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Blood, Vol. 95 No. 6 (March 15), 2000:
pp. 1911-1917
CHEMOKINES
From the University of Texas Medical Branch, Department of Internal
Medicine, Division of Allergy and Immunology, Galveston, TX; and
Laboratory of Medical Allergology, National University Hospital,
Copenhagen, Denmark.
Eotaxin and other CC chemokines acting via CC chemokine receptor-3
(CCR3) are believed to play an integral role in the development of
eosinophilic inflammation in asthma and allergic inflammatory diseases.
However, little is known about the intracellular events following
agonist binding to CCR3 and the relationship of these events to the
functional response of the cell. The objectives of this study were to
investigate CCR3-mediated activation of the mitogen-activated protein
(MAP) kinases extracellular signal-regulated kinase-2 (ERK2), p38, and
c-jun N-terminal kinase (JNK) in eosinophils and to assess the
requirement for MAP kinases in eotaxin-induced eosinophil cationic
protein (ECP) release and chemotaxis. MAP kinase activation was studied
in eotaxin-stimulated eosinophils (more than 97% purity) by Western
blotting and immune-complex kinase assays. ECP release was measured by
radioimmunoassay. Chemotaxis was assessed using Boyden microchambers.
Eotaxin (10
Eosinophils are crucial effector cells in the
inflammatory reactions associated with asthma, allergic-inflammatory
diseases, and parasitic infections. Eosinophils are recruited to sites
of inflammation by locally released chemotactic agents. The CC
chemokines eotaxin, RANTES (regulated upon activation, normal T cell
expressed and secreted), and monocyte chemotactic
protein-2, -3, and -4 are potent stimulators of eosinophil chemotaxis.
They also induce mediator release from eosinophils and are believed to
play an integral role in the development of eosinophilic
inflammation.1-10 CC chemokines are generally promiscuous
and bind to more than 1 CC chemokine receptor (CCR). The exceptions
appear to be eotaxin and eotaxin-2, which bind exclusively to
CCR3.11-13 Compared to other CCRs, CCR3 is expressed in
high numbers on eosinophils.14 Blocking of CCR3 has been
shown to inhibit eosinophil response to eotaxin, RANTES, and monocyte
chemotactic protein-2, -3, and -4 by more than 95%, indicating that
CCR3 is the most important eosinophil receptor for these
chemokines.15 Both CCR3 and the other CCRs belong to the
family of serpentine receptors. These receptors traverse the plasma
membrane 7 times and are linked to heterotrimeric G proteins. CCR3 is
sensitive to pertussis toxin, indicating linkage to Gi.
Further, agonist binding to CCR3 has been demonstrated to induce
an increase of the intracellular
Ca++-concentration.11,12,14 Apart from the
above, the signaling pathways through CCR3 and the involvement of
relevant signaling molecules in the functional response of the cell are unknown.
We hypothesize that mitogen-activated protein (MAP) kinases play an
important role in CCR3 signaling. In the present study, we investigated
the involvement of extracellular signal-regulated kinase-2 (ERK2), p38,
and c-jun N-terminal kinase (JNK) in the signal transduction mechanism
of eosinophil degranulation and chemotaxis.
Donors
Eosinophil purification
Stimulation, preparation of cytosolic extracts, and immunoprecipitation PD98 059 (New England BioLabs, Beverly, MA) and SB202 190 (CalBiochem, La Jolla, CA) were dissolved in dimethyl sulfoxide (DMSO). The highest final concentration of DMSO was 0.1%. Where appropriate, eosinophils were preincubated with PD98 059 for 30 minutes at 37°C, monoclonal anti-CCR3 blocking antibody (clone 7B11, a gift from Dr Poul D. Ponath, LeukoSite Inc, Cambridge, MA), isotype-specific control antibody (clone GE-1, Sigma Chemical Co, St. Louis, MO) for 30 minutes on ice, or pertussis toxin (Sigma) for 1 hour at 37°C. For detection of ERK2 with anti-phosphotyrosine and anti-ERK2 antibodies, 2 × 106 eosinophils were incubated with human recombinant eotaxin (eotaxin-1, Peprotech, Rocky Hills, NJ) or medium at 37°C. The reaction was stopped by the addition of 4 volumes of ice-cold 1.25 × lysis buffer (62.5 mM Tris HCl, pH 7.4; 1.25% NP-40; 313 mM NaCl; 1.25 mM EDTA; 0.625 mM phenylmethylsulfonylfluoride; 1.25 µg/mL aprotinin; 1.25 µg/mL leupeptin; 1.25 µg/mL pepstatin; 1.25 mM Na3VO4; and 1.25 mM NaF) and immediate transfer to ice. After incubation on ice for 20 minutes, the detergent-insoluble materials were sedimented by centrifugation (12 000g, 4°C). The supernatants were precleared by incubation with 20 µL of Protein A/G Plus agarose (Santa Cruz Biotechnology, Santa Cruz, CA). The protein concentration of the cytosolic extracts was determined using the microBCA assay (Pierce Chemical Co, Rockford, IL), and the protein content (200 µg) and concentration of the samples was equalized. For immunoprecipitation, the samples were incubated with 7 µg/mL of anti-ERK2 (Santa Cruz Biotechnology) for 1 hour at 4°C, followed by incubation with 20 µL of Protein A/G Plus agarose for 2 hours at 4°C. The immunoprecipitates were washed 4 times with ice-cold 1 × lysis buffer and boiled with 20 µL of 2 × Laemmli buffer for 4 minutes. Samples used for Western blotting were denatured prior to immunoprecipitation by boiling with 6% glycerol; 0.8% -mercaptoethanol; 1.7% sodium dodecyl sulfate
(SDS); 58 mM Tris-HCl, pH 6.8; and 0.002% bromphenol blue for 4 minutes.
SDS gel electrophoresis and Western blotting SDS-polyacrylamide (10%) gels were prepared according to the Laemmli protocol. The gels were blotted onto Hybond ECL membranes (Amersham Corp, Arlington Heights, IL). Excess binding sites were blocked by incubation with 10% BSA (bovine serum albumin) in TBS-T 20 mM Tris base, 137 mM NaCl, and 0.05% Tween-20, pH 7.6, for 1 hour. For detection of ERK2 and JNK, the membranes were then incubated with 0.1 µg/mL of primary antibody for 2 hours at room temperature (monoclonal anti-phosphotyrosine antibody, clone 4G10, Upstate Biotechnology, Inc, Lake Placid, NY, or polyclonal anti-ERK2 antibody, monoclonal anti-phospho-ERK2 antibody, or monoclonal anti-phospho-JNK antibody from Santa Cruz Biotechnology). The membranes were washed 5 times with TBS-T, incubated with horseradish peroxidase-conjungated secondary antibody (1:10 000 dilution of anti-mouse immunoglobulin (Ig) from Sigma or 0.04 µg/mL of anti-rabbit IgG antibodies from Santa Cruz Biotechnology) for 20 minutes and washed 5 times with TBS-T. The blots were visualized by the enhanced chemiluminescence system (Amersham) according to the manufacturer's instructions. Detection of p38 was performed as described above, except for the incubations with primary antibody (1:1000 dilution of polyclonal anti-p-p38, New England BioLabs) and secondary antibody (0.2 µg/mL horseradish peroxidase-conjungated anti-rabbit Ig, Santa Cruz Biotechnology), which were performed overnight at 4°C and for 1 hour at room temperature, respectively.Immune-complex kinase assay Immunoprecipitates of ERK2 were prepared as described above. Immunoprecipitates of p38 were prepared using anti-p38 antibodies from Santa Cruz Biotechnology. The precipitates were washed 2 times in ice-cold 1 × lysis buffer and 2 times in ice-cold kinase buffer (10 mM HEPES, 50 mM NaCl, 10 mM MgCl2, 100 µM Na3VO4, 500 µM dithiothreiol, 25 mM-glycerophosphate) and incubated with 40 µL of kinase buffer
containing 2.5 µM of adenosine triphosphate (ATP; Pharmacia and
Upjohn), 10 µCi of  -[32P]-ATP
(Amersham), and 50 µg/mL of myelin basic protein (MBP, Sigma) for
detection of ERK2 activity or 12.5 µg/mL of activating transcription
factor (ATF-2, Santa Cruz Biotechnology) for detection of p38 activity
for 17 minutes at 30°C. After centrifugation for 6 minutes at room
temperature, the reaction was stopped by boiling 30 µL of supernatant
with 30 µL of 2 × Laemmli buffer. A 20-µL sample was loaded
on 15% (for MBP detection) or 10% (for ATF-2 detection)
SDS-polyacrylamide gels. The gels were either dried or blotted onto
polyvinylidine difluoride membranes (Millipore Corp, Bedford, MA). MBP
or ATF-2 phosphorylation was visualized by autoradiography.
ECP release Ninety-six-well plates were coated with 3% HSA in Hank's balanced salt solution (HBSS) for 2 hours at 37°C and washed 3 times with HBSS without Ca++ and Mg2+ (Life Technologies). Purified eosinophils were suspended at 5 × 105 cells/mL in RPMI 1640 with 0.1% HSA. Eosinophils (5 × 104 cells/well) were preincubated with medium, PD98 059, or SB202 190 for 1 hour at 37°C and stimulated with eotaxin (10 7 mol/L) for 4 hours in a total volume of
110 µL. The cell suspensions were then transferred to microfuge tubes
that had been coated with 3% HSA, and the supernatants were collected
after centrifugation. The eosinophil cationic protein (ECP)
concentration was measured by a radioimmunoassay kit (Pharmacia,
Piscataway, NJ) according to the manufacturer's instructions.
Chemotaxis assay The chemotaxis assay was performed in a 48-well Boyden microchamber (Neuro Probe, Gaithersburg, MD) technique. Briefly, eotaxin was diluted in RPMI 1640 with 0.5% pooled human serum and placed in the lower wells (25 µL) at 108 mol/L concentration; 50 µL
of the cell suspension at 1 × 106 cells/mL were
added to the upper well of the chamber, which was separated from the
lower well by a 5-µm-pore-size, polycarbonate, polyvinylpyrolidone-free membrane (Nucleopore, Pleasanton, CA). The
cells were freshly isolated eosinophils or eosinophils incubated with
reagents as indicated. The chamber was incubated for 60 minutes at
37°C in an atmosphere containing 5% carbon dioxide. The membrane was then carefully removed, fixed in 70% methanol, and stained for 5 minutes in Coomassie brilliant blue. The cells that migrated and
adhered to the lower surface of the membrane were counted from 5 fields
by light microscopy. The chemotactic response to buffer (less than 20 cells/5 fields) was subtracted from that induced with eotaxin with or
without the inhibitors.
Statistical analyses ECP release and chemotaxis in the presence and absence of inhibitors was compared by ANOVA. P < .05 was considered statistically significant.
Eotaxin induces rapid activation of ERK2 and p38, but not of JNK, in eosinophils MAP kinases constitute 3 major groups of serine/threonine kinases: ERK1/ERK2, p38, and JNK.17 We examined the activation of MAP kinases by eotaxin in eosinophils. Eosinophils (more than 97% purity) were incubated with medium or stimulated with eotaxin (108 mol/L) for 0.5, 1, 2.5, 5, 15, or 30 minutes.
To study the activation of ERK2, cytosolic extracts were
immunoprecipitated with anti-ERK2 antibodies. Western blotting with
anti-phosphotyrosine antibodies revealed tyrosine phosphorylation of
ERK2 reaching a maximum at about 1 minute and returning to baseline
levels by 2.5 to 5 minutes (Figure 1A).
Reprobing the membrane with the anti-ERK2 antibodies showed a
concomitant motility shift of ERK2 (Figure 1B). To test whether
tyrosine phosphorylation of ERK2 was paralleled by activation, we
assessed the kinase activity of ERK2 immunoprecipitates in the
immune-complex kinase assay using MBP as the substrate. The kinetics of
ERK2 activation were similar to those of ERK2 phosphorylation (Figure
1C). Activation of MAP kinases requires simultaneous phosphorylation of
select threonine and tyrosine residues.17 To investigate the activation of p38 and JNK, cytosolic extracts of eotaxin-stimulated eosinophils were used for Western blotting with antibodies directed toward dual threonine-tyrosine-phosphorylated p38 and JNK. Dual phosphorylation of p38 reached a maximum at about 1 minute and returned
to baseline after 2.5 to 5 minutes (Figure
2A). To verify that the phosphorylation of
p38 was accompanied by activation, we immunoprecipitated p38 from
cytosolic extracts. The immunoprecipitates were tested in the
immune-complex kinase assay using ATF-2 as the substrate. The kinetics
of activation (Figure 2B) and phosphorylation of p38 were in agreement.
Under basal conditions, some threonine-tyrosine phosphorylation of JNK
was detected. Stimulation with eotaxin did not increase JNK
phosphorylation at any of the tested time points (Figure 2C).
Eotaxin activates ERK2 and p38 in a concentration-dependent manner To test whether the activation of ERK2 and p38 by eotaxin was concentration-dependent, we incubated eosinophils with medium or stimulated them with various concentrations of eotaxin for 1 minute. ERK2 was immunoprecipitated from cytosolic extracts. Western blotting with anti-phosphotyrosine antibodies followed by reprobing of the membrane with anti-ERK2 antibodies showed a concentration-dependent phosphorylation of ERK2 (Figure 3A and 3B). The result was verified by the immune-complex kinase assay (Figure 3C). Likewise, Western blotting of dual threonine-tyrosine-phosphorylated p38 revealed concentration-dependent activation (Figure 3D).
Activation of ERK2 and p38 by eotaxin is mediated through CCR3 Eotaxin is known to activate eosinophils through CCR3.11,12,14 To establish whether the activation of ERK2 and p38 by eotaxin was mediated through this receptor, we preincubated eosinophils with monoclonal anti-CCR3 blocking antibodies (7B11) or an isotype-specific control antibody. The cells were then incubated with medium or stimulated with eotaxin (108 mol/L) for 1 minute. Western blotting with anti-dual-phosphorylated ERK1/ERK2 and
p38 antibodies revealed that the anti-CCR3 blocking antibody caused a
marked inhibition of ERK2 and p38 phosphorylation, indicating that the
activation of the MAP kinases was mediated through CCR3 (Figure
4). CCR3 is linked to pertussis
toxin-sensitive heterotrimeric G proteins.11,12,14 To
further confirm the receptor dependence of MAP kinase activation, we
preincubated eosinophils with pertussis toxin (100 ng/mL) for 1 hour
before stimulation with eotaxin (108 mol/L) for 1 minute. As assessed by Western blotting with antibodies against
dual-phosphorylated ERK2 and p38, pertussis toxin inhibited the
phosphorylation of both MAP kinases (Figure
5), confirming that eotaxin exerts its
effect through a receptor linked to a pertussis toxin-sensitive G
protein.
MEK inhibitor PD98 059 inhibits ERK2 activation by eotaxin MEK (MAP ERK kinase) is a dual-specificity kinase that acts immediately upstream of ERK2 and catalyzes the simultaneous phosphorylation of tyrosine and threonine residues necessary to activate ERK2.18 PD98 059 is a selective MEK inhibitor.19-21 To test the involvement of MEK in eotaxin/CCR3-mediated activation of ERK2, eosinophils were preincubated with medium or 0.5, 5.0, or 50 µM of PD98 059 for 30 minutes at 37°C and stimulated with eotaxin (108
mol/L) for 1 minute. The cytosolic extracts underwent Western blotting with the anti-phospho-ERK1/ERK2 antibody (Figure
6A). The membrane was then stripped and
reprobed with the anti-ERK1/ERK2 antibody (Figure 6B). PD98 059
induced concentration-dependent inhibition of ERK2 phosphorylation,
which was detectable at 0.5 µM, clearly visible at 5 µM, and
virtually complete at 50 µM.
ERK2 and p38 play a role in eotaxin-induced ECP release and chemotaxis Next, we tested the functional relevance of ERK2 and p38 in eosinophil release of ECP induced by eotaxin. We used the MEK inhibitor PD98 059 to inhibit ERK2 activation. Eosinophils were preincubated with the MEK inhibitor PD98 059 (0-50 µM) and the p38 inhibitor SB202 190 (0-10 µM) and stimulated with eotaxin (107 mol/L) for 4 hours prior to measurement of ECP
in the supernatants. Both inhibitors caused concentration-dependent
inhibition of ECP release (Figure 7).
SB202 190 appears to have a greater inhibitory effect than PD98 059
on ECP secretion. The effect of the inhibitors was also studied on
eosinophil chemotaxis in Boyden microchambers. Previous studies from
other laboratories20,21 as well as our own experiment with
ECP release suggested that 10- and 1-µM concentrations of PD98 059
and SB202 190, respectively, effectively inhibited MAP
kinase-dependent biologic functions of various cells. For this reason,
we used these concentrations of the inhibitors in the chemotaxis
experiment. Cells were preincubated with the inhibitors and then
applied in the upper chambers. The lower chambers contained eotaxin
(108 mol/L). Both inhibitors significantly
(P < .05) and nearly completely blocked eosinophil
chemotaxis (Figure 8).
Eotaxin and other CC chemokines acting via CCR3 are believed to play an integral role in the development of eosinophilic inflammation in asthma and allergic inflammatory diseases. In this study, we looked into the mechanisms of action of eotaxin and demonstrated that in eosinophils (1) eotaxin induced phosphorylation and activation of ERK2 and p38 but not JNK MAP kinases, (2) PD98 059 and SB202 190 inhibited eotaxin-induced ECP release, and (3) both inhibitors blocked eosinophil chemotaxis in response to eotaxin. We further confirmed that eotaxin activates eosinophils via CCR3 by a pertussis toxin-sensitive mechanism. These findings imply that both ERK2 and p38 are essential for eosinophil degranulation and locomotion.
Submitted May 4, 1999; accepted November 16, 1999.
Supported by grants from the National Institutes of Health (AI35137), John Sealy Memorial Foundation, the University of Texas Medical Branch, and Fonden til Allergiforskning paa Rigshospitalet, Copenhagen, Denmark. G.T.K. was supported by a travel grant from Fhv. Direktør Leo Nielsen og Hustru Karen Margrethe Nielsens Legat for Lægevidenskabelig Grundforskning.
Reprints: Rafeul Alam, Division of Allergy and Immunology, Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX 77555-0762; e-mail: Ralam{at}utmb.edu.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Presented in part at the annual meetings of the American Academy of Allergy, Asthma, and Immunology in Washington, DC, March 13-18, 1998, and in Orlando, FL, February 26-March 3, 1999.
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Top
Abstract
Introduction
-PY) antibody. (B) Concomitant
bandshifting was detected by reprobing the membrane with the anti-ERK2
antibody. This also verified that the tyrosine-phosphorylated band was
ERK2 (n = 3). (C) ERK2 immunoprecipitates were assayed for kinase
activity using MBP as the substrate. The kinase activity following
eotaxin stimulation paralleled the tyrosine phosphorylation profile of
ERK2. The numbers on the left side of the panels indicate the position
of the molecular weight markers.
), JNKs, ERK5, ERK6, and ERK7.30, 31-33 We found that activation of ERK2 and p38 was
induced by nanomolar concentrations of eotaxin consistent with the
concentrations necessary to induce chemotaxis and mediator release in
vitro.
RI in mouse bone marrow-derived mast cells. In these
cells, PD98 059 inhibited serotonin release
significantly.
heat shock protein 27 pathway as well as the changes in F-actin content induced by the
secretagogue cholecystokinin.