CC Chemokine Receptor-3 Undergoes Prolonged Ligand-induced
Internalization*
Nives
Zimmermann
,
Juliana J.
Conkright§, and
Marc E.
Rothenberg
¶
From the
Division of Pulmonary Medicine, Allergy, and
Clinical Immunology and the § Division of Pulmonary Biology
and Neonatology, Department of Pediatrics, Children's Hospital Medical
Center, Cincinnati, Ohio 45229
 |
ABSTRACT |
CC chemokine receptor-3 (CCR-3) is a major
receptor involved in regulating eosinophil trafficking; therefore,
elucidation of ligand-induced CCR-3 events has important implications
in understanding the biological and pathological properties of
eosinophils. Previous studies have demonstrated that unique receptor
events occur in different cell types supporting investigation of
CCR-3-mediated events in eosinophilic cells. We now report biochemical
characterization of CCR-3 internalization following exposure of
eosinophils to CCR-3 ligands. Treatment of freshly isolated human
eosinophils with CCR-3 ligands resulted in marked and differential
internalization of CCR-3 in a dose-dependent manner.
Exposure to 100 ng/ml eotaxin reduced surface expression to 43, 43, and
76% at 15 min, 1 h, and 3 h, respectively. RANTES (reduced
on activation T cell expressed and secreted) treatment induced more
significant and prolonged internalization of CCR-3 than eotaxin;
following 100 ng/ml of RANTES, 29, 24, and 47% of the receptor was
expressed at 15 min, 3 h, and 18 h, respectively. Confocal
microscopy demonstrated that receptor modulation involved receptor
internalization by an endocytic pathway shared with the transferrin
receptor. Receptor internalization was accompanied by partial
degradation of CCR-3, and reexpression of CCR-3 was dependent in part
upon de novo protein synthesis. Internalization was not
blocked by pretreatment of eosinophils with pertussis toxin.
Furthermore, staurosporine did not inhibit internalization although it
blocked phorbol 12-myristate 13-acetate-induced CCR-3 down-modulation.
These results demonstrate that CCR-3 ligands induce differential
receptor internalization that is not dependent upon
Gi-protein coupling, calcium transients, or protein
kinase C.
 |
INTRODUCTION |
Eosinophils are bone marrow-derived granulocytes associated
with numerous diseases including allergic disorders, parasitic infections, and malignancies (1-3). Elucidating the processes that regulate eosinophil tissue accumulation is fundamental, since these cells markedly increase in tissue locations and cause potent proinflammatory effects in numerous diseases.
Recent studies have focused on the orchestration of eosinophil tissue
accumulation by chemokines, especially eotaxin, the most selective
eosinophil chemoattractant identified to date (4, 5). Chemokines are
grouped into the CXC, CC, C, and CX3C subfamilies on the
basis of the arrangement of the conserved cysteine residues (6-8). The
CXC and CC groups, in contrast to the C and CX3C groups, contain many members and have been studied in detail. The specific effects of chemokines are mediated by a family of seven-transmembrane spanning G-protein-coupled receptors
(GPCR)1 (9, 10). To date, 17 chemokine receptors have been described: receptors for the CXC
chemokines CXCR-1 through -5; the CC chemokines CCR-1 through -10; the
receptor for CX3C chemokine CX3CR-1; and the C
chemokine XCR-1. Most CXC chemokines are active on neutrophils, while
CC chemokines have variable potencies for monocytes, lymphocytes, eosinophils, and basophils. In addition to mediating leukocyte chemoattraction and activation, selected chemokine receptors
(e.g. CXCR-4, CCR-2, CCR-3, and CCR-5) serve as co-receptors
for the entry of human immunodeficiency virus type 1 into cells
(11).
The major chemokine receptor operational in eosinophils is CCR-3. This
receptor appears to play a central role in allergic responses, since it
is not only expressed on eosinophils but also on basophils and Th2
lymphocytes, other cells central in allergic responses (12, 14-17).
CCR-3 binds multiple ligands including eotaxin, RANTES, MCP-2, MCP-3,
and MCP-4. Of these chemokines, only eotaxin signals exclusively
through CCR-3. Eotaxin has been shown to be responsible for eosinophil
trafficking during base-line and inflammatory processes (18-23).
Following ligand binding to GPCRs, cellular responses are rapidly
attenuated. This may be particularly important in eosinophils, since
treatment of eosinophils with eotaxin, a chemokine specific for CCR-3,
for only 10 min in vitro results in chemokine
unresponsiveness for at least 8 h when the cells are adoptively
transferred in vivo (21). A variety of mechanisms may be
responsible for signal attenuation including receptor desensitization,
endocytosis, and down-regulation (24). Each receptor and each cell type
utilize unique mechanisms indicating the importance of elucidating
individual receptor events in the appropriate cell type (25). It is
therefore important to analyze CCR-3-mediated events in eosinophilic
cells rather than CCR-3-transfected heterologous cells. We now report biochemical analysis of CCR-3 internalization following exposure of
human eosinophils to CCR-3 ligands.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
The AML14.3D10 cell line (kindly provided by
C. Paul and M. Baumann, Dayton Veterans Affairs Medical Center, Dayton,
OH) was grown in RPMI 1640 (Life Technologies, Inc.) containing 10%
fetal calf serum (Life Technologies), 50 µM
2-mercaptoethanol (Sigma), 0.1 mM nonessential amino acids
(Life Technologies), 1 mM sodium pyruvate (Sigma), and
penicillin-streptomycin (Life Technologies). Initially, the pH of the
media was titrated to 7.8, but after it was established that CCR-3
expression does not depend upon the pH of the media (data not shown)
this practice was discontinued. A stock of 50 mM butyric
acid (Sigma) in phosphate-buffered saline (PBS) was prepared and stored
at 4 °C prior to use. IL-5 (R&D, Minneapolis, MN) was prepared as a
stock of 100 µg/ml and stored at
80 °C prior to use. Induction
experiments were performed by growing cells in six-well plates on glass
coverslips, starting with a concentration of ~105 cells
in 3 ml/well. Cells were induced with 0.5 mM butyric acid. 2 days later, 10 ng/ml IL-5 was added, and experiments were performed on day 7. Medium was not replenished during the induction period. These
cells will be referred to as dAML.
Eosinophil Purification--
Eosinophils were purified from
healthy or mildly atopic volunteers by negative immunomagnetic
selection based on the method of Hansel et al. (26).
Briefly, granulocytes were isolated from heparin-anticoagulated whole
blood by dextran sedimentation, Percoll centrifugation, and hypotonic
lysis of red blood cells. Cells were resuspended in Hanks' buffered
salt solution (Life Technologies, Inc.) with 2% fetal calf serum and
incubated with 0.75 µl/106 cells of anti-CD16-conjugated
microbeads (MACS; Miltenyi Biotech Inc., Sunnyvale, CA) for 30 min at
4 °C. The cell suspension was then applied onto a CS MACS column,
and negative populations were collected through a magnetic field. The
isolates routinely contained >95% eosinophils with viability of
>95% as assessed by trypan blue exclusion. For internalization
experiments, freshly isolated eosinophils were plated at 0.5 × 106/ml in the same medium used for AML cells but
supplemented with 50 pM IL-5. Cells were cultured
(37 °C, 5% CO2) for a maximum of 22 h, and
viability was >95%.
Intracellular [Ca2+] Measurement--
Cells
(2 × 106/ml) were loaded with 5 µM
Fura-2/AM (Molecular Probes, Inc., Eugene, OR) in Hanks' buffered salt
solution with 1% fetal calf serum for 60 min at 37 °C in the dark.
After two washes in flux buffer (145 mM NaCl, 4 mM KCl, 1 mM NaHPO4, 0.8 mM MgCl2, 1.8 mM CaCl2,
25 mM Hepes, and 22 mM glucose), cells were
resuspended at 2 × 106 cells/ml and maintained on
ice. Cells (2 ml) were prewarmed to 37 °C and stimulated in a
cuvette with a continuously stirring magnetic bar using a RatioMaster
fluorimeter (Photon Technology, Inc., South Brunswick, NJ). Data were
recorded as the relative ratio of fluorescence emitted at 510 nm after
excitation at 340 and 380 nm (y axis) over time
(x axis).
Flow Cytometry--
Cells (5 × 105) were
washed with FACS buffer (2% bovine serum albumin, 0.1% sodium azide
in PBS) and incubated with 0.5 µg of anti-human CCR-3 antibody (clone
7B11, kindly provided by Dr. Paul Ponath, Leukosite, Cambridge, MA),
0.5 µg of anti-CD18 antibody (clone TS1/18, ATCC), or the mouse
isotype-matched control IgG2a or IgG1, respectively (Pharmingen, San
Diego, CA) for 30 min at 4 °C. After two washes in FACS buffer,
cells were incubated with 0.5 µg of fluorescein
isothiocyanate-conjugated isotype-specific secondary antibody
(Pharmingen) for 30 min at 4 °C in the dark. After two washes,
labeled cells were subjected to flow cytometry on a FACScan flow
cytometer (Becton Dickinson) and analyzed using the CELLQuest software
(Becton Dickinson). Internalization of surface CCR-3 was assayed by
incubating cells at 37 °C for the indicated lengths of time with
0-1000 ng/ml human eotaxin or human RANTES (Peprotech, Rocky Hill,
NJ). In other experiments, eosinophils were exposed to pertussis toxin
(List Laboratories, Campbell, CA) at a dose of 20-1000 ng/ml for
3 h, and chemokine was added for the last hour of the incubation
period. In other experiments, eosinophils were exposed to 1-100 ng/ml
staurosporine (Sigma) for 3 h, and chemokine or phorbol
12-myristate 13-acetate (PMA; Sigma) was added for the last hour of the
3-h incubation period. Following chemokine exposure, cells were
immediately placed on ice and washed with at least twice the volume of
cold FACS buffer. Receptor density (percentage) was calculated as
100 × (mean channel fluorescence of chemokine
mean
channel fluorescence of isotype-matched control)/(mean channel
fluorescence of medium
mean channel fluorescence of
isotype-matched control). Results are expressed as mean ± S.E.
Western Blotting--
Whole cell lysates were prepared from
eosinophils by washing twice in cold PBS and lysing in radioimmune
precipitation assay buffer (1% Nonidet P-40, 0.5% sodium
deoxycholate, and 0.1% SDS in PBS) with 10 µg/ml aprotinin, 10 µg/ml antipain, 10 µg/ml chymostatin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A (all from Boehringer Mannheim) and 2 mM
phenylmethylsulfonyl fluoride (Sigma). Detergent-insoluble material was
removed by centrifugation at 12,000 × g for 15 min at
4 °C. Supernatants were stored in siliconized tubes and either used
immediately or stored at
80 °C. The protein concentration was
determined using the bicincholic acid assay (Pierce), and 50 µg were
separated by electrophoresis on a 10% SDS-polyacrylamide gel and
transferred to a nitrocellulose membrane. Equal loading was verified by
staining with Ponceau S (Sigma). After blocking the membrane for 1 h at room temperature in Tris-buffered saline with 0.2% Tween 20 (TBST) with 5% dry milk, the anti-CCR-3 polyclonal rabbit antiserum
(kindly provided by Dr. Bruce Daugherty, Merck) was added for 1 h
at room temperature (1:5000 in TBST), followed by goat anti-rabbit
horseradish peroxidase-conjugated secondary antibody (1:10,000 in TBST;
Calbiochem). The signal was developed using enhanced chemiluminescence
(Amersham Pharmacia Biotech) according to the manufacturer's
instructions. Antibody specificity was determined on lysates obtained
from HOS.CD4 cells transfected with CCR-1 or CCR-3 (AIDS Research and
Reference Reagent Program, Rockville, MD) (27, 28). Cycloheximide
(Sigma) was used at 10 µg/ml for 3 h. Inhibition of protein
synthesis (>80%) was verified by [35S]methionine
incorporation for 3 h in the presence or absence of cycloheximide.
Protein was precipitated by trichloroacetic acid, and radioactivity
measured in a
-counter.
Confocal Microscopy--
Induced AML cells were cultured in
six-well plates on glass coverslips. For experiments, the chemokine was
added to the growth medium for 3 h at 37 °C. To stop the
reaction, cells were placed on ice and fixed with 3% paraformaldehyde
in PBS. The fixed cells were washed with PBS, quenched with 15 mM glycine in PBS, and permeabilized with 0.2% saponin in
permeabilization buffer (1% cold fish gelatin and 1% bovine serum
albumin in PBS). Staining was achieved with the 7B11 antibody (0.75 µg/coverslip) in 1% cold fish gelatin and 3% bovine serum albumin
in PBS for 1 h at room temperature. Following three washes in 1%
cold fish gelatin in PBS, Texas Red-conjugated anti-mouse IgG (Jackson
Immunoresearch Laboratories, Inc., West Grove, PA) was added for 1 h at room temperature. Finally, cells were stained with fluorescein
isothiocyanate-conjugated anti-CD71 (transferrin receptor) antibody
(Research Diagnostics, Inc., Flanders, NJ). Cells were washed three
times with 1% cold fish gelatin, twice with PBS, and once with water.
Coverslips were mounted onto slides, sealed, and stored at
20 °C
until analysis on a Leica DMIRBE inverted microscope equipped with a
confocal laser scanner. Images were analyzed with Metamorph (Universal Imaging Corporation, West Chester, PA) and printed in Adobe Photoshop (Adobe Systems Inc., Mountain View, CA). In some experiments, fresh
human eosinophils were treated with chemokine for 15 min, cytocentrifuged, and stained for CCR-3 as above.
 |
RESULTS |
Ligand-induced Internalization of CCR-3--
In order to
investigate whether CCR-3 attenuation involves modulation of receptor
expression, we examined receptor internalization following ligand
binding. First, the surface expression of CCR-3 over 18 h
following exposure of eosinophils to eotaxin was investigated by FACS
analysis. Eotaxin (100 ng/ml) caused receptor loss, which was
detectable after 15 min, remained reduced at 3 h, and returned to
base line at 18 h. The combined results are shown in Fig.
1, which demonstrate only 43 ± 9, 43 ± 2, and 76 ± 4% of the original receptor level present
on the surface after 15 min, 1 h, and 3 h, respectively. A
representative experiment is shown in Fig. 2. Exposure of eosinophils to RANTES (100 ng/ml), another CCR-3 ligand, also internalized CCR-3. Only 29 ± 6, 24 ± 2, 24 ± 6, and 47 ± 7% of the receptor
remain on the cell surface after 15 min, 1 h, 3 h, and
18 h, respectively (Fig. 1). A representative experiment for
RANTES is shown in Fig. 2.

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Fig. 1.
CCR-3 cell surface expression after
stimulation of human eosinophils with CCR-3 ligands. Freshly
isolated peripheral blood eosinophils were cultured with 100 ng/ml
eotaxin (solid line) or RANTES (dashed
line) for the indicated lengths of time. Cell surface
expression of CCR-3 was measured by FACS analysis and compared with
CCR-3 expression of eosinophils not treated with the chemokine. Results
are expressed as mean ± S.E. of the mean of three separate
experiments. The difference in CCR-3 expression after eotaxin and
RANTES treatment was statistically significant for all time points with
p < 0.05 using the paired Student's t
test.
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Fig. 2.
CCR-3 cell surface expression after
stimulation with eotaxin or RANTES. Eosinophils were incubated
with eotaxin (A-D) or RANTES (E-H) for 15 min
(A, E), 1 h (B, F),
3 h (C, G), and 18 h (D,
H) and the cell surface expression was assessed by FACS
analysis. Results are of a representative experiment (n = 3). The isotype-matched control is depicted as the filled
histogram, CCR-3 expression without chemokine as a
solid line, and expression with chemokine as a
dashed line.
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In all instances, RANTES induced a greater magnitude of CCR-3
internalization and had a longer duration compared with eotaxin (p < 0.05). We were interested in determining if this
difference could be related to different potencies of these two
chemokines. Therefore, several doses of both chemokines were analyzed.
As shown in Fig. 3, RANTES and eotaxin
induced a dose-dependent internalization of CCR-3. At all
doses, eotaxin was less potent than RANTES. At the highest dose of
eotaxin (1000 ng/ml), there was still reduction in CCR-3 surface
expression at 18 h. At 100-250 ng/ml RANTES, there was also
reduction in CCR-3 surface expression at 18 h.

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Fig. 3.
Dose-dependent ligand-induced
CCR-3 internalization on eosinophils. Peripheral blood eosinophils
were cultured with eotaxin (A) or RANTES (B) for
3 h (solid line) or 18 h
(dashed line). Cell surface expression of CCR-3
was measured by FACS analysis and compared with CCR-3 expression of
eosinophils not treated with the chemokine.
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We were interested in determining the effect of chemokine removal on
the surface expression of CCR-3 following receptor internalization. Eosinophils were exposed to eotaxin or RANTES for 15 min, washed extensively to remove chemokine, and then monitored for CCR-3 expression over the subsequent 2 h. After exposure to chemokine, CCR-3 expression was 49, 43, 47, and 88% for eotaxin and 37, 33, 42, and 86% for RANTES at 0, 30, 60, and 120 min, respectively (n = 2-3 for each time point).
Since detection of the receptor was performed using an antibody that
might be blocked by ligand binding, we were interested in verifying
that ligand binding alone was not sufficient for detection of reduced
CCR-3 expression. We therefore performed FACS analysis following
chemokine exposure at 4 °C. This temperature allows ligand binding
but prevents receptor internalization (29, 30). At 4 °C, there was
no internalization of surface CCR-3, whereas replicate cells at
37 °C demonstrated receptor internalization (n = 2, data not shown). To demonstrate that this phenomenon was specific for
CCR-3, the expression of CD18, an unrelated cell surface molecule, was
monitored after treatment with eotaxin (100 ng/ml for 3 h). The
CD18 expression level did not change with eotaxin treatment
(n = 3, data not shown).
Analysis of CCR-3 Internalization by Confocal Microscopy--
In
order to directly demonstrate CCR-3 ligand-induced receptor
internalization, confocal microscopy was performed. In these experiments, an immature eosinophilic myelocytic cell line was induced
to express CCR-3 by differentiation with IL-5 and butyric acid. Using
methods established for differentiation of HL-60 cells, these AML cells
express levels of CCR-3 comparable with human eosinophils.2 Additionally,
the CCR-3-expressing cells are functionally active in eotaxin-induced
calcium flux and chemotaxis assays. These cells, unlike fresh
eosinophils, express the transferrin receptor, which was useful for
co-localization studies. Induced eosinophilic cells were grown on glass
coverslips and treated with eotaxin or medium alone for 3 h at
37 °C. This treatment resulted in 36 ± 6% (n = 12) CCR-3 expression. Subsequently, the cells were fixed,
permeabilized, and stained with the anti-CCR-3 antibody. Nine sections
at 0.8 µm were taken through each cell. In cells cultured in medium
alone, the majority of the CCR-3 staining was at the cell surface (Fig. 4A). Upon exposure to eotaxin,
staining with the anti-CCR-3 resulted in a different pattern; the
majority of the staining was associated with a cytoplasmic granular
staining (Fig. 4B). Experiments were conducted to further
delineate CCR-3 localization. Transferrin receptor is a well
established marker of early endosomes (31). Two-color staining was used
to identify CCR-3 with Texas Red (depicted in red) and the
transferrin receptor with fluorescein isothiocyanate (depicted in
green). Co-localization of both markers produces a
yellow color. Untreated cells had co-localization
of both markers predominantly on the membrane (Fig. 4C). In
contrast, following eotaxin treatment, CCR-3 and the transferrin
receptor co-localized to a predominantly intracellular location (Fig.
4D). Eotaxin-treated cells did not stain for CCR-3 alone,
suggesting that the major intracellular location overlapped with that
used by the transferrin receptor. Experiments were also conducted with
fresh human eosinophils in order to verify that ligand-induced receptor
internalization, rather than antigen blockade by chemokine, was
occurring. These experiments revealed translocation of CCR-3 staining
predominantly from a membrane-associated pattern to a granular pattern
in a perinuclear location consistent with endosomes (data not shown).

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Fig. 4.
Confocal microscopy. Cells (dAML) were
grown on glass coverslips and incubated in medium alone (A
and C) or with eotaxin (B and D) for
3 h at 37 °C. Subsequently, cells were fixed, permeabilized,
and stained with anti-CCR-3 antibody (A and B).
Alternatively, cells were co-stained with the anti-transferrin receptor
antibody anti-CCR-3 (C and D). Staining was
analyzed using confocal microscopy. CCR-3 staining is depicted in
red, and transferrin receptor staining is shown in
green. Co-localization is shown as yellow.
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Ligand-induced Modulation of CCR-3 Protein
Level--
Internalization of CCR-3 may induce receptor degradation.
Therefore, it was of interest to examine total cell protein for the
level of CCR-3 protein by Western blot analysis. The conditions for
Western blot analysis were first established from control cells: human
eosinophils, untransfected HOS.CD4 cells, or HOS.CD4 cells transfected
with CCR-1 or CCR-3. A polyclonal rabbit anti-human CCR-3 serum
detected multiple background bands but showed a strong, large band
between 50 and 60 kDa in HOS.CD4 cells transfected with CCR-3 but not
with CCR-1 (Fig. 5A). Human
eosinophils had a strong band between 50-60 kDa, and this band was not
present in granulocytes or mononuclear cells (Fig. 5B). In
all cases, detection of immunoreactive CCR-3 protein was lost if the
protein extract was boiled prior to electrophoresis. Eosinophils were then treated with medium alone or medium supplemented with eotaxin (250 ng/ml) or RANTES (100 ng/ml) for 3 h, conditions that promote optimal chemokine-induced internalization. Western blot analysis of
whole cell lysates revealed that the level of CCR-3 protein decreased
by ~30% (Fig. 5C). The decreased level of CCR-3 was further reduced (by ~60%) when eosinophils were treated with
cycloheximide for the 3 h during the exposure to the chemokine.
Cells treated with cycloheximide alone had no change in the level of
immunoreactive CCR-3 protein compared with cells incubated in medium
alone (Fig. 5C). Since the antibody used in the Western blot
recognizes the carboxyl-terminal region of CCR-3, the absence of
degradation products may be due to the loss of this epitope in the
degradation products. These experiments demonstrate that
internalization of CCR-3 is accompanied by receptor degradation and
that de novo synthesis of CCR-3 protein significantly
contributes to the total level of CCR-3 protein following chemokine
binding. In order to investigate if reexpression of CCR-3 on the cell
surface is dependent upon protein synthesis, we monitored CCR-3
expression by FACS analysis on cells treated with eotaxin in the
presence or absence of cycloheximide. Following treatment with 100 ng/ml eotaxin for 3 h, the receptor was beginning to reappear on
the cell surface. In contrast, on cells treated with eotaxin in the
presence of cycloheximide, the receptor expression on the cell surface
remained low (Fig. 5D). These data suggest that reexpression
of CCR-3 on the surface depends in part upon de novo protein
synthesis.

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Fig. 5.
CCR-3 protein level with eotaxin
treatment. The specificity of the polyclonal anti-CCR-3 antibody
was first assessed on untransfected HOS.CD4 cells or HOS.CD4 cells
transfected with CCR-1 or CCR-3 (A) and fractions of
peripheral blood leukocytes (B). C, the effect of
treating eosinophils with eotaxin (250 ng/ml), RANTES (100 ng/ml), or
chemokine plus cycloheximide (CHX, 10 µg/ml) for 3 h
was assessed. Whole cell lysates (50 µg) were electrophoresed in a
10% SDS-polyacrylamide gel, transferred to nitrocellulose, and stained
with the polyclonal anti-CCR-3 antibody. The results are representative
of three separate experiments. Molecular weight standards are shown on
the side of each panel. D, cells were
treated with eotaxin (100 ng/ml) with and without cycloheximide and
stained for surface expression of CCR-3 by FACS analysis. Cells treated
with eotaxin alone are shown with a solid line,
cells treated with eotaxin plus cycloheximide are shown with a
dashed line, and untreated cells are shown with a
filled histogram.
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Effect of Pertussis Toxin on CCR-3 Internalization--
We were
interested in determining if G-protein coupling was required for CCR-3
internalization. CCR-3-induced calcium transients are known to be
inhibited by pertussis toxin, suggesting that CCR-3 couples to
Gi-proteins (32). We first determined a dose of pertussis
toxin that was able to completely inhibit eotaxin-induced calcium
transients in eosinophils. Treatment of eosinophils with 100-1000
ng/ml of pertussis toxin for 3 h inhibited calcium flux, but we
were surprised to see that these doses also reduced CCR-3 expression
down to 30% (data not shown). At a lower dose of 20 ng/ml pertussis
toxin, inhibition of eotaxin-induced calcium transients was maintained
(Fig. 6, A and B),
but the level of CCR-3 was not significantly reduced. Exposure of
eosinophils to this dose of pertussis toxin (20 ng/ml) did not
block eotaxin (100 ng/ml)-induced receptor internalization
(Fig. 6, C and D). There was also no evidence of
an effect of pertussis toxin using eotaxin at 10 or 500 ng/ml (data not
shown). These data indicate that CCR-3 internalization is not dependent
upon Gi-protein coupling or calcium transients in human
eosinophils.

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Fig. 6.
Effect of pertussis toxin on CCR-3
internalization. Eosinophils were cultured for 3 h in medium
alone (A, C) or medium containing pertussis toxin
(20 ng/ml) (B, D), and calcium transients
(A, B) induced by eotaxin treatment (500 ng/ml)
are shown. Data are presented as the relative ratio of fluorescence
emitted at 510 nm after excitation at 340 and 380 nm (y
axis) over time (x axis). Replicate cells were also exposed
to eotaxin for the last 1 h of the culture, and the level of CCR-3
expression was determined (C, D). Cell surface
expression of CCR-3 was measured by FACS analysis and compared between
eotaxin-treated cells (dashed histogram) and
non-chemokine-treated cells (boldface histogram).
The level of isotype matched control antibody expression is indicated
with the filled histogram. The insets
in C and D represent data expressed as percentage
of CCR-3 expression.
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Involvement of Protein Kinase C in CCR-3 Internalization--
We
were next interested in determining if protein kinase C (PKC) was
involved in ligand-induced CCR-3 internalization, since the
internalization of some GPCRs is dependent upon PKC. We first treated
human eosinophils with PMA in order to determine if pharmacological activation of PKC was able to cause CCR-3 down-regulation. As shown in
Fig. 7A, treatment of
eosinophils with PMA for 1 h resulted in a
dose-dependent down-modulation of CCR-3 surface expression. We next determined the nontoxic dose of staurosporine that would effectively inhibit PMA-induced CCR-3 down-regulation. As shown in Fig.
7B, pretreatment of eosinophils with 10 ng/ml of
staurosporine for 2 h prior to PMA completely inhibited PMA
induced CCR-3 down-modulation. We next determined the effect of
staurosporine treatment on chemokine-induced CCR-3 internalization. As
shown in Fig. 7C, staurosporine had no effect on the level
of eotaxin (100 ng/ml)-induced CCR-3 internalization. There was also no
evidence of an effect of staurosporine using eotaxin at 10 or 500 ng/ml
(data not shown). These results indicate that although PMA induces
CCR-3 down-modulation, PKC is not involved in ligand-induced CCR-3
internalization.

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Fig. 7.
Effect of PMA and staurosporine on CCR-3
internalization. Eosinophils were treated with increasing doses of
0.1 ng/ml PMA (solid line) and 1 ng/ml PMA
(dashed line), and the level of CCR-3 was
analyzed by FACS analysis (A). Staurosporine (ST,
10 ng/ml, depicted as a dashed line) inhibited
the PMA-induced down-modulation of CCR-3 expression (solid
line) (B). C, the eotaxin-induced
reduction of CCR-3 surface expression (solid
line) was not inhibited by staurosporine treatment (10 ng/ml, dashed line). The level of CCR-3
expression in untreated cells is indicated by the filled
histogram. The insets in B and
C represent data expressed as percentage of CCR-3
expression.
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DISCUSSION |
We report that CCR-3 is rapidly removed from the cell surface into
an intracellular endocytic compartment following ligand binding. The
two CCR-3 ligands studied, eotaxin and RANTES, induced a different
magnitude and duration of receptor internalization. In all cases,
RANTES was more potent and induced a longer duration of internalization
than eotaxin although it was not a stronger chemoattractant in previous
studies (15) and in eosinophil transmigration assays (data not shown).
This suggests that internalization of CCR-3 involves molecular events
that are dissociated, at least in part, from the signals involved in
triggering chemotaxis. Consistent with this, chemokine-induced
internalization was not dependent upon G-protein coupling or calcium
transients although both of these processes are involved in
chemoattraction. Interestingly, a derivative of RANTES that functions
as an antagonist, aminooxypentane-RANTES, induces a stronger and more
prolonged internalization of CCR-5 in transfected Chinese hamster ovary
cells compared with RANTES itself (33). Additionally, IL-8 derivatives
that have equal chemotactic activity can have differential ability to
induce elastase release, suggesting that they trigger independent
events involving the same receptor (34).
We also demonstrate that the half-life of CCR-3 is prolonged (>3 h),
since treatment of eosinophils with cycloheximide did not change the
level of CCR-3 protein in the whole cell lysate. However, following
3 h of chemokine treatment, the level of cell-associated CCR-3
protein was decreased in a manner augmented by cycloheximide treatment.
This indicates that chemokine treatment induces protein degradation and
that new protein synthesis is involved in maintaining the level of the
CCR-3 protein following chemokine treatment. Concomitantly, the surface
expression of CCR-3 was further decreased with cycloheximide treatment,
indicating that reexpression of the receptor on the cell surface is
dependent upon de novo protein synthesis. Ligand-induced
modulation of chemokine receptor expression has only been examined for
a limited number of chemokine receptors, and variable pathways have
been reported. IL-8 induces rapid internalization of its receptor in
neutrophils (29). Stromal cell-derived factor-1
induces a decrease
of cell surface CXCR-4 in the CEM T-cell line, HeLa cells, and
peripheral blood mononuclear cells (35, 36). Additionally, CCR-5
ligands induce receptor internalization in lymphocytes,
monocytes/macrophages, and CCR-5-transfected Chinese hamster ovary
cells (33). In these cases, the receptors enter an endocytic pathway
but recycle by 3 h after ligand binding. In contrast, CXCR-2
undergoes internalization and does not recycle, since it enters a
degradative lysosomal pathway (37). Rapid ligand-induced
internalization of CCR-1 in transfected Chinese hamster ovary cells
(38) and CCR-2b in transfected HEK-293 cells (39) have been described,
but their intracellular processing was not studied. The diverse
mechanisms of ligand-induced modulation of chemokine receptors indicate
the importance of dissecting these processes for each chemokine receptor.
Because most experiments were done by FACS analysis utilizing an
antibody against CCR-3, it remained possible that the apparent decrease
in the level of CCR-3 expression was dependent upon blockade of the
CCR-3 immunoreactive epitope by the chemokine. Therefore, experiments
were conducted at 4 °C in order to block internalization. These
experiments showed no modulation of CCR-3 expression, indicating that
ligand binding alone was not sufficient to decrease CCR-3 detection.
Furthermore, confocal microscopy was performed in order to directly
demonstrate ligand-induced CCR-3 internalization. These experiments
were performed on an eosinophilic cell line that expressed high levels
of functionally active CCR-3. This cell line was useful especially
because it maintained expression of the transferrin receptor, which
allowed co-localization studies to be performed. On resting cells, most
CCR-3 was demonstrated to be on the cell membrane; however, following
ligand treatment, the majority of the CCR-3 protein was detected in an
intracellular location. Co-staining studies with the transferrin
receptor revealed that intracellular CCR-3 was predominantly
co-localized with the transferrin receptor, which is known to recycle
via the early endosome compartment. This pathway exists in numerous
cells and is commonly involved in receptor endocytosis. It is
interesting that the cytoplasmic motif (YXXF) required for
internalization of other receptors (e.g. transferrin
receptor) is conserved in CCR-3 (40).
We have begun to elucidate mechanisms of CCR-3 internalization.
Experiments conducted with pertussis toxin demonstrated that Gi-protein coupling is not necessary for internalization.
Interestingly, high doses of pertussis toxin alone down-regulated the
level of surface CCR-3 expression. The dissociation of G-protein
coupling and GPCR internalization has been reported for other selected GPCRs such as CXCR-4 (35, 36). We also tested the involvement of PKC in
mediating ligand-induced CCR-3 internalization. Using staurosporine to
inhibit PKC, we demonstrated that ligand-induced CCR-3 internalization
was not dependent upon PKC. However, in these experiments, CCR-3
down-modulation was induced by activation of PKC with PMA. Similarly,
although PMA and ligands induce rapid phosphorylation of CXCR-2, only
PMA-induced phosphorylation is inhibited by staurosporine (37). These
data indicate that at least two pathways exist for down-modulating
CCR-3 expression: one mediated by PKC and another mediated by
chemokines and independent of PKC. The latter pathway may be
dependent upon G-protein-related kinases, such as GRK-2, which has
been shown to be involved in CCR-5 internalization (41).
The present results have several biological implications,
especially since CCR-3 events have been investigated specifically in
eosinophils. Conclusions concerning eosinophil chemokine receptor events have often been drawn from investigation of heterologous cell
lines transfected with CCR-3. Since individual cell types often utilize
distinct signaling events, it is important to examine chemokine-triggered biochemical events in eosinophils. For example, fMLP inhibits adenylate cyclase in fMLP receptor-transfected cells but
not in human neutrophils (25). We have therefore performed all
experiments in eosinophilic cells. The finding that chemokines induce
CCR-3 to undergo significant and prolonged receptor internalization has
mechanistic implications for understanding eosinophil trafficking in vivo. It is widely accepted that chemokines induce
cellular activation and chemoattraction. However, it is unclear if
chemokines are also involved in stopping leukocyte movement. For
example, eotaxin has been demonstrated to be required for the
maintenance of gastrointestinal eosinophils at base line (23). If
eotaxin also induces receptor internalization with subsequent cellular hyporesponsiveness, then eosinophils would likely home into the intestine and become localized there, since they would no longer be
responsive to other chemokine gradients operating through CCR-3. In
contrast, eosinophils in hematopoietic organs, tissues that do not
express eotaxin, would remain responsive to the induction of subsequent
chemokine gradients. Additionally, the observed inefficiency of
receptor recycling following ligand binding, especially for RANTES, may
have mechanistic implications concerning the difficulty in expressing
CCR-3 on the surface of transfected cell lines. In one study, only
2-5% of CCR-3-transfected cells expressed CCR-3 on their surface
although substantial intracellular protein could be detected in most
transfected cells (15), suggesting a problem with CCR-3 protein
trafficking. Last, the current study sheds light on possible mechanisms
by which eotaxin may block cellular entry of CCR-3 trophic human
immunodeficiency virus type 1 strains (13). Besides directly blocking
human immunodeficiency virus binding to CCR-3, eotaxin may inhibit
human immunodeficiency virus uptake by causing internalization of the
CCR-3 co-receptor. Further studies are under way analyzing the long
term biological consequences of exposing eosinophils to chemokines and
a further elucidation of the biochemical events associated with CCR-3 signaling.
 |
ACKNOWLEDGEMENTS |
We thank John Heile for technical assistance,
Drs. Stephen Liggett and Tim Weaver for helpful discussions, and Dr.
Raphael Hirsch for the use of the FACScan. We are grateful to Drs. C. Paul and D. Baumann for the AML14.3D10 cell line, Dr. Paul Ponath for
the CCR-3 antibody and helpful discussions, and Dr. Bruce Daugherty for
the polyclonal CCR-3 antibody. We thank Drs. Ann Richmond, Michael
Fiedler, Gurjit Hershey, and Paul Foster for critical review of this
manuscript. The pc.CCR-1 plasmid and HOS.CD4 cells were obtained
through the AIDS Research and Reference Reagent Program, Division of
AIDS, NIAID, National Institutes of Health, from Dr. Nathaniel Landau.
RANTES and macrophage inflammatory protein-1
were also obtained
through the AIDS Research and Reference Reagent Program, Division of
AIDS, NIAID, National Institutes of Health, from Peprotech.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Division of
Pulmonary Medicine, Allergy and Clinical Immunology, Department of Pediatrics, Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229. Tel.: 513-636-7177; Fax: 513-636-3310; E-mail: rothm0{at}chmcc.org.
2
N. Zimmermann and M. E. Rothenberg,
unpublished results.
 |
ABBREVIATIONS |
The abbreviations used are:
GPCR, G-protein-coupled receptor;
CCR, CC chemokine receptor;
CXCR, CXC
chemokine receptor;
FACS, fluorescence-activated cell sorter;
IL, interleukin;
MCP, monocyte chemotactic protein;
PBS, phosphate-buffered
saline;
PKC, protein kinase C;
RANTES, reduced on activation T cell
expressed and secreted;
PMA, phorbol 12-myristate 13-acetate.
 |
REFERENCES |
-
Gleich, G. J.,
and Adolphson, C. R.
(1986)
Adv. Immunol.
39,
177-253[Medline]
[Order article via Infotrieve]
-
Weller, P. F.
(1991)
N. Engl. J. Med.
324,
1110-1118[Medline]
[Order article via Infotrieve]
-
Rothenberg, M. E.
(1998)
N. Engl. J. Med.
338,
1592-1600[Free Full Text]
-
Jose, P. J.,
Griffiths-Johnson, D. A.,
Collins, P. D.,
Walsh, D. T.,
Moqbel, R.,
Totty, N. F.,
Truong, O.,
Hsuan, J. J.,
and Williams, T. J.
(1994)
J. Exp. Med.
179,
881-887[Abstract]
-
Luster, A. D.,
and Rothenberg, M. E.
(1997)
J. Leukocyte Biol.
62,
620-633[Abstract]
-
Baggiolini, M.,
Dewald, B.,
and Moser, B.
(1994)
Adv. Immunol.
55,
97-179[Medline]
[Order article via Infotrieve]
-
Rollins, B. J.
(1997)
Blood
90,
909-928[Free Full Text]
-
Luster, A. D.
(1998)
N. Engl. J. Med.
338,
436-445[Free Full Text]
-
Murphy, P. M.
(1994)
Annu. Rev. Immunol.
12,
593-633[CrossRef][Medline]
[Order article via Infotrieve]
-
Gerard, C.,
and Gerard, N. P.
(1994)
Curr. Opin. Immunol.
6,
140-145[CrossRef][Medline]
[Order article via Infotrieve]
-
Bates, P.
(1996)
Cell
86,
1-3[Medline]
[Order article via Infotrieve]
-
Combadiere, C.,
Ahuja, S. K.,
and Murphy, P. M.
(1995)
J. Biol. Chem.
270,
16491-16494[Abstract/Free Full Text]; Correction (1996) J. Biol. Chem. 271, 11034
-
Choe, H.,
Farzan, M.,
Sun, Y.,
Sullivan, N.,
Rollins, B.,
Ponath, P. D.,
Wu, L. J.,
Mackay, C. R.,
Larosa, G.,
Newman, W.,
Gerard, N.,
Gerard, C.,
and Sodroski, J.
(1996)
Cell
85,
1135-1148[Medline]
[Order article via Infotrieve]
-
Daugherty, B. L.,
Siciliano, S. J.,
DeMartino, J. A.,
Malkowitz, L.,
Sirotina, A.,
and Springer, M. S.
(1996)
J. Exp. Med.
183,
2349-2354[Abstract]
-
Ponath, P. D.,
Qin, S.,
Post, T. W.,
Wang, J.,
Wu, L.,
Gerard, N. P.,
Newman, W.,
Gerard, C.,
and Mackay, C. R.
(1996)
J. Exp. Med.
183,
2437-2448[Abstract]
-
Sallusto, F.,
Mackay, C. R.,
and Lanzavecchia, A.
(1997)
Science
277,
2005-2007[Abstract/Free Full Text]
-
Uguccioni, M.,
Mackay, C. R.,
Ochensberger, B.,
Loetscher, P.,
Rhis, S.,
LaRosa, G. J.,
Rao, P.,
Ponath, P. D.,
Baggiolini, M.,
and Dahinden, C. A.
(1997)
J. Clin. Invest.
100,
1137-1143[Abstract/Free Full Text]
-
Gonzalo, J.-A.,
Lloyd, C. M.,
Kremer, L.,
Finger, E.,
Martinez-A, C.,
Siegelman, M. H.,
Cybulsky, M.,
and Guitierrez-Ramos, J.-C.
(1996)
J. Clin. Invest.
98,
2332-2345[Abstract/Free Full Text]
-
Humbles, A. A.,
Conroy, D. M.,
Marleau, S.,
Rankin, S. M.,
Palframan, R. T.,
Proudfoot, A. E.,
Wells, T. N.,
Li, D.,
Jeffery, P. K.,
Griffiths-Johnson, D. A.,
Williams, T. J.,
and Jose, P. J.
(1997)
J. Exp. Med.
186,
601-612[Abstract/Free Full Text]
-
Lamkhioued, B.,
Renzi, P. M.,
Abi-Younes, S.,
Garcia-Zepeda, E. A.,
Allakhverdi, Z.,
Ghaffar, O.,
Rothenberg, M. E.,
Luster, A. D.,
and Hamid, Q.
(1997)
J. Immunol.
159,
4593-4601[Abstract]
-
Teixeira, M. M.,
Wells, T. N.,
Lukacs, N. W.,
Proudfoot, A. E.,
Kunkel, S. L.,
Williams, T. J.,
and Hellewell, P. G.
(1997)
J. Clin. Invest.
100,
1657-1666[Abstract/Free Full Text]
-
Rothenberg, M. E.,
MacLean, J. A.,
Pearlman, E.,
Luster, A. D.,
and Leder, P.
(1997)
J. Exp. Med.
185,
785-790[Abstract/Free Full Text]
-
Matthews, A. N.,
Friend, D. S.,
Zimmermann, N.,
Sarafi, M. N.,
Luster, A. D.,
Pearlman, E.,
Wert, S. E.,
and Rothenberg, M. E.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
6273-6278[Abstract/Free Full Text]
-
Bohm, S. K.,
Grady, E. F.,
and Bunnett, N. W.
(1997)
Biochem. J.
322,
1-18[Medline]
[Order article via Infotrieve]
-
Uhing, R. J.,
Gettys, T. W.,
Tomhave, E.,
Snyderman, R.,
and Didsbury, J. R.
(1992)
Biochem. Biophys. Res. Commun.
183,
1033-1039[Medline]
[Order article via Infotrieve]
-
Hansel, T. T.,
De Vries, I. J. M.,
Iff, T.,
Rihs, S.,
Wandzilak, M.,
Betz, S.,
Blaser, K.,
and Walker, C.
(1991)
J. Immunol. Methods
145,
105-110[CrossRef][Medline]
[Order article via Infotrieve]
-
Deng, H. K.,
Liu, R.,
Ellmeier, W.,
Choe, S.,
Unutmaz, D.,
Burkhart, M.,
Dimarzio, P.,
Marmon, S.,
Sutton, R. E.,
Hill, C. M.,
Davis, C. B.,
Peiper, S. C.,
Schall, T. J.,
Littman, D. R.,
and Landau, N. R.
(1996)
Nature
381,
661-666[CrossRef][Medline]
[Order article via Infotrieve]
-
Landau, N. R.,
and Littman, D. R.
(1992)
J. Virol.
66,
5110-5113[Abstract]
-
Samanta, A. K.,
Oppenheim, J. J.,
and Matsushima, K.
(1990)
J. Biol. Chem.
265,
183-189[Abstract/Free Full Text]
-
Chuntharapai, A.,
and Kim, K. J.
(1995)
J. Immunol.
155,
2587-2594[Abstract]
-
Eskelinen, S.,
Kok, J. W.,
Sormunen, R.,
and Hoekstra, D.
(1991)
Eur. J. Cell Biol.
56,
210-222[Medline]
[Order article via Infotrieve]
-
Tenscher, K.,
Metzner, B.,
Schopf, E.,
Norgauer, J.,
and Czech, W.
(1996)
Blood
88,
3195-3199[Abstract/Free Full Text]
-
Mack, M.,
Luckow, B.,
Nelson, P. J.,
Cihak, J.,
Simmons, G.,
Clapham, P. R.,
Signoret, N.,
Marsh, M.,
Stangassinger, M.,
Borlat, F.,
Wells, T. N.,
Schlondorff, D.,
and Proudfoot, A. E.
(1998)
J. Exp. Med.
187,
1215-1224[Abstract/Free Full Text]
-
Clark, L. I.,
Schumacher, C.,
Baggiolini, M.,
and Moser, B.
(1991)
J. Biol. Chem.
266,
23128-23134[Abstract/Free Full Text]
-
Amara, A.,
Gall, S. L.,
Schwartz, O.,
Salamero, J.,
Montes, M.,
Loetscher, P.,
Baggiolini, M.,
Virelizier, J. L.,
and Arenzana-Seisdedos, F.
(1997)
J. Exp. Med.
186,
139-146[Abstract/Free Full Text]
-
Forster, R.,
Kremmer, E.,
Schubel, A.,
Breitfeld, D.,
Kleinschmidt, A.,
Nerl, C.,
Bernhardt, G.,
and Lipp, M.
(1998)
J. Immunol.
160,
1522-1531[Abstract/Free Full Text]
-
Mueller, S. G.,
Schraw, W. P.,
and Richmond, A.
(1995)
J. Biol. Chem.
270,
10439-10448[Abstract/Free Full Text]
-
Solari, R.,
Offord, R. E.,
Remy, S.,
Aubry, J. P.,
Wells, T. N.,
Whitehorn, E.,
Oung, T.,
and Proudfoot, A. E.
(1997)
J. Biol. Chem.
272,
9617-9620[Abstract/Free Full Text]
-
Franci, C.,
Gosling, J.,
Tsou, C. L.,
Coughlin, S. R.,
and Charo, I. F.
(1996)
J. Immunol.
157,
5606-5612[Abstract]
-
Mellman, I.
(1996)
Annu. Rev. Cell Dev. Biol.
12,
575-625[CrossRef][Medline]
[Order article via Infotrieve]
-
Aramori, I.,
Ferguson, S. S.,
Bieniasz, P. D.,
Zhang, J.,
Cullen, B.,
and Cullen, M. G.
(1997)
EMBO J.
16,
4606-4616[Abstract/Free Full Text]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.