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Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2365-2373
Eosinophil Apoptosis Is Mediated by Stimulators of Cellular Oxidative
Metabolisms and Inhibited by Antioxidants: Involvement of a
Thiol-Sensitive Redox Regulation in Eosinophil Cell Death
By
Bettina Wedi,
Julia Straede,
Britta Wieland, and
Alexander Kapp
From the Department of Dermatology and Allergology, Hannover Medical
University, Hannover, Germany.
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ABSTRACT |
The mechanisms for induction of eosinophil apoptosis remain
uncertain. The role of oxidative stress has not been investigated. The
present study was undertaken to determine the role of reactive oxygen
species (ROS) and selective antioxidants in eosinophil apoptosis.
Eosinophils were cultured with sodium arsenite (SA) known to induce
intracellular oxidative metabolites. There was a significant increase
in the rate of eosinophil apoptosis with low concentrations of
arsenite, whereas high concentrations showed rates of apoptosis similar
to control medium. Investigating the role of intracellular oxidants by
flow cytometry, we found that while inducing apoptosis, SA more than
anti-Fas resulted in a significant dose-dependent production of
intracellular H2O2. In contrast, the
extracellular release of superoxide decreased after stimulation with SA
or anti-Fas as assessed by lucigenin-dependent chemiluminescence. Coincubation experiments demonstrated that arsenite-induced apoptosis can be nearly completely prevented by
selective antioxidants such as glutathione (GSH) and N-acetyl-cysteine (NAC), but not dimethyl sulfoxide (DMSO) or taurine (TAUR). Moreover, GSH and NAC significantly reduced eosinophil apoptosis mediated by a
monoclonal antibody directed to Fas antigen. Next it was shown that GSH
and NAC, but not DMSO or TAUR, were able to significantly delay
spontaneous apoptosis in unstimulated eosinophils. Taken together,
these data point to an important role of oxygen-dependent mechanisms in
the regulation of eosinophil survival and apoptosis. We propose that
eosinophil apoptosis may be related to the ability of the cell to
maintain an appropriate oxidant-antioxidant balance.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE LAST FEW YEARS have provided
increasing evidence that apoptosis plays a major role in promoting
resolution of the acute and chronic inflammatory response. The
eosinophil granulocyte is able to release a variety of proinflammatory
mediators such as basic proteins (eosinophil cationic protein, major
basic protein), enzymes (eosinophil peroxidase), several cytokines, and
potent reactive oxygen species (ROS), and it is widely accepted that eosinophils contribute to tissue destruction during allergic
inflammation.1-3 In contrast to necrotic eosinophils,
apoptotic eosinophils are recognized by macrophages by a specific
process without disgorgement of their histotoxic
contents.4,5 Thus, to prevent tissue injury and
inflammation, it is important to determine the mechanisms by which
eosinophils undergo apoptosis. Known major triggers involved in
eosinophil apoptosis are glucocorticosteroids,6,7
theophylline,8 transforming growth
factor- ,9,10 abrogation of Fas/CD95 by its ligand or a
monoclonal antibody (MoAb),6,11 or CD69 perturbation with
MoAb.12 However, the cellular mechanisms involved in
eosinophil apoptosis are far from being clear. Recently, it was
suggested that bcl-2 family members and interleukin-1 converting
enzyme (ICE)-like proteases may be implicated in the regulation of
eosinophil apoptosis.13,14 The role of ROS has not been
investigated, although there are emerging data indicating that ROS can
serve as potent intracellular second messengers.15-17 ROS
are highly reactive metabolites generated during normal cell
metabolism; the cell contains many systems to limit their damaging
effects.18 Many agents, which induce apoptosis, are either
oxidants or stimulators of cellular oxidative metabolisms, whereas many
inhibitors of apoptosis show antioxidant activities.19 For
example, thiol antioxidants such as N-acetylcysteine (NAC) and
glutathione (GSH) completely block the activation-induced cell death of
T-cell hybridomas.20 Antioxidants, such as dimethyl
sulfoxide (DMSO) and o-phenanthroline, inhibited endotoxin-mediated
endothelial cell apoptosis.21 In human monocytes, Fas, a
potent mediator of apoptosis in many cell types, was shown to mediate
its apoptotic effect by a ROS-dependent pathway.22
Recently, it was shown that in naturally Fas resistant tumor cell lines
a reduction in intracellular O2
concentration induced sensitivity to Fas, whereas an increased intracellular superoxide anion concentration abrogated fas-mediated cell death.23 All of these data point to a major role of
intracellular oxidative metabolites in the regulation of apoptosis.
Therefore, we studied the role of the heavy metal salt sodium arsenite
(SA), which represents not only a tumor enhancer, but also a potent inducer of stress responses.24 SA is known to disturb the
oxygen metabolism in mitochondria, which are major sites of ROS
production. Previous studies demonstrated that SA-induced oxidative
stress resulted in apoptosis mediated by ROS in several cell
systems.25-27 Apart from the induction of intracellular
oxidative metabolites, SA also regulates intracellular GSH
levels.28 Furthermore, the effect of several antioxidants
in SA-, Fas-mediated, and spontaneous eosinophil apoptosis was
assessed, and we analyzed the possibility that SA and anti-Fas may
modulate the production of cellular oxidants.
In the present report, we provide clear evidence that antioxidants such
as NAC and GSH, but not taurine (TAUR) or DMSO, can completely block
SA- and Fas-induced apoptosis of eosinophils. In addition, these
antioxidants also inhibited spontaneous eosinophil apoptosis
implicating a major role for oxidative stress in eosinophil apoptosis.
Induction of apoptosis by SA and anti-Fas MoAb correlated with enhanced
production of intracellular ROS and decreased release of extracellular
ROS. These results point to oxidative stress as a central mechanism
regulating eosinophil apoptosis.
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MATERIALS AND METHODS |
Purification of eosinophils.
Peripheral blood eosinophils were separated by Ficoll density gradient
centrifugation and an improved immunomagnetic negative selection
procedure using anti-CD16 antibody-coated Dynabeads (Dynal A.S., Oslo,
Norway) as described in detail.29,30 Purity and viability
were 96% or greater after isolation, as assessed by Kimura staining
and trypan blue dye exclusion, respectively.
Cultures for eosinophil survival assay.
Eosinophils were resuspended in culture medium RPMI-1640 with 10%
fetal calf serum (FCS), 1% penicillin/streptomycin (Life Technologies,
Eggenstein, Germany) to 1 × 106/mL, and 1-mL aliquots
were added to 24-well flat-bottomed tissue culture plates and
stimulated with an equal volume of culture medium or the test factors
at 37°C, 5% CO2. SA, GSH, NAC, DMSO, and TAUR (all
from Sigma Chemicals, Deisenhofen, Germany) were diluted in
phosphate-buffered saline (PBS) in concentrations as indicated.
Anti-Fas MoAb (2 µg/mL), known to significantly induce apoptosis in
eosinophils,6 served as positive control (Immunotech, Marseille, France), and interleukin-3 (IL-3) (100 U/mL) was used to significantly inhibit eosinophil apoptosis (Genzyme Corp,
Cambridge, MA).
Eosinophil viability.
Viability of cultured eosinophils was assessed at different time points
by flow cytometric analysis monitoring cell size and ethidium bromide
(Ethbr; Molecular Probes, Eugene, OR) uptake. Briefly, after indicated
incubation times, cells received Ethbr (1 µg/mL), were incubated at
room temperature for 5 minutes, and analyzed on a FACScan flow
cytometer (Becton Dickinson Immunocytometry System, San Jose, CA).
Ethbr penetrates and intercalates into the DNA of nonviable cells,
causing them to fluoresce red with ultraviolet (UV) light (620 nm).
Identification of cells undergoing apoptosis by light and
fluorescence microscopy.
Light microscopic appearance of apoptotic eosinophils induced by SA and
anti-Fas and its modification by antioxidants and IL-3 was studied on
cytocentrifuge smears stained with Mayer's 0.1% hematoxylin.
Apoptotic eosinophils are smaller than normal eosinophils with
condensed nuclei, which are round in shape, but frequently the nuclear
structures become undiscernible among the numerous granules and the
cell diameter was strongly reduced as previously
described.31,32 Fluorescence microscopic appearance of
apoptotic eosinophils was studied on cytocentrifuge smears stained with
propidium iodide (PI) solution (100 µg/mL in PBS).
Cytofluorometric analysis of eosinophil apoptosis.
The proportion of apoptotic eosinophils displaying a hypodiploid DNA
peak was determined using a modification of the protocol of Nicoletti
et al.33-35 In brief, 1 × 105 eosinophils
were gently resuspended in 200 µL of hypotonic fluorochrome solution
(prodium iodide, 50 µg/mL in 0.1% sodium citrate plus 0.1% Triton
X-100) and incubated for 2 hours at 4°C. The red fluorescence of PI
of individual nuclei was measured using a FACScan flow cytometer. The
forward scatter and side scatter of cells were measured simultaneously. Cell debris was excluded from analysis by appropriately raising the
forward scatter threshold, but no gates were set. The PI fluorescence of individual nuclei with an acquisition of FL2 was plotted against forward scatter, and the data were registered on a logarithmic scale.
The minimum number of 5,000 events was collected. Apoptotic eosinophil
nuclei were distinguished by their hypodiploid DNA content from the
diploid DNA content of normal eosinophil nuclei.
DNA fragmentation assay.
The classical biochemical method for demonstrating apoptosis is the
presence of oligonucleosome-sized fragments of DNA, which when run on
agarose gels, produce a "ladder" pattern. DNA fragmentation assay
was performed as described.29 Electrophoresis of the
nucleosomal DNA fragments was performed in 1.8% agarose gels using 0.5 × Tris borate/EDTA electrophoresis buffer containing 0.1 µg/mL
ethidium bromide. After electrophoresis, fragments were visualized by
UV light and photographed.
Detection of hydrogen peroxide (H2O2).
To measure changes in intracellular H2O2, we
used the oxidation-sensitive fluorescent probe dihydrorhodamine (DHR).
DHR (Molecular Probes) is nonfluorescent, uncharged, and accumulates
within cells, whereas R123, the product of DHR oxidation, is
fluorescent, positively charged, and trapped within cells (emission at
525 nm).36 Eosinophils (1 × 106/mL) were
incubated with control medium or SA (50 µmol/L), anti-Fas (2 µg/mL), IL-3 (100 U/mL), or phorbol myristate acetate (PMA) (107 mol/L) for 20 hours at 37°C. Thereafter,
the cells were labeled with 1 µmol/L DHR for another 10 minutes at
37°C. Probes were placed on ice and green fluorescence (FL 1) was
measured immediately using a FACScan flow cytometer (Becton Dickinson
Immunocytometry System).
Chemiluminescence.
Highly sensitive lucigenin-dependent chemiluminescence representing a
reliable measurement for the release of ROS, which is independent from
the release of peroxidase, was quantitated by a single-photon imaging
system (MTP reader; Hamamatsu Photonics, Herrsching, Germany) as
described previously.37,38 After stimulation (in RPMI-1640
as indicated), eosinophils were gently pelleted and resuspended at 5 × 104/mL in Hanks' Balanced Salt Solution (HBSS), pH
7.4, containing 200 µmol/L lucigenin and 1 mg/mL bovine serum albumin
(BSA). One hundred-microliter aliquots were placed in flat-bottom,
white microtiter plates (Microfluor; Dynatech Deutschland,
Denkendorf, Germany). Measurements were performed in triplicate at
37°C. Integral counts were obtained from a 0- to 60-minute
incubation interval immediately after addition of stimuli and were
indicated as intensity counts in the figure.
Statistical analysis.
Unless otherwise stated, all data are presented as mean ± standard
error of mean (SEM). Analysis of variance (one-way ANOVA) was used for
comparing experimental group with control value. If global test for
differences was significant, pair-wise tests for differences between
groups were applied (Student's t-test for paired data or
Mann-Whitney rank sum test), a statistical software package (SigmaStat
for Windows; Jandel Scientific, Erkrath, Germany) being used. A
P value < .05 was considered statistically significant.
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RESULTS |
Effects of SA on eosinophil apoptosis.
To study the potential involvement of ROS in eosinophil apoptosis, we
assessed the effect of the heavy metal salt, SA, known to induce
intracellular oxygen metabolites and to regulate glutathione levels.39
SA significantly promoted apoptosis in a dose-dependent manner from 50 µmol/L up to 100 µmol/L, whereas higher concentrations (>200
µmol/L) were ineffective (Fig 1).
Anti-Fas MoAb consistently enhanced eosinophil apoptosis, whereas IL-3
stimulation resulted in a significant inhibition (not shown). After a
19-hour incubation, enhancement of apoptosis by SA (50 µmol/L) was
significantly higher compared with Fas-mediated apoptosis, whereas
after 42 hours, both stimuli resulted in similar numbers of apoptotic
nuclei (Fig 2). Moreover, time kinetic
studies showed that SA-mediated apoptosis occurred after about 8 hours
(Fig 3), similar to Fas-mediated apoptosis
(not shown). This time-delayed effect suggests that both arsenite and
anti-Fas trigger a series of events leading to DNA fragmentation.
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| Fig 1.
Effect of SA incubation with eosinophils for 19 hours on
percent apoptotic nuclei compared with medium control. The data were
determined by PI uptake (modified Nicoletti's protocol) and are given
as mean ± SEM values of 8 separate experiments with eosinophils of
different subjects. Fifty and 100 µmol/L SA significantly enhanced
eosinophil apoptosis, whereas at higher concentrations (200 to 500 µmol/L), this apoptosis inducing effect was reversed. **P < .01 versus control medium.
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| Fig 2.
Comparison of the effect of IL-3 (100 U/mL,  ),
anti-Fas MoAb (FAS, 1 µg/mL,  ), SA (50 µmol/L,  ), and medium
control (Med,  ) on eosinophil apoptosis. Data are presented as mean ± SEM of 8 separate experiments. Whereas IL-3 significantly inhibited
eosinophil apoptosis, FAS and SA significantly enhanced it. After 19 hours of incubation, SA resulted in a significantly higher enhancement
of apoptosis compared with Fas. **P < .01, ***P<
.001 versus Med.
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| Fig 3.
(A) Side scatter and forward scatter characteristics of
unstimulated eosinophils. (B) Time course for the SA-mediated
eosinophil apoptosis. Eosinophils were incubated with medium control or
50 µmol/L SA for 2 to 19 hours as indicated. Original flow cytometric
histograms demonstrating hypodiploid DNA peaks representing apoptotic
eosinophil nuclei are demonstrated. SA-mediated apoptosis (unfilled
graph) occurred after about 8 hours similar to Fas-mediated eosinophil
apoptosis (not shown) when compared with spontaneous apoptosis (medium
control, shown by the filled graph).
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To confirm that apoptosis is the mechanism of SA-mediated death,
eosinophils were analyzed by light microscopy of cytospin preparations,
by flow cytometry after incubation with Ethbr, by fluorescence
microscopy, and by DNA fragmentation formation (not shown). All of
these assays showed that SA-mediated cell death was due to apoptosis
and not to necrosis.
Effects of SA and anti-Fas on intracellular release of ROS.
We then analyzed the possibility that production of cellular oxidants
may be controlled by Fas or SA using the DHR method. Intracellular ROS,
primarily hydrogen peroxide H2O2, oxidate DHR, which rapidly accumulates within the cells to the fluorescent R123.
After 19 hours, the intracellular R123 fluorescence of eosinophils was
increased by eosinophil treatment with SA in a dose-dependent manner
(Fig 4A). The optimum concentration of SA
was 50 µmol/L, whereas higher concentrations resulted in a decrease
of the fluorescence. The dose response of oxidation using the DHR
method was similar to the dose-response effects of SA on apoptosis (Fig
4B).
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| Fig 4.
(A) Dose response of intracellular oxidation by SA.
Eosinophils were incubated with medium control or the indicated
concentration of SA (µmol/L). The mean green fluorescence
representing the intracellular release of H2O2
as determined by augmented R123 fluorescence (FL 1-H) is indicated. (B)
Intracellular H2O2 increase (A) was associated
with increased percentages of apoptotic nuclei. Eosinophils were
incubated for 19 hours with medium control or the indicated
concentration of SA and the hypodiploid DNA peak was measured using the
PI method. Nuclei were flow cytometrically analyzed and data plotted on
log histograms as red fluorescence intensity (x-axis) versus relative
cell number (y-axis). Percentages of hypodiploid DNA peaks
corresponding to apoptotic nuclei are indicated.
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In comparison to anti-Fas (2 µg/mL), 50 µmol/L SA resulted in a
significant higher augmentation of the cellular R123 fluorescence, indicating intracellular production of H2O2
(Fig 5).
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| Fig 5.
Comparison of SA-, anti-Fas-, and PMA-mediated
intracellular release of H2O2 as determined by
augmented R123 fluorescence (FL 1). Eosinophils were stimulated with
medium control or SA (50 µmol/L) or anti-Fas (2 µg/mL) for 19 hours, thereafter the cells were labeled with DHR (for details see
Materials and Methods). Stimulation with PMA (107 mol/L)
served as positive control. One representative of 4 experiments is
shown. The thin line represents the medium control, whereas the bold
line represents the stimulus indicated.
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Effects of SA and Fas on eosinophil respiratory burst.
Stimulation of eosinophils with anti CD95 MoAb at concentrations of 0.1 to 2 µg/mL for 20 hours resulted in a significant reduction of
respiratory burst (Fig 6A). This inhibition
was similar when the eosinophils were activated with f-Met-Leu-Phe
(fMLP, 107 mol/L, Fig 6) or PMA
(107 mol/L, not shown). SA alone both inhibited (10, 50, and 100 µmol/L) and potentiated (500 µmol/L, not shown)
superoxide generation after 10 minutes (Fig 6B).
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| Fig 6.
(A) Stimulation with anti-CD95 (anti-Fas) MoAb (0.1 to 2 µg/mL) for 19 hours resulted in a significant reduction of
respiratory burst in unstimulated () and fMLP (107
mol/L) activated ( ) eosinophils as determined by lucigenin-dependent
chemiluminescence. (B) Stimulation with 10 to 100 µmol/L SA ( ) for
10 minutes significantly blocked fMLP (107
mol/L)-induced eosinophil respiratory burst, whereas 500 µmol/L
resulted in a significant enhancement (not shown). Data are presented
as mean ± SEM intensity counts of 8 different experiments. *P
< .05, **P < .01.
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Inhibition of SA-, anti-Fas MoAb-mediated, and spontaneous eosinophil
apoptosis by antioxidants.
To address a possible involvement of ROS in SA- and Fas-mediated
apoptosis, eosinophils were incubated with selective antioxidants such
as NAC, GSH, TAUR, and DMSO. NAC can directly reduce ROS and increase
the intracellular level of glutathione, GSH is an endogenous
antioxidant reducing the intracellular H2O2
load, TAUR plays a functional role in the regulation of the oxidative
pathway, and the cell-membrane permeable DMSO represents a potent
scavenger .OH.
As shown in Figs 7 and
8, NAC and GSH inhibited SA- and
Fas-mediated apoptosis in a dose-dependent manner. Optimal inhibition was obtained at 10 mmol/L of each, demonstrating a role for cellular oxidants in SA- and FAS-mediated apoptosis.
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| Fig 7.
Effect of antioxidants on SA-induced eosinophil
apoptosis. Eosinophils were coincubated for 19 hours with SA (50 µmol/L) and GSH (), NAC (), or TAUR ( ) at the indicated
concentrations. Data are presented as mean ± SEM of 11 separate
experiments. NAC more than GSH inhibited SA-mediated eosinophil
apoptosis. Optimal concentration of GSH and NAC were 10 mmol/L each. In
contrast, TAUR and DMSO (not shown) showed no effect on SA-mediated
eosinophil apoptosis. *P < .05, **P < .01, ***P < .001 versus control (without stimulus).
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| Fig 8.
Effect of antioxidants on Fas-mediated eosinophil
apoptosis. Eosinophils were coincubated for 19 hours with anti-Fas MoAb
(2 µg/mL) and IL-3 (), GSH (), NAC (), or TAUR () at the
concentrations indicated. Data are presented as mean ± SEM of percent
apoptotic nuclei as compared with eosinophils incubated with control
medium (n = 8 separate experiments). NAC and GSH inhibited
Fas-mediated eosinophil apoptosis in a similar manner (optimal
concentrations 5 to 10 mmol/L). Higher concentrations of GSH and NAC
(15 to 20 mmol/L) induced apoptosis (not significant). Interestingly,
TAUR at 10 mg/mL also significantly blocked Fas-mediated apoptosis.
DMSO (0.5% to 4%) had no inhibitable effect (not shown). **P < .01 versus control (without stimulus). n.s., not significant.
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Additional metabolic inhibitors such as DMSO and TAUR did not alter SA-
or Fas-mediated eosinophil apoptosis, although they were reported to
block apoptosis in other cell types.21,40,41 However, after
19 hours (Fig 8), but not after 42 hours, taurine at 10 mg/mL reversed
Fas-mediated eosinophil apoptosis.
Finally, it was shown that NAC and GSH were also able to block
spontaneous apoptosis in unstimulated eosinophils
(Fig 9). This effect was most evident after
incubation for 42 hours in vitro. Thus, ROS may play a major role in
the regulation of eosinophil apoptosis.
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| Fig 9.
Effect of antioxidants on spontaneous eosinophil
apoptosis in vitro. Eosinophils were incubated with control medium,
GSH, NAC, DMSO, or TAUR for 19 hours () and 42 hours () at the
concentrations indicated. Data are presented as mean ± SEM of 8 separate experiments. GSH (optimal concentration, 5 to 10 mmol/L) more than NAC (optimal concentration, 10 mmol/L) inhibited
spontaneous eosinophil apoptosis. In contrast, higher concentrations of
NAC and GSH (20 mmol/L) and DMSO (>1%) significantly induced
eosinophil apoptosis. With TAUR, only 5 mg/mL significantly inhibited
spontaneous eosinophil apoptosis after 19 hours, but this effect was
not confirmed after incubation for 42 hours and was not dose-dependent.
*P < .05, **P < .01, ***P < .001 versus unstimulated control. n.s., not significant.
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DISCUSSION |
In contrast to necrosis, apoptosis represents a kind of cell death in
which cells are phagocytosed without provocation of any inflammatory
response. The eosinophil represents a major proinflammatory cell,
particularly in the allergic response. Parasitic infections and
allergic diseases, such as allergic rhinitis, bronchial asthma, and
atopic dermatitis, are often associated with peripheral blood and/or
tissue eosinophilia. Previously, we have shown that regulation of
eosinophil apoptosis may be pivotal in atopic diseases such as inhalant
allergy and atopic dermatitis.29 Moreover, it was shown
that eosinophil apoptosis is significantly delayed in eosinophil-rich nasal polyps of nonatopic subjects with aspirin-sensitive bronchial asthma,42 and that eosinophil apoptosis may be clinically
relevant in asthma.43 However, the cellular mechanisms of
eosinophil apoptosis are not clear, and only a few promoters with
potential therapeutical use have been described so far.
Herein we showed that apoptosis is significantly induced in human
eosinophils by the heavy metal salt, SA, known to generate cellular
oxidative metabolites. Previous studies demonstrated that oxidative
stress induced by SA resulted in apoptosis in neutrophils, endothelial
cells, and Chinese hamster ovary cells by generation of
ROS.25-27 In this study at low concentrations (10 to 100 µmol/L), SA induced apoptosis, whereas at high concentrations,
apoptosis was inhibited (>200 µmol/L). The demonstration of such
paradoxical actions is characteristic, even predictable, of systems
sensitive to redox regulation.
Analyzing the role of SA and Fas with regard to the production of
intracellular ROS in eosinophils, we found that induction of apoptosis
was associated with a dose-dependent increase in the intracellular
production of H2O2. A similar increase in
intracellular H2O2 was demonstrated previously
in myeloid cells treated with oxidants, but not in myeloid cells
treated with cytokines.44
In contrast, spontaneous, receptor-dependent (by FMLP), and receptor
independent stimulation (by PMA) of the extracellular release of ROS,
namely superoxide anion, was significantly reduced during apoptosis. In
this context, it may be noted that cytokines known to enhance
eosinophil survival and delay apoptosis such as granulocyte-macrophage
colony-stimulating factor (GM-CSF), IL-3, or IL-5 were described to
significantly enhance the release of
O2.45-47
That SA-induced eosinophil apoptosis may be mediated through an
oxygen-dependent mechanism was confirmed by the fact that it could be
prevented by low concentrations (5 to 10 mmol/L) of selective
antioxidants such as NAC and GSH. Interestingly, higher concentrations
of NAC and GSH (>15 mmol/L) resulted in apoptosis. This effect may be
explained by the fact that antioxidants not only function as
antioxidants, but that intrinsically they have prooxidant action as well.
Moreover, comparing SA-mediated apoptosis with apoptosis induced by the
MoAb to Fas antigen (anti-Fas), our data suggest similar mechanisms.
Both stimuli resulted in apoptosis after 8 hours, implying a
time-delayed mechanism resulting in DNA fragmentation. The
antioxidants, GSH and NAC, displayed at similar concentrations a
dose-dependent ability to reverse SA- and Fas-mediated apoptosis.
Furthermore, we clearly showed that ROS may represent key signals not
only in SA- and Fas-mediated, but also in spontaneous eosinophil
apoptosis. Spontaneous eosinophil apoptosis in culture was
significantly inhibited by the antioxidants NAC and GSH. This effect
became statistically significant after 19 hours and most evident after
42 hours. Interestingly, only NAC and GSH prevented spontaneous and SA-
as well as Fas-mediated eosinophil apoptosis, whereas DMSO and TAUR
were ineffective. In contrast, in neutrophils, not only GSH and NAC,
but also TAUR, inhibited SA-mediated apoptosis27 and it was
demonstrated that Escherichia coli (E coli) induces neutrophil apoptosis through an oxygen-dependent mechanism inhibitable by antioxidants such as DMSO (1%), GSH (25 mmol/L), and NAC (25 mmol/L).41 Moreover, TAUR and DMSO prevented apoptosis in
human endothelial cells.21,40 Interestingly, in
neutrophils, NAC and GSH have been shown to be ineffective in delaying
spontaneous apoptosis.48,49 In contrast, our data show
significant inhibition of spontaneous eosinophil apoptosis by NAC and
GSH. Whereas in neutrophils, 25 mmol/L of GSH and NAC were protective
in SA-mediated apoptosis, in eosinophils similar concentrations induced
apoptosis. These data implicate a species- or cell-specific sensitivity
to different oxidant species even between different granulocyte types.
TAUR is a semiessential amino acid contained in eosinophils, but in
lower concentrations compared with neutrophils.50 Our data
indicate that in eosinophils, TAUR represents an antioxidant not
modulating apoptosis. The cell-membrane permeable antioxidant, DMSO,
specifically removes .OH, but was not protective in
eosinophil apoptosis. GSH and NAC, which clearly blocked apoptosis,
represent thiol antioxidants modulating the intracellular level of GSH.
Thus, a thiol-dependent redox state may define if the eosinophil
survives or if it undergoes apoptosis or necrosis. This is confirmed by
the fact that SA, which significantly induced apoptosis, regulates
intracellular GSH levels.28 A role for intracellular GSH in
apoptosis has been recently demonstrated in T cells and in neural
cells.51-53
The major source of ROS in most cell types is probably the single
electron reduction of molecular oxygen to superoxide ions. Superoxide
ions are subsequently converted to hydrogen peroxide (H2O2) by the action of
Cu2+/Zn2+-dependent or
Mn2+-dependent superoxide dismutases (Cu/ZnSOD or
MnSOD), and H2O2 is then detoxified
by catalase or GSH peroxidase. However, H2O2 can also react to generate the highly damaging hydroxyl radical by the
Fe2+-dependent Fenton reaction or the
Fe2+-catalyzed Haber-Weiss reaction.18 The
oxygen metabolites .OH and H2O2
have been shown to induce apoptosis in a variety of cells. However, ROS
may not result in apoptosis per se, rather an oxidative shift in the
cellular redox state may modulate a stimulatory signal resulting in
apoptosis. It should be noted that eosinophils have various endogenous
enzymes, which can detoxify ROS such as catalase, GSH peroxidase, and
eosinophil peroxidase. Different contents of these enzymes may define
the intracellular concentration of ROS, thereby resulting in survival,
apoptosis, or necrosis.
Taken together, our data show that SA, which generates intracellular
oxidative metabolites, induces eosinophil apoptosis. It was shown that
spontaneous Fas- and SA-mediated eosinophil apoptosis can be blocked by
specific antioxidants modulating the GSH content. Our data
suggest that eosinophil apoptosis may be regulated by an
intracellular thiol-sensitive redox system. Moreover, we demonstrated
that eosinophil apoptosis is associated with a decrease in the
releasability of superoxide anion and with an increase in intracellular
hydrogen peroxide. Hebestreit et al34 showed
that nitric oxide (NO) inhibits Fas-mediated apoptosis in eosinophils,
indicating that reactive nitrogen species (RNS) may block apoptosis.
Thus, we hypothesize that ROS may induce and RNS may inhibit apoptosis.
Emerging data suggest that a balance between NO and superoxide
generation may be a critical determinant in the etiology of
many human diseases including chronic inflammation, atherosclerosis, neurodegenerative disorders, and cancer54; therefore, it is not astonishing that a balance between these radicals
may regulate eosinophil survival and cell death.
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FOOTNOTES |
Submitted July 14, 1998; accepted May 26, 1999.
Supported by Grants No. We-1912/3-1 and Ka-578/7-1 from the German
Research Foundation (DFG).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Bettina Wedi, MD, Department
of Dermatology and Allergology, Hannover Medical University, Ricklinger
Str 5, D-30449 Hannover, Germany.
| |
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