Regulation of the Fas Death Pathway by FLICE-Inhibitory Protein in Primary Human B Cells1 Ana Hennino,* Marion Berard,* Montserrat Casamayor-Palleja`,* Peter H. Krammer,† and Thierry Defrance2* The Fas/Fas ligand (L) system plays an important role in the maintenance of peripheral B cell tolerance and the prevention of misguided T cell help. CD40-derived signals are required to induce Fas expression on virgin B cells and to promote their susceptibility to Fas-mediated apoptosis. In the current study, we have analyzed the early biochemical events occurring upon Fas ligation in CD40L-activated primary human tonsillar B cells with respect to Fas-associated death domain protein (FADD), caspase-8/FADD-like IL-1-converting enzyme (FLICE), and c-FLICE inhibitory protein (FLIP). We report here that Fas-induced apoptosis in B cells does not require integrity of the mitochondrial Apaf-1 pathway and that caspase-8 is activated by association with the death-inducing signaling complex (DISC), i.e., upstream of the mitochondria. We show that both FADD and the zymogen form of caspase-8 are constitutively expressed at high levels in virgin B cells, whereas c-FLIP expression is marginal. In contrast, c-FLIP, but neither FADD nor procaspase-8, is strongly up-regulated upon ligation of CD40 or the B cell receptor on virgin B cells. Finally, we have found that c-FLIP is also recruited and cleaved at the level of the DISC in CD40L-activated virgin B cells. We propose that c-FLIP expression delays the onset of apoptosis in Fas-sensitive B cells. The transient protection afforded by c-FLIP could offer an ultimate safeguard mechanism against inappropriate cell death or allow recruitment of phagocytes to ensure efficient removal of apoptotic cells. The Journal of Immunology, 2000, 165: 3023–3030.
F
as (CD95/APO-1) belongs to the death receptor subfamily, which is part of the TNFR superfamily. Its cardinal deathsignaling function is ensured by the presence of a cytoplasmic protein-protein interaction motif called the death domain (DD)3 (see Ref. 1 for review). Clustering of Fas induces association of the cytoplasmic adaptor protein Fas-associated DD-containing protein (FADD) with the oligomerized DD of the receptor (2, 3). FADD in turns recruits the proenzymatic form of the initiator caspase-8/FADD-like IL-1-converting enzyme (FLICE) (4 – 6), thus leading to the formation of the death-inducing signaling complex (DISC) described as the most receptor-proximal element of the signal transduction via Fas (7). The Fas/FADD and FADD/ caspase-8 associations are mediated via homophilic DD and death effector domain (DED) interactions, respectively. Recruitment of the zymogen form of caspase-8 to the DISC leads to its autoproteolytic cleavage and the release of its active enzymatic form in the cytosol, which initiates the cascade of caspases leading to apoptosis. Genetic studies have established that the functions exerted by
*Institut National de la Sante´ et de la Recherche Me´dicale Unite´ 404, “Immunite´ et Vaccination,” Lyon, France; and †Tumor Immunology Program, German Cancer Research Center, Heidelberg, Germany Received for publication February 16, 2000. Accepted for publication June 26, 2000. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 M.B. and M.C.-P. are recipients of a grant from “La Ligue Nationale Contre le Cancer” and “l’Association pour la Recherche sur le Cancer,” respectively. This work was supported by funding from the Re´gion Rhoˆnes-Alpes (convention no. H098730000). 2 Address correspondence and reprint requests to Dr. Thierry Defrance, Institut National de la Sante´ et de la Recherche Me´dicale Unite´ 404, Avenue Tony Garnier, 69365, Lyon, Cedex 07, France. E-mail address:
[email protected] 3 Abbreviations used in this paper: DD, death domain; DED, death effector domain; FADD, Fas-associated DD-containing protein; FLICE, FADD-like IL-1-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.
Copyright © 2000 by The American Association of Immunologists
FADD and caspase-8 in mediating death signaling by the DDcontaining receptors are nonredundant. Both FADD⫺/⫺ (8, 9) and caspase-8⫺/⫺ (10) cells are resistant to the cytotoxic effect exerted by most death receptor but remain fully susceptible to death induced by other pathways, such as irradiation, serum deprivation, or treatment with anti-cancer drugs. Two cell types that each preferentially use one of two different Fas signaling pathways have been identified (11). In type I cells, cleavage of caspase-8 occurs at the level of the DISC before the loss of mitochondrial transmembrane potential (⌬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 (12–19). 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 0022-1767/00/$02.00
3024 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 (23–25). 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 Fassensitive 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.
Materials and Methods Reagents and Abs 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 g/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 g/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 -actin mAbs were purchased from Transduction Laboratories (Lexington, KY) and Sigma, respectively. The caspase-8 colorimetric substrate Ac-IETDpNA 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 M throughout the study. The caspase inhibitors (z-VADfmk, 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 M.
REGULATORY ROLE OF FLIP IN HUMAN 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 g/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 ⫻ 107 cells/well in six-well plates. For secondary cultures, viable B blasts recovered by density gradient centrifugation were seeded at 5 ⫻ 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 FLAGFasL 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 ⫻ 106 per sample) were washed twice with cold PBS, resuspended in 100 l 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 ⫻ 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 g 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  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 ⫻ 107 B blasts were treated with the anti-APO-1 mAb (3 g/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 ⫻ 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 ⫻ 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 M 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 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.
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 FITCconjugated avidin (Immunotech, Marseille, France) used at 2.5 g/ml. Immunofluorescence stainings were analyzed on a FACScan flow cytometer
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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 ⫻ (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 ⫻ (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 g/ml streptomycin, and 2.5 M 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.
Results Ligation of Fas on CD40L-activated B cells induces activation of caspase-8 Because CD40 ligation induces susceptibility of mature B cells to Fas-mediated apoptosis (23–25), 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 antiFADD and caspase-8 mAbs or tested for caspase-8 enzymatic activity using the caspase-8-specific colorimetric substrate IETDpNA. As illustrated by Fig. 1A, 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
FIGURE 1. Fas ligation on CD40L-activated B blasts promotes activation of caspase-8. A, Viable virgin B blasts were recovered by density gradient centrifugation after 48 h of activation with CD40L. They were then recultured either with the agonistic anti-Fas mAb 7C11 (⫹) or with its isotypic control (⫺) for the indicated periods of time. Equal amounts of whole cell lysates were separated on 12% SDS-PAGE. The blots were successively probed with the anti-caspase-8, the anti-FADD, and the anti- actin Abs and revealed with the appropriate HRP-conjugate. The proenzymatic form (55 kDa) as well as the active enzymatic form of caspase-8 (18 kDa) are indicated (arrow heads). The first lane on the left (0) corresponds to the extracts of B blasts recovered from the primary culture with CD40L. B, B blasts cultured as in A were lysed and incubated for 6 h with the Ac-IETD pNA substrate. OD were read on a spectrophotometer at 405 nm. Representative of four distinct experiments.
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. Caspase-8 is activated upstream of the mitochondria in Fas-sensitive B cells 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 CD40Lactivated B cells. The cathepsin B inhibitor z-FA and the broadrange 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
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REGULATORY ROLE OF FLIP IN HUMAN B CELLS
FIGURE 2. Blockade of caspase-8 activity prevents the Fas-induced disruption of the mitochondrial transmembrane potential in CD40L-activated B blasts. Viable virgin B blasts were recovered by density gradient centrifugation after 48 h of stimulation with CD40L. They were next recultured for 24 h with: 1) the anti-Fas mAb 7C11 or its isotypic control, and 2) soluble FLAG-FasL (50 ng/ml) aggregated with an anti-FLAG mAb (1 g/ml) or with the anti-FLAG mAb alone. Cultures were further supplemented with the fluoromethylketone derivatives of the following peptides: z-FA, z-VAD, z-IETD, or z-LEHD, all used at 50 M. Cells were processed for analysis of the mitochondrial transmembrane potential (⌬m) by DiOC6 staining and for PS exposure as revealed by the binding of annexin V. Results are expressed as means of the percent specific apoptosis calculated from duplicate determinations. SDs never exceeded 10% of the mean values. Representative of three distinct experiments.
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. c-FLIP is recruited and cleaved at the level of the DISC in Fas-sensitive B cells 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  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
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FIGURE 3. dATP depletion prevents neither apoptosis nor cleavage of caspase-8 promoted by Fas triggering on CD40L-activated B blasts. A, Viable CD40L-induced B blasts were recultured for 4 and 7 h with or without the anti-Fas mAb 7C11 or its isotypic control in glucose-containing medium or in glucosefree medium supplemented with oligomycin (2.5 M). Intracellular ATP levels were determined using the luciferin-luciferase method. Results are expressed as percent of the ATP content of the control samples, i.e., cells cultured in glucose-containing medium without oligomycin in the absence of the anti-Fas mAb. B, Same culture conditions as in A. PS externalization and ⌬m were monitored as described in the legend of Fig. 3. Results are expressed as means of the percent specific apoptosis calculated from duplicate determinations. SDs never exceeded 10% of the mean values. C, Whole cell extracts were prepared from the same cultures and equal amounts of proteins were separated on 12% SDS-PAGE. The blots were probed with the anti-caspase-8 mAb C15. The zymogen form of caspase-8 (p55), its intermediate cleavage product (p43/p41), and its active cleaved form (p18) are indicated by arrowheads. Oligomycin treatment by itself induced neither PS exposure nor ⌬m drop within the time frame of the experiment. Representative of three independent experiments.
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. To examine the composition of the DISC in sensitive B blasts, Fas was immunoprecipitated from CD40L-activated B blasts after 3 and 4 h of stimulation with the anti-Fas mAb APO-1. The immunoprecipitates were analyzed by Western blotting using monoclonal or polyclonal Abs directed against Fas, FADD, c-FLIP, and caspase-8. Fas immunoprecipitates from unstimulated B blasts were used as negative controls. As illustrated by Fig. 6, 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.
Discussion In this study, we have explored the nature of the biochemical events elicited by ligation of Fas on human B cells. In agreement with our current knowledge of Fas signaling in human T lymphocytes (11, 28, 35), we show here that induction of apoptosis through Fas in human B cells relies on the cleavage of caspase-8 at a receptor proximal level, i.e., upstream of the mitochondria. This assertion is supported by two lines of evidence: 1) the caspase-8 inhibitory peptide z-IETD-fmk prevents the Fas-induced perturbations of the mitochondrial function, and 2) caspase-8 is recruited and cleaved at the level of the DISC following Fas triggering. Besides, we found that integrity of the Apaf-1 mitochondrial pathway is not mandatory for the activation of caspase-8 in response to Fas ligation in human B cells. Likewise, it has been
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REGULATORY ROLE OF FLIP IN HUMAN B CELLS
FIGURE 4. c-FLIP is up-regulated upon B cell activation. A, Whole cell extracts were prepared from freshly isolated virgin B cells before (first lane on the left) and after stimulation for 24 h with: 1) CD40L, 2) anti-Ig Abs, and 3) CD40L and anti-Ig Abs. A total of 40 g of proteins was loaded and separated on 12% SDS-PAGE. The blot was successively probed with the anti-FLIP mAb NF6, HRP-conjugated goat antimouse Igs Ab and revealed with an ECL kit. The blot was next stripped and reprobed with the anti-FADD and caspase-8 Abs. The long and short forms of c-FLIP migrate as a 55-kDa and 28-kDa band, respectively. B, Virgin B blasts recovered after 48 h of CD40 stimulation were recultured with the anti-Fas mAb 7C11 (⫹) or its isotypic control (⫺) for 4 h. Whole cell lysates were separated on 12% SDS-PAGE and probed with the anti-FLIP mAb NF6. The cleaved form of c-FLIPL migrates as a 43-kDa band. ⴱⴱ, Nonspecific staining. Representative of three experiments.
demonstrated that Fas-induced cleavage of caspase-8 in Jurkat cells is not affected by dATP depletion, whereas activation of caspase-8 in response to certain chemotherapeutic drugs, such as staurosporine, can be blocked by oligomycin in the same cells (36). Nevertheless, our present data do not definitely rule out participation of the mitochondria in execution of the Fas death program in B cells because other apoptogenic factors, such as caspases 2, 3, and 9 (37, 38), or the apoptosis-inducing factor (AIF) (39), are also released from damaged mitochondria in addition to cytochrome c. Generally speaking, the role of the members of the Bcl-2 family and of the mitochondrial pathway in Fas-in-
duced apoptosis is still a controversial issue. For example, Alam and colleagues (40) have shown that overexpression of Bcl-2 protects the Burkitt lymphoma cell line Ramos from Fas-mediated apoptosis. By contrast, the same approach applied to the SKW6 human B lymphoblastoid cell line and to murine CH1 lymphoma cells by another group (34, 41) has led to the conclusion that neither Bcl-2 nor Bcl-xL can promote resistance of these cells to Fasinduced killing. Similarly, whereas Schneider et al. (42) report that B cells from Bcl-x-transgenic mice are resistant to Fas-mediated killing, Newton and Strasser (43) show that both resting and activated B lymphocytes from Bcl-2 transgenic mice are as sensitive
FIGURE 5. The degree of susceptibility of CD40L-activated B blasts to Fas-induced apoptosis correlates with the level of c-FLIP expression. A, Whole cell extracts were prepared from viable virgin B blasts recovered after 12, 24, 48, or 72 h of CD40L stimulation. Equal amounts of proteins (30 g) were loaded and separated on 12% SDS-PAGE. The blot was successively probed with the anti-c-FLIP and -actin mAbs. B, The blot shown in A was subjected to densitometry scanning analysis. The graph shows the relative intensities of the c-FLIPL and c-FLIPS bands, i.e., the ratios between intensity of the c-FLIP and  actin bands. C and D, CD40L-induced virgin B blasts were recovered after 24 or 72 h of stimulation and recultured for the indicated times with the agonistic anti-Fas mAb 7C11 or its isotypic control. Cells were processed for analysis of PS exposure (annexin V staining) (C) or activation of caspase-3 by a PE-conjugated anti-active caspase-3 Ab (D). Results are expressed as means ⫾ SD of the percent specific apoptosis calculated from duplicate determinations. Representative of three experiments.
The Journal of Immunology
FIGURE 6. Immunoprecipitation of the DISC in CD40L-activated B cells. Fas was immunoprecipitated from 1 ⫻ 107 CD40L-induced B blasts that had been stimulated for 3 h or 4 h with the anti-Fas mAb APO-1. For the immunoprecipitation control (0), unstimulated B blasts were first lysed and then supplemented with the anti-APO-1 mAb. Immunoprecipitates were washed, subjected to 12% SDS PAGE, and successively probed with the anti-c-FLIP, caspase-8 FADD and Fas Abs. Whole cell lysate of CD40L-induced B blasts treated for 4 h with the anti-Fas mAb 7C11 have been loaded on the last lane on the right (ctrl). It was used as a migration control for Fas, FADD, caspase-8, and c-FLIP. Representative of three experiments.
as their wild-type counterparts to killing by FasL. The reasons for these discrepancies are unclear, but it has recently been suggested that suboptimal aggregation of Fas by soluble agonistic Abs might not elicit the same biological response as the membrane-anchored FasL (34). However, it is unlikely that our observation that the Apaf-1 pathway is not involved in the Fas-induced death of activated human B cells results from inefficient cross-linking of Fas in our experimental conditions. Indeed, the caspase-9-inhibitory peptide z-LEHD failed to revert Fas-induced apoptosis of B blasts either when the Fas receptor was engaged by the agonistic mAb 7C11 or by an aggregated form of the FasL. Furthermore, our present findings are consistent with genetic studies demonstrating that the susceptibility to Fas-induced apoptosis is impaired neither in embryonic fibroblasts from caspase-9-deficient mice (44, 45) nor in Apaf-1-deficient T cells (46). An alternative explanation could be that transduction of the Fas death signal in mature human B cells requires cooperation between certain mitochondrial proapoptotic molecules and the direct caspase-dependent pathway initiated by cleavage of caspase-8 at the level of the DISC. It remains that our observation that ATP-depletion enhances both the mitochondrial and membrane manifestations of apoptosis in response to Fas triggering in B cells is puzzling. It evokes the possibility that partial or total blockade of the mitochondrial pathway could be compensated by amplification of the caspase-dependent pathway during Fas-induced apoptosis. 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 Fassensitive 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
3029 DISC in response to Fas triggering of sensitive B cells. One possible explanation for this finding would be that association of cFLIP 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 CD40Lactivated 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).
Acknowledgments We thank Marek Kubin (Immunex) for useful suggestions and helpful discussion.
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