficiently inhibited apoptosis. Altogether these results suggest
that signaling through Fas and HLA class I involve caspase(s), targeted
by zVAD-fmk, which act upstream of ceramide generation and
mitochondrial events, whereas interleukin-1 converting enzyme-like and CPP32-like proteases act downstream of the
mitochondria.
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INTRODUCTION |
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Apoptosis is a process that occurs in physiological or pathological conditions, in many different cell types, which serves as a fundamental control during the development of multicellular organisms. In the immune system, apoptosis induced by surface receptors is a central mechanism for the homeostasis of T and B lymphocytes.
With regard to human T cells, apoptosis can be triggered by several
membrane receptors including three members of the tumor necrosis factor
receptor (TNF-R)1 family (1),
Fas (CD95) (2-4), TNF-RI (5, 6), and CD30 (7), but also CD2 (8, 9),
CD45 (10), HLA class I (11, 12), and CTLA4 (13) molecules. We recently
described two monoclonal antibodies (mAbs) that bind to the 1 domain
of HLA class I heavy chain and induce apoptosis of activated, but not
resting, T and B lymphocytes (14, 15). Apoptosis induced by anti-HLA
class I mAbs did not result from Fas/Fas-L interaction and distinct though partly overlapping populations of activated T cells were susceptible to Fas- and HLA class I-mediated apoptosis, respectively (15).
Regarding apoptosis signaling, TNF-RI and Fas have been shown to
trigger rapid sphingomyelin hydrolysis into ceramide. Mutations in the
TNF-RI cytoplasmic domain that abolished acidic sphingomyelinase and
NF-B activation in response to TNF
, also prevented cell death
(16). Furthermore, in a Fas-resistant tumor cell line which expresses a
death domain-defective Fas splice isoform, Fas ligation does not
activate acidic sphingomyelinase although neutral sphingomyelinase and
ERK-2 activities are still intact (17). In addition to TNF
and
Fas-ligand (Fas-L), anti-IgM, ionizing radiation, heat shock,
ultraviolet light, and oxidative stress (18-22) have been shown to
induce ceramide production by cells in which they initiate apoptosis,
suggesting that ceramide might be a general mediator of apoptosis.
Recent studies have provided compelling evidence that a cascade of
Asp-directed cysteine proteases, renamed caspases (23), plays a pivotal
role in transduction of apoptotic signals. Particularly,
interleukin-1 converting enzyme (ICE)-like proteases have been
implicated in Fas- and TNF
-induced cell death (24-29). Caspases can
be clustered into three groups according to their specificities
and their biological functions: group I/ICE (caspase-1),
TX/ICH2/ICErel-II (caspase-4), ICErel-III/TY (caspase-5), group II/Yama/CPP-32/apopain (caspase-3),
Mch3/ICE-LAP3/CMH-1 (caspase-7), and 3/ICH-1/Nedd2 (caspase-2), and
group III/Mch2 (caspase-6), MACH/FLICE/Mch5 (caspase-8) ICE-LAP-6/Mch6
(caspase-9), and Mch4 (caspase-10) (reviewed in Refs. 23 and 30). The
specificity of the caspase family has been defined by cleavage
sequences in their respective substrates, which permitted the
generation of specific inhibitory peptides, such as Ac-DEVD-CHO, which
inhibits CPP32-like protease activity but also other members of caspase family (31), Ac-YVAD-CHO, a specific inhibitor of ICE-like protease activity (27), and Ac-zVAD-fmk, a pan-caspase inhibitor.
The relationship between ceramide production and caspases
activation in apoptosis signaling is still not well defined. Recently, a control of ceramide production by caspases has been proposed in
REAPER- and TNF-induced apoptosis models (32, 33), but remains to be
demonstrated in other models of apoptosis.
In the present study we investigated the HLA class I signaling pathway
leading to apoptosis of activated T lymphocytes, and we examined the
possible connection between ceramide and caspases in both Fas- and HLA
class I-apoptosis pathways. Our results point toward activation of
caspases upstream and downstream of ceramide production, leading to
reduction of mitochondrial transmembrane potential (m)
and subsequent propagation of the death signal.
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EXPERIMENTAL PROCEDURES |
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Cell Preparation and Culture-- Peripheral blood lymphocytes (PBL) were collected from healthy donors in the presence of sodium citrate. Blood was defibrinated, then mononuclear cells were isolated by centrifugation on a layer of Histopaque® (Sigma). Those cell suspensions referred to as PBL contained 1.8 ± 0.4% monocytes as defined by expression of CD14. PBL were resuspended in RPMI 1640 (Sigma) supplemented with 10% fetal calf serum, 2 mM L-glutamine, and antibiotics (penicillin, 100 units/ml; streptomycin, 100 µg/ml). Cells (1 × 106/ml) were incubated in the presence of phytohemagglutinin (PHA), 5 µg/ml (Sigma). Cultures were maintained in a humid atmosphere containing 5% CO2 for 72 h.
Antibodies and Reagents--
MAb90 (anti-HLA-A, -B, -C, mouse
IgG1) was produced as described previously (15) and
purified from ascites fluids by DEAE chromatography. The YTH862 mAb
(anti-HLA-A, -B, and -C, rat IgG2b) was produced in Prof. H. Waldmann's laboratory (Oxford, United Kingdom). W6/32 mAb (anti-HLA-A,
-B, and -C, IgG2a) was obtained from ATCC (Rockville, MD).
Epitope specificity of the different anti-HLA class I antibodies was
analyzed by using transfected C1R cells that express HLA-A2.1 or hybrid
mouse/human exon-shuffled HLA-A2.1/H-2Dd genes (34) and has
been previously reported (15). Briefly, mAb90 and YTH862 bind to
closely related or identical epitopes of the 1 domain, whereas W6/32
binds to a non-polymorphic epitope of the
2 and
3 domains.
Purified anti-Fas mAb agonist (IgM, clone CH11) and FITC anti-Fas mAb
(IgG1, clone UB2) were purchased from Immunotech
(Marseille, France). IgG1 control mAb (for bioassays) and
FITC IgG control mAbs (for flow cytometric analyses) were from Dako
(Glostrup, Denmark). The phosphatidylcholine-specific phospholipase C
(PC-PLC) inhibitor D609, genistein, chelerythrine, okadaic acid, and
cytochalasin B and D were purchased from Sigma. The tetrapeptide
ICE-like protease inhibitor Ac-YVAD-CHO and CPP32-like protease
inhibitor Ac-DEVD-CHO were from Neosystem (Strasbourg, France),
zVAD-fmk was from Bachem (Voisins le Bretonneux, France). Cyclosporin A
(CsA) was kindly supplied by Sandoz Corp. (Basel, Switzerland).
Measurement of Apoptosis--
After 3 days of culture,
PHA-activated PBL were harvested. Dead cells were removed by
centrifugation on a layer of Histopaque (Sigma) and washed in Hank's
balanced salt solution. Viable cells (106/ml) were
incubated in 96-well microplates with various inhibitors and then
treated with mAb90. After 24 h of incubation, cell death was
evaluated by fluorescence microscopy after staining with Hoechst 33342 (Sigma) at 10 µg/ml following previously described methods (35).
Nuclear fragmentation and/or marked condensation of the chromatin with
reduction of nuclear size were considered as typical features of
apoptotic cells. Based on these measurements, results were expressed as
percentage of specific apoptosis according to the following formula, % specific apoptosis = (% of apoptotic treated cells % of
apoptotic control cells) × 100/100
of apoptotic control cells.
Evaluation of Mitochondrial Transmembrane Potential--
To
evaluate the mitochondrial transmembrane potential (36), cells (2 × 105/ml) were incubated with 3,3'-dihexyloxacarbocyanine
(3), 40 nM in phosphate-buffered saline (Molecular Probes,
Inc., Eugene, OR) for 15 min at 37 °C, followed by analysis on a
cytofluorometer (Becton Dickinson, Mountain View, CA; excitation maximum 488 nM,
emission maximum 525 nm).
Ceramide Assay-- Ceramide was quantified by the diacylglycerol kinase assay as 32P incorporated upon phosphorylation of ceramide to ceramide 1-phosphate by diacylglycerol kinase from Escherichia coli (Biomol, Plymouth Meeting, PA) (37). Briefly, after 3 days of culture, 2 × 107 PBL were starved for 2 h in RPMI containing 2% bovine serum albumin and then treated with the different mAbs for indicated times. Ceramide 1-phosphate was resolved by TLC using CHCl3/CH3OH/CH3COOH (65:15:5, v/v) as solvent. Authentic ceramide 1-phosphate was identified by autoradiography at RF 0.25. The level of ceramide was determined by comparison to concomitantly run standard curve comprised of known amounts of ceramide (Sigma) and normalized to [3H]triglyceride introduced during lipid extraction.
ICE-like Protease Activity Assay-- Three-day PHA-stimulated PBL were permeabilized using the osmotic shock method as followed: 107 PBL/ml were incubated with 50 µM ICE substrate DABCYL-YVADAPV-EDANS (Bachem, Voisins le Bretonneux, France) and distilled water (1:1, v/v). After 5 min incubation at 37 °C, appropriate volumes of 5 × phosphate-buffered saline in 50% fetal calf serum were added to bring the osmolarity to normal level. Then cells (106/ml) were incubated with different mAbs for indicated times and analyzed by FACS Star Plus analysis (Becton Dickinson, Pont de Claix, France) using an excitation wavelength of 360 nm and emission wavelength of 488 nm.
Determination of Poly(ADP-ribose) Polymerase (PARP)
Cleavage--
Cleavage of PARP was determined by Western blotting. The
cells (1 × 106) were washed twice in
phosphate-buffered saline, pelleted, and lysed in 100 µl of lysis
buffer containing 62.5 mM TRIS, pH 6.8, 2% SDS, 10%
glycerol, 2% -mercaptoethanol. After 10 min on ice, 20 µl of
5 × sample buffer was added, and samples were heated for 5 min at
95 °C. Thirty µl of the lysate were subjected to 7.5%
SDS-polyacrylamide gel electrophoresis gel and transferred to
nitrocellulose membrane. Blots were probed using anti-PARP mAb C-2-10
(Biomol, TEBU, Le Perray en Yvelines, France). Bound antibodies were
detected with rabbit anti-mouse peroxidase-conjugated antibodies
(Bio-Rad, Ivry sur Seine, France). An enhanced chemiluminescence system
(Amersham, France) was used for detection.
Fluorescence Energy Transfer Experiments-- Three-day PHA-activated PBL were stained with tetramethylrhodamine isothiocyanate-conjugated (TRITC) mAb90 and FITC anti-Fas mAb UB2 or FITC anti-CD25 mAb. Fluorescence energy transfer experiments have been done by measurement of decrease fluorescence anisotropy by a confocal microscope Acas 570 (Meridian) as described previously (38, 39). When resonance energy transfer occurred between a donor-acceptor pair, the following phenomena could be observed: quenching of the fluorescence emission of the donor molecule; sensitization of the emission of the acceptor; and a reduction in the anisotropy of the acceptor emission, which was dependent on the ratio of excitation through direct energy transfer from the donor to the acceptor.
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RESULTS |
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Inhibition of HLA Class I-mediated Apoptosis by Cytochalasins and Okadaic Acid-- A series of inhibitors of various activation pathways was used for comparison of Fas and HLA class I-mediated apoptosis. PBL from healthy donors were activated for 3 days with PHA and then treated with various inhibitors before exposure to mAb90, YTH862, or the anti-Fas mAb CH11. Genistein, chelerythrine, EGTA, or CsA failed to interfere with HLA class I and Fas-mediated apoptosis, suggesting that neither protein tyrosine kinase nor PKC activation, Ca2+ influx, or phosphatase 2B were involved in HLA class I apoptotic signaling (Fig. 1A). In contrast, okadaic acid which inhibits phosphatase 1, 2A, and 2C significantly decreased HLA class I- and Fas-mediated apoptosis (Fig. 1, A and B). The inhibition of mAb 90 and YTH862-induced apoptosis was dose-dependent and reached 90% at 10 ng/ml (Fig. 1B). Unlike Fas-, HLA class I-mediated cell death was strongly inhibited by cytochalasin B and D (Fig. 1, A and C), suggesting that HLA class I but not Fas-dependent apoptosis requires cytoskeletal rearrangement and possibly association with other molecules.
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MAb90 and YTH862 Induce Ceramide Production under the Control of a
Phosphatidylcholine-specific Phospholipase C--
Okadaic acid has
been reported to inhibit ceramide-induced apoptosis, possibly by
blocking a ceramide-activated protein phosphatase which belongs to the
heterodimeric subfamily of the phosphatases 2A group (40). Since
okadaic acid inhibits HLA class I-induced apoptosis (Fig. 1,
A and B), we investigated whether HLA class I
engagement would generate ceramide production. Lipids were extracted from PHA-activated PBL treated by W6/32, mAb90, YTH862, anti-Fas mAb
CH11 or control IgG1, and endogenous ceramide production
was measured by a diacylglycerol kinase assay. While W6/32 and control IgG1 did not induce ceramide production, the two anti-HLA
class I 1 domain, mAb90 or YTH862 which trigger apoptosis, increased the basal level of ceramide production by 159 and 166%, respectively (Fig. 2A). As control,
anti-Fas mAb increased the basal level of ceramide production by 162%.
Ceramide production by mAb 90 was obtained after 10 min and decreased
rapidly thereafter (Fig. 2B). We also tested an inhibitor of
PC-PLC, the xanthate D609 which subsequently blocks the activation of
acidic sphingomyelinase but not neutral sphingomyelinase (17, 18) on
HLA class I-induced ceramide production. Results in Fig. 2C
show that D609 reduced the production of ceramide induced by mAb90 to
the control level, indicating that HLA class I-mediated ceramide
generation may depend upon PC-PLC activation.
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Anti-HLA Class I mAbs Activate ICE-like Proteases and Induce PARP
Cleavage--
Several studies have demonstrated the activation of
caspases in different pathways of apoptosis (25, 26, 31). To directly address the involvement of ICE-like proteases in HLA class I-mediated apoptosis, we studied the kinetics of ICE-like protease activity after
anti-HLA class I treatment. Permeabilized PHA-activated PBL were
incubated with the fluorogenic ICE substrate DABCYL-YVADAPV-EDANS, which contains the cleavage site of interleukin-1 precursor (41) and
then treated with W6/32, mAb90, YTH862, and anti-Fas mAb CH11. As shown
in Fig. 3A, mAb90, YTH862, and
anti-Fas mAb induced ICE-like protease activity with a peak at 1 h
for anti-HLA class I mAbs and 2 h for anti-Fas mAb. In parallel,
cleavage of PARP to its signature 89-kDa fragment (42) was studied by
immunoblotting. PHA-activated PBL were treated for 0, 2, and 6 h
in the presence of CH11, mAb90, or IgG1 as a control, PARP
cleavage was evident within 2 h with both mAb90 and the anti-Fas
mAb CH11, but not with control IgG1 (Fig. 3B).
These results suggest that, like Fas cross-linking, HLA class
I-mediated apoptosis involves activation of at least ICE-like and
CPP32-like family proteases.
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Inhibition of Caspases Prevents HLA Class I-mediated Apoptosis-- Caspases are specifically inhibited in vitro and in vivo by cell-permeable tetrapeptides designed to mimic cleavage sites of their respective substrates. We used such inhibitory peptides to test whether caspases, other than ICE-like proteases, are involved in HLA class I-mediated apoptosis. Preincubation of PHA-activated PBL, for 3 h, with zVAD-fmk, Ac-DEVD-CHO, or Ac-YVAD-CHO, before treatment with antibodies, inhibited both HLA class I- and Fas-mediated apoptosis (Fig. 4). However, Ac-DEVD-CHO significantly decreased apoptosis at 1 µM and achieved nearly complete (96%) inhibition of HLA class I, but partial (75%) diminution of Fas-mediated apoptosis at 100 µM (Fig. 4B), while Ac-YVAD-CHO was not or weakly active at 1 µM and inhibited about 50 to 60% of apoptosis at 100 µM (Fig. 4A). zVAD-fmk was the most efficient inhibitor, reducing by 50-80% at 1 µM and 100% at 100 µM both HLA class I- and Fas-mediated apoptosis. These results suggest that HLA class I-mediated apoptosis involves at least two different caspases, an ICE-like protease and another protease(s) inhibited by the CPP32-like inhibitor Ac-DEVD-CHO.
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HLA Class I Molecules Do Not Associate with Fas--
Since HLA
class I molecules may associate with other cell surface functional
molecules such as the interleukin-2 receptor (43), it could be
hypothesized that HLA class I may trigger apoptosis by associating
itself with death signaling molecules such as Fas. This type of
association might be induced by PBL activation or, alternatively, only
by engagement of HLA class I molecules with mAbs which induce
apoptosis, such as mAb 90 and YTH862. To investigate these hypotheses,
fluorescence energy transfer experiments have been performed by
measurement of fluorescence anisotropy after incubation of
PHA-activated PBL with TRITC-conjugated mAb90 or W6/32 and
FITC-conjugated anti-Fas mAb UB2 or anti-CD25 mAb as positive control.
When two molecules are physically associated, resonance energy transfer
occurs between the donor-acceptor pair, which leads to the quenching of
fluorescence emission by the donor molecule, sensitization of the
emission by the acceptor, and a reduction in the anisotropy of the
acceptor molecule. Results in Table I
show that FITC anti-CD25 mAbs decrease the fluorescence anisotropy
induced by TRITC-mAb90 or TRITC-W6/32 alone confirming the association
between HLA class I and IL-2 receptor, whereas FITC anti-Fas mAbs do
not. Moreover, mAb90 precipitated the 45-kDa heavy chain and 12-kDa
2m from the surface biotinylated PHA blasts but not a 48-kDa protein
corresponding to Fas (data not shown). These results show that HLA
class I molecules do not associate with Fas upon PHA activation or upon
binding of the apoptosis-inducing mAb90, further excluding a
contribution of cell surface Fas molecules in HLA class I-mediated
apoptosis, and reinforcing the hypothesis of an intracellular
interaction between Fas and HLA class I signaling cascades.
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zVAD-fmk, but Not Ac-DEVD-CHO or AC-YVAD-CHO, Inhibits Ceramide Production Induced by Anti-Fas and Anti-HLA Class I mAbs-- Knowing that the peptides zVAD-fmk, Ac-DEVD-DHO, and Ac-YVAD-CHO inhibit both HLA class I- and Fas-mediated apoptosis, we tested whether these three inhibitory peptides would also inhibit ceramide production. In these experiments PHA-activated PBL were treated for 2 h with each peptide at 100 µM before the addition of mAb90, YTH862, or CH11 mAbs. After 10 min in the presence of CH11, mAb90, or YTH862 mAbs, phospholipids were extracted and endogenous ceramide production was measured by a diacylglycerol kinase assay. As shown in Fig. 5, zVAD-fmk, but not Ac-YVAD-CHO or Ac-DEVD-CHO, inhibited ceramide production induced by either anti-Fas or anti-HLA class I mAbs. Pretreatment of cells with zVAD-fmk (100 µM) resulted in 60-70% inhibition of ceramide production induced by CH11 and mAb90 mAbs, and 34% inhibition in the case of YTH862 mAb (Fig. 5A). A dose-response experiment with the zVAD-fmk inhibitory peptide on ceramide production induced by CH11 mAb showed a very weak inhibition at 1 µM (<10%), whereas at 10 or 100 µM zVAD-fmk reduced ceramide production to the level observed in presence of the tetrapetide alone (Fig. 5B). In contrast pretreatment with either Ac-YVAD-CHO or AC-DEVD-CHO did not block Fas- and HLA-class I-induced ceramide production, but significantly increased it (Fig. 5, C and D). These results show that the generation of ceramide requires at least one functional caspase which could be different from ICE-like or CPP32-like proteases.
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zVAD-fmk, but Not Ac-DEVD-CHO or Ac-YVAD-CHO, Inhibits Reduction of
Mitochondrial Transmembrane Potential Induced by Anti-Fas and Anti-HLA
Class I mAbs--
Before cells exhibit signs of nuclear apoptosis
(chromatin condensation and DNA fragmentation), they undergo a
reduction of m, due to the opening of mitochondrial
permeability transition pores (44). We tested the effect of the three
inhibitory peptides on
m modification induced in both HLA
class I and Fas apoptosis. Pretreatment of PHA-activated PBL, for
3 h, with 100 µM zVAD-fmk, before addition of CH11
or mAb90 significantly reduced
m disruption induced by
both CH11 and mAb90 (Fig. 6). The
protective effect of zVAD-fmk on
m was not observed with
similar doses of Ac-DEVD-CHO or Ac-YVAD-CHO. However, the three
tetrapeptides efficiently inhibited chromatin condensation and nuclear
fragmentation evaluated by fluorescence microscopy after staining
with Hoechst 33342 (Fig. 4). Finally
m disruption induced
by addition of exogenous C2-ceramide, but not the inactive control
DHC2, was not inhibited by any of the peptides (Fig. 6). These results
suggest that zVAD-fmk targets a protease which acts upstream of
ceramide generation and mitochondrial events, whereas ICE-like
and CPP32-like proteases act downstream of mitochondria (Fig.
7).
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DISCUSSION |
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This study was undertaken to define the molecular events involved
in HLA class I-mediated apoptosis and examine the relationship between
caspases and ceramides, two mediators implicated in different apoptosis
pathways. We show here that cell death induced by HLA class I molecules
involves activation of caspases and ceramide production, similarly to
what is observed during Fas apoptosis, despite the fact that HLA class
I-mediated apoptosis is independent of Fas/Fas-L interaction (15).
Recently two other reports described induction of apoptosis by HLA
class I molecules (11, 12). Apoptosis induced by the anti-HLA class I
3 domain, 5H7, was not inhibited by zVAD-fmk and was triggered in
cell lines derived from patients with Niemann-Pick disease that lack
acidic sphingomyelinase activity, suggesting that neither ceramide nor
caspases were involved in 5H7-mediated apoptosis (11). Furthermore,
Skov et al. (12) demonstrated that herbimycin A inhibits
rabbit anti-
2m-induced apoptosis of Jurkat T cells, suggesting that
TPK might be involved in this type of apoptosis. The difference with
our results may be explained by the fact that our antibodies (mAb90 and
YTH862) recognize the
1 domain of HLA class I molecules and do not
need to be cross-linked to induce apoptotic cell death (15).
Several reports have demonstrated that activation induced by
cross-linking of HLA class I molecules depends on an association between these molecules and other cell surface proteins such as interleukin-2R or CD3/TCR (45). We further observed that inhibition of
cytoskeleton rearrangement by cytochalasin B or D, which may inhibit
association of cell surface molecules, prevents HLA class I- but not
Fas-mediated apoptosis. However, HLA class I-induced apoptosis seems to
be independent of associated cell surface molecules. Indeed we showed
that monovalent Fab' fragments from mAb90 were nearly as efficient as
intact IgG to induce apoptosis (15). Furthermore, using two different
methods, energy transfer and immunoprecipitation, we failed to
demonstrate any physical association between HLA class I and Fas
molecules. This argues in favor of a death signal transduced by the HLA
class I molecule itself which differ from Fas-mediated apoptosis at
least by cytoskeleton rearrangement requirement. The cytoplasmic region
of the chain of the HLA class I molecule which comprises 33 amino
acids does not bear the typical death domain found in the intracellular
C terminus region of both Fas and TNF-RI. So we can speculate that HLA
class I molecules will interact with specific intracellular proteins that can initiate cell killing, and then converge in a route common with Fas. HLA class I-specific associated proteins that are involved in
proximal events which control HLA class I cell death signaling remain
to be identified.
The two anti-HLA class I mAbs which induce apoptosis, mAb90 and YTH862,
increased ceramide levels as did the anti-Fas mAb (Fig. 2, Ref. 17).
Evidence for the requirement of a PC-PLC/acidic sphingomyelinase
pathway in this process was suggested by using the xanthate D609 which
inhibits ceramide production induced by Fas (17) and anti-HLA class I
mAbs. Indeed D609 specifically blocks the PC-PLC and subsequent
activation of acidic sphingomyelinase but not neutral sphingomyelinase
(46, 47). The complete inhibition of ceramide production in the
presence of D609 suggests that anti-HLA class I mAbs activate only
acidic sphingomyelinase and not neutral sphingomyelinase. The major
role for ceramide in apoptosis signaling was recently demonstrated by
genetic models. Santana et al. (48) reported that
lymphoblasts from Niemann-Pick patients, which bear an inherited
deficiency of acidic sphingomyelinase activity, as well as cells from
acidic sphingomyelinase knockout mice, failed to respond to ionizing
radiations with ceramide generation and apoptosis. Finally our results
extend the list of apoptotic stimuli which use ceramides for the
propagation of their death signal since TNF (18), Fas-L (17, 19),
anti-IgM (20), and stress-induced apoptosis (21, 22) increase ceramide
levels.
It is clear that caspases play a major role in apoptosis signaling.
Expression of crmA, a cowpox virus encoding a serpin that is
a specific inhibitor of ICE-like proteases, suppressed TNF-, Fas-,
and granzyme B-induced apoptosis (28, 29, 49). By measuring the
cleavage of a fluorogenic substrate specific for ICE-like proteases as
well as PARP cleavage, and using synthetic inhibitors specific for
ICE-like (Ac-YVAD-CHO) and CPP32-like (Ac-DEVD-CHO) proteases, we found
that these two caspases subfamilies that are involved in Fas-mediated
apoptosis, are also activated during HLA class I-induced
apoptosis. More importantly our data demonstrate that the large
spectrum caspases inhibitor, zVAD-fmk, inhibits ceramide production
induced by both anti-HLA class I and anti-Fas mAbs, suggesting that
zVAD-fmk targets (a) protease(s) acting upstream of ceramide
generation. In agreement with this hypothesis zVAD-fmk has no effect on
the decrease of mitochondrial transmembrane potential induced by
addition of exogenous ceramide (Fig. 6). Control of ceramide production
by caspases was already proposed in other models. The Drosophila
melanogaster protein REAPER, which is critical for the
Drosophila embryo, was found to induce generation of
ceramide and apoptosis, both being blocked by the zVAD-fmk peptide
(32). More recently it was demonstrated that Ac-YVAD-CHO and CrmA, two
potent inhibitors of ICE-like proteases, inhibited ceramide generation
and prevented TNF-
-induced cell death in MCF-7 breast carcinoma
cells (33). Despite the observation that, in Fas- and HLA class
I-mediated apoptosis, a different pattern of inhibition is obtained
with the tetrapeptide Ac-YVAD-CHO compared with TNF-
-mediated
apoptosis, our results support the role of caspases in controlling
ceramide generation in cell death signaling. Furthermore, the strong
accumulation of ceramides observed when cells were pretreated with
either Ac-DEVD-CHO or Ac-YVAD-CHO suggests that blockade of ICE-like or
CPP32-like proteases inhibits or delays ceramide consumption. It was
recently reported that CD95- and ceramide-induced apoptosis requires
GD3 ganglioside, newly synthesized from ceramide (50). The
authors documented that a DEVD- and/or YVAD-sensitive caspase controls
GD3 accumulation. According to these data, a possible control of GD3
synthase by caspases can be proposed and certainly deserves further
investigations.
A possible candidate for the proximal caspase targeted by zVAD-fmk peptide, could be the recently identified FLICE protease (51, 52) proposed to be the most receptor-proximal caspase activated in Fas signaling (53, 54). The recruitment of FLICE in the death-inducing complex is mediated by the N terminus prodomain of the molecule, which interacts with the death effector domain of FADD. However, FLICE was also shown to be activated by granzyme B (51), indicating that FLICE can be recruited and activated by proteins which do not possess a death domain. Whether FLICE is also activated in HLA class I-mediated apoptosis remains to be demonstrated. Recent data from Medema et al., (53) on the B lymphobastoid cell line SKW6.4 showed that in an in vitro cleavage assay, where 35S-labeled FLICE is added to immunoprecipitates of the death-inducing signaling complex, autocatalytic cleavage of FLICE is blocked by the peptide inhibitors zVAD-fmk or DEVD-fmk but not by CrmA or Ac-YVAD-CHO. In the cell, however, inhibition of FLICE was only documented with zVAD-fmk. It is possible that DEVD-fmk (or Ac-DEVD-CHO) would not inhibit FLICE activity in vivo because of a higher affinity for other proteases such as CPP32. This would explain why we did not see any effect of the tetrapeptide Ac-DEVD-CHO on ceramide generation, despite its capacity to inhibit HLA class I and Fas-mediated apoptosis.
According to our results, we propose a model (Fig. 7) where a first set
of proteases targeted by the inhibitory peptide zVAD-fmk control
generation of ceramide. Whether proteases directly affect PC-PLC or
acidic sphingomyelinase activities remains to be investigated. One
consequence of ceramide production would be a loss in mitochondrial membrane potential as suggested by the major decrease of
m in cells treated by exogenous ceramides (Fig. 6). We
described above that anti-Fas and anti-HLA class I mAbs partially
reduced
m. The decrease of
m is inhibited
by zVAD-fmk but not by Ac-DEVD-CHO and Ac-YVAD-CHO, suggesting that
DEVD- and YVAD-specific proteases act downstream of mitochondrial
events. Cytochrome c was recently reported to be released
from mitochondria in apoptotic cells, to activate DEVD-specific
caspases and induce apoptotic effects in cell-free systems containing
cytosol (55, 56). However, in these cell-free assays, release of
cytochrome c from the mitochondria was not accompanied by
changes in
m. In Jurkat cells undergoing apoptosis after
Fas ligation, a loss of cytochrome c function was also
reported (57). Interestingly this cytochome c inactivation
was accompanied by a slow and partial decrease of
m and
inhibited by zVAD-fmk peptide. Therefore the loss of cytochrome
c activity during Fas-mediated apoptosis is likely to
involve a step dependent on a zVAD-specific protease. Altogether these
results support a close relationship between caspases and ceramide in
the signaling of apoptosis mediated by Fas and HLA class I
molecules.
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ACKNOWLEDGEMENTS |
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We thank Zohair Mishal (Center of Flow cytometry, CNRS-UPS 47, 94801 Villejuif, France) for expert assistance in energy transfer experiments, Prof. Herman Waldmann (University of Oxford, United Kingdom) for the gift of YTH862 mAb, and D. R. Green for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported in part by a contract from the Région Rhône-Alpes (H0987 30000), European Biotech Program In Vitro Immunotoxicology Grant Bio 2. CT 92-0316, and ARC Grant 9607.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: La Jolla Institute for Allergy and Immunology, 10355 Science Center Dr., San Diego, CA 92121.
** To whom correspondence should be addressed. Tel.: 33-4-72-11-01-77; Fax: 33-4-72-33-00-44; E-mail: bonnefoy{at}inserm.lyon151.fr.
1
The abbreviations used are: TNF-R, tumor
necrosis factor receptor; CsA, cyclosporin A; m,
mitochondrial transmembrane potential; Fas-L, Fas ligand; ICE,
interleukin-1 converting enzyme; mAb, monoclonal antibody; PBL,
peripheral blood lymphocytes; PC-PLC, phosphatidylcholine-specific
phospholipase C; PARP, poly(ADP-ribose) polymerase; PHA,
phytohemagglutinin; FITC, fluorescein isothiocyanate; TRITC,
tetramethylrhodamine B isothiocyanate.
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REFERENCES |
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