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PHAGOCYTES
From the Institute for Lung Health, University of
Leicester, Glenfield Hospital, Leicester, United Kingdom, and the
Medical Research Council Toxicology Unit, University of Leicester,
Leicester, United Kingdom.
The apoptosis and subsequent clearance of eosinophils without
histotoxic mediator release is thought to be crucial in the resolution
of airway inflammation in asthma. Interleukin-5 (IL-5) is a potent
suppressor of eosinophil apoptosis. The mechanism by which
IL-5 inhibits spontaneous eosinophil apoptosis was investigated. Freshly isolated eosinophils constitutively expressed the
conformationally active form of Bax in the cytosol and nucleus. During
spontaneous and staurosporine-induced apoptosis, Bax underwent a
caspase-independent translocation to the mitochondria, which was
inhibited by IL-5. Eosinophil apoptosis was associated with the release
of cytochrome c from the mitochondria, which was also
inhibited by IL-5. IL-5 and the cell-permeable caspase inhibitor,
benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone
(z-VAD.fmk), prevented phosphatidylserine (PS) externalization,
although only IL-5 inhibited loss of mitochondrial membrane
potential ( Eosinophils play a pivotal role in the pathogenesis
of asthma and allergic disease.1,2 Apoptosis and efficient
clearance of apoptotic cells without histotoxic mediator release
is thought to be crucial in the resolution of airway
inflammation,3 and delayed eosinophil apoptosis has an
association with asthma, inhalant allergy, and atopic
dermatitis.4,5 The accumulation and persistence of
eosinophils at sites of inflammation are mediated at least in part by
extended survival in response to circulating hematopoietins interleukin-3 (IL-3), IL-5, and granulocyte-macrophage
colony-stimulating factor (GM-CSF). Eosinophils rapidly undergo
apoptosis unless exposed to these cytokines in vitro6,7
and in vivo.8,9 The mechanism by which IL-5
prevents apoptosis in eosinophils is largely unknown.
The Bcl-2 homologues are critical regulators of the apoptotic pathway,
with interactions between proapoptotic (Bax, Bik, Bim, Bak) and
antiapoptotic (Bcl-2, Bcl-xL) proteins controlling the release of apoptogenic factors from mitochondria10,11 and
subsequent activation of caspases, the conserved death proteases of the
cell. Previous reports investigating the role of Bcl-2 homologues in eosinophils have concentrated on the level of protein expression. Human
peripheral blood eosinophils endogenously express relatively high
levels of proapoptotic Bax and antiapoptotic Bcl-xL, with little or no detectable antiapoptotic Bcl-2
expression,12,13 although there is some evidence of higher
Bcl-2 expression in eosinophils derived from patients with asthma and
hypereosinophilic syndrome,14,15 and stimulation in vitro
with IL-5 resulted in detectable up-regulation of Bcl-2 expression in
some12,16 but not all studies.17,18 It has
been proposed that susceptibility to cell death is determined by the
ratio of death agonist to death antagonist and their subsequent
interactions by homodimerization and heterodimerization, via conserved
BH3 domains.11 The, at best, small increase in Bcl-2 in
IL-5-stimulated eosinophils,12 would suggest
up-regulation of Bcl-2 alone is unlikely to be solely responsible for
the potent survival-enhancing activity of IL-5. This implicates other
regulatory mechanisms, possibly involving the well-expressed Bax or
Bcl-xL.12,18 Bax is a monomeric, cytosolic
protein that has been shown in cell lines to undergo a
conformational change and translocate from the cytosol to the outer
mitochondrial membrane during apoptosis,19 facilitating the release of cytochrome c20,21 and subsequent
activation of caspases. The conformation change allows both
oligomerization and membrane insertion of Bax, but also reveals an
N-terminal epitope recognized by the monoclonal antibody
6A7.22
Caspases, aspartate-specific cysteine proteases, regulate the
execution phase of apoptosis, being responsible for most of the
biochemical and morphologic changes associated with the apoptotic phenotype.23 Distinct caspase cascades are initiated
dependent on the death stimulus.24,25
"Receptor-mediated" apoptosis involves ligation of death receptors,
such as CD95 (Fas/Apo-1) and tumor necrosis factor receptor-1 within
the plasma membrane of many cell types, and direct processing of
caspase-8 on recruitment to the receptor complex.26 A
target of caspase-8 is the proapoptotic Bcl-2 homologue, Bid, which on
proteolysis translocates from the cytosol to mitochondria, and by a
mechanism possibly involving interaction with Bax, potentiates the
release of cytochrome c.27 In
"chemical/stress-induced" apoptosis, cellular signals induce perturbations of the mitochondria and the release of apoptosis mediators including cytochrome c,28
apoptosis-inducing factor (AIF),29 and
Smac/Diablo.30,31 Release of cytochrome c
allows formation of the "apoptosome," a caspase-9-activating
complex, involving cytochrome c, Apaf-1, deoxyadenosine
triphosphate (dATP), and procaspase-9.32,33 The activation
of "initiator" caspase-8 and -9, results in direct or indirect
activation of "effector" caspases, such as caspase-3, -6, and -7.
In this study, we investigated the mechanism by which IL-5 inhibits
spontaneous apoptosis in eosinophils. We report that IL-5 prevented Bax
translocation and cytochrome c release and mediated eosinophil survival by inhibiting perturbation of the mitochondrial membrane and caspase activation.
Antibodies and reagents
Cell lines and culture
Isolation of peripheral blood eosinophils and cell culture Heparinized peripheral venous blood was taken from healthy volunteers with peripheral blood eosinophilia of less than 0.5 × 106/mL. Eosinophils were purified by a 2-step method of density gradient centrifugation, followed by negative immunomagnetic selection as described previously.35 Briefly, removal of erythrocytes by dextran sedimentation was followed by centrifugation of the leukocyte-rich supernatant (100g, 15 minutes at room temperature). The cell pellet was resuspended in Hanks balanced salt solution without Ca++ and Mg++, supplemented with 1% bovine serum albumin (BSA) and 20 mM EDTA. The cell suspension was centrifuged on Histopaque 1083 and the mononuclear cell layer carefully removed, prior to lysis of erythrocytes contaminating the granulocyte pellet by hypotonic shock using sterile, ice-cold water. Eosinophils were separated from neutrophils by negative immunomagnetic selection using anti-CD16-coated magnetic beads (Miltenyi Biotec, Auburn, CA). Purity and viability after isolation were routinely more than 99% as assessed by morphology after Kimura stain and trypan blue exclusion, respectively. Peripheral blood eosinophils were cultured in RPMI 1640 supplemented with 1% BSA and 1% Glutamax in the presence of IL-5 (10 10 M), staurosporine (STS; 105 M), or
z-VAD.fmk (100 µM) where indicated. Preliminary experiments were
performed to determine optimum concentrations.
Immunoblotting Cells (5 × 105) were washed with ice-cold phosphate-buffered saline (PBS) and snap-frozen in dry ice before storage at 70°C. Samples were resuspended in sample buffer with
freshly added 5%  -mercaptoethanol and boiled for 5 minutes.
Proteins were then separated on 10%/15% sodium dodecyl
sulfate-polyacrylamide gels and electrotransferred onto presoaked
Hybond-C nitrocellulose filter (Amersham Life Science, Bucks, United
Kingdom). Immunoblotting was performed as described
previously12 and proteins detected by enhanced
chemiluminescence according to the manufacturer's instructions
(Amersham Life Science).
Assessment of phosphatidylserine exposure and mitochondrial membrane potential The exposure of phosphatidylserine (PS) has been shown to be a sensitive marker of apoptosis in eosinophils and other cell types.36,37 Cultured cells were washed once in PBS and incubated with annexin V-FITC (1:1000) according to the manufacturer's instructions. Cells were then incubated with 50 µg/mL propidium iodide for 2 minutes on ice prior to analysis using the FACScan (Becton Dickinson, Oxford, United Kingdom), excitation at 488 nm and detection between 515 and 550 nm. To measure mitochondrial membrane potential (  m), cells were loaded with 75 nM TMRE for 30 minutes at 37°C.
Alterations of m can be determined from the fluorescence
intensity of TMRE because the transmembrane distribution of this
cationic dye is dependent on membrane potential.38 Cells
were washed once and resuspended in PBS for FACScan analysis,
excitation at 488 nm and detection at 610 nm. Freshly isolated
eosinophils were treated with the mitochondrial membrane uncoupler
carbonylcyanide m-chlorophenyl hydrazone (mCCP) at 1 µM for 15 minutes at 37°C as a positive control for dissipation of
m.
Apoptotic morphology Eosinophils were stained with Kimura stain39 and examined under light microscopy for the characteristic morphologic changes associated with apoptosis, namely, condensation of the nucleus and cytoplasm.36 Two hundred and fifty cells were counted per treatment in a blinded fashion.Immunocytochemistry and confocal analysis Cells were incubated with 75 nM Mitotracker Red CMXRos for 45 minutes at 37°C and washed in RPMI without BSA, and cytospins were performed (650 rpm for 6 minutes) on silane-coated slides at 1 × 106 cells/mL. Slides were fixed in 2% paraformaldehyde for 15 minutes at room temperature and washed 5 times in PBS. Cells were permeabilized for 10 minutes at room temperature in blocking buffer (3% BSA in PBS) plus 0.1% Triton X-100 followed by blocking of nonspecific binding in blocking buffer for 1 hour at room temperature. Cells to be stained with anti-Bax 6A7 were not permeabilized because the recognized Bax conformation has been shown to be sensitive to nonionic detergent.40 Cells were incubated overnight at 4°C with 5 µg/mL primary antibody diluted in blocking buffer. Cells were then incubated for 20 minutes at room temperature with 0.2% chromotrope-2R, which binds to highly basic eosinophil granules thereby reducing nonspecific binding of the secondary antibody. Cells were then incubated with species-specific Alexa 488-conjugated secondary antibody diluted 1:300 in blocking buffer for 50 minutes at room temperature in the dark. The nuclei were stained with Hoechst 33258 (250 ng/mL) for 10 minutes at room temperature in the dark prior to mounting with fluoromount (Dako). Images were collected by confocal laser microscopy (model TCS 4D, Leica, Heidelberg, Germany). The 488- and 568-nm lines of the krypton/argon laser were used for the excitation of Alexa 488 and Mitotracker Red CMXRos, respectively. Excitation of Hoechst 33258 was by UV laser. Cytochrome c release and Bax translocation were quantified on these cytospins by counting at least 300 cells in a blinded fashion in 4 random visual fields and assessing evidence of diffuse cytochrome c or punctate Bax staining.Statistical analysis Comparisons between treated and untreated cells were made at individual time points using the Student paired t test.
Caspase expression and activation in peripheral blood eosinophils The primary aim of this project was to identify the point in the apoptotic pathway at which IL-5 exerts its antiapoptotic effect. Initial investigation explored the role of caspases in eosinophil apoptosis. Peripheral blood eosinophils endogenously expressed both the "initiator" caspase-8 and -9, and the "effector" caspase-3, -6, and -7 (Figures 1 and 2). Treatment of eosinophils for 2 hours with the protein kinase C inhibitor, STS (105 M),
was sufficient to induce activation of caspase-3, -6, -7, -8, and -9, as evidenced either by the generation of the immunoreactive, catalytically active, large subunits resulting from processing of the
caspase proforms, or direct loss of the proform itself (Figures 1
and 2).
Procaspase-3 is expressed as an inactive 32-kd zymogen, which on activation is cleaved initially at D175 (single-letter amino acid codes) generating a p20 subunit, followed by further proteolytic cleavage at D9 and D28 to generate p19 and p17 subunits, respectively.41 In freshly isolated eosinophils, caspase-3 was present as a 32-kd proform (Figure 1A, lane 1). Exposure to STS for 2 hours resulted in the formation of 20-kd (p20), 19-kd (p19), and 17-kd (p17) immunoreactive fragments (Figure 1A, lane 4). After 6 hours, the proform was almost completely processed (Figure 1A, lane 7). Jurkat T cells exposed to etoposide for 4 hours exhibited similar caspase-3 processing (Figure 1A, lane 12). Procaspase-6 was expressed in eosinophils as its 34-kd zymogen (Figure 1B, lane 1). Processing of caspase-6 generates p18 and p11 subunits due to sequential cleavage at D179 and D193.42 Due to lack of immunoreactivity of these subunits, processing of caspase-6 was indicated by the time-dependent loss of the proform after 6 hours of STS treatment (Figure 1B, lane 7). A slight loss of caspase-6 proform was observed in Jurkat cells treated with etoposide for 4 hours (Figure 1B, lane 12). In eosinophils, caspase-7 was expressed as its 35-kd proform (Figure 1C, lane 1). Treatment with STS for 2 hours resulted in formation of the immunoreactive 19-kd (p19) fragment (Figure 1C, lane 4), corresponding to the large catalytically active subunit generated due to cleavage at D198 and removal of the prodomain.34 After 6 hours of STS treatment, procaspase-7 was almost completely lost (Figure 1C, lane 7). Similar caspase-7 processing was observed in Jurkat T cells (Figure 1C, lane 12). Caspase-8 was detectable in freshly isolated eosinophils and Jurkat T cells as the 55- kd proform (Figure 1D, lanes 1 and 12). However, shorter exposure revealed that caspase-8 was expressed as a doublet of 55 and 53 kd, corresponding to the proposed occurrence of 2 isoforms, caspase-8a and caspase-8b.43 After STS treatment for 2 hours, cleavage of the proform resulting in the formation of 43-kd (p43) and 41-kd (p41) subunits was observed corresponding to cleavage between the large and small subunits (Figure 1D, lane 4). After 6 hours of STS treatment, an 18-kd (p18) fragment was observed due to removal of the death effector domains from p43 and p41 (Figure 1D, lane 7). Generation of the p45 and p43 subunits was observed in Jurkat T cells treated with etoposide for 4 hours, although the p18 subunit was not marked (Figure 1D, lane 12). Caspase-9 is the apical caspase of stress-induced apoptosis. Activated on association with Apaf-1 in the presence of cytochrome c, caspase-9 activates downstream "effector" caspase-3, -6, and -7.44,45 Procaspase-9 was detectable in eosinophils as its 46-kd zymogen (Figure 2E, lane 1), and time-dependent formation of 35-kd and 37-kd (p35 and p37) subunits was observed after 2 and 6 hours of STS treatment, respectively, due to cleavage at D315 and D330 (Figure 2E, lanes 4 and 8). Detection of caspase processing in eosinophils after treatment with STS for 2 hours was commonly associated with approximately 35% to 50% apoptosis assessed by PS exposure and 10% to 15% cells showing evidence of nuclear condensation by morphology after Kimura stain (Figure 1). IL-5 inhibits spontaneous eosinophil apoptosis upstream of caspase processing In the absence of IL-5 for 24 hours, approximately 65% apoptosis assessed by PS externalization was associated with processing of caspase-3, -7, -8, and -9 in eosinophils (Figures 1 and 2). After 2 and 6 hours of IL-5 deprivation, subunit detection was difficult due to low levels of caspase processing. However, longer exposure revealed the presence of caspase-3 subunits p20, p19, and p17 after 6 hours in the absence of IL-5 (approximately 15% apoptosis by PS exposure). Processing of caspase-9 was less marked compared with caspase-3, -7, and -8, but the p37 subunit was detectable after 24 hours of IL-5 deprivation (Figure 2E, lane 10). Processing of caspase-3, -7, -8, and -9 was completely inhibited by IL-5 (1010 M) over the
24-hour culture period (Figure 1A,C,D, lane 9; Figure 2E, lane 11).
Lack of immunoreactivity of caspase-6 subunits made determination of
processing in the absence of IL-5 difficult. A slight loss of proform
in IL-5-deprived eosinophils compared with IL-5-stimulated
eosinophils after 24 hours was observed, but was not marked (Figure 1B,
lanes 8 and 9). The broad-spectrum, cell-permeable caspase inhibitor,
z-VAD.fmk, inhibited the processing of each caspase in IL-5-deprived
eosinophils after 24 hours (Figure 2A-D, lane 13). However, inhibition
was not absolute because caspase-9 processing after 24 hours in the
presence of z-VAD.fmk was observed, evidenced by the detection of the
p35 subunit (Figure 2E, lane 13).
The results demonstrate that caspases were activated during spontaneous eosinophil apoptosis and IL-5 acted at a point prior to caspase activation in inhibiting spontaneous eosinophil apoptosis. The events that precede the activation of caspases in the apoptotic pathway were then investigated. The mitochondria are pivotal in the regulation of apoptotic cell death, with apoptogenic factor release required for the activation of caspase-9. The role of mitochondria in eosinophil apoptosis was investigated. IL-5 inhibits the loss of mitochondrial membrane potential in eosinophil apoptosis Apoptosis is characterized by the exposure of PS at the cell membrane,36,37 and a reduction inm.46
Less than 5% of freshly isolated eosinophils exhibited PS
externalization or loss of m (Figure
3A,B). Uncoupling of mitochondrial
respiration with mCCP as a positive control caused a marked loss of
m (Figure 3A, 0 hour, open histogram). Eosinophils undergoing
spontaneous apoptosis exhibited an increase in both the percentage of
cells with dissipated m and the percentage of cells with PS
externalization after 24 hours (Figure 3A,B, IL-5). The increase in
spontaneous eosinophil apoptosis assessed by both criteria was observed
after 6 hours, but was very marked after 24 hours (Figure
4). IL-5 significantly inhibited loss of
m and externalization of PS compared with medium control (Figure
3A, 25% versus 48%, and 13% versus 66% respectively; and Figure
3B). Therefore, spontaneous eosinophil apoptosis was associated with a
time-dependent externalization of PS and loss of m, and the
antiapoptotic effect of IL-5 acted at or prior to the loss of m.
There was no significant difference between the progression of PS
externalization and dissipation of m during spontaneous
eosinophil apoptosis (Figure 4). Apoptosis assessed by both these
criteria was significantly increased as early as 2 hours exposure to
STS and was almost maximal after 6 hours (Figure 4).
z-VAD.fmk inhibits eosinophil PS exposure after loss of mitochondrial membrane potential In eosinophils cultured in the absence of IL-5 for 24 hours, incubation with z-VAD.fmk significantly inhibited PS externalization (Figure 3A, 46% versus 66%, and Figure 3B), although less efficiently than IL-5, but exhibited little or no effect on loss ofm
compared with medium control (Figure 3A, 44% versus 48%, and Figure
3B). Thus during spontaneous eosinophil apoptosis PS externalization was dependent on caspase activation, whereas the perturbations of the
mitochondria leading to loss of m were independent of caspases.
Prevention of caspase activation inhibited apoptosis assessed by PS
externalization, thereby implicating caspases as major effectors of the
execution of spontaneous eosinophil apoptosis. To further elucidate the
role of the mitochondria in eosinophil apoptosis, cytochrome
c release was investigated. Cytochrome c release from
mitochondria is an early, pivotal event in the apoptosis of many cell
types.47
Release of cytochrome c from mitochondria during eosinophil apoptosis Spontaneous eosinophil apoptosis was associated with caspase-dependent release of mitochondrial cytochrome c, which was inhibited by IL-5. In freshly isolated peripheral blood eosinophils, cytochrome c exhibited a punctate distribution, which colocalized with mitochondria (Figure 5A). No staining was observed with the isotype-matched negative control (data not shown). Due to the relatively low level of constitutive cytochrome c expression and number of mitochondria in eosinophils, cells that had undergone cytochrome c release generally appeared diffuse or devoid of green staining entirely (Figure 5B,D, indicated by arrows). Release of cytochrome c was observed in eosinophils cultured in the absence of IL-5 for 6 hours (Figure 5B) and was marked after 24 and 48 hours (Figure 6A). Cytochrome c release was commonly associated with eosinophils exhibiting evidence of apoptotic nuclear condensation (Figure 5B). Stimulation with IL-5 for 6 hours maintained the association of cytochrome c with the mitochondria (Figure 5C), and inhibited cytochrome c release compared with medium control after 24 and 48 hours (Figure 6A). z-VAD.fmk had no inhibitory effect on cytochrome c release compared with medium control (Figure 6A). Cytochrome c release was also observed in eosinophils treated with STS for 2 hours (Figure 5D), and after 6 hours of STS treatment almost 100% of eosinophils exhibited cytochrome c release, which was not inhibited by z-VAD.fmk (Figure 6B). Due to downstream proteolytic degradation of the cell, accurate determination of cytochrome c release beyond 6 hours of STS treatment was not possible. Therefore, spontaneous and STS-induced eosinophil apoptosis involved caspase-independent cytochrome c release. IL-5 inhibited spontaneous eosinophil apoptosis at or prior to release of cytochrome c. The mechanism by which cytochrome c is released from the mitochondria during apoptosis is unclear. We investigated the possible role of Bax in eosinophil apoptosis because its translocation to the mitochondrial membrane facilitates the release of cytochrome c in many models of apoptosis.20,21
Cytosol-to-mitochondria translocation of Bax during eosinophil apoptosis Spontaneous eosinophil apoptosis was associated with a caspase-independent redistribution of Bax from the cytosol to the mitochondria, which was inhibited by IL-5. Anti-Bax 6A7 detected diffuse cytosolic and nuclear Bax distribution in the majority of freshly isolated eosinophils (Figure 7A), indicating constitutive expression of the conformationally altered form of Bax. Interestingly, Bax expression was observed to localize predominantly to the nucleus compared with the cytosol in freshly isolated eosinophils (Figure 7A). Isotype-matched mouse negative control showed essentially no evidence of nonspecific staining (Figure 7B). Punctate Bax distribution that colocalized with the mitochondria was observed in eosinophils deprived of IL-5 for 6 hours (Figure 7C). This punctate distribution was commonly perinuclear and consistently associated with cells exhibiting characteristic apoptotic morphology of condensed nucleus and cytoplasm. Stimulation of eosinophils with IL-5 for 6 hours maintained the cytosolic and nuclear distribution observed in the freshly isolated eosinophils (Figure 7D), and prevented Bax translocation to the mitochondria observed during spontaneous apoptosis after 24 and 48 hours (Figure 6C). In contrast, although z-VAD.fmk inhibited spontaneous apoptosis assessed by PS externalization, it increased the percentage of eosinophils showing Bax translocation to the mitochondria (Figure 6C). Treatment of eosinophils with STS for 2 hours also resulted in the marked appearance of punctate Bax distribution (Figure 7E), and similarly to cytochrome c release, nearly 100% of eosinophils exhibited translocated Bax after 6 hours (Figure 6D). STS-induced Bax redistribution was not inhibited by z-VAD.fmk (Figure 6D). To determine whether translocation of other proapoptotic Bcl-2 homologues occurred during apoptosis, the expression of Bik, Bak, and Bim was also investigated. Expression of Bik and Bak was undetectable by either immunoblotting or immunocytochemistry in peripheral blood eosinophils (data not shown). Bim expression was detected both by immunoblotting and immunocytochemistry, where it was localized to the cytoplasm. However, Bim did not redistribute during spontaneous or STS-induced apoptosis (data not shown).
We have shown for the first time that IL-5 exhibits its potent antiapoptotic effect in eosinophils by preventing Bax translocation to the mitochondria, cytochrome c release, increased mitochondrial membrane permeability, and subsequent caspase activation. In nonapoptotic cells, Bax is a soluble, cytosolic monomer, with an N-terminus proposed to mask its hydrophobic C-terminus.22 During the apoptosis of murine thymocytes, COS-7, and murine fibrosarcoma cell lines, it has been shown that a conformational change exposes the BH3 domain and C-terminus, facilitating oligomerization of Bax and translocation from the cytosol to the mitochondrial membrane, respectively.19,22,48 Bax oligomers have channel-forming activity and trigger the release of cytochrome c from human melanoma cells21 and isolated mitochondria.20,49,50 We, and others, have previously shown that human peripheral blood eosinophils express high levels of Bax.12,13 We now provide evidence that freshly isolated eosinophils constitutively express the conformationally altered form of Bax, detected by the monoclonal antibody 6A7 that specifically recognizes an N-terminal epitope inaccessible in soluble, cytosolic Bax.22 Interestingly, despite diffuse Bax expression being detectable throughout the entire cell, expression was predominantly observed in the nucleus (Figure 7A). Lack of nuclear and cytosolic staining in the isotype-matched negative control would suggest that the staining was not nonspecific (Figure 7B). An association of Bax with the nuclear membrane and matrix in nonapoptotic cells has been previously reported in MCF-7 cells51,52 and a number of human lung cancer cell lines.53 However, the relevance of nuclear Bax expression is currently unclear. During both spontaneous and STS-induced apoptosis, Bax underwent a caspase-independent translocation to the mitochondria in human eosinophils. This colocalization was consistently observed in eosinophils exhibiting the condensed nuclear morphology characteristic of apoptosis and was inhibited by IL-5. Interestingly, Bax redistribution to the mitochondria seemingly increased with z-VAD.fmk (Figure 6C), probably due to the delayed onset of the caspase-mediated execution phase of apoptosis and the consequent proteolytic degradation of cells cultured for up to 48 hours, thereby prolonging the association of Bax with the mitochondria. This is the first report of Bax translocation to the mitochondria during spontaneous apoptosis of human granulocytes due to cytokine deprivation. A recent report has demonstrated that Bax inserts into the mitochondrial membrane and cytochrome c is released during apoptosis of neutrophils, induced by a 15°C to 37°C temperature transition.54 Similarly, in this temperature arrest system, Bax translocation to the mitochondria was shown to be of crucial importance in the execution of granulocyte apoptosis. Caspase-independent release of cytochrome c from
mitochondria was observed in eosinophils undergoing STS-induced and
spontaneous apoptosis, with the latter inhibited by IL-5. The mechanism
by which cytochrome c is released from the mitochondria
remains unclear. It has been proposed that Bax induces the opening of
the permeability transition pore complex via interaction with the
adenine nucleotide translocator, resulting in mitochondrial membrane
depolarization and cytochrome c release.55
Spontaneous eosinophil apoptosis clearly involved
caspase-independent loss of Receptor-mediated and chemical/stress-induced caspase activation are associated with apical processing of caspase-8 and -9, respectively. Although recent studies have reported activation of caspases in eosinophils in response to Fas, growth factor withdrawal, and dexamethasone,17,56,57 caspase processing has not been investigated in detail. We have demonstrated processing of caspase-3, -7, -8, and -9, and possible caspase-6 activation during spontaneous eosinophil apoptosis. Immunoblot analysis seemingly indicated caspase-3 processing proximal to caspase-8 processing. In receptor-mediated apoptosis, perturbations of the mitochondrial membrane are dependent on the activation of caspase-8. The time scale of activation of caspase-3 and -8, coupled with the observation that mitochondrial perturbations were not blocked by z-VAD.fmk, suggested that spontaneous eosinophil apoptosis is mediated by caspase-independent mitochondrial events and the consequent activation of the caspase-9/-3/-7 pathway. The processing and activation of caspase-8 observed after 24 hours may form an amplification loop mediated by caspase-3 and -6 as previously described.45 The processing of caspase-9 was not as marked as the other caspases tested. This may be due to the relative efficacy of the antibodies used, although it must be noted that caspase-9 can be activated without proteolytic cleavage.58 We have demonstrated that caspases are major effectors of the execution phase of spontaneous eosinophil apoptosis, and IL-5 induces eosinophil survival by inhibiting apoptosis before caspase activation. Ligation of the IL-5 receptor induces rapid tyrosine phosphorylation
and activation of juxtamembranous tyrosine kinases. The signal is
propagated via the Janus kinase2 (Jak2)/signal transducers and
activators of transcription (STAT) and Ras-Raf-1-mitogen-activated protein kinase (MAPK) cascades (Figure
8).59,60 Activation of Jak2,
Lyn, and Syk tyrosine kinases, and Src homology 2 phosphatase 2 tyrosine phosphatase (SHPTP-2) is crucial for IL-5-induced eosinophil survival.60,61 SHPTP-2 activation and association
with the adaptor protein Grb2 is proposed to couple the IL-5 receptor
to the Ras signaling pathway,61 supported by the
requirement for Raf-1 serine/threonine kinase activation in
IL-5-mediated suppression of eosinophil apoptosis.60 The
mechanism by which these signaling cascades integrate into the
apoptotic pathway, thereby regulating Bax translocation, is unknown and
the subject of future investigation. Interactions with other proteins,
such as the apoptosis suppressing Bcl-xL, the expression of
which is down-regulated during spontaneous apoptosis, but
maintained or up-regulated on stimulation with GM-CSF and IL-5, may be
involved.18
Elucidating the point in the signaling cascade that IL-5 inhibits apoptosis would enable specific inhibitors of growth factor-mediated eosinophil survival to be developed. This could have important therapeutic implications for asthma and related eosinophilic disorders.
We would like to thank Kul Sikand for his expert technical assistance with the confocal microscopy.
Submitted December 11, 2000; accepted May 30, 2001.
Supported by a grant from the National Asthma Campaign.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Andrew J. Wardlaw, Division of Respiratory Medicine, Institute of Lung Health, Clinical Sciences Bldg, Glenfield Hospital, Leicester, LE3 9QP, United Kingdom; e-mail: aw24{at}le.ac.uk.
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Human eosinophils express Bcl-2 family proteins: modulation of Mcl-1 expression by IFN- |
Top
Abstract
Introduction
), STS (10
), or medium alone (
) for the
indicated times. Apoptosis was assessed by PS exposure and 
P < .01) and was almost maximal after 6 hours
(*P < .05). Results are expressed as mean ± SE of 3 separate experiments.
.
Am J Respir Cell Mol Biol.
1998;18:315-322