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(Journal of Leukocyte Biology. 2001;70:767-775.)
© 2001 by Society for Leukocyte Biology

Involvement of caspases and of mitochondria in Fas ligation-induced eosinophil apoptosis: modulation by interleukin-5 and interferon-{gamma}

Séverine Létuvé*, Anne Druilhe*, Martine Grandsaigne*, Michel Aubier*,{dagger} and Marina Pretolani*

* Institut National de la Santé et de la Recherche Médicale U408, Faculté de Médecine Xavier Bichat, and
{dagger} Service de Pneumologie, Hôpital Bichat, Paris, France

Correspondence: Marina Pretolani, Ph.D., INSERM U408, Faculté de Médecine Xavier Bichat, 16, rue Henri Huchard, 75018 Paris, France. E-mail: mpretol{at}bichat.inserm.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we examined the relative importance of caspases and mitochondria in Fas-mediated eosinophil apoptosis. Stimulation of human peripheral blood eosinophils with an agonistic anti-human Fas monoclonal antibody, but not with control IgM, induced a time-dependent increase in their apoptosis, which was associated with a loss in mitochondrial transmembrane potential ({Delta}{Psi}m) and with caspase-8 and caspase-3 activation. Interleukin (IL)-5 and interferon (IFN)-{gamma}, two cytokines known to prolong eosinophil survival, inhibited Fas-mediated apoptosis and caspase activation but poorly affected the decrease in {Delta}{Psi}m. Eosinophil incubation with bongkrekic acid, an inhibitor of the mitochondrial permeability transition pore (MPTP) opening, failed to modify Fas-mediated loss in {Delta}{Psi}m, caspase activation, and apoptosis. In contrast, caspase inhibitors markedly reduced eosinophil apoptosis without significantly affecting {Delta}{Psi}m dissipation. We conclude that caspase-8 and caspase-3 activation, but not MPTP opening, mediate Fas-induced eosinophil apoptosis and are the main targets for the protective effect of IL-5 and IFN-{gamma}.

Key Words: cell death • asthma • granulocytes • {Delta}{Psi}m


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Several studies have shown that the accumulation and activation of eosinophils recruited to inflammatory sites are related to the occurrence of tissue injury, a common feature of many atopic diseases, particularly bronchial asthma [1 ]. The persistence of eosinophils in inflammatory tissues may result from increased survival and/or decreased death [2 3 4 ]. Apoptosis is the most common form of physiological cell death, which is involved in the resolution of inflammatory reactions [4 , 5 ].

Factors that extend the life span of tissue eosinophils by favoring their survival and inhibiting their apoptosis include T helper-1 and T helper-2–derived cytokines such as interferon (IFN)-{gamma} [6 , 7 ] and interleukin (IL)-5 [8 , 9 ], respectively. Enhanced expression and production of IL-5 in asthmatic airways has widely been documented [10 ]. Although the role of IFN-{gamma} in tissue eosinophilia and in allergic and inflammatory diseases is less defined than that of IL-5, it has been suggested that this cytokine is involved in the exacerbation of asthma observed in respiratory virus–infected patients [11 ].

Pro-apoptotic signals include the activation of Fas antigen, a transmembrane protein belonging to the tumor necrosis factor receptor family [12 ]. Fas expression by human peripheral blood and tissue eosinophils [13 14 15 16 17 ], as well as its ability to facilitate the elimination of airway eosinophils in vivo [15 , 18 ], has been extensively reported.

Fas ligation in several cell types involves the activation of caspases, a family of cysteine proteases with aspartate substrate specificity. Caspases are produced as inactive zymogens and activated by proteolytic cleavage [19 ]. Once activated, caspases cleave cytoplasmic and nuclear components, leading to cell dismantling, DNA degradation, and ultimately cell death [19 ].

Two different intracellular pathways have been described during Fas-mediated apoptosis. These depend on the requirement of mitochondria in the propagation of the death signal [20 ]. Accordingly, Fas cross-linking by its natural ligand, Fas-ligand, or by an agonistic antibody activates caspase-8, which, in turn, initiates a caspase cascade, either by directly cleaving caspase-3 or by altering mitochondrial homeostasis [20 , 21 ]. This latter phenomenon is characterized by the disruption of transmembrane potential, defined as {Delta}{Psi}m, and by the opening of the mitochondrial permeability transition pore (MPTP), which is thought to be responsible for releasing apoptogenic factors, such as cytochrome c [19 , 21 , 22 ], into the cytosol. Cytochrome c, in turn, activates cytosolic factors that cleave caspase-3 [21 , 22 ]. Scaffidi et al. [20 ] suggested that activation of the mitochondria-independent or mitochondria-dependent pathway during the apoptotic process relates to the cell type. Although this concept has been established for numerous cell lines, little is known about the precise mechanisms that regulate Fas-induced apoptosis in eosinophils.

We wanted to determine the relative importance of caspase- and mitochondria-dependent pathways in mediating Fas-induced eosinophil apoptosis. In addition, we investigated the ability of IL-5 and IFN-{gamma}, two cytokines known to prolong eosinophil survival and inhibit their apoptotic death [6 7 8 9 ], to interfere with these phenomena.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Eosinophil purification
Human peripheral venous blood (50 mL) was obtained from hypereosinophilic volunteers (>=500 eosinophils/µL). Each patient provided a written consent, and each experiment was performed with cells obtained from a single donor. Eosinophils were isolated by immunomagnetic negative selection using a modification of the method of Hansel et al. [23 ]. Briefly, phosphate-buffered saline (PBS)-diluted blood was centrifuged on Lymphocytes Separation Medium (Eurobio, Les Ulis, France; density 1.077 g/mL) to remove mononuclear cells. We lysed the remaining erythrocytes by incubating the granulocyte pellet with an ice-cold isotonic ammonium-chloride solution. In some experiments, a further elimination of mononuclear cells with Percoll (Pharmacia Biotech, Orsay, France; density 1.082 g/mL) was required. Mixed granulocytes were then incubated with magnetic microbead-conjugated anti-CD16 monoclonal antibody (mAb) (Miltenyi Biotec, Paris, France) before unlabeled eosinophils were eluted through a magnetic separation column (MACS system, Miltenyi Biotec). The purity of the resulting cell population (>98% eosinophils) was evaluated after cytospin preparations were stained with Diff-Quik® dye (Merz Dade, Baxter Dade AG, Duedingen, Switzerland).

Cell culture
Eosinophils were resuspended in RPMI 1640 (Life Technologies, Cergy Pontoise, France), supplemented with antibiotic–antimycotic solution (Life Technologies) and 10% fetal calf serum (Hyclone, Logan, UT), and incubated at 37°C with 5% CO2 in a humidified atmosphere for 0–24 h.

Eosinophils were stimulated with either 3–3000 ng/mL of agonistic mouse anti-human Fas antigen mAb (clone 7C11, Beckman Coulter, Villepinte, France) or 3000 ng/mL of its isotype matched mouse IgM (Dako, Trappes, France), in the absence or presence of 0.0005–5 ng/mL of recombinant human IL-5 (PeproTech Inc., Rocky Hill, NJ) or of 0.1–1,000 U/mL recombinant human IFN-{gamma} (R&D Systems Europe Ltd., Oxon, U.K.). In a separate series of experiments, eosinophils were preincubated for 2 h with 30 µM of the following permeant and irreversible caspase inhibitors: Z-Ile-Glu-Thr-Asp-fluoromethylketone (Z-IETD-fmk, caspase-8 inhibitor), Z-Asp-Glu-Val-Asp-fmk (Z-DEVD-fmk, caspase-3 inhibitor), or Z-Val-Ala-Asp-fmk (Z-VAD-fmk, broad spectrum caspase inhibitor, all from Calbiochem, Bad Soden, Germany), or with their vehicle (i.e., a 0.4% solution of dimethyl sulfoxide [DMSO]). Alternatively, 62 µM of bongkrekic acid (Calbiochem) or its vehicle (i.e., a 0.02 N NH4OH solution [Sigma, St. Quentin Fallavier, France]) was added to eosinophil cultures 2 h before stimulation. This concentration of bongkrekic acid was selected on the basis of previous studies showing its ability to prevent the loss in {Delta}{Psi}m in neutrophilic granulocytes [24 ].

Viability and apoptosis determination by flow cytometry
For eosinophil viability, cells (0.15x106) were washed twice in PBS and resuspended in PBS containing 2.5 µg/mL propidium iodide (Sigma) immediately before analysis. For apoptosis determination, eosinophils (0.15x106) were resuspended in a hypotonic solution containing 0.1% (w/v) sodium citrate, 0.1% (v/v) Triton X-100, and 50 µg/mL propidium iodide, as described [25 ]. In both cases, cells were applied to an Epics XL flow cytometer (FL-3 photomultiplier, Beckman Coulter). A total of 5000 cells were analyzed with Expo 32 software (Beckman Coulter). The percent of propidium iodide-unstained (viable) cells or propidium iodide-low stained apoptotic nuclei, respectively, was determined.

Determination of {Delta}{Psi}m by flow cytometry
Changes in {delta}{Psi}m were evaluated by detecting the uptake of the lipophilic cationic dye, 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3), Molecular Probes Inc., Eugene, OR), which accumulates into polarized mitochondria under the influence of {Delta}{Psi}m [26 ]. Eosinophils (0.3 x 106) were washed in PBS and incubated at 37°C for 30 min in the presence of 40 nM of DiOC6(3) in RPMI [27 ]. Propidium iodide, at the final concentration of 2.5 µM, was added immediately before cytofluorimetric analysis to exclude necrotic cells. Negative control experiments were performed by preincubating cells (10 min, 37°C) with 200 µM of the mitochondrial uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP, Sigma), which collapses the {Delta}{Psi}m [27 , 28 ]. A total of 5000 events were analyzed with an Epics XL flow cytometer (DiOC6(3) emission in FL-1 and propidium iodide in FL-3) using Expo 32 software. The percent of DiOC6(3)-high stained cells was determined.

Apoptosis determination by electron microscopy
Ultrastructural changes induced by Fas stimulation were determined by electron microscopy. Briefly, IgM- or anti-Fas (3000 ng/mL)-stimulated cytokine-untreated or cytokine-treated eosinophils were fixed in 1.6% glutaraldehyde and postfixed in 2% osmic acid (both from Sigma), as previously described [13 ]. After acetone dehydration, pellets were embedded into epoxy resin. Ultrathin sections were contrasted with uranyl acetate and lead citrate and examined with a JEM-100 CX II transmission electron microscope (Jeol, Tokyo, Japan).

Preparation of eosinophil extracts
Eosinophils were lysed by sonication in 10 mM N-(2-hydroxyethyl)piperazine-N'-(2-ethane-sulfonic acid), pH 8.0, 1% nonidet P-40, 150 mM NaCl, 500 mM sucrose, 1 mM Na2EDTA, 2 mM phenylmethylsulfonyl fluoride, 4% ß-mercaptoethanol, 10 µg/mL aprotinin, 10 µM leupeptin, and 10 µM pepstatin A (all from Sigma) and subsequently centrifuged for 15 min at 12,000 g. Protein contents in clarified supernatants were determined by comparison with an ovalbumin standard curve (ICN Biomedical, Costa-Mesa, CA), using the Biorad protein assay (Biorad, München, Germany).

Evaluation of caspase-8 and caspase-3 expression by Western blot
Protein extracts (25 µg) were resolved by 13% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroblotted on polyvinylidene difluoride membranes (Biorad), and next reacted with a 1:10 dilution of mouse anti-human caspase-8 Ab (clone C15, a generous gift from Dr. M. E. Peter, German Cancer Research Center, Heidelberg, Germany), a 1:1000 dilution of rabbit anti-human caspase-3 Ab (BD Biosciences, Le Pont de Claix, France), or with the mouse anti-ß-actin mAb (clone AC-74, Sigma) at a 1:4000 dilution. Immunoblots were detected with peroxidase-conjugated donkey anti-rabbit or sheep anti-mouse Abs, both at a 1:4000 dilution, using the ECL Western blotting detection system (all from Amersham, Les Ulis, France). The intensities of the expression of zymogen caspase-8, zymogen and cleaved-caspase-3, and ß-actin were quantified using a densitometer (CCD-COHU, Japan) and Gel Analyst software (Claravision, Orsay, France). Results are expressed as a ratio, defined as the optic density (OD) values of specific caspase bands/OD values of the corresponding ß-actin bands.

In vitro fluorogenic caspase-8 and caspase-3-like cleavage assay
The following protocol was adapted from Nicholson et al. [29 ]. Briefly, cleavage of 50 µM of the caspase-8 substrate, Z-IETD-7-amino-4-(trifluoromethyl)coumarin (Z-IETD-AFC, Calbiochem), and the caspase-3 substrate, acetyl-DEVD-7-amino-4-methyl coumarin (Ac-DEVD-AMC, Promega, Charbonnieres, France), were measured in a caspase reaction buffer consisting of 10 mM N-(2-hydroxyethyl)piperazine-N'-(2-ethane-sulfonic acid), pH 7.5, 10% sucrose, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Sigma). Aliquots (10 and 20 µg, for caspase-3 and caspase-8, respectively) of eosinophil lysates were preincubated with 50 µM of the caspase-8 inhibitor Ac-IETD-aldehyde (Ac-IETD-CHO, Calbiochem) or the caspase-3 inhibitor Ac-DEVD-CHO (Promega), or with their vehicle (i.e., a 2% DMSO solution) for 30 min at 37°C before the addition of 50 µM of Z-IETD-AFC or Ac-DEVD-AMC. AFC and AMC releases were monitored using a Fluostar II spectrofluorimeter with filter settings at 380 or 355 nm, respectively, for excitation and 510 or 460 nm, respectively, for emission using the Biolise software (both from BMG Labtechnologies, Champigny-sur-Marne, France). Results are expressed as picomoles of free AFC or AMC, by comparison with standard curves of AFC (Calbiochem) and AMC (Promega).

Statistical analysis
Data were analyzed statistically using the StatView SE+Graphics program for Macintosh (Abacus Concepts, Berkeley, CA). If ANOVA was significant, a Student’s t test for paired values was used to assess comparability between the means (p values of 0.05 or less were considered significant). The results are expressed as means ± SEM of the indicated number of experiments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IL-5 and IFN-{delta} inhibit Fas ligation-induced loss in viability and apoptosis in human eosinophils
In preliminary experiments, eosinophil cross-linking during 12 and 24 h with 3–3000 ng/mL of murine anti-human IgM agonistic anti-Fas mAb, but not with 3000 ng/mL of its isotype-matched control IgM, time-dependently augmented the number of apoptotic nuclei, as assessed by flow cytometry (Fig. 1A ). Although a plateau was observed for the concentration of 30 ng/mL of the anti-Fas mAb, particularly at 24 h (Fig. 1A) , the intensity of the apoptotic response varied depending on the eosinophil preparations, and, in some cases, a clear apoptotic effect could only be observed with concentrations of anti-Fas above 300 ng/mL. No significant increment in the number of apoptotic nuclei was detected after 3 and 6 h of Fas stimulation (data not shown).



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  		Figure 1. Kinetics and dose–response study of inhibition by IL-5 and IFN-{gamma} of anti-Fas-induced apoptosis in human peripheral blood eosinophils. (A) Eosinophils were incubated for 0–24 h with 3000 ng/mL of isotype-matched mouse IgM or with 3–3000 ng/mL of anti-Fas mAb. Apoptotic nuclei were quantified by flow cytometry after cell permeabilization and DNA staining with propidium iodide. (B) Eosinophils were treated for 12 and 24 h with 3000 ng/mL of mouse IgM or of anti-Fas mAb in the absence or presence of 0.0005–5 ng/mL IL-5 or of 0.1–1000 U/mL IFN-{gamma}. The percent inhibition of Fas-induced apoptosis by the indicated concentrations of cytokines was determined. Results are the means ± SEM of 3–4 experiments. *P < 0.05, as compared with IgM-treated eosinophils.

 
We then did some testing to see whether increasing concentrations of IL-5 and IFN-{gamma} interfered with anti-Fas (3000 ng/mL)-induced eosinophil apoptosis. Eosinophil incubation with 0.0005–5 ng/mL IL-5 or with 0.1–1000 U/mL IFN-{gamma} resulted in dose-dependent inhibition of Fas-mediated apoptosis at 12 and 24 h. The maximum effect was observed with the highest concentrations of both cytokines (Fig. 1B) .

In view of these results, the concentrations of 3000 ng/mL of anti-Fas mAb, 5 ng/mL IL-5, and 1000 U/mL IFN-{gamma} were selected for further study.

Using flow cytometry, we also established that the increase in the number of apoptotic nuclei in IgM (and, to higher extent, in anti-Fas-treated eosinophils) paralleled a decrease in their viability (Fig. 2A and B ). The addition of IL-5 (5 ng/mL) or IFN-{gamma} (1000 U/mL) to the culture medium reduced the loss in cell viability and the increase in the percent of apoptotic nuclei in both IgM- and anti-Fas-treated eosinophils at 12 and, to a lesser extent, at 24 h (Fig. 2A and 2B) .



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  		Figure 2. Effect of IL-5 and IFN-{gamma} on IgM- and anti-Fas-induced changes in viability (A) and apoptosis (B) in cultured human blood eosinophils. Eosinophils were stimulated for 0–24 h with 3000 ng/mL of isotype-matched mouse IgM or agonistic anti-Fas mAb in the absence or presence of 5 ng/mL IL-5 or of 1000 U/mL IFN-{gamma}. Viable cells and apoptotic nuclei were quantified by flow cytometry in intact cells or after permeabilization and staining of DNA with propidium iodide, respectively. Results are the means ± SEM of 8 (for cell viability) and 26 (for cell apoptosis) experiments.*P < 0.05, between IgM-treated cells and values obtained at time 0; {dagger} P < 0.05, as compared with IgM-stimulated cells; {ddagger} P < 0.05, as compared with anti-Fas-treated cells.

 
These observations were confirmed by analyzing eosinophil ultrastructural changes by electron transmission microscopy. When stimulated for 12 h with 3000 ng/mL of anti-Fas mAb, most eosinophils showed characteristic features of apoptosis, notably condensation of nuclear chromatin and simplification and rounding of the nuclear structures. In contrast, IgM-treated cells exhibited a normal morphology (Fig. 3A and B ). The addition of 5 ng/mL IL-5 or 1000 U/mL IFN-{gamma} restored a normal phenotype in most eosinophils (Fig. 3C and 3D) .



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  		Figure 3. Electron transmission microphotographs showing purified human eosinophils incubated for 12 h with 3000 ng/mL isotype matched mouse IgM (A) or with 3000 ng/mL anti-Fas mAb, in the absence (B) or presence of 5 ng/mL IL-5 (C) or of 1000 U/mL IFN-{gamma} (D). The eosinophils indicated by the arrows show morphologic features of apoptosis. Magnification x 1500.

 
Inhibition of Fas-mediated apoptosis by IL-5 and IFN-{gamma} was unrelated to changes in Fas antigen expression at the cell surface at any time point (data not shown).

The effect of IL-5 and IFN-{gamma} was specific since a loss in their inhibitory activity against spontaneous and Fas-induced-eosinophil apoptosis was noted when they were heated at 100°C for 1 h (data not shown).

Effect of IL-5 and IFN-{gamma} on Fas ligation-induced {Delta}{Psi}m alterations in human eosinophils
To determine whether Fas-induced apoptotic death involved {Delta}{Psi}m alterations, we used flow cytometry to assess the ability of eosinophils to incorporate the lipophilic cationic fluorochrome DiOC6(3) into their mitochondria. Freshly purified human eosinophils exhibited high fluorescence staining with DiOC6(3) (Fig. 4A and B ). A moderate and not significant reduction in {Delta}{Psi}m was noted when eosinophils were incubated for 3 and 12 h with control IgM (Fig. 4A and 4B) . In contrast, the addition of anti-Fas mAb induced a significant loss in DiOC6(3) uptake, which occurred as early as 3 h of stimulation and further decreased at 12 h (Fig. 4A and 4B) . Specific staining of polarized mitochondria was confirmed by the absence of accumulated DiOC6(3) in eosinophils previously treated with the oxidative phosphorylation uncoupling agent CCCP (Fig. 4A) [28 ].



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  		Figure 4. Effect of IL-5 and IFN-{gamma} on Fas ligation-induced eosinophil {Delta}{Psi}m dissipation. (A) Eosinophils were stimulated for 12 h with 3000 ng/mL of mouse IgM or of agonistic anti-Fas mAb, in the absence or presence of 5 ng/mL IL-5 or 1000 U/mL IFN-{gamma}. Cells were incubated with 40 nM of DiOC6(3) and 2.5 µM of propidium iodide. Negative control experiments were performed by treating the cells with 200 µM of the mitochondrial uncoupler, CCCP. Events were recorded on gated propidium iodide negative population. These flow cytometry profiles are representative of 3–4 different experiments. (B) Eosinophils were stimulated and incubated with DiOC6(3) and propidium iodide, as described above and DiOC6(3) high stained cells were quantified. Results are the means ± SEM of 3–4 experiments.* P < 0.05, as compared with IgM-treated cells; {dagger} P < 0.05, as compared with anti-Fas-treated cells.

 
Adding IL-5 or IFN-{gamma} to the culture medium significantly reversed Fas-induced loss in {Delta}{Psi}m at 3 h (Fig. 4A and 4B) . Whereas IL-5 still attenuated by 50% anti-Fas induced mitochondrial depolarization at 12 h, IFN-{gamma} had only a minor effect (approximately 12%; Fig. 4A and 4B ). Neither IL-5 nor IFN-{gamma} modified DiOC6(3) uptake in IgM-treated eosinophils at 3 h, although they slightly increased that observed at 12 h (Fig. 4A and 4B) .

Effect of IL-5 and IFN-{gamma} on Fas-induced caspase-8 and caspase-3 activation
Next, we investigated the expression and activation of caspase-8 and caspase-3 following Fas engagement in human eosinophils by Western blot analysis. Freshly purified eosinophils displayed detectable amounts of the zymogen forms of caspase-8 and caspase-3 of 53–55 (p53–55) and 32 (p32) kDa, respectively, but not the caspase-3-processed form of 17 kDa (p17) (Fig. 5A and B ). Eosinophil incubation with control IgM failed to modify the levels of p53–55 and p32 at 12 h but allowed us to detect the p17 active subunit (Fig. 5A and 5B) . Cross-linking with the anti-Fas mAb was followed by a marked reduction in the expression of both caspase-8 and caspase-3 inactive precursors and an increase in the amounts of p17 at 12 h (Fig. 5A and 5B) . The caspase-8-processed form of 18 kDa was undetectable under our experimental conditions.



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  		Figure 5. Effect of IL-5 and IFN-{gamma} on Fas ligation-induced caspase-8 and caspase-3 proteolysis in human eosinophils. Eosinophils were cultured for 0–12 h with 3000 ng/mL of isotype-matched mouse IgM or of anti-Fas mAb, in the absence or presence of 5 ng/mL IL-5 or of 1000 U/mL IFN-{gamma}. Panels A and B show representative Western blot analysis for caspase-8 and caspase-3 expression, respectively, from 3–4 different cell preparations. Blots were probed with mouse anti-caspase-8, rabbit anti-caspase-3, and anti-ß-actin Abs. The intensities of the expression of the caspase-8 zymogen form of 53–55 kDa and the caspase-3 zymogen (32 kDa) and processed (17 kDa) forms were determined by densitometry and expressed as the ratio OD p53–55, or p32 or p17 corresponding band/OD ß-actin band values. Numbers under blots are the means of OD ratios of 3–4 different experiments.

 
Both IL-5 and IFN-{gamma} attenuated Fas-induced caspase-8 and caspase-3 proteolysis (Fig. 5A and 5B) , a finding that was confirmed by densitometric analysis (Fig. 5A and 5B) . Caspase-8 and caspase-3 activation during anti-Fas-mediated eosinophil apoptosis was further supported by the ability of protein extracts to cleave the specific caspase-8 and caspase-3 substrates, Z-IETD-AFC and Z-DEVD-AMC, respectively (Fig. 6 ). These proteolytic activities augmented markedly 12 h after anti-Fas stimulation and declined at 24 h (Fig. 6A and 6B) . Lysates from IgM-treated eosinophils also displayed moderate IETD-ase and DEVD-ase activities, possibly as a reflection of spontaneous apoptosis (Fig. 5A and 5B) . IL-5 and IFN-{gamma} inhibited both spontaneous and Fas-induced increases in caspase-8- and caspase-3-like activities in eosinophil lysates at 12 h (Fig. 6) .



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  		Figure 6. Kinetics of Fas-induced caspase-8 and caspase-3 activities in eosinophils (A and B) and their modulation by IL-5 and IFN-{gamma} (C and D). Eosinophils were stimulated for 0, 3, 12, or 24 h with 3000 ng/mL of mouse IgM, or of agonistic anti-Fas mAb in the absence or presence of 5 ng/mL IL-5, or 1000 U/mL IFN-{gamma}. Whole-cell lysates were tested for their ability to cleave the caspase-8 and caspase-3 substrates, Ac-IETD-AFC (A and C) and Ac-DEVD-AMC (B and D), respectively. Results are expressed as picomoles of free AFC and AMC and represent the means ± SEM of 3–10 experiments. * P < 0.05, between IgM-treated cells and values obtained at time 0; {dagger} P < 0.05, as compared with IgM-treated cells; {ddagger} P < 0.05, as compared with anti-Fas-stimulated cells.

 
Preincubation of protein extracts with the caspase-8 and caspase-3 inhibitors, Z-Ac-IETD-CHO and Ac-DEVD-CHO, respectively, abrogated Z-IETD-AFC and Ac-DEVD-AFC cleavage, indicating that the protease activities present in eosinophil lysates were attributable to caspase-8 and caspase-3 (data not shown).

Role of mitochondria and caspases during Fas ligation-induced human eosinophil apoptosis
To determine the role of mitochondrial disruption in Fas ligation-induced eosinophil apoptosis, cells were pretreated with bongkrekic acid, an inhibitor of the MPTP component, adenine nucleotide translocator (ANT) [30 ]. At a concentration of 62 µM, bongkrekic acid did not modify the loss in {Delta}{Psi}m in either IgM- or anti-Fas-treated eosinophils at 3 and 12 h (Fig. 7A ). Under these conditions, bongkrekic acid failed to reduce eosinophil apoptotic death after 24 h of IgM or anti-Fas stimulation (Fig. 7B) , although a moderate but not significant inhibitory effect was observed at 12 h (Fig. 7B) . Bongkrekic acid was also ineffective against Fas-induced loss in eosinophil viability at 12 and 24 h (data not shown) and caspase-8 and caspase-3 activation at 12 h (Fig. 8A and B ).



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  		Figure 7. Effect of bongkrekic acid on Fas ligation-induced loss in {Delta}{Psi}m and apoptosis in human eosinophils. Eosinophils were incubated for 2 h with 62 µM bongkrekic acid (BA) or with its vehicle (0.02 N NH4OH) prior to stimulation with 3000 ng/mL of mouse IgM or of anti-Fas mAb for 0–24 h. In panel A, {Delta}{Psi}m was determined by flow cytometry, as described in the legend of Fig. 3 and the population of DiOC6(3)-high stained cells was quantified. In panel B, apoptotic nuclei were determined by flow cytometry after cell permeabilization and propidium iodide DNA staining. Results are the means ± SEM of 3 experiments. * P < 0.05, as compared with IgM-treated cells.

 


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  		Figure 8. Effect of bongkrekic acid on Fas ligation-induced caspase-8 and caspase-3 activation. Eosinophils were incubated for 2 h with 62 µM bongkrekic acid (BA) or with its vehicle (0.02 N NH4OH) prior to stimulation with 3000 ng/mL of mouse IgM, or of anti-Fas mAb for 12 h. Whole-cell lysates were tested for their ability to cleave the caspase-8 (A) or caspase-3 (B) substrates. Results are expressed as picomoles of free AFC and AMC and represent the means ± SEM of 3 experiments. * p < 0.05, as compared with IgM-stimulated BA-treated cells.

 
Finally, we determined the role of caspases during Fas-induced {Delta}{Psi}m alteration and apoptosis by preincubating the eosinophils with the cell-permeant and irreversible inhibitors of caspase-8 and caspase-3, Z-IETD-fmk, and Z-DEVD-fmk, respectively, or with the broad spectrum caspase inhibitor, Z-VAD-fmk. At a concentration of 30 µM, Z-VAD-fmk, Z-IETD-fmk, and, to a lesser extent, Z-DEVD-fmk significantly inhibited anti-Fas mAb-induced mitochondrial disruption at 3 h but not at 12 h (Fig. 9A ). This was accompanied by a marked reduction in Fas-mediated apoptosis at 12 h, and, to a lesser extent, at 24 h (Fig. 9B) .



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  		Figure 9. Effect of caspase inhibitors on Fas ligation-induced loss in {Delta}{Psi}m and apoptosis in human eosinophils. Eosinophils were incubated for 2 h with 30 µM of Z-VAD-fmk (VAD), Z-IETD-fmk (IETD) or Z-DEVD-fmk (DEVD) or their vehicle, 0.4% DMSO, and then stimulated for 0–24 h with 3000 ng/mL of mouse IgM or anti-Fas mAb. Flow cytometric analysis was performed to evaluate (A) mitochondrial disruption using DiOC6(3)/propidium iodide staining and (B) apoptosis, after permeabilization of the cells and propidium iodide DNA staining. Results are the means ± SEM of 3–11 experiments. * P < 0.05, as compared with IgM-treated cells; {dagger} P < 0.05, as compared with anti-Fas-treated cells.

 
Under these conditions, Z-VAD-fmk, Z-IETD-fmk, and Z-DEVD-fmk failed to alter significantly spontaneous eosinophil apoptosis (28.5%, 12.0%, and 8.1% inhibition, respectively, at 12 h, and 26.4%, 8.2%, and 6.3% inhibition, respectively, at 24 h).

Similar results were obtained when the effect of caspase inhibitors was evaluated against Fas-induced loss in eosinophil viability at 12 and 24 h (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The concept of different pathways downstream of Fas antigen stimulation was recently proposed [20 ]. Accordingly, Fas ligation-dependent apoptosis may be propagated by a caspase cascade initiated by the activation of caspase-8, followed by a rapid cleavage of other caspases, including caspase-3, which, in turn, degrade many proteins involved in DNA fragmentation and cell dismantling [20 , 21 ]. In contrast, in certain cell types, the caspase cascade cannot be propagated directly but needs to be amplified via the mitochondria [20 , 21 ]. During the apoptotic process, indeed, disruption of the mitochondrial inner transmembrane potential and/or opening of the MPTP lead to the release in the cytosol of pro-apoptotic factors, such as cytochrome c and apoptosis-inducing factor [22 , 30 ].

To identify the intracellular signals involved in Fas-mediated apoptosis in human eosinophils, we investigated the effect of Fas ligation on mitochondrial transmembrane potential and caspase activation.

In this study, we show that activation of the Fas receptor with an agonistic antibody induced a rapid decrease in eosinophil mitochondrial potential, which occurred before the loss in cell viability and the induction of DNA fragmentation. These latter phenomena were associated with a cleavage of the zymogen forms of caspase-8 and caspase-3 and their functional activation, as ascertained by the ability of eosinophil lysates to digest specific substrates. The fact that caspase-like activities augmented at 12 h and decreased thereafter suggests that apoptotic death induced by prolonged Fas stimulation may result in a nonspecific degradation of cellular constituents.

To define the role of mitochondria in Fas-induced eosinophil apoptosis, we investigated how MPTP contributed to this process. This channel is formed by a complex containing ANT [22 , 30 ], a protein involved in the induction of apoptotic death [31 ]. Inhibition of ANT by bongkrekic acid blocks MPTP opening [22 , 30 ]. We show here that bongkrekic acid, at a concentration sufficient to inhibit anti-Fas-induced mitochondrial disruption and apoptosis in human neutrophils [24 ], fails to stabilize mitochondrial transmembrane potential and to modify the extent of apoptosis in Fas-stimulated eosinophils. These observations suggest that the Fas ligation-induced loss in {Delta}{Psi}m is unrelated to the opening of the MPTP, similar to findings previously reported by Marchetti et al. in murine thymocytes [32 ]. Very recently, bongkrekic acid has been shown to decrease {Delta}{Psi}m in eosinophils [33 ], a conflicting finding that may result from the higher and probably toxic concentration (100 µM) used in this study.

Our kinetics studies disclosed that caspase-8 and caspase-3 activation preceded and/or coincided with Fas-induced DNA fragmentation, which suggests that Fas activation promoted apoptosis most likely by activating these caspases. To prove this causality, we tested the ability of the potent irreversible and cell-permeant inhibitors of caspase-8 and caspase-3, Z-IETD-fmk and Z-DEVD-fmk, respectively, to interfere with Fas-induced effects. Although both compounds prevented eosinophil apoptosis induced by Fas ligation at early time points, the broad spectrum caspase inhibitor, Z-VAD-fmk, extended its protective effect over the whole time course of Fas stimulation. This result indicates that caspases other than caspase-8 and caspase-3 are presumably activated upon Fas cross-linking in human eosinophils.

We then attempted to determine the hierarchical implication and the functional connections between MPTP opening and caspase activation. Adding bongkrekic acid during anti-Fas stimulation failed to modulate caspase activation, which suggests that opening of the MPTP is not a prerequisite for generating the caspase cascade in eosinophils. In contrast, caspase-8, and, to a lesser extent, caspase-3 inhibitors preserved mitochondria from anti-Fas-induced loss in transmembrane potential at early stages of eosinophil stimulation, while they became ineffective at later time points. This observation suggests that activation of caspases, in particular caspase-8, may probably account for early, but not late, mitochondrial depolarization.

Next, we investigated the potential modulatory properties of IL-5 and IFN-{gamma} on Fas-induced apoptosis and the associated mitochondrial disruption and caspase activation. Exposure of Fas-stimulated eosinophils to IL-5 inhibited the loss in viability and the increase in apoptosis at 12 h, and, to a lesser extent, at 24 h. These findings are in agreement with those previously reported, which showed that IL-5 failed to modify the degree of apoptosis of murine lung and human peripheral blood eosinophils after 48–72 h of Fas stimulation [14 , 18 , 34 ]. These observations, together with those presented in this study, suggest that eosinophil exposure to Fas cross-linking for more than 24 h may overcome the protective effect of IL-5.

Similar to IL-5, IFN-{gamma} inhibited Fas-induced loss in eosinophil viability and increase in apoptosis. This result extends previous findings showing that IFN-{gamma} enhanced human eosinophil survival [6 , 7 , 35 ] and decreased spontaneous apoptotic death [7 , 35 ], and they further identify IFN-{gamma} as an eosinophil-protecting cytokine.

The decreased sensitivity of IL-5- or IFN-{gamma}- treated eosinophils in response to anti-Fas triggering in the presence of IL-5 or IFN-{gamma} was unrelated to changes in Fas receptor expression. Our results are thus consistent with the failure of IL-5 to modify the expression of this antigen on eosinophil surface [14 , 16 ], but they differ from those previously reported showing a moderate increasing effect of IFN-{gamma} on Fas expression after 24 h of stimulation [16 ].

IL-5-mediated attenuation of Fas-induced apoptosis was associated with the preservation of mitochondrial potential at 3 h, and, to a lesser extent, at 12 h. Similar results were obtained in human neutrophils, where GM-CSF was shown to inhibit Fas-induced apoptosis by preventing the loss in {Delta}{Psi}m [24 ]. Unlike IL-5, the inhibition by IFN-{gamma} of Fas-mediated eosinophil apoptosis paralleled a limited effectiveness in stabilizing mitochondria, particularly after 12 h of stimulation. Together, these findings indicate that attenuation of eosinophil apoptotic death by surviving stimuli, such as IL-5 and IFN-{gamma}, is not necessarily associated with a similar degree of protection against mitochondrial depolarization.

Our kinetics studies disclosed the ability of IL-5 and IFN-{gamma} to inhibit Fas ligation-induced caspase-8 and caspase-3 activation at 12 h. Zangrilli et al. [34 ] recently reported a limited inhibition by IL-5 of Fas-induced caspase-8 and caspase-3 cleavage after 24 h of eosinophil stimulation. As for apoptosis, this discrepancy may result from a reduced effectiveness of IL-5 after long-term Fas stimulation. Inhibition of Fas-induced caspase-8 and caspase-3 activation in neutrophils has previously been associated with the apoptosis-rescuing effect of GM-CSF [24 ], which supports a role for caspases as potential targets for stimuli that influence granulocyte apoptosis.

In conclusion, our results demonstrate the requirement of caspase activation, but not of MPTP opening, in the propagation of apoptotic signals downstream of Fas activation in human peripheral blood eosinophils. Nevertheless, mechanisms involving mitochondrial permeabilization, but independent from the MPTP opening, may operate in amplifying caspase activation and in inducing DNA fragmentation. These may involve Bcl-2 family proteins, which have been shown to tightly regulate mitochondrial homeostasis [22 , 30 ]. In particular, cleavage of the Bcl-2 family member, Bid, and subsequent mitochondrial depolarization and/or release of apoptogenic factors induced by activated caspase-8, establishes a link between caspases and mitochondrial alterations during Fas-induced apoptosis [21 , 30 ]. Whether Bid is expressed by human eosinophils and is a target for stimuli that modulate mitochondrial permeabilization, such as IL-5 and IFN-{gamma}, is an interesting area for future research.

By identifying caspases as the predominant intracellular effector molecules involved in Fas-mediated apoptosis, we have helped elucidate the molecular mechanisms governing the cell death program in human eosinophils. Our work may have important implications for therapeutic considerations, particularly in the search for new ways to treat more selectively chronic inflammatory diseases in which the presence of excessive numbers of eosinophils in blood and tissues plays a critical role.


   ACKNOWLEDGEMENTS
 
This work was supported by the "Fonds de Recherche Hoechst Marion Roussel", by the "Société de Pneumologie de Langue Française", by the "Legs Poix" of the University Chancellery, and by the "Caisse d’Assurance Maladie des Professions Indépendantes", Paris, France. Séverine Létuvé is a recipient of a fellowship from the "Fondation pour la Recherche Médicale", Paris, France. The authors are grateful to Dr. Sefik Alkan (Aventis Pharmaceuticals, Bridgewater, USA) for his support, and to Dr. Marcus E. Peter (Tumor Immunology Program, German Cancer Research Center, Heidelberg, Germany) for the kind gift of the anti-caspase-8 mAb.

Received April 5, 2001; revised July 3, 2001; accepted July 9, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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