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*
Neurocrine Biosciences, Inc., San Diego, CA 92121; and
Laboratory of Immunology and Vascular Biology, La Jolla Institute for Experimental Medicine, La Jolla, CA 92037
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Numerous CC chemokines attract eosinophils, including eotaxin,
eotaxin-2, MCP-2,3
MCP-3, MCP-4, RANTES, and MIP-1
(5). Eotaxin is unique,
however, because in vivo protein administration studies have
demonstrated that it is chemotactic primarily for eosinophils (6, 7). CCR3 appears to be the primary eotaxin receptor, although
eotaxin has recently been shown to bind, but not to activate, CXCR3
(8, 9, 10, 11, 12). Both receptors are seven-transmembrane-spanning G
protein-coupled receptors (8, 9, 10, 11, 12).
Endothelial cell-bound chemokines are thought to provide a gradient to circulating leukocytes, such as eosinophils, that induces a signaling cascade culminating in migration toward the source of the chemoattractant (13, 14). Binding of chemokines, including eotaxin, to their receptors leads to activation of heterotrimeric G proteins that initiate multiple signaling events, including a transient intracellular rise in Ca2+, and tyrosine phosphorylation (6, 10, 11). Furthermore, it has been shown for certain chemokines that receptor binding activates the ERK (extracellular signal-regulated kinase)/MAPK pathway in multiple cell types (15, 16, 17, 18, 19, 20, 21). Three families of MAPKs have been identified, the ERKs (which include p44 (ERK1) and p42 (ERK2)), c-Jun amino-terminal kinase, and the p38 subgroup of kinases, and can be distinguished by their tripeptide dual phosphorylation motif required for activation (22). MAPK kinases or ERK kinases (MEKs) are dual specificity kinases that can phosphorylate ERK1 and ERK2 on tyrosine and threonine residues, thereby inducing ERK activity (23, 24). The MEK1 and MEK2 inhibitor PD98059 can specifically block activation of ERK/MAPK (25).
The chemotaxis process in response to chemokine stimulation of leukocytes requires cytoskeletal rearrangement involving both microtubules, which impart directionality and stabilize the cell (26), and the actin-myosin system, which is thought to provide the contractile forces necessary for directional migration (27). Additionally, changes in the actin-myosin complex regulate the avidity and membrane localization of multiple adhesion molecules, including very late Ag-4 (VLA-4) and L-selectin (CD62L) (28, 29, 30, 31). Thus, cytoskeletal remodeling has a direct effect on leukocyte rolling, firm adhesion, and transendothelial migration by controlling the location and affinity state of cell surface adhesion molecules for their counter-receptors as well as cell shape.
Because eosinophils play a pivotal role in the pathogenesis of allergic diseases, a better understanding of the signal transduction pathways, which regulate eosinophil chemotaxis and their sequestration to sites of inflammation in the tissue, is of considerable importance. Since a number of chemoattractants can activate MAPK in different cell types, we wanted to explore whether the chemokine eotaxin could activate the p42/p44 MAPK signaling pathway. We demonstrate in this study that eotaxin stimulation of eosinophils leads to p42/p44 MAPK phosphorylation and activation that can be inhibited by the MEK antagonist PD98059. Functionally, eosinophils stimulated with eotaxin undergo MAPK-dependent rolling and chemotaxis, changes in the filamentous actin content and cellular distribution patterns, and increased CD11b cell surface expression. The significance of these results indicate that a critical step involved in eotaxin-induced eosinophil rolling and migration is the activation of ERK1 and ERK2, which play a key role in regulating the actin cytoskeleton rearrangements required for chemotaxis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All reagents were purchased from Sigma (St. Louis, MO) unless otherwise noted. The animal care and use committee approved all animal experimentation before implementation.
Eosinophil isolation
Eosinophils were purified from the peripheral blood of allergic donors (32). Anticoagulated blood was diluted 1/1 in PBS (Mediatech, Tustin, CA) and 0.1 mM EDTA and layered onto Percoll (density, 1.087 g/ml; pH 7.4; Pharmacia Biotech, Uppsala, Sweden). Following centrifugation, mononuclear cells were aspirated and RBC were lysed using 150 mM NH4Cl. The cells were centrifuged again, and the resulting cell pellet was incubated with anti-CD16 microbeads (Miltenyi Biotech, Burlingame, CA) to deplete neutrophils. Following Ab incubation, the cells were magnetically sorted using the MACs system (Miltenyi Biotech) to retain the positively stained neutrophils. The eluate was then washed, and viability was assessed by staining cells with trypan blue. Cytospin slides were stained with Diff-Quik (Baxter Healthcare, Miami, FL) to determine eosinophil purity, which was routinely >95%. Further RBC lysis was performed if necessary using 0.2% NaCl. For the in vivo experiments, eosinophils were fluorescently labeled with carboxyfluorescein diacetate (Molecular Probes, Eugene, OR) as previously described (33). Eosinophils were resuspended in PBS containing 0.01% glucose at a concentration of 1 x 107 cells/ml and kept at room temperature in the dark until use.
Western blot analysis
Eosinophils were resuspended at 1 x 107 cells/ml in PBS/0.1% BSA (fraction V, low endotoxin). Stimulation with eotaxin (R&D Systems, Minneapolis, MN) was conducted at 37°C and was halted by addition of cold PBS. The cells were then centrifuged at 4°C and resuspended in lysis solution consisting of 1% Nonidet P-40 (Calbiochem, La Jolla, CA), 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM PMSF, and 1 mM sodium orthovanadate. The cells were vortex mixed briefly, incubated on ice for 15 min, and subsequently centrifuged to pellet insoluble material. The supernatants were isolated, and boiled for 5 min in 2x Laemmli sample buffer, then loaded onto SDS/Tris-glycine polyacrylamide gels (Novex, San Diego, CA). Protein was transferred to a nitrocellulose membrane using the XCELL II system (Novex). Following transfer, the nitrocellulose membrane was washed briefly in water, and nonspecific protein binding sites were blocked by incubating the membrane in 2% BSA, 0.5% OVA, 2.5% nonfat dry milk (Bio-Rad, Hercules, CA), 10 mM Tris (pH 8), 150 mM NaCl, and 0.2% thimerserol for 1 h at 25°C. The membrane was incubated at 4°C overnight with p42/p44 anti-phospho-MAPK Ab (New England Biolabs, Beverly, MA) diluted 1/1000. The immunoblots were subsequently washed in TTBS (20 mM Tris-HCl (pH 7.6), 137.5 mM NaCl, and 0.1% Tween-20), three times for 5 min each time, and incubated with peroxidase-conjugated goat anti-rabbit IgG (Kirkegaard & Perry, Gaithersburg, MD) diluted 1/5000 for 1 h at 25°C. The filter was washed again three times for 5 min each time in TTBS, three times for 5 min each time in TBS (20 mM Tris-HCl (pH 7.6) and 137.5 mM NaCl), and once for 5 min in H20. Phospho-MAPK was visualized using the chemiluminescent peroxidase substrate Super Signal Ultra (Pierce, Rockford, IL). For subsequent blotting the membrane was stripped using 7 M guanidine, 50 mM glycine, 50 µM EDTA, 100 mM KCl, and 20 mM 2-ME, pH 10.8, for 10 min at 25°C. The anti-MAPK blot was conducted in the same manner, except the primary Ab was a rabbit IgG anti-human ERK-1 (p44) at 1 µg/ml (Santa Cruz Biotechnology, Santa Cruz, CA), which cross-reacts with ERK-2 (p42).
In vitro kinase assay
Eosinophils were stimulated as described, and the cell pellets
were resuspended in 20 µl of lysing solution (1% Nonidet P-40, 50 mM
Tris-HCl (pH 8.0), 150 mM NaCl, 0.25% deoxycholate, 5 mM EDTA, 1 mM
PMSF, 10 µg/ml each of leupeptin and aprotinin, 1 mM sodium
orthovanadate, 1 mM EGTA, 100 µM 
-glycerophosphate, 10 mM sodium
fluoride, and 1 mM tetrasodium phosphate) and incubated on ice for 20
min. The lysates were centrifuged to remove cell debris, 4 µg of
agarose-conjugated ERK1 and ERK2 polyclonal Abs (Santa Cruz
Biotechnology) was added, and the cell lysate was incubated rotating
for 1 h at 4°C. Immunoprecipitated ERK1 and ERK2 were washed
once with lysing solution, once in lysing solution containing 0.5 M
NaCl, and once in MAPK washing buffer (25 mM Tris-HCl (pH 7.4), 40 mM
MgCl2, 137 mM NaCl, and 10% glycerol). ERK
enzyme activity was assessed in an in vitro kinase assay using 20 µl
of buffer (42.5 mM HEPES, 42.5 mM MgCl2, 0.21 mM
ATP, 50 mM myelin basic protein (MBP; Upstate Biotechnology, Lake
Placid, NY) and 50 µCi of [
-32P]ATP
(>3000 Ci/mmol sp. act.; Amersham, Arlington Heights, IL)) for 30 min
at 30°C. Following incubation, an equal volume of 2x Laemmli sample
buffer (Novex) was added, the samples were boiled for 5 min, cooled on
ice, and loaded on 16% Tris-glycine gels (Novex). Proteins were then
transferred to Immobilon P membranes (Millipore), and phosphorylation
of the MBP substrate was visualized by autoradiography. Equal loading
of the immunoprecipitates was subsequently assayed by Western analysis
of the membrane using ERK1 and ERK2 polyclonal Abs (Santa Cruz
Biotechnology).
Chemotaxis assay
All chemotaxis assays were performed in a 48-well modified Boyden chamber (Neuroprobe, Cabin John, MD) (34). Eosinophils were resuspended in DMEM (Mediatech) with 10% FBS (HyClone, Logan, UT) at 2 x 106 cells/ml. Twenty-five microliters of medium or medium containing eotaxin (R & D Systems) at varying concentrations was added to the bottom of the chamber. A 5-µm pore size polyvinylene difluoride polycarbonate filter (Osmotics, Livermore, CA) was overlayed, and 1 x 105 eosinophils were added to the upper chamber. Where indicated eosinophils were preincubated with PD98059 (Calbiochem) at 10 µM for 15 min at 25°C. Cells were allowed to migrate for 1 h at 37°C, after which time the filter was washed twice in PBS (Mediatech), fixed in methanol for 5 min, and stained in Diff-Quik (Baxter). Migrating cells were quantified by microscopy, manually counting four fields under high power (x400).
Animal preparation and superfusion of the mesenteric vascular bed with eotaxin
The circulation of 5- and 6-carboxyfluorescein diacetate
(CFDA)-labeled human eosinophils in postcapillary venules of
IL-1-stimulated mesenteric blood vessels of New Zealand White
rabbits was examined by intravital microscopy as described previously
(33, 35). The ability of eotaxin to alter the flux of
rolling eosinophils in inflamed blood vessels was determined as
previously described (36). Between 6 and 10 h after
IL-1 stimulation (i.p.), portions of the exposed mesentery were
superfused with 50 nM eotaxin or vehicle alone using a constant flow
infusion syringe pump (Harvard Apparatus, South Natick, MA). The flow
of syringe pump was adjusted to match the flow of superfusion buffer.
CFDA-labeled eosinophils (0.2–0.5 x 107
cells/ml) were administered into the mesenteric circulation 5–10 min
after the superfusion had ensued. These cells were injected
successively through the side branch of the terminal superior
mesenteric artery cannulated with a PE10 polyethylene catheter, and
their ability to roll in the mesenteric circulation was determined.
The ability of PD98059 to alter the flux of eotaxin-induced eosinophil
rolling in IL-1-stimulated mesenteric venules was determined.
Eosinophils were incubated ex vivo with increasing concentrations of
PD98059 (0–37 µM) for 15 min at room temperature before injecting
them into the rabbit mesentery, which was continuously superfused with
eotaxin (50 nM). As a control, eosinophils were treated with vehicle
alone.
Intravital microscopy and image analysis
The passage of CFDA-labeled eosinophils in the inflamed venules was made visible by stroboscopic epi-illumination as previously described, and the images were recorded using an SVHS video recorder (33). The video images were analyzed off-line by manually counting the total number of CFDA-labeled eosinophils passing through a reference point in a venular segment (total flux). The tapes were rewound, and only those cells found to be visibly rolling along the venular wall were counted (rolling flux). The rolling fraction (RF) was calculated as the percentage of rolling cells in the total flux of eosinophils passing through a venular segment during a given injection. The interaction between eosinophils and venular endothelium in vivo before and after treatment with the MAPK inhibitor PD98059 was analyzed by Student’s t test using a statistical software package (SigmaStat, Jandel Scientific). For all tests, p values less than 5% were considered significant. Data are presented as the mean ± SD.
Actin polymerization assay
Eosinophils were resuspended in DMEM (Mediatech) and 10% FBS (HyClone) at a concentration of 5 x 105 cells/sample (37). A portion of the eosinophils was treated with PD98059 (Calbiochem) at 10 µM for 30 min at 25°C. The cells were then stimulated with 100 nM eotaxin (R & D Systems) for the indicated times at 37°C. Cells were fixed with 4% paraformaldehyde for 10 min. Cells were washed twice in PBS (Mediatech) and subsequently permeabilized with 0.1% Triton X-100 (Calbiochem) for 5 min. Cells were washed again twice in PBS. Incubating the cells in PBS/1% BSA for 10 min blocked nonspecific binding sites. Oregon Green phalloidin (325 nM; Molecular Probes) was added to samples and incubated for 20 min. Cells were washed twice in PBS and resuspended in PBS/1% BSA. Samples were analyzed by flow cytometry using a FACScan II analyzer (Becton Dickinson, Mountain View, CA).
Actin staining of eosinophils
Freshly isolated eosinophils were incubated in the presence or the absence of eotaxin and/or the MEK inhibitor PD98059 for various time points. Following stimulation at 37°C, the cells were fixed in 3.7% formaldehyde for 10 min and permeabilized with 0.1% Triton X-100 (Calbiochem). The eosinophils were subsequently stained with rhodamine-labeled phalloidin according to the manufacturer’s protocol (Molecular Probes).
Flow cytometric analysis
Isolated eosinophils were prepared and stained for flow cytometry as previously described (38). Briefly, the cells were incubated on ice for 30 min with unconjugated isotype control Abs to block nonspecific binding sites (PharMingen, San Diego, CA). The cells were then washed in 2 ml of FACS buffer (PBS containing 1% BSA and 0.1% sodium azide). The cells were subsequently incubated on ice and stained with 10 µl of the appropriate Ab, which were obtained from PharMingen. The Abs used for this study include CD18 (clone 6.7), CD62L (clone Dreg 56), CD11a (clone G43-25B), CD11b (clone ICRF44), CD11c (clone B-ly6), CD4 (clone RPA-T4), CD49d (clone 9F10), CD49e (clone VC5), CD49f (clone GoH3), HLA-DR, DP, and DQ (clone TU39). The cells were washed again in cold FACS buffer and analyzed using a FACScan II analyzer (Becton Dickinson) and CellQuest software (Becton Dickinson).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been previously shown that stimulation of a number of
different hemopoietic cell types with chemoattractants leads to the
activation of p42/p44 MAPKs (15, 16, 17, 18, 19, 20, 21). Therefore, we
investigated whether eotaxin induced MAPK activation in eosinophils.
Phosphorylation of ERKs is essential for inducing kinase activity.
Accordingly, eosinophils were incubated with 100 nM eotaxin for varying
lengths of time, and cell lysates were subjected to immunoblotting
using Abs specific for the phosphorylated forms of ERK1 (p44) and ERK2
(p42) (Fig. 1
, A and
B). A low basal level of ERK phosphorylation was detectable
in unstimulated eosinophils, particularly in p42 MAPK. Following
eotaxin treatment, ERK phosphorylation increased dramatically, with
peak phosphorylation observed between 5 and 15 min (Fig. 1A). The ERKs returned to nearly basal levels of
phosphorylation by 30 min (Fig. 1A). Because p42 and p44 are
rapidly phosphorylated, we wanted to further examine earlier time
points. Eosinophils from separate donors were isolated, and a fraction
was pretreated with 10 µM of the MEK inhibitor PD98059 for 15 min.
The eosinophils were then stimulated with 100 nM eotaxin for 1, 3, or 5
min (Fig. 1B). Eotaxin induced the phosphorylation of p42
and p44 MAPK as early as 1 min poststimulation, and this was blocked by
PD98059. We again noticed baseline phosphorylation of p42 MAPK, and
this intrinsic level of activation was not down-regulated by the
PD98059 pretreatment of eotaxin-treated cells (Fig. 1B).
These data suggest that eotaxin rapidly induces the phosphorylation of
the MAPKs p42 and p44 in human eosinophils.
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As an independent measure of MAPK activation, we directly
monitored MAPK activity in eosinophils. Eosinophils were treated with
eotaxin, ERK1 and ERK2 were then immunoprecipitated, and their kinase
activity was assessed in vitro. A slight baseline kinase activity was
observed; however, eotaxin greatly increased MAPK activity at all
concentrations tested over a 15-min stimulation period, particularly at
the 10- and 100-nM concentrations (Fig. 2, left panels). This
correlated with an increase in the amount of phospho-MAPK detected by
immunoblotting. Consistent with the phosphorylation results shown in
Fig. 1
B; a dose of 10 nM eotaxin maximally induced MAPK
activity as early as 1 min (Fig. 2, right panels). Since
phosphorylation and activation of ERKs are mediated by MEK, the
MEK-specific inhibitor, PD98059 (25), should inhibit
eotaxin-induced MAPK activation. As shown in Fig. 2, PD98059 blocked
kinase activity following stimulation with 10 nM eotaxin at 15 min,
demonstrating that p42/p44 MAPK is phosphorylated and activated via a
MEK-dependent signaling pathway. Collectively, these data demonstrate
that p42/p44 MAPK activation in eosinophils is induced by eotaxin in a
time- and dose-dependent manner.
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Since eotaxin induces MAPK activation and triggers chemotaxis
(6, 10, 11), blockade of MAPK activation may have
functional consequences for eosinophils. The effect of MAPK inhibition
on eotaxin-mediated chemotaxis of eosinophils was examined by
incubating the cells in a modified Boyden chamber with various
concentrations of eotaxin with or without 10 µM of the MEK inhibitor
PD98059. Eotaxin induced eosinophil migration in a dose-dependent
manner, which was maximal at 10 nM eotaxin (Fig. 3, A and B).
Strikingly, addition of PD98059 (Fig. 3, A and B)
almost completely abrogated eotaxin-induced eosinophil chemotactic
migration, suggesting a role for the ERK pathway in regulating
eosinophil chemotaxis in response to eotaxin stimulation. Treatment of
eosinophils with 100 ng/ml pertussis toxin before eotaxin stimulation
blocked all chemotaxis (Fig. 3B), confirming earlier
observations that CCR3 is linked to the Gi
family of G proteins (11).
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Given that in vitro eosinophil chemotaxis in response to eotaxin
was strongly inhibited by the blockade of MAPK activation, we wanted to
investigate whether earlier steps in the leukocyte migration pathway
were similarly affected. This was addressed by examining the behavior
of fluorescently labeled human eosinophils to roll in inflamed blood
vessels of the rabbit mesentery by intravital microscopy. CFDA-labeled
human eosinophils were observed to efficiently roll along the walls of
IL-1-stimulated postcapillary venules (RF, 16.4 ± 6.7%).
However, superfusion of the mesentery with 50 nM eotaxin resulted in a
3-fold increase in the flux of rolling eosinophils (RF, 48.3 ±
7.2; Fig. 4A). Pretreatment of
eosinophils with the MEK inhibitor PD98059 for 15 min resulted in a
dose-dependent decrease in the flux of rolling eosinophils (Fig. 4B). Maximum inhibition of rolling was observed when
eosinophils were treated with PD98059 at a concentration of 37 µM
(
60% inhibition; p < 0.01 vs control). The rolling
of vehicle-treated eosinophils was not effected. Together with the in
vitro data, this suggests that the p42/p44 MAPK pathway regulates
multiple steps (rolling and chemotaxis) in the migration process of
eotaxin-stimulated eosinophils.
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Actin polymerization controls a number of processes regulating
eosinophil migration. First, the polymerization of actin provides the
contractile forces necessary for chemotaxis of the cell
(27). Second, the actin cytoskeleton regulates the
affinity state of multiple adhesion molecules toward their
counter-receptors and their positioning on the eosinophil microvilli
(28, 29, 30, 31). Therefore, we examined the effect of
eotaxin-induced MAPK activation on actin polymerization by using the
MEK inhibitor PD98059. Eotaxin-induced actin polymerization was
strongly inhibited, as there was a 59.1% decrease in the F-actin
content of the PD98059 and eotaxin-stimulated eosinophils compared with
that of eotaxin alone-treated eosinophils (Fig. 5).
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,
upper right panel). In contrast, the addition of 10 nM
eotaxin dramatically changed both the actin distribution and
polymerization into bundles after just 45 s (Fig. 6, lower
left panel). This effect on intracellular actin rearrangement was
blocked by PD98059 (Fig. 6, lower right panel). Thus, actin
polymerization and redistribution in eosinophils appear to be
MAPK-regulated processes. This suggests that MAPK activation could
regulate eosinophil migration by controlling both the location on the
cell membrane and avidity of adhesion molecules as well as the changes
in cell structure necessary for diapedesis to occur.
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To further investigate the role of ERK activation in
eotaxin-induced eosinophil migration, we examined the expression of
cell surface molecules. Eotaxin treatment of eosinophils increased
CD11b cell surface expression; however, PD98059 had a minimal effect on
this up-regulation, indicating that the p42/p44 MAPK pathway is
probably not involved (Fig. 7). Induction
of CD11b expression appeared to be unique in that expression of LFA-1,
CD18, CD11c, CD62L, VLA-4, VLA-5, VLA-6, CD4, or HLA class II was not
modulated by eotaxin treatment (data not shown). As a control,
eosinophils treated with 10 ng/ml IL-5 for 45 min shed L-selectin
(CD62L; data not shown) (39). CD11b has previously been
shown not to play a significant role in eosinophil tethering and
rolling in response to chemoattractants (33). Consistent
with these previous results, CD11b did not appear to play a major
part in in vivo rolling in this study, as eotaxin induced its
expression, which was not affected by PD98059 treatment, yet in vivo
rolling was substantially decreased by MEK inhibitor treatment
(Fig. 4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-stimulated mesenteric venules under conditions of blood flow by
intravital microscopy. Previous results have shown that VLA-4 acts in
concert with L-selectin in promoting eosinophil rolling, leading to
firm adhesion in vivo (33). We demonstrate that blockade of p42/p44 MAPK activation strongly inhibits eosinophil rolling in vivo. Once the cell is stably arrested to the endothelium, the leukocyte can diapedesis through the vessel wall and migrate toward the source of the chemoattractant, a process involving cell motility. Our results examining the polymerization and redistribution of the eosinophil actin cytoskeleton in response to eotaxin stimulation point to multiple mechanisms in which MAPK activation can regulate chemotaxis. First, eotaxin-induced MAPK activation induces an immediate rise in the cellular F-actin content of the eosinophil, which may allow for proper adhesion molecule positioning within the microvilli (30). Second, intracellular actin distribution and orientation control the avidity of VLA-4 and L-selectin (CD62L) toward their counter-receptors expressed on the luminal surface of the vessel (28, 29, 30, 31). Third, actin rearrangement may govern the physical process of chemotaxis by directly regulating changes in cell shape and motility (27). The finding that the MEK-specific inhibitor PD98059 constrains rolling and chemotaxis, presumably by impeding ERK activation and ultimately actin reorganization, is consistent with studies showing that cytochalasin B also inhibits cell migration (28). Thus, a critical event controlling eosinophil trafficking in vivo is p42/p44 MAPK-regulated intracellular actin reorganization of the cytoskeleton.
The eosinophils from many of the allergic donors in this study varied
in their basal activation state of both ERK1 and ERK2 (data not shown).
Because activation of p42/p44 MAPK appears to regulate chemotaxis, this
illustrates the difficulty in controlling eosinophil tissue
accumulation in allergic diseases. We consistently observed in our
studies with eosinophils a baseline phosphorylation of ERK2, previously
noted by others (17). Treatment of eosinophils with 10
µM PD98059 did not reduce the basal level of p42 MAPK
phosphorylation; however, this concentration of PD98059 maintained
specificity for the MEK/ERK pathway. The lack of reduction in the basal
level of p42 MAPK phosphorylation may be explained by the fact that
PD98059 inhibits MEK2 activity with an IC50 of
50 µM, which is 20-fold less potent than its effect on MEK1
(IC50, 2.4 µM) (25). This
suggests that the baseline level of phosphorylation of p42 MAPK after
pretreatment with PD98059 may be specifically due to the kinase
activity of MEK2. This raises the possibility that MEK1 activity is
critical for the functional changes observed, and furthermore, that
MEK1 or MEK2 may preferentially phosphorylate either ERK1 or ERK2. It
has already been shown that the p42 and p44 MAPK isoforms
differentially regulate eosinophil function (43).
Specifically, activation of p44 MAPK results in the activation of the
Elk-1 transcription factor, whereas ERK2 activation stimulates c-Myc
(43).
In the broader context of chemokine-regulated inflammation, this study
along with others (15, 16, 17, 18, 19, 20, 21) raise the question of whether
chemokines binding to their receptors in multiple cell types initiates
a common signaling pathway leading to ERK1 and ERK2 activation and
culminating functionally in chemotaxis. Both CC and CXC chemokines have
been shown to activate the p42/p44 MAPKs in eosinophils, T lymphocytes,
thymocytes, monocytes, and neutrophils. In addition, the chemokines
RANTES, MCP-3, and eotaxin all increase F-actin levels in eosinophils
following stimulation (44). Signaling leading to MAPK
activation has been best studied in neutrophils stimulated with IL-8
(15). These studies demonstrated that ERK activation
followed Ras GTP loading and activation of Raf-1 and B-Raf
(15). A role for phosphatidylinositol 3-kinase in
regulating p42/p44 MAPK activation was also implicated. Similar
observations were made with eosinophils stimulated with MIP-3
(unpublished observations), IL-5, or platelet-activating factor
(17), and monocytes treated with MCP-1 (16).
Our results with PD98059 demonstrate that MEK-1 and -2 act upstream in
the ERK activation cascade, while actin polymerization occurs
downstream of p42/p44 MAPK activation. The chemokine pathway leading to
ERK activation may be different from those signal transduction pathways
triggered by nonchemokine chemoattractants such as C5a and FMLP
(45, 46). Specifically, in neutrophils FMLP-induced
F-actin polymerization and subsequent chemotaxis are not blocked by the
MEK inhibitor PD98059 (47). This again suggests that there
multiple signaling pathways leading to leukocyte chemotaxis, a finding
suggested by our results using eosinophils.
In summary, understanding the intracellular signaling pathways triggered by chemoattractants that govern eosinophil trafficking to sites of inflammation may offer additional molecular targets for antagonism of eosinophil-mediated allergic diseases. This study demonstrates that eotaxin-stimulated eosinophils activate p42/p44 MAPK, which leads to changes in the intracellular actin organization and polymerization that may directly affect eosinophil chemotaxis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stefen A. Boehme, Neurocrine Biosciences, Inc., 10555 Science Center Drive, San Diego, CA 92121. E-mail address:
3 Abbreviations used in this paper: MCP, monocyte chemoattractant protein; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK ERK kinase; VLA-4, very late Ag-4; MBP, myelin basic protein; RF, rolling fraction; F-actin, filamentous actin; CFDA, 5- and 6-carboxyfluorescein diacetate.
Received for publication March 1, 1999. Accepted for publication May 14, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chemokine, stromal cell-derived factor-1, binds to the transmembrane G-protein-coupled CXCR-4 receptor and activates multiple signal transduction pathways. J. Biol. Chem. 273:23169. induces migration and activation of human thymocytes. Blood 91:2905.1 and 2 integrin avidity by chemoattractants in eosinophils. Proc. Natl. Acad. Sci. USA 93:10939.-actinin: receptor positioning in microvilli does not require interaction with -actinin. J. Cell Biol. 129:1155.This article has been cited by other articles:
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  S. A. Boehme, K. Franz-Bacon, E. P. Chen, T. W. Ly, Y. Kawakami, and K. B. Bacon Murine bone marrow-derived mast cells express chemoattractant receptor-homologous molecule expressed on T-helper class 2 cells (CRTh2) Int. Immunol., June 1, 2009; 21(6): 621 - 632. [Abstract] [Full Text] [PDF]
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S. A. Boehme, E. P. Chen, K. Franz-Bacon, R. Sasik, L. J. Sprague, T. W. Ly, G. Hardiman, and K. B. Bacon Antagonism of CRTH2 ameliorates chronic epicutaneous sensitization-induced inflammation by multiple mechanisms Int. Immunol., January 1, 2009; 21(1): 1 - 17. [Abstract] [Full Text] [PDF]
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 J. H. Kang, D. H. Lee, H. Seo, J. S. Park, K. H. Nam, S. Y. Shin, C.-S. Park, and I. Y. Chung Regulation of Functional Phenotypes of Cord Blood Derived Eosinophils by {gamma}-Secretase Inhibitor Am. J. Respir. Cell Mol. Biol., November 1, 2007; 37(5): 571 - 577. [Abstract] [Full Text] [PDF]
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 P. Magni, E. Dozio, M. Ruscica, H. Watanobe, A. Cariboni, R. Zaninetti, M. Motta, and R. Maggi Leukemia Inhibitory Factor Induces the Chemomigration of Immortalized Gonadotropin-Releasing Hormone Neurons through the Independent Activation of the Janus Kinase/Signal Transducer and Activator of Transcription 3, Mitogen-Activated Protein Kinase/Extracellularly Regulated Kinase 1/2, and Phosphatidylinositol 3-Kinase/Akt Signaling Pathways Mol. Endocrinol., May 1, 2007; 21(5): 1163 - 1174. [Abstract] [Full Text] [PDF]
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 V. Dolgachev, M. Thomas, A. Berlin, and N. W. Lukacs Stem cell factor-mediated activation pathways promote murine eosinophil CCL6 production and survival J. Leukoc. Biol., April 1, 2007; 81(4): 1111 - 1119. [Abstract] [Full Text] [PDF]
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H. Chunsong, H. Yuling, W. Li, X. Jie, Z. Gang, Z. Qiuping, G. Qingping, Z. Kejian, Q. Li, A. E. Chang, et al. CXC Chemokine Ligand 13 and CC Chemokine Ligand 19 Cooperatively Render Resistance to Apoptosis in B Cell Lineage Acute and Chronic Lymphocytic Leukemia CD23+CD5+ B Cells J. Immunol., November 15, 2006; 177(10): 6713 - 6722. [Abstract] [Full Text] [PDF]
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H. C. Lane, A. R. Anand, and R. K. Ganju Cbl and Akt regulate CXCL8-induced and CXCR1- and CXCR2-mediated chemotaxis Int. Immunol., August 1, 2006; 18(8): 1315 - 1325. [Abstract] [Full Text] [PDF]
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U. Sela, N. Mauermann, R. Hershkoviz, H. Zinger, M. Dayan, L. Cahalon, J. P. Liu, E. Mozes, and O. Lider The Inhibition of Autoreactive T Cell Functions by a Peptide Based on the CDR1 of an Anti-DNA Autoantibody Is via TGF-{beta}-Mediated Suppression of LFA-1 and CD44 Expression and Function J. Immunol., December 1, 2005; 175(11): 7255 - 7263. [Abstract] [Full Text] [PDF]
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Y. Kobayashi, S. Ueki, G. Mahemuti, T. Chiba, H. Oyamada, N. Saito, A. Kanda, H. Kayaba, and J. Chihara Physiological Levels of 15-Deoxy-{Delta}12,14-Prostaglandin J2 Prime Eotaxin-Induced Chemotaxis on Human Eosinophils through Peroxisome Proliferator-Activated Receptor-{gamma} Ligation J. Immunol., November 1, 2005; 175(9): 5744 - 5750. [Abstract] [Full Text] [PDF]
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P. Joubert, S. Lajoie-Kadoch, I. Labonte, A. S. Gounni, K. Maghni, V. Wellemans, J. Chakir, M. Laviolette, Q. Hamid, and B. Lamkhioued CCR3 Expression and Function in Asthmatic Airway Smooth Muscle Cells J. Immunol., August 15, 2005; 175(4): 2702 - 2708. [Abstract] [Full Text] [PDF]
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S. R. White, R. Tse, and B. A. Marroquin Stress-Activated Protein Kinases Mediate Cell Migration in Human Airway Epithelial Cells Am. J. Respir. Cell Mol. Biol., April 1, 2005; 32(4): 301 - 310. [Abstract] [Full Text] [PDF]
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G. Badr, G. Borhis, D. Treton, and Y. Richard IFN{alpha} enhances human B-cell chemotaxis by modulating ligand-induced chemokine receptor signaling and internalization Int. Immunol., April 1, 2005; 17(4): 459 - 467. [Abstract] [Full Text] [PDF]
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U. Sela, R. Hershkoviz, L. Cahalon, O. Lider, and E. Mozes Down-Regulation of Stromal Cell-Derived Factor-1{alpha}-Induced T Cell Chemotaxis by a Peptide Based on the Complementarity-Determining Region 1 of an Anti-DNA Autoantibody via Up-Regulation of TGF-{beta} Secretion J. Immunol., January 1, 2005; 174(1): 302 - 309. [Abstract] [Full Text] [PDF]
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S. Oka, S. Ikeda, S. Kishimoto, M. Gokoh, S. Yanagimoto, K. Waku, and T. Sugiura 2-Arachidonoylglycerol, an endogenous cannabinoid receptor ligand, induces the migration of EoL-1 human eosinophilic leukemia cells and human peripheral blood eosinophils J. Leukoc. Biol., November 1, 2004; 76(5): 1002 - 1009. [Abstract] [Full Text] [PDF]
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 Z. Qiuping, X. Jei, J. Youxin, J. Wei, L. Chun, W. Jin, W. Qun, L. Yan, H. Chunsong, Y. Mingzhen, et al. CC Chemokine Ligand 25 Enhances Resistance to Apoptosis in CD4+ T Cells from Patients with T-Cell Lineage Acute and Chronic Lymphocytic Leukemia by Means of Livin Activation Cancer Res., October 15, 2004; 64(20): 7579 - 7587. [Abstract] [Full Text] [PDF]
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S. A. Boehme, F. M. Lio, L. Sikora, T. S. Pandit, K. Lavrador, S. P. Rao, and P. Sriramarao Cutting Edge: Serotonin Is a Chemotactic Factor for Eosinophils and Functions Additively with Eotaxin J. Immunol., September 15, 2004; 173(6): 3599 - 3603. [Abstract] [Full Text] [PDF]
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W. Duan, J. H. P. Chan, C. H. Wong, B. P. Leung, and W. S. F. Wong Anti-Inflammatory Effects of Mitogen-Activated Protein Kinase Kinase Inhibitor U0126 in an Asthma Mouse Model J. Immunol., June 1, 2004; 172(11): 7053 - 7059. [Abstract] [Full Text] [PDF]
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 M. J. Smit, P. Verdijk, E. M. H. van der Raaij-Helmer, M. Navis, P. J. Hensbergen, R. Leurs, and C. P. Tensen CXCR3-mediated chemotaxis of human T cells is regulated by a Gi- and phospholipase C-dependent pathway and not via activation of MEK/p44/p42 MAPK nor Akt/PI-3 kinase Blood, September 15, 2003; 102(6): 1959 - 1965. [Abstract] [Full Text] [PDF]
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 L. Sikora, S. P. Rao, and P. Sriramarao Selectin-dependent rolling and adhesion of leukocytes in nicotine-exposed microvessels of lung allografts Am J Physiol Lung Cell Mol Physiol, September 1, 2003; 285(3): L654 - L663. [Abstract] [Full Text] [PDF]
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 S. Kishimoto, M. Gokoh, S. Oka, M. Muramatsu, T. Kajiwara, K. Waku, and T. Sugiura 2-Arachidonoylglycerol Induces the Migration of HL-60 Cells Differentiated into Macrophage-like Cells and Human Peripheral Blood Monocytes through the Cannabinoid CB2 Receptor-dependent Mechanism J. Biol. Chem., June 27, 2003; 278(27): 24469 - 24475. [Abstract] [Full Text] [PDF]
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A. Holub, J. Byrnes, S. Anderson, L. Dzaidzio, N. Hogg, and A. Huttenlocher Ligand density modulates eosinophil signaling and migration J. Leukoc. Biol., May 1, 2003; 73(5): 657 - 664. [Abstract] [Full Text] [PDF]
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A. P. Curnock, Y. Sotsios, K. L. Wright, and S. G. Ward Optimal Chemotactic Responses of Leukemic T Cells to Stromal Cell-Derived Factor-1 Requires the Activation of Both Class IA and IB Phosphoinositide 3-Kinases J. Immunol., April 15, 2003; 170(8): 4021 - 4030. [Abstract] [Full Text] [PDF]
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V. E. L. Stubbs, P. Schratl, A. Hartnell, T. J. Williams, B. A. Peskar, A. Heinemann, and I. Sabroe Indomethacin Causes Prostaglandin D2-like and Eotaxin-like Selective Responses in Eosinophils and Basophils J. Biol. Chem., July 12, 2002; 277(29): 26012 - 26020. [Abstract] [Full Text] [PDF]
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H. Tachimoto, M. Kikuchi, S. A. Hudson, C. A. Bickel, R. G. Hamilton, and B. S. Bochner Eotaxin-2 Alters Eosinophil Integrin Function via Mitogen-Activated Protein Kinases Am. J. Respir. Cell Mol. Biol., June 1, 2002; 26(6): 645 - 649. [Abstract] [Full Text] [PDF]
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T. Adachi, R. Vita, S. Sannohe, S. Stafford, R. Alam, H. Kayaba, and J. Chihara The Functional Role of Rho and Rho-Associated Coiled-Coil Forming Protein Kinase in Eotaxin Signaling of Eosinophils J. Immunol., October 15, 2001; 167(8): 4609 - 4615. [Abstract] [Full Text] [PDF]
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B.-S. Youn, Y. J. Kim, C. Mantel, K.-Y. Yu, and H. E. Broxmeyer Blocking of c-FLIPL-independent cycloheximide-induced apoptosis or Fas-mediated apoptosis by the CC chemokine receptor 9/TECK interaction Blood, August 15, 2001; 98(4): 925 - 933. [Abstract] [Full Text] [PDF]
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C. Bandeira-Melo, A. Herbst, and P. F. Weller Eotaxins . Contributing to the Diversity of Eosinophil Recruitment and Activation Am. J. Respir. Cell Mol. Biol., June 1, 2001; 24(6): 653 - 657. [Full Text] [PDF]
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H. Sano, X. Zhu, A. Sano, E. E. Boetticher, T. Shioya, B. Jacobs, N. M. Munoz, and A. R. Leff Extracellular Signal-Regulated Kinase 1/2-Mediated Phosphorylation of Cytosolic Phospholipase A2 Is Essential for Human Eosinophil Adhesion to Fibronectin J. Immunol., March 1, 2001; 166(5): 3515 - 3521. [Abstract] [Full Text] [PDF]
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S. A. Boehme, F. M. Lio, D. Maciejewski-Lenoir, K. B. Bacon, and P. J. Conlon The Chemokine Fractalkine Inhibits Fas-Mediated Cell Death of Brain Microglia J. Immunol., July 1, 2000; 165(1): 397 - 403. [Abstract] [Full Text] [PDF]
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O. T. Lynch, M. A. Giembycz, I. Daniels, P. J. Barnes, and M. A. Lindsay Pleiotropic role of lyn kinase in leukotriene B4-induced eosinophil activation Blood, June 1, 2000; 95(11): 3541 - 3547. [Abstract] [Full Text] [PDF]
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M. E. Bates, V. L. Green, and P. J. Bertics ERK1 and ERK2 Activation by Chemotactic Factors in Human Eosinophils Is Interleukin 5-dependent and Contributes to Leukotriene C4 Biosynthesis J. Biol. Chem., April 6, 2000; 275(15): 10968 - 10975. [Abstract] [Full Text] [PDF]
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G. T. Kampen, S. Stafford, T. Adachi, T. Jinquan, S. Quan, J. A. Grant, P. S. Skov, L. K. Poulsen, and R. Alam Eotaxin induces degranulation and chemotaxis of eosinophils through the activation of ERK2 and p38 mitogen-activated protein kinases Blood, March 15, 2000; 95(6): 1911 - 1917. [Abstract] [Full Text] [PDF]
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N. Zimmermann, B. L. Daugherty, J. M. Stark, and M. E. Rothenberg Molecular Analysis of CCR-3 Events in Eosinophilic Cells J. Immunol., January 15, 2000; 164(2): 1055 - 1064. [Abstract] [Full Text] [PDF]
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J. A. Burger, M. Burger, and T. J. Kipps Chronic Lymphocytic Leukemia B Cells Express Functional CXCR4 Chemokine Receptors That Mediate Spontaneous Migration Beneath Bone Marrow Stromal Cells Blood, December 1, 1999; 94(11): 3658 - 3667. [Abstract] [Full Text] [PDF]
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