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J. Biol. Chem., Vol. 278, Issue 32, 30302-30310, August 8, 2003
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¶
From the
Sanquin Research at CLB, the Landsteiner
Laboratorium and the Department of Hematology,
Academic Medical Center, University of Amsterdam, Amsterdam 1066 CX, The
Netherlands
Received for publication, May 7, 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(CXCL12) is the major chemokine
involved in the homing of primary human CD34+ cells but is also a
chemoattractant for other leukocytes, such as monocytes and B and T
lymphocytes
(3–5). SDF-1 is produced by several stromal cell types, including those of the bone marrow (3), and signals through the G-protein-coupled receptor CXCR4 (6). It has been shown that SDF-1 stimulates integrin-mediated arrest of CD34+ cells and also of T lymphocytes on vascular endothelium under flow, and that SDF-1 is constitutively expressed on human bone marrow endothelium (2, 7). Moreover, SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature CD34+ cells (8). Thus, the interplay between SDF-1, presented on the bone marrow endothelium, and adhesion molecules on the CD34+ cells represents the main driving force for the homing process.
Although directional migration of leukocytes is accompanied by polarization
of the cell body and of the actin cytoskeleton, redistribution of chemokine
receptors has been the subject of contradictory reports. Studies with C5a
receptor-GFP1 fusion
proteins in neutrophils or cAMP receptor-GFP fusion proteins in
Dictyostelium cells show uniform distribution of these receptors over
the plasma membrane in cells that migrate to chemoattractants
(9,
10). Recent evidence indicates
that differential distribution of G
-subunits leads to
amplification of the chemokine receptor signaling
(11). Other chemokine
receptors, such as the formylmethionylleucylphenylalanine receptor on
neutrophils and CCR2 and CCR5 on T lymphocytes, are distributed to the leading
edge upon exposure to their corresponding chemokines
(12,
13). In B lymphocytes, SDF-1
induces polarization of CXCR4 to the leading edge of the cell
(14). In addition, it has been
reported that SDF-1 can bind to fibronectin and subsequently induces a
polarized distribution of CXCR4 on adherent T lymphocytes
(15). However, these studies
were all performed with artificial substrates, and to our knowledge it is
unclear whether leukocytes that adhere to activated endothelial cells
redistribute chemokine receptors, such as CXCR4, over their surface to drive
directional migration. In addition, there is no information on the
distribution and localization of CXCR4 in human hematopoietic progenitor
cells.
To study SDF-1-CXCR4 signaling and the distribution of CXCR4 during the interaction of human hematopoietic progenitor cells with human bone marrow endothelium in more detail, we generated a CXCR4-GFP fusion construct and retrovirally expressed the protein into the CXCR4-deficient CD34+ progenitor cell model KG1a. The CXCR4-GFP protein was functionally expressed at the cell surface and mediated SDF-1-dependent actin polymerization, ERK1/2 activation, adhesion to endothelium under flow, and directional migration. By using this cell model, we found that SDF-1, immobilized on activated human bone marrow endothelium to mimic the in vivo situation, induced formation of lamellipodia and membrane ruffles and a marked redistribution of surface-expressed CXCR4 to the leading edge of the cell. In addition, CXCR4 co-localized with lipid rafts at the leading edge, at the sites of contact with the endothelial apical surface. In conclusion, these findings suggest that SDF-1, when presented by the endothelium, induces CXCR4 polarization during directional and transendothelial migration of human hematopoietic progenitor cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-) was from PeproTech (Rocky Hill, NJ); calcein-acetoxymethyl,
Texas Red phalloidin, membrane-dye DiI, cholera toxin subunit B (CTB)-Alexa
594, Alexa 488/568-labeled goat anti-mouse Ig, and Alexa 488/568-labeled goat
anti-rabbit Ig secondary antibodies (Abs) were from Molecular Probes (Leiden,
The Netherlands). Phospho-ERK1/2 mAb and ERK2 polyclonal Ab were purchased
from Santa Cruz Biotechnology (Heerhugowaard, The Netherlands). Recombinant
human SDF-1 was purchased from Strathmann Biotech GmbH (Hannover, Germany).
Pooled human serum, human serum albumin, fibronectin (FN), and control
PE-labeled Abs IgG1 and IgG2a were obtained from the CLB (Amsterdam, The
Netherlands). Fetal calf serum was from Invitrogen. Basic fibroblast growth
factor was from Roche Applied Science. CXCR4 expression on the cell surface
was detected with PE-labeled anti-human CXCR4; for CLSM imaging, unlabeled
anti-human CXCR4 mAb (both 12G5) from Pharmingen was used when indicated.
Methyl--cyclodextrin (CD) was from Sigma. ICAM-3 mAb conjugated to PE
was obtained from Beckman Coulter (Mijdrecht, The Netherlands). Isolation of CD34+ Hematopoietic Progenitor Cells—Cord blood was collected after delivery, according to the guidelines of Eurocord (Leiden, The Netherlands). Cord blood CD34+ cells were isolated with the VarioMacs system (Miltenyi Biotec GmbH, Gladbach, Germany) as described (4). At least 95% of the cells from cord blood expressed CD34 as determined by fluorescence-activated cell sorter with CD34 mAb (number 581, Beckman Coulter).
Cell Cultures—The human bone marrow endothelial cell line-60
(HBMEC) has been described previously
(16). The cells were cultured
in FN-coated culture flasks (NUNC, Invitrogen) in Medium 199 (Invitrogen),
supplemented with 10% (v/v) pooled, heat-inactivated human serum, 10% (v/v)
heat-inactivated fetal calf serum, 1 ng/ml basic fibroblast growth factor, 5
units/ml heparin, 300 µg/ml glutamine, 100 units/ml penicillin, and 100
µg/ml streptomycin. After reaching confluency, the endothelial cells were
passaged by treatment with trypsin/EDTA solution (Invitrogen). In all
experiments, HBMEC monolayers were pretreated with TNF- for 4 h. HL60
and KG1a cell lines were obtained from ATCC (Manassas, VA) and were maintained
in Iscove's modified Dulbecco's medium (BioWhittaker, Brussels, Belgium)
containing L-glutamine, 100 units/ml penicillin, 100 µg/ml
streptomycin, and 10% fetal calf serum. All cells were cultured at 37
°Cat5%CO2. All experiments were performed in Iscove's modified
Dulbecco's medium with 0.25% bovine serum albumin (BSA), hereafter termed
assay medium.
Construction of the CXCR4-GFP—The full-length cDNA encoding CXCR4 was obtained from the National Institutes of Health AIDS Research and Reference Reagent Program, operated by McKesson Bioservices (Rockville, MD). The CXCR4 cDNA was subcloned as an EcoRI-ApaI fragment into the N2pEGFP vector (Clontech, Palo Alto, CA). The insert was then cloned into the EcoRI-NotI sites of the modified vector LZRS-IRES-Zeocin (17). The resulting construct, LZRS-CXCR4-GFP-IRES-Zeocin, was transfected into amphotropic Phoenix packaging cells (18) by means of the calcium-phosphate transfection system (Invitrogen) to produce retroviruses. KG1a cells were transduced with virus-containing supernatant in the presence of 10 µg/ml N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (Roche Applied Science) and were centrifuged twice at 2000 rpm for 30 min. After 6 h, the supernatant was replaced with fresh medium, and the cells were allowed to recover overnight. This procedure was repeated twice on 2 consecutive days. Transduced cells were sorted for GFP expression by FACStar (BD Biosciences).
Transmigration Assay—Transmigration assays were performed in
6.5-mm, 5-µm pore Transwell plates (Corning Costar, Cambridge, MA). HBMEC
were plated at 50,000 cells/Transwell on FN-coated filters. Non-adherent cells
were removed after 18 h. HBMEC were cultured to confluency and stimulated
overnight with TNF- (10 ng/ml). Freshly isolated CD34+ cells
or cell line cells (100,000) were added to the upper compartment, and the
assay was performed as described previously
(19). After the assays, the
filters were fixed and stained with Texas Red-phalloidin to confirm the
integrity of the HBMEC monolayer by CLSM.
Immunocytochemistry—Cells were fixed with 2%
paraformaldehyde and immunostained with Abs or DiI as indicated in figure
legends, followed by staining with fluorescently labeled secondary Abs (10
µg/ml) when necessary. Cells were permeabilized with 0.5% Triton X-100 when
indicated. F-actin was visualized with Texas Red-phalloidin (1 unit/ml).
Images were recorded with a Zeiss LSM510 confocal laser-scanning microscope
(CLSM) with appropriate filter settings. Cross-talk between the green and red
channel was avoided by the use of sequential scanning. Time-lapse microscopy
was performed at 37 °C in a temperature-controlled incubation chamber.
FN-coated glass coverslips or confluent endothelial monolayers cultured on
FN-coated glass coverslips and stimulated overnight with TNF- (10
ng/ml) were used. FN or endothelium was preincubated for 30 min with 100 ng/ml
SDF-1 at 37 °C, and medium was refreshed at the start of the experiments,
as described previously (2,
8). SDF-1 presence on
endothelium was checked by CLSM with anti-SDF-1 polyclonal Ab (PeproTech).
200,000 KG1a wild type (wt) or KG1a CXCR4-GFP cells were added to the
incubation chamber, and cell migration was recorded for at least 30 min.
Imaging interval between recordings was 
10 s.
Quantification of CXCR4 Polarization—Cells were incubated on activated endothelium, which was coated or not with SDF-1, for 30 min at 37 °C. Then cells were fixed with 2% paraformaldehyde and immunostained with ICAM-3-PE mAbs. ICAM-3 was used to determine the back end in polarized cells, and four different microscopic fields per glass coverslip were counted. By determine the back end with ICAM-3 expression and the leading edge with CXCR4-GFP expression, the amount of polarized CXCR4-GFP cells was quantitated.
CXCR4 Internalization—Following the indicated preincubations, the cells were fixed and not permeabilized, subsequently washed and kept in PBS containing 0.25% BSA and 1 mM calcium with CXCR4-PE Ab (1:4) for 30 min at 4 °C, and then washed with a 30-fold excess of ice-cold PBS with 0.25% BSA. The relative fluorescence intensity was measured by flow cytometry (FACScan, BD Biosciences).
Adhesion of Cells under Flow—Perfusions under steady flow
(0.4 dynes/cm2) were performed in a modified version of a
transparent parallel plate perfusion chamber as described previously by Ulfman
et al. (20). The
endothelial monolayer was cultured to confluency on a gelatin-coated glass
coverslip. Monolayers were pretreated overnight with TNF- (10 ng/ml).
Additional incubation with SDF-1 (100 ng/ml) was done at 37 °C for 30 min.
Migration medium with 2 x 106 KG1a wt or KG1a CXCR4-GFP
cells/ml was perfused. After 5 min, the chamber was flushed with migration
medium to wash out all non-adhered cells, and thereafter at least 20 areas of
the perfusion surface were analyzed with the image analysis software Optimas
6.1 (Media Cybernetics Systems, Silver Spring, MD). Adhesion of CD-treated
cells to TNF--treated endothelium was determined under static
conditions, after three washing steps with migration medium and by counting
the adhered cells per field of view, and 4 fields of view were analyzed per
experiment.
Actin Polymerization Assay—Cells were washed three times in assay medium and resuspended in a concentration of 1 x 106 cells/ml. Prior to the experiment, the cells and SDF-1 were incubated separately at 37 °C for 10 min. SDF-1 (100 ng/ml) was added to the cell suspension, and at indicated time points 100 µl of cell solution was transferred to 100 µl of fixation solution (Intraprep Permeabilization Reagent, Beckman Coulter). Cells were incubated in the fixation solution for at least 15 min. Thereafter, the cells were centrifuged and resuspended in 100 µlof permeabilization reagent (Intraprep Permeabilization Reagent). After 5 min, 1 unit/ml Texas Red-phalloidin was added to visualize the F-actin. After 20 min the cells were centrifuged and resuspended in PBS with 0.25% BSA. Mean fluorescence intensity was measured by FACScan. The distribution of F-actin was analyzed on cytospins of 50,000 cells. Images were analyzed by CLSM.
Statistics—Student's t test for paired samples (two-tailed) was used for statistical analysis. Student's t test for independent samples was used where indicated.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemokine-induced intracellular signaling via G-protein-coupled receptors, including CXCR4, has been studied extensively (9, 22). To confirm that the transduced CXCR4-GFP fusion protein was functional in mediating intracellular signaling, we analyzed the phosphorylation of ERK1/2 following SDF-1 stimulation (23). We observed rapid maximal phosphorylation of ERK1/2 in the KG1a CXCR4-GFP cells after 1 min of SDF-1 treatment (Fig. 2). Medium changes did not induce increased phosphorylation of ERK1/2 in either cell type, whereas fetal calf serum, used as a positive control, induced ERK1/2 phosphorylation in the KG1a wt cells as well as in the KG1a CXCR4-GFP-transduced cells (data not shown). p38 mitogen-activated protein kinase phosphorylation was observed in KG1a CXCR4-GFP cells after 15–30 min of SDF-1 stimulation, whereas the wt cells did not show p38 phosphorylation upon SDF-1 stimulation (data not shown). These findings indicate that the transduced fusion protein is capable of inducing intracellular signaling toward ERK1/2 and p38.
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Chemokine-induced directional migration is accompanied by localized actin polymerization, mainly at the leading edge of the cell, to induce lamellipodial protrusions and to drive directional migration (24, 25). The KG1a CXCR4-GFP cells showed a rapid and significant actin polymerization upon exposure to SDF-1, whereas the KG1a wt cells did not (Fig. 3A). The kinetics and extent of SDF-1-induced actin polymerization in the KG1a CXCR4-GFP cells were similar as in primary human CD34+ cells (24). CLSM analysis confirmed these results (Fig. 3B) and underscored the observation that only the CXCR4-GFP-positive cells responded to SDF-1 by polymerizing and polarizing their actin cytoskeleton. The above data show that transduction of CXCR4 is sufficient to restore SDF-1-responsiveness in these cells.
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Several groups have reported that SDF-1 can be efficiently presented by fibronectin to migrating T lymphocytes and induces redistribution of CXCR4 to the leading edge of the cell, suggesting that immobilized SDF-1 promotes directional cell migration and receptor polarization (5, 7, 15). Analysis of CXCR4-GFP redistribution on immobilized SDF-1 on fibronectin-coated surfaces showed polarization of CXCR4-GFP (Fig. 4A). Labeling of the cells with the membrane probe DiI showed that the polarization of CXCR4-GFP is not simply due to accumulation of plasma membrane, because large parts of the cell that stained strongly positive for DiI were negative for CXCR4-GFP (Fig. 4B). Thus, SDF-1, immobilized on fibronectin, induces redistribution of CXCR4 at the plasma membrane.
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The expression levels of adhesion molecules such as VLA-4, VLA-5,
2 integrins, and selectins, known to be involved in the
process of homing of primary CD34+ cells, were not affected by the
CXCR4-GFP transduction into the KG1a cells (data not shown) and were
comparable with those on primary CD34+ cells (data not shown and
see Refs. 26 and
27). Peled and colleagues
(7) reported that SDF-1 is
constitutively expressed on human bone marrow endothelium and induces firm
adhesion of primary human CD34+ cells to the endothelium under
shear stress. Moreover, on primary human CD34+ cells, SDF-1
activates the integrins LFA-1, VLA-4, and VLA-5
(8). To test whether the
SDF-1-induced adhesion of CD34+ cells to the endothelium is
specifically mediated by CXCR4, the KG1a CXCR4-GFP cells and the KG1a wt cells
were exposed to TNF--stimulated and SDF-1-incubated endothelium under
physiological shear stress. Adhesion of KG1a CXCR4-GFP cells to activated
endothelium was significantly enhanced in the presence of SDF-1
(Fig. 5). KG1a wt cells did not
show a significant increase in adhesion, indicating that the SDF-1-mediated
increase in adhesion to endothelium was mainly dependent on CXCR4.
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The final step in the homing of CD34+ cells is the migration to the bone marrow stroma. In Transwell-based in vitro migration assays, the KG1a wt cells showed no increase in transmigration across FN-coated filters to SDF-1, whereas KG1a CXCR4-GFP cells showed a significant increase. Primary human CD34+ cells migrated for almost 50% across FN-coated filters to SDF-1 (Fig. 6A). Migration experiments with CXCR4-deficient KG1a cells and CXCR4-expressing and SDF-1-responsive HL60 cells, which both were transduced with GFP-actin, showed that the transduction procedure itself did not alter the migratory capacities or SDF-1 responsiveness of these cells.
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Dose-response experiments showed optimal migration of the KG1a CXCR4-GFP
cells across fibronectin at 70 ng/ml SDF-1
(Fig. 6B). A decline
in migration was observed at high SDF-1 concentrations (1000 ng/ml, n
= 1; data not shown). Migration of the KG1a CXCR4-GFP cells across
TNF--stimulated HBMEC revealed an optimal concentration for SDF-1 of 30
ng/ml and a bell-shaped dose-response curve
(Fig. 6C). Although
the absolute migration of the cells across the endothelium was lower than
across FN, the SDF-1 concentration, required for maximal migration, was
reduced. This suggests that the endothelium presents the chemokine more
efficiently than the FN or that the adhesion to the endothelium promotes SDF-1
responsiveness, compared with the adhesion to FN. Previous experiments
performed with primary human CD34+ cells showed similar results in
migration across endothelium versus FN
(26). Thus, migration of
KG1a-CXCR4-GFP cells to SDF-1 across FN and HBMEC was comparable with that of
primary human CD34+ cells and was dependent on CXCR4.
Under physiological flow, the role of soluble chemokines to create a
gradient will be limited. It has been published that chemokines can bind to
proteoglycans and glycosaminoglycans on the endothelium
(28,
29). Moreover, Middleton and
colleagues (30) have reported
that intradermally injected chemokines are presented on the apical side of the
endothelium in vivo. Based on the results that showed immobilized
SDF-1 to induce firm adhesion to activated endothelium
(Fig. 5), we analyzed the
distribution of CXCR4-GFP on untreated or SDF-1-treated TNF--activated
endothelium in real time (Fig.
7). These experiments showed that in the absence of SDF-1, KG1a
CXCR-4-GFP cells adhered and migrated on TNF--stimulated endothelial
monolayers. Moreover, upon adhesion, the cells formed membrane ruffles and
lamellipodia. However, no prominent CXCR4-GFP localization was observed in
these ruffles, but CXCR4-GFP seemed to accumulate in the back of the cell
(Fig. 7A, see Supplemental Material for 4 Quick-Time movies).
However, not all CXCR4-GFP-expressing cells showed such CXCR4-GFP localization
in the back of the cell but a more uniform distribution over the plasma
membrane. These differences might be due to the different expression levels of
CXCR4-GFP per cell. Addition of soluble SDF-1 to the medium did not induce a
redistribution of CXCR4-GFP in already adherent cells (data not shown). In
contrast, immobilization of SDF-1 onto TNF--stimulated endothelium
induced increased adhesion and lamellipodia formation by the migrating KG1a
CXCR4-GFP cells. Moreover, under these conditions CXCR4-GFP was redistributed
to the leading edge of the cell, in particular to the newly formed
lamellipodia (Fig. 7B, see Supplemental Material). These results
clearly show that immobilized SDF-1 on activated endothelium induces a
redistribution of CXCR4 toward the leading edge of the migrating cell.
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To determine whether the CXCR4-GFP was localized intracellularly or on the
cell surface, the KG1a CXCR4-GFP cells were fixed but not permeabilized and
stained with a CXCR4 antibody, following their incubation on
TNF--stimulated endothelium, with or without immobilized SDF-1.
Confocal microscopy of non-permeabilized cells confirmed that a fraction of
the CXCR4-GFP was localized to the cell surface at the leading edge, whereas
the CXCR4-GFP fraction at the back of the cell was not stained by the antibody
(Fig. 8A). By using
the PE-conjugated CXCR4 antibody, also primary human CD34+ cells
showed a polarized CXCR4 localization after exposure to immobilized SDF-1
(Fig. 8A). Real time
live cell analysis with the PE-conjugated antibody to CXCR4 revealed that
SDF-1, immobilized on the endothelium, induced CXCR4 distribution to the front
of the migrating KG1a CXCR4-GFP cell (Fig.
8B). CXCR4-GFP that did not co-localize with the antibody
staining was present in the back of the cell, similar to fixed cells as
described above. Moreover, the change of cell shape and the directionality of
migration were paralleled by a corresponding CXCR4 redistribution over the
cell surface to a newly formed lamellipodium, whereas the KG1a wt cell shape
did not change (Fig.
8B). Although the antibody used (clone 12G5) is described
as a blocking antibody, our results indicate that responses induced by
immobilized SDF-1 were not inhibited by this antibody. Reports by others
(31,
32) already showed that this
antibody only partially blocks SDF-1 responses. However, on cells that are
treated in suspension, the 12G5 antibody is still capable of blocking SDF-1
responses.2 Together
these results showed that, upon exposure to immobilized SDF-1 on endothelium,
CXCR4-GFP is present on the cell surface at the front of the cell, whereas
CXCR4-GFP at the back of the cell is localized intracellularly. To confirm
that the antibody recognized the CXCR4-GFP fusion protein, SDF-1-stimulated
KG1a CXCR4-GFP cells were fixed, permeabilized, and stained for CXCR4 with the
CXCR4 antibody. Confocal analysis showed a 100% co-localization of staining by
the antibody with the CXCR4-GFP fusion protein
(Fig. 8C).
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To quantify SDF-1-induced CXCR4-GFP polarization at the leading edge of
KG1a CXCR4-GFP cells on activated endothelium, the cells were incubated on
SDF-1-coated and TNF--activated endothelium and stained for ICAM-3,
which localizes to the back of a polarized cell
(Fig. 9A)
(14). The other side of the
cell therefore represents the leading edge. After exposure to immobilized
SDF-1, detailed examination of ICAM-3 and of CXCR4-GFP distribution showed a
significant increase in cells (73%) that expressed CXCR4-GFP at the leading
edge, whereas in the absence of SDF-1 only 34% of the cells showed a
redistribution of CXCR4-GFP to the leading edge
(Fig. 9B). Thus, in
order to amplify signaling, induced by immobilized SDF-1 on
TNF--stimulated endothelium, migrating cells redistribute CXCR4 to the
leading edge.
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To organize chemokine-induced signaling, migrating and polarized cells associate glycosphingolipids with cholesterol to cluster membrane microdomains or so-called lipid rafts. Several reports have shown that depletion of cholesterol from the plasma membrane, which disturbs lipid raft integrity, results in an impaired responsiveness to extracellular signals (33, 34). Visualizing the lipid rafts with CTB showed that in resting KG1a CXCR4-GFP cells CXCR4 co-localized with these rafts (Fig. 10A). Stimulation with soluble SDF-1 resulted in a rapid clustering of lipid rafts and polarization of CXCR4-GFP. Besides the fact that CXCR4-GFP still co-localized with the clustered lipid rafts on the cell surface, we also observed a rapid CXCR4-GFP internalization after SDF-1 exposure. Depletion of cholesterol from the membrane by CD significantly prevented SDF-1-induced CXCR4 internalization, as was measured by flow cytometry (Fig. 10B). In addition, SDF-1-driven transmigration of the KG1a CXCR4-GFP cells across FN-coated filters was also significantly inhibited upon depletion of cholesterol by CD (Fig. 10C). Moreover, pretreatment of the KG1a CXCR4-GFP cells with CD significantly decreased firm adhesion to activated endothelium (Fig. 10D). These results indicate that the complete inhibition of SDF-1-induced migration of CD-treated KG1a CXCR4-GFP cells is partially due to less adhesion to the endothelium but also due to deficient CXCR4 signaling.
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SDF-1, immobilized on activated endothelium, induced lipid raft redistribution to the leading edge of migrating cells. Detailed three-dimensional analysis revealed that the lipid rafts co-localized with CXCR4-GFP at the leading edge of the cell, at sites of contact with the apical surface of the endothelium. The back of the cell did not show co-localization between lipid rafts and CXCR4-GFP (Fig. 10E). In order to determine whether lipid rafts are involved in SDF-1-induced CXCR4 polarization, the cells were pretreated with CD and subsequently incubated on activated endothelium, on which SDF-1 was immobilized. These results showed that lipid rafts support SDF-1-induced polarization of CXCR4 (Fig. 10F). In conclusion, proper lipid raft organization is required for efficient SDF-1-mediated responses.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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By using this newly transduced cell line, we were able to document the redistribution of CXCR4 in response to SDF-1. Several studies already analyzed the behavior and redistribution of chemokine receptors. From these studies, it was clear that not all chemokine receptors behave similarly in response to their cognate ligands. Studies with GFP-tagged fusion proteins, such as the C5a receptor and the cAMP receptor, have shown that these receptors did not polarize upon activation (10, 36). However, Meili et al. (37) showed that the pleckstrin homology domain of protein kinase B/Akt fused to GFP polarizes upon exposure to cAMP, indicating that polarized signaling from G-protein-coupled receptors is involved in sensing and responding to chemokine gradients. With fluorescent antibodies, it was shown that the chemokine receptors CCR5, CCR2, and CXCR4 polarize upon chemokine activation under static conditions (13–15). These conflicting results were obtained either with Dictyostelium as a model or with mammalian cells adhered to an artificial matrix. Moreover, chemokines were added in solution or via a micropipette to study the mechanism that controls cell polarization.
In vivo, human leukocytes crawl over the endothelium of the vessel wall in response to signals from the underlying tissue. Peled et al. (7) have shown that the chemokine SDF-1 is expressed on human bone marrow endothelium. In addition, Middleton et al. (30) reported that chemokines, such as interleukin-8 and regulated on activation normal T cell expressed and secreted (RANTES), could be presented at the apical surface of endothelial cells. Moreover, because SDF-1 is not only produced by bone marrow stroma cells, but also by other tissues, and because also other human leukocytes, such as monocytes and T and B lymphocytes express CXCR4, SDF-1 presentation on the endothelium might represent a general mechanism to promote CXCR4-mediated chemotaxis. Here we show that CXCR4 rapidly redistributes to the leading edge of the cell in response to immobilized SDF-1, both on FN (this paper and Ref. 15) and on a physiological relevant substrate such as activated human endothelium. Complementary ICAM-3 staining, to visualize the back of the cell, revealed that CXCR4 was mainly polarized to the leading edge. Moreover, we observed that the shape of the KG1a wt cells did not change on activated endothelium, in the presence or absence of immobilized SDF-1, whereas the KG1a CXCR4-GFP cells did spread and migrate over activated endothelium, even in the absence of SDF-1. This observation might indicate that not only the underlying tissue but also the human bone marrow endothelium itself is able to produce and subsequently present chemokines, in this case SDF-1, in order to mediate chemotaxis, although this amount of chemokine, present at the surface of activated endothelium, is not sufficient to induce clear polarization of CXCR4 to the leading edge of the migrating cell.
It has been suggested that cells cluster signaling receptors into so-called
lipid rafts to respond rapidly to environmental changes. Previous reports
(34,
38) suggest that lipid rafts
localize to the leading edge of a migrating cell, although this topic is the
subject of conflicting reports. Here we show that SDF-1, immobilized on
activated endothelium, induces clustering of lipid rafts. Three-dimensional
analysis suggests that CXCR4 co-localizes with the lipid rafts at the
basolateral side of the cell. Recently, Shamri et al.
(39) showed that
G-protein-coupled receptor-mediated activation of
41 integrin depends on the formation of
cholesterol-raft platforms. The specific localization of CXCR4 in the lipid
rafts at the basolateral side of the cell suggests that in lipid rafts,
integrins may cluster with G-protein-coupled receptors on leukocytes, which
would likely favor chemokine-induced integrin-mediated activation.
Finally, chemokine receptor distribution can be studied by means of GFP fusion proteins or specific antibodies. Here we show that CXCR4-GFP redistributes to the cell surface at the leading edge of the cell, and we underscore these findings by using specific antibodies, directed to the receptor. However, CXCR4-GFP was also detected at the rear of the cell, where it was not recognized by these antibodies on non-permeabilized cells, indicating that this fraction of the receptor pool was localized intracellularly. This observation shows that GFP fusion protein distribution does not necessarily represent cell surface localization.
In conclusion, we have generated and characterized a suitable model cell line for human hematopoietic progenitor cells to be used in studies on SDF-1-induced chemotaxis and stem cell homing. Moreover, we have shown that the CXCR4 receptor displays substrate- and chemokine-dependent polarization to the leading edge and that the cells redistribute the receptor to lipid rafts. These data provide new insights in the mechanisms that govern cell polarization and migration and underscore the crucial role of cellular context for the responses to chemokines.
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FOOTNOTES |
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The on-line version of this article (available at
http://www.jbc.org)
contains 4 movies for Fig. 7 available as Quick Time files. Movie 1, Fig
7AGFP.mov; movie 2, Fig 7Apha.mov; movie 3, Fig 7BGFP.mov; and movie 4, Fig
7Bpha.mov.
¶ To whom correspondence should be addressed: Dept. of Experimental Immunohematology, Sanquin Research at CLB, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands. Tel.: 31205123347; Fax: 31205123474; E-mail: p.hordijk{at}sanquin.nl.
1 The abbreviations used are: GFP, green fluorescent protein; TNF-,
tumor necrosis factor-; CTB, cholera toxin subunit B; Ab, antibody;
mAb, monoclonal Ab; FN, fibronectin; PE, phycoerythrin; CD,
methyl--cyclodextrin; HBMEC, human bone marrow endothelial cell line-60;
BSA, bovine serum albumin; CLSM, confocal laser-scanning microscope; wt, wild
type; PBS, phosphate-buffered saline; DiI,
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine.
2 J. D. van Buul and C. Voermans, unpublished observations.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) but not in KG1a wt cells
(
). Untreated KG1a CXCR4-GFP cells are indicated by 
. Data are
mean ± S.D. of at least three independent experiments. *, p
< 0.01. B, following the actin polymerization assay, cytospins
were prepared from the fixed KG1a CXCR4-GFP cells and analyzed by CLSM.
CXCR4-GFP is shown in a and c and F-actin in b and
d. Exposure of SDF-1 for 15 s induced a rapid polarization of F-actin
(c and d). a and b show KG1a CXCR4-GFP
cells without SDF-1. CLSM analysis revealed that SDF-1-induced actin
polymerization and polarization only occurred in CXCR4-GFP-positive cells,
indicated by the asterisks. Bar, 10 µm.
