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Department of Experimental Immunohematology, CLB and Laboratory for Experimental and Clinical Immunology, and
Department of Hematology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. Stromal cell-derived factor-1-induced
migration was dependent on VCAM-1 and ICAM-1, even in the absence of
VE-cadherin function. Cross-linking of ICAM-1 to mimic the
leukocyte-endothelium interaction induced actin stress fiber formation
but did not induce loss of endothelial integrity, whereas cross-linking
of VCAM-1 increased the HBMEC permeability and induced gaps in the
monolayer. In addition, VCAM-1-mediated gap formation in HBMEC was
accompanied by and dependent on the production of reactive oxygen
species. These data suggest that modulation of VE-cadherin function
directly affects the efficiency of transendothelial migration of
CD34+ cells and that activation of ICAM-1 and, in
particular, VCAM-1 plays an important role in this process through
reorganization of the endothelial actin cytoskeleton and by modulating
the integrity of the bone marrow endothelium through the production of
reactive oxygen species. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, the first powerful chemoattractant for hematopoietic stem
cells (CD34+ cells) has been described and
identified as stromal cell-derived factor-1
(SDF-1).3 SDF-1
is produced by several types of stromal cell, including those of the
bone marrow (2, 3, 4, 5), and signals through a G
protein-coupled receptor called Fusin, leukocyte-derived
seven-transmembrane domain receptor, or CXCR-4
(6, 7, 8, 9). SDF-1-driven homing of
CD34+ cells has been suggested to be a multistep
process similar to the extravasation process of leukocytes at
inflammatory sites (10) and is mediated by adhesion
molecules both on CD34+ cells (11)
and on bone marrow endothelial cells. In the final stage of homing,
CD34+ cells migrate across the bone marrow
endothelium, presumably via the intercellular junctions. Therefore,
endothelial cell-cell adhesion is most likely an important regulatory
factor in the homing of CD34+ cells.
Endothelial cell-cell adhesion is largely dependent on the homotypic
cell-cell adhesion molecule vascular endothelial (VE)-cadherin
(cadherin-5, CD144). VE-cadherin is a transmembrane protein that, like
other members of the cadherin family (12), associates via
its cytoplasmic tail with various cytosolic proteins, including -,
-, and 
-catenin (plakoglobin) and p120/p100. These proteins link
VE-cadherin to the cortical actin cytoskeleton (13, 14, 15).
The role of VE-cadherin in leukocyte transendothelial migration was
first described by Gotsch et al. (16), who showed an
accelerated extravasation of neutrophils in a mouse peritonitis model
in vivo upon i.v. injection of a mAb against mouse VE-cadherin.
Transfection experiments and gene inactivation studies have shown that
VE-cadherin expression reduces monolayer permeability and promotes cell
aggregation, motility, and growth, and that VE-cadherin is required for
the organization of vascular-like structures in embryoid bodies
(17, 18, 19). Moreover, VE-cadherin, together with
-catenin, seems to be involved in cell survival (20).
Regulation of VE-cadherin, and thereby of endothelial cell-cell
adhesion, may occur through tyrosine or serine phosphorylation
(21, 22, 23, 24), association with regulatory proteins
(20), and modulation of the endothelial actin cytoskeleton
(17, 25). In addition, several studies have proposed a
role for leukocyte adhesion-induced signaling, e.g., through activation
of myosin light chain kinase in the endothelial cells, which may
indirectly regulate VE-cadherin function in the process of
transendothelial migration (26, 27, 28, 29).
A role for specific adhesion molecules in endothelial cell signaling has been suggested by studies on VCAM-1 (CD106) and ICAM-1 (CD54). Lorenzon et al. (30) concluded that VCAM-1 and endothelial selectins, in addition to their role as adhesion receptors, mediate endothelial stimulation by adherent leukocytes. Moreover, activation of ICAM-1 on endothelial cells after binding of T cells has been reported to induce tyrosine phosphorylation of the actin-binding protein cortactin (31). In line with this observation, it was suggested that ICAM-1 mediates cell shape changes through coupling to the p21Rho GTPase and by inducing phosphorylation of cytoskeletal proteins and transcription factors (32).
In the present study, the role of VE-cadherin in the control of
permeability of bone marrow endothelium and of the transmigration of
primary CD34+ cells was investigated. For this
purpose, we used immortalized human bone marrow endothelial
cells (HBMECs) (33) and primary
CD34+ cells isolated from cord blood (CB) or from
peripheral blood (PB) of healthy untreated volunteers. In this study,
we show that transendothelial migration of primary
CD34+ cells is promoted by reduced
VE-cadherin-mediated cell-cell adhesion and is accompanied by a focal
loss of VE-cadherin at sites of transmigration. Cross-linking of
VCAM-1, but not ICAM-1, was found to induce loss of endothelial
cell-cell adhesion and increased permeability. VCAM-1-mediated gap
formation was accompanied by and dependent on the generation of
reactive oxygen species (ROS). These data indicate that activation of
Ig-like adhesion molecules, in particular VCAM-1, regulate VE-cadherin
function, which might facilitate SDF-1-driven transendothelial
migration of primary CD34+ cells across the bone
marrow endothelium.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mAbs against VE-cadherin (cl75) and -catenin were from BD
Transduction Laboratories (Amsterdam, The Netherlands). VE-cadherin Ab
7H1 was from BD PharMingen (San Diego, CA). Hybridoma supernatant
TEA1.31 was a kind gift from Dr. E. Dejana (Instituto di Ricerche
Farmacologiche Mario Negri, Milan, Italy). Polyclonal Ab against
VE-cadherin (C19) was from SanverTech (Heerhugowaard, The Netherlands).
Recombinant human IL-1 was from PeproTech (Rocky Hill, NJ);
calcein-acetoxymethyl, Texas-Red phalloidin, FITC-Dextran 3000,
ALEXA 488-labeled goat anti-mouse Ig (GM-Ig), and ALEXA
488-labeled goat anti-rabbit Ig secondary Abs were from Molecular
Probes (Leiden, The Netherlands). PE-labeled secondary Abs and
botulinum C3 toxin were from DAKO (Glostrup, Denmark). Pooled human
serum, human serum albumin, fibronectin (FN), and control Abs
IgG1 and IgG2a were obtained from the CLB (Amsterdam, The
Netherlands). FCS was from Life Technologies (Paisley, U.K.). Basic
fibroblast factor was from Boehringer Mannheim (Mannheim, Germany).
CXCR-4 expression was quantitated with PE-labeled anti-human Fusin
(12G5; BD PharMingen). mAbs against ICAM-1 (84H10) and VCAM-1 (1G11)
were purchased from Immunotech (Marseille, France). Additional mAbs
against ICAM-1 (15.2; CLB) and VCAM-1 (4B2; PeproTech) were also
used to exclude epitope dependence of the effects. Cross-linking
studies were performed with F(ab')2 of GM-IgG
from Jackson ImmunoResearch Laboratories (Baltimore, MD). Thrombin and
N-acetyl-cysteine (N-AC) were from
Sigma-Aldrich (St. Louis, MO).
Isolation of CD34+ hematopoietic progenitor cells
CB was collected after delivery, according to the guidelines of Eurocord, and PB from healthy volunteers was obtained from the local blood bank. These volunteers were normal donors that were not treated with G-CSF or with chemotherapy. Mononuclear leukocytes from PB (500 ml) and from CB were enriched by density gradient centrifugation over Ficoll-Paque (1.077 g/ml; Pharmacia Biotech, Uppsala, Sweden). Then the PB mononuclear fraction was purified from thrombocytes by elutriation and further processed, similar to CB CD34+ cell isolation, with the VarioMacs system (Miltenyi Biotec, Gladbach, Germany) as described (2). At least 95% of the cells from CB and >90% of the cells from PB expressed CD34 as determined by FACS analysis with a CD34 Ab (no. 581; Immunotech).
Cell cultures
The HBMEC line has been described previously (33).
The cells were cultured in FN-coated culture flasks (Nunc, Roskilde,
Denmark; Life Technologies) in Medium 199 (Life Technologies)
supplemented with 10% (v/v) pooled, heat-inactivated human serum, 10%
(v/v) heat-inactivated FCS, 1 ng/ml basic fibroblast factor, 5 U/ml
heparin, 300 µg/ml glutamine, 100 U/ml penicillin, and 100 µg/ml
streptomycin. After reaching confluency, the cells were passaged by
treatment with trypsin/EDTA (Life Technologies). In all experiments,
HBMEC monolayers were pretreated with IL-1 for 4 h. HL-60 and
KG-1a cell lines were obtained from the American Type Culture
Collection (Manassas, VA) and were maintained in IMDM (BioWhittaker,
Brussels, Belgium) containing L-glutamine, 100 U/ml
penicillin, 100 µg/ml streptomycin, and 10% FCS. All cell lines were
cultured at 37°C at 5% CO2.
Permeability assay
Permeability of HBMEC monolayers, cultured on 5-µm-pore, 6.5-mm Transwell filters (Costar, Cambridge, MA), was assayed using FITC-labeled 3000 Dextran as described (17). The permeability response to thrombin (1 U/ml) after 30 min was set to 100%. In some experiments, monolayers were pretreated with Abs (10 µg/ml) for 30 min. Unbound blocking Abs against VCAM-1 and ICAM-1 were washed away before the start of the permeability or transmigration assays. Cross-linking Abs and Abs to VE-cadherin were present during the permeability or transmigration assay. Botulinum C3 toxin was added to the HBMEC monolayers 6 h before the assay. In our previous publication (17), the incubation time for C3 was 18 h. However, we found that a 6-h incubation of HBMEC with C3 was already sufficient to inhibit actin polymerization. The 6-h incubation with C3 did not affect the VE-cadherin localization, whereas the 18-h incubation with C3 resulted in diffuse staining of VE-cadherin (17). Kinetics of VCAM-1-mediated increase in permeability was performed as described by Corada et al. (34). In brief, at indicated time points, 50 µl were taken from the lower compartment and fluorescence was measured. After the assay, filters were washed with ice-cold Ca2+- and Mg2+-containing PBS and then fixed with 2% paraformaldehyde and 1% Triton X-100-containing PBS and stained with Texas-Red phalloidin to inspect the HBMEC monolayer by confocal laser scanning microscopy.
Transendothelial migration assay
Migration assays were performed in Transwell plates of 6.5-mm
diameter with 5-µm pore filters. Endothelial cells were plated at
20,000–30,000 cells/Transwell on FN-coated filters. Nonadherent cells
were removed after 18 h. The adherent cells were cultured for 2–3
days to obtain confluent endothelial monolayers. Monolayers of
endothelial cells were pretreated for 4 h with IL-1. Before
adding CD34+ cells to the upper compartment, the
endothelial monolayers were washed three times with assay medium (IMDM
with 0.25% (w/v) BSA (fraction V; Sigma-Aldrich)). Freshly isolated
CD34+ cells (50,000–100,000) were added to the
upper compartment in 0.1 ml of assay medium, and 0.6 ml of assay medium
with or without the indicated concentrations of recombinant human
SDF-1 (Strathmann Biotech, Hannover, Germany) was added to the lower
compartment. A 0.1-ml sample containing cells in assay medium was
diluted in 0.5 ml of assay medium and was kept as input control for
quantitation of the number of migrated cells. The Transwell
plates were incubated at 37°C, 5% CO2, for
4 h. Preliminary experiments showed that after 4 h, a
substantial fraction of the CD34+ cells had
migrated. Cells that had migrated to the lower compartment were
collected in a FACS tube to which a fixed number of control cell line
cells (kG-1a) labeled with calcein-acetoxymethyl was added. FACS
analysis was used to determine the ratio between labeled and unlabeled
cells, with characteristic light scatter parameters, in the migrated
fraction as described before (2). By comparison of this
ratio to that of the input control, the number of migrated cells was
quantitated. Using this method, we were able to determine reliably a
minimum number of 200 migrated cells. In blocking experiments, HBMECs
were preincubated for 30 min at 37°C with mAbs (10 µg/ml), followed
by washing. As controls, IgG1 and IgG2a isotypes were used. Blocking
Abs against VCAM-1 and ICAM-1 were not present during the
transendothelial migration assay. However, blocking Abs against
VE-cadherin (cl75) were present during the transendothelial migration
assay. After the assay, the filters were fixed and stained with
Texas-Red phalloidin to inspect HBMEC monolayers by confocal
laser scanning microscopy.
Immunocytochemistry
HBMECs were cultured on FN-coated glass coverslips and were
fixed and immunostained as described (17) with mAb against
VE-cadherin (7H1, 10 µg/ml) or anti--catenin (10 µg/ml)
followed by staining with fluorescently labeled secondary Abs (10
µg/ml). Filamentous actin (F-actin) was visualized by Texas-Red
phalloidin (1 U/ml). In some experiments, cells were pretreated with
mAb against VE-cadherin (blocking cl75, 10 µg/ml; partially blocking
TEA1.31; hybridoma supernatant 1/2 dilution; or the nonblocking 7H1, 10
µg/ml) for 30 min. In the analysis after the permeability
experiments, a polyclonal Ab against VE-cadherin was used for
immunostaining. Images were recorded with a confocal microscope with
appropriate filter settings (LSM510; Zeiss, Oberkochen, Germany).
Cross-talk between the green and red channels was avoided by use of
sequential scanning.
Analysis of ROS production
To measure intracellular ROS production, HBMECs were cultured on
FN-coated six-well plates and loaded with di-hydro-rhodamine-1,2,3
(DHR; 30 µM; Molecular Probes) for 60 min in the presence of catalase
(60 µg/ml) and NaN3 (2 mM) at 37°C. Adhesion
molecules were subsequently cross-linked at 37°C as described in
Permeability assay. After 30 min, cells were
incubated for 1 min with trypsin at 37°C, collected on ice, and
washed with ice-cold Ca2+- and
Mg2+-containing PBS, and DHR fluorescence was
quantitated by FACS in the FL-2 channel (
EX,
488 nm; EM, 585 nm) for 10,000 counted cells
per incubation. For the kinetics of the production of ROS, the
endothelial cells were cultured on FN-coated glass coverslips and
loaded as described above. Then ROS production was recorded and
quantitated by time-lapse confocal microscopy of DHR-loaded HBMECs.
Intensity values are shown as the percentage of increase relative to
the values at the start of the experiment. In some experiments, cells
were pretreated for 18 h with 5 mM of the oxygen radical scavenger
N-AC to prevent ROS-mediated signaling. FACS analysis showed
that N-AC preincubation had no effect on the
IL-1-mediated up-regulation of adhesion molecules such as VCAM-1 and
ICAM-1.
Statistics
All results that were performed at least three times were expressed as the mean ± SD or SEM, as indicated in the legend. Differences were tested by using the Student t test. A value of p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the initial series of experiments, the role of VE-cadherin in
the control of monolayer integrity of HBMECs was examined. Ab-mediated
inhibition of VE-cadherin function using two independent blocking Abs
resulted in increased permeability of HBMEC monolayers, whereas a
nonblocking, isotype-matched VE-cadherin Ab and an irrelevant IgG1 did
not have any effect on the permeability of HBMECs (Fig. 1
a). Immunofluorescent
staining of IL-1-prestimulated HBMECs after treatment with the
nonblocking VE-cadherin Ab showed a jagged distribution of VE-cadherin
(Fig. 1bA) and normal F-actin stress fiber formation (Fig. 1bB). At the ends of the F-actin stress fibers,
colocalization of VE-cadherin with actin was observed (Fig. 1bC), as previously described for primary HUVECs (17, 34, 35). Pretreatment of HBMEC monolayers with a blocking Ab
against VE-cadherin (cl75) resulted in a redistribution of VE-cadherin
over the cell surface (Fig. 1bD) and a marked reorganization
of the actin cytoskeleton (Fig. 1bE). Although the partially
blocking TEA1.31 Ab to VE-cadherin did increase the HBMEC permeability
(Fig. 1a), it did not dramatically affect the localization
of VE-cadherin (Fig. 1bG) or the actin cytoskeleton of the
HBMECs (Fig. 1bH), in agreement with published results
obtained with primary HUVECs (35). These findings
underscore the essential role of VE-cadherin in the regulation of the
integrity of HBMEC monolayers and show that its function (i.e., control
of permeability) and localization can be modulated differentially by
different blocking Abs.
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Purified primary CD34+ cells from CB and PB
and the human leukemic cell line HL-60, which all express CXCR-4, were
tested for their ability to migrate across FN or HBMECs to a gradient
of SDF-1. HL-60 cells migrated across FN with a similar efficiency
as primary PB CD34+ cells to 30 ng/ml SDF-1,
whereas primary CB CD34+ cells showed a higher
migration efficiency (Fig. 2a). However, CXCR-4
expression of PB and CB CD34+ was comparable
(data not shown). Because of the limited supply of primary
CD34+ cells, CXCR-4-expressing HL-60 cells were
used as a model in dose response studies of SDF-1-induced migration
across IL-1-prestimulated HBMECs. The results show a bell-shaped
response with optimal migration at 70 ng/ml SDF-1 (Fig. 2b). When VE-cadherin was blocked with the cl75 Ab on
IL-1-prestimulated HBMECs, a shift of the optimal concentration for
migration from 70 to 30 ng/ml SDF-1 and a significant increase in
migration across HBMECs were observed (Fig. 2b). Moreover,
migration of primary CB CD34+ cells to 30 ng/ml
SDF-1 across HBMECs pretreated with either the partially blocking
TEA1.31 or the blocking cl75 Ab resulted in a significant increase in
transmigration efficiency (Fig. 2c). These findings show
that VE-cadherin function is an important regulator of efficient
migration of primary CD34+ cells across bone
marrow endothelium.
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a) where stress fibers seemed
to converge at the periphery of the transmigrating
CD34+ cell (Fig. 3, b and
c). The cell is fixed during its passage through the filter
and the endothelial junctions (Fig. 3d). Similarly, also
-catenin was redistributed over the surface of HBMECs at such sites
(data not shown). These observations suggest a coordinated interaction
between the actin cytoskeleton and VE-cadherin at sites of
transmigrating CD34+ cells.
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Blocking VE-cadherin function by the cl75 Ab was accompanied by a
reorganization of the actin cytoskeleton (Fig. 1
b). Together
with the results shown in Fig. 3, these findings indicate that the
actin cytoskeleton, in concert with VE-cadherin, regulates barrier
function in HBMECs. Regulation of cytoskeletal contractility in
endothelial cells can be mediated by changes in cAMP levels
(36) and by the small GTPase p21Rho (37, 38).
p21Rho is required for actin stress fiber formation in response to
extracellular stimuli in many cell types and has also been implicated
in the organization of cadherin-based cell-cell adhesion in epithelial
cells (39, 40). Moreover, inactivation of p21Rho by
pretreatment of primary HUVECs with the botulinum C3 toxin causes
cytoskeletal reorganization, mainly reflected in a loss of actin stress
fibers (17). To investigate the role of p21Rho in the
control of transendothelial migration of CD34+
cells and endothelial permeability, HBMECs were pretreated with the C3
toxin. As a result, a loss of actin stress fibers (Fig. 4a), but no loss of
VE-cadherin localization (Fig. 4b), was observed. When, in
addition to C3 pretreatment, the blocking cl75 Ab was used (Fig. 4d), VE-cadherin was diffusely distributed over the cell
surface (Fig. 4e) and the cells had lost cell-cell contact
as a consequence of reduced VE-cadherin function. This result also
indicates that cellular contractility is not an absolute requirement
for the Ab-mediated loss of VE-cadherin-mediated cell-cell adhesion.
Inactivation of p21Rho by C3 did not significantly affect the migration
of primary CD34+ cells across HBMECs (Fig. 4g), indicating a minor role for p21Rho in the control of
transmigration of primary CD34+ cells. Moreover,
inhibition of active p21Rho by C3 did not affect the permeability of
HBMECs either (Fig. 4h).
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It was previously shown by our group and others that the 1
integrins very late Ag (VLA)-4 and VLA-5 are required for efficient
transendothelial migration of CD34+ cells
(2, 11, 41, 42). Therefore, we investigated which adhesion
molecules that are known ligands for VLA-4 and VLA-5 mediate the
migration of CD34+ cells across HBMECs. ICAM-1 is
highly expressed on IL-1-prestimulated HBMECs, whereas VCAM-1 is
expressed at a lower level. Pretreatment of the HBMEC monolayers with
blocking Abs to ICAM-1 inhibited the transmigration of PB and CB
CD34+ cells across HBMECs. Blocking Abs against
VCAM-1 also inhibited the transmigration of PB and CB
CD34+ cells. The combination of Abs against
ICAM-1 and VCAM-1 inhibited the transmigration of PB and CB
CD34+ cells significantly (Fig. 5, a and b,
respectively). After pretreatment of the HBMECs with the blocking cl75
Ab to VE-cadherin, the transmigration of untreated PB
CD34+ (Fig. 5a) and CB
CD34+ cells (Fig. 5b) remained
dependent on ICAM-1 and VCAM-1. Other blocking mAbs to VCAM-1 and
ICAM-1 showed similar results.
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As was shown in Figs. 1 and 2, loss of VE-cadherin function
increased the permeability of HBMEC monolayers and promoted
transendothelial migration of CD34+ cells. To
establish a role for intracellular signaling in the control of
VE-cadherin-mediated cell-cell adhesion, induced by specific adhesion
molecules, permeability assays in combination with Ab-mediated
cross-linking were performed. Cross-linking of ICAM-1 resulted
in a significant decrease in the HBMEC permeability (Fig. 6a). In contrast,
cross-linking of VCAM-1 induced a significant increase in the
permeability of HBMEC monolayers (Fig. 6a). Cross-linking of
ICAM-1 induced stress fiber formation (Fig. 6bD), which was
not accompanied, however, by altered VE-cadherin distribution at
cell-cell junctions (Fig. 6bE) or discernible gaps in the
HBMEC monolayer (Fig. 6bF). In contrast, cross-linking of
VCAM-1 did induce loss of cell-cell contacts of endothelial cells
(Figs. 6bG and 7d) and concomitant loss of
VE-cadherin localization at sites of gap formation (Fig. 6, bH and bI). Kinetics of VCAM-1-induced
permeability showed maximal increase in permeability already after 90
min (Fig. 6a, inset).
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Cross-linking of VCAM-1 on activated HUVECs has been reported to
be accompanied by the production of low levels of ROS
(43). Moreover, ROS have been described to mediate
intercellular gap formation, cell shape change, and F-actin
cytoskeletal reorganization in endothelial cells (44). In
line with these data, we found that incubation with the oxygen radical
scavenger N-AC significantly inhibited the VCAM-1-mediated
increase in permeability (Fig. 6a). In addition,
cross-linking of VCAM-1, but not ICAM-1, on HBMECs induced ROS
production (Fig. 7a). Kinetic
analysis, performed by time-lapse confocal microscopy, revealed a rapid
increase in ROS production in HBMECs within 5–15 min after VCAM-1
cross-linking (Fig. 7b). Moreover, transmigration of primary
CB CD34+ cells across HBMECs was significantly
diminished when HBMECs were pretreated with N-AC, indicating
a role for oxygen radicals during transendothelial migration (Fig. 7c). In addition, pretreatment of HBMECs with
N-AC, which by itself did not affect the actin cytoskeleton
or VE-cadherin distribution, prevented VCAM-1-induced gap formation
(Fig. 7d).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Primary CD34+ cells induced a focal loss of
VE-cadherin and of -catenin at sites of transmigration. This focal
loss of VE-cadherin was reversible. The normal jagged pattern of
VE-cadherin distribution was restored after the transmigration assays
(data not shown). A similar phenomenon was also reported by Allport et
al. (35), who studied monocyte migration across HUVECs
under flow, albeit in the absence of a chemotactic gradient. One of the
proposed mechanisms for the loss of VE-cadherin function during
leukocyte passage is the trapdoor mechanism: the VE-cadherin-catenin
complex is mechanically pushed aside by the migrating leukocyte and
simply re-adheres after leukocyte passage (35). However,
despite the fact that the VE-cadherin distribution was lost at sites of
CD34+ cell transmigration, the migration across
HBMECs remained dependent on VCAM-1- and ICAM-1-mediated adhesion in
the absence of VE-cadherin function. It is important to note that the
transendothelial migration assay is performed under static conditions.
Although the VE-cadherin distribution during transmigration under
static conditions was similar to what Allport and colleagues
(35) found using shear stress, the effect of blocking Abs
to VCAM-1 and ICAM-1 on the transendothelial migration of
CD34+ cells under flow remains to be
investigated. Recently, Cinamon et al. (45) showed that
shear stress itself induced migration of lymphocytes across
endothelium. Therefore, shear stress might also have its effect on stem
cell homing and on the role of specific adhesion molecules in this
process.
Conflicting results on the role of VCAM-1 and ICAM-1 in the transmigration of CD34+ cells have been published (46, 47). Therefore, we tested the hypothesis that leukocyte-endothelium interactions, mediated by VCAM-1 and ICAM-1, induce endothelial signaling that might control endothelial cell-cell adhesion. Cross-linking experiments supported a role for VCAM-1 in the regulation of endothelial cell-cell junctions. In contrast, ICAM-1 failed to induce changes in endothelial cell-cell junctions or formation of gaps between the cells after cross-linking, despite the induction of actin stress fibers. Similar findings have been reported by Del Maschio et al. (28). Moreover, activation of the small GTPase p21Rho by ICAM-1 cross-linking in HUVECs has been suggested (32). Our studies also indicate that in HBMECs, ICAM-1 may activate Rho, as deduced from the induction of stress fibers. However, this response was not sufficient to induce loss of HBMEC integrity. In line with this notion, inhibition of p21Rho by the C3 toxin did not affect the transmigration of CD34+ cells or the integrity of HBMEC monolayers, suggesting only a minor role for the p21Rho GTPase in the control of CD34+ cell transmigration. Although several reports suggest a signaling role for ICAM-1 during leukocyte transmigration, e.g., through activation of p21Rho (36, 48, 49), these findings indicate a differential, possibly cell-type-specific role for a p21Rho signaling pathway in the control of endothelial monolayer integrity during leukocyte transmigration. Moreover, in the absence of p21Rho activity, the endothelial cell-cell contacts are still reduced upon Ab-mediated loss of VE-cadherin function. This finding shows that gap formation, induced by anti-VE-cadherin Ab or cross-linking of VCAM-1, might occur independently of cellular contractility.
The increased HBMEC permeability after VCAM-1 cross-linking and the accompanying formation of endothelial gaps suggest that this Ig-like cell adhesion molecule can act as a bona fide signaling receptor. Lorenzon et al. (30) already described a prominent role for VCAM-1 in the induction of cytoskeletal changes in endothelial cells, although these authors did not analyze changes in endothelial permeability or VE-cadherin localization. Our present work adds new data to this issue in that the production of ROS appears to be a crucial signaling event in VCAM-1-induced gap formation. The fact that VCAM-1-induced loss of cell-cell adhesion is dependent on the production of ROS represents a new mechanism by which cell surface molecules can modulate cadherin-mediated cell-cell adhesion. However, activation of ICAM-1 did not increase significant generation of ROS or gap formation or changes of VE-cadherin localization in HBMECs. Therefore, ICAM-1 seems to function mainly as an endothelial adhesion molecule for CD34+ cells rather than as a signaling molecule in HBMECs. In contrast, VCAM-1 displays, besides its adhesive role in the interaction between the CD34+ cells and the bone marrow endothelium, a function as a signaling receptor that modulates HBMEC cell-cell contact, presumably through the production of ROS. Interestingly, ICAM-1-mediated production of ROS in HUVECs has recently been implicated in cytoskeletal remodeling (50). Although we did not observe ROS production upon ICAM-1 activation in HBMECs, this might very well represent a general signaling event, involved in adhesion-mediated signaling in endothelial cells. Decreased migration of CD34+ cells across HBMECs, which were pretreated with the oxygen radical scavenger N-AC, supports the role for intracellular endothelial ROS production during transmigration.
Obviously, there remain many unresolved issues with respect to this signaling pathway. First, the nature of the oxidase-generating system in endothelial cells is obscure, although it has been suggested that endothelial cells express a similar NADPH oxidase, as is also found in neutrophils (51). However, additional oxidase-like enzymes have recently been described that may be expressed in endothelial cells as well (52). Second, the pathway through which the endothelial oxidase is activated by cell surface receptors is unknown. Studies on the neutrophil NADPH oxidase suggest an extremely complex mechanism of activation involving Ser/Thr kinases, small GTPases, and correct intracellular targeting and assembly of the NADPH oxidase complex (53, 54, 55). Finally, although an important role for ROS has been described in the regulation of endothelial barrier function (44), the components of the pathway by which ROS lead to altered cadherin function are unclear.
An intriguing possibility is that ROS production plays a more general role in receptor-mediated modulation of endothelial cell-cell adhesion. Our own recent results, which showed the importance of ROS production for endothelial cell migration and for thrombin-mediated loss of cell-cell adhesion (J. D. van Buul, S. van Wetering, and P. L. Hordijk, unpublished observations), underscore the relevance for this signaling event in endothelial cell function.
Our present results suggest that the transendothelial migration of primary CD34+ cells requires VCAM-1 and ICAM-1 for firm adhesion to the bone marrow endothelium and that the binding to VCAM-1 may lead to the focal loss of VE-cadherin function through the production of ROS. Detailed analysis of VCAM-1-induced signaling to VE-cadherin-mediated cell-cell adhesion thus will be of relevance, not only for our understanding of the role of the endothelium in transmigration of hematopoietic stem cells, but also for our insight in endothelial cell function, which is important in a wide range of (patho)physiological conditions.
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Acknowledgments |
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2 Address correspondence and reprint requests to Dr. Peter L. Hordijk, Department of Experimental Immunohematology, CLB, Academic Medical Center, University of Amsterdam, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands. E-mail address: Hordijk{at}clb.nl
3 Abbreviations used in this paper: SDF-1, stromal cell-derived factor-1; VE, vascular endothelial; HBMEC, human bone marrow endothelial cell; CB, cord blood; PB, peripheral blood; ROS, reactive oxygen species; GM-Ig, goat anti-mouse Ig; FN, fibronectin; N-AC, N-acetyl-cysteine; F-actin, filamentous actin; DHR, di-hydro-rhodamine-1,2,3; VLA, very late Ag.
Received for publication July 23, 2001. Accepted for publication November 16, 2001.
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, Control; 
, VCAM-1 cross-linking.
b, Effect of cross-linking of adhesion molecules on the
morphology of HBMECs. HBMECs were grown to confluency on FN-coated
glass coverslips, treated as indicated, fixed, and stained for
VE-cadherin and F-actin. The images show F-actin in red
(A, D, and G) and
VE-cadherin (VE-cad) in green, immunostained with a polyclonal Ab
(B, E, and H).
Colocalization appears in yellow (C, F,
and I). HBMEC monolayers were incubated for 30 min with
mAbs against ICAM-1 (X-ICAM-1), followed by 30 min of cross-linking
with G
, VCAM-1
cross-linking. Data represent the mean of two independent experiments.
c, Role for ROS in SDF-1