From the Theodor-Kocher Institute, University of
Bern, P. O. Box 99, CH-3000 Bern 9, Switzerland and the
¶ Biomedical Research Centre and Department of Biochemistry and
Molecular Biology, University of British Columbia, Vancouver,
British Columbia V6T 1Z3, Canada
Received for publication, June 26, 2000, and in revised form, November 9, 2000
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ABSTRACT |
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Th1 and Th2 lymphocytes express a different
repertoire of chemokine receptors (CCRs). CXCR3, the receptor for I-TAC
(interferon-inducible T cell Chemokines are small secretory proteins produced by tissue cells
and leukocytes that regulate leukocyte migration in inflammation and
immunity (1-5). Two main subfamilies (CXC and CC chemokines) are
distinguished depending on the arrangement of the first two cysteines,
which are separated by one amino acid (CXC) or are adjacent (CC). All
chemokines act via seven-transmembrane-domain receptors coupled to G
proteins, which also form two subfamilies, CXC and CC chemokine
receptors (CXCRs and CCRs).1
Depending on function and pathophysiological roles, it is possible to
distinguish between inflammatory and homing chemokines. Inflammatory chemokines are produced in most tissues under pathological conditions upon stimulation by cytokines and bacterial toxins, whereas homing chemokines are produced constitutively at homing sites.
In the past few years it was found that lymphocytes can express most
chemokine receptors in relation to their state of maturation, activation, and differentiation. CCR3, CCR5, and CXCR3, for instance, are up-regulated in T cells by treatment with IL-2 and are expressed differentially in Th1 and Th2 cells (1-3). These observations explain
how T cells with appropriate cytokine production and effector properties can be attracted specifically into diseased tissues. CCR3-expressing Th2 cells are recruited together with eosinophils, which express the same receptor, to sites of allergic inflammation as
shown by immunochemical analysis of nasal polyps and atopic dermatitis
lesions (6, 7). In such infiltrates, Th2 cells are believed to promote
inflammation by releasing IL-4 and IL-5 as priming and survival factors
for eosinophil and basophil leukocytes (8). CCR5, on the other
hand, is characteristic for Th1 cells, which also express high levels
of CXCR3. Th1 cells accumulate in delayed-type hypersensitivity
reactions and autoimmune inflammation (9, 10).
CCR3 binds many different CC chemokines, namely eotaxin, eotaxin-2,
eotaxin-3, RANTES, MCP-2, MCP-3, and MCP-4 (4). The eotaxins are highly
selective for CCR3, whereas RANTES and the MCPs recognize additional CC
chemokine receptors. Eotaxin is expressed in a wide variety of cells,
including eosinophils, lymphocytes, macrophages, and endothelial and
epithelial cells, and is critically involved in the regulation of the
basal and inflammation-dependent traffic of eosinophils
(11, 12). In eotaxin-deficient mice and in animals treated with
antibodies that neutralize eotaxin, eosinophil infiltration of the
airways is markedly reduced (13-15). CXCR3 is highly expressed on T
cells activated with IL-2 and binds selectively I-TAC, Mig, and IP10
(4). Of the three ligands, I-TAC has the highest receptor affinity and
is the most potent agonist, as shown by chemotaxis and Ca2+
mobilization assays (16). A notable feature of I-TAC, Mig, and IP10 is
that their production is induced by interferon- While studying the activities of several chemokines on Th1 and Th2
cells, we observed that I-TAC, Mig, and IP10 act as antagonists for
CCR3. These data suggest that CXCR3 agonists in addition to attracting
CXCR3-bearing cells have the capacity to inhibit responses mediated via CCR3.
Chemokine Synthesis--
All chemokines and chemokine analogs
were synthesized chemically using tBoc (tertiary butyloxycarbonyl)
solid-phase chemistry (19). They were purified by high pressure liquid
chromatography and analyzed by electron spray mass
spectrometry. For each chemokine used, the mass determined by mass
spectrometry corresponded to the expected value.
Cell Preparation and Culture--
Eosinophils (>98% pure) were
purified from venous blood of healthy volunteers (20). A human Th2 cell
line generated from cord blood cells was kindly provided by Dr. C. Chizzolini (University Hospital, Geneva, Switzerland). These cells were
expanded periodically by restimulation with phytohemagglutin in the
presence of feeder cells (21).
Chemokine Receptor Transfectants--
Murine pre-B 300-19 cells
that stably express chemokine receptors were generated by transfection.
cDNAs for CXCR1 (22), CXCR2 (22), CXCR4 (23), CCR1 (24) (kindly
provided by Dr. P. Murphy, National Institutes of Health, Bethesda,
MD), CCR2 (25), CCR3 (6, 26), CCR5 (68), CCR6 (27), XCR1 (28), and
CX3CR1 (29) were cloned into the SR Receptor Binding and Functional Assays--
Competition binding
assays were performed with CCR3-B300-19 cells using
125I-eotaxin labeled by the Bolton-Hunter procedure (37).
Briefly, the maximal binding of labeled eotaxin was determined by
measuring binding at saturating concentrations; the bindability was
about 20%. 5 × 106 cells were incubated with 4 nM 125I-eotaxin and increasing concentrations
of unlabeled competitor (10 Receptor Internalization--
Chemokine-induced internalization
was assayed as described (40). Briefly, CCR3-B300-19 or CXCR3-B300-19
cells were incubated for 40 min at 37 °C with the chemokine to be
tested at a 1 µM concentration. After washing twice with
phosphate-buffered saline, surface-bound ligands were removed by
exposure to 50 mM glycine buffer, pH 3.0, containing 100 mM NaCl for 1 min followed by washing with
phosphate-buffered saline. Receptor expression was then determined by
flow cytometry (see "Receptor Binding and Functional
Assays"), and the relative fluorescence intensity was
calculated (41).
Inhibition of Chemotaxis--
The migration of CCR3-B300-19 cells
and of human eosinophils in response to eotaxin was inhibited by I-TAC,
Mig, and IP10, as shown for I-TAC in Fig.
1, A and B. The
inhibition of chemotaxis induced by optimum concentrations of eotaxin
was concentration-dependent and was complete at 100-1000
nM (Fig. 1, C and D). I-TAC and Mig were equally effective and somewhat more potent than IP10. None of the
CXCR3 ligands induced chemotaxis of CCR3-B300-19 cells or eosinophils
(not shown).
Inhibition of Ca2+ Mobilization--
As shown in Fig.
2, the [Ca2+]i rise
induced by eotaxin in CCR3-bearing cells was decreased in a
concentration-dependent manner by pretreatment with I-TAC,
which was completely inhibitory between 100 and 1000 nM.
I-TAC, on the other hand, did not induce [Ca2+]i
changes in CCR3-B300-19 cells or eosinophils, even at high
concentrations, confirming that it is devoid of agonistic activity on
CCR3 and indicating that it is a pure antagonist. Experiments with Th2
cells, which express CCR3 and CXCR3, show that blockade of CCR3 can
occur concomitantly with the activation of CXCR3 (Fig. 2B).
Marked [Ca2+]i changes were observed after
stimulation with increasing concentrations of I-TAC, which in turn
prevented the response to eotaxin. Ca2+ mobilization
induced by eotaxin in CCR3-B300-19 cells and Th2 cells was inhibited in
a concentration-dependent manner by all three
CXCR3-selective chemokines (Fig. 2, C and D).
I-TAC was the most potent antagonist, followed by Mig and IP10. In
agreement with the chemotaxis assays, these results show that I-TAC,
Mig, and IP10 significantly inhibit CCR3-dependent
responses of Th2 cells at concentrations as low as 1 nM (Fig. 2B). CCR3-B300-19 cells were less
susceptible, presumably because they express higher numbers of
receptors (Fig. 2C).
As several chemokines bind and activate CCR3, it was important to test
the effect of the antagonists on responses induced by different
ligands. As shown in Fig. 3, I-TAC
prevented the [Ca2+]i changes elicited in
CCR3-B300-19 cells by eotaxin, eotaxin-2, MCP-2, MCP-3, MCP-4, and
RANTES, demonstrating that the antagonistic effect is not dependent on
the CCR3 agonist used. It was also important to test the specificity of
the antagonists to block chemokine receptor-mediated responses. In a
panel of 14 receptor-transfected B300-19 cell lines, I-TAC (the most
potent of the three CCR3 antagonists) abrogated the
[Ca2+]i changes in response to stimulation with
the appropriate chemokine only in CCR3-expressing cells (Fig.
4). I-TAC was slightly inhibitory on CCR5
(30-40% decrease of the [Ca2+]i rise in three
experiments) but had no effect on all other receptors, indicating that
its antagonistic activity is restricted largely to CCR3.
Effect of Eotaxin and MCP-4 on CXCR3-mediated Responses--
It
has been reported that eotaxin and MCP-4, which are agonists for CCR3,
bind to CXCR3 and that eotaxin prevents [Ca2+]i
changes induced by IP10 (42). In view of the observed antagonism for
CCR3, we tested the effect of eotaxin and MCP-4 on CXCR3-B300-19 cells
that were stimulated with I-TAC, Mig, and IP10. Neither eotaxin nor
MCP-4 inhibited Ca2+ mobilization or chemotaxis induced by
I-TAC, Mig, and IP10, indicating that their effect on CXCR3 was
negligible (data for I-TAC are shown in Fig.
5).
CCR3 Antagonist Obtained by Chemokine Modification--
It has
been suggested that chemokines dock onto receptors by interacting with
the loop region that follows the second cysteine and that the docking
facilitates a triggering of the receptor by the
NH2-terminal domain (43-46). In the attempt to enhance the antagonistic effect, we synthesized a hybrid chemokine by substituting the NH2-terminal region of eotaxin with that of I-TAC. As
shown in Fig. 6, I-TAC/EoH1,
corresponding to eotaxin with the first eight amino acids of I-TAC, was
about 5-fold more potent than I-TAC itself as an inhibitor of
CCR3-dependent [Ca2+]i changes and
chemotaxis. I-TAC/EoH1 was also tested on CXCR3-bearing cells and was
found to have moderate agonistic and no antagonistic activity (data not
shown).
CCR3 Binding Studies--
The relative affinities of I-TAC,
I-TAC/EoH1, Mig, and IP10 to CCR3 were determined by binding
competition assays with 125I-labeled eotaxin (Fig.
7). All antagonists fully displaced
labeled eotaxin. I-TAC/EoH1 was the most potent competitor
(Kd 4.5 ± 1.0 nM,
n = 3) followed by eotaxin (Kd
13.5 ± 1.9 nM, n = 3) and I-TAC
(Kd 65.0 ± 7.7 nM,
n = 3), whereas the affinity of Mig
(Kd 4065 ± 1, 231 nM,
n = 3) and IP10 (Kd 1582 ± 154 nM, n = 3) was comparatively low. Overall,
the binding data are in agreement with the observed antagonistic activities. The affinity of Mig, however, was lower than expected.
CCR3 Internalization--
Binding of chemokines leads to a rapid
receptor internalization, which is not observed on binding of
antagonists (40, 47). Internalization was determined in cells
expressing either CCR3 or CXCR3 by flow cytometry measurements of
surface receptors before and after ligand exposure. As shown in Fig.
8, in CCR3-bearing cells, receptor uptake
was induced by eotaxin only. In CXCR3-bearing cells, on the other hand,
receptor uptake was observed with I-TAC, Mig, IP10, and I-TAC/EoH1 but
not with eotaxin. Together with the functional data, these results show
that the CXCR3 ligands lack agonistic activity and act on CCR3 as pure
antagonists.
This paper shows that I-TAC, Mig, and IP10 are potent antagonists
for CCR3 and prevent the responses of Th2 cells and eosinophils to
CCR3-binding chemokines.
The search for chemokine antagonists began several years ago when
chemokine receptor blockade was recognized as a possible therapeutic
approach for inflammatory diseases. It is well established that
antagonists can be generated by modifying the NH2-terminal region of natural chemokines (48). Studies were performed first with
IL-8 and other ELR chemokines yielding antagonists for CXCR1 and
CXCR2 (49, 50). The same principle proved valid for CC chemokines, as
shown by the effects obtained upon NH2-terminal truncation
of MCP-1, MCP-3, and RANTES (37, 51, 52). These studies indicate that,
as a rule, receptor recognition depends on structural motifs located
in the loop region of chemokines that follows the second
cysteine. Extensive structure-activity studies of SDF-1 (stromal
cell-derived factor-1) led to the proposal of a two-step interaction of
chemokines with their receptors, involving specific docking via the
loop region and subsequent triggering by the NH2-terminal
region preceding the first cysteine (43, 53). In some cases, however,
NH2-terminal truncation did not yield derivatives with
antagonistic properties. This result was observed with eotaxin
and IP10, which lose the capacity to bind to CCR3 and CXCR3,
respectively, when only a few NH2-terminal residues are
deleted.2 It was also shown
that dipeptidyl-peptidase IV (CD26) reduces the activity of eotaxin by
cleaving off the first two NH2-terminal residues (54).
The observation that the naturally occurring I-TAC, Mig, and IP10 block
CCR3 was unexpected, because normally CXC and CC chemokines discriminate precisely between CXC and CC chemokine receptors. Our data
suggest that CCR3 and CXCR3, despite their overall sequence identity of
only 34%, share sufficient structural similarity within domains that
determine the binding of I-TAC, Mig, and IP10, on the one hand, and the
binding and triggering by eotaxins, MCPs, and RANTES on the other. The
existence of binding-relevant homology between CXCR3 and CCR3 is
suggested in particular by the observation that the potency ranking for
CCR3 antagonism, I-TAC > Mig > IP10, as shown in the
present study, and the potency ranking for agonistic activity via CXCR3
as determined by Cole et al. (16) are the same. It is also
noteworthy that NH2-terminal truncation of IP10 leads to a
loss of agonistic activity on CXCR3 as well as a loss of
antagonistic effects on CCR3 (data not shown). We found that CXCR3
ligands block CCR3, but we were unable to demonstrate the converse;
this contrasts with the report of Weng et al. (42) and
suggests that CCR3 ligands are unlikely to exert biologically relevant
effects via CXCR3. It has been reported that murine secondary lymphoid tissue chemokine has agonistic activity on murine CXCR3 (55).
Human secondary lymphoid tissue chemokine, on the other hand, is
inactive on human and murine CXCR3 (Ref. 56; data not shown).
Attempts to design high affinity antagonists by
NH2-terminal truncation of eotaxin have been unsuccessful.
This paper presents an alternative approach. After realizing that CXCR3
and CCR3 receptors may have homologous binding domains, we synthesized
a hybrid by replacing the NH2-terminal region of eotaxin
with that of I-TAC. Eotaxin was chosen because it has the highest
affinity for CCR3 and I-TAC because it is the best CCR3 antagonist.
Substitution of the NH2-terminal region led to the loss
of receptor triggering activity, whereas retaining high affinity
binding yielded a chemokine analog with higher affinity to CCR3 than
eotaxin and I-TAC.
Several lines of evidence indicate that I-TAC, Mig, IP10, and
I-TAC/EoH1 are pure CCR3 antagonists. These ligands did not induce chemotaxis or [Ca2+]i changes in
CCR3-bearing cells and did not induce CCR3 internalization, which is an
agonist-mediated event due to the phosphorylation of the receptor by G
protein-coupled receptor kinases and subsequent uptake in
clathrin-coated pits (57, 58). Several chemokine receptors, including
CXCR1 and CXCR2 (59), CXCR4 (40, 60, 61), CCR1 (62), CCR2 (63), and
CCR5 (40, 41, 47, 64), are known to be internalized after agonist binding. Different chemokines that bind to the same receptor can induce
differential internalization as recently reported for CCR3 (65, 66) and
CCR5 (47). In agreement with these findings, CXCR3 was internalized to
different extents by I-TAC, Mig, IP10, and I-TAC/EoH1.
Inflammatory and immune reactions are characterized by the production
of chemokines in the affected tissues, leading to the infiltration of
leukocytes that bear the appropriate receptors. The local expression of
chemokines is often induced by cytokines. In the context of this paper,
it is important to realize that I-TAC, Mig, and IP10, which attract
CXCR3-bearing cells, are induced by interferon--chemoattractant), Mig (monokine
induced by
-interferon), and IP10 (interferon-inducible protein 10),
is expressed preferentially on Th1 cells, whereas CCR3, the receptor
for eotaxin and several other CC chemokines, is characteristic of Th2
cells. While studying responses that are mediated by these two
receptors, we found that the agonists for CXCR3 act as antagonists for
CCR3. I-TAC, Mig, and IP10 compete for the binding of eotaxin to
CCR3-bearing cells and inhibit migration and Ca2+
changes induced in such cells by stimulation with eotaxin, eotaxin-2, MCP-2 (monocyte chemottractant protein-2), MCP-3, MCP-4, and RANTES (regulated on activation normal T cell expressed and secreted). A
hybrid chemokine generated by substituting the first eight
NH2-terminal residues of eotaxin with those of I-TAC bound
CCR3 with higher affinity than eotaxin or I-TAC (3- and 10-fold,
respectively). The hybrid was 5-fold more potent than I-TAC as an
inhibitor of eotaxin activity and was effective at concentrations as
low as 5 nM. None of the antagonists described induced the
internalization of CCR3, indicating that they lack agonistic effects
and thus qualify as pure antagonists. These results suggest that
chemokines that attract Th1 cells via CXCR3 can concomitantly block the
migration of Th2 cells in response to CCR3 ligands, thus enhancing the
polarization of T cell recruitment.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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, a cytokine that is
typically associated with Th1 responses (16-18). IP10, for
instance, is expressed in skin lesions caused by delayed-type hypersensitivity, psoriasis, and tuberculoid leprosy, where
interferon-
expression is enhanced.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
puro vector (kindly provided by Dr. F.Arenzana-Seisdedos, Pasteur Institute, Paris). The
cells (5 × 106) were transfected by electroporation
with 20 µg of linearized SR
puro receptor plasmid DNA. Clones of
receptor-expressing B300-19 cells were established by limiting dilution
in the presence of 1.5 µg/ml puromycin (Sigma). The following
receptor-transfected murine pre-B 300-19 cells were generated and
characterized previously: CXCR3-B300-19 (30), CXCR5-B300-19 (31),
CCR4-B300-19 (32), CCR7-B300-19 (33), and CCR8-B300-19 (34). Receptor
expression was determined by RNA dot plot analysis and flow cytometry
(FACScan, Becton Dickinson, Mountain View, CA) using monoclonal
antibodies to CXCR4 (12G5) (kindly provided by Dr. J. Hoxie, University
of Pennsylvania, Philadelphia) and CCR3 (7B11) (35), CCR5 (5C7) (36),
and CXCR3 (1C6.2) (10), all kindly provided by LeukoSite Inc.
(Cambridge, MA). Labeling was done with phycoerythrin-conjugated goat
anti-mouse IgG (Dako). Receptor function was assessed by Ca2+ mobilization in response to the appropriate chemokine.
9-3 × 10
5 M) in 200 µl of RPMI 1640 containing Hepes (25 mM, pH 7.4), bovine serum albumin (10 mg ml
1), and sodium azide (0.1%). The
incubations were carried out for 30 min at 4 °C, and cell-associated
radioactivity was separated immediately by spinning the cells through a
2:3 mixture of diacetylphthalate and dibutylphthalate. The cpm that
specifically bound to cells was calculated by subtracting the
nonspecific cpm (the cpm bound in the presence of 100-fold molar excess
of unlabeled eotaxin) from the total cpm that was bound to the cells.
Dissociation constants (K values) were determined by
Scatchard analysis using the computer program LIGAND (38).
Ca2+ mobilization was assayed in Fura-2-loaded cells after
single or sequential stimulation with chemokines or chemokine analogs by recording [Ca2+]i-related fluorescence changes
(39). The rate of the change was expressed as the percentage of Fura-2
saturation/s. Chemotaxis was assessed in 48-well Boyden microchambers
(Neuro Probe Inc., Cabin John, MD) using polyvinylpyrrolidone-free
polycarbonate membranes (Poretics Corp., Livermore, CA) with 5-µm
pores (30). Cell suspensions and chemokine dilutions were made in RPMI
1640 containing 1% pasteurized plasma protein (Swiss Red Cross
Laboratory, Bern, Switzerland) and 20 mM Hepes, pH 7.4. Migration was allowed to proceed for 2 h, and migrated cells were
counted at a ×1000 magnification in 5 fields/well. All determinations
were performed in triplicate.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Fig. 1.
Inhibition of CCR3-mediated chemotaxis by
I-TAC, Mig, and IP10. A and B, migration of
CCR3-B300-19 cells (A) and human eosinophils (B)
in response to eotaxin in the presence or absence of 1,000 nM I-TAC. C and D, migration of
CCR3-B300-19 cells (C) and human eosinophils (D)
in response to 10 nM eotaxin in the presence of increasing
concentrations of I-TAC, Mig, or IP10. Shown are the average numbers of
migrating cells per five high power fields in triplicate wells. The
data are representative of four independent experiments.
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Fig. 2.
Inhibition of CCR3-mediated
[Ca2+]i changes by I-TAC, Mig, and IP10.
CCR3-B300-19 cells (A and C) and Th2 cells
(B and D) loaded with Fura-2 were exposed to
increasing concentrations of I-TAC, Mig, or IP10 and stimulated after
60 s with 3 nM eotaxin.
[Ca2+]i-dependent fluorescence
changes (A and B) and initial rates of the
[Ca2+]i rise expressed as a percentage of Fura-2
saturation/s (C and D) are shown. The results are
representative of three to five independent experiments.
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Fig. 3.
Inhibition of [Ca2+]i
changes induced by different CCR3 ligands. CCR3-B300-19 cells
loaded with Fura-2 were left untreated or exposed to 300 nM
I-TAC and then stimulated with 3 nM eotaxin, eotaxin-2, or
MCP-4 or 50 nM RANTES, MCP-2, or MCP-3.
[Ca2+]i-dependent fluorescence
changes are shown. The results are representative of three independent
experiments.
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Fig. 4.
Effect of I-TAC on
[Ca2+]i changes in chemokine receptor
transfected B300-19 cells. The cells loaded with Fura-2 were left
untreated (buffer) or exposed to 1000 nM I-TAC
and stimulated after 60 s with a 10 nM
receptor-specific chemokine.
[Ca2+]i-dependent fluorescence
changes are shown. The results are representative of two to three
experiments.
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Fig. 5.
Effect of eotaxin and MCP-4 on CXCR3-mediated
[Ca2+]i changes and chemotaxis induced by
I-TAC. A, CXCR3-B300-19 cells loaded with Fura-2 were
exposed to buffer, 3000 nM eotaxin, or MCP-4 and stimulated
after 60 s with 1 nM I-TAC.
[Ca2+]i-dependent fluorescence
changes are shown. B, migration of CXCR3-B300-19 cells in
response to I-TAC in the presence or absence of 3000 nM
eotaxin or MCP-4. Shown are the average numbers of migrating cells/five
high power fields in triplicate wells. The data are representative of
three independent experiments.
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Fig. 6.
Inhibition of CCR3-mediated
[Ca2+]i changes and chemotaxis by I-TAC and
I-TAC/EoH1. A, CCR3-B300-19 cells loaded with Fura-2
were exposed to increasing concentrations of I-TAC or I-TAC/EoH1 and
stimulated after 60 s with 3 nM eotaxin. Initial rates
of the [Ca2+]i rise expressed as the percent of
Fura-2 saturation/s are shown. B and C, migration
of CCR3-B300-19 cells (B) and human eosinophils
(C) in response to 10 nM eotaxin in the presence
of increasing concentrations of I-TAC or I-TAC/EoH1. Shown are the
average numbers of migrating cells/per five high power fields in
triplicate wells. The data are representative of three to four
independent experiments.
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Fig. 7.
Displacement of 125I-eotaxin from
CCR3-B300-19 cells by eotaxin, I-TAC, Mig, IP10, and I-TAC/EoH1.
The cells were incubated with 4 nM 125I-eotaxin
in the presence of increasing concentrations of unlabeled competing
ligand. The values were normalized by setting the specific binding of 4 nM 125I-eotaxin to 100%. The data are
representative of three independent experiments.
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Fig. 8.
Agonist-dependent receptor
internalization. CCR3-B300-19 (A) and CXCR3-B300-19
(B) cells were incubated for 40 min at 37 °C with or
without 1000 nM eotaxin, I-TAC, Mig, IP10, or I-TAC/EoH1,
and surface expression of CCR3 and CXCR3 was determined by flow
cytometry and expressed as relative fluorescence intensity. Mean values
from two independent experiments are shown.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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(16-18), whereas
eotaxin, a specific agonist for CCR3, is induced by IL-4 (67). The
infiltrate observed in the presence of interferon-
is rich in Th1
cells, whereas Th2 cells predominate under the influence of IL-4. The
present observations suggest that CXCR3-selective chemokines enhance
this polarization by acting as antagonists of CCR3 and thus inhibiting
the infiltration of Th2 cells, in addition to their effect as
attractants of Th1 cells via CXCR3. This paper describes a new
mechanism for the regulation of leukocyte recruitment by chemokines
based on the combination of agonistic and antagonistic effects.
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ACKNOWLEDGEMENTS |
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We thank Regula Stuber, T. McLeod, G. Wasney, and P. Owen for expert technical assistance, Drs. U. Forssmann and P. Ogilvie for help in isolating eosinophils, Dr. C. Chizzolini (University Hospital, Geneva, Switzerland) for the human Th2 cell line, and Dr. P. Murphy (National Institutes of Health, Bethesda, MD) for the CCR1 cDNA. Donor blood buffy coats were provided by the Swiss Central Laboratory Blood Transfusion Service, Bern, Switzerland.
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FOOTNOTES |
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* This work was supported by the Protein Engineering Network Centers of Excellence (Canada) and the Arthritis Society (Canada) and by Grant 31-55996.98 from the Swiss National Science Foundation.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. Tel.: +41 31 631 4164; Fax: +41 31 631 3799; E-mail: pius.loetscher@tki.unibe.ch.
Present address: Cytos Biotechnology AG, 8952 Zurich-Schlieren, Switzerland.
Published, JBC Papers in Press, November 10, 2000, DOI 10.1074/jbc.M005652200
2 I. Clark-Lewis, P. Loetscher, M. Uguccioni, U. Forssmann, J.-H. Gong, M. Loetscher, and M. Baggiolini, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
CCR, CC chemokine
receptor;
CXCR, CXC chemokine receptor;
I-TAC, interferon-inducible T cell -chemoattractant;
I-TAC/EoH1, I-TAC-eotaxin hybrid-1;
Mig, monokine induced by
-interferon;
IP10, interferon-inducible protein 10;
RANTES, regulated on activation normal
T cell expressed and secreted;
MCP, monocyte chemoattractant protein;
IL, interleukin.
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1. | Baggiolini, M. (1998) Nature 392, 565-568[CrossRef][Medline] [Order article via Infotrieve] |
2. | Sallusto, F., Mackay, C. R., and Lanzavecchia, A. (2000) Annu. Rev. Immunol. 18, 593-620[CrossRef][Medline] [Order article via Infotrieve] |
3. | Loetscher, P., Moser, B., and Baggiolini, M. (2000) Adv. Immunol. 74, 127-180[Medline] [Order article via Infotrieve] |
4. |
Murphy, P. M.,
Baggiolini, M.,
Charo, I. F.,
Hebert, C. A.,
Horuk, R.,
Matsushima, K.,
Miller, L. H.,
Oppenheim, J. J.,
and Power, C. A.
(2000)
Pharmacol. Rev.
52,
145-176 |
5. | Zlotnik, A., and Yoshie, O. (2000) Immunity 12, 121-127[Medline] [Order article via Infotrieve] |
6. | Gerber, B. O., Zanni, M. P., Uguccioni, M., Loetscher, M., Mackay, C. R., Pichler, W. J., Yawalkar, N., Baggiolini, M., and Moser, B. (1997) Curr. Biol. 7, 836-843[Medline] [Order article via Infotrieve] |
7. |
Yawalkar, N.,
Uguccioni, M.,
Scharer, J.,
Braunwalder, J.,
Karlen, S.,
Dewald, B.,
Braathen, L. R.,
and Baggiolini, M.
(1999)
J. Invest. Dermatol.
113,
43-48 |
8. | Corrigan, C. J., and Kay, A. B. (1992) Immunol. Today 13, 501-507[CrossRef][Medline] [Order article via Infotrieve] |
9. | Loetscher, P., Uguccioni, M., Bordoli, L., Baggiolini, M., Moser, B., Chizzolini, C., and Dayer, J. M. (1998) Nature 391, 344-345[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Qin, S.,
Rottman, J. B.,
Myers, P.,
Kassam, N.,
Weinblatt, M.,
Loetscher, M.,
Koch, A. E.,
Moser, B.,
and Mackay, C. R.
(1998)
J. Clin. Invest.
101,
746-754 |
11. | Gutierrez-Ramos, J. C., Lloyd, C., and Gonzalo, J. A. (1999) Immunol. Today 20, 500-504[CrossRef][Medline] [Order article via Infotrieve] |
12. | Rothenberg, M. E., Zimmermann, N., Mishra, A., Brandt, E., Birkenberger, L. A., Hogan, S. P., and Foster, P. S. (1999) J. Clin. Immunol. 19, 250-265[CrossRef][Medline] [Order article via Infotrieve] |
13. |
Rothenberg, M. E.,
MacLean, J. A.,
Pearlman, E.,
Luster, A. D.,
and Leder, P.
(1997)
J. Exp. Med.
185,
785-790 |
14. |
Gonzalo, J. A.,
Lloyd, C. M.,
Kremer, L.,
Finger, E.,
Martinez, C.,
Siegelman, M. H.,
Cybulsky, M.,
and Gutierrez-Ramos, J. C.
(1996)
J. Clin. Invest.
98,
2332-2345 |
15. |
Gonzalo, J. A.,
Lloyd, C. M.,
Wen, D. Y.,
Albar, J. P.,
Wells, T. N. C.,
Proudfoot, A.,
Martinez, C.,
Dorf, M.,
Bjerke, T.,
Coyle, A. J.,
and Gutierrez-Ramos, J. C.
(1998)
J. Exp. Med.
188,
157-167 |
16. |
Cole, K. E.,
Strick, C. A.,
Paradis, T. J.,
Ogborne, K. T.,
Loetscher, M.,
Gladue, R. P.,
Lin, W.,
Boyd, J. G.,
Moser, B.,
Wood, D. E.,
Sahagan, B. G.,
and Neote, K.
(1998)
J. Exp. Med.
187,
2009-2021 |
17. | Baggiolini, M., Dewald, B., and Moser, B. (1997) Annu. Rev. Immunol. 15, 675-705[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Sauty, A.,
Dziejman, M.,
Taha, R. A.,
Iarossi, A. S.,
Neote, K.,
Garcia-Zepeda, E. A.,
Hamid, Q.,
and Luster, A. D.
(1999)
J. Immunol.
162,
3549-3558 |
19. | Clark-Lewis, I., Vo, L., Owen, P., and Anderson, J. (1997) Methods Enzymol. 287, 233-250[Medline] [Order article via Infotrieve] |
20. | Rot, A., Krieger, M., Brunner, T., Bischoff, S. C., Schall, T. J., and Dahinden, C. A. (1992) J. Exp. Med. 176, 1489-1495[Abstract] |
21. |
Loetscher, P.,
Seitz, M.,
Clark-Lewis, I.,
Baggiolini, M.,
and Moser, B.
(1994)
FASEB. J.
8,
1055-1060 |
22. | Loetscher, P., Seitz, M., Clark-Lewis, I., Baggiolini, M., and Moser, B. (1994) FEBS Lett. 341, 187-192[CrossRef][Medline] [Order article via Infotrieve] |
23. |
Loetscher, M.,
Geiser, T.,
O'Reilly, T.,
Zwahlen, R.,
Baggiolini, M.,
and Moser, B.
(1994)
J. Biol. Chem.
269,
232-237 |
24. | Gao, J.-L., Kuhns, D. B., Tiffany, H. L., McDermott, D., Li, X., Francke, U., and Murphy, P. M. (1993) J. Exp. Med. 177, 1421-1427[Abstract] |
25. | Charo, I. F., Myers, S. J., Herman, A., Franci, C., Connolly, A. J., and Coughlin, S. R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2752-2756[Abstract] |
26. | Moser, B., Barella, L., Mattei, S., Schumacher, C., Boulay, F., Colombo, M. P., and Baggiolini, M. (1993) Biochem. J. 294, 285-292[Medline] [Order article via Infotrieve] |
27. |
Power, C. A.,
Church, D. J.,
Meyer, A.,
Alouani, S.,
Proudfoot, A. E. I.,
Clark-Lewis, I.,
Sozzani, S.,
Mantovani, A.,
and Wells, T. N. C.
(1997)
J. Exp. Med.
186,
825-835 |
28. |
Yoshida, T.,
Imai, T.,
Kakizaki, M.,
Nishimura, M.,
Takagi, S.,
and Yoshie, O.
(1998)
J. Biol. Chem.
273,
16551-16554 |
29. | Imai, T., Hieshima, K., Haskell, C., Baba, M., Nagira, M., Nishimura, M., Kakizaki, M., Takagi, S., Nomiyama, H., Schall, T. J., and Yoshie, O. (1997) Cell 91, 521-530[Medline] [Order article via Infotrieve] |
30. | Loetscher, M., Gerber, B., Loetscher, P., Jones, S. A., Piali, L., Clark-Lewis, I., Baggiolini, M., and Moser, B. (1996) J. Exp. Med. 184, 963-969[Abstract] |
31. |
Legler, D. F.,
Loetscher, M.,
Roos, R. S.,
Clark-Lewis, I.,
Baggiolini, M.,
and Moser, B.
(1998)
J. Exp. Med.
187,
655-660 |
32. |
Arenzana-Seisdedos, F.,
Amara, A.,
Thomas, D.,
Virelizier, J. L.,
Baleux, F.,
Clark-Lewis, I.,
Legler, D. F.,
Moser, B.,
and Baggiolini, M.
(1998)
Science
281,
487a |
33. | Willimann, K., Legler, D. F., Loetscher, M., Roos, R. S., Delgado, M. B., Clark-Lewis, I., Baggiolini, M., and Moser, B. (1998) Eur. J. Immunol. 28, 2025-2034[CrossRef][Medline] [Order article via Infotrieve] |
34. |
Roos, R. S.,
Loetscher, M.,
Legler, D. F.,
Clark-Lewis, I.,
Baggiolini, M.,
and Moser, B.
(1997)
J. Biol. Chem.
272,
17251-17254 |
35. |
Heath, H.,
Qin, S.,
Rao, P.,
Wu, L.,
LaRosa, G.,
Kassam, N.,
Ponath, P. D.,
and Mackay, C. R.
(1997)
J. Clin. Invest.
99,
178-184 |
36. |
Wu, L. J.,
LaRosa, G.,
Kassam, N.,
Gordon, C. J.,
Heath, H.,
Ruffing, N.,
Chen, H.,
Humblias, J.,
Samson, M.,
Parmentier, M.,
Moore, J. P.,
and Mackay, C. R.
(1997)
J. Exp. Med.
186,
1373-1381 |
37. |
Gong, J. H.,
Uguccioni, M.,
Dewald, B.,
Baggiolini, M.,
and Clark-Lewis, I.
(1996)
J. Biol. Chem.
271,
10521-10527 |
38. |
Clark-Lewis, I.,
Schumacher, C.,
Baggiolini, M.,
and Moser, B.
(1991)
J. Biol. Chem.
266,
23128-23134 |
39. | von Tscharner, V., Prod'hom, B., Baggiolini, M., and Reuter, H. (1986) Nature 324, 369-372[Medline] [Order article via Infotrieve] |
40. |
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 |
41. |
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 |
42. |
Weng, Y.,
Siciliano, S. J.,
Waldburger, K. E.,
Sirotina-Meisher, A.,
Staruch, M. J.,
Daugherty, B. L.,
Gould, S. L.,
Springer, M. S.,
and DeMartino, J. A.
(1998)
J. Biol. Chem.
273,
18288-18291 |
43. |
Crump, M. P.,
Gong, J. H.,
Loetscher, P.,
Rajarathnam, K.,
Amara, A.,
Arenzana-Seisdedos, F.,
Virelizier, J. L.,
Baggiolini, M.,
Sykes, B. D.,
and Clark-Lewis, I.
(1997)
EMBO J.
16,
6996-7007 |
44. |
Crump, M. P.,
Rajarathnam, K.,
Kim, K. S.,
Clark-Lewis, I.,
and Sykes, B. D.
(1998)
J. Biol. Chem.
273,
22471-22479 |
45. | Clark-Lewis, I., Kim, K. S., Rajarathnam, K., Gong, J. H., Dewald, B., Moser, B., Baggiolini, M., and Sykes, B. D. (1995) J. Leukocyte Biol. 57, 703-711[Abstract] |
46. |
Monteclaro, F. S.,
and Charo, I. F.
(1996)
J. Biol. Chem.
271,
19084-19092 |
47. |
Blanpain, C.,
Migeotte, I.,
Lee, B.,
Vakili, J.,
Doranz, B. J.,
Govaerts, C.,
Vassart, G.,
Doms, R. W.,
and Parmentier, M.
(1999)
Blood
94,
1899-1905 |
48. |
Baggiolini, M.,
and Moser, B.
(1997)
J. Exp. Med.
186,
1189-1191 |
49. |
Moser, B.,
Dewald, B.,
Barella, L.,
Schumacher, C.,
Baggiolini, M.,
and Clark-Lewis, I.
(1993)
J. Biol. Chem.
268,
7125-7128 |
50. |
Jones, S. A.,
Dewald, B.,
Clark-Lewis, I.,
and Baggiolini, M.
(1997)
J. Biol. Chem.
272,
16166-16169 |
51. | Gong, J. H., and Clark-Lewis, I. (1995) J. Exp. Med. 181, 631-640[Abstract] |
52. | Arenzana-Seisdedos, F., Virelizier, J. L., Rousset, D., Clark-Lewis, I., Loetscher, P., Moser, B., and Baggiolini, M. (1996) Nature 383, 400-400[CrossRef][Medline] [Order article via Infotrieve] |
53. |
Loetscher, P.,
Gong, J. H.,
Dewald, B.,
Baggiolini, M.,
and Clark-Lewis, I.
(1998)
J. Biol. Chem.
273,
22279-22283 |
54. |
Struyf, S.,
Proost, P.,
Schols, D.,
De Clercq, E.,
Opdenakker, G.,
Lenaerts, J. P.,
Detheux, M.,
Parmentier, M.,
De, M., I,
Scharpe, S.,
and Van Damme, J.
(1999)
J. Immunol.
162,
4903-4909 |
55. |
Soto, H.,
Wang, W.,
Strieter, R. M.,
Copeland, N. G.,
Gilbert, D. J.,
Jenkins, N. A.,
Hedrick, J.,
and Zlotnik, A.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
8205-8210 |
56. |
Jenh, C. H.,
Cox, M. A.,
Kaminski, H.,
Zhang, M.,
Byrnes, H.,
Fine, J.,
Lundell, D.,
Chou, C. C.,
Narula, S. K.,
and Zavodny, P. J.
(1999)
J. Immunol.
162,
3765-3769 |
57. | Koenig, J. A., and Edwardson, J. M. (1997) Trends Pharmacol. Sci. 18, 276-287[CrossRef][Medline] [Order article via Infotrieve] |
58. |
Lefkowitz, R. J.
(1998)
J. Biol. Chem.
273,
18677-18680 |
59. |
Samanta, A. K.,
Oppenheim, J. J.,
and Matsushima, K.
(1990)
J. Biol. Chem.
265,
183-189 |
60. |
Signoret, N.,
Oldridge, J.,
Pelchen-Matthews, A.,
Klasse, P. J.,
Tran, T.,
Brass, L. F.,
Rosenkilde, M. M.,
Schwartz, T. W.,
Holmes, W.,
Dallas, W.,
Luther, M. A.,
Wells, T. N.,
Hoxie, J. A.,
and Marsh, M.
(1997)
J. Cell Biol.
139,
651-664 |
61. |
Forster, R.,
Kremmer, E.,
Schubel, A.,
Breitfeld, D.,
Kleinschmidt, A.,
Nerl, C.,
Bernhardt, G.,
and Lipp, M.
(1998)
J. Immunol.
160,
1522-1531 |
62. |
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 |
63. | Franci, C., Gosling, J., Tsou, C. L., Coughlin, S. R., and Charo, I. F. (1996) J. Immunol. 157, 5606-5612[Abstract] |
64. |
Aramori, I.,
Zhang, J.,
Ferguson, S. S. G.,
Bieniasz, P. D.,
Cullen, B. R.,
and Caron, M. G.
(1997)
EMBO J.
16,
4606-4616 |
65. |
Zimmermann, N.,
Conkright, J. J.,
and Rothenberg, M. E.
(1999)
J. Biol. Chem.
274,
12611-12618 |
66. |
Elsner, J.,
Mack, M.,
Bruhl, H.,
Dulkys, Y.,
Kimmig, D.,
Simmons, G.,
Clapham, P. R.,
Schlondorff, D.,
Kapp, A.,
Wells, T. N.,
and Proudfoot, A. E.
(2000)
J. Biol. Chem.
275,
7787-7794 |
67. |
Mochizuki, M.,
Bartels, J.,
Mallet, A. I.,
Christophers, E.,
and Schröder, J. M.
(1998)
J. Immunol.
160,
60-68 |
68. | Samson, M., Labbe, O., Mollereau, C., Vassart, G., and Parmentier, M. (1996) Biochemistry 35, 3362-3367[CrossRef][Medline] [Order article via Infotrieve] |