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The Journal of Immunology, 2004, 172: 5405-5414.
Copyright © 2004 by The American Association of Immunologists

Cathepsin-Dependent Apoptosis Triggered by Supraoptimal Activation of T Lymphocytes: A Possible Mechanism of High Dose Tolerance1

Marie-Cécile Michallet*, Frédéric Saltel{dagger}, Monique Flacher*, Jean-Pierre Revillard2,* and Laurent Genestier3,*,{ddagger}

* Laboratoire d’Immunopharmacologie, Institut National de la Santé et de la Recherche Médicale Unité 503, Centre d’Etudes et de Recherche en Virologie et Immunologie, Lyon, France; {dagger} Laboratoire de Biologie Moléculaire et Cellulaire, Unité Mixte de Recherche 5665 Centre National de la Recherche Scientifique/Ecole Normale Supérieur, Lyon, France; and {ddagger} Institut National de la Santé et de la Recherche Médicale Unité 404, Centre d’Etudes et de Recherche en Virologie et Immunologie, Institut Fédératif No 128 BioSciences Lyon-Gerland, Lyon, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
High doses of Ag can paradoxically suppress immune responses in vivo. This Ag-specific unresponsiveness (termed high dose tolerance) involves extrathymic mechanisms in mature T lymphocytes. To investigate these mechanisms, we used the in vitro model of PBL activated with anti-CD3 or PHA. In these conditions, increasing mitogen concentrations resulted in a reduction of the proliferative response, associated with an increased percentage of apoptotic cells. Apoptosis did not require prior exposure to IL-2, it was not the consequence of CD178/CD95 or TNF/TNFR interactions, and was therefore clearly distinct from activation-induced cell death. Although the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) decreased DNA fragmentation, cytochrome c release and caspase-9 and caspase-3 activation were not implicated, suggesting that this apoptosis did not primarily involve the intrinsic mitochondrial pathway. E64d, a cysteine protease inhibitor, as well as specific inhibitors of cathepsin B and cathepsin L conferred protection. We further demonstrated that cathepsin B and cathepsin L were released from the lysosomes and catalytically active in the cytosol. Release of cathepsin B and cathepsin L was the consequence of lysosomal membrane permeabilization without complete disruption of the cytosol-lysosome pH gradient. These results demonstrate a role for cathepsins in supraoptimal activation-induced apoptosis in vitro and suggest their possible participation in high dose tolerance in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Administration of high doses of Ag can paradoxically suppress the immune response to the same Ag (1, 2). This type of Ag-specific unresponsiveness was termed "high dose suppression" or "high dose tolerance". It involves extrathymic mechanisms in mature T lymphocytes, but its precise molecular mechanisms and possible contribution to natural "peripheral tolerance" are poorly understood as yet. The role of high concentrations of Ag has been investigated using in vitro models and the reports showed a suppression of the proliferative response, which was the consequence of the induction of anergy (3) or apoptosis (4) mediated by CD95/CD178 interaction (5) of CD4+ T cell, whereas in CD8+ effector CTL, apoptosis was mediated by TNFRII/TNF interaction (6, 7). In models using TCR transgenic mice, supraoptimal peptide-MHC stimulation in vivo induced CD4+ or CD8+ T cell deletion by apoptosis (4, 8, 9). In most of these models, deletion of specific T cells occurs after an initial phase of expansion and is thought to result from re-exposure of cycling T lymphocytes to Ag. This process called activation-induced cell death (AICD)4 is mainly mediated through CD95/CD178 or TNFRII/TNF interactions (10). In other experimental models, mature T lymphocytes have been shown to die by apoptosis after TCR stimulation by processes that may involve IFN-{gamma} (11, 12) or lymphokine withdrawal when TCR stimulation disappears (10). In contrast to AICD, which triggers the extrinsic apoptotic pathway involving death receptors and subsequently caspase-activating protein complexes such as death-inducing signaling complex (DISC) (13), lymphokine withdrawal results from the elimination of the Ag and activates the intrinsic apoptotic pathway that involves cytoplasmic activation of caspases regulated by the mitochondrion and proteins from the Bcl-2 family (14).

Caspases are cysteine proteases that are highly conserved through evolution and that have an absolute specificity for an aspartic acid in the P1 position of the substrate (15). Cleavage of their major cellular substrates accounts for the characteristic features of apoptosis (15). Several studies with caspase inhibitors or mice deficient for various caspases have supported the involvement of caspases in various forms of apoptosis including that induced by CD178, TNF, or lymphokine withdrawal (16). However, other cysteine proteases distinct from caspases have been suggested to be involved in apoptosis. Calpain, a calcium-dependent cysteine protease, is required for neuronal apoptosis triggered by amyloid {beta}-peptide (17). Cathepsins, especially the cysteine cathepsins B and L and the aspartyl cathepsin D, participate in both caspase-dependent and caspase-independent apoptosis induced by several stimuli, including death receptors of the TNFR family, B cell receptors, the p53 tumor suppressor gene, camptothecin, bile salt oxidants, and retinoids (18, 19, 20, 21, 22, 23, 24, 25). Depending on the cell type and the stimuli, cathepsins may function upstream (20, 26) or downstream of caspases (21, 25), or even completely independently of them (21).

In this report, using PBL stimulated with increasing concentrations of anti-CD3 mAb or PHA, we show that apoptotic death may occur as a result of stimulation with high concentrations of these mitogens. When investigating the mechanism of this apoptosis, we found that a previously unrecognized effect of supraoptimal activation was to release cathepsin B and cathepsin L from the lysosomes into the cytosol. Those cathepsins may therefore be considered as the main candidate mediators of supraoptimal activation-induced apoptosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Abs and reagents

PHA, anti-Flag mAb (clone M2), rapamycin, protein G, staurosporine, and pepstatin A were purchased from Sigma-Aldrich (St. Quentin Fallavier, France). Cyclosporin A was kindly supplied by Novartis Pharmaceuticals (St. Quentin Fallavier, France). Anti-CD3 mAb (IgG2a, clone OKT3) was obtained from Janssen-Cilag (Issy-Les-Moulineaux, France), and anti-CD28 mAb (IgG1, clone CD28.2) from BD Biosciences (Le Pont de claix, France). Purified anti-CD95 mAb agonist (IgM, clone 7C11) and antagonist (IgG1, clone ZB4) were purchased from Immunotech (Marseille, France). TNFR p55 Ig fusion protein was kindly provided by Dr. H. Waldmann (University of Oxford, Oxford, U.K.), and TNF-{alpha} was obtained from R&D Systems (Abingdon, U.K.). Recombinant soluble human CD178 was obtained from Alexis (San Diego, CA). The broad spectrum caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), and L-trans-epoxysuccinyl-Leu-3-methylbutylamide ethyl ester (E-64d), z-Phe-Phe-diazomethylketone (zFF-dmk), z-Phe-DL-Ala-fluoromethylketone (zFA-fmk), and z-Phe-Lys-2,4,6-trimethylbenzoyloxymethylketone (zFK-mbmk) were purchased from Bachem (Voisins-Le-Bretonneux, France). N-benzyloxycarbonyl-L-leucylnorleucinal (Calpeptin) and N-acetyl-Leu-Leu-Methional (Calpain inhibitor II) were obtained from Tebu (Le Perray-en-Yvelines, France), and PD150606 from Calbiochem (Meudon, France). The lysosomotropic fluorochrome used was acridine orange (AO) from International Biotechnologies (New Haven, CT).

Cell preparation and culture

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 (Dutcher, Brumath, France). PBL were resuspended in RPMI 1640 (Sigma-Aldrich) supplemented with 10% FCS, 2 mM L-glutamine, and antibiotics (penicillin 100 U/ml, streptomycin 100 µg/ml), and cultivated in a humid atmosphere containing 5% CO2. PBL were activated in 96-well tissue culture plates with various concentrations of PHA or anti-CD3 coated in 50 mM Tris, pH 9.0. Costimulation through CD28 was performed using anti-CD28 at 5 µg/ml in the presence of protein G (5 µg/ml). Activated T lymphocytes were obtained by activation of PBL for 3 days with PHA (5 µg/ml). At this stage, dead cells were removed and viable cells (106/ml) were treated with the different apoptotic stimuli such as staurosporine, anti-CD95, and CD178. After 3 days of stimulation with PHA, cells were further incubated for 11 days with IL-2 (50 U/ml). In these conditions, T lymphoblasts were susceptible to apoptosis induced by TNF-{alpha}. For proliferation assay, cells were pulsed during the indicated time with [methyl-3H]thymidine (Amersham, Les Ulis, France) at 0.5 µCi/well. [methyl-3H]Thymidine uptake was measured using a Packard direct beta counter (Packard Instrument, Meriden, CT) after harvesting.

Measurement of apoptosis

Phosphatidylserine (PS) exposure was quantified by surface binding of annexin V. Cells were resuspended in annexin V binding buffer containing FITC-conjugated annexin V for 15 min following instructions of the manufacturer (Bender MedSystems, Vienna, Austria). Propidium iodide (1 µg/ml) was then added and cell suspension was immediately analyzed by flow cytometry using a FACSCalibur and the CellQuest software (BD Biosciences). ssDNA fragmentation was detected using F7-26 mAb from Alexis (Apostain, Laufelfingen, Switzerland), according to the manufacturer’s instructions.

Western blot analysis

Treated cells were washed with PBS and pellets were lysed in 10 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% Triton X-100, 10 mM EDTA and the protease inhibitor mixture for caspase-8, and 62.5 mM Tris-HCl pH 6.8, 2% SDS, 0.72 M 2-mercaptoethanol, 7% glycerol for caspase-3, caspase-7, caspase-9, poly(ADP-ribose) polymerase (PARP), and FLIP. To measure cytochrome c (Cyt c) and cathepsin release in the cytosol, cytosolic fractions (S-100) were prepared as previously described (27). Proteins (30 µg) were separated on SDS-PAGE and transferred to nitrocellulose membrane (Schleicher & Schüll, Dassel, Germany). Blots were incubated overnight at 4°C with anti-caspase-3, anti-caspase-7, anti-Cyt c (BD Biosciences), anti-caspase-9 (New England Biolabs, Beverly, MA), anti-PARP mAb C-2-10 (Biomol, Plymouth Meeting, PA), anti-cytochrome oxidase (Molecular Probes, Eugene, OR), anti-cathepsin B, anti-cathepsin L (R&D Systems), anti-actin (Sigma-Aldrich), anti-caspase-8, or anti-FLIP Abs. Anti-caspase-8 and anti-FLIP Abs were kindly provided by Dr. P. Krammer (German Cancer Research Center, Heidelberg, Germany), and anti-Lamp-1 Ab by Dr. S. Meresse (Institut National de la Santé et de la Recherche Médicale U136, Marseilles-Luminy, France). Detection was achieved with the appropriate secondary Abs coupled to HRP, followed by ECL Western blotting (Amersham), and visualized by autoradiography.

Lysosomal stability assessment

Cells were assessed for lysosomal stability at different times following PHA or anti-CD3 treatments using the AO-uptake method. Cells were exposed to an AO solution (5 µg/ml) in complete medium for 15 min at 37°C, washed twice in complete medium, and red AO fluorescence was determined by flow cytometry (FACSCalibur; BD Biosciences).

Measurement of cytosolic caspase and cathepsin activities

For measurement of caspase activities, treated cells were washed in PBS and pellets were lysed for 15 min at 4°C in lysis buffer (10 mM HEPES KOH pH 7.4, 250 mM sucrose, 2 mM EDTA, 0.1% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), 5 mM DTT, and the protease inhibitor mixture). After 15 min centrifugation at 12,000 x g and 4°C, supernatant was recovered to measure caspase activity. Caspase-3, caspase-7, and caspase-8 activities were estimated on 30 µg of proteins by adding 400 µM of DEVD-AMC (Ac-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin) or IEPD-AMC (Ac-Ile-Glu-Pro-Asp-7-amido-4-methylcoumarin), respectively. To measure cathepsin activities in the cytosol, cytosolic fractions were prepared as previously described. Cathepsin B and cathepsin L activities were estimated as described by Foghsgaard et al. (21) on 30 µg of cytosolic proteins by adding 50 µM of zRR-AMC (z-Arg-Arg-7-amido-4-methylcoumarin) (Bachem) or zFR-AMC (z-Phe-Arg-7-amido-4-methylcoumarin) (Bachem), respectively. The liberation of 7-amido-4-methylcoumarin (AMC, excitation 380 nm, emission 442 nm) was measured at indicated times at 37°C with a fluorometer.

Immunofluorescence and confocal laser scanning microscopy

Treated cells were seeded on glass coverslips by cytospin method, fixed in paraformaldehyde 3.7%, permeabilized with 0.2% Triton X-100, and stained with anti-CD8 mAb (20 µg/ml), anti-CD4 mAb (5 µg/ml; Sigma-Aldrich), anti-cathepsin B Ab (1:50; Oncogen Research Products, Darmstadt, Germany), anti-cathepsin L Ab (1:100; R&D Systems) and Alexa Fluor 488-conjugated secondary Abs (Molecular Probes) or cyanin 5-labeled anti-mouse (Jackson ImmunoResearch Laboratories, West Grove, PA). Cells were observed under a LSM 510 laser scanning confocal microscope (Zeiss, Oberkochen, Germany), and images were processed with Adobe Photoshop software 6.0 (Adobe Systems, San Jose, CA). Staining with LysoTracker red (Molecular Probes) was used to characterize lysosomal membrane alterations. Treated cells were incubated for 30 min in the presence of 50 nM of LysoTracker red, washed two times, and visualized by fluorescent microscopy.

Transmission electron microscopy

After two washes with PBS, cells were fixed in 2% osmium tetroxide in 0.1 M cacodylate buffer, pH 7.4, dehydrated and embedded in epon. Thin sections were cut, and following lead to citrate and uranyl acetate contrasting, were observed in a Jeol 100CS electron microscope.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
High concentrations of PHA or anti-CD3 induce apoptosis of mature T lymphocytes

The in vitro model of PBL activated by PHA or anti-CD3 was used to investigate the effect of mitogen concentration on T cell proliferation and survival. Increasing concentrations of PHA and anti-CD3 resulted in a progressive increase of thymidine incorporation, reaching a maximum at 5 µg/ml PHA and 1 µg/ml anti-CD3, followed by a decrease at high concentrations of PHA (50–100 µg/ml) and anti-CD3 (5–50 µg/ml) (Fig. 1A). To ensure that the lack of response observed at high concentrations was not the result of a very early proliferation that was completed by the time of pulsing with [methyl-3H]thymidine, a cell division experiment was performed using CFSE-labeled cells activated with PHA and anti-CD3 at optimal or high concentrations. No cell division at high PHA or anti-CD3 concentrations was observed at any time (24–72 h) in contrast to optimally activated controls, reflecting an immediate arrest of the proliferation in the former conditions (data not shown).



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  		FIGURE 1. High concentrations of PHA or anti-CD3 induce apoptosis of PBLs. A, PBLs were incubated with PHA or solid-phase anti-CD3 over a range of concentrations. Apoptosis (•) was measured by surface binding of annexin V after 24 h of culture in the presence of PHA and after 48 h in tissue culture wells precoated with anti-CD3. [methyl-3H]Thymidine (3[H]TdR) uptake ({circ}) was measured 72 h after addition of the mitogens. Results are expressed as the mean ± SEM from triplicate measurements of one experiment representative of several independent experiments. B, PBL were incubated for 24 h in the absence (control) or presence of PHA (50 µg/ml), and for 48 h in medium alone (control) or with solid phase anti-CD3 (10 µg/ml). DNA fragmentation was analyzed by flow cytometry staining using the F7-26 mAb. The percentage of cells with fragmented DNA is indicated for each histogram. Results shown are representative of four independent experiments. C, The morphologic features of cell following treatment with medium alone, PHA, or solid phase anti-CD3 were observed using transmission electron microscopy.

 
To determine whether this decrease of proliferation was the consequence of cell death, two apoptotic parameters were analyzed: PS exposure, quantified by surface binding of annexin V, and DNA fragmentation. As shown in Fig. 1A, the percentage of apoptotic cells in PBL treated by PHA or anti-CD3 increased in a dose-dependent manner, reaching 58% at 50 µg/ml PHA and 30% at 10 µg/ml anti-CD3. In agreement with these results, ~40% and ~30% of cells activated during 24 h with 50 µg/ml PHA and 48 h with 10 µg/ml anti-CD3, respectively, exhibited fragmented DNA (Fig. 1B). We also demonstrated by transmission electron microscopy that cells treated with high concentrations of PHA or anti-CD3 exhibit the typical features of apoptotic cells (Fig. 1C).

Supraoptimal activation-induced apoptosis does not require G1 to S phase transition and is not inhibited by CD28 costimulation

To assess whether supraoptimal activation-induced apoptosis requires IL-2 signaling and therefore entry into S phase of the cell cycle, experiments were performed in the presence of cyclosporin A and rapamycin, which interfere with the IL-2 expression and signaling, respectively, and inhibit G1 to S phase transition. Although cyclosporin A and rapamycin inhibit [methyl-3H]thymidine uptake of T lymphocytes treated with mitogenic concentrations of PHA or anti-CD3 (data not shown), apoptosis induced by high concentrations of these mitogens was not significantly decreased (Fig. 2A), suggesting that G1 to S phase transition is not required for supraoptimal activation-induced apoptosis.



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  		FIGURE 2. Supraoptimal activation-induced apoptosis of T cells is independent of G1 to S phase transition and CD28 costimulation. PBL were incubated with medium alone, solid phase anti-CD3 10 µg/ml for 48 h, or PHA 50 µg/ml for 24 h in the presence of cyclosporin A (CsA) (1 µg/ml) or rapamycin (RPM) (500 nM) (A), in the presence of anti-CD28 (5 µg/ml) and protein G (5 µg/ml) (B). Apoptosis was measured by surface binding of annexin V. Results shown are representative of two independent experiments.

 
Absence of costimulation has been reported to trigger anergy and/or cell death of TCR-activated T lymphocytes (28). To assess whether costimulation may inhibit supraoptimal activation-induced apoptosis, PBL were treated with high concentrations of PHA or anti-CD3 in the absence or presence of a cross-linked anti-CD28 mAb, which in these conditions increases proliferation induced by suboptimal concentrations of anti-CD3 (data not shown). As shown in Fig. 2B, costimulation by anti-CD28 did not inhibit PHA- or anti-CD3-induced apoptosis.

AICD is not implicated in supraoptimal activation-induced apoptosis

In vitro stimulation of previously activated mature T lymphocytes or T cell lines leads to AICD, which involves death receptor/ligand interactions (i.e., CD95/CD178 or TNFR/TNF) (10) or IFN-{gamma} (11, 12). To investigate the possible contribution of these interactions in supraoptimal activation-induced apoptosis, experiments were performed in the presence of CD95 antagonist mAb (ZB4), soluble TNFR (TNFR-Ig), or neutralizing anti-IFN-{gamma}. ZB4 and TNFR-Ig completely inhibited apoptosis of activated lymphocytes induced by CD178 and TNF-{alpha}, respectively (Fig. 3B), whereas in T cells undergoing apoptosis induced by PHA or anti-CD3, addition of ZB4, TNFR-Ig, or neutralizing anti-IFN-{gamma} did not decrease the percentage of annexin V-positive cells (Fig. 3A and data not shown). Although both CD95 and TNFR play a major role during AICD, we could not exclude the implication of other death receptors belonging to the TNFR family such as TRAIL-R or DR3. Triggering of apoptosis through the death receptors requires the formation of an active DISC in which caspase-8 or caspase-10 is activated in the absence of FLIP (13). Therefore caspase-8 processing and FLIP expression from PBL treated with high concentrations of PHA or anti-CD3 were analyzed by Western blotting in our model. Twenty-four hour treatment with PHA at high concentrations (10–50 µg/ml) or at the optimal mitogenic concentration (5 µg/ml) increased processing of pro-caspase-8 into p41/p43, but never into the p18 active subunit, which was readily cleaved from caspase-8 only in the presence of anti-CD95 mAb (Fig. 3C). Cleaved forms p43 and p41 were weakly detectable in cells treated 48 h with anti-CD3 at low concentrations (0.01–0.1 µg/ml) but never at higher concentrations (1–10 µg/ml). In vitro enzyme assay for caspase-8 revealed the absence of caspase-8 activity in cell extract from PHA- or anti-CD3-treated T lymphocytes whereas anti-CD95 mAb highly increased this activity in activated T lymphocytes (Fig. 3D). Furthermore, FLIP short (FLIPS) expression was up-regulated in cells cultured with supraoptimal as well as mitogenic concentrations of PHA and anti-CD3 (Fig. 3E and data not shown). Altogether these results indicate that supraoptimal activation-induced apoptosis of T cells is completely independent of death receptor/ligand interactions.



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  		FIGURE 3. Apoptosis of T lymphocytes at high concentrations of mitogens is not the consequence of an AICD process. A, PBL were activated by PHA or solid phase anti-CD3 at the indicated concentrations in the presence or absence of ZB4 (2.5 µg/ml) and TNFR-Ig (20 µg/ml), and apoptosis was measured at 24 h (PHA) or 48 h (anti-CD3). B, Activated lymphoblasts were treated for 24 h with medium alone, CD178 (100 ng/ml) in the presence or absence of ZB4 (2.5 µg/ml), and TNF-{alpha} (50 ng/ml) in the presence or absence of TNFR-Ig (20 µg/ml). A and B, Apoptosis was measured by a double-staining annexin V-propidium iodide, and percentages of annexin V–/propidium iodide–, annexin V+/propidium iodide–, and annexin V+/propidium iodide+ are indicated on the upper left of each dot plot. C, Analysis of caspase-8 processing in the presence of PHA or anti-CD3. PBL were treated with PHA for 24 h or solid phase anti-CD3 for 48 h. As positive control, activated lymphoblasts were treated with anti-CD95 (clone 7C11, 1 µg/ml) for 6 h. D, Measurement of caspase-8 activity in the presence of PHA or anti-CD3. PBL were treated with PHA for 12 h or solid phase anti-CD3 for 24 h. As positive control, activated lymphoblasts were treated with anti-CD95 (clone 7C11, 1 µg/ml) for 3 h. Caspase-8 activity was measured in cell lysates using the fluorogenic substrate IEPD-AMC. E, Kinetics of FLIPS expression in PBL treated with PHA (50 µg/ml) or solid phase anti-CD3 (10 µg/ml). C and E, Cells were lysed after a wash with PBS, and proteins were loaded and separated on 12% SDS-PAGE followed by Western blotting with the anti-caspase-8 mAb (C) or the anti-FLIP mAb (E). Amounts of loaded proteins have been controlled for homogeneity by probing membranes with an anti-{beta}-actin mAb. Results from one typical experiment among two showing similar results.

 
Supraoptimal activation-induced apoptosis of T lymphocytes triggers a late Cyt c release

In contrast to AICD, numerous proapoptotic signals such as lymphokine withdrawal activate the intrinsic apoptotic pathway that converge on mitochondrial membranes to induce their permeabilization (14). In this context, some proteins that are normally strictly confined to the mitochondrial intermembrane space, in particular Cyt c and apoptosis-inducing factor (AIF) are released in the cytosol (29, 30). Thus, to assess the possible role of the mitochondrion, kinetics of apoptosis were compared with that of Cyt c release measured by Western blotting in cytosolic extracts. In the cytosol of PBL treated with PHA or anti-CD3 at high concentrations, Cyt c was released only after 24 h and 48 h, respectively (Fig. 4), whereas DNA fragmentation was detectable as soon as 6 h in the presence of PHA (15% of cells exhibit fragmented DNA vs 3% in the medium alone) and 12 h with anti-CD3 (12% of apoptotic cells). Therefore Cyt c release in the cytosol appears rather a consequence than a cause of supraoptimal activation-induced apoptosis.



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  		FIGURE 4. Kinetics of Cyt c release during supraoptimal activation-induced apoptosis of T cells. PBL were incubated with medium alone, PHA (50 µg/ml), or solid phase anti-CD3 (10 µg/ml). At the indicated time, cytosolic extracts were prepared and analysis by Western blotting was performed with the anti-Cyt c mAb or the anti-cytochrome oxidase mAb. Cytochrome oxidase (Cyt ox) served as a marker of mitochondrial contamination of the extracts. Amounts of loaded proteins have been controlled for homogeneity by probing membranes with an anti-{beta}-actin mAb. The percentage of cells with fragmented DNA is indicated for different times. Results from one typical experiment among two showing similar results.

 
Supraoptimal activation-induced DNA fragmentation of T lymphocytes is inhibited by the pan-caspase inhibitor zVAD-fmk

As a first approach to assess the role of caspases in supraoptimal AICD, we examined the effect of the pan-caspase inhibitor zVAD-fmk on PS exposure. Percentage of annexin V-positive cells was measured in PBL treated with PHA 50 µg/ml or anti-CD3 10 µg/ml at indicated times. As shown in Fig. 5A, zVAD-fmk does not inhibit the PS exposure induced by PHA or anti-CD3 at high concentrations, whereas it blocks externalization of PS induced by anti-CD95. We next studied the effect of zVAD-fmk on DNA fragmentation (Fig. 5B). zVAD-fmk strongly inhibited PHA-induced DNA fragmented cells (18.2 vs 46.6%), and only partially after treatment with anti-CD3 (20.7 vs 37.7%). These results indicate that DNA fragmentation induced by high concentrations of PHA and anti-CD3 involves caspases, whereas PS exposure does not.



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  		FIGURE 5. Effect of the caspase inhibitor zVAD-fmk on PS exposure and DNA fragmentation of lymphocytes exposed to high concentrations of PHA or anti-CD3. The caspase inhibitor zVAD-fmk (100 µM) was added to PBL (A and B) or 3-day-activated lymphoblasts (A) for 1 h and then cells were incubated with PHA 50 µg/ml and solid phase anti-CD3 10 µg/ml or anti-CD95 (clone 7C11, 1 µg/ml), respectively. A, Apoptosis was measured at the indicated times by surface binding of annexin V in the presence or absence of zVAD-fmk. B, DNA fragmentation was analyzed at the indicated times by using the F7-26 mAb. The percentage of cells with fragmented DNA is indicated for each histogram. Results shown are representative of three independent experiments.

 
Processing of caspase-3 and caspase-7 is associated with activation rather than apoptosis

We next investigated whether caspases were activated in the course of PHA- and anti-CD3-induced apoptosis. We first used an anti-human caspase-3 Ab, which recognizes both the proform and the processed forms to assess the precise kinetics of caspase-3 activation. The p20 subunit of caspase-3 was detected as soon as 3 h in PHA- or anti-CD3-treated cells and progressively increased to reach a maximum after 24 h of culture, but the p17 cleaved form that accounts for enzymatic activity was never detected (Fig. 6A), in contrast to anti-CD95-treated activated T lymphocytes (Fig. 6B). To assess whether caspase-3 was active in these conditions, cleavage of PARP, a caspase-3 substrate, was studied by Western blotting in cell lysates of PBL treated with high concentrations of PHA or anti-CD3 after 24 h of culture. The p85 cleavage product of PARP was only detected in anti-CD95-treated cells, but never in PHA- and anti-CD3-treated PBL (Fig. 6C). The absence of caspase-3 activity was further demonstrated by using an in vitro enzyme assay for caspase-3 (Fig. 6D). Taken together, these results suggest that during supraoptimal activation-induced apoptosis, caspase-3 was only partially cleaved but never activated in contrast to death receptor triggering (Fig. 6D). Because it was recently demonstrated that some caspases were activated during T lymphocyte activation and proliferation (31, 32), we investigated the activation of caspase-3, caspase-7, and caspase-9 in PBL treated with PHA or anti-CD3 at increasing concentrations during 24 and 48 h, respectively. As shown in Fig. 6E, we could not detect caspase-9 cleavage into p35, whatever the dose of PHA or anti-CD3, whereas p35 was readily demonstrable in lysates from staurosporine-treated cells. In contrast to caspase-9, caspase-3, and caspase-7 were cleaved into p20 and p21/p17, respectively. However this phenomenon seems to be associated with cell activation rather than apoptosis because these cleavage products were detected at mitogenic concentrations of PHA and anti-CD3, but decreased at the higher concentrations of mitogens, which trigger cell death (Fig. 6F).



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  		FIGURE 6. Analysis of caspase-3, caspase-7, and caspase-9 processing in PHA- or anti-CD3-treated PBL. A, Kinetics of caspase-3 processing during PHA- and anti-CD3-induced apoptosis. PBL were cultured with PHA 50 µg/ml or solid phase anti-CD3 10 µg/ml, and at the indicated times, cells were lysed and proteins were separated on 12% SDS-PAGE followed by Western blotting with the anti-caspase-3 mAb. B, Cleavage of caspase-3 in 3-day-activated PBL treated by anti-CD95 (clone 7C11; 1 µg/ml). Caspase-3 cleavage was studied by Western blotting as described in A. C, Analysis of PARP cleavage is shown. Resting PBL were treated by PHA 50 µg/ml or solid phase anti-CD3 10 µg/ml and activated cells by anti-CD95 (clone 7C11; 1 µg/ml). At 24 h, cells were lysed and proteins were separated on SDS-PAGE. The p85 band corresponds to the caspase-3 cleavage product of PARP. D, Measurement of caspase-3 and caspase-7 activities in the presence of PHA or anti-CD3. PBL were treated with PHA for 12 h or solid phase anti-CD3 for 24 h. As positive control, activated lymphoblasts were treated with anti-CD95 (clone 7C11, 1 µg/ml) for 3 h. Caspase activities were measured in cell lysates using the fluorogenic substrate DEVD-AMC. E, Analysis of caspase-9 cleavage. Cells were treated with PHA or solid phase anti-CD3, lysed at 24 h for PHA and 48 h for anti-CD3, and proteins were separated on SDS-PAGE by Western blotting with an anti-caspase-9 Ab, which recognizes the p35 cleaved form of caspase-9. As control, 3-day-activated PBL were treated with staurosporine (STS) (0.5 µM) for 12 h. F, Caspase-3 and caspase-7 processing in PBL treated by dose-responses of PHA or anti-CD3. Cells were treated by dose-responses of PHA or solid phase anti-CD3, lysed at 24 h for PHA and 48 h for anti-CD3, and proteins were loaded and separated on SDS-PAGE. Amounts of loaded proteins have been controlled for homogeneity by probing membranes with an anti-{beta}-actin mAb. All the data are from one typical experiment among two showing similar results.

 
Supraoptimal activation-induced apoptosis is impaired by inhibitors of cathepsins B and L

Because we could not detect the activation of caspase-3, caspase-7, and caspase-9 specifically associated with apoptosis induced at high concentrations of PHA- or anti-CD3, we examined whether the protective effect of zVAD-fmk could be due to the inhibition of proteases other than caspases. For instance, zVAD-fmk has been shown to inhibit lysosomal cysteine proteases (33), which have been recently reported to contribute to TNF-induced apoptosis (20). Thus, we studied the effect of a panel of protease inhibitors on PHA- and anti-CD3-induced apoptosis. Compounds reported to inhibit lysosomal cysteine proteases (E64d) as well as those more specific for cathepsin B (zFA-fmk and zFK-mbmk) and cathepsin L (zFA-fmk and zFF-dmk) conferred significant protection against PHA- and anti-CD3-induced apoptosis in T cells, whereas inhibitors of calpains (PD 150606, calpeptin, and calpain inhibitor II) or aspartic proteases including cathepsin D (pepstatin A) did not prevent apoptosis measured by surface binding of annexin V or propidium iodide staining (Fig. 7A). A more specific cathepsin B inhibitor, CA-074-Me, was also tested but could not be used because of its toxicity for T lymphocytes. Finally, zFA-fmk and zFF-dmk completely inhibited DNA fragmentation induced by PHA or anti-CD3 at high concentrations (Fig. 7B). These results suggest that cathepsins B and L are required for supraoptimal activation-induced apoptosis.



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  		FIGURE 7. Inhibition of cathepsin B and cathepsin L protects T lymphocytes from supraoptimal activation-induced apoptosis. PBL were pretreated for 1 h with indicated protease inhibitors before the addition of mitogens (PHA 50 µg/ml or solid phase anti-CD3 10 µg/ml). A, The percentage of apoptotic cells was measured 24 h later for PHA and 48 h later for anti-CD3 by surface binding of annexin V and propidium iodide staining. B, DNA fragmentation was analyzed at the indicated times by using the F7-26 mAb. The percentage of cells with fragmented DNA is indicated for each histogram. Results are mean ± SEM from a triplicate experiment and are representative of three independent experiments.

 
Cathepsin B and cathepsin L are released and catalytically active in the cytosol during supraoptimal activation-induced apoptosis

Prevention of apoptosis by cathepsin B and L inhibitors suggested that these cathepsins could be released from the lysosome to the cytosol. To assess this release, cathepsins B and L were measured by immunoblot analysis on cytosolic extracts. As shown in Fig. 8A, the p30 cathepsin B active fragment as well as the p36 and p28 active forms of cathepsin L were detectable in the cytosol of cells treated with PHA 50 µg/ml or anti-CD3 10 µg/ml whereas cells exposed to mitogenic concentrations of PHA or anti-CD3 (5 µg/ml or 0.1 µg/ml, respectively) only slightly release cathepsins B and L into the cytosol. Whereas cathepsin L release in the cytosol was almost completed after treatment of cells with high concentrations of mitogens, only part of cathepsin B was released as compared with the amount of cathepsin B recovered after solubilization of cell membrane with SDS (Fig. 8A). To provide independent evidence for the translocation of cathepsins from lysosomes to cytosol, confocal microscopy was used to directly visualize the cellular redistribution of cathepsins. In cells treated with mitogenic concentrations of PHA or anti-CD3, the distribution of cathepsins B and L was localized in granules (Fig. 8B). After treatment with PHA or anti-CD3 at high concentrations, the fluorescence was still present in some granules but also diffusely distributed in the cytoplasm, showing that cathepsins B and L were partially released from the lysosomes into the cytosol (Fig. 8B). Thus these results are consistent with a translocation of cathepsins B and L from a vesicular compartment into the cytoplasm during exposure to high concentrations of PHA or anti-CD3.



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  		FIGURE 8. Cathepsins B and L are translocated from lysosomes into the cytosol during supraoptimal activation-induced apoptosis. A, Cells were treated with PHA or solid phase anti-CD3, and cytosolic extracts were prepared at indicated times. Proteins (30 µg) were separated on SDS-PAGE, and cathepsin B release and cathepsin L release from lysosomes were studied by immunoblot analysis. Lamp-1 served as a marker of lysosomal contamination of the extracts. B, Visualization of cathepsin B release and cathepsin L release from lysosomes to cytosol in PBL treated by PHA or solid phase anti-CD3 by confocal microscopy. Cells were treated for 12 h in the presence of PHA (5 or 50 µg/ml) or 24 h anti-CD3 (0.1 or 10 µg/ml). C and D, Cells were treated with PHA or solid phase anti-CD3, and cytosolic extracts were prepared at indicated times. Equal amounts of protein were analyzed for cathepsin B and cathepsin L activities using fluorogenic proteases substrates zFR-AMC (C) and zRR-AMC (D). Results are mean ± SEM from a triplicate experiment. All the data are from one typical experiment among two showing similar results.

 
We next addressed whether cathepsins B and L were catalytically active in the cytosol of cells treated with PHA at high concentrations. Cathepsin B and L activity was measured in cytosolic extracts using the fluorogenic protease substrate zFR-AMC. As expected, cathepsin B and cathepsin L activity was detected at 12 h in PBL treated by PHA at 50 µg/ml whereas it was not detectable at the optimal mitogenic concentration of PHA (Fig. 8C). We then specifically examined the kinetics of cathepsin B activation using the fluorogenic substrate zRR-AMC. Cytosolic cathepsin B activity reached a maximum after 6 h of treatment at high PHA concentrations and then progressively decreased, whereas anti-CD3-induced cathepsin B activation was maximal at 24 h and only partially decreased after 48 h of treatment (Fig. 8D). As control, we also measured calpain activity using the specific fluorogenic substrate Suc-LLVY-AMC, but we never detected any activity in the cytosol of PHA- and anti-CD3-treated cells (data not shown).

Cathepsin B and cathepsin L release are the consequence of lysosomal membrane permeabilization (LMP)

To determine whether the release of cathepsins B and L was the consequence of LMP, we used a lysosomotropic fluorescence probe, AO. Cells with intact lysosomes exhibit a marked red AO fluorescence whereas it decreased when lysosomes are permeabilized. Treatment of PBL with high concentrations of PHA or anti-CD3 for 24 h triggered LMP as demonstrated by the decrease red AO fluorescence in 36.7% and 21.7% of the cells, respectively (Fig. 9A). LMP was detected as soon as 1 h with 50 µg/ml PHA and 12 h with 10 µg/ml anti-CD3 (Fig. 9B). Although supraoptimal activation-induced apoptosis was associated with LMP, the cytosol-lysosome pH gradient seemed to be preserved in PHA- or anti-CD3-treated cells because LysoTracker red, an acidic organelle-specific probe, still accumulated in granular cellular structures (Fig. 9C), suggesting that only limited perturbations of lysosomal membrane would allow release of lysosomal proteins such as cathepsins B and L.



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  		FIGURE 9. LMP is required for release in the cytosol of active cathepsins during supraoptimal activation-induced apoptosis. A and B, Cells were treated with PHA or solid phase anti-CD3 before being exposed to AO and analyzed by cytofluorometry at 24 h in (A) and at indicated times in (B). The percentage of cells indicated for each histogram corresponds to AO low cells with reduced numbers of intact lysosomes. C, Cells were treated with PHA or solid phase anti-CD3, and staining with Lysotracker red (acidic organelle-selective probe) was used to characterize lysosomal membrane alterations and visualized by fluorescent microscopy. All data are from one typical experiment among two showing similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we investigated the effects of the magnitude of initial T cell activation on the survival and proliferation of mature human T cells. We indeed hypothesized that increasing Ag load to achieve high dose unresponsiveness could result in clonal deletion by apoptosis. We could demonstrate an intrinsic tuning event that triggers apoptosis of activated T cells when the number of engaged TCR (i.e., the concentration of mitogen) was raised beyond a death threshold. This novel mechanism differs from the two major pathways that restrict clonal expansion, namely AICD and death by neglect, by several features, of which the most typical is the involvement of cathepsins B and L released from the lysosomes into the cytosol.

Recent models of lysosome-dependent apoptosis have shown the requirement of Bax translocation to mitochondria (34, 35, 36). However the requirement of cathepsins for Bax translocation and the consequences of this translocation to mitochondrial membrane permeability are still debated (34, 36). Indeed, in a first model, mitochondrial membrane permeabilization leads to classical mitochondrial pathway with Cyt c release and caspase-dependent apoptosis (34). In the second model, mitochondrial membrane permeability does not trigger Cyt c release but rather AIF, which leads to caspase-independent death (36). In our present study, LMP occurs very rapidly (during the first hour for PHA), followed by cathepsin B and cathepsin L release (cytosol activity at maximum after 6 h for PHA) and finally DNA is fragmented (between 6 and 12 h for PHA) before the Cyt c release from mitochondria (at 24 h for PHA). These data clearly eliminate the possibility that during supraoptimal activation-induced apoptosis, cathepsins B and L trigger the classical mitochondrial pathway. Whether the second hypothesis, i.e., release of cathepsins B and L would trigger Bax translocation, AIF relocalization, and caspase-independent apoptosis, would account for supraoptimal activation-induced apoptosis is still an opened question and would deserve additional experiments.

Several important differences are apparent between supraoptimal activation-induced apoptosis and AICD. First, CD95/CD178 or TNFRII/TNF interactions, which have been demonstrated to mediate AICD of CD4+ or CD8+ T cells, respectively (10), do not play a role in this study. Second, an active DISC, which is the signature of death receptor triggering (37), was not formed during supraoptimal activation-induced apoptosis because caspase-8 was only partially processed but never active and FLIPS expression rapidly up-regulated. Third, in contrast to AICD, which requires prior exposure to IL-2 (38), apoptosis described in this study was not inhibited by cyclosporin A or rapamycin, which interfere with IL-2 synthesis or signaling, respectively (39). Finally, our data clearly demonstrate that T lymphocytes undergo apoptosis within 24–48 h following activation, without any cell division. Such kinetics markedly differ from those of AICD and others models of activation at high Ag concentrations in which deletion of specific T cells requires re-exposure of previously activated T lymphocytes to Ag or occurs after an initial phase of expansion (4, 8). Although the kinetics of apoptosis observed in our model are quite similar to those reported after strong TCR ligation of naive murine CD4+ T cells by Kishimoto and Sprent (5), the CD95 dependency of TCR-mediated apoptosis in the latter model was clearly different from supraoptimal activation-induced apoptosis in human T lymphocytes.

No more than the death receptor pathway, the intrinsic mitochondrial pathway does seem to primarily play any role in supraoptimal activation-induced apoptosis. Indeed the mitochondrial pathway involves Cyt c release from mitochondria that will lead to the formation of a so called apoptosome, a ternary complex of Cyt c, APAF-1, and caspase-9 that triggers the autocatalytic activation of caspase-9, and by cascade, of the executioner caspases such as caspase-3 and caspase-7 (40). Now, our results clearly demonstrate that caspase-9 is not processed and caspase-3 and caspase-7 activities were not increased during supraoptimal activation-induced apoptosis. Furthermore, Cyt c release is very late and occurs after the completion of cell death, suggesting that this release is a consequence rather than the cause of apoptosis.

Our results provide evidence that caspase-3, caspase-7, and caspase-8 are at least partially cleaved after treatment with anti-CD3 and PHA whereas caspase-9 is not. However, dose-response studies clearly demonstrate that caspase processing is selectively associated with activation rather than apoptosis because p20 of caspase-3, p21/p17 of caspase-7 and p41/43 of caspase-8 are only detectable at optimal mitogenic concentrations of anti-CD3 and PHA, but not or less at the higher concentrations that trigger apoptosis. All these results are in accordance with previous studies (31, 32) demonstrating that caspase-3, caspase-6, caspase-7, and caspase-8 were processed during activation through the TCR, but not caspase-9, which remains as a proenzyme.

Even if caspase-3, caspase-7, caspase-8, and caspase-9 do not play a pivotal role in supraoptimal activation-induced apoptosis, it remains that zVAD-fmk inhibits DNA fragmentation of cells treated with high concentrations of anti-CD3 or PHA. This could mean as a first hypothesis that other caspases may be involved, but as a very downstream event. Indeed cathepsins have been shown to activate the caspase cascade either by directly cleaving and activating caspases (41, 42) or through Bid-mediated release of Cyt c (41, 43). Consistent with this observation, in cell-free extracts prepared from TNF-{alpha}-treated hepatocytes, active cathepsin B from purified lysosomes can be released and may in turn increase cytosol-induced release of Cyt c from mitochondria and caspase-9 and caspase-3 activation (20). In fact this indirect activation of caspases by cathepsins B or L involving Cyt c release is not very likely during supraoptimal activation-induced apoptosis because of the late occurrence of Cyt c at time when DNA fragmentation was already detectable (Fig. 4).

A second possibility that may account for the effect of zVAD-fmk on DNA fragmentation could be the inhibition of proteases other than caspases. Indeed, zVAD-fmk has been shown to inhibit lysosomal cysteine proteases (33). In this case, zVAD-fmk could inhibit cathepsins B and L, which are known to be directly connected to proteins that possess endonuclease activity (23) responsible for the DNA fragmentation (42). In such case, the apoptosis reported in this study would be caspase-independent. Although there is evidence that caspase-independent death can also lead to some of the typical features of apoptosis, such as PS exposure, chromatin condensation, and early phagocytic removal (44, 45, 46), the classical apoptotic morphology has been closely linked to caspase activation. However recent evidences are emerging regarding the ability of other proteases, including the cathepsin D (22) and cathepsin B (21), to induce all the classical features of apoptosis in the absence of caspase activation. Furthermore there is now increasing evidence for caspase-independent programs of T cell apoptosis during normal T cell activation (reviewed in Ref.46).

Engagement of TCR by MHC-peptide complexes on Ag-presenting cells and subsequent aggregation of TCR is the key initial event that controls T cell activation and, ultimately, clonal expansion and differentiation into effector and/or memory T cells. Based on both in vivo and in vitro models, major advances have been achieved in delineating the role of different parameters in the outcome of TCR engagement, according to the stage of differentiation of T cells. As regards naive and memory peripheral T cells, the use of T cell clones and TCR transgenic mice has been instrumental in analyzing the role of coactivation signals and that of parameters that control the first signal such as dose and structure of the peptide. Substitutions in the amino acid sequences that could modify the affinity of the peptide for MHC or TCR may result in incomplete T cell activation, clonal anergy, and apoptosis (47, 48, 49). In addition to affinity, the number of TCR engaged initially or sequentially during T cell activation also appears to control the outcome of activation (50). Hence, the concept of a minimal threshold in the number of engaged TCR (e.g., between 800 and 1500 per mature T cell) for inducing full activation and proliferation was proposed by Viola and Lanzavecchia (51). We now propose that when the number of engaged TCR was raised beyond a death threshold, activated T cells are primed to apoptosis mediated through lysosomal perturbation and cathepsin B and cathepsin L release. This mechanism would ensure that potential autoreactive peripheral T cells are removed at the onset of the activation before proliferation and differentiation into effector helper or CTLs.


   Acknowledgments
 
We thank Jean-Pierre Rouault for the measurement of cytosolic cathepsin activity by fluorometry, H. Waldmann for the kind gift of TNFR-Ig, and P. Krammer for anti-caspase-8 and anti-FLIP Abs. We thank T. Defrance and Y. Leverrier for critical reading of the manuscript. We dedicate this work to Dr. J. P. Revillard who died on June 2, 2003.


   Footnotes
 
1 This work was supported by Institut National de la Santé et de la Recherche Médicale Grant 98046 and région Rhône-Alpes Grant 00816945. M.-C.M. is a recipient of a fellowship from the Ministère de l’Education Nationale et de la Recherche. Back

2 Deceased on June 2, 2003. Back

3 Address correspondence and reprint requests to Dr. Laurent Genestier, Institut National de la Santé et de la Recherche Médicale Unité 404, Centre d’Etudes et de Recherche en Virologie et Immunologie, Institut Fédératif No 128, BioSciences Lyon-Gerland, 21 Ave Tony Garnier, 69365 Lyon cedex 07, France. E-mail address: genestier{at}cervi-lyon.inserm.fr Back

4 Abbreviations used in this paper: AICD, activation-induced cell death; DISC, death-inducing signaling complex; AO, acridine orange; Cyt c, cytochrome c; PARP, poly(ADP-ribose) polymerase; PS, phosphatidylserine; AMC, 7-amido-4-methylcoumarin; AIF, apoptosis-inducing factor; LMP, lysosomal membrane permeabilization; FLIPS, FLIP short. Back

Received for publication October 23, 2003. Accepted for publication February 13, 2004.


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 Introduction
 Materials and Methods
 Results
 Discussion
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