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Blood, Vol. 95 No. 5 (March 1), 2000:
pp. 1551-1559
CHEMOKINES
GCP-2-induced internalization of IL-8 receptors: hierarchical
relationships between GCP-2 and other ELR+-CXC chemokines
and mechanisms regulating CXCR2 internalization and
recycling
Rotem Feniger-Barish,
Dan Belkin,
Alon Zaslaver,
Shira Gal,
Mally Dori,
Maya Ran, and
Adit Ben-Baruch
From the Department of Cell Research and Immunology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.
 |
Abstract |
The chemotactic potencies of ELR+-CXC chemokines
during acute inflammation are regulated by their binding affinities and
by their ability to activate, desensitize, and internalize their specific receptors, CXCR1 and CXCR2. To gain insight into the fine
mechanisms that control acute inflammatory processes, we have focused
in this study on the highly potent ELR+-CXC chemokine
Granulocyte Chemotactic Protein 2 (GCP-2), and on its ability to
control the cell surface expression of CXCR1 and CXCR2. Although GCP-2
has been considered an effective ligand for both CXCR1 and CXCR2, our
findings demonstrated that it was a potent inducer of CXCR2
internalization only. A functional hierarchy was shown to exist between
GCP-2 and 2 other ELR+-CXC chemokines, IL-8 and NAP-2, in
their abilities to induce CXCR1 and CXCR2 internalization, according to
the following: IL-8 > GCP-2 > NAP-2. By the use of pertussis toxin
(PTx), it was demonstrated that the actual events of
G i-coupling to CXCR2 do not have a major role in the
regulation of its internalization. Rather, CXCR2 internalization was
shown to be negatively controlled by induction of signaling events, as
indicated by the promotion of CXCR2 internalization following exposure
to wortmannin, a potent inhibitor of phosphatidylinositol (PI) 3 kinases and PI4 kinases. Furthermore, our results suggest that
rab11+-endosomes participate in the trafficking of CXCR2
through the endocytic pathway, to eventually allow its recycling back
to the plasma membrane. To conclude, our findings shed light on the
interrelationships between GCP-2 and other ELR+-CXC
chemokines, and determine the mechanisms involved in the regulation
of GCP-2-induced internalization and recycling of CXCR2.
(Blood. 2000;95:1551-1559)
© 2000 by The American Society of Hematology.
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Introduction |
Human Granulocyte Chemotactic Protein 2 (GCP-2) is a
member of the ELR-expressing CXC subfamily of chemokines
(ELR+-CXC) and acts as a potent chemoattractant of
neutrophils in the course of acute inflammation.1-5 The
high chemotactic potency of GCP-2 is well illustrated in mice, where it
is the predominant neutrophil chemoattractant.4,6,7
Moreover, GCP-2 is highly produced by MG-63 osteosarcoma cells and
induces neovascularization,4,5,7-11 suggesting that it may
be involved in tumor development and metastasis formation.
As a member of the ELR+-CXC subfamily of chemokines, human
GCP-2 is one of several CXC chemokines that differ in their ability to
attract and activate neutrophils.4,5,10-18 The differential functional capabilities of these chemokines are in part the result of
their divergent abilities to bind and activate 2 receptors, CXCR1 and
CXCR2, which are expressed on neutrophils.19-26 Interleukin 8 (IL-8), the most potent of all human ELR+-CXC chemokines,
binds to both receptors with high affinity, whereas most other
ELR+-CXC chemokines (such as Neutrophil Activating Protein
2 [NAP-2]), bind with high affinity to CXCR2 only.21-26
In that respect, GCP-2 is of major interest, being the only
ELR+-CXC chemokine, except for IL-8, that is an effective
ligand for CXCR1, in addition to CXCR2.24-26 These
characteristics of GCP-2 may allow it to serve as a "backup
chemokine" in case inappropriate down-regulation of IL-8 expression
has occurred.
The redundancy in ELR+-CXC chemokines and in their
receptors may provide multiple levels of regulation that allow for
chemokine- and receptor-specific control of inflammatory processes.
Fine tuning of ELR+-CXC chemokine-induced responses could
also be achieved by the different abilities of these chemokines to
regulate the induction of intracellular events, which follow the
binding of each of them to its specific receptors, CXCR1 and/or CXCR2.
CXCR1 and CXCR2 belong to a superfamily of G protein-coupled receptors
(GPCR), whose signaling is mediated by their coupling to heterotrimeric
G proteins, resulting in the exchange of GDP for GTP on the  subunit
of the G protein.27-29 Studies of CXCR1 and CXCR2 have
demonstrated that the cascade of events that follows leads to specific
cellular responses, which are strictly regulated by homologous
desensitization. The process of homologous desensitization is induced
by high concentrations of ligands and is mediated primarily by receptor
phosphorylation.30-34 In addition, one of the major determinants that may control the functionality of CXCR1 and CXCR2 is
the level of their expression, determined by rapid dynamics of
internalization and recycling back to the plasma
membrane.31,32,34-38
Since the fine control of neutrophil responses results from the
coordinated activity of several ELR+-CXC chemokines, it is
of major importance to elucidate the regulation of CXCR1 and CXCR2 by
members of this subfamily. In that respect, the aim of our study is to
gain insight into the regulation of these receptors by the highly
potent neutrophil chemoattractant, GCP-2. Because receptor expression
is a principal regulator of the functionality of CXCR1 and CXCR2, we
have focused on the hierarchical relationships between GCP-2 and other
ELR+-CXC chemokines and their abilities to induce receptor
internalization, and on the intracellular events that are involved in
GCP-2-induced CXCR2 internalization and recycling. The results of our
study shed light on the coordinated interaction of ELR+-CXC
chemokines with their receptors and on the tight regulation of their activities.
| |
Materials and methods |
DNAs for human CXCR1 and human CXCR2
Wild type receptor DNAs were generated using polymerase chain
reaction, shuttled into the expression vector pRc/CMV (Invitrogen, San
Diego, CA), and subjected to full length sequencing as previously described.35,39
Cell cultures, transfections, and characterization of receptor
expression by transfected cells
Human embryonal kidney 293 cells (HEK 293 cells) were grown and
stably transfected as previously described.35,39 Scatchard analysis showed that CXCR1 and CXCR2 bound IL-8 with similarly high
affinity. A higher binding affinity of IL-8 to CXCR1 as compared to
NAP-2 was observed, whereas both ligands showed a similarly high
affinity of binding to CXCR2.23 The binding characteristics of GCP-2 to CXCR1 and CXCR2 were determined as previously
described,24,25 indicating the moderately higher binding
capability of GCP-2 to CXCR2 than to CXCR1. To further increase the
expression level of transfected receptors, stable cell lines expressing
CXCR1 or CXCR2 were subjected to cytofluorometric sorting, using
monoclonal antibodies to the amino terminus of CXCR1 or
CXCR2.35,39 Fluorescence-activated cell sorting (FACS)
analyses showed that a high percentage (over 90%) of the transfected
cells expressed the receptor on the cell surface with high mean
fluorescence value. Control transfections were performed with the
vector (pRc/CMV) alone, and the resulting cells did not specifically
bind IL-8, NAP-2, and GCP-2, or antibodies specific for human CXCR1 or
CXCR2.23,24,35,39
Analysis of receptor downmodulation by FACS analysis
This analysis was performed as previously described.35
Briefly, aliquots of stable HEK 293 transfectants were removed and supplemented with GCP-2, IL-8, or NAP-2 (GCP-2, NAP-2: PeproTech, Inc,
Rocky Hill, NJ; IL-8: Dainippon, Japan), while no chemokines were added
to control tubes. The cells were incubated at 37°C for the indicated
time periods. The cells were washed in CSB (phosphate-buffered saline
[PBS] containing 1% fetal calf serum, 0.02% NaN3, and
25 mmol/L Hepes) and incubated with monoclonal mouse anti-CXCR1 or anti-CXCR2 antibodies (R&D Systems, Minneapolis, MN; IgG2a,
1 µg/5 × 105 cells). Baseline staining was obtained by
adding CSB to the cells instead of anti-CXCR1 or anti-CXCR2 antibodies.
Following incubation and washings, the cells were incubated with
fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG
antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA),
washed, and resuspended. The effects of PTx and of wortmannin on
internalization levels were determined by pre-incubating the cells with
the compounds for 2 hours and 1 hour, respectively, at 37°C.
FACSort® (Becton Dickinson, San Jose, CA), was used to
analyze 5000 live cell events. Percent receptor downmodulation was
calculated from the mean channel fluorescence values of cells treated
with ligand at 37°C vs the mean channel fluorescence of cells not
treated with ligand, under similar conditions. P values were
calculated by Student t test.
Determination of receptor re-expression on the plasma membrane
The procedure used to determine receptor re-expression was similar
to that used to evaluate receptor downmodulation, but
matched samples were allowed to undergo a receptor recovery process
that was performed as previously described.35 To determine
the contribution of de novo protein synthesis to receptor reappearance
on the cell membrane, cell samples matching those undergoing receptor
recovery were incubated with medium containing 10 µg/mL cycloheximide
(during the recovery phase) (Sigma, St Louis, MO).
Analysis of the cells following cycloheximide treatment indicated that
cell viability was not affected by this treatment. Mean fluorescence
values of cells that were not exposed to GCP-2, and of cells exposed to GCP-2 and allowed to undergo recovery, were used to determine the
level of receptor re-expression. P values were calculated by
Student t test.
Chemotaxis assays
The migration of CXCR2-expressing HEK 293 cells was assessed by a
48-well microchemotaxis chamber technique as previously described.39 Briefly, the lower compartment of the chamber
was loaded with aliquots of medium, or 100 ng/mL GCP-2 diluted in medium, while the upper compartment of the chamber was loaded with
cells (resuspended in a similar medium). The 2 compartments were
separated by a 10 µm pore-sized polycarbonate PVPF coated with 50 µg/mL rat collagen type I (Collaborative Biomedical
Products, Bedford, MA). Following 5 to 6 hours
incubation at 37°C, the filter was removed, fixed, and stained. The
effects of pertussis toxin (PTx) and of wortmannin on the migration of
the cells were determined by pre-incubating the cells with the
compounds for 2 hours and 1 hour, respectively, at 37°C, followed by
washings. The migrated cells in 3 high-power fields (in each of the
triplicates performed) were counted by light microscopy. The
statistical significance of the number of cells migrating in response
to stimuli vs to BSA medium was evaluated using Student t test.
Confocal analyses of receptor downmodulation
Aliquots of stable CXCR2-expressing HEK 293 cells were supplemented
with 1000 ng/mL GCP-2, while no chemokines were added to control tubes.
The cells were incubated at 37°C for the indicated time points, fixed
in 4% paraformaldehyde, and centrifuged onto 0.5% gelatin-coated
slides. From this stage on, the entire procedure was performed at room
temperature. Cells were permeabilized in 0.2% triton X-100 for 30 minutes, and blocking was performed for 1 hour in blocking buffer (PBS
supplemented with 0.25% gelatin, 0.15% saponin, and 3% goat serum).
Primary antibodies were added for 1.5 hours, after diluting them in
washing buffer (PBS supplemented with 0.25% gelatin, 0.15% saponin,
and 0.1% goat serum). The primary antibodies included polyclonal
rabbit antibodies against human CXCR2 (0.5 µg/60 µL; Santa Cruz
Biotechnology, Santa Cruz, CA) and monoclonal mouse antibodies against
human rab11 (2 µg/60 µL; Transduction Laboratories,
Lexington, KY). After rinsing the cells in washing buffer, incubations
were performed for 1 hour with secondary antibodies:
rhodamine-conjugated goat anti-rabbit IgG (3.5 µg/60 µL) and
FITC-conjugated goat anti-mouse IgG (8 µg/60 µL) (both antibodies
were purchased from Jackson ImmunoResearch Laboratories). No
cross-reactivity of the rhodamine-conjugated goat antibodies with mouse
anti-rab11 antibodies, or of the FITC-labeled goat antibodies with
rabbit anti-CXCR2 antibodies, was distinguished. Following additional
washings, stained cells were analyzed using a Zeiss confocal laser
scanning microscope.
| |
Results |
Characteristics of GCP-2 induced downmodulation of CXCR1 and
CXCR2
To determine the effect of GCP-2 on cell surface expression of CXCR1
and CXCR2, we first exposed CXCR1- and CXCR2-expressing HEK 293 cells
to high concentrations of this chemokine for 2 hours at 37°C (high
concentrations of ligands were previously shown to be required for
optimal induction of receptor internalization31,32,35). The
expression of CXCR1 and CXCR2 was determined by anti-CXCR1 or
anti-CXCR2 specific antibodies, using FACS analysis. Similar experiments that were performed at 4°C indicated that the
pre-incubation of the cells with chemokines did not prevent the
antibodies from binding to cell surface-expressed CXCR1 and CXCR2
(data not shown).
As shown in Figure 1, 3000 ng/mL GCP-2 were
required to potently downmodulate CXCR1 expression, at the level of
33.7 ± 0.9% (Figure 1A) (This value is the mean ± SD of 4 independent experiments). One thousand ng/mL GCP-2 did not induce a
significant downmodulation of CXCR1 (data not shown). These results
indicate that GCP-2 differs from IL-8 in its ability to induce
reduction in cell surface expression of CXCR1, since IL-8 was
previously shown to elicit 71.8 ± 5.3% downmodulation of CXCR1
expression in concentrations of 1100 ng/mL.35 (To rule out
the possibility that GCP-2-induced low levels of CXCR1 internalization
were due to an artifact, experiments with IL-8 were performed in
parallel and on the same batch of cells, and indicated that IL-8
induced high levels of CXCR1 downmodulation.) However, GCP-2 proved to
be a more potent inducer of CXCR1 internalization when compared to
NAP-2, since high concentrations of NAP-2 (2000 ng/mL) did not induce
any significant reduction in CXCR1 expression.35
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| Fig 1.
GCP-2-induced downmodulation of CXCR1 and CXCR2
expression.
CXCR1- or CXCR2-transfected HEK 293 cells incubated with 3000 and 1000 ng/mL GCP-2, respectively, for 2 hours at 37°C.
The cells were washed and stained with anti-CXCR1 or anti-CXCR2
specific antibodies, and subjected to fluorescence-activated cell
sorting (FACS) analysis as described in "Materials and methods."
(A) CXCR1-expressing cells. (B) CXCR2-expressing cells. Counts indicate
relative cell number; baseline, cells stained with cell sorter buffer
(CSB) instead of antibodies to CXCR1 or CXCR2. A representative
experiment of 4 to 5 performed is shown.
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Exposure of CXCR2-expressing cells to 1000 ng/mL GCP-2 resulted in 65 ± 2.3% downmodulation of CXCR2 cell surface expression (Figure 1B).
(This value is the mean ± SD of 5 independent
experiments.) In that respect, the downmodulation-inducing potencies of
GCP-2 and IL-8 were similar since 1000 ng/mL of IL-8 induced 78.9 ± 2.4% downmodulation of CXCR2 expression.35 On the other
hand, GCP-2 proved to be more potent than NAP-2 in induction of CXCR2 downmodulation, because the latter chemokine induced only 37.4 ± 6.6% reduction in receptor expression on exposure to 2000 ng/mL.35
Since relatively high levels of receptor downmodulation were observed
only in GCP-2-treated CXCR2-expressing cells, dose response and
kinetics experiments were performed on GCP-2-induced downmodulation of
CXCR2 only. Exposure of CXCR2-expressing cells to 50 ng/mL GCP-2 did
not induce significant reduction in receptor expression (Figure
2). At 250 ng/mL of GCP-2, moderate levels
of CXCR2 downmodulation (34.5 ± 8.3%) were observed, and plateau
levels were reached on exposure to 1000 to 2000 ng/mL of this chemokine
(65 ± 2.3% and 72 ± 3.5%, respectively) (Figure 2). Further
analysis of GCP-2-induced reduction in cell surface expression of
CXCR2 indicated that on exposure to 1000 ng/mL GCP-2 for 5 minutes, 27 ± 6.1% downmodulation was observed, increasing to 55.6 ± 1.8%
following 30 minutes' exposure, and reaching a plateau level following
90 minutes' exposure to GCP-2 (65.8 ± 4.4%).
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| Fig 2.
Dose response of GCP-2-induced downmodulation of CXCR2.
CXCR2-expressing HEK 293 cells were incubated with various
concentrations of GCP-2 for 2 hours at 37°C. The cells were washed
and stained with anti-CXCR2-specific antibodies, and subjected to
fluorescence-activated cell sorting (FACS) analysis as described in
"Materials and methods." Each value represents the mean ± SD of
5 independent experiments.
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Following determination of the ability of GCP-2 to induce CXCR1 and
CXCR2 downmodulation, confocal analyses were performed to determine
whether receptor downmodulation resulted from GCP-2-induced internalization of both receptors. These analyses indicated that exposure to high concentrations of GCP-2 induced internalization of
CXCR2 (Figure 3C, D). As shown in Figure
3A, the majority of the
receptors in CXCR2-expressing cells that were not exposed to GCP-2 were
localized on the cell surface. However, on exposure to 1000 ng/mL GCP-2
for 1 hour, the distribution of CXCR2 was different, as indicated by
the fact that the majority of the receptor-specific staining was
associated with a cytoplasmic granular localization (Figure 3C, D).
Although GCP-2 induced only 33.7% reduction in CXCR1 cell surface
expression (Figure 1), the process could be observed by confocal
analysis in a moderate number of cells, and was characterized as
receptor internalization (data not shown).
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| Fig 3.
Localization of CXCR2 expression and of the expression of
rab11+ endosomes by confocal analysis.
CXCR2-expressing HEK 293 cells were incubated with or without 1000 ng/mL GCP-2, at 37°C for 1, 5, and 60 minutes. The GCP-2-exposed
cells were subdivided into 2 groups. One group of cells was washed and
stained immediately after exposure to GCP-2 with anti-CXCR2 and
anti-rab11-specific antibodies, as described in "Materials and
methods." The cells of the other group were washed and allowed to
recover at 37°C for 90 minutes, in the presence or absence of 10 µg/mL cycloheximide. The cells were washed and stained with
anti-CXCR2 and anti-rab11-specific antibodies, and subjected to
confocal analysis as described in "Materials and methods." In all
the pictures shown, the red color represents the expression of CXCR2,
as distinguished by the staining with rabbit antibodies against CXCR2,
followed by rhodamine-conjugated antibodies against rabbit IgG. The
green color, in all the pictures shown, represents the expression of
rab11, as distinguished by the staining with mouse antibodies against
rab11, followed by fluorescein isothiocipnate (FITC)-conjugated
antibodies against mouse IgG. The yellow color indicates the
colocalization of CXCR2 with rab 11 expression. (A) The expression of
CXCR2 and rab11 prior to exposure to GCP-2. (B, C, D) The localization
of CXCR2 and rab11 expression following the exposure of the cells to
GCP-2 for 1, 5, and 60 minutes, respectively. (E) The expression of
CXCR2 and rab11 after induction of internalization and recovery at
37°C for 1.5 hours in the absence of cycloheximide. (F) The same as
in (E), in the presence of cycloheximide. Reference bars in the lower
right corners of selected sections represent 10 µm.
A representative experiment of 2 to 4 performed is
shown.
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A functional hierarchy exists between IL-8, GCP-2, and NAP-2, as
manifested by their abilities to induce receptor internalization
Previous studies on the interactions of IL-8 and NAP-2 with CXCR2
have indicated that the 2 chemokines have similar affinities to CXCR2
and that IL-8 very potently displaced NAP-2, as well as binding to this receptor. However, despite their similarly high
affinities for CXCR2, NAP-2 could not effectively block IL-8 binding to
this receptor.22,23 The different displacement abilities of
IL-8 and NAP-2 were shown to be functionally relevant, as indicated by
the ability of IL-8 to functionally dominate the ability of NAP-2 to
induce CXCR2 internalization.35 However, the entire functional hierarchical relationships between the 3 closely related ELR+-CXC chemokines, IL-8, GCP-2, and NAP-2, was not yet established.
To further characterize the functional relationships between IL-8,
GCP-2, and NAP-2, we have extended our study to determine the hierarchy
between the 3 chemokines with regard to their ability to induce
receptor internalization. To this end, 2 approaches were taken: (1)
Based on the ability of IL-8 to induce higher CXCR1 internalization
than GCP-2 (Figure 1A and Feniger-Barish et al35), the
ability of IL-8 to functionally compete with or displace GCP-2 was
determined using CXCR1-expressing cells exposed to GCP-2. (2) Based on
the ability of GCP-2 to induce higher CXCR2 internalization as compared
to NAP-2 (Figure 1B and Feniger-Barish et al35), the
potential of GCP-2 to functionally compete with or displace NAP-2 was
determined by its ability to induce CXCR2 internalization in
CXCR2-expressing cells that were exposed to NAP-2.
As suggested in approach no. 1 above, to determine the functional
hierarchy between IL-8 and GCP-2, CXCR1-expressing cells were exposed
to 2 different procedures. In the first, the ability of IL-8 to compete
with GCP-2 was assayed by the exposure of the cells to concomitant
stimulation with both IL-8 and GCP-2. In the second, the ability of
IL-8 to displace GCP-2 was evaluated by exposing the cells to GCP-2,
followed by their treatment with IL-8. As shown in Figure
4A, exposure of the cells to IL-8 in the
presence, or following the administration of GCP-2, resulted in the
induction of potent internalization of CXCR1 that was significantly higher than GCP-2-induced internalization (P  .001), and
similar to internalization levels induced by IL-8 alone, suggesting the functional dominance of IL-8 over GCP-2.
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| Fig 4.
The functional hierarchy between IL-8, GCP-2, and NAP-2,
as manifested by their abilities to induce CXCR1 or CXCR2
internalization.
(A) IL-8 functionally competes for and displaces GCP-2-induced
internalization of CXCR1 expression. CXCR1-transfected HEK 293 cells
were incubated with IL-8 (1000 ng/mL) or GCP-2 (3000 ng/mL) for the
indicated time at 37°C. GCP-2 + IL-8 (1 hour) indicates a concomitant
exposure to both chemokines for 1 hour. GCP-2 (2 hours) + IL-8 (1 hour)
indicates an exposure to GCP-2 for 2 hours, to which IL-8 was added
during the second hour of incubation with GCP-2. The cells were washed
and stained with anti-CXCR1-specific antibodies, and subjected to
fluorescence-activated cell sorting (FACS) analysis as described in
"Materials and methods." Each value represents the mean ± SD of 3 independent experiments. *P = .001 for GCP-2
exposure for 2 hours vs the GCP-2 (2 hours) + IL-8 (1 hour) treatment.
**P < .001 for GCP-2 exposure for 1 hour vs the GCP-2 + IL-8
(1 hour) treatment. (B) GCP-2 functionally competes for and displaces
NAP-2-induced downmodulation of CXCR2 expression. CXCR2-transfected
HEK 293 cells were incubated with GCP-2 (1000 ng/mL) or NAP-2 (2000 ng/mL) for the indicated time at 37°C. NAP-2 + GCP-2 (1 hour)
indicates a concomitant exposure to both chemokines for 1 hour. NAP-2
(2 hours) + GCP-2 (1 hour) indicates an exposure to NAP-2 for 2 hours,
to which GCP-2 was added during the second hour of incubation with
NAP-2. The cells were washed and stained with anti-CXCR2-specific
antibodies, and subjected to FACS analysis as described in
"Materials and methods." Each value represents the mean ± SD of
3 independent experiments. * P = .01 for GCP-2
exposure for 1 hour vs the NAP-2 + GCP-2 (1 hour) treatment. **
P = .006 for NAP-2 exposure for 2 hours vs the NAP-2
(2 hours) + GCP-2 (1 hour) treatment.
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When similar experiments were performed on CXCR2-expressing cells
(Figure 4B), determining the ability of GCP-2 to compete with and
displace NAP-2 (as suggested in approach no. 2 above), it was observed
that exposure of the cells to GCP-2, in the presence or following the
administration of NAP-2, resulted in the ability of GCP-2 to induce the
same levels of internalization that were observed without the prior
exposure to NAP-2, and induce levels significantly higher than those
induced by NAP-2 alone (P = .01 and P = .006
depending on the assay conditions; see the legend to Figure 4B). These
results demonstrate that GCP-2 is functionally dominant over NAP-2. In
addition, although both GCP-2 and IL-8 induced high levels of CXCR2
internalization, the somewhat higher levels of CXCR2 internalization
induced by IL-8 as compared to GCP-2 allowed us to analyze the
hierarchy between the 2 chemokines, indicating that IL-8 is
functionally dominant over GCP-2 in the context of CXCR2 (data not
shown). Therefore, our findings suggest a functional hierarchy,
according to which IL-8 > GCP-2 > NAP-2 in their ability to induce
receptor internalization.
The role of G protein coupling and of signaling events in
GCP-2-induced internalization of CXCR2
Analysis of the dose response curve of GCP-2-induced
internalization of CXCR2 (Figure 2) indicated that potent
internalization of CXCR2 was induced by high (>1000 ng/mL) and
partially desensitizing concentrations of the chemokine
(38.2 ± 7.2% of chemotactic desensitization was induced by such
concentrations of GCP-2; data not shown and Wolf et al26).
On the other hand, significantly lower levels of internalization were
induced by activating doses of GCP-2 (ie, 100 ng/mL). Activation was
determined by chemotaxis assays (Table 1,
Wuyts et al,25 and Wolf et al26) (Figure 2).
This observation is in agreement with other studies,40-43
suggesting the significant interdependence between GPCR desensitization
and internalization. These findings raise the possibility that
GCP-2-induced G protein coupling to the receptors, and/or the
resulting intracellular signaling events, should be partially turned
off to allow for potent and maximal internalization processes to take
place.
To determine the role of G protein coupling in the regulation of
GCP-2-induced internalization of CXCR2, CXCR2-expressing cells were
exposed to PTx. PTx was shown to efficiently modify members of the
G i subclass of G proteins, which were shown to be key
mediators of IL-8-, as well as of GCP-2-induced migration of
neutrophils.24,44,45 First, the ability of the toxin to uncouple the binding of Gi to CXCR2 was determined, as
manifested by its ability to inhibit GCP-2-induced migration of
CXCR2-expressing cells. Different concentrations of PTx, in the range
of 100 to 1000 ng/mL, were assayed for their inhibitory potential, and
all proved to abolish GCP-2-induced chemotactic responses. As shown in
Table 1, 100 ng/mL PTx completely inhibited GCP-2-induced migration of
CXCR2-expressing cells (P < .001), indicating that in our
system this concentration of PTx was a potent inhibitor of
GCP-2-induced coupling of Gi to CXCR2.
Following determination of the ability of PTx to inhibit
Gi coupling, the effect of 100 ng/mL PTx on
GCP-2-induced CXCR2 internalization was evaluated. When
CXCR2-expressing cells were exposed to 360 ng/mL GCP-2, a concentration
that potently induced G protein coupling and effectively activated
cellular migration (similar to that induced by 100 ng/mL; data not
shown and Wolf et al26), PTx did not significantly affect
the level of CXCR2 internalization (P > .05; Figure
5). However, a minimal, but statistically significant inhibitory effect of the toxin was observed on CXCR2 internalization, when the cells were exposed to 1000 ng/ml GCP-2 (Figure 5; reduction of 16.3%; P < .002), a concentration
that partly desensitized migration and induced optimal
internalization of the receptor. Therefore, our data suggest that
the actual event of Gi protein coupling has only a minor
role, if at all, in the regulation of GCP-2-induced
internalization of CXCR2.
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| Fig 5.
The role of G protein coupling in the regulation of
GCP-2-induced CXCR2 internalization.
CXCR2-expressing HEK 293 cells were incubated with 360 ng/mL or 1000 ng/mL GCP-2 for 2 hours at 37°C. Prior to (for 2 hours) and during
the exposure to GCP-2, the cells were treated with 100 ng/mL pertussis
toxin (PTx) at 37°C. The cells were washed and stained with
anti-CXCR2-specific antibodies, and subjected to fluorescence
activated cell sorting (FACS) analysis as described in "Materials
and methods." Each value represents the mean ± SD of over 4 independent experiments. * P < .002 for internalization
induced by 1000 ng/mL GCP-2 in the absence of, vs in the presence of,
pertussis toxin (PTx).
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PTx, as a potent inhibitor of Gi coupling to CXCR2,
blocks the initiation of Gi-dependent signaling events
in response to GCP-2 stimulation. The fact that the toxin had only a
minimal effect on GCP-2-induced internalization of CXCR2 does not
exclude the possibility that signaling events, once initiated, regulate the internalization process. To examine this possibility, the effect of
wortmannin on GCP-2-induced internalization of CXCR2 was evaluated.
Wortmannin is a potent inhibitor of phosphatidylinositol (PI) 3 kinases
and PI4 kinases, major mediators of IL-8-induced signaling.46,47 First, the ability of wortmannin to inhibit signals that are required for GCP-2-induced migration was determined. The results shown in Figure 6A indicate
that wortmannin induced a dose-dependent and significant reduction in
GCP-2-induced migratory responses of CXCR2-expressing cells (reduction
of 53 ± 17.4% in migration by 1000 nmol/L
wortmannin. This value is the mean ± SD of 4 independent experiments;
P < .01). Although potent and significant, the inhibitory
effect of wortmannin on GCP-2-induced migration of CXCR2-expressing
cells was partial, as expected from the possible participation of other
signaling events, except for PI3 and PI4 kinases, in this process.
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| Fig 6.
The role of signaling events in the regulation of
GCP-2-induced CXCR2 internalization.
(A) Inhibition of GCP-2-induced chemotaxis of CXCR2-expressing HEK 293 cells by wortmannin. The cells were treated with 10, 100, or 1000 nmol/L wortmannin for 1 hour at 37°C, and washed and
subjected to a chemotaxis assay in response to 100 ng/mL GCP-2, as
described in "Materials and methods." A representative experiment
of 4 performed is shown. *P < .05, **P < .01 for
the response after the treatment, vs without treatment, by wortmannin.
(B) Enhancement of GCP-2-induced internalization of CXCR2-transfected
HEK 293 cells by wortmannin. CXCR2-transfected cells were incubated
with 360 ng/mL GCP-2 for 2 hours at 37°C. Prior to (for 1 hour) and
during the exposure to GCP-2, the cells were treated with 10, 100, or
1000 nmol/L wortmannin, washed, stained with
anti-CXCR2-specific antibodies, and subjected to fluorescence-activated
cell sorting (FACS) analysis, as described in "Materials and
methods." Each value represents the mean ± SD of 4 to 6 independent experiments. ***P < .001 for the response in the
presence, vs the response in the absence of wortmannin.
|
|
Similar doses of wortmannin were analyzed for the regulation of
GCP-2-induced internalization of CXCR2. Internalization was induced by
concentrations of GCP-2 that provoked potent activation of migration
(360 ng/mL), as well as by doses that partially desensitized GCP-2-induced chemotactic responses (1000 ng/mL). As shown in Figure
6B, when internalization was induced by 360 ng/mL GCP-2, a notable
increase, of 49 ± 13%, in receptor internalization was induced by
1000 nmol/L wortmannin (This value is the
mean ± SD of 4 independent experiments; P < .001). The
dose response of wortmannin-mediated inhibition of chemotaxis was in
full agreement with that of wortmannin-induced enhancement of CXCR2
internalization. The potent, but partial, increase in CXCR2
internalization that was induced following the treatment with
wortmannin may be the result of the only partial inhibition of
GCP-2-induced migratory responses that was induced by this compound
(Figure 6A). Similar stimulating effects of wortmannin on CXCR2
internalization were observed when receptor downmodulation was induced
by 1000 ng/mL GCP-2 (data not shown). These results suggest that
PI3/PI4 kinase-mediated signaling events, once initiated following
receptor activation, are effective regulators of the internalization process.
Determination of mechanisms regulating CXCR2 re-expression on
the cell surface
Studies of neutrophils, as well as our previous findings on CXCR1-
and CXCR2-expressing HEK 293 cells, have shown that CXCR1 and CXCR2 are
recycled back to the plasma membrane on free ligand (IL-8) removal and
recovery of the cells at 37°C.35,37 To determine the
mechanisms involved in such a process, we first analyzed by FACS the
kinetics of re-expression of CXCR2 on GCP-2 removal, and recovery of
the cells at 37°C. These experiments were performed in the presence
or in the absence of cycloheximide, to determine whether receptor
recycling or receptor de novo synthesis accounted for receptor
re-expression on the plasma membrane. Receptor expression levels
following ligand removal were compared to the levels of their
expression in control cells in which GCP-2-induced CXCR2 internalization was not elicited.
As shown in Figure 7, following the removal
of GCP-2 and recovery of the cells at 37°C, CXCR2 was re-expressed on
the cell membrane. On incubation at 37°C for 90 minutes, CXCR2
expression was restored to 81.9 ± 5.3% of the control level in cells
that had no exposure to GCP-2. Recovery of receptor expression to 92.1 ± 5.1% of control cells was observed following 4 hours of
incubation. Treatment with cycloheximide did not affect the recovery of
CXCR2 at 90 minutes (re-expression of 74.1 ± 5.2%;
P = .14 when recovery in the absence of cycloheximide was
compared to recovery in the presence of the compound), but
significantly reduced the level of receptor re-expression when
incubations were extended to 4 hours (re-expression of
62.7 ± 12.3%, P = .03). (All the above values are the
mean ± SD of 3 independent experiments.)
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| Fig 7.
Re-expression of CXCR2 on the cell membrane.
CXCR2-expressing HEK 293 cells were either not incubated with GCP-2
(control cells) or incubated with 1000 ng/mL GCP-2 at 37°C for 1 hour. The GCP-2-exposed cells were subdivided into 2 groups. One group
of cells was washed and stained immediately after exposure to GCP-2
with anti-CXCR2-specific antibodies, as described in "Materials and
methods." The cells of the other group were washed and allowed to
recover at 37°C for various intervals, in the presence or absence of
10 µg/mL cycloheximide. The cells were washed and stained with
anti-CXCR2-specific antibodies, and subjected to fluorescence-activated
cell sorting (FACS) analysis as described in "Materials and
methods." No recovery indicates cells undergoing internalization and
no recovery at 37°C; 90 minutes and 4 hours indicate the time of
recovery at 37°C. Counts indicates relative cell number; and
baseline, cells stained with cell sorter buffer instead of antibodies
to CXCR2. A representative experiment of 3 performed is
shown.
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|
These results indicate that the membrane expression of CXCR2 is quickly
recovered following GCP-2-induced internalization, and that
cycloheximide affected the recovery of CXCR2 only at later time points
subsequent to the internalization process. Therefore, "short-term" recovery of CXCR2 expression was mainly the result of receptor recycling.
To further investigate the regulation of receptor re-expression on the
cell surface following GCP-2-induced internalization of CXCR2, we
determined the endocytic machinery involved in the recycling of these
receptors to the plasma membrane. The intracellular localization of
CXCR2 was determined by confocal analysis in relation to the
distribution of recycling endosomes, identified by antibodies directed
against rab11 (a protein that regulates receptor trafficking through
recycling endosomes and serves as a marker of early/recycling endosomes48-52). The localization of CXCR2 and of
rab11+ endosomes was determined prior to and after the
induction of receptor internalization, and subsequently after removal
of the ligand and recovery of CXCR2-expressing cells at 37°C for 90 minutes (the time point at which receptor recycling accounted for
receptor re-expression on the cell surface).
As shown in Figures 3A1 and 3A2, prior to
GCP-2-induced CXCR2 internalization, CXCR2 was localized primarily on
the cell membrane and showed notable colocalization with the expression
of rab11+ endosomes. Internalization of CXCR2 was induced
by the exposure of CXCR2-expressing cells to 1000 ng/mL GCP-2 for
different time points, 1, 5, 15, and 60 minutes. At 1 minute following
induction of internalization, the majority of CXCR2 and rab11 staining
was still localized on the cell membrane (Figures 3B1 and
3B2). However, starting at 5 minutes after induction
of CXCR2 internalization, the receptor could be distinguished in the
cytoplasmic regions of the cell (Figure 3C1). The
number of cells in which intracellular localization of the receptor
could be observed was increased as the exposure time to GCP-2 was
elongated. Analysis of CXCR2 internalization at the 5-, 15-, and
60-minute time points indicated that the internalization of the CXCR2
and its cytoplasmic localization were accompanied by the cytoplasmic
expression and colocalization of rab11+ endosomes (Figures
3C1-2, 3D1-3; the data for 15 minutes are not shown).
Following removal of the ligand, and recovery of the cells at 37°C
for 90 minutes, the expression of CXCR2 on the plasma membrane was
restored (Figure 3E1). However, at this stage, the distribution of
rab11+ endosomes was primarily cytoplasmic and only in part
membranous (Figure 3E2). The expression of CXCR2 on the cell membrane
and the dissociation of its localization from that of rab11 were
distinguished not only in the absence, but also in the presence of
cycloheximide (Figure 3F). To conclude, the colocalization of CXCR2
with rab11+ endosomes prior to and after the induction of
internalization by GCP-2 suggests that rab11+ endosomes are
involved in CXCR2 intracellular trafficking.
| |
Discussion |
To gain insight into the coordinated activity of
ELR+-CXC chemokines, and to determine the fine tuning of
their ability to interact with their receptors, our study focused on
the regulation of cell surface expression of CXCR1 and CXCR2 by GCP-2.
Due to possible intracellular interactions between CXCR1 and CXCR2
following their interaction with ELR+-CXC chemokines, we
utilized a well-characterized model system in which each of the
receptors was solely expressed. This system was shown to highly
resemble neutrophils, in terms of ligand binding affinity, activation,
homologous desensitization, and internalization of CXCR1 and
CXCR2.23,30,35,39
Our study provides evidence for the following novel findings:
(1) Although GCP-2 is considered as an effective ligand of both CXCR1
and CXCR2, the interaction of this chemokine with the 2 receptors is
different. This is manifested by the low ability of GCP-2 to induce
CXCR1 internalization, whereas a highly potent CXCR2 internalization is
induced by this chemokine. These results are supported by previous
observations, demonstrating that GCP-2 is a less potent activator of
CXCR1 than of CXCR2.24-26 In contrast to GCP-2, IL-8 is a
potent inducer of activation, as well as of internalization, of both
CXCR1 and CXCR2.22,23,35,53,54 The divergent abilities of
IL-8 and GCP-2 to induce chemotactic signals and receptor
internalization may represent the existence of fine control mechanisms
that regulate the migration of neutrophils to inflammatory sites.
(2) A distinct functional hierarchy exists between GCP-2 and 2 closely
related ELR+-CXC chemokines, IL-8 and NAP-2, in their
abilities to interact with CXCR1 and CXCR2, as manifested by their
relative abilities to induce receptor internalization. Our previous
results35 and the present study (Figure 4B) indicated that
in the context of CXCR2, IL-8 and GCP-2 competed with and displaced
NAP-2, suggesting that both IL-8 and GCP-2 are functionally dominant
over NAP-2. Our results also indicated that IL-8 dominated the
GCP-2-induced internalization of CXCR2. In addition, when the
regulation of CXCR1 was studied, IL-8 proved to govern GCP-2-induced
responses (Figure 4A). On the whole, a hierarchy between these
chemokines emerges, and its dominance order is IL-8 > GCP-2 > NAP-2. Therefore, our results suggest that the interrelationships
between these chemokines are highly regulated, and that on the
concomitant exposure of neutrophils to these 3 chemokines, the potency
of the response will be dictated primarily by IL-8. However, if
conditions at a certain inflammatory site do not allow for IL-8
expression, GCP-2 will be the dominant chemokine, resulting in a potent
interaction with IL-8 receptors, primarily with CXCR2 and evoking a
sufficiently effective acute inflammatory response. Although dominated
by the other 2 ELR+-CXC chemokines, NAP-2 may have a
pathophysiological role, being a cleavage product of Connective Tissue
Activating Peptide III that reaches high serum concentrations during
injury.18
(3) Our findings are the first to suggest the contribution of
signaling events to the regulation of CXCR2 internalization. The
regulation of CXCR2 internalization by G protein coupling was shown to
be clearly dissociated from the downstream signaling events that follow
receptor activation. Whereas the actual event of Gi
coupling to CXCR2 did not have a major role in the regulation of its
internalization, the subsequent signaling cascade that was induced by
chemokine-receptor interactions had a noticeable contribution to the
regulation of CXCR2 internalization. The ability of wortmannin to
promote CXCR2 internalization was correlated with its ability to
inhibit signaling events that give rise to chemotactic responses. These
results suggest that once induced, potent activation of certain
signaling components results in a partial decrement of internalization
processes. Such a mechanism may explain our findings, as well as
observations made by other researchers, demonstrating the tightly
regulated inter-relationships between the processes of internalization
and homologous desensitization of GPCR.40-43
Our observations on the enhancing effect of wortmannin on GCP-2-induced
CXCR2 internalization is supported by similar findings on the
internalization-stimulating activities of this compound with regard to
receptors that are not G protein-coupled.55,56 The optimal
internalization-promoting ability of wortmannin was induced by 1000 nmol/L of the compound. Such high concentrations of
wortmannin are considered to preferentially inhibit PI4
kinases.55,57,58 In that respect, it is interesting to note
that Ins-1,4,5-P3, and primarily PI(4,5)P2,
have been implicated to be most potent inhibitors of the organization
of AP-2 adaptin complexes.59 AP-2 adaptins are major
participants in the internalization of receptors via clathrin-coated
pits.60,61 Indeed, IL-8-induced internalization of CXCR2
was previously shown to be mediated by clathrin-coated
structures.62 Therefore, our results suggest that the
potent activation of CXCR2 by GCP-2 results in activation of PI3
kinases and PI4 kinases, whose products partially inhibit the
self-association activity of AP-2, and therefore do not allow for
maximal levels of clathrin-coated pits-mediated internalization of
CXCR2 to occur. However, on exposure to desensitizing concentrations of
GCP-2, the activation of these kinases is partially turned off, and the
ability of their products to inhibit AP-2 organization is reduced,
giving rise to maximal and potent internalization of CXCR2.
(4) This study is the first to suggest evidence for the nature of the
endocytic machinery involved in the recycling of the chemokine receptor
CXCR2. A significant membranous colocalization of CXCR2 and rab11 was
identified before induction of internalization, followed by complete
cytoplasmic colocalization of the 2 components after elicitation of
receptor internalization. Since the majority of internalized CXCR2 is
targeted for re-expression on the plasma membrane, our results suggest
that rab11+ endosomes participate in the first stages of
receptor trafficking to the cell surface. In agreement with this idea,
rab11 was partially co-localized with the membranous CXCR2 expression
following receptor recycling back to the plasma membrane. Therefore,
the potential involvement of rab11+ endosomes in the
recycling process of CXCR2 was demonstrated. However, the partial
dissociation between rab11 expression and the localization of CXCR2
following re-expression of the receptor on the cell membrane suggests
that additional endosomes, not expressing the rab11+
phenotype, may also mediate the final stages of CXCR2 delivery to the
cell surface.
| |
Acknowledgments |
The authors greatly appreciate the assistance of Dr
Leonide Mittelman in the performance of confocal analysis.
| |
Footnotes |
Submitted September 13, 1999; accepted November 5, 1999.
Reprints: Adit Ben-Baruch, Department of Cell Research and
Immunology, The George S. Wise Faculty of Life Sciences, Tel Aviv
University, Tel Aviv 69978, Israel; e-mail: aabb{at}post.tau.ac.il.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
References |
1.
Ben-Baruch A, Michiel DF, Oppenheim JJ.
Signals and receptors involved in recruitment of inflammatory cells.
J Biol Chem.
1995;270:11,703[Free Full Text].
2.
Ward SG, Westwick J.
Chemokines: understanding their role in T-lymphocyte biology.
Biochem J.
1998;333:457.
3.
Zlotnik A, Morales J, Hedrick JA.
Recent advances in chemokines and chemokine receptors.
Critical Rev Immunol.
1999;19:1[Medline]
[Order article via Infotrieve].
4.
Van Damme J, Wuyts A, Froyen G, et al.
Granulocyte chemotactic protein-2 and related CXC chemokines: from gene regulation to receptor usage.
J Leukoc Biol.
1997;62:563[Abstract].
5.
Froyen G, Proost P, Ronsse I, et al.
Cloning, bacterial expression and biological characterization of recombinant human granulocyte chemotactic protein-2 and differential expression of granulocyte chemotactic protein-2 and epithelial cell-derived neutrophil activating peptide-78 mRNAs.
Eur J Biochem.
1997;243:762[Medline]
[Order article via Infotrieve].
6.
Wuyts A, Haelens A, Proost P, et al.
Identification of mouse granulocyte chemotactic protein-2 from fibroblasts and epithelial cells: functional comparison with natural KC and macrophage inflammatory protein-2.
J Immunol.
1996;157:1736[Abstract].
7.
Smith JB, Rovai LE, Herschman HR.
Sequence similarities of a subgroup of CXC chemokines related to murine LIX: implications for the interpretation of evolutionary relationships among chemokines.
J Leukoc Biol.
1997;62:598[Abstract].
8.
Strieter RM, Polverini PJ, Arenberg DA, et al.
Role of C-X-C chemokines as regulators of angiogenesis in lung cancer.
J Leukoc Biol.
1995;57:752[Abstract].
9.
Rovai LE, Herschman HR, Smith JB.
Cloning and characterization of the human granulocyte chemotactic protein-2 gene.
J Immunol.
1997;158:5257[Abstract].
10.
Proost P, Wuyts A, Conings R, et al.
Human and bovine granulocyte chemotactic protein-2: complete amino acid sequence and functional characterization as chemokines.
Biochemistry.
1993;32:10,170[Medline]
[Order article via Infotrieve].
11.
Proost P, De Wolf-Peeters C, Conings R, Opdenakker G, Billiau A, Van Damme J.
Identification of a novel granulocyte chemotactic protein (GCP-2) from human tumor cells: in vitro and in vivo comparison with natural forms of GRO, IP-10 and IL-8.
J Immunol.
1993;150:1000 |
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Abstract
Introduction