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Blood, Vol. 92 No. 3 (August 1), 1998:
pp. 778-783
RAPID COMMUNICATION
Role for Bcl-xL in Delayed Eosinophil Apoptosis
Mediated by Granulocyte-Macrophage Colony-Stimulating Factor and
Interleukin-5
By
Birgit Dibbert,
Isabelle Daigle,
Doris Braun,
Corinna Schranz,
Martina Weber,
Kurt Blaser,
Uwe Zangemeister-Wittke,
Arne N. Akbar, and
Hans-Uwe Simon
From the Swiss Institute of Allergy and Asthma Research (SIAF),
University of Zurich, Davos, Switzerland; Clinic for Dermatology and
Allergy, Davos, Switzerland; the Department of Internal Medicine, the
Division of Oncology, University Hospital, Zurich, Switzerland; and the
Department of Clinical Immunology, The Royal Free Hospital School of
Medicine, London, UK.
 |
ABSTRACT |
Eosinophils are potent inflammatory cells involved in allergic
reactions. Inhibition of apoptosis of purified eosinophils by certain
cytokines has been previously shown to be an important mechanism
causing tissue eosinophilia. To elucidate the role of Bcl-2 family
members in the inhibition of eosinophil apoptosis, we examined the
expression of the known anti-apoptotic genes Bcl-2, Bcl-xL,
and A1, as well as Bax and Bcl-xS, which promote apoptosis in other systems. We show herein that freshly isolated human
eosinophils express significant amounts of Bcl-xL and Bax,
but only little or no Bcl-2, Bcl-xS, or A1. As assessed by
reverse transcription-polymerase chain reaction, immunoblotting, flow
cytometry, and immunocytochemistry, we show that spontaneous eosinophil
apoptosis is associated with a decrease in Bcl-xL mRNA and
protein levels. In contrast, stimulation of the cells with
granulocyte-macrophage colony-stimulating factor (GM-CSF) or
interleukin-5 (IL-5) results in maintenance or upregulation of
Bcl-xL mRNA and protein levels. Moreover, Bcl-2 protein is not induced by GM-CSF or IL-5 in purified eosinophils. Bcl-2 protein is
also not expressed in tissue eosinophils as assessed by
immunohistochemistry using two different eosinophilic tissue models.
Furthermore, Bcl-xL antisense but not scrambled
phosphorothioate oligodeoxynucleotides can partially block the
cytokine-mediated rescue of apoptotic death in these cells. These data
suggest that Bcl-xL acts as an anti-apoptotic molecule in
eosinophils.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
EOSINOPHILIC INFILTRATION into tissues is
usually followed by elimination of these cells by apoptosis.
Cytokine-mediated inhibition of apoptosis contributes to the
accumulation of eosinophils in tissues.1 Such eosinophilia
is often observed in patients with chronic allergic diseases such as
bronchial asthma.2,3 In the past few years there has been
some progress in defining the tyrosine kinases that are activated by
the interleukin-3 (IL-3)/IL-5/granulocyte-macrophage colony-stimulating
factor (GM-CSF) receptor  -subunit. In contrast, no information is
available regarding the genetic control of eosinophil apoptosis in
allergic diseases.
Members of the Bcl-2 family of proteins are important regulators of
apoptosis in many cellular systems.4 The first member of
the family, Bcl-2, was originally cloned from the breakpoint of a
t(14;18) translocation present in many human B-cell lymphomas. Increased production of Bcl-2 protein as a result of t(14;18) translocation contributes to neoplastic B-cell expansion by preventing B-cell death.4 Besides Bcl-2, there are other members of
the family that inhibit apoptosis. For instance, the proteins encoded by Bcl-xL4,5 and A16 genes are also
potent blockers of apoptosis. In contrast, other members of the Bcl-2
family promote cell death. For example, Bax7 and
Bcl-xS5 are such pro-apoptotic proteins.
There have been some reports on the expression of Bcl-2 in eosinophils.
Whereas two groups8,9 obtained evidence for Bcl-2 expression, a third group10 did not observe significant
Bcl-2 levels. Therefore, the expression of Bcl-2 and other members of the Bcl-2 family in eosinophils does not appear to be clear at the
moment. Moreover, all previously published work analyzed gene expression, but no functional data are currently available on Bcl-2
family members in eosinophils. To better understand the regulation of
eosinophil apoptosis in chronic eosinophilic inflammation, we have
investigated the expression of Bcl-2, Bcl-xL, A1, Bax, and
Bcl-xS in freshly purified ex vivo eosinophils from control individuals and patients with atopic dermatitis as well as in tissue
eosinophils that reflect the in vivo situation of chronic eosinophilic
inflammation. Moreover, we measured the expression of these genes in
eosinophils cultured in the presence or absence of GM-CSF and IL-5 in
vitro. Furthermore, this study provides functional evidence for a role
of Bcl-xL in the control of eosinophil apoptosis.
| |
MATERIALS AND METHODS |
Antibodies.
Anti-Bcl-2 monoclonal antibody (MoAb), control IgG1 MoAb, swine
anti-rabbit fluorescein isothiocyanate (FITC)-conjugated secondary IgG
antibody, and control rabbit IgG were from Dako (Zurich, Switzerland). FITC-conjugated anti-Bcl-2 MoAb was from Ancell Corp (Bayport, MN).
Polyclonal rabbit antibodies against Bcl-x and Bax were purchased from
Santa Cruz Biotechnology, Inc (Santa Cruz, CA). Polyclonal rabbit
anti-Bcl-xS antibody was from Calbiochem-Novabiochem Corp (San Diego, CA). Goat anti-rabbit and anti-mouse horseradish peroxidase (HRP)-labeled secondary antibodies were obtained from Amersham International (Bucks, UK). FITC-conjugated control IgG1 MoAb was from
Coulter (Hialeah, FL). Anti-IL-3 and anti-GM-CSF MoAbs were purchased
from Genzyme (Cambridge, MA). Anti-IL-5 MoAb (5A5) was a kind gift
from Dr J. Tavernier (University of Gent, Gent, Belgium). Anti-eosinophil cationic protein (ECP) MoAb (EG1) was from Pharmacia (Uppsala, Sweden). Anti-CD16 MoAb microbeads were from Miltenyi Biotec
(Bergisch-Gladbach, Germany).
Eosinophil purification and cell cultures.
Eosinophils were purified from patients with atopic dermatitis and
healthy normal individuals as previously described.11-14 Eosinophils were cultured at 1 × 106/mL (expression
experiments) or 0.5 × 106/mL (antisense experiments)
for the indicated times using complete culture medium (RPMI 1640 supplemented with 10% fetal calf serum [FCS]). GM-CSF was a kind
gift from Dr T. Hartung (University of Konstanz, Konstanz, Germany).
IL-5 was obtained from Genzyme. The final cytokine concentrations were
25 ng/mL. Phosphorothioate oligodeoxynucleotide were synthesized by
Genset S.A. (Paris, France). Sequences used were as follows: antisense
Bcl-xL, 5 -TGT ATC CTT TCT GGG AAA GC-3; and
scrambled Bcl-xL, 5-TAA GTT CCG ATG CGA CTT
GT-3. These antisense molecules were selected from a panel of
different oligodeoxynucleotides as previously described.15 The oligodeoxynucleotides were given to purified eosinophils that had
been cultured for 20 hours in complete culture medium (at this time,
the cells did not express detectable Bcl-xL protein levels), and were delivered in the form of complexes with the cationic
lipid DOTAP (Boehringer Mannheim, Mannheim, Germany). Equal volumes of
oligodeoxynuleotide (6 µmol/L) and DOTAP (0.2 mmol/L) were mixed and
allowed to complex for 10 minutes at room temperature. The final
oligodeoxynucleotide concentrations were 0.45 µmol/L. The initial
phase (first 4 hours) of incubation with the oligodeoxynucleotides was
performed in medium without serum to increase the uptake of these
molecules by the eosinophils. Cells were then cultured again in
complete culture medium in the presence or absence of GM-CSF or IL-5
for another 24 hours (apoptosis assay) or 36 hours (cell death assay).
Therefore, cell viability was determined after the eosinophils had been
in culture for a total of 60 hours.
Reverse transcription-polymerase chain reaction (RT-PCR).
mRNA expression of Bcl-2 family members was studied using Southern blot
analysis linked to RT-PCR.1,11,12 Primers for Bcl-2
(5-ACA ACA TCG CCC TGT GGA TGA C-3 and 5-ATA GCT
GAT TCG ACG TTT TGC C-3), Bcl-x (5-GGA ATT CTT GGA CAA
TGG ACT GGT TGA-3 and 5-CCC AAG CTT GTA GAG TGG ATG GTC
AGT G-3), Bax (5-GGA ATT CTG ACG GCA ACT TCA ACT
GGG-3 and 5-GGA ATT CTT CCA GAT GGT GAG CGA GG-3),
and A1 (5-GAA GAT GAC AGA CTG TGA AT-3 and 5-TCA
ACA GTA TTG CTT CAG GAG-3) amplifications were synthesized (Bcl-2: HSC Biotechnology Service Centre, Toronto, Ontario, Canada; Bcl-x and Bax: R & D Systems, Abingdon, UK; A1: Microsynth, Balgach, Switzerland) according to previously published
sequences.6,16 For positive controls, PCR amplifications
were performed at the same time using cDNAs from HL-60 (Bcl-2, Bcl-x,
Bax) and Jurkat (A1) cells. For negative controls, PCR reactions were
performed without template DNA. Primers for -actin control
amplification were obtained from Clontech (Palo Alto, CA). The cDNAs
used for the probes were cloned by PCR amplification of positive
template DNA (cDNAs from HL-60, Jurkat cells, and neutrophils), and
their specificities confirmed by sequencing.
Immunofluorescence.
Purified eosinophils (0.1 × 106) were stained with
FITC-conjugated anti-Bcl-2 or control MoAb for 15 minutes at room
temperature after a 45-minute permeabilization of the cell membrane
with Permeafix (Ortho Diagnostic Systems, Raritan, NJ). To determine
Bcl-x and Bax expression, cells were initially incubated with 10 µg/mL anti-Bcl-x, anti-Bcl-xS, anti-Bax, or control
rabbit antibody for 15 minutes at room temperature, washed, and then
incubated with FITC-conjugated purified swine anti-rabbit IgG antibody
for 15 minutes at room temperature. Directly or indirectly stained
cells were washed, and fixed in phosphate-buffered saline
(PBS)-buffered 2% paraformaldehyde solution. Eosinophils were analyzed
by flow cytometry in an EPICS XL (Coulter).
Immunocytochemistry.
Bcl-x, Bcl-xS, and Bax protein expression was also
investigated by immunocytochemistry using a commercial kit (Histostain SP kit; Zymed Laboratories, San Francisco, CA) according to the manufacturer's instructions. Briefly, cytospins were prepared from 0.1 × 106 purified eosinophils. Slides were fixed in
freshly made and filtered PBS-buffered 4% paraformaldehyde solution
for 20 hours at room temperature in the dark. After washing with
H2O and PBS, slides were incubated with Peroxo-Block (Zymed
Laboratories) for 10 minutes to quench endogenous peroxidase activity.
After blocking with nonimmune serum, 1 µg/mL primary antibody was
added for 1 hour. This incubation was followed by addition of
biotinylated secondary antibody and streptavidin-peroxidase conjugate.
Bound peroxidase was detected by addition of substrate chromogen
mixture, followed by hematoxylin counterstaining. Slides were mounted,
and examined under a Zeiss Axioscope microscope (Oberkochen, Germany)
at a magnification of ×1,000.
For Bcl-2 staining, the alkaline phosphatase-anti-alkaline phosphatase
(APAAP) method was used.11 Staining was performed with a
commercial kit (Dako) according to the manufacturer's instructions.
Immunoprecipitation, gel electrophoresis, and immunoblotting.
Immunoprecipitation, gel electrophoresis, and immunoblotting were
performed as previously described,13 using anti-Bcl-x antibody.
Immunohistochemistry.
Immunohistochemistry was performed using nasal polyp and bladder cancer
tissues as previously described.1 Anti-ECP, anti-Bcl-2, anti-IL-3, anti-IL-5, or anti-GM-CSF MoAb immunostainings were performed using the APAAP method with a commercial kit (Dako) according
to the manufacturer's instructions. In additional experiments, sections were stained with anti-Bcl-x and anti-Bax antibody. These immunostainings were performed using a kit for rabbit primary antibody
(Zymed Laboratories). Sections were examined under a Zeiss Axioscope
microscope at a magnification of ×400 or ×1,000.
Determination of eosinophil death and apoptosis.
Cell death of eosinophils was assessed by uptake of 1 µmol/L ethidium
bromide and flow cytometric analysis (EPICS XL) as previously described.12-14 To determine whether eosinophil death was
apoptosis, eosinophils were morphologically examined.17
Cytospin preparations were made, stained with Diff-Quik (Baxter,
Düdingen, Switzerland), and analyzed as well as photographed
under a Zeiss Axioscope microscope at a magnification of ×1,000.
| |
RESULTS AND DISCUSSION |
Bcl-xL, but not Bcl-xS, is significantly
expressed by eosinophils.
We first measured mRNA levels by RT-PCR in freshly isolated as well as
cultured eosinophils in the presence or absence of the survival factors
GM-CSF or IL-5. Because the PCR primers used bind to sequences shared
by Bcl-xL and Bcl-xS, this technique allowed
simultaneous identification of both Bcl-x mRNAs. As shown in
Fig 1, freshly purified eosinophils
expressed Bcl-xL but not Bcl-xS mRNA. The
expression of Bcl-xL appeared to increase after GM-CSF
stimulation of the eosinophils in vitro but rapidly decreased when
eosinophils were cultured without cytokine support (Fig 1). To better
quantify the expression of Bcl-xL mRNA in response to eosinophil hematopoietins, RT-PCR was performed using different numbers
of cycles. These experiments clearly showed that Bcl-xL mRNA expression in eosinophils is downregulated in the absence and
upregulated in the presence of eosinophil survival factors such as
GM-CSF (Fig 2D) or IL-5 (not
presented).
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| Fig 1.
mRNA expression of Bcl-2 family members by purified blood
eosinophils as assessed by RT-PCR using 20 cycles of amplification. Freshly isolated peripheral blood eosinophils (0) expressed significant levels of mRNA for Bcl-xL and Bax, but not for Bcl-2,
Bcl-xS, and A1. When eosinophils were cultured in medium
without cytokine support, the levels of Bcl-xL rapidly
decreased, but were maintained or slightly increased after GM-CSF
stimulation in vitro. In contrast, Bax mRNA levels remained unchanged
when eosinophils were cultured in the presence or absence of GM-CSF.
The same results were observed when eosinophils were cultured with IL-5
(not presented). Furthermore, Bcl-2 and A1 mRNA levels did not increase
after incubation with eosinophil hematopoietins. As a control, human
-actin cDNA was amplified and PCR products were stained by ethidium
bromide in an agarose gel. Positive control amplifications were
performed using cDNAs from HL-60 (Bcl-x, Bax, Bcl-2) and Jurkat (A1)
cells. All data are representative of eight independent experiments.
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| Fig 2.
Bcl-xL, but not Bcl-xS,
protein is significantly expressed by purified blood eosinophils. (A)
Immunocytochemistry. Bcl-x (the antibody used reacts with both
Bcl-xL and Bcl-xS proteins) proteins were
expressed in freshly isolated peripheral blood eosinophils (0), and
levels rapidly decreased under conditions of cytokine withdrawal in
vitro, but were maintained in the presence GM-CSF. (B) Flow cytometry.
The Bcl-x protein expression observed in freshly isolated peripheral
blood eosinophils (0) was undetectable in the absence of eosinophil
hematopoietins and appeared to be increased by cytokine exposure in
cultured eosinophils after 20 hours. (C) Immunoprecipitation and
immunoblot. Bcl-xL (arrow), but not Bcl-xS (arrowhead), protein expression was observed in freshly isolated peripheral blood eosinophils (0). Bcl-xL protein expression
was increased after GM-CSF stimulation but decreased after cytokine withdrawal in vitro. (Right) The positions of molecular size standards. (D) Semiquantitative RT-PCR. Bcl-xL protein expression
correlates with the expression of Bcl-xL mRNA. Cytokine
withdrawal in vitro decreased Bcl-xL mRNA in eosinophils,
whereas stimulation with GM-CSF or IL-5 (not presented) increased
Bcl-xL mRNA expression. (Top) Numbers of PCR cycles. (E and
F) Immunocytochemistry and flow cytometry. No detectable
Bcl-xS (the antibody used reacts specifically with
Bcl-xS) protein expression was observed in freshly isolated
peripheral blood eosinophils (0). (A, B, E, and F) are representative
of eight independent experiments, (C and D) of three independent
experiments.
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To determine whether the expression of Bcl-xL and
Bcl-xS mRNAs correlates with the expression of their
proteins, we performed immunocytochemistry, flow cytometry, and
immunoblotting following immunoprecipitation using anti-Bcl-x (reacts
with both Bcl-xL and Bcl-xS proteins) and
specific anti-Bcl-xS antibodies. As shown in Fig 2A and B,
and Fig 3A, freshly purified blood and
tissue eosinophils expressed Bcl-x protein. Moreover, levels of Bcl-x protein were maintained or increased in GM-CSF-stimulated eosinophils. In contrast, levels of Bcl-x protein decreased under conditions of
cytokine withdrawal in vitro (Fig 2A through C). Immunoblotting studies
after anti-Bcl-x immunoprecipitation showed that the Bcl-x expression
observed by immunocytochemistry and flow cytometry was totally caused
by Bcl-xL expression, because no protein from the
Bcl-xS splice form was detected (Fig 2C). In agreement with these data and recently published work,10
Bcl-xS expression was also not seen using a specific
anti-Bcl-xS antibody as assessed by immunocytochemistry
(Fig 2E) and flow cytometry (Fig 2F). Together, these results suggest
that expression of Bcl-xL transcripts correlates with the
patterns of Bcl-xL protein expression in both freshly isolated or in vitro cultured eosinophils. Moreover, Bcl-x function does not appear to be regulated at the level of splicing in
eosinophils.
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| Fig 3.
Bcl-x and Bax, but not Bcl-2, proteins are significantly
expressed by eosinophils in eosinophilic tissues. (A)
Immunohistochemical staining with the indicated antibody using nasal
polyp tissues. Bcl-x and Bax, but not Bcl-2, proteins were detectable
in tissue eosinophils. In the Bcl-2 panel, some representative
eosinophils are marked with arrows. The Bcl-2+ control was
bone marrow tissue from a patient with a B-cell lymphoma. Moreover,
IL-5 protein was highly expressed in nasal polyp tissues. (B) Staining
of tissue infiltrated by bladder cancer cells as well as eosinophils.
Again, eosinophils expressed no detectable Bcl-2 protein (see cells
marked with an arrow in the third panel), although high levels of the
eosinophil hematopoietins were present in this tissue.
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Bax is expressed at high levels by eosinophils.
As shown in Fig 1, Bax mRNA was highly expressed in freshly isolated
eosinophils. In addition, Bax mRNA levels remained unchanged when
eosinophils were cultured in the presence or absence of GM-CSF (Fig 1)
or IL-5 (not presented).
To determine Bax protein expression, an anti-Bax antibody was used.
This antibody was able to stain tissue and purified blood eosinophils
(Fig 3A, and Fig 4A and B). Moreover, we
had no evidence for a change in Bax protein expression after in vitro
cell cultures of eosinophils in the presence or absence of cytokines
with anti-apoptotic properties (Fig 4A and B).
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| Fig 4.
Bax protein is expressed by purified blood eosinophils.
Bax protein levels remained unchanged when eosinophils were cultured in
the presence or absence of eosinophil hematopoietins, as seen by
immunocytochemistry (A) and flow cytometry (B). Each figure is
representative of eight independent experiments.
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Bcl-2 and A1 are expressed at very low levels by eosinophils.
We again used RT-PCR to determine Bcl-2 and A1 mRNA levels. As shown in
Fig 1, freshly isolated as well as cultured eosinophils in the presence
or absence of GM-CSF from normal control individuals and allergic
patients do not express Bcl-2 and A1 mRNA. Note that positive signals
for Bcl-2 and A1 were only obtained when RT-PCR with a higher number of
cycles (>25) was performed.
To determine whether eosinophils express Bcl-2 protein under conditions
where they are exposed to eosinophil hematopoietins, we investigated
tissues with significant eosinophilic infiltration using
immunohistochemistry. We have previously shown that delayed eosinophil
apoptosis occurs in nasal polyp tissues.1 Although high
levels of IL-5 were present in these tissues, Bcl-2 expression was not
observed in eosinophils (Fig 3A). Moreover, we had the chance to
investigate eosinophil-infiltrated tissue from a patient with bladder
cancer. Immunohistochemical examination of this tissue showed that the
eosinophil hematopoietins IL-3, IL-5, and GM-CSF were highly expressed,
especially by the cancer cells (Fig 3B). Again, the eosinophils under
these pathologic conditions did not appear to express significant
amounts of Bcl-2 protein (Fig. 3B). Furthermore, we used
immunocytochemistry and flow cytometric analysis to measure Bcl-2
protein levels in purified blood eosinophils. We did not observe
immunoreactive Bcl-2 in resting or GM-CSF-stimulated eosinophils (not
presented). Together, these results show that Bcl-2 and A1 are either
not present or are present at very low levels in human eosinophils.
Role for Bcl-xL in the regulation of eosinophil apoptosis
by cytokines.
Bcl-xL is an anti-apoptotic protein that regulates
apoptosis in many cellular systems,4,5 perhaps by
regulating the electrical and osmotic homeostasis of
mitochondria.17 Although the expression of
Bcl-xL in mature eosinophils might be little compared with umbilical cord blood-derived eosinophils as assessed by
immunoblotting,10 our in vivo and in vitro expression
studies suggested that Bcl-xL might be a good candidate
involved in the anti-apoptotic pathway mediated by cytokines in
eosinophils. To test this hypothesis, we determined the effect of
decreasing the levels of Bcl-xL expression. Eosinophils
that had been cultured for 20 hours did not express detectable levels
of Bcl-xL protein (Fig 2B). Exposure of these eosinophils
to an optimal dose of phosphorothioate-derivatized Bcl-xL
antisense oligodeoxynucleotides for another 28 hours clearly inhibited
GM-CSF- or IL-5-mediated upregulation of Bcl-xL protein levels, whereas scrambled control oligodeoxynucleotides had no effect
(Fig 5A).
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| Fig 5.
Bcl-xL antisense but not scrambled
oligodeoxynucleotides partially inhibit cytokine-mediated rescue of
spontaneous eosinophil apoptosis. (A) Eosinophils lost the expression
of Bcl-xL protein after in vitro culture for 24 hours (see
Fig 2B). In these cells, IL-5-induced Bcl-xL expression
within 24 hours (total cell culture time: 48 hours). Whereas scrambled
Bcl-xL oligodeoxynucleotides (sc Bcl-xL) had no
effect on IL-5-mediated upregulation of Bcl-xL protein
levels, no significant induction of Bcl-xL protein
expression was observed in eosinophils treated with Bcl-xL
antisense molecules (as Bcl-xL). The same data were
observed using GM-CSF to upregulate Bcl-xL protein
expression (not presented). This figure is representative of five
independent experiments. (B) Bcl-xL antisense but not scrambled oligodeoxynucleotides partially inhibited the effects of
GM-CSF and IL-5 on eosinophil viability (*; P < .001). In
addition, no significant effects of the oligodeoxynucleotides on
spontaneous eosinophil death were observed (not presented). Means ± SEM of six independent experiments are presented. (C)
Bcl-xL antisense and scrambled
oligodeoxynucleotides-treated eosinophils were cultured in the presence
of IL-5 as described in Materials and Methods for a total of 48 hours.
Cells were stained with Giemsa-May-Grünwald (Diff-Quik). Apoptotic eosinophils are characterized by
reduced cell volume as well as nuclear condensation. Some cells
demonstrate secondary necrosis (lower panel, the two cells in the right
margin; these cells show complete nuclear fragmentation). In these
experiments, we did not observe any cell death that was the result of
primary necrosis. The Bcl-xL antisense-treated cell
populations demonstrated much more apoptosis. Data are representative
of three independent experiments.
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The ability of antisense oligodeoxynucleotides to specifically inhibit
the upregulation of Bcl-xL protein allowed exploration of
its role in the prevention of apoptosis by eosinophil hematopoietins in
this cellular system. Apoptosis assays were performed after a total
cell culture time of 48 hours and cell death assays after 60 hours. No
significant effects of the oligodeoxynucleotides on the viability of
eosinophils in the absence of IL-5 or GM-CSF were observed (not
presented). In contrast, antisense but not scrambled Bcl-xL
oligodeoxynucleotides consistantly inhibited by approximately 34% the
ability of IL-5 or GM-CSF to prevent eosinophil death (Fig 5B). The
effect of the antisense molecules was highly significant
(t-test, P < .001). We also investigated whether the
observed cell death was apoptosis. Using analysis of the eosinophil
morphology,18 we observed much more pycnosis of the nucleus
and cell shrinkage in cells that had been treated with antisense
Bcl-xL oligodeoxynucleotides (Fig 5C). Quantitative analysis (counting of 300 cells) showed similar results as observed in
the cell death experiments: The antisense molecules reduced the
anti-apoptotic effect of IL-5 in average by 33% (not presented). Furthermore, no differences were observed between eosinophils from
normal control individuals and allergic patients.
These data suggest that Bcl-xL is functionally active
within the intracellular anti-apoptotic pathway mediated by cytokines such as IL-5 or GM-CSF in eosinophils. However, the inhibitory effect
of Bcl-xL antisense oligodeoxynucleotides on GM-CSF- or IL-5-mediated delay of eosinophil apoptosis was only partial. This
could be due in part to the fact that, although the antisense molecules
significantly blocked Bcl-xL protein synthesis, some Bcl-xL expression was still induced by IL-5. It is possible
that these relatively little Bcl-xL levels were enough to
promote GM-CSF or IL-5 responses in the majority of the eosinophils.
Another explanation for the incomplete block of cytokine-mediated
inhibition of eosinophil apoptosis by Bcl-xL antisense
oligodeoxynucleotides would be the involvement of other, as yet
unidentified, gene products. Therefore, further definition of the
genetic control of eosinophil apoptosis is required.
| |
FOOTNOTES |
Submitted March 17, 1998;
accepted May 19, 1998.
Supported by grants from the Swiss National Science Foundation
(32-49210.96) and the OPO-Foundation, Zurich, Switzerland (both to
H.-U.S.).
Address reprint requests to Hans-Uwe Simon, MD, Swiss Institute of
Allergy and Asthma Research, University of Zurich, Obere Strasse 22, CH-7270 Davos, Switzerland; e-mail: hus{at}siaf.unizh.ch.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank T. Hartung (University of Konstanz, Konstanz, Germany) for
GM-CSF, J. Tavernier (University of Gent, Gent, Belgium) for anti-IL-5
MoAb, A. Schapowal (Clinic for Allergy and Dermatology Davos, Davos,
Switzerland) for nasal polyp tissues, and St. Gratzl (Kantonsspital Chur, Chur, Switzerland) for the cancer tissue.
| |
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Abstract
Introduction
Abstract