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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 2 (July 15), 1998:
pp. 547-557
Role for Tyrosine Phosphorylation and Lyn Tyrosine Kinase in Fas
Receptor-Mediated Apoptosis in Eosinophils
By
Hans-Uwe Simon,
Shida Yousefi,
Birgit Dibbert,
Holger Hebestreit,
Martina Weber,
Donald R. Branch,
Kurt Blaser,
Francesca Levi-Schaffer, and
Gary P. Anderson
From the Swiss Institute of Allergy and Asthma Research (SIAF),
University of Zurich, Davos, Switzerland; the Research Section,
Canadian Red Cross Society, Toronto, Ontario, Canada; the Department of
Pharmacology, School of Pharmacy, Hebrew University-Hadassah Medical
School, Jerusalem, Israel; and the Department of Pharmacology,
University of Melbourne, Parkville, Victoria, Australia.
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ABSTRACT |
Fas ligand/Fas receptor molecular interactions have been implicated
as having an important function for the regulation of eosinophil
apoptosis. The purpose of the present study was to investigate
biochemical events triggered by the engagement of the Fas receptor in
freshly isolated human and mouse eosinophils. Activation of the Fas
receptor on eosinophils with the agonistic anti-Fas monoclonal antibody
(MoAb) resulted in increased tyrosine phosphorylation of several
intracellular proteins. The tyrosine kinase inhibitors lavendustin A
and genistein inhibited Fas receptor-induced cell death in both human
and mouse eosinophils in vitro and prevented, at least partially, Fas
receptor-mediated resolution of eosinophilic inflammation in a mouse in
vivo model of lung eosinophilia. In addition, in freshly purified human
eosinophils, lavendustin A prevented anti-Fas MoAb-induced proteolytic
cleavage of lamin B, suggesting that tyrosine kinases may amplify the
proteolytic signaling cascade within interleukin-1 converting enzyme
(ICE) family proteases. Moreover, the tyrosine kinase Lyn was
identified as being involved in Fas receptor-mediated cell death.
Collectively, these results demonstrate that tyrosine phosphorylation
is an important step in the generation of the Fas receptor-linked
transmembrane death signal in eosinophils and that Lyn participates in
this pathway.
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INTRODUCTION |
THE Fas RECEPTOR (CD95/APO-1) is a widely
expressed 45-kD transmembrane protein.1,2 Fas is a member
of the tumor necrosis factor (TNF)/nerve growth factor (NGF) receptor
family, which mediates apoptosis in many but not all
systems.3,4 It has been shown that Fas receptor/Fas ligand
molecular interactions are critical for peripheral T-cell
homeostasis.5-9 Therefore, it is possible that the Fas
receptor contributes to the regulation of cell death in a multicellular
organism to maintain correct cell numbers.
Functional Fas receptors have been recently described on human and
murine eosinophils.10-13 These studies suggested that Fas receptor/Fas ligand molecular interactions are involved in the regulation of eosinophil apoptosis. Moreover, functional defects in Fas
receptor-mediated eosinophil apoptosis may contribute to the chronic
eosinophilic inflammation often observed in allergic and asthmatic
patients. Therefore, it is important to identify the signaling pathways
transduced via the Fas receptor that lead to induction of apoptosis in
eosinophils.
Protein tyrosine kinases play a key role in the transduction of signals
through cell surface receptors.14 Protein tyrosine phosphorylation has also been shown to be involved in Fas receptor transmembrane signaling in many cellular systems,15-20
although it is controversial whether tyrosine kinase activation is
necessary for Fas receptor-mediated apoptosis.20-23
However, a role of tyrosine phosphorylation is supported by the
observation that expression of the hematopoietic cell phosphatase is a
prerequisite for Fas receptor-induced apoptosis in several lymphoid
cell lines.24,25 Another protein-tyrosine phosphatase,
FAP-1, has been shown to associate with the Fas receptor and to exert a
negative influence on Fas receptor signal transduction.26
In this study, we show that activation of the Fas receptor on
eosinophils results in increased protein-tyrosine phosphorylation of
several cellular proteins. Blockers of tyrosine kinases inhibit Fas
receptor-mediated cell death in both human and murine eosinophils. Moreover, we demonstrate that the proteolytic degradation of lamin B is
prevented by inhibition of tyrosine kinase activity, suggesting that
tyrosine kinases are part of a signaling pathway leading to
amplification of the proteolytic cascade within the interleukin-1 converting enzyme (ICE) proteases family. We also show that the tyrosine kinase Lyn is functionally active within the death pathway initiated by anti-Fas monoclonal antibody (MoAb) treatment of these cells. In addition, the activation of Lyn does not
appear to be dependent on early activition of ICE-like
proteases. Taken together, these findings suggest that
activation of tyrosine kinases, including Lyn, is important for Fas
receptor-mediated transmembrane signaling events leading to eosinophil
apoptosis.
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MATERIALS AND METHODS |
Antibodies.
Anti-Lyn MoAb and RC-20 Ab were obtained from Transduction
Laboratories (Lexington, KY). Antilamin B MoAb was from
Calbiochem-Novabiochem Corp (San Diego, CA). MoAb against
antiphosphotyrosine (ptyr), clone 4G10, and Fas receptor, clone CH-11
(IgM), were from Immunotech (Bühlmann Laboratories AG, Basel,
Switzerland). Control IgM and IgG2b MoAb were obtained from Dako
(Zurich, Switzerland). Anti-Fas MoAb (IgG3,  ) was a kind gift of Dr
P.H. Krammer (German Cancer Research Center, Heidelberg, Germany). This
MoAb recognizes an epitope on the extracellular part of the Fas
receptor.27 Anti-JNK Ab (C-17) was from Santa Cruz
Biotechnology (Santa Cruz, CA). GST-fusion protein of c-Jun (GST-c-Jun,
1-79) was obtained from Biomol (Plymouth Meeting, PA). Goat antimouse
horseradish peroxidase (HRP)-labeled secondary Ab was obtained from
Amersham International (Amersham, Bucks, UK). Anti-CD16 MoAb microbeads
were from Miltenyi Biotec (Bergisch-Gladbach, Germany). For the mouse
experiments, antimouse Fas receptor MoAb (clone Jo2, Armenian Hamster
IgG MoAb; Pharmingen, Palo Alto, CA) and irrelevant control Ab
(Armenian Hamster IgG; Pharmingen) were used. Biotinylated antimouse
B220, CD19, Thy1.2, CD4, CD8, and TER-119 MoAbs were also obtained from Pharmingen.
Purification of human blood eosinophils.
Human eosinophils were purified as previously
described.9,13,28-31 Briefly, peripheral blood mononuclear
cells (PBMC) were separated from peripheral blood of patients with
moderate eosinophilia (8% to 16%) by centrifugation on Ficoll-Hypaque
(Seromed-Fakola AG, Basel, Switzerland). The remaining cell population,
mainly granulocytes and erythrocytes, was treated with erythrocyte
lysis solution (155 mmol/L NH4Cl, 10 mmol/L
KHCO3, and 0.1 mmol/L EDTA, pH 7.3). The resulting
granulocyte population contained mainly neutrophils. To purify
eosinophils, the granulocyte population was incubated with anti-CD16
MoAb microbeads. CD16+ neutrophils were depleted by passing
the granulocytes through a magnetic cell separation (MACS; Miltenyi
Biotec) with column type C and an attached 21-gauge needle in the field
of a permanent magnet. The resulting cell populations contained 99%
eosinophils as determined by staining with Diff-Quik (Baxter,
Düdingen, Switzerland) and light microscopy.
Immunoprecipitation.
Human eosinophils (3 to 5 × 106/mL) were stimulated
in CG medium (Vitromex GmbH, Vilshofen, Germany) in the absence of
fetal calf serum (FCS) with 1 µg/mL anti-Fas MoAb (CH-11, IgM) or
control IgM MoAb for the indicated times at 37°C. In some
experiments, cells were pretreated with tyrosine kinase inhibitors at
the indicated concentrations for 1 hour at 37°C before anti-Fas
receptor stimulation. In other experiments, cells were pretreated with
the ICE-inhibitor II (N-Acetyl-Tyr-Val-Ala-Asp-chloromethylketone;
Ac-YVAD-cmk; Bachem, Bubendorf, Switzerland) at 100 nmol/L for 1 hour
at 37°C before the addition of anti-Fas MoAb. The reaction was
stopped by the addition of ice-cold phosphate-buffered saline
(PBS) containing 0.5 mmol/L
Na3VO4. Cells were immediately pelleted at
4°C and lysed with 1 mL of ice-cold 0.5% Triton X-100 lysis buffer
(50 mmol/L Tris-HCl, pH 7.4, 25 mmol/L KCl, 5 mmol/L MgCl2,
1 mmol/L EGTA, 1 mmol/L Na3VO4, 10 µg/mL
pepstatin A, 18 µg/mL aprotinin, 1 mmol/L phenylmethylsulfonyl
fluoride [PMSF], 1 µg/mL leupeptin, and 1 mmol/L
benzamidine) on ice for 10 to 15 minutes. Insoluble material was
removed by centrifugation at 4°C for 15 minutes at 15,800g.
Cell lysates were precleared with 100 µL of Pansorbin (Calbiochem-Novabiochem; 10% vol/vol solution of fixed Staphylococcus aureus Cowan I, prewashed 3 times in lysis buffer without proteinase inhibitors) at room temperature for 1 hour or at 4°C overnight. Lysate supernatants were further incubated with 50 µL of packed Sepharose 4B (Pharmacia, Uppsala, Sweden) coupled to bovine serum albumin (BSA) at room temperature for 1 hour. Supernatants were transferred to 25 to 30 µL protein G-Sepharose (Pharmacia) that had
been cross-linked with 4G10 MoAb, control IgG2b MoAb, anti-Fas IgG3
MoAb, or anti-Lyn MoAb overnight at 4°C and washed 4 times with
lysis buffer. Tubes were rotated at 4°C overnight and
immunocomplexes were washed 4 times with cold lysis buffer. The
proteins were eluted by adding Laemmli sample buffer plus 0.1 mol/L
dithiothreitol and heating at 95°C for 5 minutes before gel
electrophoresis.
Gel electrophoresis and immunoblotting.
Electrophoresis was conducted in 10% sodium dodecyl sulfate (SDS)
polyacrylamide gels, and the separated proteins were transferred to a
polyvinylidene fluoride filter (Immobilon-P; Millipore AG, Volketswil,
Switzerland). In the RC-20 experiments, the filters were incubated in
blocking solution I (10 mmol/L Tris-HCl, pH 7.5, 100 mmol/L NaCl, 0.1%
Tween 20, and 5% BSA; Sigma, Buchs, Switzerland). In the Fas receptor
and Lyn experiments, the filters were incubated in blocking solution II
(5% low fat milk, 10 mmol/L Tris-HCl, pH 7.5, 100 mmol/L NaCl, 0.1%
Tween 20). These blocking steps were performed at room temperature for
1 hour. In the lamin B experiments, a new rapid immunodetection method
without a blocking step was used according to the manufacturer's
instructions. Filters were incubated with HRP-labeled RC-20 Ab or
primary anti-Fas, anti-Lyn, or lamin B MoAb at room temperature for 1 to 2 hours. Binding of primary MoAb was detected by additional
incubation of the filters with goat antimouse HRP-labeled secondary Ab
at room temperature for 1 hour. Blots were developed by an enhanced chemiluminescence technique (ECL kit; Amersham) according to the manufacturer's instructions. Lamin B proteolysis was analyzed densitometrically using an automated scanner system supported by the
NIH-image program.
Determination of JNK activation.
Determination of JNK activation was performed as previously
described.32 Briefly, freshly purified human eosinophils
were cultured in the presence or absence of 16 µg/mL lavendustin A for 1 hour at 37°C and then stimulated with anti-Fas MoAb for another 1 hour at 37°C. Cells were lysed on ice for 15 minutes with
1 mL of ice-cold 0.5% Triton X-100 lysis buffer (0.5% Triton X-100,
50 mmol/L Tris-HCl, pH 7.4, 25 mmol/L KCl, 5 mmol/L MgCl2, 1 mmol/L EGTA, 1 mmol/L Na3VO4, 1 µg/mL
pepstatin A, 18 µg/mL aprotinin, 1 mmol/L PMSF, 1 µg/mL leupeptin,
and 1 mmol/L benzamidine). Insoluble material was removed by
centrifugation at 4°C for 15 minutes at 15,800g.
Supernatants were incubated on ice for 1 hour with 1 µg/mL anti-JNK
Ab. Forty microliters of protein A-Sepharose beads were added and tubes
were rotated at 4°C for 1 hour. The bead pellets were washed,
resuspended in 30 µL kinase buffer (20 mmol/L HEPES, pH 7.5, 2 mmol/L
dithiothreitol, 5 mmol/L MgCl2, 10 µmol/L
PMSF, 0.2 mmol/L Na3VO4, and 1 mmol/L
benzamidine), and incubated with 20 µg GST-c-Jun (1-79) and 1 µL
[ -32P]ATP (Amersham) at 30°C for 20 minutes. The
reaction was stopped by the addition of 40 µL 2× SDS, 2-ME
sample buffer. The samples were heated to 95°C for 5 minutes.
Electrophoresis was conducted in 15% SDS polyacrylamide gels, and
phosphorylation of c-Jun was detected by autoradiography.
Antisense oligodeoxynucleotides.
Eosinophils (106/mL) were cultured in RPMI 1640 plus 10%
FCS (complete culture medium) in the presence of phosphorothioate oligodeoxynucleotides (MWG-Biotech, Münchenstein, Germany) at 10 µmol/L. Sequences used were as follows: antisense, Lyn
5 -CATATTTCCCGCTCG-3; and sense, Lyn
5-CGAGCGGGAAATATG-3. Treatment of
eosinophils31 and neutrophils33 with
phosphorothioate-derivatized Lyn antisense oligodeoxynucleotide for 6 hours has been previously shown to significantly reduce Lyn protein
levels.
Determination of eosinophil death.
Human and mouse eosinophils (106/mL) were pretreated in the
presence or absence of tyrosine kinase inhibitors (1 hour) or
phosphorothioate oligodeoxynucleotides (6 hours). The following
tyrosine kinase inhibitors (Life Technologies Trading AG, Basel,
Switzerland) were used: genistein (10 to 50 µg/mL), herbimycin A (1 to 20 µg/mL), and lavendustin A (1 to 50 µg/mL). In other
experiments, human eosinophils were preincubated with the ICE-inhibitor
Ac-YVAD-cmk for 1 hour at the indicated concentrations. Cells were then
cultured in complete culture medium in the presence or absence of
1 µg/mL (human eosinophils) or 0.1 µg/mL (mouse eosinophils)
anti-Fas MoAb for the indicated times. Cell death of human eosinophils was assessed by uptake of 1 µmol/L ethidium bromide and flow
cytometric analysis (EPICS XL; Coulter Corp, Hialeah, FL), as
previously described.9,13,29,31 In the mouse experiments,
eosinophil death was determined by trypan blue exclusion using a
Neubauer chamber (Assistent GmbH, Sondheim, Germany) to
enumerate the cells. We have previously demonstrated that the Jo2
anti-Fas MoAb induces apoptosis in mouse eosinophils under identical
conditions.11
Determination of eosinophil apoptosis.
To determine whether eosinophil death was apoptosis, human eosinophils
were morphologically examined. Cells were pretreated in the presence or
absence of 16 µg/mL lavendustin A for 1 hour. Cells were then
stimulated with 1 µg/mL anti-Fas IgM MoAb in complete culture medium
for 8 hours. Cytospin preparations were made, stained with Diff-Quik,
and photographed under a Zeiss Axioscope microscope (Oberkochen, Germany) at 1,000× magnifications.
Mice and in vivo experimental procedures.
Eosinophilic inflammation was elicited as previously
described.11,34,35 Briefly, male Balb/c mice (16 to 20 g,
approximately 6 weeks old) were immunized
intraperitoneally with 10 µg of ovalbumin (OA; grade V;
Sigma, St Louis, MO) in 4 mg of alum (Serva, Heidelberg, Germany) on
days 0 and 14. Sham-immunized mice received 2 injections of alum alone.
Seven to 10 days after the last immunization, animals were challenged
with antigen by exposure to an aerosol of OA (50 mg/mL) for 20 minutes
using a Devilbiss ultrasonic nebulizer whose particle size
characteristics had been previously determined to produce 90%
particles less than 10 µm in diameter. Seventy-two hours after
allergen inhalation, mice were used for in vivo experiments or
eosinophil purification. To determine the effects of tyrosine kinase
inhibitors, mice were lightly anesthetized with 2.5% vol/vol enflurane
(Parke-Davis, Frankfurt/Main, Germany) in air delivered into a 200-mL
perspex narcosis chamber. During spontaneous breathing, 2× 10 µL aliquots of inhibitors (10 µg, total dose) or vehicle dimethyl
sulfoxide (DMSO; 0.1% in PBS) were administered to the lungs via nares. Mice are obligate nose breathers, and it has been
previously reported that this method effectively delivers greater than
90% of the complete drug volume to the lungs. After 1 hour, 35 µg/35
µL anti-Fas or control Ab was delivered to the lungs. Twenty-four
hours after Ab administration, mice were deeply anesthetized with 0.3 mL of 14% wt/vol urethane (Fluka, Buchs, Switzerland), the trachea was
cannulated, and the lungs were lavaged with 4× 0.3 mL aliquots of
PBS at room temperature. Total cell counts on individual
bronchoalveolar lavage (BAL) fluid aliquots were determined.
Differential BAL cell counts on 500 cells were performed according to
standard morphological criteria using cytospin preparations stained
with Diff-Quik and light microscopy. All procedures conformed to
international standards of animal care and welfare and were approved by
the Federal Animal Health Department (Basel, Switzerland).
Purification of mouse lung eosinophils.
Mouse eosinophils were purified from BAL fluids of immunized and
challenged mice using lectin affinity negative selection. Briefly, mice
were deeply anesthetized, and the lungs were lavaged. BAL cells
(106/mL) were incubated with 10 µg/mL biotinylated
Bandierea (Griffonia) simplifora isolectin I (Vector Labs, Burlingame,
CA), which preferentially binds alveolar macrophages and monocytes,
together with biotinylated rat antimouse MoAb directed against the
lymphocyte (B220, CD19, Thy1.2, CD4, CD8) and erythrocyte (TER-119)
surface antigens. Biotin-bound cells were removed using
streptavidin-coupled paramagnetic beads (Dynal, Hamburg, Germany). The
resulting cell populations contained 98% to 99% eosinophils as
determined by staining with Diff-Quik and light microscopy.
Statistical analysis.
Data are presented as the mean ± SEM. Statistical analysis was
performed by using ANOVA followed by multiple comparison corrected t-test. A probability value of less than .05 was considered
statistically significant.
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RESULTS |
Anti-Fas MoAb induces increased tyrosine phosphorylation in freshly
isolated human eosinophils.
It has been recently shown that eosinophils express the Fas
receptor10-13 and that granulocyte apoptosis is regulated
by tyrosine phosphorylation.29,31,33 To determine whether
tyrosine phosphorylation also participates in early signaling mediated
by the Fas receptor in human eosinophils, we analyzed intracellular
tyrosine phosphorylated proteins before and after stimulation of the
cells with anti-Fas MoAb. As shown in Fig
1, anti-Fas MoAb treatment was followed by increased tyrosine
phosphorylation of proteins of approximately 48, 60, 72, 82, 88, and 98 kD within 2 minutes. Phosphorylation of the 48-kD protein peaked at 2 minutes and that of all other proteins at 5 to 10 minutes.
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| Fig 1.
Fas receptor activation induces tyrosine phosphorylation
of several cellular proteins in human eosinophils. Freshly isolated eosinophils were stimulated with anti-Fas MoAb for the indicated times.
The cell lysates were immunoprecipitated with 4G10 MoAb, and the ptyr
immunoprecipitates were analyzed by immunoblotting with a MoAb to ptyr,
RC20. Increased tyrosine phosphorylation of 48-, 60-, 72-, 82-, 88-, and 98-kD proteins (arrows) was observed. The position of molecular
size standards (left). Data are representative of three independent
experiments.
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Furthermore, a series of tyrosine-phosphorylated proteins ranging from
56 to 110 kD coimmunoprecipitated with the Fas receptor. As
demonstrated in Fig 2A, a prominant 56-kD
protein appears to physically associate with the receptor in
nonstimulated eosinophils. In addition, a 98-kD protein and, in some
cases, a 82-kD protein associated with the receptor under these
conditions. Moreover, 88- and 110-kD tyrosine-phosphorylated proteins
inducibly coimmunoprecipitated with the Fas receptor. These
phosphorylation events were best detected between 1 and 10 minutes
after Fas receptor stimulation (Fig 2A, upper panel). In contrast,
stimulation of the eosinophils with control MoAb did not result in
increases in tyrosine phophorylation or in the appearance of 88- and
110-kD proteins (Fig 2A, lower panel). The Fas receptor could also be
coimmunoprecipitated with anti-ptyr but not control MoAb (Fig 2B),
further suggesting a physical association of the Fas receptor with
tyrosine-phosphorylated proteins.
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| Fig 2.
The Fas receptor is physically associated with
a series of tyrosine-phosphorylated proteins in human eosinophils. (A)
Cells (5 × 106) were stimulated with anti-Fas MoAb
(IgM) for the indicated times. The cell lysates were immunoprecipitated
with anti-Fas MoAb (IgG3). The immunoprecipitates were analyzed by
immunoblotting with an MoAb to ptyr, RC20. (Upper panel) A prominent
56-kD protein and two additional tyrosine-phosphorylated proteins
associated with the receptor in unstimulated cells. In addition, 88- and 110-kD tyrosine-phosphorylated proteins inducibly
coimmunoprecipitated with the Fas receptor. (Lower panel) Stimulation
of 3 × 106 eosinophils with control MoAb did not result
in an increase in either the quantity or the degree of tyrosine
phosphorylation of proteins associated with the Fas receptor. (B) Cells
(3 × 106) were stimulated with anti-Fas MoAb (IgM)
for the indicated times. The cell lysates were immunoprecipitated with
4G10 MoAb (upper panel) or control IgG2b MoAb (lower panel), and the
immunoprecipitates were examined for the presence of Fas receptor
protein. The Fas receptor inducibly coimmunoprecipitated with ptyr
proteins (upper panel). The positions of molecular size standards for
(A) and (B) are on the left. All data are representative
of at least three independent experiments.
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Tyrosine kinase blockers inhibit Fas receptor-mediated apoptosis in
human eosinophils.
The role of protein tyrosine kinases in the transmembrane signaling
cascade leading to eosinophil apoptosis was deduced from the ability of
tyrosine kinase blockers to inhibit Fas-mediated apoptosis. As an
initial control, we determined the effect of tyrosine kinase blockers
on anti-Fas MoAb-induced levels of tyrosine phosphorylation.
Eosinophils were pretreated with the inhibitors for 1 hour and then
stimulated for the indicated times with anti-Fas MoAb. Genistein at 20 µg/mL and lavendustin A at 16 µg/mL prevented the inducible
tyrosine phosphorylation of proteins after Fas receptor activation,
suggesting that both compounds indeed inhibit tyrosine kinases in human
eosinophils (data not presented).
We then measured the effect of several tyrosine kinase inhibitors on
Fas-mediated cell death and apoptosis. Pretreatment of freshly isolated
human eosinophils with lavendustin A for 1 hour inhibited, in a
dose-dependent manner, the death effect of anti-Fas MoAb treatment in
these cells (Fig 3A, right panel). Optimal
concentrations of lavendustin A (16 µg/mL) completely abrogated the
specific cell death initiated by Fas receptor cross-linking (Fig 3A,
left and right panel). Furthermore, the tyrosine kinase blockers
genistein (20 µg/mL) and herbimycin A (4 µg/mL) inhibited
Fas-mediated cell death by approximately twofold (Fig 3A, left panel).
This inhibitory effect did not go beyond these values if higher
concentrations of genistein or herbimycin A were used (data not
presented). In addition, no cytotoxic effect of lavendustin A and
genistein (up to 50 µg/mL) or herbimycin A (up to 20 µg/mL) was
observed (data not presented).
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| Fig 3.
Tyrosine kinase blockers inhibit Fas receptor-mediated
apoptosis in human eosinophils. (A) (Left panel) Freshly isolated
eosinophils were preincubated with lavendustin A (16 µg/mL),
genistein (20 µg/mL), or herbimycin A (4 µg/mL) and then stimulated
with anti-Fas MoAb for 30 hours. Lavendustin A completely, and the
other two kinase blockers partially, abrogated Fas receptor-mediated
eosinophil death. *P < .05; **P < .01. (Right
panel) Dose-dependent effect of lavendustin A in a time course
experiment. (B) Fas receptor-mediated cell death in human eosinophils
is due to apoptosis. Untreated or lavendustin A-pretreated eosinophils
were stimulated with anti-Fas MoAb. Cells were stained with
Giemsa-May-Grünwald (Diff-Quik). Apoptotic eosinophils are
characterized by reduced cell volume as well as nuclear condensation
and fragmentation. The lavendustin A-pretreated cell populations
demonstrated less apoptosis. Data are from six independent experiments.
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We next investigated whether the observed cell death was apoptosis.
Using analysis of the eosinophil morphology, we demonstrate that the
type of cell death induced by activation of the Fas receptor is
apoptotic and that lavendustin A inhibits Fas-mediated apoptosis (Fig
3B). Apoptotic eosinophils are characterized by several features of
apoptosis, including condensation of nuclear chromatin and reduced cell
volume.36 Fas-induced apoptosis was visible 8 hours after Fas receptor stimulation (Fig 3B).
Tyrosine kinase blockers inhibit Fas receptor-mediated cell death in
mouse eosinophils in vitro and resolution of eosinophilic inflammation
of the airways in vivo.
Because there are some discrepancies among the various reports
regarding the role of tyrosine phosphorylation in Fas receptor signaling,15-26 we also investigated the effects of
tyrosine kinase inhibitors on Fas receptor-mediated cell death in mouse
eosinophils. As shown in Fig 4A,
pretreatment of freshly purified mouse BAL eosinophils with 16 µg/mL
lavendustin A or 20 µg/mL genistein significantly inhibited the death
induced by anti-Fas MoAb treatment, suggesting that tyrosine kinase
activation is also involved in the death process after activation of
the Fas receptor in mouse eosinophils.
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| Fig 4.
Tyrosine kinase blockers inhibit Fas receptor-mediated
apoptosis in mouse eosinophils in vitro and in vivo. (A) Freshly
purified BAL eosinophils were preincubated with lavendustin (16 µg/mL) or genistein (20 µg/mL) and then stimulated with anti-Fas
MoAb for 24 hours. Both blockers significantly inhibited Fas
receptor-mediated death. Data are from a total of 45 animals.
*P < .05. (B) OA-immunized mice were challenged with antigen
to cause lung eosinophilia. The lungs of these mice were pretreated
with tyrosine kinase blocker for 1 hour and then with anti-Fas MoAb via
the intranasal route. BAL was performed 24 hours later, and the numbers
of eosinophils were determined. Anti-Fas MoAb-treated mice demonstrated
significantly reduced BAL eosinophils. This treatment had no
significant effect on BAL eosinophil numbers when mice were pretreated
with lavendustin A. *P < .05; **P < .01. (C)
Examples for BAL cytology showing disappearance of eosinophils as a
consequence of anti-Fas MoAb treatment and abrogation of this effect by
lavendustin A. Data are from 6 to 8 animals per condition.
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We have previously reported that allergen provocation of OA-immunized
mice results in eosinophil infiltration of the lungs 72 hours later and
that activation of the Fas receptor by anti-Fas MoAb leads to
eosinophil apoptosis and the resolution of eosinophilic inflammation.11 Using this in vivo model, 72 hours after
allergen provocation, the same tyrosine kinase inhibitors used in the
in vitro systems were administered to the lungs via the intranasal route. Control mice received an equal volume of DMSO. One hour after
inhibitor administration, anti-Fas or control Ab was administered to
the lungs. BAL was performed 24 hours later and the cellular composition determined. As shown in Fig 4B and C (and previously reported11), anti-Fas MoAb treatment resulted in a
resolution of the eosinophilic lung inflammation in control mice that
had received DMSO. This effect has been shown to be due to the
induction of eosinophil apoptosis.11 There was no change in
the number of BAL eosinophils after control Ab treatment compared with
before Ab administration (not presented). Moreover, pretreatment of the mice with 10 µg of tyrosine kinase inhibitor for 1 hour prevented, at
least partially, the reduction of BAL eosinophil numbers induced by
anti-Fas MoAb administration (Fig 4B and C). Thus, these data suggest
that tyrosine kinase inhibitors exert similar blocking effects on Fas
receptor-mediated cell death in human and mouse eosinophils.
Tyrosine kinase activation is required for Fas receptor-mediated
lamin protease(s) and JNK activation in human eosinophils.
A critical event in the Fas receptor-mediated apoptotic program seems
to be the activation of ICE-like proteases.37,38 The
proteolytic cleavage of the nuclear lamins by ICE family members facilitates the nuclear events of apoptosis.39,40 For
example, lamin cleavage seems to be required for packaging of the
condensed chromatin into apoptotic bodies.41 Therefore,
lamin cleavage is important for the nuclear events of apoptosis and
constitutes an indirect marker for the activation of ICE family
proteases.41,42 Because lavendustin A completely blocked
the death signal via the Fas receptor in human eosinophils, we
investigated the role of tyrosine kinases in ICE activation. To measure
activation of ICE family proteases in lavendustin A-pretreated and
untreated cells, we assessed Fas receptor-mediated proteolytic cleavage of lamin B. Lamin B is cleaved into a fragment of 46 kD, which can be
visualized by Western blotting.39
As shown in Fig 5A, partial lamin B
proteolysis is observed in freshly isolated human eosinophils, as
demonstrated by a clearly visible 46-kD fragment. This implies that ICE
proteases are active in eosinophils to facilitate spontaneous
apoptosis. Treatment of human eosinophils with anti-Fas MoAb was
followed by increased cleavage of lamin B clearly visible 8 hours upon
stimulation (Fig 5A, upper panel). In contrast, when these cells were
pretreated with lavendustin A before the anti-Fas MoAb stimulation,
increased lamin B degradation did not occur before 18 hours (Fig 5A,
lower panel). Moreover, as analyzed by densitometry, approximately 70% to 75% (compared with freshly isolated cells) of the lamin B was degraded in Fas receptor-activated eosinophils which have not been
pretreated, whereas lamin B reduction was only about 20% to 25% in
lavendustin A-pretreated cells after 18 hours (Fig 5A). In addition,
increased lamin B degradation in untreated and lavendustin A-treated
eosinophils also did not occur before 18 hours in culture (data not
presented), indicating that lavendustin A prevents Fas receptor-mediated but not spontaneous lamin B cleavage in these cells.
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| Fig 5.
Increased proteolytic cleavage of lamin B and
phosphorylation of c-jun after stimulation of the Fas receptor are
tyrosine kinase-dependent events in human eosinophils. (A) Freshly
isolated lavendustin A (16 µg/mL) -pretreated and untreated
eosinophils were stimulated with anti-Fas MoAb for the indicated times.
The cell lysates were analyzed by immunoblotting with an antilamin B
MoAb. A proteolytic fragment of lamin B (arrowhead) was already present
in freshly purified eosinophils. After 8 hours, a reduction in the
amount of lamin B was observed in untreated but not in lavendustin
A-pretreated cells. The intensity (od-Bkg/mm2) of the lamin
B signal was analyzed by densitometry. (Upper panel) 1,787 (100%),
1,626 (91%), 1,881 (105%), 775 (43%), and 496 (28%). (Lower panel)
1,459 (100%), 1,551 (106%), 1,375 (94%), 1,618 (111%), and 1,156 (79%). The position of molecular size standards (left). (B)
Lavendustin A abrogated phosphorylation of c-Jun. Freshly isolated
lavendustin A (16 µg/mL) -pretreated and untreated eosinophils were
stimulated with anti-Fas MoAb for 60 minutes. The cell lysates were
immunoprecipitated with an anti-JNK Ab, and phosphorylation of
GST-c-Jun (1-79) was observed by an in vitro kinase assay. No increase
in phosphorylation of c-Jun was found in lavendustin A-pretreated
eosinophils, suggesting that tyrosine kinase(s) activation is essential
for Fas receptor-mediated JNK activation. All data are from at least
three independent experiments.
|
|
Besides ICE family proteases, we also searched for other possible
molecules involved within the signaling cascade transduced by
cytoplasmic tyrosine kinases. Previously published work has suggested
that JNK is a Fas receptor-activated kinase.23,32,43,44 Therefore, phosphorylation of GST-c-Jun (1-79) was used to measure JNK
activity by an immune-complex assay. As shown in Fig 5B, anti-Fas MoAb
stimulation of eosinophils for 60 minutes induced phosphorylation of
c-Jun. In contrast, this increase was abolished by pretreatment with
lavendustin A. Together, these data suggest a link between tyrosine
kinase(s), lamin protease(s), JNK activation, and apoptosis in Fas
receptor signal transduction in freshly purified human eosinophils.
The tyrosine kinase Lyn is involved in Fas receptor-mediated death in
human eosinophils.
Because the Fas receptor does not contain an intrinsic kinase
activity,1,2 our data suggested that cytoplasmic tyrosine kinases are activated rapidly after cross-linking of the Fas receptor. The prominent 56-kD protein that is physically associated with the Fas
receptor (Fig 2A) could represent a member of the Src family of
tyrosine kinases. It has been previously shown that a member of this
family, the tyrosine kinase Lyn, is highly expressed in human
eosinophils31,45,46 and neutrophils.33,47,48 Therefore, we attempted to determine whether this tyrosine kinase is
also involved in the Fas receptor signaling pathway. To do so, Lyn
immunoprecipitates were examined. As shown in
Fig 6A, Fas receptor stimulation by
anti-Fas MoAb induced increases in tyrosine phosphorylation of Lyn
within 3 minutes, suggesting activation of Lyn in human eosinophils
after initiation of the death signal by receptor cross-linking.
View larger version (25K):
[in this window]
[in a new window]
| Fig 6.
Important role for Lyn tyrosine kinase in Fas
receptor-mediated signaling in human eosinophils. (A) Freshly isolated
eosinophils were stimulated with anti-Fas MoAb for the indicated times.
The cell lysates were immunoprecipitated with an anti-Lyn MoAb and then
analyzed by immunoblotting with a MoAb to ptyr, RC20. Increases in
tyrosine phosphorylation of Lyn were observed within 3 minutes. The
same Lyn immunoprecipitates were used for Western blotting with
anti-Lyn MoAb to check for immunoprecipitation efficiency (bottom). The
positions of molecular size standards (left). Data are representative
of three independent experiments. (B) (Right panel) Eosinophils were
cultured in the presence of 10 µmol/L Lyn antisense or sense
oligodeoxynucleotides for 6 hours. Decreased Lyn expression in
eosinophils cultured with Lyn antisense was observed. Lyn sense
oligodeoxynucleotides had no or only little effects on Lyn protein
expression. (Left panel) Cells were then stimulated with anti-Fas MoAb
for 18 hours. Statistically significant inhibition of Fas
receptor-mediated cell death was observed in Lyn antisense but not
sense treated eosinophils. Data are from six independent experiments.
*P < .05. (C) (Left panel) The ICE inhibitor Ac-YVAD-cmk
blocked in a dose-dependent manner Fas receptor-mediated but not
spontaneous eosinophils death. Purified eosinophils were cultured for
24 hours. Cell viability was assessed by uptake of 1 µmol/L ethidium
bromide and flow cytometric analysis. Values are means ± SEM of
three independent experiments. (Right panel) Ac-YVAD-cmk did not
prevent activation of Lyn following Fas receptor cross-linking. Freshly
isolated Ac-YVAD-cmk (0.1 µmol/L) -pretreated and untreated
eosinophils were stimulated with anti-Fas MoAb for the indicated times.
The cell lysates were immunoprecipitated with an anti-Lyn MoAb and then
analyzed by immunoblotting with a MoAb to ptyr, RC20. Increases in
tyrosine phosphorylation of Lyn were observed in both untreated and
Ac-YVAD-cmk-treated eosinophils. The same Lyn immunoprecipitates were
used for Western blotting with anti-Lyn MoAb to check for
immunoprecipitation efficiency (bottom).
|
|
To examine the specific role of Lyn in Fas receptor-mediated eosinophil
death, we decreased the level of Lyn expression by antisense
oligodeoxynucleotides. Antisense oligodeoxynucleotides corresponding to
the Lyn tyrosine kinase have been previously used to inhibit Lyn
protein expression in eosinophils,31
neutrophils,33 and B-lineage lymphoma cells.49
As previously reported31,33 and demonstrated in Fig 6B
(right panel), eosinophils exposed to 10 µmol/L of
phosphorothioate-derivatized Lyn antisense oligodeoxynucleotides for 6 hours expressed reduced Lyn protein, whereas Lyn sense
oligodeoxynucleotides did not significantly alter Lyn protein levels.
The ability of antisense oligodeoxynucleotides to specifically decrease
the expression of Lyn allowed exploration of the role of Lyn in Fas
receptor-mediated cell death of eosinophils. Lyn antisense and sense
oligodeoxynucleotides were added to freshly isolated human eosinophils
6 hours before the cells were exposed to anti-Fas MoAb. As shown in Fig
6B (left panel), Lyn antisense oligodeoxynucleotides significantly
reduced the ability of anti-Fas MoAb to induce eosinophil death. In
contrast, Lyn sense oligodeoxynucleotides failed to inhibit Fas
receptor-mediated death. These data suggest a role for Lyn in the
apoptotic signaling pathway induced by activation of the Fas receptor
in human eosinophils.
We next investigated whether ICE-like proteases activation is required
for tyrosine kinase, in particular Lyn, activation. As demonstrated in
Fig 6C (left panel), the ICE-inhibitor II Ac-YVAD-cmk completely
abrogates Fas receptor-mediated death in human eosinophils. However,
this inhibitor did not block tyrosine phosphorylation of Lyn after
engagement of the Fas receptor (Fig 6C, right panel). These data
suggest that Lyn activation does not depend on activation of ICE-like
proteases.
| |
DISCUSSION |
Allergic diseases of the respiratory tract are associated with a marked
eosinophilic inflammation.50-52 Several groups have studied
the mechanisms of this eosinophil accumulation. For example, mechanisms
of selective recruitment in tissue sites of allergic diseases have been
suggested based on the identification of specific eosinophil
chemotactic factors53-55 or on specific eosinophil adhesion to cytokine-activated endothelial cells.56-61 Moreover,
cytokines such as interleukin-3 (IL-3), IL-5, granulocyte-macrophage
colony-stimulating factor (GM-CSF), or interferon- (IFN-)
dramatically increase the life-span of purified eosinophils by
inhibiting their apoptotic cell death in vitro.29,31,62-67
Furthermore, delayed eosinophil apoptosis as a mechanism causing tissue
eosinophilia has been recently demonstrated in nasal polyp
tissues,68 suggesting that this process also occurs in vivo
under certain pathological conditions. Thus, besides specific
eosinophil adhesion and chemotaxis, inhibition of eosinophil apoptosis
also contributes to the eosinophilia observed in patients with chronic
eosinophilic disorders.
Besides cytokines, eosinophil apoptosis also seems to be regulated by
at least one member of the TNF/NGF receptor superfamily, namely the Fas
receptor.10-13 Cross-linking of the Fas receptor is
associated with the induction of apoptosis in eosinophils even in the
presence of eosinophil hematopoietins.10,11 These studies suggested that eosinophil apoptosis does not only occur passively after
the withdrawal or absence of eosinophil survival factors, but can be
actively induced via activation of the Fas receptor. Although it is
still not clear whether cognate interactions between eosinophils and
other cells expressing Fas ligand actually occur in vivo, it has been
shown that eosinophils from eosinophilic donors often do not express
functional Fas receptors, although Fas protein is normally expressed in
these cells.13 Therefore, it is important to identify the
signaling pathways that are transduced via the Fas receptor in
eosinophils and to unravel the molecular mechanisms by which Fas
receptor-mediated apoptosis can be inhibited.
Recently, there has been some progress in the understanding of the
signal transduction pathways activated via the Fas receptor in cell
lines expressing functional Fas receptors. A series of intracellular
proteins (CAPs) inducibly coimmunoprecipitate with the Fas receptor to
form a death-inducing signaling complex (DISC).69 CAP1 and
2 have been identified and represent FADD/MORT1.69-71 Molecular characterization of CAP4 demonstrated that the molecule is an
ICE-like protease, FLICE/MACH.72,73 Proteases of the ICE
family play a key role within a protease cascade responsible for the
structural changes of apoptosis.37,38 The identification of
FLICE as a member of the DISC suggests a direct pathway where Fas
receptor activation leads to triggering of the protease cascade.
In the present study, we have demonstrated that tyrosine
phosphorylation is an important event involved in transmembrane signal transduction via the Fas receptor in human and mouse eosinophils. The
Fas receptor is physically associated with a number of
tyrosine-phosphorylated proteins, as shown by coimmunoprecipitation
studies with Fas receptor. Also, the Fas receptor coimmunoprecipitated
with tyrosine phosphorylated proteins, confirming this link. These data
are in agreement with previously published work demonstrating increased
tyrosine phosphorylation after Fas receptor cross-linking in several
cellular systems.15,16,19,20 Moreover, phosphorylation of
both tyrosine residues within the intracellular part of the human Fas
receptor has been recently demonstrated.19 Interestingly,
these two tyrosine residues are also present in the amino acid sequence
predicted for the murine Fas receptor cDNA,2 consistent
with the suggestion that these tyrosine residues are important for
signal transduction.
Tyrosine kinase activation was also reported in response to stimulation
of other members of the TNF/NGF receptor family. Extensive work has
elucidated a role for tyrosine phosphorylation via CD40, a molecule
that also belongs to this family of receptors and induces apoptosis
under certain conditions.74 CD40 activation results in
increases in tyrosine phosphorylation as well as activation of Lyn,
Syk, Fyn, and Blk in B cells.75-77 Increased tyrosine
phosphorylation has also been observed in TNF-treated
neutrophils78 and endothelial cells.79
Therefore, tyrosine phosphorylation appears to play a major role in
Fas, TNF, and CD40 receptor-mediated transmembrane signaling.
Because activation of the Fas receptor stimulates eosinophil
apoptosis10-13 and increases tyrosine phophorylation in
human eosinophils (Figs 1, 2, and 6), we examined the effect of
tyrosine kinase inhibitors on Fas receptor-mediated cell death. Because a direct involvement of tyrosine phosphorylation in Fas
receptor-induced apoptosis is still controversial,20-23 we
performed these experiments in both human and mouse systems. Among the
tyrosine kinase blockers tested in this study, lavendustin A had the
highest potency to inhibit Fas receptor-mediated death in both cultured
human blood eosinophils and mouse lung eosinophils. To complement our
studies of purified cell populations, we used an in vivo approach
analyzing eosinophilic lung inflammation induced by allergen exposure
in mice.11 Administration of anti-Fas MoAb resulted in a
significant reduction in the number of eosinophils in the lung after 24 hours. In contrast, if the mice were pretreated with tyrosine kinase inhibitor, the resolution of eosinophilic inflammation was less prominent. Moreover, in this in vivo system, we also observed a similar
potency of tyrosine kinase inhibitors compared with the in vitro
experiments (lavendustin A > genistein). Taken together, these
results strongly implicate tyrosine kinase activation as likely
involved in the death response after cross-linking of the Fas receptor
in eosinophils.
Recent studies suggested that there is a direct physical connection
between the Fas receptor and the basal death machinery, the proteases
of the ICE family.44,69-73 To determine whether tyrosine
kinase activation contributes to the protease cascade initiated by
receptor cross-linking, we observed the effects of inhibition of
tyrosine kinases on lamin B cleavage. Lamins represent key substrates
for ICE family proteases activated during the apoptotic process.39-42 Therefore, lamin B cleavage was used in this
study as a marker to measure Fas receptor-mediated activation of ICE family members. We observed increased cleavage of lamin B in
eosinophils after Fas receptor stimulation. This increased cleavage was
prevented by lavendustin A, suggesting that tyrosine kinase(s)
activation may be a prerequisite for full activation of the cascade of
ICE family proteases.
Besides activation of tyrosine kinases and ICE family proteases,
multiple other signaling events have been reported to be involved in
Fas receptor-mediated apoptosis. For example, another signaling pathway
may involve activation of Ras16 and, consequently, as
demonstrated in this study, activation of JNK. Because lavendustin A
completely blocked Fas receptor-mediated apoptosis, it is possible that
amplification of the ICE family proteases cascade via tyrosine kinases,
Ras, and JNK is essential for the apoptotic process in eosinophils.
This assumption is further supported by very recent observations
demonstrating that activation of CPP32-like proteases occurs distal to
MAPK and JNK activation.80,81 Furthermore, it has been
demonstrated that JNK activation correlates with cell death.23,32,43,44,80,82-84
The demonstration of tyrosine phosphorylation of the Fas
receptor,19 which does not itself contain an intrinsic
kinase activity,1,2 suggests that a cytoplasmic tyrosine
kinase is associated with the receptor. The prominent
tyrosine-phosphorylated 56-kD protein that coimmunoprecipitated with
the Fas receptor provides evidence that at least one member of the Src
family of tyrosine kinases may associate with, and act as signal
tranducer for, this death receptor. Lyn has been previously
demonstrated to be highly expressed in both
neutrophils33,47,48 and eosinophils.31,45,46 To characterize the role of Lyn, Lyn immunoprecipitates were examined to
assay tyrosine phosphorylation of the kinase. After exposure to
anti-Fas MoAb, we observed a rapid increase in Lyn tyrosine phosphorylation, suggesting activation of this kinase. To specifically examine the possibility that Lyn participates in the death pathway after Fas receptor activation in eosinophils, we determined the effect
of decreasing the level of expression of Lyn in this system. In
agreement with earlier studies in granulocytes,31,33 Lyn antisense oligodeoxynucleotides significantly reduced Lyn protein expression after 6 hours. Similar to activated B cells from
Lyn-deficient mice,18 we observed a reduced susceptibility
to Fas receptor-mediated death in eosinophils partially lacking Lyn.
These data suggest that Lyn is involved in the transmembrane
death-signaling cascade via the Fas receptor in eosinophils.
Previous studies have suggested that Lyn is required for the prevention
of apoptosis by cytokines in both eosinophils31 and
neutrophils.33 Thus, Lyn emerges as a signaling molecule capable of inducing two mutually exclusive cellular functions, cell
survival and cell death. Similar observations have been previously reported for other signaling molecules. For example, ceramide, a second
messenger of the sphingomyelin pathway, can induce both mitogenesis and
apoptosis.85 Moreover, a number of growth factors are known
to have antiapoptotic effects on cells and many of them, including IL-5
in eosinophils, signal via Ras.45,46,86,87 However,
activation of Ras was also seen in apoptotic pathways initiated by
lymphokine deprivation88 or Fas receptor
activation.16 Furthermore, not only signaling molecules but
also cell surface receptors, including the Fas receptor itself, can
signal both proliferation and apoptosis.89 Our results
suggest that Lyn may have a similar ability to influence both cell
survival and cell death pathways. However, it is currently unclear
under which conditions Lyn activation is an antiapoptotic or
proapoptotic event. A recent study provided evidence that both
activation of the Lyn-Ras-Raf-1-MAP kinase and Jak-STAT pathways are
obligatory events for cytokine-mediated delayed eosinophil
apoptosis.90 Therefore, Lyn might only be able to
facilitate proapoptotic activities under conditions in which Jak-STAT
activation does not occur. Although further studies are needed to
completely explain the striking phenomenon of transduction of both
antiapoptotic and proapoptotic signals by Lyn, the data in this study
further support an important role for this tyrosine kinase as a common
element responsible for signal transduction after granulocyte
stimulation with widely different agonists.48
Although it is clear that Fas receptor activation results in increased
tyrosine phosphorylation, a requirement for tyrosine kinase activation
for Fas receptor-mediated apoptosis is controversial, especially in T
cells.20-23 In contrast, as this study demonstrates, tyrosine phosphorylation appears to modulate the functional death response not only in eosinophils but also in B cells18 and
neutrophils.17 It has been suggested earlier that the
efficacy of the death signal mediated by the Fas receptor may be
dependent on a critical level of surface receptor
expression.91 Because the level of Fas receptor surface
expression is very low in eosinophils and neutrophils compared with T
cells,13 it is possible that, in granulocytes, tyrosine
kinase activation is required for optimal signal transduction via the
Fas receptor. In contrast, an optimal interaction between the receptor
and second messenger molecules might already be present in activated T
cells and, therefore, the activation of tyrosine kinases might not be
functionally relevant in these systems. Thus, in this model, the role
of tyrosine kinase activation could be to decrease the threshold of
needed receptor molecules per cell for induction of apoptosis in
granulocytes and B cells. This assumption is supported by the fact that
tyrosine kinase activation appears to be independent from ICE-like
proteases, because an inhibitor of ICE, YVAD, completely blocked Fas
receptor-mediated death, but did not abrogate the activation of Lyn in
eosinophils.
Regardless of the exact mechanism, tyrosine kinase activation appears
to increase the death response after Fas receptor cross-linking in
eosinophils. It is still not clear under which circumstances tyrosine
kinase activation is necessary or facilitates Fas receptor-mediated apoptosis. However, tyrosine kinase activation seems to be required for
the signaling cascade within ICE family proteases after stimulation of
the death receptor, at least in some cellular systems such as
eosinophils.
| |
FOOTNOTES |
Submitted October 28, 1997;
accepted March 11, 1998.
Supported by Grant No. 32-49210.96 from the Swiss National Science
Foundation.
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 |
The authors are grateful to all clinicians in Davos who provided blood
samples from patients. We thank P.H. Krammer (Heidelberg, Germany) for
affinity-purified anti-Fas MoAb (IgG3, ) and M. Roth (Basel,
Switzerland) for densitometry measurements.
| |
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Abstract
Introduction
Abstract