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Chemoattractant (CXCL11)1
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Division of Rheumatology, Allergy, and Immunology, Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129; and
Immunology and Allergy Division, University Hospital, Lausanne, Switzerland.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-activated human
saphenous vein endothelial cells (HSVEC), but not with resting HSVEC.
Similar results were obtained using human CXCR3-transfected murine
300-19 B cells. CXCR3 down-regulation was significantly more pronounced
when T cells were in contact with HSVEC than with their supernatants,
suggesting that CXCR3 ligands were efficiently displayed on the surface
of HSVEC. Using neutralizing mAbs to IFN-induced protein-10
(CXCL10), monokine induced by IFN- (CXCL9), and IFN-inducible
T cell 
chemoattractant (I-TAC; CXCL11), we found that even though
I-TAC was secreted from IFN--activated HSVEC to lower levels than
IFN-induced protein-10 or the monokine induced by IFN-, it was the
principal chemokine responsible for CXCR3 internalization. This
correlated with studies using recombinant chemokines, which revealed
that I-TAC was the most potent inducer of CXCR3 down-regulation and of
transendothelial migration. Known inhibitors of chemokine-induced
chemotaxis, such as pertussis toxin or wortmannin, did not reduce
ligand-induced internalization, suggesting that a distinct signal
transduction pathway mediates internalization. Our data demonstrate
that I-TAC is the physiological inducer of CXCR3 internalization and
suggest that chemokine receptor internalization occurs in physiological
settings, such as leukocyte contact with an activated
endothelium. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(Mig; CXCL9), and
IFN-inducible T cell chemoattractant (I-TAC; CXCL11) are unique in
that they are all induced by IFN- in a wide variety of cell types,
including endothelial cells (2, 3, 4, 5), and act through a
unique chemokine receptor, CXCR3. CXCR3 is expressed on activated T
cells, preferentially of the Th1 phenotype, NK cells, and on a
significant fraction of circulating CD4+ and
CD8+ T cells (
20–40%; Ref.
6, 7, 8). The majority of peripheral
CXCR3+ T cells express CD45RO (memory T cells) as
well as 
1 integrins (8) which are
implicated in the binding of lymphocytes to endothelial cells and the
extracellular matrix (9). In addition, CXCR3 has been
reported to be expressed on plasmacytoid dendritic cells
(10), leukemic B cells (11, 12), eosinophils
(13), and dividing microvascular endothelial cells
(14).
CXCR3+ T cells accumulate at sites of Th1-type
inflammation where IFN- is highly expressed, including
atherosclerosis (5), sarcoidosis (15),
inflammatory bowel diseases (8), and rheumatoid arthritis
(8, 16). IP-10 has been found to be highly expressed in a
number of Th1-type inflammatory diseases, including psoriasis
(17), tuberculoid leprosy (18), sarcoidosis
(15), and viral meningitis (19). In addition,
our finding that IFN--stimulated endothelial cells and endothelium
from atherosclerotic lesions are a rich source of IP-10, Mig, and I-TAC
suggest that these chemokines play an important role in the
transendothelial migration and local retention of
CXCR3+ T cells found in atherosclerotic lesions
(5). In support of this hypothesis, IP-10 and Mig induce
the rapid adhesion of IL-2-activated T cells to immobilized VCAM-1 and
ICAM-1, and IP-10, Mig, and I-TAC are potent chemotactic agents for
activated T cells. Both of these activities are dependent on CXCR3
activation (20).
Chemokine receptors are seven transmembrane-spanning G protein-coupled cell surface receptors that are pertussis toxin (PTX)-sensitive, indicating that they are linked to the Gi class of heterotrimeric G proteins (21). Activation of chemokine receptors induces G protein-dependent inhibition of adenylate cyclase, activation of phosphoinositol 3-kinase (PI3K), phospholipase C, protein kinase C (PKC), and protein kinase A, the generation of inositol triphosphate, and a transient rise in intracellular calcium (22). Overall, the biological responses resulting from these different cascades include rapid activation of integrin-dependent cell adhesion and directed cell migration (chemotaxis).
As is the case in many biological systems, chemokine receptor
activation also turns on a program to limit its own responses. In the
chemokine system, this takes the form of receptor desensitization and
receptor internalization. These two processes likely play an important
role in allowing a cell to continuously sense small changes in an
existing, and perhaps even changing, concentration gradient. Receptor
desensitization is a very rapid response, rendering a cell transiently
unresponsive to a subsequent stimulation through that receptor, and
usually results from uncoupling the receptor from heterotrimeric G
proteins (23). Receptor internalization is a distinct
process where activated receptors are removed from the cell surface for
degradation or recycling and leads to a more prolonged state of
cellular unresponsiveness to the internalized receptors’ agonists
(24, 25, 26). The role of receptor internalization in the
physiological responses induced by chemokines, such as transendothelial
migration and chemotaxis, is unclear. To begin to address these
questions, we studied the fate of CXCR3 following contact between
CXCR3+ T cells with a resting or
IFN--activated endothelium.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant human IP-10, I-TAC, Mig, IFN-, and IL-2 were
obtained from PeproTech (Rocky Hill, NJ). Neutralizing murine mAb to
human IP-10 and Mig were purchased from R&D Systems (Minneapolis, MN).
Neutralizing murine mAb to human I-TAC was a kind gift from K. Neote
(Pfizer, Groton, CT). FITC-conjugated and nonlabeled mouse
anti-human CXCR3 mAb (49801.111; IgG1) was purchased from R&D
Systems. PE-conjugated mouse anti-CXCR4 mAb (12G5, IgG2a),
irrelevant-purified and FITC-conjugated IgG1, and PE-conjugated IgG2a
mouse Abs (isotype controls) were obtained from BD PharMingen (San
Diego, CA). Texas Red-conjugated goat anti-mouse (GAM) Ab was
obtained from Southern Biotechnology Associates (Birmingham, AL).
Bispecific anti-CD3/CD8 was a kind gift from J. Wong (Massachusetts
General Hospital, Boston, MA; Ref. 27). PTX and
calcium ionophore were purchased from Calbiochem (La Jolla, CA).
Genestein, wortmannin, PMA, staurosporin, and propidium iodine were
obtained from Sigma-Aldrich (St. Louis, MO). T cell enrichment columns
were purchased from R&D Systems.
Cell isolation and culture
Human vascular endothelial cells were isolated from saphenous veins from seven different donors by collagenase treatment as described elsewhere (5). Cells were maintained in M199 medium (BioWhittaker, Walkersville, MD) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin (BioWhittaker), 5% FCS (Atlanta Biologicals, Norcross, GA), 100 µg/ml heparin (Sigma-Aldrich), and 50 mg/ml endothelial cell growth factor (Pel-Freez Biologicals, Rogers, AR). Human microvascular endothelial (HMEC)-1 cells, a human microvascular cell line, were cultured in complete endothelial growth medium (Clonetics, Walkersville, MD) supplemented with 5% FCS, gentamicin, amphotericin, bovine brain extract (12 µg/ml) and hydrocortisone (1 µg/ml), and epidermal growth factor (10 ng/ml).
CXCR3+ T cells were obtained from different sources. In some experiments, PBMC isolated by a Ficoll gradient were incubated with 200 ng/ml IL-2 and bispecific anti-CD3/CD8 or anti-CD3/CD4 for 4 days in RPMI 1640 (Cellgro and Mediatech, Herndon, VA) supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin (Mediatech), and 2 mM of L-glutamine (Mediatech). Proliferating CXCR3+ CD4+ or CD8+ T cells were kept in IL-2 only for an additional 4–10 days. In other experiments, CD3+ T cells were derived from PBMC passed through a T cell enrichment column (R&D Systems) and incubated for 4 days with 5 µg/ml PHA (Sigma-Aldrich) and 200 ng/ml IL-2 and then in IL-2 only for 4–10 days. Human CXCR3+-transfected 300-19 cells were a gift from B. Moser (Theodor Kocher Institute, Bern, Switzerland; Ref. 6).
Coculture experiments
Human saphenous vein endothelial cells (HSVEC) were used from
passage two to passage four and were cultured in 0.1%
gelatin-precoated wells from 6-well plates (Costar, Cambridge, MA).
When cells reached confluence, they were treated with or without 100
ng/ml IFN- for 16 h in fresh complete M199 medium. Following
removal of the supernatant and two washes with prewarmed HBSS,
CXCR3+ T cells (1 x
106) in 1 ml of complete RPMI were then added per
well. HSVEC-conditioned medium (250 µl) was used to resuspend a
separate aliquot of 0.5 x 106
CXCR3+ T cells pelleted in an Eppendorf. After
various times, CXCR3+ cells were collected by
being gently washed off of the endothelial monolayer in 6-well plates
or by centrifugation of CXCR3+ cells incubated
with HSVEC supernatants. Cells were analyzed for CXCR3 surface
expression by flow cytometry. In control experiments, we found that
IFN- treatment of T cells had no direct effect on CXCR3
expression.
Measurement of CXCR3 surface expression
CXCR3+ cells (0.25–0.5 x 106) were washed into an ice-cold FACS buffer (HBSS without Ca2+ and Mg2+ supplemented with 1% BSA, 2% FCS, and 0.1% sodium azide) and resuspended in 90 µl of FACS buffer and 10 µl of FITC-conjugated anti-CXCR3 mAb, PE-conjugated anti-CXCR4, or isotype controls. Cells were incubated for 30 min on ice in the dark. After two washings with the ice-cold FACS buffer, cells were resuspended in FACS buffer with propidium iodine (0.3 µg/ml, and only when CXCR3 was studied alone) to gate on live cells. FACS analysis was performed on 104 cells using a FACSCaliber flow cytometer (BD Biosciences, San Jose, CA) using CellQuest software (San Jose, CA). Mean fluorescence intensity (MFI) values were obtained by subtracting the MFI of the isotype control from the MFI of the positively stained sample. CXCR3 surface expression was expressed as a percent of baseline expression using the formula: (MFI of tested cells/MFI of untreated cells) x 100.
Regulation of CXCR3 cell surface expression
CXCR3+ T cells or CXCR3+ 300-19 cells (0.25 x 106) were incubated with various concentrations of rIP-10, I-TAC, or Mig for different times as indicated. In some experiments, T cells were preincubated for 2 h with 1 µM of wortmannin, 100 µM of genestein, 10 nM of staurosporin, or 0–1000 ng/ml PTX at 37°C, 5% CO2 before the addition of I-TAC, IP-10, or Mig (250 ng/ml), or PMA (0.1 nM) for an additional 30 min. T cells were also incubated with different concentrations of PMA and calcium ionophore for 30 min. At the end of these experiments, ice-cold FACS buffer was added, then cells were studied for cell surface expression of CXCR3.
Indirect immunofluorescence
CXCR3+ T cells or 300-19 cells
(107 cells/ml) were incubated with anti-CXCR3
(10 µg/ml in PBS) for 30 min on ice. Cells were then washed twice in
ice-cold PBS and then incubated for 30 min with 500 ng/ml I-TAC diluted
in complete RPMI prewarmed at 37°C. Alternatively, they were exposed
for 30 min to a monolayer of HSVEC treated or not with IFN- for
16 h and washed twice with prewarmed PBS. Cells were then
resuspended in the ice-cold FACS buffer, centrifugated, and immediately
fixed in 3.7% paraformaldehyde for 20 min on ice. After
permeabilization with 0.1% saponin in FACS buffer, cells were
incubated with Texas Red-conjugated GAM diluted in 0.1% saponin buffer
for 30 min on ice and in complete darkness. CXCR3 expression was
assessed by immunofluorescence microscopy (Axiophot microscope; Zeiss,
Oberkochen, Germany).
Role of heparin
Confluent HSVEC were incubated with complete M199 supplemented
with 0–500 µg/ml heparin with or without 100 ng/ml IFN-
for 16 h. After removal of the supernatant, cells were washed
twice with prewarmed HBSS (37°C). HSVEC and their supernatants were
examined for their ability to induce CXCR3 internalization on T cells
as mentioned above. In another set of experiments, 100 ng/ml IP-10 or
I-TAC in either complete RPMI, complete M199 (100 µg/ml heparin), or
complete M199 without heparin were used to resuspend
CXCR3+ T cells. Following incubation for 2 h
at 37°C, 5% CO2, T cells were stained for
CXCR3 surface expression and analyzed by FACS.
Effect of exogenous CXCR3 ligands added on HSVEC
Confluent HSVEC were incubated with complete M199 (including 100 µg/ml heparin) with or without 250 µg/ml I-TAC, IP-10, or Mig, or the three of them for 1 h at 37°C, 5% CO2. After removal of the supernatant, cells were washed twice with prewarmed HBSS (37°C) to remove unbound chemokines. CXCR3+ T cells were then added onto HSVEC monolayers or resuspended into HSVEC supernatants and incubated for 30 min before CXCR3 surface expression was analyzed by FACS.
CXCR3 recycling
CXCR3+ T cells (0.75 x 106) were treated with I-TAC (500 ng/ml) in complete RPMI for 30 min (37°C, 5% CO2). Following two washes with prewarmed (37°C) HBSS to remove unbound chemokines, they were resuspended in 750 µl of complete RPMI for up to 3 h. CXCR3 surface expression was determined at baseline in untreated cells (-30 min) and at times 0, 1, and 3 h following I-TAC stimulation.
Neutralizing HSVEC chemokine activity
When tested for their ability to inhibit CXCR3 internalization
induced by recombinant chemokines, all neutralizing mAb were effective
in a dose-dependent manner. Fifty micrograms per milliliter
neutralizing mAb significantly reduced CXCR3 surface disappearance
induced by 250 ng/ml Mig (43.4 vs 18%), IP-10 (47 vs 18%), and I-TAC
(65 vs 19%). IFN--treated HSVEC supernatants were pretreated with
50 µg/ml neutralizing anti-Mig, anti-IP-10, or anti-I-TAC
mAb or control mAb for 1 h at 37°C. CXCR3+
T cells were then incubated with these supernatants for 30 min and
CXCR3 surface expression examined as described above. The ability of
neutralizing mAb to inhibit the activity of HSVEC monolayers was also
studied. After removal of their supernatant and two washes, HSVEC
monolayers (in 24-well plates) were treated with 50 µg/ml
neutralizing mAb or control mAb for 1 h at 37°C before the
addition of CXCR3+ T cells.
Transendothelial migration
HMEC-1 cells were trypsinized and added (5 x 105 in 50 µl of complete endothelial cell growth medium) on the top of the filter (polycarbonate, pore size: 5 µm) of a 96-well ChemoTx plate (NeuroProbe, Gaithersburg, MD). After 96 h, cell adherence and confluence were confirmed by staining with Diff-Quick and acetyl/low density lipoprotein-DiI (Biomedical Technologies, Stoughton, MA), 10 µg/ml in DMEM 1% BSA. Indicated concentrations of chemokines were added in the bottom chamber (in 31 µl of RPMI/1% BSA). After gentle washing of the HMEC-1 cells, 2.5 x 104 of human CXCR3+ 300-19 cells in 25 µl were added on top of HMEC-1 cells and incubated for 5 h at 37°C, 5% CO2. The filter was then removed and the migrated cells were counted. Each experiment was performed in triplicate.
Data analysis
Experiments were performed using at least five different donors for HSVEC and CXCR3+ T cells. Results are shown as the mean of at least three independent experiments ± SE. Statistical analysis was calculated using a paired Student’s t test and statistical significance was considered with p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-activated HSVEC
Initial experiments showed that overnight coincubation of freshly
isolated CD3+ T cells with IFN--stimulated,
but not resting, HSVEC led to the disappearance of CXCR3 surface
expression on T cells (Fig. 1
A). We found this for both
CD8+ and CD4+
CXCR3+ T cells (data not shown). Time-course
experiments showed that CXCR3 surface expression decreased from 100 to
30–35% within the first 15 min of coincubation (Fig. 1B).
To assess whether this process was specific for CXCR3, we studied CXCR4
expression during these kinetic experiments and found no significant
decrease in CXCR4 expression (Fig. 1B). Thus, IFN-
activated, but not resting, HSVEC quickly and selectively
down-regulates CXCR3 expression on T cells.
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The most likely explanation for selective chemokine receptor
internalization is selective chemokine receptor activation
(26). IFN--activated endothelial cells secrete the
three known CXCR3 ligands: IP-10, Mig, and I-TAC (5, 28).
Following overnight culture, we have found 100 ng/ml IP-10, 10 ng/ml
Mig, and 1 ng/ml I-TAC in the conditioned medium of IFN-
activatedHSVEC (28). Although chemokines are
secreted from cells, they are basic proteins that under physiological
conditions have an affinity for cell surface negatively charged heparan
sulfate proteoglycans (HSPG) and accumulate on the surface of
endothelial cells (29). Therefore, we compared the ability
of HSVEC-conditioned medium with that of direct cell contact at
inducing CXCR3 down-regulation. Coincubation of
CXCR3+ T cells with IFN--activated HSVEC for
2 h induced an 80% decrease in CXCR3 expression compared with a
25% decrease following coincubation with unstimulated HSVEC controls
(Fig. 1
C). In contrast, supernatants collected from these
same IFN--activated HSVEC decreased CXCR3 expression on T cells by
only 50%, while supernatants collected from unstimulated HSVEC
cultures had no effect on CXCR3 expression. Thus, IFN--activated and
control HSVEC were significantly more potent at inducing CXCR3
down-regulation than their respective conditioned media
(p < 0.015 and p < 0.03,
respectively).
CXCR3 down-regulation induced by CXCR3 ligands: IP-10, Mig, and I-TAC
Because we recently showed that IFN--stimulated HSVEC produced
IP-10, Mig, and I-TAC, the three known ligands for CXCR3 (5, 28), we sought to determine whether these chemokines induced
CXCR3 internalization on T cell surfaces. CXCR3+
T cells and CXCR3+ 300-19 cells were treated with
500 ng/ml IP-10, Mig, or I-TAC for various times and then analyzed by
FACS for CXCR3 surface expression (Fig. 2A). We found that maximum
receptor internalization occurred within the first 15 min with all
three ligands, as shown for HSVEC-mediated internalization. For both
cell types, I-TAC was more potent than IP-10 or Mig, but this
difference was much greater for T cells compared with CXCR3-transfected
300-19 B cell line. Indeed, CXCR3 expression on T cells diminished by
75–85% with I-TAC, by 35–40% with IP-10, and by 30% with
Mig. In addition, we observed that in presence of the ligand, there was
no significant CXCR3 reappearance on T cells for up to 120 min. In
contrast, CXCR3 reappeared on 300-19 cells by 120 min despite the
continuous presence of the ligands (Fig. 2A). The
differences in the magnitude and kinetics of CXCR3 internalization and
recycling in T cells compared with transfected 300-19 B cells is not
clear. However, we found that simply washing
CXCR3+ 300-19 cells for flow cytometric analysis
or into fresh medium increased CXCR3 expression 1.5-fold and
2.5-fold above baseline, respectively. This effect was not affected
by cycloheximide, indicating that these treatments induced the
mobilization of an intracellular pool of receptors in these transfected
cells (data not shown).
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10 ng/ml, only a minor decrease in CXCR3 expression
was seen for all three ligands on T cells (Fig. 2B). At
higher concentrations, I-TAC was much more potent than IP-10 or Mig,
and IP-10 was more potent than Mig. Representative results from FACS
analysis performed on T cells treated with different doses of I-TAC are
shown in the bottom panel of Fig. 2B. A similar
attenuated pattern was also found with CXCR3+
300-19 cells (Fig. 2B). These results demonstrate that
IP-10, Mig, and I-TAC have differential abilities to down-regulate
CXCR3 internalization on T cells, and that among these three
IFN--inducible CXCR3 agonists, I-TAC is the most potent inducer of
CXCR3 down-regulation. Internalization of CXCR3 following ligand binding
To study CXCR3 internalization, cells were treated with an
anti-CXCR3 mAb and the fate of CXCR3 was followed after exposure to
HSVEC cells and rI-TAC. Immunofluorescence microscopy using a Texas
Red-conjugated GAM revealed CXCR3 at the cell surface and in a
perinuclear vesicular distribution in untreated
CXCR3+ T cells and 300-19 cells (Fig. 3). Following I-TAC exposure as well as
exposure to IFN--treated HSVEC, an increase in CXCR3 internalization
occurred, discernible as a decrease of cell surface expression and an
increase in intracellular accumulation. In contrast, the cellular
distribution of CD4 was unaffected by CXCR3 ligand exposure,
demonstrating the specificity of I-TAC-induced CXCR3 internalization
(data not shown).
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To determine whether CXCR3 is recycled back to the cell surface
after agonist-induced internalization, CXCR3+ T
cells were treated for 30 min with I-TAC (250 ng/ml) and then washed
into fresh medium without I-TAC (Fig. 4).
CXCR3 surface expression was assessed at times 0, 1, and 3 h.
Following agonist stimulation, receptor recycling occurred rapidly and
by 3 h, CXCR3 cell surface expression reached 79 ± 4% of
its prestimulation levels.
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To begin to dissect the mechanisms responsible for
chemokine-induced CXCR3 internalization, we investigated the role of
different intracellular signaling pathways using selective inhibitors
and activators. Activation of chemokine receptors is known to activate
multiple intracellular signaling pathways, including heterotrimeric G
proteins, PI3K, tyrosine kinases, and PKC. To investigate the role of
PI3K and tyrosine kinases, T cells were pretreated for 2 h with
wortmannin to inhibit PI3K or genestein to inhibit tyrosine kinases.
The expression of CXCR3 was then determined by FACS analysis following
30 min incubation with 250 ng/ml I-TAC or IP-10 (Fig. 5A). Neither wortmannin nor
genestein inhibited the ability of I-TAC or IP-10 to induce CXCR3
internalization, suggesting that PI3K and tyrosine kinases are not
mediating the internalization signal.
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1
inhibitor, on I-TAC-induced CXCR3 internalization. T cells were
therefore pretreated with PTX (250, 500, or 1000 ng/ml) for
2 h at 37°C and then stimulated with I-TAC (250 ng/ml; Fig. 5B). PTX pretreatment at concentrations that inhibited
I-TAC- and IP-10-induced chemotaxis and calcium flux did not inhibit
I-TAC- or IP-10-induced CXCR3 internalization. This suggests that
agonist-induced CXCR3 internalization is not dependent on
G1 activation.
Because other chemokine receptors, such as CXCR4 (30) and
CCR3 (31) have been shown to be internalized following
activation of PKC, we determined whether direct activation of PKC by
PMA or calcium ionophore (A12387) would induce CXCR3
internalization. We found that both PKC activators induced a
dose-dependent CXCR3 down-regulation (Fig. 5C), suggesting
that global activation of PKC can induce CXCR3 internalization in T
cells and might be involved in agonist-induced CXCR3 internalization.
Therefore, we pretreated CXCR3+ T cells for
2 h with 10 µM of staurosporin, a PKC inhibitor, prior to
exposure to 250 ng/ml I-TAC, Mig, or IP-10 (Fig. 5D).
Although staurosporin completely inhibited PMA (nonagonist)-induced
CXCR3 internalization, it did not affect the effect of IP-10 and Mig
and very mildly affected the effect of I-TAC. Therefore, PKC activation
does not seem to be involved in agonist-induced CXCR3
internalization.
Effect of heparin on ligand-induced CXCR3 internalization
Chemokines are basic proteins that bind negatively charged
glycosaminoglycan molecules, such as heparin and HSPG, at physiological
salt concentrations. This interaction can significantly modulate the
activity of chemokines and influence chemokine binding to their
receptors and subsequent signaling. In particular, IP-10 has a
relatively high affinity for heparin (25 nM) and can bind to HSPG on
endothelial cells. Heparin has been shown to displace an IP-10
alkaline-phosphatase fusion protein bound to endothelial cells
(29). Therefore, we sought to determine whether heparin
could affect the down-regulation of CXCR3 induced by incubation of T
cells with HSVEC monolayers or their supernatants. To study this, HSVEC
were incubated overnight with or without IFN- (100 ng/ml) in medium
containing various concentrations of heparin (0–500 µg/ml). HSVEC
were then washed to remove the heparin, and
CXCR3+ T cells were added to theHSVEC
monolayers or resuspended in HSVEC supernatants for 30 min. We found
that heparin, in a dose-dependent manner, was able to very modestly
decrease CXCR3 internalization on T cells incubated with HSVEC
monolayers or their supernatants (13 ± 3% vs 24 ± 3%,
p < 0.007, and 33 ± 5% vs 47 ± 10%, NS,
for monolayers and supernatants, respectively, at 500 ng/ml heparin)
(Fig. 6A). These results
suggest that heparin, at least at these concentrations, only moderately
displaced CXCR3 ligands fromHSVEC surfaces.
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B).
This is in contrast to experiments using HSVEC supernatants, and
suggests that IP-10 or I-TAC secreted fromHSVEC or sequestered on
HSVEC surfaces might be already bound to HSPGs in such a way that does
not inhibit their activity on CXCR3.
To examine whether exogenous chemokines would efficiently bind to
resting HSVEC and induce CXCR3 internalization on T cells, IP-10, Mig,
and I-TAC were individually added (250 ng/ml) to the medium of HSVEC
for 1 h and then unbound chemokines were removed by washing. We
found that I-TAC, IP-10, and Mig remained bound to HSVEC surfaces after
washing because they induced CXCR3 sequestration in T cells, with a
potency order (I-TAC > IP-10 > Mig) identical to soluble
recombinant proteins (Fig. 7). It is
worth noting that the simultaneous addition of all three ligands was
not significantly different from I-TAC alone and that I-TAC (250 ng/ml)
was similar to the effect of IFN--stimulated HSVEC monolayers. These
data suggest that the level of I-TAC found in HSVEC supernatants might
not be an accurate reflection of the amount of biologically active
I-TAC bound to HSVEC surfaces following IFN- treatment.
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Because IP-10, Mig, and I-TAC are produced to varying levels from
IFN--activated HSVEC (IP-10 > Mig > I-TAC) and
exogenously added recombinant ligands can, to varying degrees, induce
CXCR3 internalization (I-TAC > IP-10 > Mig), we sought to
determine the contribution of these ligands to the ability of
IFN--activated HSVEC monolayers and their supernatants to induce
CXCR3 internalization on T cells. This was achieved using neutralizing
Abs. Anti-IP-10 and anti-Mig mAbs (50 µg/ml), which were able to
completely inhibit IP-10- and Mig- (250 ng/ml) induced CXCR3
down-regulation, had no effect on HSVEC monolayer- or
supernatant-induced CXCR3 down-regulation (Fig. 8). In contrast, anti-I-TAC mAb (50
µg/ml), which was able to inhibit I-TAC (250 ng/ml)-induced CXCR3
down-regulation, was able to partially inhibit the ability of HSVEC
supernatants from down-regulating CXCR3 expression on T cells (Fig. 8).
The addition of all three mAbs was no better than the I-TAC mAb alone
(Fig. 8). These results suggest that I-TAC is the physiological inducer
of CXCR3 internalization produced by IFN--activated HSVEC. The fact
that anti-I-TAC did not completely inhibit HSVEC-induced CXCR3
down-regulation suggests that I-TAC bound to HSPG on HSVEC may not be
completely accessible to mAb neutralization, or that the surface of
HSVEC has an I-TAC independent mechanism for inducing CXCR3
internalization.
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The ability of IP-10, Mig, and I-TAC to induce transendothelial
cell migration of CXCR3+ 300-19 cells was
compared with the ability of these ligands to induce chemotaxis across
bare filters. Both assays used 5 µM-pore size filters with
transendothelial cell migration being performed with filters coated
with HMEC-1 endothelial cells (Fig. 9, A and B). Chemotaxis across uncoated filters
essentially confirmed published studies, demonstrating comparable
chemotaxis for all three ligands. However, in contrast, I-TAC was
markedly more potent and more efficacious at inducing migration across
endothelial cells. Although the presence of HMEC-1 increased the number
of cells that migrated across the filter for all ligands, this effect
was more pronounced for I-TAC, especially at low concentrations. For
example, at 10 ng/ml I-TAC there was an 33-fold increase in the
number of cells that migrated across HMEC-1 coated filters compared
with bare filters, while HMEC-1 cells only caused an 5-fold increase
to IP-10 and an 6-fold increase to Mig.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-activated HSVECs and that cell contact is more efficient at
inducing internalization than conditioned medium collected from these
cells. Furthermore, although IFN--activated HSVEC express more IP-10
and Mig than I-TAC (28), I-TAC is the factor present in
HSVEC that is responsible for inducing CXCR3 internalization.
These findings are consistent with our observation that rI-TAC was the
most potent inducer of CXCR3 internalization. This type of functional
hierarchy in terms of chemokine-induced receptor internalization
(I-TAC > IP-10 > Mig) has been described for other
chemokines, such as CCR5 ligands
(amino-oxypentane-RANTES > wild-type RANTES; Ref.
34), CCR3 ligands(RANTES > eotaxin;
Ref. 31), and CXCR2 ligands (IL-8 > granulocyte
chemotactic protein-2 > neutrophil-activating peptide-2; Ref.
35). CXCR3 ligands, as well as IFN--activated HSVECs,
specifically induced CXCR3, but not CXCR4, surface down-regulation on T
cells. This finding indicates that IFN--activated HSVEC express
CXCR3 ligands but not the CXCR4 ligand SDF-1. It also reflects the fact
that CXCR3 agonists are not globally activating a pathway that
down-regulates chemokine receptors but are specifically targeting CXCR3
for internalization following activation. These data also imply that
different chemokines differentially activate the same receptor
resulting in different biological outcomes.
Our data also revealed that the signal transduction pathway mediating agonist-induced CXCR3 internalization is distinct from the pathway that mediates CXCR3-induced chemotaxis. We found that wortmannin, as well as PTX, did not inhibit agonist-induced CXCR3 internalization as was reported for CCR3. This is in contrast to agonist-induced chemotaxis, which is inhibited by both wortmannin and PTX (35). The finding that genestein, a tyrosine kinase inhibitor, could not block CXCR3 internalization is consistent with previous reports on other chemokine receptors, suggesting that phosphorylation of serine/threonine residues, but not of tyrosine residues, by specific G protein-receptor kinases is required for receptor sequestration. CXCR3 sequestration was induced by direct activation of PKC with PMA and calcium ionophore, suggesting that intracellular signaling pathways that activate PKC may lead to agonist-independent internalization of CXCR3, a mechanism that was reported for CXCR4 (30) and CCR3 (31). In contrast, PKC activation was not significantly involved in agonist-induced CXCR3 internalization.
We found that anti-I-TAC mAb only partially blocked HSVEC
monolayer- and supernatant-mediated CXCR3 internalization in T cells
and that anti-Mig and anti-I-TAC had no effect on this process.
This was observed despite the fact that these mAbs can neutralize
recombinant protein-induced internalization, chemotaxis, and calcium
flux. The inability of these Abs to neutralize HSVEC-induced CXCR3
internalization is unclear but may reflect the fact that these
chemokines are secreted from cells bound to proteoglycans (A. D.
Luster and L. Wagner, unpublished observations; Ref.
36) or bound to proteoglycans on cells which limit
the ability of the mAbs to neutralize them. Alternatively, additional
ligands may be present on HSVEC cells or in their supernatants or there
may even be ligand-independent mechanisms that contribute to a loss of
CXCR3 surface expression. For example, it has recently been shown that
CXCR1 and CXCR2 undergo surface cleavage following activation of
neutrophils with TNF- and LPS (37), a process
inhibited by metalloproteinase inhibitors. This process would have to
be specific for certain chemokine receptors because CXCR4 surface
expression on T cells was unaffected by coculture with HSVECs while
CXCR3 was down-regulated.
To explore the functional consequences of CXCR3 down-regulation, we examined the ability of CXCR3 ligands to induce chemotaxis of CXCR3–300-19 cells across uncoated filters and filters coated with endothelial cells. We found that the presence of endothelial cells preferentially increased the potency and efficacy of I-TAC-induced transendothelial migration compared with IP-10 and Mig. Although the reason for this cannot be ascertained from these experiments, in light of our data, it is interesting to speculate that preferential receptor internalization induced by I-TAC plays a role in its marked ability to induce transendothelial migration. It will be interesting to determine whether receptor internalization plays a role in transendothelial cell migration.
Our findings that activated endothelial cells retain functional CXCR3
ligands is consistent with Piali et al. (20), who found
that T cells adhered to IFN- and TNF--stimulated HUVEC, and that
this effect was partially blocked by an anti-CXCR3 mAb. Our study
suggests that following the induction of chemokine-induced firm
adhesion, CXCR3 is internalized on T cells and that this may play a
role in transendothelial cell migration.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Andrew D. Luster, Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital, Building 149, 13th Street, Charlestown, MA 02129. E-mail address: luster{at}helix.mgh.harvard.edu
3 Abbreviations used in this paper: IP-10, IFN-induced protein of 10 kDa; Mig, monokine induced by IFN-; I-TAC, IFN-inducible T cell chemoattractant; PTX, pertussis toxin; PI3K, phosphoinositol 3-kinase; PKC, protein kinase C; GAM, goat anti-mouse; HMEC, human microvascular endothelial cells; HSVEC, human saphenous vein endothelial cells; MFI, mean fluorescence intensity; HSPG, heparan sulfate proteoglycan; SDF, stromal cell-derived factor.
Received for publication December 20, 2000. Accepted for publication October 4, 2001.
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, +HMEC-1 cells; •, -HMEC-1 cells. Data is
representative of four independent experiments and is presented as the
mean ± SE of one representative experiment performed in
triplicate.