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Originally published online as doi:10.1189/jlb.0803404 on April 1, 2004

Published online before print April 1, 2004
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(Journal of Leukocyte Biology. 2004;75:1045-1055.)
© 2004 by Society for Leukocyte Biology

Membrane receptor-mediated apoptosis and caspase activation in the differentiated EoL-1 eosinophilic cell line

Mohammed W. Al-Rabia, Morgan G. Blaylock, Darren W. Sexton and Garry M. Walsh1

Department of Medicine & Therapeutics, Institute of Medical Sciences, University of Aberdeen, Foresterhill, United Kingdom

1 Correspondence: Department of Medicine & Therapeutics, IMS Building, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK. E-mail: g.m.walsh{at}abdn.ac.uk


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ABSTRACT
 
Caspases are key molecules in the control of apoptosis, but relatively little is known about their contribution to eosinophil apoptosis. We examined caspase-3, -8, and -9 activities in receptor ligation-dependent apoptosis induction in the differentiated human eosinophilic cell line EoL-1. Differentiated EoL-1 exhibited bi-lobed nuclei, eosinophil-associated membrane receptors, and basic granule proteins. Annexin-V fluorescein isothiocyanate binding to EoL-1 revealed significant (P<0.01) apoptosis induction in cells cultured for 20 h with monoclonal antibodies (mAb) specific for CD45 (71%±4.3), CD45RA (58%±2.3), CD45RB (68%±2.4), CD95 (47%±2.6), and CD69 (52%±2.1) compared with control (23%±1.6) or CD45RO mAb (27%±3.9). The pan-caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (fmk) and inhibitors of caspase-8 (Z-Ile-Glu-Thr-Asp-fmk) and caspase-9 (Z-Leu-Glu-His-Asp-fmk) significantly inhibited mAb-induced apoptosis of EoL-1 but had no effect on constitutive (baseline) apoptosis at 16 and 20 h. Caspase activity was analyzed using the novel CaspaTagTM technique and flow cytometry. EoL-1 treated with pan-CD45, CD45RA, CD45RB, and CD95 mAb exhibited caspase-3 and -9 activation at 12 h post-treatment, which increased at 16 and 20 h. Activated caspase-8 was detected 12 and 16 h after ligation with CD45, CD45RA, CD45RB, and CD95 mAb followed by a trend toward basal levels at 20 h. CD69 ligation resulted in caspase-3 activation, a modest but significant activation of caspase-8, and a loss in mitochondrial transmembrane potential but had no significant effect on activation of caspase-9. Thus, the intrinsic and extrinsic caspase pathways are involved in controlling receptor ligation-mediated apoptosis induction in human eosinophils, findings that may aid the development of a more targeted, anti-inflammatory therapy for asthma.

Key Words: asthma • cell-surface molecules • eosinophils • programmed cell death


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INTRODUCTION
 
Asthma is a complex disorder characterized by reversible airway obstruction, bronchial hyper-responsiveness, and airway inflammation. Much of the inflammation in asthma is thought to be a consequence of the inappropriate accumulation of eosinophils and the subsequent release of their potent, proinflammatory arsenal that includes such diverse elements as granule-derived basic proteins, mediators, cytokines, and chemokines [1 , 2 ]. Apoptosis or programmed cell death is a critical factor for a functional immune system [3 ] and is a tightly controlled, central, and essential process in the resolution of inflammation. Eosinophil removal from the tissues is thought to be dependent on their death by apoptosis, followed by their recognition and phagocytosis by macrophages [4 ] or resident bronchial epithelial cells [5 , 6 ]. Thus, it can be envisaged that apoptosis induction in eosinophils followed by their recognition and removal by phagocytes is a rational avenue for the development of novel therapy for asthma [7 ], a view supported by clinical studies [8 , 9 ]. There is therefore much interest in furthering our understanding of the intracellular pathways that control the induction and execution of apoptosis in human eosinophils.

Decisive events during the apoptotic process involve mitochondrial permeabilization and caspase activation. Caspases, aspartate-specific cysteine proteases, regulate the execution phase of apoptosis, being responsible for most of the biochemical and morphological changes associated with the apoptotic phenotype [10 ]. The initiation of distinct caspase cascades is dependent on the death stimulus [11 , 12 ]. Two main pathways have been shown to trigger caspase activation in cells undergoing apoptosis [13 ]. Schematically, the intrinsic pathway involves the disruption of the outer mitochondrial membrane barrier function, thus permitting the release of proapoptotic molecules from the mitochondria to the cytosol. One of these molecules is cytochrome c, which once in the cytosol, oligomerizes the adaptor molecule Apaf-1 to recruit and activate the initiator caspase, caspase-9. In turn, caspase-9 cleaves and activates downstream effector enzymes such as caspase-3. The extrinsic pathway to cell death involves a plasma membrane death receptor. In response to their engagement, these receptors trimerize and recruit the adaptor molecule Fas-associated death domain protein, which in turn, interacts with and activates an initiator enzyme, usually caspase-8. This enzyme, directly or through the previously described mitochondrial pathway, activates downstream effector enzymes including caspase-3 [14 ]. In both pathways, effector caspases trigger the limited proteolytic cleavage of intracellular structural and regulatory proteins, thus leading to membrane blebbing, chromatin condensation, and nuclear DNA fragmentation, which characterize apoptosis.

In eosinophils, apoptosis can be induced by treatment with corticosteroids [15 ] or ligation of membrane receptors by specific monoclonal antibodies (mAb) for Fas (CD95) [16 , 17 ], CD69 [18 ], and CD45 [19 ]. Previous work with eosinophils derived from healthy and asymptomatic, allergic individuals demonstrated involvement of caspase-3 and -8 in Fas-induced [20 ] or glucocorticoid-induced apoptosis [21 ]. In contrast, others have reported that dexamethasone-induced apoptosis failed to induce specific caspase-3 and -8 activity in eosinophils compared with spontaneous apoptosis [22 ]. Thus, much remains to be understood about the role that caspases play in controlling apoptosis in human eosinophils. In the present study, we have used the differentiated human eosinophilic leukaemiac cell line EoL-1 to investigate differential activation of caspases following mAb-dependent membrane receptor ligation. EoL-1 can be induced to differentiate into eosinophilic granule-containing cells by a number of stimuli and have been used as models of eosinophil function including chemotaxis, receptor expression, mediator release, and apoptosis induction. [23 24 25 26 27 ]. In our hands, differentiated EoL-1 were characterized by a bi-lobed nucleus, granules containing eosinophil cationic protein (ECP) and major basic protein (MBP), together with the expression of the membrane receptors very late antigen-4 (VLA-4), CD11b, CD18, pan-CD45, CD45 isoforms, CD95, and CD69. They were therefore used to investigate patterns of caspase-3, -9, and -8 activation following mAb-dependent ligation of CD45 and its isoforms CD69 or CD95.


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MATERIALS AND METHODS
 
Antibodies
CD45 mAb (Bra-55) and CD45RB mAb (Bra11) were supplied by Stratech Scientific (Cambridgshire, UK). CD45RA mAb (MB1), CD45RB mAb (MT4), and CD45RO mAb (UCHL-1) were obtained from Immuno-Quality Products (Groningen, The Netherlands). Mouse immunoglobulin G1 (IgG1) mAb isotype-matched control (DAK-GO1), mouse IgG2a mAb isotype-matched control (DAK-GO5), rabbit anti-mouse Ig, and alkaline phosphatase antialkaline phosphatase (APAAP) mouse mAb were all from Dako (Cambridgeshire, UK). CD69 was obtained from Autogen Bioclear (Wiltshire, UK). FAS (B-10) was purchased from Santa Cruz Biotechnology (Calne, UK). Anti-human ECP mAb (clone EG2) was supplied by Pharmacia AB (Uppsala, Sweden). The caspase peptide inhibitor Z-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk) was from Promega (Madison, WI). Mouse monoclonal anti-human MBP (clone BMK-13) was purchased from Serotec (Oxon, UK). 5,5',6,6'-Tetrachloro-1,1', 3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1) was from Molecular Probes (Eugene, OR). Caspase Inhibitor Set IV was obtained from Calbiochem (San Diego, CA).

Cell culture and apoptosis induction
EoL-1 cells were maintained in RPMI-1640 medium (supplemented with 10% fetal calf serum, 100 IU/ml penicillin, 100 mg/ml streptomycin, and L-glutamine) at 37°C with 5% carbon dioxide in a humidified atmosphere. EoL-1 cells were differentiated by culturing with 0.1 mM dibutyryl cyclic adenosine monophosphate (dbcAMP) for 9 days or with 0.5 mM butyric acid (BA) for 7 days. Cell number readjustment to 5 x 105/ml was performed every 3 days.

Immunocytochemical labeling
Immunostaining for ECP or MBP was performed as described previously [28 ]. Briefly, cytospins were prepared from differentiated and undifferentiated EoL-1, fixed for 10 min in 99% methanol, and stored at –20°C until required. Slides of cultured cells were washed and incubated with primary mouse mAb against MBP (BMK13), ECP (EG2), or an isotype-matched (IgG1) mouse control antibody. Immunoreactivity to MBP, ECP, and IgG1 was detected using rabbit anti-mouse IgG and APAAP. Positive cells stained red after development with Fast Red (Sigma, Dorset, UK). The number of positively stained cells was calculated by counting at least 200 cells per slide, and the proportion was expressed as a percentage.

Expression of surface antigens on EoL-1 cells
Immunostaining and flow cytometry were performed as described previously [19 ]. Briefly, differentiated and undifferentiated EoL-1 cells (2x105) were immunostained with the relevant antibodies or controls at saturating concentrations for 30 min on ice. After a wash and staining with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG mAb, the cells were analyzed on a flow cytometer (FACScan, Becton Dickinson, Oxon, UK). Flow cytometry was performed on eosinophils gated on the basis of their forward- and side-scatter profile with any cell debris excluded from analysis. Nonspecific fluorescence was controlled for by incubation with isotype-specific control antibodies.

Determination of EoL-1 apoptosis by flow cytometry
Eol-1 differentiated with dbcAMP (2x105 cells/well) were incubated for 20 h in 96-well, flexible, flat-bottomed plates (Becton Dickinson), alone or with experimental antibodies and isotype-matched controls. All control and experimental mAb were used at a final concentration of 10 µg/ml for 20 h in the presence or absence of interleukin (IL)-5 (R&D Systems, Oxon, UK) at a final concentration of 10–11 M. For some experiments, Eol-1 were treated with the pan-caspase inhibitor z-VAD-fmk at a final concentration of 100 µM, caspase-8 [Z-Ile-Glu-Thr-Asp-fmk (z-IETD-fmk)] or -9 [Z-Leu-Glu-His-Asp-fmk (z-LEHD-fmk)] irreversible inhibitors at a final concentration of 20 µM, or the negative control [Z-Phe-Ala-fmk (z-FA-fmk)] added at the same time as the experimental or control mAb. Preliminary experiments established 100 µM z-VAD-fmk and 20 µM caspase-8 or -9 inhibitors, respectively, to be the optimal concentrations (data not shown).

Analysis of differentiated EoL-1 apoptosis and viability was performed with a combination of annexin V–FITC and phosphatidylinositol (PI). We have demonstrated that assessment of human eosinophil apoptosis with this technique is accurate and sensitive [29 ]. Briefly, differentiated EoL-1 cultured in the presence of experimental mAb with or without IL-5 were washed in cold phosphate-buffered saline (PBS) and resuspended in binding buffer (HEPES-buffered PBS supplemented with 2.5 mmol/L calcium chloride) before the addition of FITC-conjugated annexin V (Bender MedSystems, Loughborough, UK) for 10 min at room temperature. Cells were washed and resuspended in binding buffer, and PI was added to a final concentration of 1 mg/ml; cells were immediately analyzed on a flow cytometer as described above.

Assay of caspase activity
Caspase activities were determined using the CaspaTagTM caspase-3 [Asp-Glu-Val-Asp (DEVD)], caspase-9 (LEHD), and caspase-8 (IETD) activity kits (Intergen, Purchase, NY). These are fluorescein-labeled caspase inhibitors that are cell-permeable and nontoxic and covalently bind only active caspases. Analysis of caspase activity was performed using flow cytometry. Briefly, differentiated EoL-1 cells were incubated at 37°C with 5% CO2 in the presence of experimental mAb (20 µg/ml); following this, the cells were washed and resuspended in warmed, complete RPMI, supplemented with fluorochrome-peptide-fmk for 1 h at 37°C under 5% CO2. Cells were washed twice and resuspended in fixing buffer. The cells were analyzed immediately by flow cytometry.

Effect of mAb-dependent CD69 ligation on mitochondrial membrane potential ({Delta}{Psi}m)
The {Delta}{Psi}m in differentiated EoL-1 cells was measured using the cationic dye JC-1, which exhibits potential, dependent accumulation in mitochondria, indicted by a fluorescence emission shift from green (~525 nm) to red (~590 nm). Consequently, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence-intensity ratio. Briefly, differentiated EoL-1 cells were incubated at 37°C with 5% CO2 in the presence of CD69 mAb (10 µg/ml) for 16 h; following this, the cells were washed and resuspended in warmed, complete RPMI supplemented with 10 µg/ml JC-1 for 20 min at room temperature in the dark. Cells were washed twice, resuspended in PBS, and analyzed immediately by flow cytometry.

Statistical analysis
All data are presented as mean (±SEM), and where n is given, this represents the number of experiments. Statistical analysis was performed with the unpaired two-tailed Student’s t-test, and a P value of <0.05 was considered significant.


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RESULTS
 
Characterization of Eol-1 differentiated with BA or dbcAMP
We first determined which differentiation agents would result in EoL-1 cells with the closest eosinophil-specific phenotype. Cells were cultured for 7 days in the presence of BA or dbcAMP for 9 days. Stimulation with BA or dbcAMP increased membrane receptor expression of VLA-4, CD11b, and CD18 pan-CD45 and its isoforms but decreased the expression of CD69 and CD95 (Table 1 ). All EoL-1 differentiated in BA or dbcAMP had bi-lobed nuclei, and the majority of dbcAMP-treated Eol-1 had positive immunostaining for MBP and ECP (Table 2 ). Eosinophil phenotype, morphology, and granule protein content was greatest in EoL-1 differentiated with dbcAMP. As these cells had the greatest resemblance to peripheral blood eosinophils, particularly with respect to expression of the membrane receptors examined in this study—CD45, CD45 isoforms, CD69, and CD95 [30 ]—they were therefore used in all subsequent experiments.


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  		Table 1. Receptor Expression* by EOL-1 Cells


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  		Table 2. The Proportion of EoL-1 Cells with Positive Immunostaining for MBP and ECP

Ligation of CD45 and the CD45RA and CD45RB isoforms, CD95, and CD69 accelerates EoL-1-constitutive apoptosis
A representative experiment showing simultaneous measurement of apoptosis and secondary necrosis 20 h after ligation with mAb against CD45 and the isoforms RA, RB, and RO, CD95 (Fas), and CD69, together with appropriate controls, is shown in Figure 1 . The dot plots have a typical sigmoid shape: As differentiated EoL-1 cells ligated with CD45, CD45RA, CD45RB, CD95 (Fas), or CD69 become apoptotic, they bind annexin V–FITC. Although apoptotic, over time, these cells exhibited secondary necrosis, as their membrane integrity was lost, and they failed to exclude PI. The cells are in the late apoptotic stage, that is, they are annexin V- and PI-positive. Washed, differentiated EoL-1 (i.e., t=0) excluded PI and did not bind annexin V–FITC (data not shown). Differentiated EoL-1 cultured alone for 20 h exhibited constitutive apoptosis (19%±3.3), which was comparable with that of undifferentiated cells (data not shown). Significant (P<0.01) enhancement of EoL-1 apoptosis was observed following mAb treatment with CD45 (71%±4.3), CD45RA (58%±2.3), CD45RB (68%±2.4), CD95 (47%±2.6), and CD69 (52%±2.1). CD45RO or control mAb did not significantly accelerate EoL-1 apoptosis induction.



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  		Figure 1. Flow cytometric analysis of apoptosis in EoL-1 cells. Representative experiment showing flow cytometric analysis of differentiated EoL-1 cells binding of annexin V–FITC (FL1-H) and uptake of PI (FL3-H) after incubation for 20 h with mAb against pan-CD45 (Bra55), CD45RB (Bra11), CD45RA, CD45RB (MT4), CD45RO, FAS (CD95), CD69, or an isotype control. Numbers in the upper-right quadrant refer to annexin+/PI– (early apoptosis) and annexin+/PI+ (late apoptosis) EoL-1.

Ligation of CD45, CD95, and CD69: dose response and time-course
A series of experiments was subsequently performed to assess the effect on differentiated EoL-1 apoptosis of the mAb concentration and duration of CD45, CD95 (Fas), and CD69 ligation. Figure 2A shows the effect of saturating and suboptimal concentrations of representative CD45, CD45RA, CD45RB, CD95 (Fas), and CD69 mAb compared with the effect of isotype mAb. We demonstrated a statistically significant, dose-dependent increase in apoptosis with increasing mAb concentration, which was maximal at the saturating concentration (20 µg/mL) for each mAb. Differentiated EoL-1 cells were also cultured for 0, 5, 10, and 20 h in the presence of CD45, CD95, or CD69 mAb before assessment of apoptosis induction by annexin V–FITC staining. Time-course experiments are shown in Figure 2B . At 20 h post-receptor ligation, a clear difference was seen between differentiated EoL-1 incubated with CD45, CD45RA, CD45RB, CD95, or CD69 compared with cells ligated with control mAb or untreated cells.



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  		Figure 2. Dose response and kinetics of EoL-1 apoptosis. (A) Dose response of effect of increasing concentration of representative pan-CD45 (Bra55), CD45RB (Bra11), CD45RA, CD45RB (MT4), FAS (CD95), and CD69 mAb compared with isotype control after 20 h post-ligation on the constitutive rate of differentiated EoL-1 cell apoptosis as defined by annexin V–FITC binding. Each point represents mean ± SEM of four experiments. (B) Time-course of effect of mAb-dependent ligation of pan-CD45 (Bra55), CD45RB (Bra11), CD45RA, CD45RB (MT4), FAS (CD95), and CD69 on the constitutive rate of differentiated EoL-1 cell apoptosis compared with isotype control and untreated cells. Each bar represents the mean ± SEM of three experiments (*, P<.05; **, P<.005, compared with isotype control in each case).

The cytokine IL-5 has been shown to prolong eosinophil survival in vitro by delaying apoptosis. We observed the addition of IL-5 at a concentration (10–11 M) had no significant effect on CD45, CD45RA, CD45RB, and CD95 receptor-mediated apoptosis induction. In contrast, significant inhibition of differentiated EoL-1 apoptosis by addition of IL-5 was demonstrated in the presence of isotype control and CD45RO (data not shown).

Caspase activity in mAb-induced apoptosis of EoL-1 cells
We first demonstrated a role for caspase-dependent signaling following mAb ligation-induced EoL-1 apoptosis. Differentiated EoL-1 cells were treated with optimal concentrations of CD45, CD95, and CD69 mAb in the presence of the broad-spectrum caspase inhibitor z-VAD-fmk (final concentration, 100 µM; determined in preliminary experiments) or irreversible inhibitors of caspase-8 (z-IETD-fmk) and caspase-9 (z-LEHD-fmk; final concentration, 20 µM in each case, as determined in preliminary experiments). z-VAD-fmk, z-IETD-fmk, and z-LEHD-fmk significantly inhibited CD69 or CD45 mAb-induced apoptosis of EoL-1 cells as determined by annexin V binding. In addition, we observed a modest but significant increase in apoptosis inhibition in mAb-ligated EoL-1 when the caspase-8 and caspase-9 inhibitors were added together (Fig. 3A and 3B ). We next investigated the role of caspase-3, -8, and -9 in mAb-induced apoptosis of differentiated EoL-1 cells using CaspaTagTM with analysis by flow cytometry. Figures 4A 5A, and 6A are representative experiments demonstrating differential activation of caspase-3, -8, and -9 in EoL-1 treated for 20 h with optimal concentrations of mAb specific for CD45, CD45RA, CD45RB, CD45RO, CD95, and CD69. In general, the pan CD45, CD45RA, CD45RB, and CD95 mAb ligation resulted in modest caspase-3 and -9 activation after 12 h post-treatment. This trend in caspase-3 and -9 activation continued to increase through to the 16- and 20-h time-points (Figs. 4B and 6B) . It is interesting that the (upstream) activated caspase-8 was detected at a high level following ligation with mAb specific for CD45, CD45RA, CD45RB, and CD95 after 12 and 16 h of incubation, which was followed by a trend toward basal levels at the 20-h time-point (Fig. 5B) . CD69 activated caspase-8 at 16 h post-ligation but had no significant effect on caspase-9 activation at any time-point tested, and CD45RO had no significant effect on activation of any of the caspases tested.



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  		Figure 3. Effect of z-VAD-fmk and caspase-8 and -9 inhibitors on EoL-1 apoptosis. Effect of presence or absence of pan-caspase inhibitor (z-VAD-fmk, 100 µM; A) or (B) the specific caspase-8 (z-IETD-fmk), caspase-9 inhibitors (z-LEHD-fmk) or negative control (z-FA-fmk) on apoptosis induction as defined by annexin V–FITC binding in differentiated EoL-1 cells ligated with CD45, CD95, and CD69 mAb for 16 h. Results are expressed as the mean ± SEM of four experiments. (*, P<.05; **, P<.005, compared with no treatment in each case).



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  		Figure 4. Caspase-3 activation in EoL-1. (A) Representative data from flow cytometric analysis of differentiated EoL-1 cells after incubation for 20 h with mAb against pan-CD45 (Bra55), CD45RB (Bra11), CD45RA, CD45RO, FAS (CD95), CD69, or an isotype control. The frequency histograms of number of events (y-axis) versus fluorescence intensity (x-axis) show two peaks appearing at different fluorescence intensities. Caspase-3-negative (–) cells occur within the first log decade of the x-axis (FL1), whereas caspase-3-positive (+) cells are within the second and third log decade (M1). (B) Time-course of effect of mAb-dependent ligation of pan-CD45 (Bra55), CD45RB (Bra11), CD45RA, CD45RO, FAS (CD95), and CD69 on differentiated EoL-1 caspase-3 activation compared with isotype control and untreated cells. Each bar represents the mean ± SEM of three experiments (*, P<.05; **, P<.005, compared with isotype control in each case).



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  		Figure 5. Caspase-8 activation in EoL-1. (A) Representative experiment showing flow cytometric analysis of differentiated EoL-1 cells after incubation for 20 h with mAb against pan-CD45 (Bra55), CD45RB (Bra11), CD45RA, CD45RO, FAS (CD95), CD69, or an isotype control. The frequency histograms of number of events (y-axis) versus fluorescence intensity (x-axis) show two peaks. Caspase-8-negative (–) cells occur within the first log decade of the x-axis (FL1), whereas caspase-8-positive (+) cells are within the second and third log decade (M1). (B) Time-course of effect of mAb-dependent ligation of pan-CD45 (Bra55), CD45RB (Bra11), CD45RA, CD45RO, FAS (CD95), and CD69 on differentiated EoL-1 caspase-8 activation compared with isotype control and untreated cells. Each bar represents the mean ± SEM of three experiments (*, P<.05; **, P<.005, compared with isotype control in each case).



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  		Figure 6. Caspase-9 activation in EoL-1. (A) Representative flow cytometry experiment showing caspase-9 activity in differentiated EoL-1 cells after incubation for 20 h with mAb against pan-CD45 (Bra55), CD45RB (Bra11), CD45RA, CD45RO, FAS (CD95), CD69, or an isotype control. The frequency histograms of number of events (y-axis) versus fluorescence intensity (x-axis) show two peaks. Caspase-9-negative (–) cells occur within the first log decade of the x-axis (FL1), whereas caspase-9-positive (+) cells are within the second and third log decade (M1). (B) Time-course of effect of mAb-dependent ligation of pan-CD45 (Bra55), CD45RB (Bra11), CD45RA, CD45RO, FAS (CD95), and CD69 on differentiated EoL-1 caspase-9 activation compared with isotype control and untreated cells. Each bar represents the mean ± SEM of three experiments (*, P<.05; **, P<.005, compared with isotype control in each case).

CD69 induces loss of {Delta}{Psi} m
Use of the specific inhibitor z-LEHD-fmk demonstrated caspase-9 involvement in CD69-dependent apoptosis induction in Eol-1. However, we were unable to detect caspase-9 activation in Eol-1 ligated with CD69 Ab using CaspaTagTM. We therefore further assessed caspase-9 involvement by measuring {Delta}{Psi}m in Eol-1 ligated with CD69 mAb. {Delta}{Psi}m is reduced very early in apoptosis as a result of the opening of permeability-transition pores, which may result in mitochondrial swelling and rupture of the outer mitochondrial membrane with release of cytochrome c from the intermembrane space of the mitochondria into the cytoplasm [31 ]. To study these events in CD69-induced apoptosis in differentiated EoL-1 cells, the cells were ligated with 10 µg/ml CD69 for 16 h, and the involvement of mitochondrial permeability transition was assessed by flow cytometry. The fluorescence intensity of JC-1 was decreased in CD69-treated EoL-1 cells compared with untreated cells (Fig. 7 ).



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  		Figure 7. Analysis of {Delta}{Psi}m in EoL-1. Bivariate JC-1 analysis of {Delta}{Psi}m in differentiated EoL-1 cells by flow cytometry. Distinct populations of cells with different extents of mitochondrial depolarization are detectable following apoptosis-inducing treatment with 10 µg/ml CD69 mAb for 16 h. Numbers indicate the percentage change in {Delta}{Psi}m. This is a representative example of four experiments that gave near-identical results.


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DISCUSSION
 
In the present study, we have used the differentiated eosinophilic cell line EoL-1 to examine apoptosis induction and caspase activation following mAb-dependent ligation of the membrane receptors CD45, CD45 isoforms, CD95, and CD69. To date, the pattern of caspase activation following mAb-dependent receptor ligation in peripheral blood eosinophils or the differentiated EoL-1 cell line has not been defined. EoL-1 cells have been used extensively as a functional model of peripheral blood eosinophils, as they exhibit many characteristics of these cells including a bi-lobed nucleus and expression of granule proteins together with cytokine, adhesion, and Fc{gamma} receptors [23 24 25 26 27 ]. Here, we confirm that EoL-1 differentiated with dbcAMP express ECP and MBP together with the receptors VLA-4, CD11b, CD18, CD45, CD45RA, CD45RB, CD45RO, CD95, and CD69. The latter was constitutively expressed, unlike peripheral blood eosinophils, which require activation with cytokines including IL-3 and IL-5 before CD69 is expressed [32 ]. In contrast, CD95, CD45, and CD45 isoform expression by differentiated EoL-1 was comparable with that of resting peripheral blood eosinophils [30 ]. We found that differentiated EoL-1 had comparable levels of constitutive apoptosis with peripheral blood eosinophils and were sensitive to the apoptosis-inducing effects of mAb against CD45 and the isoforms CD45RA and CD45RB, CD69, or CD95. These findings are similar to those reported in our previous study using peripheral blood eosinophils [19 ]. Furthermore, our observations with CD95 mAb confirm a previous report in which CD95 ligation induced apoptosis in undifferentiated EoL-1 cells [27 ], although the pattern of caspase activation following Fas mAb treatment was not examined in this study. CD45RO did not induce significant apoptosis in EoL-1, also confirming our observations with peripheral blood eosinophils [19 ]. To our knowledge, the only clone currently available for CD45RO is UCHL-1, and its failure to induce apoptosis in EoL-1 might be a consequence of its inability to initiate full activity of its ligand after binding. Thus, the possibility cannot be excluded that another CD45RO mAb might have induced apoptosis in EoL-1 if it were available.

A number of studies have demonstrated a role for caspases in corticosteroid or CD95 (Fas) ligation-dependent apoptosis in human peripheral blood eosinophils [20 21 22 ]. Here, we have defined the pattern of differential caspase activation in differentiated EoL-1 cells following mAb-dependent membrane receptor ligation of pan-CD45, CD45RA, CD45RB, CD45RO, CD95, and CD69. Our initial studies with the pan-caspase inhibitor z-VAD-fmk or caspase-8 (z-IETD-fmk) and caspase-9 (z-LEHD-fmk) inhibitors demonstrated a role for these caspases in membrane receptor ligation-induced apoptosis in EoL-1 cells. These findings are in agreement with a previous report showing that Fas-induced apoptosis in blood eosinophils was prevented by the general caspase inhibitor z-VAD-fmk and specific caspase-3 and caspase-8 inhibitors, z-DEVD-fmk and z-IETD-fmk, respectively [20 ]. However, z-VAD-fmk, z-IETD-fmk, and z-LEHD-fmk did not totally abolish EoL-1 apoptosis following mAb-dependent receptor ligation nor did they prevent spontaneous baseline apoptosis in EoL-1 cells incubated with control mAb.

To date, the majority of published studies has determined the levels of caspase activation in eosinophils using antibodies that bind the caspase precursor and the cleaved portion. However, levels of cleaved fragment are relatively small in eosinophils; therefore, some workers have used the reduction in the levels of precursor as an indication of caspase activity. Typically, these experiments were conducted at later time-points (24 h and above), where the levels of induced activation are comparable with spontaneous activation of caspases [21 ]. In the present study, we have used a novel and powerful technique (CaspaTagTM) to analyze caspase activity in human differentiated EoL-1 cells. The technique uses fluorescein-labeled caspase inhibitors that are cell-permeable and nontoxic and covalently bind active caspase-3 (DEVD), caspase-9 (LEHD), and caspase-8 (IETD). The degree of fluorescence and therefore caspase activation is directly and rapidly analyzed by flow cytometry. We also used the differentiated eosinophilic cell line EoL-1, as this has many characteristics of peripheral blood eosinophils together with the advantages associated with access to large numbers of cells. Furthermore, apoptosis induction in human peripheral blood eosinophils has been reported to vary greatly between individuals [33 ], an observation in agreement with our own experience (M. G. Blaylock, D. W. Sexton, and G. M. Walsh, unpublished observation). Moreover, many groups use eosinophils isolated from asthmatic and allergic individuals, and these have been reported to have a degree of resistance to apoptosis induction [34 , 35 ]. These considerations emphasize the advantages of conducting studies of this nature in an eosinophilic cell line. However, we cannot rule out the possibility that there might be a synergistic effect between dbcAMP treatment and mAb-dependent receptor ligation on apoptosis induction and caspase activation in differentiated Eol-1.

In this study, we demonstrate differences in the magnitude and kinetics of specific caspases following receptor ligation of differentiated EoL-1 cells by mAb specific for different receptors. Caspase-3 was significantly activated by all mAb tested, with the exception of CD45RO. Ligation of CD69 gave modest activation of caspase-3 at 16 h with comparable activation observed at 20 h after ligation with the other mAb tested. Activation of caspase-8 was markedly augmented by the addition of CD45, CD45RA, CD45RB, and CD95 at 12 and 16 h time-points but then decreased thereafter. Analysis of casapase-9 in differentiated EoL-1 revealed a similar pattern to that observed for caspase-3, with the exception of CD69 mAb-dependent ligation, which was not associated with activation of this caspase. Moreover, CD69 mAb-dependent ligation of EoL-1 cells was only associated with significant but modest caspase-8 activation at the 16-h time-point. However, we observed inhibition of CD69 mAb-induced apoptosis in Eol-1 by specific inhibitors of caspase-8 and -9. These contradictory results are likely explained by the fact that CaspaTagTM monitors caspase activation over a short time-period (1 h), whereas the caspase inhibitors are present throughout the experimental period. The involvement of mitochondria during Fas ligation, irradiation, or dexamethasone-induced apoptosis has been reported in a number of inflammatory and immune cells, including neutrophils, eosinophils, thymocytes, and splenocytes [10 , 20 , 31 , 36 37 38 ]. We therefore investigated the involvement of mitochondrial alterations during CD69-induced, differentiated EoL-1 apoptosis to provide indirect evidence for the involvement of caspase-9 activation. Ligation with CD69 was followed by mitochondrial permeabilization, as demonstrated by the marked decrease in {Delta}{Psi}m. Although we were unable to demonstrate caspase-9 activity using CaspaTagTM following CD69-induced apoptosis in differentiated EoL-1 cells, our data showing decreased {Delta}{Psi}m and the data from the caspase-9 inhibition studies do suggest its involvement in CD69-induced apoptosis in EoL-1.

In summary, we have demonstrated that the differentiated EoL-1 eosinophilic cell line is highly sensitive to apoptosis induction by mAb-dependent receptor ligation. We identified caspase-3, -8, and -9 as important, intracellular effector molecules involved in pan-CD45-, CD45RA-, CD45RB-, CD69-, and CD95-mediated apoptosis in EoL-1 cells. These observations increase our knowledge of the caspase-dependent mechanisms controlling receptor-mediated apoptosis in human eosinophils and may aid the development of more targeted, anti-inflammatory therapy for asthma.


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ACKNOWLEDGEMENTS
 
The Saudi Arabia Ministry of Higher Education (M. W. A.), a Scottish Hospitals Endowments Research Trust Fellowship (M. G. B.), and Tenovus Scotland (G. M. W.) supported this work.

Received August 28, 2003; revised February 11, 2004; accepted February 19, 2004.


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