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-Inducible Protein-10/CXCL10-Specific Receptor Expressed by Epithelial and Endothelial Cells That Is Neither CXCR3 Nor Glycosaminoglycan1
Department of Adult Oncology, Dana-Farber Cancer Institute, and Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-inducible protein-10 (IP-10)/CXCL10 is a CXC
chemokine that attracts T lymphocytes and NK cells through activation
of CXCR3, the only chemokine receptor identified to date that binds
IP-10/CXCL10. We have found that several nonhemopoietic cell types,
including epithelial and endothelial cells, have abundant levels of a
receptor that binds IP-10/CXCL10 with a Kd
of 1–6 nM. Surprisingly, these cells expressed no detectable CXCR3
mRNA. Furthermore, no cell surface expression of CXCR3 was detectable
by flow cytometry, and the binding of 125I-labeled
IP-10/CXCL10 to these cells was not competed by the other high affinity
ligands for CXCR3, monokine induced by IFN-/CXCL9, and I-TAC/CXCL11.
Although IP-10/CXCL10 binds to cell surface heparan sulfate
glycosaminoglycan (GAG), the receptor expressed by these cells is not
GAG, since the affinity of IP-10/CXCL10 for this receptor is much
higher than it is for GAG, its binding is not competed by platelet
factor 4/CXCL4, and it is present on cells that are genetically
incapable of synthesizing GAG. Furthermore, in contrast to IP-10/CXCL10
binding to GAG, IP-10/CXCL10 binding to these cells induces new gene
expression and chemotaxis, indicating the ability of this receptor to
transduce a signal. These high affinity IP-10/CXCL10-specific receptors
on epithelial cells may be involved in cell migration and, perhaps, in
the spread of metastatic cells as they exit from the vasculature. (All
of the lung cancer cells we examined also expressed CXCR4, which has
been shown to play a role in breast cancer metastasis.) CXCR3-negative
endothelial cells may also use this receptor to mediate the angiostatic
activity of IP-10/CXCL10, which is also expressed by these cells in an
autocrine manner. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Leukocytes are not the only cell types that respond to chemokines. For
example, although the CXC chemokine IFN--inducible protein-10
(IP-10)3/CXCL10 was initially
characterized as a chemoattractant for T lymphocytes (15, 16), it also has antiangiogenic activities that appear to be
mediated by its direct effects on endothelial cells
(17, 18, 19). The only known functional receptor of
IP-10/CXCL10 is CXCR3 (20), which also binds the CXC
chemokines Mig (monokine induced by IFN-)/CXCL9 (20)
and I-TAC/CXCL11 (21) and the murine CC chemokine
SLC/CCL21 (22, 23) with equal affinities. But, even though
IP-10/CXCL10 has direct effects on endothelial cell preparations such
as HUVECs, some endothelial cells express no detectable CXCR3
(24, 25, 26), suggesting that there may be other
functional IP-10/CXCL10 receptors. IP-10/CXCL10 can also bind heparan
sulfate glycosaminoglycan (GAG), but this interaction does not result
in signal transduction (18).
In addition to endothelial cells, chemokine receptors have also been found on epithelial cells, although their functional significance is not clear (27, 28, 29, 30). Recently, it has been suggested that these receptors may be involved in patterns of metastatic spread (31, 32). In a search for chemokine receptor expression by normal lung epithelial and lung cancer cells, we tested several normal and malignant cells for their ability to bind chemokines and to express chemokine receptors. To our surprise, we found that all of these cells, as well as a wide variety of other cell types including endothelial cells, display high affinity binding sites for IP-10/CXCL10. Furthermore, binding is due neither to CXCR3 nor to GAG. Engagement of this apparently novel receptor results in signal transduction, which suggests that it may be involved in IP-10/CXCL10-mediated effects that could range from tumor cell migration or metastasis to regulation of angiogenesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBMC were isolated from heparinized human blood using
Ficoll-Hypaque (Pharmacia, Piscataway, NJ). Normal human bronchial
epithelial (NHBE) cells and HUVECs were obtained from Clonetics
(Walkersville, MD). Nonsmall cell lung cancer (NSCLC) cell lines, COS
cells, and pgsA-745 Chinese hamster ovary (CHO) cells (33)
were purchased from American Type Culture Collection (ATCC, Manassas,
VA). NHBE cells were cultured in bronchial epithelial cell growth
medium (BEGM; Clonetics, San Diego, CA) supplemented with 52 µg/ml
bovine pituitary extract, 0.5 µg/ml hydrocortisone, 0.5 ng/ml human
recombinant epidermal growth factor, 0.5 µg/ml epinephrine, 10
µg/ml transferrin, 5 µg/ml insulin, 0.1 ng/ml retinoic acid, and
6.5 ng/ml triiodothyronine. HUVECs were cultured in microvascular
endothelial cell growth medium (Clonetics) supplemented with 10 ng/ml
epidermal growth factor, 1 µg/ml hydrocortisone, 12 µg/ml bovine
brain extract, and 2% FBS. All NSCLC cells and COS cells were
maintained in DMEM with 10% FCS (Sigma, St. Louis, MO). PgsA-745 cells
were grown in Ham’s F12K medium with 10% FCS. Recombinant human
stromal cell-derived factor-1
(SDF-1)/CXCL12, IP-10/CXCL10, and
Mig/CXCL9 were obtained from R&D Systems (Minneapolis, MN). Plasmids
containing cDNA clones of CXCR3 and orphan receptors Apj, DEZ, GPR1,
and GPR15 were kindly provided by C. Gerard, Children’s Hospital
(Boston, MA). The plasmid, pBR322-pc-fos[human]-1, was
purchased from ATCC, and a XhoI/NcoI fragment was
used as a probe for Northern blot analysis of c-fos
expression.
RNase protection assay (RPA)
Total cellular RNA was isolated using RNeasy kits (Qiagen,
Hilden, Germany). Fifty micrograms of total RNA were treated with 5 U
RNase-free DNase I (Life Technologies, Gaithersburg, MD) in the
presence of RNasin (Promega, Madison, WI) for 15 min at 37°C.
Multiprobe template sets hCR5 and hCR6 were purchased from BD
PharMingen (San Jose, CA). DNA templates were used to synthesize probes
incorporating [-32P]UTP (3000 Ci/mmol; Life
Science Products, Boston, MA) using T7 RNA polymerase (Promega).
Hybridization with 15 µg of each target RNA was performed overnight,
followed by digestion with RNase A and T1 (Boehringer Mannheim,
Mannheim, Germany), according to the BD PharMingen protocol. The
samples were treated with a proteinase K-SDS mixture, extracted with
phenol:chloroform:isoamyl alcohol (50:49:1), and then precipitated with
ethanol in the presence of ammonium acetate. Protected RNA was loaded
on a 5% acrylamide-urea sequencing gel next to the labeled probes, and
electrophoresed at 50 W in 0.5x Tris-borate/EDTA electrophoresis
buffer.
Reverse-transcription PCR
Total RNA was isolated and treated with DNase I, as described above. Reverse transcription was performed by using a GeneAmp RNA PCR kit (Perkin-Elmer, Branchburg, NJ). PCR was performed on cDNA samples, using intron-straddling primers. Primer sequences for CXCR3 were 5'-AACCACAAGCACCAAAGCAG-3' (forward) and 5'-TGATGTTGAAGAGGGCACCT-3' (reverse); for CXCR4, 5'-ATCTGGAGAACCAGCGGTTA-3' (forward) and 5'-GGAAGTTCCCAAAGTACCAG-3' (reverse); and for CCR5, 5'-GAAGAGCTGAGACATCCGTT-3' (forward) and 5'-CGATTGTCAGGAGGATGATG-3' (reverse). PCR conditions were as follows: 95°C for 5 min; 95°C for 1 min, 60°C for 1 min, and 72°C for 1 min (40 cycles); and 72°C for 7 min. Amplified products were electrophoresed on 2% agarose gels and stained with ethidium bromide.
Flow cytometry
Cell staining was performed using mouse anti-human chemokine receptor mAbs, followed by FITC-conjugated, affinity-purified, multiply adsorbed, polyclonal goat anti-mouse Abs (BD PharMingen). The mAbs used in this study were directed against CXCR2 (6C6, IgG1), CXCR3 (1C6, IgG1), CCR2 (5A11, IgG1), and CCR5 (2D7, IgG1), all generous gifts from Millennium Pharmaceuticals (Cambridge, MA), and CXCR4 (12G5, IgG2a; BD PharMingen). The isotype control Abs were purchased from BD PharMingen. Samples were analyzed by FACScan (BD Biosciences, Mountain View, CA).
Chemokine-binding assay
Binding assays were performed using 1–2 x 105 NSCLC cells, NHBE cells, or COS cells transfected with receptor expression plasmids using a standard calcium phosphate transfection procedure (34). Cells were plated in 24-well culture plates and incubated overnight in standard medium. For the binding assay, cells were washed twice with washing buffer (0.5 M NaCl, 50 mM HEPES, 1 mM CaCl2, 5 mM MgCl2, and 0.5% BSA, pH 7.2), once with binding buffer (washing buffer without NaCl), and then incubated in duplicate with a constant concentration (85 pM) of 125I-labeled chemokines (Life Science Products) in the presence of increasing concentrations (up to 800-fold molar excess) of unlabeled chemokines. Incubations took place in 200 µl binding buffer. After incubation at room temperature for 90 min, binding buffer was aspirated, and cells were washed three times with the washing buffer and then were lysed in 0.5 ml 1 N NaOH. Radioactivity was determined using a gamma counter. Data were analyzed using MacLigand software (35). These salt conditions have been optimized for chemokine binding and minimize the potential for chemokine self-association (36). The low salt-binding conditions did not induce osmotic lysis.
Northern blot analysis
NSCLC cells were grown to subconfluence in DMEM with 10% FCS,
and then medium was changed to DMEM without FCS for 24 h. Cells
were treated with 100 ng/ml chemokine or 0.1% BSA-PBS for 30 min,
followed by RNA extraction, as described above. Since BEGM does not
contain serum, NHBE cells were grown to subconfluence in BEGM and
directly treated with chemokines. Fifteen micrograms of total RNA were
electrophoresed through a 1.2% agarose/2.2 M formaldehyde gel with
3-[N-morpholino]propane sulfonic acid/EDTA buffer and
transferred to nylon filters (Nytran; Schleicher & Schuell, Dassel,
Germany), in 10x SSC (1x SSC is 0.15 M NaCl/0.015 M sodium citrate,
pH 7), using a TurboBlotter (Schleicher & Schuell). RNA was covalently
fixed to the membrane by UV cross-linking using a Stratalinker
(Stratagene, La Jolla, CA). A c-fos cDNA probe was
radiolabeled with [-32P]dCTP (3000 Ci/mmol;
Life Science Products) by random primer labeling (High Prime;
Boehringer Mannheim). Hybridization was conducted in ExpressHyb
hybridization solution (Clontech Laboratories, Palo Alto, CA) under
stringent conditions.
F-actin staining
Cells (1 x 106) were incubated in
a Chamber Slide (Nunc, Naperville, IL) at 37°C in FCS-free DMEM with
100 ng/ml SDF-1/CXCL12 or IP-10/CXCL10 for 2, 4, 8, 15, 30, or 60
min. A negative control with 0.1% BSA in DMEM was analyzed in the same
manner. After incubation and washing with PBS, cells were fixed,
permeabilized, and stained in a single step by addition of 0.5 ml 40%
paraformaldehyde-PBS containing 0.2 µM Oregon Green 488 phalloidin
(Molecular Probes, Eugene, OR) and 100 µg/ml lysophosphatidylcholine
(Sigma), and the mixture was incubated for 20 min at 4°C. Cells were
photographed using an Eclipse E800 camera (Nikon, Tokyo, Japan)
with a x40 water immersion lens.
Chemotaxis
Cells were washed with PBS, then washed with chemotaxis buffer (DMEM containing 12 mM HEPES (pH 7.4) and 0.1% BSA) and resuspended in the same buffer. A total of 3 x 104 cells was placed in the upper wells of a 48-well chemotaxis chamber (Neuroprobe, Cabin John, MD). Lower wells contained varying amounts of IP-10/CXCL10 in the same buffer. Lower and upper wells were separated by polycarbonate filter with 8-µm pores that had been precoated with fibronectin. After incubation at 37°C for 6 h, the filter was removed, cells were scraped from the upper surface, and cells that remained adherent to the lower surface were fixed in methanol and stained with Diff Quik (Baxter, McGaw Park, IL). Cells were counted in four high power fields in each of two duplicate wells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In an attempt to identify chemokine receptors on epithelial cells,
we screened 11 NSCLC cell lines and NHBE cells for their ability to
bind radiolabeled chemokines. While several chemokines showed no
binding activity, IP-10/CXCL10 bound with high affinity to all examined
cells. Fig. 1
shows a typical example of
a displacement curve generated by this analysis, and the results from
all of the binding experiments are collected in Table I. All 11 NSCLC cell lines as well as
NHBE cells had a single class of high affinity binding sites for
IP-10/CXCL10 (Kd ranging from 1.2 nM
to 5.6 nM) present at 40,000–600,000 sites/cell.
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20–100,000 sites/cell), the two-site model implied the existence
of more than 2,000,000 low affinity sites/cell, suggesting that this
model was biologically inaccurate. Molecular analysis of chemokine receptor expression in NSCLC and NHBE cells
Having documented high affinity binding sites for IP-10/CXCL10, we
next sought evidence for expression of CXCR3, the only functional
IP-10/CXCL10 receptor identified to date. A multiplex RPA was unable to
detect CXCR3 mRNA expression by any of the NSCLC cell lines or NHBE
cells (Fig. 2A). In contrast,
other chemokine receptors were expressed, including CXCR4 by 8 of 10
NSCLC cell lines, CXCR2 by 2 cell lines, and both CCR1 and CCR2a by 1
line (Fig. 2, A and B).
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shows that only NCI-H661 cells expressed detectable CXCR3 mRNA. Thus,
RT-PCR generally confirmed the RPA results, indicating that despite
their ability to bind IP-10/CXCL10, all cell lines but one expressed no
detectable CXCR3. Surface expression of CXCR3 was also not detected by
fluorescence-activated cell sorting using anti-CXCR3 (data not
shown). The increased sensitivity of RT-PCR compared with RPA also
revealed that all cell lines, rather than just 8 of 10, expressed CXCR4
mRNA (Fig. 3).
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The absence of detectable CXCR3 mRNA expression by 10 cell lines
suggested that the IP-10/CXCL10 receptor on these cells was not CXCR3.
We tested this idea by attempting to compete radiolabeled IP-10/CXCL10
binding with unlabeled Mig/CXCL9, another non-ELR
(glutamate-leucine-arginine)-containing CXC chemokine that binds to
CXCR3 with the same affinity as IP-10/CXCL10 (20). Fig. 4A shows that Mig/CXCL9 was
unable to displace radiolabeled IP-10/CXCL10 from A549 cells even when
its concentration exceeded that of IP-10/CXCL10 by 800-fold.
IP-10/CXCL10 efficiently displaced itself under the same conditions. As
a control for Mig/CXCL9 activity, nonradiolabeled Mig/CXCL9 competed
radiolabeled IP-10/CXCL10 from CXCR3 binding sites, but not non-CXCR3
sites, in transfected COS cells, as expected (data not shown). In
addition, Mig/CXCL9 was able to displace only 20% of IP-10/CXCL10 from
NCI-H661 cells (Fig. 4B), suggesting that CXCR3 comprised a
minority of IP-10/CXCL10 binding sites on these cells. This low level
of CXCR3 expression is consistent with our ability to detect it in
NCI-H661 cells only by RT-PCR, and not by RPA or flow cytometry.
Similar results were obtained using I-TAC/CXCL11, another ligand for
CXCR3 (data not shown).
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), as it does when heparan sulfate is
the receptor. In addition, radiolabeled IP-10/CXCL10 bound to pgsA-745
cells, a CHO cell mutant deficient for a glycosyltransferase required
for the production of GAG (33). In the absence of any cell
surface GAG, these cells displayed 385,000 IP-10/CXCL10 receptors with
Kd of 3.3 nM, similar to the receptor
detected on NSCLC and NHBE cells. Since we saw comparable
IP-10/CXCL10-binding characteristics using wild-type CHO cells, our
binding conditions probably do not detect IP-10/CXCL10 binding to
GAG.
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A variety of seven-transmembrane-spanning G protein-coupled receptors has been described that have no identified ligands, but do have sequence motifs shared by chemokine receptors. Several were tested for their ability to bind IP-10/CXCL10. Four receptor cDNAs were transfected into COS cells, and transfectants were tested for the presence of increased numbers of high affinity IP-10/CXCL10 binding sites. (Increased numbers of sites were monitored because untransfected COS cells express 119,000 IP-10/CXCL10 binding sites with a Kd of 1.7 nM.) While transient expression of CXCR3 resulted in a 4-fold increase in IP-10/CXCL10 binding sites, expression of Apj (37), DEZ (38), GPR1 (39), and GPR15 (40) did not. This suggests that the IP-10/CXCL10 receptor expressed by NSCLC cells is unlikely to be encoded by these orphan receptor cDNAs.
Functional activation of the non-CXCR3 IP-10/CXCL10 receptor
To determine whether the IP-10/CXCL10 receptors identified on
NSCLC cells are functional, we examined all NSCLC cell lines and NHBE
cells for physiological responses to IP-10/CXCL10. Although
IP-10/CXCL10 treatment did not increase intracellular calcium
concentration, it induced chemotactic responses in some cells. For
example, Fig. 6 shows that IP-10/CXCL10
induced migration of SK-LU-1 cells with an EC50
between 0.1 and 1 nM. Checkerboard analysis showed that this movement
was chemotactic rather than chemokinetic. (Similar results were
observed with A549 cells.) Chemotaxis to IP-10/CXCL10 was inhibited by
pretreating cells with pertussis toxin, indicating that this response
involves receptor coupling to
Gi. Consistent with the
chemotactic response, IP-10/CXCL10 also induced F-actin redistribution
in A549 and NCI-H661 cells, although not in others, and was observed as
early as 2 min after adding IP-10/CXCL10 and lasted for 30 min
in A549 cells and 60 min in NCI-H661 cells (Fig. 7). Finally, since chemokines can
modulate gene expression, IP-10/CXCL10 was tested for its ability to
induce c-fos mRNA expression. IP-10/CXCL10 induced
c-fos mRNA expression in SW900, A549, NCI-H520, and NCI-H661
cells (Fig. 8), and Calu-3 cells (not
shown), but not in others. Protooncogene induction was not inhibited by
pretreating cells with 500 nM pertussis toxin, suggesting that not all
responses are coupled to
Gi. Although all of the
cells in this study expressed CXCR4 mRNA by RT-PCR, only two of them,
NCI-H520 and A549, responded to SDF-1/CXCL12 treatment with
c-fos expression (Fig. 8).
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IP-10/CXCL10 is known to have antiangiogenic activity, and this
may be effected through CXCR3 in endothelial cells that express this
receptor (41, 42, 43, 44). However, some endothelial cell
preparations do not express CXCR3 (24, 25, 26). To determine
whether these cells express another receptor through which IP-10/CXCL10
could exert direct effects, we performed a binding assay on HUVECs.
Fig. 9A shows that HUVECs have
a single class of high affinity binding sites for IP-10/CXCL10,
characterized by a Kd of 3.1 nM and
present at 166,000 sites/cell. In addition, like the majority of NSCLC
cell lines examined above, HUVECs express no detectable CXCR3 mRNA
(Fig. 9B). Notably, these cells express IP-10/CXCL10
constitutively, suggesting that the non-CXCR3 IP-10/CXCL10 receptor may
be involved in an autocrine loop. However, we have been unable to
demonstrate IP-10/CXCL10-induced migration or c-fos
expression in HUVECs, so the physiological consequences of receptor
activation in these cells are unknown. Finally, we tested U937
myelomonocytic leukemia cells and found that they expressed 9000
receptors for IP-10/CXCL10 with a Kd
of 2.2 nM. Like monocytes, U937 cells express no CXCR3
(20), and our findings may explain, in part, reports that
monocytes respond to IP-10/CXCL10 (45).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There are several potential explanations for the presence of
IP-10/CXCL10 receptors without detectable CXCR3 mRNA expression. First,
IP-10/CXCL10 binding might be due to a functionally wild-type CXCR3
encoded by an alternatively spliced mRNA not detected by our RT-PCR
primers. However, this is unlikely because we were unable to detect any
portion of an authentic CXCR3 mRNA by RPA (Fig. 2
A) or by
Northern blotting using a full-length CXCR3 cDNA probe (data not
shown). Furthermore, the CXCR3 locus appears to be deleted
in NCI-H520 cells (Fig. 3), which, nonetheless, display IP-10/CXCL10
binding sites. Second, our cell lines may have been infected with human
herpesvirus 8, which encodes a G protein-coupled receptor that binds
multiple chemokines, including IP-10/CXCL10 (46). We
tested this possibility by performing an RT-PCR analysis to search for
expression of this receptor’s mRNA, but none was detected (data not
shown). Third, IP-10/CXCL10 could be binding to cell surface heparan
sulfate (18). However, this is unlikely to be the receptor
we have identified, since the Kd for
that interaction is 25 nM (5–20 times weaker than the interactions we
detected), IP-10/CXCL10 binding to NSCLC cells results in signal
transduction (which does not occur when IP-10/CXCL10 binds GAG),
PF4/CXCL4 did not compete for IP-10/CXCL10 binding to NSCLC cells (Fig. 5), and IP-10/CXCL10 bound to mutant CHO cells that do not synthesize
GAG. Fourth, the universal expression of CXCR4, at least at the mRNA
level, parallels that of the IP-10/CXCL10 receptor (Figs. 2A
and 3) and could theoretically be responsible for binding IP-10/CXCL10.
However, CXCR4 has not been reported to bind IP-10/CXCL10, and only one
cell line, NCI-H520, had sufficient surface expression of CXCR4 to be
detected by SDF-1/CXCL12 binding (data not shown), while all of the
cells showed significant IP-10/CXCL10 binding.
The last possibility is that the IP-10/CXCL10 receptor identified in this study may be a novel chemokine receptor. In support of this notion is the observation that, unlike CXCR3 (21, 47), this receptor does not bind Mig/CXCL9 or I-TAC/CXCL11, and a receptor with this binding profile has not yet been described. Several G protein-coupled receptors without ligand assignments, so-called orphan receptors, have been described that have signature amino acid sequences found in chemokine receptors. None of the receptors we tested bound IP-10/CXCL10 with high affinity, suggesting that the potentially novel receptor we describe in this work is not one of these.
Regardless of the identity of this receptor, it appears to
be functional; but what might its function be in epithelial cells? By
analogy to chemokine receptors on leukocytes, activation of this
receptor on NSCLC cells might control their trafficking during
metastasis. Once a malignant cell has entered the vasculature, its
subsequent metastatic invasion recapitulates the paradigm of the
multistep model of leukocyte emigration. The circulating tumor cell
must be tethered to the luminal surface of endothelial cells in the
target organ, this interaction must be transformed into firm adhesion,
and then the tumor cell must accomplish diapedesis into the
subendothelium. If a tumor cell bearing IP-10/CXCL10 receptors comes
into contact with IP-10/CXCL10 (or an as yet unidentified ligand for
this receptor) displayed by endothelial cells, its activation may
induce the tumor cell to stop and invade the tissue. Viewed in this
light, metastases to lymph nodes, for example, might not reflect a
passive filtration function of the node, but would rather be the result
of an active cell migratory process by a cancer cell that has co-opted
a system already in place for leukocyte trafficking. This function has
recently been ascribed to CXCR4, which is expressed nearly universally
by breast (31) and ovarian (32) carcinoma
cells. It is worth noting that our data extend the ubiquity of CXCR4
expression to lung carcinomas, and that this does not exclude a similar
role for an IP-10/CXCL10 receptor. It should also be noted that
IP-10/CXCL10 did not elicit the same biological responses from all
cells tested (nor did SDF-1/CXCL12), indicating that the effects of
IP-10/CXCL10 may not universally occur in all cells bearing this
receptor.
Another cancer-related function for this receptor is suggested by its presence on endothelial cells. Considerable evidence indicates that IP-10/CXCL10 exerts its angiostatic effects directly on endothelial cells, including HUVECs (17, 18, 19). Although some endothelial cells express CXCR3 (41, 42, 43, 44), we and others were unable to document the expression of CXCR3 by HUVECs, suggesting that the receptor we have identified biochemically might be the means by which IP-10/CXCL10 accomplishes this activity. Furthermore, the constitutive expression of IP-10/CXCL10 by these cells may indicate the presence of an angiostatic autocrine loop that would have to be interrupted in order for angiogenesis to occur. A variety of animal models can be used to test this hypothesis, but in the absence of reproducible in vitro correlates of antiangiogenic responses to IP-10/CXCL10, the role that this receptor might play in angiogenesis remains conjectural.
Regardless of its true function in normal physiology or disease, we have identified a widely expressed and functional receptor for IP-10/CXCL10. Although we have found extensive biochemical evidence that this is a novel, non-CXCR3 receptor, definitive characterization will require its cloning.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Barrett J. Rollins, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115. E-mail address: barrett_rollins{at}dfci.harvard.edu
3 Abbreviations used in this paper: IP-10, IFN--inducible protein-10; BEGM, bronchial epithelial cell growth medium; CHO, Chinese hamster ovary; GAG, glycosaminoglycan; IP-10, IFN--inducible protein-10; Mig, monokine induced by IFN-; NHBE, normal human bronchial epithelial; NSCLC, nonsmall cell lung cancer; PF4, platelet factor 4; RPA, RNase protection assay; SDF-1, stromal cell-derived factor-1.
Received for publication July 23, 2001. Accepted for publication October 3, 2001.
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), as described in Materials and Methods.
At each concentration of nonradiolabeled ligand, the amount of
[125I]IP-10/CXCL10 still bound to cells is expressed as a
percentage of the amount bound in the absence of competing ligand. Each
point is the mean of duplicate measurements.
