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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 7 (April 1), 1998:
pp. 2547-2557
Analysis of Signal Transduction Pathways in Human Eosinophils
Activated by Chemoattractants and the T-Helper 2-Derived Cytokines
Interleukin-4 and Interleukin-5
By
Paul J. Coffer,
René C. Schweizer,
Gerald R. Dubois,
Tjander Maikoe,
Jan-Willem J. Lammers, and
Leo Koenderman
From the Departments of Pulmonary Diseases and
Dermatology/Allergology, University Hospital Utrecht, Utrecht, The
Netherlands.
 |
ABSTRACT |
Activation and recruitment of eosinophils in allergic inflammation
is in part mediated by chemoattractants and T-helper 2 (Th2)-derived
cytokines. However, little is known concerning the signal transduction
mechanisms by which this activation occurs. We have investigated
tyrosine kinase-mediated activation of phosphatidylinositol 3-kinase
(PI3K) and compared this with the activation of the p21ras-ERK signaling pathway in human eosinophils. The related cytokines interleukin-3 (IL-3), IL-5, and granulocyte-macrophage
colony-stimulating factor (GM-CSF), all induced PI3K activity
detected in antiphosphotyrosine immunoprecipitates. Furthermore, the
chemoattractants platelet-activating factor (PAF),
RANTES, and C5a were also able to induce phosphotyrosine-associated PI3K activity. Protein kinase B (PKB) is a downstream target of PI3K
activation by growth factors. Induction of PKB phosphorylation in human
eosinophils was transiently induced on activation with the cytokines
IL-4 and IL-5, as well as the chemoattractants PAF, C5a, and RANTES
showing a broad activation profile. Surprisingly, analysis of the
activation of the mitogen-activated protein (MAP) kinases
p44ERK1 and p42ERK2, showed that ERK2, but not
ERK1, was transiently activated in human eosinophils after stimulation
with IL-5 or PAF. Activation kinetics correlated with activation of
p21ras by both cytokines and chemoattractants as measured by a novel
assay for guanosine triphosphate (GTP)-loading.
Finally, using specific inhibitors of both the p21ras-ERK and PI3K
signaling pathways, a role was demonstrated for PI3K, but not
p21ras-ERK, in activation of the serum-treated zymosan (STZ)-mediated
respiratory burst in IL-5 and PAF-primed eosinophils. In summary, these
data show that in human eosinophils, Th2-derived cytokines
differentially activate both PI3K and MAP kinase signal transduction
pathways with distinct functional consequences showing complex
regulation of eosinophil effector functions.
| |
INTRODUCTION |
ALLERGIC INFLAMMATION in bronchial
tissue in asthma is characterized by the presence of T-helper 2 (Th2)
lymphocytes and eosinophilic granulocytes.1,2 Th2
lymphocytes are characterized by production of a distinct cytokine
pattern in vitro, which includes interleukin-5 (IL-5), IL-4, and
IL-13.3 These cytokines play an important role in the
propagation of the allergic inflammatory response and in the activation
and recruitment of eosinophils.2,4 Furthermore, elevated
levels of the cytokines IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been detected in sera from
allergic asthmatic patients.5,6 In vivo, IL-5 is the terminal differentiation factor for eosinophils and an important modulator of eosinophil functions.7 In vitro, IL-5 induces chemokinesis and priming of eosinophil functions such as migration, respiratory burst activation, and platelet-activating factor (PAF) release.8-11 Transmembrane receptors for these cytokines
are tyrosine kinase-linked and associate with members of the JAK family
and agonist binding results in intracellular increases in tyrosine phosphorylation.12 The second major effector molecules
regulating both eosinophil chemotaxis and priming are chemoattractants.
These proinflammatory molecules include PAF, fMLP, C5a, as well as
chemokines such as IL-8 and RANTES.13,14 In contrast to
cytokine receptors, chemoattractant receptors are serpentine G-protein
coupled receptors. The mechanisms by which these receptors induce
tyrosine phosphorylation and regulate intracellular signal transduction
are poorly defined. Furthermore, there is significant cross-talk
between the tyrosine-kinase linked and G-protein coupled receptor
signal transduction systems, as observed during priming of granulocytes
in vitro and in vivo.15
Recently, much attention has been paid to the activation of the lipid
kinase phosphatidylinositol 3-kinase (PI3K) in the context of cytokine
signaling. At least three families of PI3K have been described in
mammalian cells.16 One is activated by tyrosine kinases and
consists of a regulatory subunit of 85 kD (p85) and a
catalytic subunit of 110 kD (p110).17,18 The second family consists of a 110-kD protein (p110 ), which is activated by the  subunit of heterotrimeric G-proteins.19,20 The third
consists of a phosphatidylinositol (PI)-specific PI3K.21
Activated PI3K phosphorylates the hydroxyl group on the D-3 position of
the inositol ring of the lipids PI, PI 4-phosphate (PI4-P), and PI
4,5-biphosphate (PI4,5-P2), which results in formation of
PI3-P, PI3,4-P2, and PI3,4,5-P3. These products
are potential second messengers and have been implicated in the
activation of several members of the protein kinase C (PKC)
family.22-25 The small guanosine triphosphate (GTP)-binding
protein Rac has also been proposed to be a target of
PI3,4,5-P3.26 Furthermore, phosphoinositide
products of PI3K-activity have also been shown to bind to pleckstrin
homology (PH) domain-containing proteins.27-29 We and
others have shown that the PH domain-containing protein kinase B/c-Akt
(PKB) is a downstream target for PI3K and has been shown to bind and be
activated by 3-phosphorylated lipids in vitro.30,31 Recent
data has indicated that constitutive activation of PI3K in a
monoblastic phagocyte cell line (GM-1) results in constitutive
activation of PKB and furthermore phosphorylation of
p47phox suggesting a potential role in regulating
the respiratory burst.32
Although activation of p21ras and the mitogen-activated protein (MAP)
kinase p44ERK1 has been recently demonstrated in human
eosinophils, this work has only investigated the cytokine
IL-5.33 The potential for activation of p21ras-ERK signal
transduction in eosinophils by chemokines or other Th2-derived
cytokines, such as IL-4, have not been investigated. Activation of MAP
kinases have been postulated to play an important role in regulating
granulocyte effector functions such as superoxide production, although
no direct evidence has been provided linking p21ras-ERK signaling to
these events in either neutrophils or eosinophils.34-36
Here we show that PI3K-PKB and p21ras-ERK signal transduction pathways
are activated in human eosinophils after stimulation with Th2-derived
cytokines. We show that while activation of PI3K is critical for both
IL-5 and PAF-primed STZ-mediated respiratory burst, activation of
p21ras-ERK is not. These data show that in human eosinophils,
Th2-derived cytokines differentially activate both PI3K and MAP kinase
signal transduction pathways with distinct functional consequences.
| |
MATERIALS AND METHODS |
Reagents.
PAF(1-0-hexadecyl-2-acetyl-sn-glycero-3-phosphoryl-choline), C5a,
L- -phosphatidylinositol (PI), and bovine serum albumin (BSA) were
purchased from Sigma (St Louis, MO). Human serum albumin (HSA) was from
the Central Laboratory of the Netherlands Red Cross Blood Transfusion
Service (Amsterdam, The Netherlands). RANTES was purchased from Pepro
Tech (Rocky Hill, NJ). Recombinant human (rh) GM-CSF (2.5 × 108 U/mg) was from Genzyme (Boston, MA). rhIL-4 was a kind
gift from Dr F. Kalthoff (Sandoz Forschungsinstitut, Vienna, Austria).
rhIL-5 was a kind gift from Dr D. Fattah (GlaxoWellcome, Stevenage,
UK). Ficoll-Hypaque and Percoll were obtained from Pharmacia (Uppsala, Sweden). Wortmannin and PD098059 were purchased from Biomol (Plymouth Meeting, PA). All other materials were reagent grade. Experiments were
performed in incubation buffer, containing 132 mmol/L NaCl, 6.0 mmol/L
KCL, 1.0 mmol/L CaCl2, 1.0 mmol/L MgSO4, 1.2 mmol/L KH2PO4, 20 mmol/L HEPES, 5 mmol/L
glucose, and 1.0% HSA (wt/vol).
The murine antiphosphotyrosine monoclonal antibody (MoAb) (4G10,
IgG2bk) and anti-p85 rabbit antiserum were obtained from UBI (Lake
Placid, NY), the peroxidase-conjugated rabbit antimouse (P 260) and
swine antirabbit antibodies were from Dakopatts (Glostrup, Denmark).
Monoclonal anti-IL-4 (clone 1-41-1) was a kind gift from Dr F. Kalthoff (Sandoz Forschungsinstitut, Vienna, Austria). Rabbit
polyclonal antisera for ERK1 (C-16) and ERK2 (C-14) were from Santa
Cruz Biotechnology Inc (Santa Cruz, CA). PKB antisera were from UBI
(Lake Placid, NY). All antibodies were stored at 4°C.
Isolation of human eosinophils.
Blood was obtained from healthy volunteers from the Blood Bank,
Utrecht, The Netherlands. Mixed granulocytes were isolated from the
buffy coat of 500 mL of blood anticoagulated with 0.4% (wt/vol)
trisodium citrate (pH 7.4) as described previously.37 To
reduce the number of neutrophils in this mixed granulocyte population,
the cells were subjected to discontinuous Percoll gradient (1.084 to
1.1 g/mL) centrifugation (20 minutes, 1,000g, room
temperature). Eosinophils were subsequently isolated by the method
described by Hansel et al.38 This isolation method is based
on the fact that, in marked contrast to neutrophils, eosinophils lack
the epitope on FcRIII recognized by the MoAb CLB-FcR-gran 1 (CD16)
directed against FcRIII.39 As a result, highly purified eosinophils can be isolated by removing neutrophils coated with CLB-FcR-gran 1 with immunomagnetic dynabeads (Dynal, Oslo, Norway). Briefly, neutrophils were coated with a MoAb against FcRIII
(CLB-FcR-gran 1, CD16, 2 µg/107 cells/mL) during 30 minutes at 4°C. The cells were washed twice and subsequently
incubated head over head in a rotator with dynabeads at a ratio of 1:2
(cells/beads) for 20 minutes at 4°C. Neutrophils were subsequently
removed by a magnetic particle concentrator (MPC-1, Dynal). Eosinophil
purity was always more than 95%.
Phosphatidylinositol 3(OH)-kinase assays.
Eosinophils were resuspended in incubation buffer and preincubated for
30 minutes at 37°C. Thereafter, the cells were stimulated with
different agonists before being stopped by the addition of 2 volumes of
ice-cold incubation buffer containing 2 mmol/L
Na3VO4. Subsequently the eosinophils were
pelleted by centrifugation at 4°C. Thereafter, the cells were
resuspended in lysis buffer (1% Triton X-100, 20 mmol/L Tris/HCl, 100 mmol/L NaCl, 10 mmol/L Na4P2O7, 2 mmol/L EDTA, 50 mmol/L NaF, 10% glycerol, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 10 µg/mL soybean tryptase inhibitor, 1 mmol/L phenylmethyl sulfonyl fluoride (PMSF), and 1 mmol/L
Na3VO4, pH 8.0) for 30 minutes on ice. After 30 minutes, detergent-insoluble material was removed by centrifugation for
10 minutes at 14,000 rpm at 4°C.
Lysates were treated for 1 hour at 4°C with the antiphosphotyrosine
MoAb 4G10 (2.5 µg/mL). Thereafter, protein A-sepharose (Pharmacia,
Uppsala, Sweden) was added for another hour and subsequently, the
protein sepharose beads were washed three times with lysis buffer and
two times with 10 mmol/L Tris-HCl (pH 7.4) containing 1 mmol/L
Na3VO4. PI3K activity was measured by adding
100 µg of sonicated PI, and 20 µCi of (-32P)
adenosine triphosphate (ATP) (ICN, Plainview, NY) in
the presence of 200 µmol/L adenosine to inhibit PI4K activity, 30 mmol/L MgCl2 and 35 µmol/L ATP in a volume of 60 µL.
Reactions were performed for 20 minutes at room temperature and stopped
by addition of 100 µL 1 mol/L HCl and 200 µL chloroform:methanol
(1:1 vol/vol). After centrifugation and taking away the upper layer, 80 µL methanol/HCl was added. After centrifugation, lipids were
separated on thin-layer chromatography (TLC) plates (Merck, Darmstadt,
Germany) using a solvent system of
chloroform:methanol:ammoniumhydroxide (45:35:10 vol/vol/vol). TLC
plates were exposed to x-ray film at  80°C. The identity of
the labeled phosphatidylinositol phosphate (PIP) spot was verified by
comigration with a PIP standard, which was visualized in a iodine
chamber (results not shown). Immunoprecipitation with polyclonal
anti-p85 antibody was used as positive control for PI3K activity
(results not shown). In Figs 1 and
2, x-ray films were scanned on a
densitometer (Molecular Dynamics, Sunnyvale, CA)
analyzed with Image Quant software (Molecular Dynamics). These results
are expressed as fold induction compared with the unstimulated control
(see legends).
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| Fig 1.
Induction of PI3K activity by cytokines and
chemoattractants in human eosinophils. (A) Eosinophils (2 × 106) were left unstimulated for 5 minutes at 37°C (lane
1) or stimulated with the cytokines IL-5 (10 9 mol/L),
IL-3 (109 mol/L), and GM-CSF (6 × 108
mol/L) for 5 minutes at 37°C (lanes 2 to 4). Densitometric analysis showed that the fold induction compared with the unstimulated control
(lane 1) was 61.7 (lane 2), 20.4 (lane 3), and 14.4 (lane 4). (B)
Eosinophils (2 × 106) were stimulated with IL-4
(108 mol/L, lane 1) or with IL-4 (108
mol/L) in combination with a MoAb directed against IL-4 (lane 2). The
fold induction compared with the unstimulated control (lane 1) was 14.7 (lane 2) and 6.2 (lane 3). (C) In lanes 1 to 3, eosinophils (2 ×106) were stimulated for 1 minute at 37°C with PAF
(106 mol/L), RANTES (106 mol/L), C5a
(108 mol/L), respectively. Lane 4 represents an
unstimulated control sample that was kept at 37°C for 1 minute. The
fold induction compared with the unstimulated control (lane 4) was 15.3 (lane 1), 6.3 (lane 2), and 3.0 (lane 3). The experiment shown is
representative of three other experiments.
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| Fig 2.
Effect of the PI3K inhibitor wortmannin on IL-5 and
PAF-induced PI3K activity in human eosinophils. All samples (2 × 106 cells) were pretreated for 15 minutes at 37°C in
the presence or absence of 30 nmol/L wortmannin (WTM). (A) Eosinophils
were stimulated with IL-5 (109 mol/L) for 5 minutes at
37°C (lanes 2 and 3). Lane 1 represents PI3K activity in
unstimulated eosinophils not treated with WTM and incubated at 37°C
for 5 minutes. Densitometric analysis showed that the fold induction
compared with the unstimulated control (lane 1) was 49.8 (lane 2) and
1.1 (lane 3). (B) Eosinophils were left untreated (lane 1, 1 minute at
37°C) or stimulated with PAF (106 mol/L) for 1 minute at 37°C (lanes 2 and 3). The fold induction compared with
the unstimulated control (lane 1) was 24.3 (lane 2) and 1.2 (lane 3).
The experiment shown is representative of three other experiments (n
= 3).
|
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p21ras Activation assay.
GST-Ras-binding domain (RBD) (aa51-131 of Raf1) fusion protein was
constructed and isolated as previously described.40 The desired amount of crude GST-RBD was incubated with glutathione-agarose beads at 4°C for 1 hour. The beads were isolated by centrifugation and washed five times with lysis buffer (50 mmol/L Tris-Cl pH 7.4, 150 mmol/L NaCl, 1% NP-40, 10% glycerol, 0.1 µmol/L aprotinin, 1 µmol/L leupeptin, and 1 mmol/L PMSF). After stimulation, eosinophils (7.5 × 106 cells) or neutrophils (107
cells) were lysed in 1 mL lysis buffer at 4°C and centrifuged to
remove nuclei. Precoupled GST-RBD beads were added and the lysates
incubated on a rotating wheel for 30 minutes at 4°C. Beads were
pelleted by centrifugation and washed three times with lysis buffer
before being resuspended on Laemmli sample buffer. Protein samples were
separated on a 15% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) gel and transferred to polyvinylidene fluoride (PVDF)-membrane. Western blots were probed with anti-Ras MoAb
Y13-259 (16 hours at 4°C) followed by horseradish peroxidase (HRP)-coupled goat antirat antiserum (Santa Cruz; 2 hours). Blots were developed by enhanced chemiluminescence (ECL;
Amersham, Amersham, UK).
MAP kinase assays.
Eosinophils and neutrophils were isolated as described above and
incubated at 37°C for 30 minutes. After stimulation, the cells were
washed twice in ice-cold phosphate-buffered saline (PBS) and lysed in
50 mmol/L Tris-HCl (pH 7.5), 100 mmol/L NaCl, 50 mmol/L NaF, 5 mmol/L
EDTA, 40 mmol/L -glycerophosphate, 1 mmol/L
Na3VO4, 1% Triton X-100, 10 µg/mL aprotinin,
10 µg/mL leupeptin, and 1 mmol/L PMSF. Lysates were precleared for 30 minutes at 4°C with protein A-sepharose. MAP kinase was
immunoprecipitated with 1 µg of either ERK1 or ERK2 polyclonal
antisera for 1 hour at 4°C on a rotating wheel. Protein A-sepharose
was then added for an additional 1 hour at 4°C. After washing twice
with lysis buffer, samples were washed twice with kinase buffer without
ATP. Precipitates were then incubated in 25 µL kinase buffer
consisting of 30 mmol/L Tris-HCL (pH 8.0), 20 mmol/L MgCl2,
2 mmol/L MnCl2, 10 µmol/L rATP, 10 µg myelin basic
protein (MBP), and 0.3 µCi of (-32P) ATP for 20 minutes at 30°C. Reactions were stopped by the addition of 5×
Laemmli sample buffer. Samples were separated by electrophoresis on
15% SDS-polyacrylamide gels. MBP phosphorylation was detected by
autoradiography.
Analysis of ERK and PKB phosphorylation.
For detection of phosphorylation of ERK1, ERK2, and PKB, cells were
washed twice in ice-cold PBS after stimulation and immediately lysed in
Laemmli buffer. After being heated for 5 minutes at 95°C, cell
lysates were run on 12.5% SDS-PAGE (173:1). After gel electrophoresis, proteins were transferred to Immobilon-P and blocked with PBS-Tween (0.3%) containing 4% low-fat milk powder for 1 hour at room
temperature. Blots were then probed in PBS-Tween containing either
ERK1, ERK2, or PKB antisera (1:2,000) for 1 hour at room temperature as
previously described.41 An additional incubation for 1 hour
with goat antirabbit peroxidase-conjugated antibody was performed.
Proteins were visualized by luminol-enhanced chemiluminescence (ECL)
using a standard kit (RPN 2109, Amersham, UK).
Measurement of oxygen consumption.
Oxygen consumption was measured at 37°C with an oxygen electrode as
described previously.42 In short, eosinophils were
resuspended (1.6 × 106 per mL) in incubation buffer.
After 5 minutes of incubation at 37°C, either IL-5
(1010 mol/L, 20 minutes) or PAF (1 µmol/L, 2 minutes) or solvent was added. For some experiments, wortmannin (100 nmol/L) or PD098059 (25 µmol/L) were preincubated for 5 or 15 minutes, respectively. Oxygen consumption was measured for 2 minutes
before and continually after addition of STZ (1 mg/mL). The maximal
rate of oxygen consumption is depicted ± standard error
(SE).
| |
RESULTS |
Activation of phosphotyrosine-associated PI3K in human eosinophils by
cytokines and chemoattractants.
The lipid kinase, PI3K, has been shown to be activated by mediators
that induce intracellular protein tyrosine
phosphorylation.16 In eosinophils, both IL-5 and PAF are
capable of inducing protein tyrosine phosphorylation11 (and
unpublished observations) and are potent primers of
eosinophil functions. Here we studied activation of PI3K by cytokines
and chemoattractants in antiphosphotyrosine immunoprecipitates derived
from lysates prepared from normal human eosinophils.
Incubation for 5 minutes at 37°C with the cytokines IL-5
(109 mol/L), IL-3 (109 mol/L),
and GM-CSF (6 × 1010 mol/L) induced PI3K
activity in human eosinophils (Fig 1A; lane 1 represents the
unstimulated control). Importantly, IL-4 (108 mol/L)
clearly also induced PI3K activity in eosinophils (Fig 1B, lane 1).
Control experiments were performed with 108 mol/L
IL-4 in combination with a blocking MoAb against IL-4, to show the
specificity of the IL-4-induced activation, as the presence of this
receptor on eosinophils has not been widely reported.43 In
the presence of anti-IL-4, PI3K activity was reduced (Fig 1B, lane 2).
This is the first example of activation of specific downstream signaling events in human eosinophils stimulated by IL-4. In addition to cytokines, incubation for 1 minute at 37°C with the
chemoattractants PAF (106 mol/L), RANTES
(106 mol/L), and C5a (108 mol/L)
also induced PI3K activity in human eosinophils (Fig 1C). This is
somewhat surprising, as previous reports have linked these G-protein
coupled receptor agonists to the nonphosphotyrosine-associated p110
isoform of PI3K.16,19,20 This shows for the first time activation of PI3K in eosinophils and furthermore that G-protein coupled receptors can activate phosphotyrosine-associated PI3K isoforms
in these cells. Control experiments were performed using the potent
PI3K inhibitor wortmannin.44 Incubation of eosinophils with
30 nmol/L wortmannin, a relatively specific PI3K inhibitor, completely
prevented PI3K activation in eosinophils stimulated by IL-5 and PAF
(Fig 2).
To further investigate the activation of PI3K in human eosinophils, we
studied the time- and dose-dependency of phosphotyrosine-associated PI3K activity induced by IL-5 and PAF. In
Fig 3, a time-dependent activation of PI3K
in antiphosphotyrosine immunoprecipitates from IL-5-(109 mol/L) and PAF (106
mol/L)-stimulated eosinophils is shown. Both compounds induced a
transient activation of PI3K with maximal activation occurring at 5 minutes after addition of IL-5 (Fig 3A) and within 1 minute after
addition of PAF (Fig 3B). This correlates with the more rapid responses
of G-protein coupled receptors versus tyrosine kinase-linked receptors
with respect to other signaling events (see below).
Figure 4 shows PI3K activity in human
eosinophils induced by different concentrations of IL-5 and PAF at the
optimal time points. PI3K activation in eosinophils was found to
increase to 109 mol/L IL-5 (Fig 4A, lane 3) and
106 mol/L PAF (Fig 4B, lane 3). While higher
concentrations yielded essentially the same result, concentrations of
PAF above 106 mol/L are toxic, and the mechanism of
PI3K-activation may involve alternative stress-related mechanisms.
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| Fig 3.
Time-course of PI3K activation in human eosinophils
treated with IL-5 and PAF. Eosinophils (1.5 × 106) were
treated with (A) IL-5 (109 mol/L) or (B) PAF
(106 mol/L) at 37°C for the indicated time
intervals. After cell lysis and immunoprecipitation, PI3K activity was
detected as previously described.
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| Fig 4.
Concentration-dependence of IL-5 and PAF on PI3K
activation in human eosinophils. PI3K activation was detected in human
eosinophils (2 × 106) after stimulation with (A) IL-5
(1011 to 109 mol/L) for 5 minutes or (B)
PAF (108 to 106 mol/L) for 1 minute at
37°C. Lane 4 represents the unstimulated control for both
experiments. The experiment shown is representative of three other
experiments.
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Phosphorylation of PKB/c-Akt in human eosinophils by cytokines and
chemoattractants.
Recently, we and others have shown that phosphorylation and activation
of PKB/c-Akt, a serine/threonine kinase, was mediated by
PI3K.30,31 We analyzed induction of PKB phosphorylation in
human eosinophils, as measured by the previously described lower
mobility of phosphorylated PKB in SDS-PAGE gels.30,31 As
shown in Fig 5A, phosphorylation of PKB
(pPKB) was transiently induced in human eosinophils on activation with
the cytokines IL-5 and IL-4. Furthermore, it can be seen that the
chemoattractants C5a (108 mol/L), PAF
(106 mol/L), and RANTES (106
mol/L) also induced phosphorylation of PKB (Fig 5B). Finally, in Fig
5C, a time course of IL-5 (109 mol/L) and PAF
(106 mol/L)-induced phosphorylation of PKB is shown,
demonstrating that PKB phosphorylation coincides with PI3K activation,
as previously described.30,31 Again, while IL-5 produces a
relatively slow phosphorylation of PKB, that of PAF is much more rapid.
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| Fig 5.
Cytokines and chemoattractants stimulate phosphorylation
of PKB in human eosinophils. Phosphorylation was measured by an
SDS-PAGE mobility shift as described in Materials and Methods. (A)
Eosinophils (2 × 106) were stimulated with the cytokines
IL-5 (109 mol/L), and IL-4 (108 mol/L) at
37°C for the indicated time intervals. (B) Eosinophils (2 × 106) were stimulated with the chemoattractants C5a
(108 mol/L), PAF (106 mol/L), and RANTES
(106) for 2 minutes at 37°C. (C) Eosinophils (2 × 106) were treated with IL-5 (109 mol/L) and
PAF (106 mol/L) at 37°C for the indicated time
intervals.
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ERK2, but not ERK1, is activated in human eosinophils after
stimulation with IL-5 and PAF.
We also analyzed the phosphorylation and activation of the MAP kinases
ERK1 and ERK2, which are both downstream targets of p21ras signal
transduction. IL-5 (1010 mol/L) induced a transient
induction of phosphorylation of ERK2 (ppERK2), which was optimal at 5 minutes (Fig 6A) and declined to basal
levels within 30 minutes. The chemoattractant PAF
(106 mol/L) induced a very rapid and transient
phosphorylation of ERK2, which was maximal within 1 minute and declined
to basal levels within 5 minutes (Fig 6B). However, in contrast to IL-5 and PAF, IL-4 (108 mol/L) did not induce
phosphorylation of ERK2 in human eosinophils (Fig 6C). IL-4 also failed
to induce ERK2 phosphorylation at other time points (data not shown).
While ERK phosphorylation generally correlates with enzyme activity, it
is itself not a direct measure of kinase activity. We thus analyzed the
activation of ERK2 after IL-5 (1010 mol/L) or PAF
(106 mol/L) stimulation by specific
immunoprecipitation and kinase assay as described in Materials and
Methods. As seen in Fig 6D, IL-5 induces relatively slow and sustained
activation of ERK2 kinase activity, lasting for more than 15 minutes.
PAF, however, stimulates a very rapid, but short-lived, activation of
ERK2 activity, returning to basal levels after several minutes.
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| Fig 6.
Effect of cytokines and chemoattractants on the
phosphorylation of ERK2 in human eosinophils. After stimulation, the
eosinophils (2 × 106) were immediately lysed in Laemmli
buffer. Phosphorylation of ERK2 (ppERK2) was detected as described in
Materials and Methods. (A) Time-course of IL-5 (5 × 1010 mol/L) induced ERK2 phosphorylation. (B) Time
course of phosphorylation of ERK2 by PAF (106 mol/L).
(C) IL-5 (5 × 1010 mol/L, 5 minutes), but not IL-4
(108 mol/L, 5 minutes) induced ERK2 phosphorylation. (D)
Time course of ERK2 activation by IL-5 (1010 mol/L) and
PAF (106 mol/L) as measured by MBP phosphorylation.
Kinase assays with 7.5 × 106 eosinophils were performed
as described in Materials and Methods.
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A recent study has suggested that ERK1, but not ERK2, can be activated
in human eosinophils.33 However, this study only analyzed
ERK phosphorylation and not ERK activity. To determine whether ERK1, as
well as ERK2, was activated in human eosinophils, we analyzed ERK1
phosphorylation as described above. Surprisingly, as shown in
Fig 7A, ERK1 was constitutively
phosphorylated in human eosinophils, and this phosphorylation was not
modified by addition of IL-5, PAF, or IL-4. As mentioned previously,
ERK phosphorylation is not a direct measure of ERK activity. Thus, we
performed immune-complex kinase assays on both ERK1 and ERK2
immunoprecipitated from human eosinophils. As shown in Fig 7B, ERK1
kinase activity was not modified by addition of IL-5, PAF, or IL-4. The
activity of ERK2, however, was induced by both IL-5 and PAF, but not by
IL-4, in agreement with the data presented in Fig 6. As a control, we
analyzed the activation of both ERK1 and ERK2 in human neutrophils
isolated from the same donor at the same time as the eosinophils.
Stimulation of neutrophils with either GM-CSF, PAF, or fMLP resulted in
activation of both ERK1 and ERK2 as shown in Fig 7C. This difference
between neutrophils and eosinophils was consistently observed with all donors tested. In contrast to previous data,33 this clearly shows that while both ERK1 and ERK2 activity can be modulated in human
neutrophils, only ERK2 activity seems to be inducible in human
eosinophils.
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| Fig 7.
Comparison of the activation of ERK1 and ERK2 in human
eosinophils and neutrophils. (A) ERK1 is constitutively phosphorylated in human eosinophils. Cells (2 × 106) were treated with
either IL-5 (1010 mol/L), PAF (106 mol/L)
or IL-4 (108 mol/L) for the times indicated before lysis
and SDS-PAGE analysis. (B) ERK1 is constitutively active, while ERK2 is
inducible in human eosinophils. Cells (7.5 × 106) were stimulated with either IL-5 (1010
mol/L), PAF (106 mol/L), or IL-4 (108
mol/L) for the times indicated. Samples were lysed and
immunoprecipitated with either ERK1 or ERK2 specific antibodies. Kinase
activity was analyzed as described in Materials and Methods. (C) ERK1
and ERK2 activity can both be induced in human neutrophils. Cells (107) were stimulated with either GM-CSF
(1010 mol/L), PAF (106 mol/L), or fMLP
(106 mol/L) for the times indicated. Kinase activity was
analyzed as described above.
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Activation of p21ras correlates with ERK2 activation by IL-5 and PAF.
Although the activation of ERKs by tyrosine kinase-linked receptors is
p21ras-dependent, this correlation has not been widely established for
other receptor families. Original studies in neutrophils have suggested
that while fMLP can activate GTP-loading of
p21ras,45 p21ras activation is not
observed for PAF in Chinese hamster ovary (CHO)
cells.46 We have used a Raf1-RBD as an activation-specific probe for p21ras GTP-loading in eosinophils after stimulation by IL-5
and PAF. This technique uses the principle that Raf1 only interacts
with active GTP-bound p21ras. Thus, a GST-fusion protein containing the
minimal Ras-Binding Domain (RBD) of Raf1 (aa51-131) is used to
"pull-down" GTP-bound p21ras.40 While not
quantitative, this methodology provides data concerning the ability and
kinetics of activation. Eosinophils were stimulated as indicated and
p21ras precipitated with GST-RBD bound to glutathione-agarose beads and identified by Western blotting using a MoAb against p21ras
(Fig 8). IL-5, which resulted in a
relatively slow activation of ERK2 in eosinophils, also produced an
optimal activation of p21ras only after several minutes (Fig 8A). In
contrast, the G-protein receptor agonist PAF, resulted in a very rapid
and short-lived p21ras-activation (Fig 8A), correlating with the
kinetics of ERK2 activation (Figs 6 and 7). Activation by PAF is in
contrast to previous studies in cell lines transfected with the PAF
receptor showing the cell-type specificity of this
response.46 The activation of p21ras in human neutrophils
was also analyzed (Fig 8B). Activation of p21ras by PAF/fMLP is very
rapid, while GM-CSF is much slower. This is similar to the activation
profiles observed in eosinophils from the same donor.
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| Fig 8.
Activation of p21ras by cytokines and chemoattractants in
human eosinophils and neutrophils. In (A) eosinophils (6 × 106) or (B) neutrophils (107) were stimulated
as indicated (IL-5, 1010 mol/L; PAF, 106
mol/L; fMLP, 106 mol/L; GM-CSF, 1010
mol/L) before lysis and incubation with GST-Raf1(RBD) to bind GTP-bound
p21ras as described in Materials and Methods. Samples were analyzed by
SDS-PAGE and immunoblotting with a MoAb against p21ras. The position of
GTP-bound p21ras is marked by an arrowhead. This is representative of
three individual experiments.
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Activation of the respiratory burst in human eosinophils by opsonized
particles is inhibited by wortmannin, but not PD098059.
To determine the functional consequences of PI3K or p21ras-ERK
activation by cytokines and chemoattractants in human eosinophils, we
used specific pharmacologic inhibitors of both signal transduction pathways. Wortmannin is a specific inhibitor of PI3K at nanomolar concentrations,44 while PD098059 has been recently
described as a specific inhibitor of MEK, a kinase mediating activation of ERK1/ERK2 by p21ras.47,48 Addition of opsonized
particles (serum-treated zymosan, STZ) to eosinophils results in
activation of the respiratory burst. Unlike human neutrophils, this
response is very sensitive for priming by both IL-5 and
PAF.49 Addition of wortmannin to eosinophils blocks
activation of PI3K by both IL-5 and PAF (Fig 2). Furthermore, addition
of PD098059 blocks both IL-5 and PAF-mediated ERK activation (data not
shown). To determine the effect of inhibiting PI3K and p21ras-ERK
signal transduction on this process, we preincubated cells with or
without either wortmannin or PD098059. Subsequently, cells were primed by treatment with IL-5 (1010 mol/L, 30 minutes) or
PAF (1 µmol/L, 2 minutes) before stimulation with STZ (1 mg/mL). As
shown in Fig 9, both IL-5 and PAF prime STZ-mediated respiratory burst (Fig 9, lanes 2 and 5). This primed respiratory burst is profoundly inhibited by preincubation with wortmannin (Fig 9, lanes 4 and 7). Inhibition of ERK activation in
eosinophils by preincubation with PD098059, however, had no effect (Fig
9, lanes 3 and 6). These data show that while activation of PI3K
appears to play a critical role in superoxide generation, a major
effector function of eosinophils, the activation of ERK is not
necessary contrary to previous suggestions for neutrophilic granulocytes.34-36
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| Fig 9.
Effect of PI3K- and MEK-inhibitors on STZ-induced
eosinophil respiratory burst. Eosinophils (1.6 × 106/mL)
were preincubated with wortmannin (100 nm, 5 minutes, lanes 4 and 7) or
PD098059 (50 µmol/L, 15 minutes, lanes 3 and 6). Cells were then
primed with IL-5 (1010 mol/L, 20 minutes, lanes 2 to 4)
or PAF (106 mol/L, 2 minutes, lanes 5 to 7) before
addition of STZ (1 mg/mL). Oxygen uptake was continually measured and
results represent the maximal rate
(nmolO2/106cells/min) of four experiments ± SE (P < .05, Student's t-test).
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DISCUSSION |
Eosinophil functioning in vivo is modulated by cytokines and
chemoattractants. Despite these well-documented effects, relatively little is known about the downstream signaling events in human eosinophils initiated on receptor activation. Recently, much attention has been paid to the activation of tyrosine kinases in human
eosinophils.50 Here, we describe the activation of
phosphotyrosine-associated PI3K and phosphorylation of PKB/c-Akt and
activation of p21ras and p42ERK2, induced by both cytokine
and serpentine receptors.
The regulation of PI3K by tyrosine kinases has been the subject of many
recent studies. The p85 regulatory subunit, of which several forms have
been cloned, contains two SH2 domains. These subunits contain a proline
rich sequence and one SH3 domain, which accounts for the adaptor
function of p85.16,17 Activation of the regulatory subunit
is essential for the activation of the p110 catalytic subunit.
Recently, a novel PI3K was cloned, which is activated by
-subunits of heterotrimeric G-proteins and thus stimulated
primarily by serpentine receptor agonists.19,20 This PI3K
lacks a p85 regulatory domain and consists of only a p110 subunit.
In our experiments, tyrosine-phosphorylated proteins were
immunoprecipitated and PI3K activity was measured in vitro. In this
way, tyrosine phosphorylation or recruitment of PI3K(s) to signaling
complexes containing tyrosine-phosphorylated proteins is measured. It
is likely that this PI3K belongs to the p85/p110 class, as we observed
that wortmannin inhibits PI3K activity in eosinophils at relatively low
concentrations (30 nmol/L, see Fig 2B), whereas it has previously been
shown in neutrophils that wortmannin inhibits p110 only at higher
concentrations.19 Thus, it appears that G-protein-coupled
receptor agonists are capable of activating phosphotyrosine-associated
PI3K isoforms in human eosinophils. Further work will be necessary to
determine the mechanisms by which these receptors, which all lack a
consensus SH2-binding sequence for p85, are able to activate
PI3K.51,52 The mechanisms by which IL-5 and IL-4 can
activate PI3K are also unclear. Recently, an 80-kD protein (p80) has
been described to directly link activated receptors for IL-3, IL-5, and
GM-CSF to PI3K signaling.53 Furthermore, the involvement of
different Src-type family protein tyrosine kinases has been described
to mediate the activation of PI3K.54,55 The IL-4 receptor,
which belongs to the cytokine receptor superfamily that makes use of a
cytokine specific -chain and a c-chain, has recently
been shown to engage IRS-1 and 4PS (IRS-2) adaptor proteins.56 These tyrosine-phosphorylated adaptor proteins
contain motifs, which could also potentially bind proteins such as p85 and Grb2.
We and others have recently identified the protein serine/threonine
kinase PKB/c-Akt as being a downstream target of PI3K activation.30,31 These studies focused on signal
transduction pathways induced only by growth factors. The present
study clearly shows that PKB activation is a widespread
phenomenon occurring not only through intrinsic tyrosine kinase
receptors, eg, the platelet-derived growth factor
(PDGF) receptor, but also through associated-tyrosine
kinase receptors, eg, the IL-5 and the IL-4 receptor, and
G-protein-coupled serpentine receptors, eg, the PAF receptor. Thus,
the activation of PKB is probably a primary target function for PI3K
activity, while its function still remains elusive. We have previously
shown that the rapamycin-sensitive p70 S6 kinase can be activated by an
oncogenic form of PKB, while PI3K inhibitors have been shown to inhibit
p70 S6 kinase activation.31,57 Interestingly, rapamycin has
been described to partially reverse the IL-5-mediated eosinophil
survival in vitro.58 It is, therefore, possible that p70 S6
kinase is activated in human eosinophils in response to cytokine
stimulation and that the PI3K-PKB pathway could play a role in
cytokine-mediated eosinophil survival. Preliminary data suggests that
PI3K inhibitors block IL-5-mediated rescue of eosinophil apoptosis
(P.J.C., unpublished data).
We have analyzed the activation of p21ras using a novel assay relying
on the specificity of p21ras-GTP, but not p21ras-GDP binding to
Raf1.40 This assay has many advantages over traditional methods of analyzing small G-protein activation including no
requirement for phosphate labeling of cells and the ability to measure
very rapid time points (<10 seconds). Activation of p21ras in human eosinophils occurs not only by tyrosine kinase-linked receptors, eg,
IL-5, but also by serpentine receptors such as PAF. Previous studies in
CHO cell lines have failed to measure an activation of p21ras by
PAF,46 while this study clearly shows activation of p21ras
in both eosinophils and neutrophils (Fig 8). This demonstrates, as was
originally shown for fMLP in neutrophils, that serpentine receptors use
similar signaling components in the activation of MAP kinases as
tyrosine kinase-linked receptors. Most striking is the dramatic
difference in kinetics of activation between IL-5 and PAF in
eosinophils. While activation by IL-5 occurs between 1 to 5 minutes and
lasts longer than 10 minutes, PAF-stimulated p21ras GTP-loading occurs
within 10 seconds and decreases to basal levels after several minutes
(Fig 8). This activation correlates with the activation of MAP kinases
for all stimuli in both neutrophils and eosinophils, suggesting either
p21ras must be GTP-bound to maintain ERK activation or that these
signals are concomitantly downregulated. The precise mechanism by which
these receptors stimulate GTP- for GDP-exchange on p21ras remains to be
identified.
It has been previously decribed that PI3K can be activated in a
p21ras-dependent mechanism.59,60 Therefore, we wished to analyze the activation of p21ras-ERK in human eosinophils by cytokines and chemoattractants. In human eosinophils, IL-5 has recently been
shown to activate several MAP kinases including a 44-kD (ERK1) and
45-kD MAP kinase.33,61 We have analyzed the activation of
p44ERK1 and p42ERK2, two members of the MAP
kinase family, in terms of phosphorylation and in vitro kinase assays
(Figs 6 and 7). Our data show that in human eosinophils, ERK2
phosphorylation and activity, but surprisingly not ERK1, is stimulated
by both cytokines and chemoattractants. In human neutrophils, however,
both ERK1 and ERK2 activities are inducible (Fig 7C). IL-4 signaling in
eosinophils did not induce activation of ERK2 (Fig 6C), indicating
important differences in signaling between the IL-4 and IL-5 receptors.
It has been reported in other cell systems that the IL-4 receptor
failed to initiate the activation of the p21ras-ERK
pathway.62,63 Our data are in marked contrast to that
recently reported by Pazdrak et al33 in which activation of
ERK1, but not ERK2, in human eosinophils was reported. However, in this
report, no specific antibodies to ERK-isoforms were used. Furthermore,
tyrosine phosphorylation of ERK isoforms was measured rather than
ERK-activity per se. Although tyrosine phosphorylation of MAP kinases
correlates with activity, it is only through immune-complex kinase
assays with specific antibodies that ERK activation can truly be
measured. Pazdrak et al use a MoAb that recognizes both
p44ERK1 and p42ERK2 thus not allowing the
distinction of which isoform is activated.
Our data indicate that there appears to be differential regulation of
the p44ERK1 and p42ERK2 MAP kinases in human
eosinophils (Figs 6 and 7). Although unusual, this differential
regulation is not unprecedented. For example, stimulation of mouse
macrophages with CSF-1 results in activation of only
ERK164; and tumor necrosis factor (TNF)- has been
reported to only activate ERK2 in mouse macrophages and
B-cells.64,65 The implications of these responses are as
yet unclear, as the specific downstream targets of MAP kinases have not
yet been identified. However, it has been reported using overexpression
of MAP kinase constructs in cell lines that ERK1 results in the
activation of the transcription factor Elk-1, while ERK2 activates
c-Myc, but not Elk-1.66 This suggests that there may be a
role for differential activation of MAP kinase isoforms in the
regulation of specific eosinophil functions.
To determine whether activation of PI3K or p21ras-ERK signal
transduction may regulate eosinophil effector function, we used specific inhibitors of these pathways. Activation of the respiratory burst is an event whereby phagocytes generate the antimicrobial oxidants via a multicomponent NADPH oxidase system.
Association with oxidase activation is the phosphorylation of cytosolic
components of this complex, eg,
p47phox.34,67 Both MAP kinases and PI3K
have been implicated in this process. Indeed, introduction of a
constitutively activated form of PI3K into a monoblastic phagocyte line
(GM-1) caused activation of PKB and furthermore phosphorylation of
p47phox.32 Using wortmannin to inhibit
PI3K and PD098059 to inhibit MEK and thus ERK activation, we show that
indeed activation of PI3K is required for STZ-mediated respiratory
burst in human eosinophils (Fig 9). Inhibition of MEK-ERK with high
concentrations of PD098059 (50 µmol/L) had no effect on oxygen
consumption and thus suggests that p21ras-ERK signal transduction is
not necessary for phagocytic killing by eosinophils.
The cytokines IL-4 and IL-5 are both potent primers of eosinophil
migration in vitro.43,68 However, because ERK2 is not activated by IL-4, the signaling pathway involving phosphorylation of
ERK2 is not essential for priming by IL-4. It is tempting to speculate
that PI3K and PKB are involved in priming of eosinophils in vivo, as
until now, no consensus has been reached on the signals involved in
this process.68 In fact, all primers of eosinophils we have
used are activators of the PI3K-PKB signaling pathway in vitro (Fig 1A
and B).
In summary, this is the first report on the activation and function of
PI3K-PKB and p21ras-ERK signal transduction pathways in human
eosinophils by Th2-derived cytokines and chemoattractants. These data
suggest that both cytokines and chemokines can use both similar
and/or distinct mechanisms for transducing an extracellular signal into an effector response. The role of these pathways, particularly p21ras-ERK, requires further work. However, it appears that PI3K plays a critical role in the activation of human phagocytes mediating host defense.
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FOOTNOTES |
Submitted April 24, 1997;
accepted November 20, 1997.
Supported by research grants from the Dutch Asthma Foundation (Grant
No. 91-36) and GlaxoWellcome bv, The Netherlands.
P.J.C. and R.C.S. contributed equally to this study.
Address reprint requests to Leo Koenderman, MD,
Department of Pulmonary Diseases, Room F 02.333, University Hospital
Utrecht, PO Box 85500, 3508 GA Utrecht, The Netherlands.
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 |
Many thanks to Prof. Hans Bos and Laura M'Rabet for the kind gift of
Raf1 GST-RBD and helpful discussions.
| |
REFERENCES |
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Abstract
Introduction
Abstract