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Originally published online as doi:10.1189/jlb.1105636 on May 9, 2006

Published online before print May 9, 2006
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(Journal of Leukocyte Biology. 2006;80:186-195.)
© 2006 by Society for Leukocyte Biology

IL-5-mediated eosinophil survival requires inhibition of GSK-3 and correlates with ß-catenin relocalization

Marcela Rosas*, Pascale F. Dijkers*, Caroline L. Lindemans*, Jan-Willem J. Lammers*, Leo Koenderman* and Paul J. Coffer*,{dagger},1

* Department of Pulmonary Diseases and
{dagger} Molecular Immunology Lab, Department of Immunology, University Medical Center, Utrecht, The Netherlands

1 Correspondence: Department of Pulmonary Diseases and Molecular Immunology Lab, Department of Immunology, University Medical Center, Lundlaan 6, 3584 EA Utrecht, The Netherlands. E-mail: p.j.coffer{at}umcutrecht.nl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin (IL)-5 is a hematopoietic cytokine able to regulate differentiation, survival, and effector functions of eosinophils. It binds specifically to its receptor, which is composed of a cytokine-specific {alpha}-chain and a ß-chain shared with the receptors for IL-3 and the granulocyte macrophage-colony stimulating factor. The molecular mechanisms by which IL-5 modulates eosinophil survival remain unclear. In this study, we demonstrate that IL-5 withdrawal induces eosinophil apoptosis through a mitochondria-dependent pathway, independently of Fas receptor activation. The lipid kinase phosphatidylinositol-3 kinase plays a crucial role in the maintenance of eosinophil survival, as inhibition of its activity results in apoptosis. IL-5 induces phosphorylation and thus, inhibition of the Forkhead transcription factor FOXO3a and glycogen synthase kinase 3 (GSK-3). We analyzed expression of FOXO3a-dependent transcriptional targets: Fas ligand or Bim (a proapoptotic Bcl-2 family member), but neither was detected in apoptotic eosinophils. We further show that GSK-3 is activated after IL-5 withdrawal, and inhibition of its activity rescues eosinophils from apoptosis. ß-catenin, a direct GSK-3 substrate, is present in the nucleus of IL-5-stimulated eosinophils, but it is translocated to the plasma membrane in the absence of cytokine in a GSK-3-dependent manner. This is the first report describing a potential role for GSK-3 and ß-catenin in regulating eosinophil survival and suggests a novel mechanism by which IL-5 inhibits the constitutive apoptotic program in these cells.

Key Words: apoptosis • PI-3K


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Asthma is a chronic inflammatory disease characterized by bronchial hyper-responsiveness, airway inflammation, and reversible obstruction of the airways [1 ]. In humans, cytokine-activated eosinophils are thought to be important players in this process, as they can release inflammatory mediators after cellular activation [2 ]. Although the role of eosinophils in asthma has been a point of controversy, recent studies about eosinophil-ablated mice have shown that eosinophils contribute and are necessary for remodeling airway [3 ] or promote asthma pathophysiology through the production of T helper cell type 2 (Th2) cytokines at the site of inflammation [4 ]. However, there are also eosinophil-independent mechanisms involved in disease pathogenesis, which remain to be characterized [3 , 5 ]. Interleukin-5 (IL-5) is a Th2-derived cytokine regulating eosinophil differentiation, survival, and effector functions [6 ]. IL-5 binds specifically to its receptor, which is composed of a cytokine-specific {alpha}-chain and a ß-chain (ßc) shared with the receptors for IL-3 and granulocyte macrophage-colony stimulating factor (GM-CSF; reviewed in ref. [7 ]).

We and others [8 , 9 ] have previously reported that the lipid kinase phosphatidylinositol-3 kinase (PI-3K) is critical for regulation of ßc-mediated survival in a variety of cell lines. Recently, Ser585 of the ßc cytoplasmic domain was identified as a critical residue regulating the activation of PI-3K, leading to nuclear factor-{kappa}B activation and expression of Bcl-2 in response to GM-CSF [10 ]. Active PI-3K also induces phosphorylation of protein kinase B (PKB/c-Akt), which can inhibit apoptosis by direct phosphorylation of various proteins containing consensus phosphorylation sites (RXRXXS/T), such as the Bcl-2 family member Bad and caspase-9 (reviewed in ref. [11 ]). Other targets of PKB-mediated phosphorylation are the Forkhead transcription factor (FOXO) and glycogen synthase kinase 3 (GSK-3). FOXO transcription factors (FOXO1, FOXO3a, and FOXO4) are characterized by the presence of a winged helix domain of 100 amino acids, which binds to DNA and is inhibited by PKB-mediated phosphorylation, resulting in export of FOXO from nucleus to cytoplasm [12 13 14 15 ]. We and others [16 17 18 ] have previously shown that cytokine withdrawal can induce FOXO dephosphorylation and expression of Fas ligand (FasL) or the proapoptotic Bcl-2 family Bim to regulate apoptosis in a variety of lymphoid and myeloid cell lines. Cell cycle arrest has also been reported to be induced by FOXO3a-mediated transcriptional activity as a result of an up-regulation of the cell cycle inhibitor p27Kip1 [19 , 20 ].

GSK-3 was initially identified as a regulator of glycogen metabolism, but it has been implicated more recently in a variety of physiological processes [21 ]. There are two isoforms, GSK-3{alpha} and GSK-3ß, sharing 85% sequence homology and expressed ubiquitously in mammalian tissues [21 , 22 ]. GSK-3 activity is inhibited by phosphorylation at an N-terminal serine residue (Ser21 in GSK-3{alpha} and Ser9 in GSK-3ß). This process is mediated directly by kinases, including PKA, PKB, and PKC (reviewed in ref. [22 ]). GSK-3 activity is also regulated by formation of protein complexes, which has been well characterized in the Wnt signaling pathway [23 ]. In the absence of Wnt, GSK-3 binds to axin, the adenomatous polyposis coli (APC) tumor suppressor protein and ß-catenin. GSK-3 induces phosphorylation of ß-catenin, which is subsequently ubiquitinated and degraded via the protosome pathway [23 , 24 ]. Following Wnt stimulation, GSK-3 is phosphorylated and inactivated, resulting in the release of nonphosphorylated ß-catenin from the complex. In nucleus, ß-catenin binds transcription factors of the T cell factor/lymphoid enhancer-binding factor (Tcf/Lef) family acting as a transcriptional coactivator and activating genes involved in cellular proliferation and survival, such as cyclin D1, c-myc, and activated protein 1 [25 ]. This pathway is evolutionary conserved and regulates proliferation, morphology, motility, and cell fate during embryonic development [24 ]. Mutations in the regulatory genes of the Wnt pathway (APC, axin, and ß-catenin) induce accumulation of nonphosphorylated ß-catenin and activation of gene transcription, promoting the development of oncogenic processes [22 ].

In this study, we have investigated the molecular mechanisms regulating IL-5-mediated eosinophil survival. We demonstrate that PI-3K is critical for IL-5-dependent survival, and inhibition of its activity resulted in eosinophil apoptosis. We also identified GSK-3 as a critical downstream mediator of PI-3K activity, which can regulate eosinophil survival. GSK-3 was phosphorylated and inhibited upon IL-5 stimulation, and inhibition of GSK-3 activity appears to be important for inhibition of the intrinsic apoptotic program. IL-5 stimulation also caused nuclear stabilization of ß-catenin. These findings suggest a novel mechanism regulating eosinophil survival and provide a better understanding of the process by which eosinophils survive during inflammatory responses.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells, reagents, and antibodies
Peripheral blood eosinophils were obtained from healthy volunteers (Blood Bank, Utrecht, The Netherlands) and isolated by modification of the method described previously by Koenderman et al. [26 ]. Briefly, granulocytes were isolated from sodium citrate anticoagulated blood by centrifugation of a Ficoll gradient. After lysis of the erythrocytes with an ice-cold NH4Cl solution, the granulocytes were washed and resuspended in phosphate-buffered saline (PBS). Immunomagnetic beads against CD3, CD14, and CD16 (Miltenyi Biotec, Germany) were added and incubated on ice for at least 30 min. The mix containing cells and beads was passed through a depletion column (Miltenyi Biotec), and only the eosinophil fraction was recovered. Cells were washed and resuspended in RPMI 1640 supplemented with 8% fetal bovine serum (FBS; Hyclone-Gibco, UK). LY294002, PD098059, and SB203580 were from Alexis (San Diego, CA). SB216763 was purchased from Biomol Research Laboratories (Plymouth Meeting, PA). Antibodies against FOXO3a and phospho-Thr32 FOXO3a were purchased from Upstate Biotechnology (Lake Placid, NY). Phospho-GSK-3 and poly(adenosine 5'-diphosphate-ribose) polymerase (PARP) antibodies were purchased from Cell Signaling Technology (Beverly, MA). FasL antibody was from Santa Cruz Biotechnology (CA), anti-Fas receptor (anti-FasR) cross-linking antibody (CH11) was from MBL International (Woburn, MA), caspase-8 antibody was from BD PharMingen (San Diego, CA), and the monoclonal antibody (mAb) against ß-catenin was a kind gift of Dr. Hans Clevers (Hubrecht Lab, Utrecht, The Netherlands).

Western blotting
Eosinophils were lysed in Laemli sample buffer [4% sodium dodecyl sulfate (SDS), 20% glycerol, and 0.12 M Tris-HCl, pH 6.8] after incubation with or without IL-5. The lysates were boiled for 5 min, and protein concentration was determined as described previously [27 ]. Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis, and Western blotting was performed using standard techniques. The blots were incubated in blocking buffer [Tris-buffered saline/Tween 20 (TBST), supplemented with 5% milk] for 1 h. For detection of FOXO3a, phospho(Thr32) FOXO3a, phospho-GSK-3, FasL, caspase-8, and PARP, the corresponding antibodies were incubated overnight in TBST, supplemented with 5% milk and detected with swine anti-rabbit or anti-mouse peroxidase-conjugated antibody (Dako, Denmark) and enhanced chemiluminescence (Amersham, UK).

Apoptosis assays
Cells were counted, washed twice with PBS, and resuspended in RPMI 1640 supplemented with 8% FBS (Hyclone-Gibco) in the presence or absence of 10–10 M human IL-5 (a kind gift of Glaxo Smithkline, Stevenage, UK). Cells were seeded in 24-well dishes (2x105 cells per well) and after 24 or 48 h incubation, were stained to detect apoptosis. Rhodamine-123 (Molecular Probes, Eugene, OR) was used to assess changes in mitochondrial membrane potential ({psi}m). Briefly, cells were incubated in medium (see above) together with 10 µg/ml Rhodamine-123 at 37°C for 30 min. Cells were washed once with PBS and analyzed by fluorescein-activated cell sorter (FACS). The percentage of cells displaying low fluorescence represents the population of apoptotic cells. The exposure of phosphatidylserine (PS) was determined by Annexin V-fluorescein isothiocyanate (FITC) binding (Bender Medsystems, Vienna, Austria). Cells were washed with PBS and resuspended in binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). Subsequently, cells were incubated with Anexin V-FITC for 15 min at room temperature in the dark, washed, and resuspended in binding buffer containing 1 µg/ml propidium iodide (PI). Percentage of apoptotic cells was detected by FACS analysis using Lysis software.

To determine the effect of pharmacological inhibitors (LY294002, PD098059, SB203580, and SB216763), eosinophils were seeded in 24-well dishes (2x105 cells per well) together with IL-5 and the corresponding inhibitors. After 24 or 48 h incubation, cells were harvested, and Rhodamine-123 staining was performed as indicated above.

Assessment of ß-catenin localization
Isolated eosinophils were incubated with RPMI 1640, supplemented with 8% FBS in the presence or absence of human IL-5 (10–10 M), with or without the inhibitor for GSK-3 (SB216763). After 48 h incubation, cells were washed once with PBS, and cytospins were prepared with 105 cells per point. Cytospins were fixed with 4% paraformaldehyde (in PBS) for 10 min and washed three times with PBS. Cells were permeabilized with PBS, supplemented with 0.25% Triton X-100 and 20 mM glycine. After three washes, cytospins were blocked with 1% bovine serum albumin (BSA)-PBS for at least 30 min at room temperature. Cells were incubated with ß-catenin antibody diluted in 0.05% BSA-PBS (1:50) for 2 h at room temperature. Cells were washed with PBS and incubated with anti-mouse-FITC (Becton Dickinson, San Jose, CA) in 1% BSA-PBS (1:100) for 1 h at room temperature. Cytospins were mounted with antifade solution (Molecular Probes, Leiden, The Netherlands) and analyzed by confocal microscopy. The percentage of cells with nuclear or plasma membrane ß-catenin staining was determined.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Eosinophils undergo apoptosis upon IL-5 withdrawal through a mitochondrial-dependent mechanism
In the absence of survival factors, eosinophils initiate a constitutive, apoptotic program. We investigated the time-dependent induction of apoptosis in cultured eosinophils after IL-5 withdrawal. First, we analyzed the externalization of PS by measuring the binding of Annexin-V-FITC [18 ]. Following cytokine withdrawal, ~40% of the cell population was Annexin-V-positive after 24 h, which increased significantly at 48 h (Fig. 1A and 1B ). To determine whether apoptosis may be initiated through an intrinsic, mitochondrial-dependent program, we analyzed the integrity of the mitochondrial membrane in eosinophils using Rhodamine-123, a dye that binds to mitochondria in a membrane potential-dependent way [28 ]. We detected a low percentage of eosinophils with loss of {psi}m after 24 h IL-5 withdrawal; this increased dramatically after 48 h cytokine depletion (Fig. 1C and 1D) . We determined the involvement of caspases in IL-5 withdrawal-mediated apoptosis. Activation of caspase-3 was assessed by detecting the cleavage of PARP, an enzyme involved in DNA repair and genomic maintenance. We found cleavage of 116 kDa PARP into an 89-kDa fragment upon 48 h IL-5 withdrawal (Fig. 2A ). In addition, we detected cleavage of procaspase-8 after 48 h IL-5 withdrawal (Fig. 2B) . Taken together, these findings indicate that eosinophils appear to undergo apoptosis through a mitochondrial-dependent mechanism, resulting in caspase activation.


Figure 1
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  		Figure 1. Eosinophil apoptosis upon IL-5 withdrawal. For all the experiments, eosinophils were cultured in RPMI-1640 medium supplemented with 8% Hyclone as indicated in Materials and Methods. (A) Eosinophils were cultured in the presence or absence of IL-5 (10–10 M) for the times indicated. Apoptosis was measured by Annexin V/PI staining as described in Materials and Methods. Data show the percentage of Annexin V-binding cells and are representative of three independent experiments. (B) FACS profiles of an individual experiment showing the PI-Annexin V binding as described above. (C) Eosinophils were incubated in the presence or absence of IL-5 (10–10 M) for the times indicated. Mitochondrial transmembrane depolarization was assessed as described in Materials and Methods. Data show the percentage of cells having lost {Psi}m and are representative of three independent experiments. (D) FACS histograms of an individual experiment showing loss of mitochondrial membrane depolarization in eosinophils after the indicated treatment.

 

Figure 2
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  		Figure 2. Activation of caspases and FasR upon IL-5 withdrawal. (A) Eosinophils were cultured in the presence or absence of IL-5 (10–10 M), with or without FasR cross-linking antibody (CH11) for 48 h. Cell lysates were prepared, and PARP cleavage was detected as described in Materials and Methods. (B) Eosinophils were cultured with or without IL-5 for 48 h. Lysates were prepared, and caspase-8 cleavage was detected by Western blotting as described in Materials and Methods. (C) Eosinophils were incubated in the presence or absence of IL-5 (10–10 M), with or without CH11 antibody for the times indicated. Loss of {Psi}m was assessed as described previously. Data are representative of three independent experiments.

 
FasR is expressed on the cell surface of eosinophils, and its activation leads to apoptosis [29 30 31 ]. To determine the functional activity of FasR in cultured human eosinophils, we induced activation by cross-linking the receptor with a specific mAb (CH11) and measured mitochondrial membrane depolarization as a marker of apoptosis. In the absence of IL-5, treatment of eosinophils with CH11 for 24 and 48 h increased apoptosis (Fig. 2C) . In the presence of IL-5, cross-linking of FasR for 24 h did not decrease basal eosinophil survival, but apoptosis increased after 48 h (Fig. 2C) . We also observed that the CH11-induced cleavage of PARP after 48 h was not prevented by IL-5 (Fig. 2A) . Activation of FasR and depletion of IL-5 have an additive effect, suggesting that IL-5 withdrawal might generate a FasR-independent mechanism of apoptosis. However, both mechanisms might involve caspase-3 activation to and cleavage of PARP to give the apoptotic phenotype.

PI-3K plays a critical role in eosinophil survival
To determine which intracellular signaling pathways are responsible for the protective effects of IL-5, we cultured eosinophils in the presence of pharmacological inhibitors for PI-3K (LY294002), extracellular signal-related kinase, mitogen-activated protein kinase (MAPK; PD098059), and p38-MAPK (SB203580). We have previously demonstrated the effectiveness of these pharmacological inhibitors in granulocytes [32 33 34 ]. After 48 h, mitochondrial membrane depolarization was measured as described previously. We observed that eosinophil survival was unaffected when cells were coincubated with IL-5 and PD098059 or SB203580, suggesting that MAPKs do not play a critical role in regulating IL-5-dependent survival (Fig. 3 ). However, LY294002 blocked the antiapoptotic effect of IL-5, indicating that PI-3K is essential for IL-5-mediated eosinophil survival.


Figure 3
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  		Figure 3. PI-3K activity is critical for eosinophil survival. Eosinophils were cultured for 48 h with IL-5 (10–10 M) in the absence or presence of the pharmacological inhibitors LY294002 (PI-3K), PD098059 (MAPK kinase), and SB203580 (p38 MAPK) at the concentrations indicated. Loss of {Psi}m was assessed as described previously. Data are representative of three independent experiments.

 
IL-5 regulates GSK-3 and FOXO3a phosphorylation
We have demonstrated previously that stimulation of human eosinophils with IL-5 results in activation of PI-3K and subsequent phosphorylation of PKB, which seems to play a critical role in the maintenance of eosinophil effector functions involved in the modulation of host defense [33 ]. Inactivation of downstream targets of PKB, including FOXO transcription factors and GSK-3, has been associated with cell survival in a variety of cells [11 ], but their role has not been investigated previously in eosinophils. First, we analyzed the IL-5-induced phosphorylation of FOXO3a. There are three putative phosphorylation sites (Thr32, Ser253, Ser315), but here we have focused on Thr32, as it has been previously shown that this residue is phosphorylated specifically by PKB in vitro and in vivo [15 ]. We observed that stimulation of human eosinophils with IL-5 resulted in a rapid and transient phosphorylation of FOXO3a (Fig. 4A ). This was inhibited completely by preincubation of cells with LY294002, demonstrating that it is PI-3K-dependent. There are three PI-3K-regulated FOXO isoforms (FOXO1, FOXO3a, FOXO4); however, although FOXO3 is likely not the only FOXO family member expressed in eosinophils, there are currently no reports demonstrating that the different FOXO isoforms have distinct transcriptional targets or are regulated independently.


Figure 4
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  		Figure 4. IL-5 induces transient phosphorylation of FOXO3a and GSK-3. Eosinophils were cultured in RPMI-1640 medium supplemented with 8% Hyclone for 16 h. Subsequently, cells were stimulated with IL-5 (10–10 M) in the presence or absence of LY294002 (LY) as indicated. Cell lysates were analyzed by Western blotting with (A) phospho-(p)FOXO3aThr32 or (B) phospho-GSK-3Ser9/21 as described in Materials and Methods.

 
Previous studies have demonstrated that GSK-3 is phosphorylated by PKB at an N-terminal serine residue (Ser21 in GSK-3{alpha} and Ser9 in GSK-3ß) [35 ]. In human eosinophils, IL-5 also induced a rapid and transient phosphorylation of GSK-3{alpha} and GSK-3ß with prevalence of the GSK-3{alpha} isoform (Fig. 4B) . Again, this was inhibited by preincubation of cells with LY294002, demonstrating PI-3K dependence. These data suggest that PI-3K-mediated regulation of eosinophil survival might be through inhibition of the PKB proapoptotic substrates such as FOXO3a or GSK-3.

IL-5 withdrawal does not result in induction of FOXO3a transcriptional targets
To determine whether FOXO transcription factors might play a role in inducing transcription of apoptotic genes, we analyzed expression of known, proapoptotic transcription targets in eosinophils after IL-5 withdrawal. It has previously been demonstrated that the promoter of the FasL gene harbors FOXO consensus-binding sites, and dephosphorylation of FOXO3a has been linked to up-regulation of FasL expression in various growth factor-dependent cells [15 ]. We have also previously demonstrated that activation of FOXO3a results in transcriptional up-regulation of the proapoptotic Bcl-2 family member Bim in lymphoid and erythroblastic cell lines [16 17 18 ]. We determined whether the FOXO transcriptional targets FasL and Bim were expressed on eosinophils following IL-5 withdrawal, as assessed by Western blot. We found that neither FasL nor Bim was expressed in the presence or absence of IL-5 (Fig. 5A and 5B ). However, FasL is expressed in LS174 and Jurkat cells (Fig. 5A) , and Bim expression was induced in TF-1 cells after cytokine withdrawal (Fig. 5B) . Taken together, these experiments indicate that FasL and Bim do not appear to be involved in IL-5 withdrawal-induced eosinophil apoptosis. Thus, activation of FOXO3a after survival factor depletion in eosinophils may not be responsible for apoptosis or if so, through additional transcriptional targets that remain to be identified.


Figure 5
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  		Figure 5. Eosinophils do not express FOXO3a transcriptional targets upon IL-5 withdrawal. (A) Eosinophils (EOS) were cultured in RPMI-1640 medium supplemented with 8% Hyclone in the presence or absence of IL-5 for 24 h. Jurkat cells were incubated for the same time in serum-free medium and LS174 cells were cultured in RPMI 1640 supplemented with 8% serum. Western blotting with anti-FasL antibody (1:1000) was performed as described in Materials and Methods. (B) Eosinophils isolated from three independent donors were cultured in RPMI-1640 medium supplemented with 8% Hyclone, with or without IL-5 for 48 h. TF-1 cells were cultured in serum-free medium, with or without IL-5 for 48 h. Western blotting with Bim antibody was performed as described in Materials and Methods.

 
Inhibition of GSK-3 activity rescues eosinophils from apoptosis upon IL-5 withdrawal
As we have described above, stimulation of eosinophils with IL-5 results in phosphorylation and thus, inactivation of GSK-3 (Fig. 4B) . To investigate a functional role of GSK-3 in eosinophil survival, we analyzed the capability of the GSK-3 inhibitor SB216763 to rescue eosinophils from apoptosis following IL-5 withdrawal. This compound has been demonstrated to be a specific GSK-3 inhibitor, and we have recently shown that it is effective in inhibiting GSK-3-mediated ß-catenin phosphorylation [18 , 36 ]. Eosinophils were cultured in the presence or absence of IL-5 and SB216763 for 24 and 48 h. Depolarization of mitochondrial membranes was assessed as detailed previously. In the presence of IL-5, GSK-3 is inactive, and as expected, SB216763 did not have any effect on eosinophil survival (Fig. 6 ). However, we observed that after IL-5 withdrawal, eosinophils incubated with SB216763 showed a dramatically reduced mitochondrial membrane depolarization compared with control cells (Fig. 6A and 6B) . This was also observed when cells were analyzed for PS exposure by Annexin V binding (Fig. 6C) . These data suggest that GSK-3 can play a critical role in the constitutive eosinophil apoptotic program, and inhibition of its activity is modulated by IL-5.


Figure 6
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  		Figure 6. Inhibition of GSK-3 rescues eosinophils from IL-5 withdrawal-induced apoptosis. Eosinophils were cultured in RPMI supplemented with 8% serum, with or without IL-5 and in the presence or absence of 10 µM SB216763 (GSK-3 inhibitor) for 24 and 48 h. (A) Loss of {Psi}m was assessed as described previously. Data are representative of three independent experiments. (B) FACS histograms of an individual experiment showing mitochondrial membrane depolarization in eosinophils after indicated treatments. (C) Eosinophils were treated as indicated. FACS histograms of an individual experiment showing Annexin V/PI staining after 48 h are shown. The percentages in each quadrant are indicated.

 
IL-5 withdrawal induces relocalization of ß-catenin in eosinophils
ß-Catenin is a 92-kDa protein, which can be found associated to the plasma membrane or soluble in cytoplasm [37 ]. At the plasma membrane, ß-catenin interacts with E-cahderin and {alpha}-catenin to modulate cell-cell interactions [37 ]. Cytoplasmic ß-catenin is in a complex with axin, APC, and GSK-3, where it is phosphorylated by GSK-3 and targeted for proteosome degradation. It has been shown that growth factors and Wnt proteins are able to induce translocation of ß-catenin into nucleus and activate ß-catenin/Tcf-mediated transcription [38 39 40 41 ]. We investigated whether the activity of GSK-3 could regulate ß-catenin relocalization. Eosinophils were incubated in the presence or absence of IL-5 and SB216763 for 48 h. Cytospins were prepared and stained with a ß-catenin antibody or only the second antibody (control) as described in Materials and Methods. IL-5 stimulation revealed a punctuate pattern in the nucleus, indicating the presence of ß-catenin in this cell compartment (Fig. 7A ). IL-5 withdrawal induced relocalization of ß-catenin from the nucleus to the plasma membrane, but this effect was abrogated when IL-5-starved eosinophils were cocultured with SB216763 (Fig. 7A and 7B) . These observations demonstrate that regulation of ß-catenin localization is GSK-3-dependent. It is interesting that ß-catenin localization at the plasma membrane and nucleus appears to be mutually exclusive, as we did not observe cells stained at both localizations (Fig. 7B) . Control slides did not show fluorescence in the nucleus or membrane, confirming the specificity of the ß-catenin antibody (Fig. 7A) . These results demonstrate that IL-5 stimulation in eosinophils leads to inactivation of GSK-3 and stabilization of ß-catenin in the nucleus, which could potentially regulate the expression of pro-survival genes.


Figure 7
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  		Figure 7. IL-5 withdrawal-induced relocalization of ß-catenin from nucleus to plasma membrane is dependent on GSK-3 activity. (A) Eosinophils were cultured in RPMI supplemented with 8% serum, with or without IL-5 and in the presence or absence of 10 µM SB216763 (GSK-3 inhibitor) for 48 h. Cytospins and ß-catenin staining were performed as described in Materials and Methods. Slides were analyzed by confocal microscopy. (B) Percentage of cells displaying nuclear or membrane localization following the indicated treatments is shown.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we provide novel insight into the molecular mechanisms underlying the constitutive eosinophil apoptotic program and how this is inhibited by IL-5. For first time, we demonstrate that activation of the PI-3K in eosinophils by IL-5 results in phosphorylation and inhibition of GSK-3 followed by stabilization of ß-catenin in the nucleus. Conversely, IL-5 withdrawal results in activation of GSK-3 and relocalization of ß-catenin from the nucleus to the plasma membrane. This may result in the transcriptional down-regulation of antiapoptotic genes.

Cells can initiate their apoptotic program through two distinct, although overlapping, pathways [42 ]. The first pathway depends on the activation of death receptors [FasR, tumor necrosis factor (TNF) receptor 1, TNF-related apoptosis-inducing ligand], of which FasR is the best characterized [40 ]. The binding of FasL to its receptor induces recruitment of proteins such as Fas-associated death domain and procaspase-8 to form the death-inducing signaling complex. In this complex, procaspase-8 is cleaved to initiate the caspase cascade, resulting in apoptosis [43 ]. In the second pathway, mitochondria play an essential role by releasing cytochrome c, which together with apoptotic protease-activating factor-1 and caspase-9, activate caspase-3, leading to apoptosis [44 ]. In eosinophils, FasR-FasL interactions have been described as an important regulating mechanism of apoptosis [45 ]. It has been shown that FasR expression can be up-regulated by interferon-{gamma} (IFN-{gamma}) and TNF-{alpha}, which increase the eosinophil susceptibility for Fas-mediated apoptosis, and IL-5 or IL-3 maintains basal levels of FasR to protect the cells from apoptosis [31 ]. We have excluded the possibility that IL-5 withdrawal induced eosinophil apoptosis through FasR-FasL interactions, as eosinophils did not express FasL (Fig. 5A) , although FasR was functionally active (Fig. 2C) . However, FasR is likely to modulate eosinophil apoptosis in vivo through interaction with other FasL-expressing cells or binding-soluble FasL [46 , 47 ]. It is surprising that we observed cleavage of procaspase-8 as a consequence of IL-5 deprivation, normally a hallmark of FasR activation (Fig. 2B) . However, we presume that activation of caspase-8 occurs in a FasR-independent manner, and its cleavage is likely attributed to other effector caspases activated in a mitochondrial-dependent manner [48 , 49 ]. Our results also demonstrate that IL-5 withdrawal induces loss of the eosinophil mitochondrial integrity (Fig. 1C and 1D) . Antiapoptotic members of the Bcl-2 family (Bcl-2, A1/Bfl-1, Bcl-w, Bcl-xL, Boo/Diva/Bcl-B, Mcl-1) have been shown to play a role in maintaining the integrity of mitochondria (reviewed in ref. [39 ]). In eosinophils, expression of Bcl-xL is induced by GM-CSF or IL-5 stimulation [50 ], but also levels of Bcl-2 and Mcl-1 have been found to be up-regulated during early eosinophil differentiation [51 ]. The main proapoptotic Bcl-2 family protein found in eosinophils is Bax, which is translocated from cytoplasm to mitochondria when eosinophils are deprived of IL-5 [52 ]. We have previously shown that cytokine withdrawal induces up-regulation of Bim expression in various cytokine-dependent cell lines [16 17 18 ]. However, we could not detect Bim expression in eosinophils after cytokine withdrawal (Fig. 5B) , which suggests that primary myeloid cells might control apoptosis by different mechanisms to those observed in transformed cell lines.

Regulation of cell survival by PI-3K has been demonstrated in neutrophils and basophils as a consequence of cytokine withdrawal [53 54 55 56 ]. In eosinophils, we have previously shown activation of the PI-3K-PKB pathway after stimulation with Th2-derived cytokines [33 ], and recent studies have demonstrated that PI-3K inhibitors are capable of blocking murine eosinophil survival [57 , 58 ]. Here, we demonstrated that inhibition of PI-3K by LY294002 could also inhibit IL-5-induced human eosinophil survival (Fig. 3) . We investigated whether activation of FOXO3a by IL-5 withdrawal had a role in the onset of eosinophil apoptosis. Our results revealed that FOXO3a is indeed phosphorylated upon IL-5 stimulation in a PI-3K-dependent manner (Fig. 4A) . However, we did not find expression of FasL or Bim in eosinophils after cytokine withdrawal (Fig. 5) . A recent study has shown that in neutrophils, formyl-Met-Leu-Phe stimulates PI-3K-mediated phosphorylation and inactivation of FOXO transcription factors, which associate with Mcl-1 to regulate survival [59 ]. In eosinophils, IFN-mediated survival also seems to be regulated by Mcl-1 [60 ], but its direct interaction with FOXO proteins remains to be established.

PKB regulates GSK-3 activity through inhibition by phosphorylation at an N-terminal serine (Ser21 in GSK-3{alpha} and Ser9 in GSK-3ß) [21 ]. It has been previously reported that activation of GSK-3 induces apoptosis via activation of the mitochondrial death pathway and induces cleavage of caspases [61 , 62 ]. GSK-3 also plays a critical role in the Wnt signaling pathway, which is essential during embryogenesis of vertebrates and invertebrates [23 ], and mutations in this pathway lead to diverse forms of cancer [24 ]. In the absence of Wnt, phosphorylated GSK-3 binds to axin, APC, and ß-catenin, resulting in ß-catenin phosphorylation. ß-catenin is subsequently ubiquitinated and degraded via the proteosome pathway [23 , 24 ]. Wnt stimulation induces inactivation of GSK-3 and release of ß-catenin from the complex. ß-Catenin accumulates in cytoplasm and is targeted to nucleus by specific nuclear proteins [pygopus (pygo) and legless (lgs)], which control binding to the Tcf/Lef family of transcription factors as well as its cotranscriptional activity [63 ]. Studies about hepatocyte growth factor/scatter factor and fibroblast growth factor-1 have demonstrated that depletion of these factors increased the destabilized pool of ß-catenin, leading to apoptosis [38 39 40 41 ]. Our results demonstrate that activation of GSK-3 by IL-5 withdrawal is at least in part responsible for induction of eosinophil apoptosis. Inhibition of GSK-3 activity by IL-5 induced phosphorylation or pharmacologically (SB216763) rescued cells from apoptosis (Fig. 6) . We observed that levels of ß-catenin in eosinophils were constant in the presence or absence of IL-5 (data not shown), but localization of the protein changed. In the presence of IL-5, ß-catenin was found in the nucleus, whereas following IL-5 withdrawal, it was found at the plasma membrane (Fig. 7) . The presence of ß-catenin at the plasma membrane is probably the result of binding to partners such as E-cadherin to constitute a membrane-associated pool antagonizing its signaling functions [25 ]. Localization of ß-catenin at the membrane has been associated with regulation of cell adhesion and migration [64 ]. It is possible that relocalization of ß-catenin to the plasma membrane upon IL-5 withdrawal might also induce changes in the adhesion properties of eosinophils.

The fact that ß-catenin is stabilized in the nucleus upon stimulation with extracellular stimuli suggests that its interaction with members of the Tcf/Lef family of transcription factors might regulate the expression of antiapoptotic genes. In the absence of cell stimulation, Tcf/Lef binds to groucho proteins (Grg), which act as transcriptional repressors of Tcf/Lef target genes [65 ]. It has been shown that the association of ß-catenin and Tcf-1 regulates Bcl-XL expression in CD4+CD8+ thymocytes, resulting in survival [66 ]. Other known target genes of the ß-catenin/Tcf complex involved in the maintenance of survival include cyclin D, Wnt-1-induced-secreted protein 1, and c-myc [67 68 69 70 71 ].

As mentioned above, the proapoptotic Bcl-2 family member Bax was translocated from the cytoplasm to the mitochondria in the absence of IL-5 [52 ]. GSK-3 activity has also been linked to conformational change of Bax and induction of cell death in primary human erythroid progenitors following growth factor withdrawal [72 ]. However, we were unable to detect changes in Bax localization in eosinophils as a consequence of IL-5 removal (data not shown).

In allergic asthma, the production of IL-5 by Th2 cells leads to eosinophilia and accumulation of eosinophils in the airways. Our data suggest that this might be mediated by up-regulation of ß-catenin-target genes linked to enhanced-eosinophil survival, as demonstrated by stabilization of ß-catenin in the nucleus as a consequence of IL-5 stimulation (Fig. 6) . However, specific target genes modulating eosinophil survival via the ß-catenin-Tcf/Lef transcription remain to be identified in further studies.


   ACKNOWLEDGEMENTS
 
We thank Leo Houben for isolation of eosinophils. M. R. was supported by a research grant from The Netherlands Asthma Foundation (NAF 98.40).

Received November 7, 2005; accepted March 21, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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N. Goplen, M. M. Gorska, S. J. Stafford, S. Rozario, L. Guo, Q. Liang, and R. Alam
A Phosphosite Screen Identifies Autocrine TGF-{beta}-Driven Activation of Protein Kinase R as a Survival-Limiting Factor for Eosinophils
J. Immunol., March 15, 2008; 180(6): 4256 - 4264.
[Abstract] [Full Text] [PDF]

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T. Adachi, S. Hanaka, T. Masuda, H. Yoshihara, H. Nagase, and K. Ohta
Transduction of Phosphatase and Tensin Homolog Deleted on Chromosome 10 into Eosinophils Attenuates Survival, Chemotaxis, and Airway Inflammation
J. Immunol., December 15, 2007; 179(12): 8105 - 8111.
[Abstract] [Full Text] [PDF]

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Y. Li, Z. Wang, D. Kong, S. Murthy, Q. P. Dou, S. Sheng, G. P. V. Reddy, and F. H. Sarkar
Regulation of FOXO3a/beta-Catenin/GSK-3beta Signaling by 3,3'-Diindolylmethane Contributes to Inhibition of Cell Proliferation and Induction of Apoptosis in Prostate Cancer Cells
J. Biol. Chem., July 20, 2007; 282(29): 21542 - 21550.
[Abstract] [Full Text] [PDF]