IP10 and MIG are two members of the CXC branch of
the chemokine superfamily whose expression is dramatically up-regulated by interferon (IFN)-
. The proteins act largely on natural killer (NK)-cells and activated T-cells and have been implicated in mediating some of the effects of IFN-
and lipopolysaccharides (LPSs), as well
as T-cell-dependent anti-tumor responses. Recently both
chemokines have been shown to be functional agonists of the same
G-protein-coupled receptor, CXCR3. We now report the pharmacological
characterization of CXCR3 and find that, when heterologously expressed,
CXCR3 binds IP10 and MIG with Ki values of 0.14 and 4.9 nM, respectively. The receptor has very modest
affinity for SDF-1
and little or no affinity for other
CXC-chemokines. The properties of the endogenous receptor expressed on
activated T-cells are similar. Surprisingly, several CC-chemokines,
particularly eotaxin and MCP-4, also compete with moderate affinity for
the binding of IP10 to CXCR3. Eotaxin does not activate CXCR3 but, in
CXCR3-transfected cells, can block IP10-mediated receptor activation.
Eotaxin, therefore, may be a natural CXCR3 antagonist.
 |
INTRODUCTION |
Chemokines are a superfamily of small secreted proteins that play
an important role in the selective trafficking of leukocytes (for
review, see Ref. 1). Most members of the superfamily can be divided
into two groups depending on the organization of the first cysteine
pair: the CC branch in which the cysteines are adjacent, and the
CXC-branch in which they are separated by a single amino acid. Two
members of the CXC branch of the superfamily, IP10 and MIG, were
initially identified because of their dramatically enhanced expression
in monocytes activated by
IFN-
1 or LPS (2-4). The
biological actions of IP10 and MIG are largely restricted to activated
T- and natural killer (NK)-cells for which both are potent
chemoattractants (5, 6). These properties suggest that the two
chemokines may mediate some of the lymphocyte-directed effects of
IFN-
and LPS, a hypothesis that is supported by the observation that
IP10 elicits a potent T-cell-dependent antitumor response
(5). IP10 and MIG are also strongly angiostatic (7), a property which
may be related to their antitumor activity.
Chemokines elicit their biological functions by binding to specific G
protein-coupled receptors expressed on the appropriate cells types.
Like the chemokines, the receptors can be largely divided into two
sub-families: the CXC receptors (CXCRs), which bind CXC chemokines, and
the CC receptors (CCRs), which bind CC-chemokines (1). Recently,
Loetscher et al. (8) identified a new member of the CXCR
subfamily, CXCR3, which when recombinantly expressed mediated
chemotaxis and Ca2+ mobilization in response to both IP10
and MIG. However, these authors were unable to show binding of either
ligand to CXCR3. We now demonstrate that CXCR3 does bind both IP10 and
MIG with affinities consistent with the concentrations of the
chemokines required to elicit cellular responses. In addition, CXCR3
has some avidity for the CXCR4 ligand, SDF-1, and rather surprisingly has considerable affinity for several CC-chemokines, particularly the
CCR3 ligands eotaxin and MCP-4. The pharmacological properties of the
recombinantly expressed receptor mirror those of the native receptor
expressed on activated human T-cells.
 |
EXPERIMENTAL PROCEDURES |
Materials--
All human chemokines were from Peprotech (Rocky
Hill, NJ) except for SDF-1
, which was from Gryphon Sciences (South
San Francisco, CA). Radioactive chemokines were from NEN Life Science
Products. Venous whole blood or plasmapheresed leukocytes from normal
human donors was obtained from the New York Blood Center or the
University of Pennsylvania Medical Center.
Cloning of CXCR3--
The cDNA encoding CXCR3 was cloned by
PCR using lymph node cDNA (CLONTECH, Palo Alto,
CA) as a template. The PCR primers were designed based on the published
sequence (8). The PCR product was digested with EcoRI and
NotI and ligated to similarly digested and linearized
pBluscript KS II+ (Promega). The sequence of the coding
region of the receptor was verified by sequencing, then excised from
pBS-CXCR3 by digestion with HindIII and NotI, and
then ligated into mammalian expression vector pBJ-neo (9).
Expression of CXCR3 in CHO and RBL-2H3
Cells--
106 CHO cells (ATCC: CCL-61) were transfected
with 20 µg of DNA using a standard calcium phosphate procedure
(Specialty Media, Lavallette, NJ). The DNA was incubated with cells at
37 °C, 6% CO2 for 6 h, whereupon the cells were
glycerol-shocked (15% glycerol shock solution, Specialty Media) and
re-fed with selection media containing 0.4 mg/ml Geneticin (Life
Technologies, Inc.). Concurrently, RBL-2H3 cells were electroporated
with the CXCR3 expression plasmid as described previously (13) and
selected in 1 mg/ml Geneticin. After 10 days, the surviving CHO or RBL
foci were pooled. Stable expression of CXCR3 was verified by
determining that an aliquot of cells bound radiolabeled IP10 (see
binding parameters below). The remaining cells were cloned by limiting
dilution in 96-well microtiter plates, and the cells were expanded.
Stable cell lines were derived from individual clones selected on the
basis of binding and functional assays.
Binding Assays--
Binding of 125I-IP10 (2200 Ci/mmol, typically 20 pM) in the presence of unlabeled
ligands was initiated by adding intact cells (75,000 cells/point) as
described previously (10). After incubation at room temperature for 30 min, the cells were filtered through GF/C filters treated with 0.33%
polyethyleneimine and washed with buffer containing 25 mM
Hepes, 0.02% NaN3, and 0.5 M NaCl, pH 7.2.
Microphysiometry--
Functional assays were performed using
microphysiometry (11, 12). Briefly, untransfected CHO-KI cells or
CHO-KI cells stably transfected with human chemokine receptor CXCR3
were seeded onto the Transwell cell capsule cups (Molecular Devices,
Sunnyvale, CA) at a density of 0.33 × 106
cells/ml/cup in Ham's F-12 medium plus 10% fetal bovine serum. Following overnight culture, the capsules were transferred to microphysiometer sensor chambers (Cytosensor, Molecular Devices) and
allowed to equilibrate for 2 h, during which time they were perfused with running medium (1 mM phosphate-buffered RPMI
1640 medium, pH 7.4 (Molecular Devices) plus 0.1% bovine serum albumin (Life Technologies, Inc.)). Once stable acidification rates were established, cells were exposed for 6 min to various concentrations of
chemokine diluted in running medium. A flow rate of 100 µl/min was
used, and acidification rates were measured at 2 min intervals.
Calcium Flux--
Measurements were carried out with transfected
cells or purified T-cells labeled with Indo-1 (Molecular Probes,
Eugene, OR) as described previously (13).
T-cell Activation--
T-cells were purified from a mononuclear
cell preparation by E-rosetting with neuraminidase-treated sheep red
blood cells, followed by overnight incubation at 37 °C. The T-cells
were washed and incubated in plastic flasks at 2 × 106/ml in media containing 400 units/ml of human
recombinant IL-2 (Biosource International, Camarillo, CA) for 1-2
days, and further maintained at a density of 2-4 × 106/ml in fresh media containing IL-2 at 200 units/ml for
the specified times.
 |
RESULTS AND DISCUSSION |
Characterization of the Binding Properties of CXCR3 on CHO Cells
Stably Expressing the Recombinant Receptor--
Because untransfected
CHO cells do not bind 125I-IP10, lines stably expressing
CXCR3 were established by monitoring the gain of this activity (see
"Experimental Procedures"). The binding properties of a
represenative CHO clone (C1.17) were assessed by competition of various
chemokines against 125I-IP10 (Fig.
1 and Table
I). The clone exhibits a single high affinity binding site (Ki = 0.14 nM) for
IP10 and expresses approximately 50,000 sites/cell. As expected, the
receptor also binds MIG, although with an affinity
(Ki = 4.9 nM) that is substantially
lower than that found for IP10. Other CXC-chemokines, notably IL-8,
which binds to both CXCR1 and CXCR2 (14, 15), and GRO
and NAP2,
which bind to CXCR2, have little or no affinity for CXCR3 (Table I).
SDF-1, the ligand for CXCR4 (16, 17), does show very slight affinity
for CXCR3 with a Ki = 400 nM.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 1.
Pharmacology of CXCR3 recombinantly expressed
in CHO cells. Increasing concentrations of unlabeled chemokines
were used to compete against a fixed concentration of
125I-IP10. Shown are the results obtained using CXC
chemokines as competitiors (A) and the results with
CC-chemokines (B). Data are from single representative
experiments. No binding of 125I-IP10 was observed in
nontransfected or mock-transfected CHO control cells.
|
|
View this table:
[in this window]
[in a new window]
|
Table I
Affinities of CXC- and CC-chemokines for CXCR3
Ki values were determined from competition binding
experiments carried out against 125I-IP10 as described in the
legends of Figures 1 and 2 and in the "Experimental Procedures"
section. All values are given in nM and are the averages of
two to three experiments. NB is no binding; ND not determined.
|
|
Rather surprisingly, a number of CC-chemokines show moderate affinity
for CXCR3, in particular the ligands for CCR3 (9, 18). Eotaxin and
MCP-4 have Ki values of 60 and 70 nM,
whereas MCP-3 and RANTES have lower affinities with
Ki values of 250 and 420 nM,
respectively. Because CCR6 and CCR7 are phylogenetically more closely
related to CXCR3 than is CCR3, we also examined the ability of their
ligands (MIP-3
and MIP-3
) to bind to CXCR3. MIP-3
does bind
with some affinity (Ki = 160 nM),
although MIP-3
shows very little activity (Ki = 1700 nM). Similarly, MIP 1
, MIP1
, and MCP-1, ligands
for CCRs 1, 2, and 5 (reviewed in Ref. 1) show little or no affinity for the receptor (data not shown).
Binding of IP10 and Other CXCR3 Ligands to Purified IL-2 Activated
T-cells--
Since a number of factors, including differences in the
endogenous complement of G-proteins or various other host cell-specific restrictions, may influence the way in which ligands bind to their receptors, we compared the pharmacology of the receptor recombinantly expressed in CHO cells to endogenously expressed CXCR3 and to receptor
expressed in RBL-2H3 cells.
It has been reported that transcripts for CXCR3 are virtually absent in
resting T-cells but are present in IL-2-activated T-cells (8). In our
hands, the ability of human T-cells to bind IP10 is consistent with
these observations. Freshly isolated T-cells show variable but low
binding activity, an activity that is substantially up-regulated by a
number of activation procedures including treatment with
anti-CD3/anti-CD28, phorbal 12-myristate 13-acetate/ionomycin, or IL-2
(data not shown). To characterize the pharmacology of CXCR3 on primary
cells, we chose to use T-cells treated with IL-2 for 6-8 days, a
protocol that generates maximal binding of IP10 (data not shown). As
shown in Fig. 2 and Table I, the
properties of the receptor on the primary cells closely mirrors that of
the recombinant molecule expressed in CHO cells. The
Ki cells are 2-3-fold lower on average, but the
receptors in both cell types bind the same ligands with the same rank
order of potencies. Similar affinities were also obtained for receptor recombinantly expressed in RBL-2H3 cells (Table I).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 2.
Microphysiometric analysis of change in
acidification rate of CXCR3 expressing CHO cells in response to
addition of IP10 (panel A: 100 nM ( ), 30 nM ( ), 10 nM ( ), 3 nM ( ),
1 nM ( ), or 100 pM ( )), MIG (panel
B): 1 µM ( ), 300 nM ( ), 100 nM ( ), 30 nM ( ), 10 nM ( ))
or eotaxin (panel B): 1 µM ( ).
Cellular responses were monitored at 2-min intervals following addition
of chemokine. No response was induced in untransfected CHO cells by
IP10 or MIG at concentrations up to 1 µM. Results with
MCP-4 and SDF-1 are not shown because these chemokines generated a
response at 1 µM in untransfected cells.
|
|
Functional Responses Induced by Chemokines through CXCR3--
The
earlier identification and characterization of CXCR3 was carried out by
assessing the ability of various CXC- and CC-chemokines to mobilize
calcium in murine 300-19 pre-B cells transfected with the receptor. In
these experiments, Loetscher et al. found IP10 and MIG to be
the only chemokines to induce a response, and as a consequence, they
referred to CXCR3 as the IP10/MIG receptor. To confirm and extend this
preliminary characterization and to determine whether the
concentrations of IP10 and MIG required for functional activation
reflect the ligands' differential affinities, we also evaluated the
functional properties of the receptor in the stably transfected CHO
cell line. Since in our hands CHO cells are refractory to the uptake of
calcium sensitive dyes, a microphysiometer was used to monitor
ligand-induced increases in cellular acidification rate as a measure of
functional activation (11).
As illustrated in Fig. 3 both IP10 and
MIG induce dose-dependent increases in the acidification
rate. As expected from the binding data, IP10 is more potent than MIG
with EC50 values of ~10 and 100-200 nM,
respectively. In fact, the ratio of EC50 values for the two
ligands (10-20) is consistent with the relative binding affinities
(~30, Table I). In comparison, eotaxin failed to generate any
response, even at a concentration of 1 µM (Fig.
3B), a value 15-fold greater than its Ki
as determined in binding studies (Table I).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 3.
Pharmacology of the IP10 receptor (CXCR3)
expressed on activated T-cells. Cells were activated for 6-8 days
with recombinant human IL-2, and the binding properties of CXCR3 were
determined as described in Fig. 1, except that the assays employed
250,000 cells/point. Panel A shows the results obtained
using CXC chemokines as competitiors, and panel B shows the
results with CC-chemokines. Data are from single representative
experiments.
|
|
We also examined the ability of IP10, MIG, and eotaxin to stimulate
functional responses in activated human T-cells and transfected RBL
cells. For these studies, increases in intracellular Ca2+
levels were used to monitor responses. The data are consistent with the
results from the recombinant CHO lines. As shown in Fig. 4, in activated primary T-cells, both
IP10 and MIG induced a flux at a concentration of 100 nM,
whereas eotaxin failed to generate a response, even at a concentration
of 1 µM. Interestingly, since a subset of Th2 T-cells has
been reported to express CCR3 (19), the primary receptor for eotaxin,
the lack of response to eotaxin also suggests that our T-cell
preparations contain a very low level of this subset. High
concentrations of eotaxin (up to 1 µM) also failed to
induce a Ca2+-flux in our stable RBL CXCR3 transfected line
(Fig. 5), whereas the EC50 of
IP10 for this response was 10 nM (data not shown).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 4.
IP10 and MIG, but not eotaxin, induce
mobilization of intracellular Ca2+ in CXCR3 expressing
IL-2-activated human T-cells. Cells were cultured as described in
the legend for Fig. 4 prior to analysis.
|
|

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 5.
Eotaxin inhibits IP10-mediated
CXCR3-dependent Ca2+ mobilization in RBL CXCR3
cells. The data represent Ca2+ fluxes mediated by 10 nM IP10 in cells pretreated 5 min before analysis with
buffer alone ( ) or a final concentration of either 1 µM ( ) or 3 µM ( ) eotaxin. No further
suppression of the IP10 response was achieved by extending the
titration of eotaxin above 3 µM. A single point
reflecting the maximal functional activation of the receptor to IP10
after pretreatment with 10 µM IL-8 ( ) shows that the
effect of eotaxin is specific since the slight diminution in response
is within the limits of experimental variability. Eotaxin at a
concentration of 3 µM ( ) does not itself induce a flux
in these cells.
|
|
The binding and functional data suggest that eotaxin, under appropriate
circumstances, should act as a receptor antagonist. To test this
hypothesis, we examined the ability of eotaxin to inhibit an
IP10-induced Ca2+-flux in the transfected RBL cells. As
shown in Fig. 5, eotaxin does inhibit the response to 10 nM
IP10 with and IC50 of about 1 µM, a potency
consistent with the 100-fold difference in binding affinities (Table
I). As a control, IL-8 had no effect at concentrations as high as 10 µM.
The chemokine system consists of more than 50 ligands and 13 receptors.
The specificities of the ligand/receptor interactions are complex as
each receptor binds multiple chemokines and most chemokines bind to
more than one receptor. However, a general rule has been that a
chemokine receptor binds either CC- or CXC-chemokines but not both. The
one exception has been DARC, a highly promiscuous, ubiquitously
expressed, nonsignaling receptor whose function is unclear (20, 21).
Thus it is surprising to find that CXCR3 has moderate affinity for
several CC-chemokines, particularly those which bind to CCR3. In fact,
those affinities are higher than for any of the CXC-chemokines examined
here, except IP10 and MIG. It would appear, therefore, that overall
protein sequence homologies are not a sufficient means from which to
predict the class of ligand (CC or CXC) a given receptor may bind.
Moreover, since the primary CXCR3 ligands, IP10 and MIG, also show
moderate affinities for CCR3 (Ki values of 100 and
30 nM,
respectively2), it is also
likely that CXCR3 and CCR3 possess structural homology that enables
them to present key interactions to shared ligands.
An important but as yet unanswered question, given the modest
affinities that the CCR3 ligands show for CXCR3 and vice versa, is
whether these overlapping ligand specificities are physiologically relevant. It is tempting to believe they are. In vivo, there
is a correlation between strong TH2 responses and the diminished accumulation of TH1 cells (22, 23). Since CCR3 is expressed on cells
characteristic of TH2 responses, including a subset of TH2 T-cells (19,
24) and whereas CXCR3 is found predominately on TH1 T-cells (24), the
putative antagonistic effects of the CCR3 ligands on CXCR3 could impair
the accumulation of TH1 cells and in part account for some of the
inhibitory activity of TH2 responses. Considerable additional evidence
is needed to support this speculation, including the direct
demonstration that CCR3 ligands do indeed antagonize the effects of
IP10 and MIG on TH1 cells. Moreover, sufficient local concentrations of
the CCR3 chemokines have to be achieved in order for such antagonism to
occur. In this regard, it is currently thought that much of the
chemokine generated in vivo is bound to surface
proteoglycan, a modality that has been argued to greatly increase the
local concentration, particularly because the interaction with the
receptor occurs on a solid phase (25).
Regardless of whether the above argument is true, our data suggest that
chemokines may play a dual regulatory role, as agonists for some
responses and antagonists for others.