Department of Anatomy and Cell Biology, The University of Western Ontario, London, Ontario, Canada
Correspondence: Martin Sandig, Department of Anatomy and Cell Biology, The University of Western Ontario, Medical Sciences Building, London, ON N6A 5C1, Canada. E-mail: msandig{at}julian.uwo.ca
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: adhesion molecules • integrins • endothelial cells • microfilaments • cell motility
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Leukocyte extravasation is a process consisting of consecutive
adhesion-mediated events [10
, 12
13
14
].
During the first step, binding of selectins to carbohydrate ligands
triggers tethering of the leukocytes to the activated endothelium
followed by intermittent rolling along the vessel wall. After
selectin-mediated rolling, arrest and firm adhesion on endothelial
cells (ECs) depend on the activation of ß1 and ß2 integrins such as
very late antigen 4 (VLA-4) and lymphocyte function-associated antigen
1 (LFA-1), which bind to endothelial VCAM-1 and ICAM-1, respectively
[13
, 15
]. The specific activation factors
necessary are not yet completely understood but involve "inside-out
signaling" initiated by inflammatory cytokines or "outside-in
signaling" through binding of the integrins to their receptors
[16
17
18
19
]. It has been shown that the activation of
endothelium with tumor necrosis factor 
, interleukin (IL)-1, and
IL-4 results in an increase of monocyte adhesion [20
]
caused by up-regulation in the expression of ICAM-1 and VCAM-1 in ECs
[21
, 22
].
Monocytes in their initial arrested state are spherical. Time-lapse microcinematography demonstrates that, after the firm binding to ECs, monocytes change their morphology (stretching) and then migrate in a lateral direction over the apical surface of ECs [20 ]. The regulatory mechanisms implicated in monocyte spreading along the endothelium have not been fully characterized, but it is generally known that a decrease in cortical tension and modulation of the cytoskeleton, such as assembly and disassembly of F-actin, are necessary to produce shape changes, protrusive forces, and polarized morphology required for cell motility. Chemotactic movements of cells involve the extension of pseudopodia and the formation of new adhesive interactions at the leading front of the cell with controlled retraction of the projections at the back of the cell, events that can push the cell forward [23 ]. The regulation of these activities has recently been shown to involve the Rho family of small GTPases, including Rho, Rac, and Cdc42. Each member of the Rho family has different effects on the formation and morphology of the actin cytoskeleton [24 , 25 ]. In macrophages interacting with extracellular matrix, RhoA induces the maintenance of cortical cell tension and a round morphology, whereas Rac induces the formation of lamellipodia and membrane ruffles, and Cdc42 regulates the formation of filopodia [26 ]. In addition, it has been shown that Rho and Rac are required for macrophage migration, and Cdc42, not being essential for cell locomotion, initiates cell polarization and chemotactic gradient detection [27 ]. Furthermore, Rho has been reported to activate ß1 and ß2 integrins [28 ].
Despite this information regarding cellular motility and locomotion on solid surfaces, little is known about the mechanisms regulating spreading and trafficking of monocytes along the apical surface of ECs. It has been demonstrated that the spreading of endothelially bound monocytes requires viable ECs and is in part an ICAM-1/VCAM-1-dependent event [29 , 30 ]. Lateral migration of monocytes on top of ECs appears to be supported by VCAM-1/VLA-4 interaction [31 ], whereas the migration of monocytes through endothelium involves, in part, LFA-1 binding to ICAM-1 as well as VLA-4 interaction with VCAM-1 [9 ]. In addition, recent studies suggest a cross talk between different integrins, leading to modulation of leukocyte diapedesis [32 ]. For example, LFA-1 down-regulates VLA-4 activity and enhances leukocyte motility on fibronectin [33 ].
Despite this abundance of information regarding the mechanisms that regulate leukocyte extravasation, the transendothelial migration process itself is poorly understood, in part because the majority of previous studies focused on quantifying leukocytes before and after diapedesis, without analyzing their behavior during the actual transmigration process. To overcome these limitations, we previously developed a culture system that allows us to examine, by laser scanning confocal microscopy (LSCM), changes in the morphology and distribution of surface molecules during diapedesis. Using this system we demonstrated that LFA-1 and F-actin are concentrated at sites of close adhesion between THP-1 cells (human acute monocytic leukemia cells) and ECs during all stages of diapedesis [34 , 35 ]. For the present report, we used time-lapse microcinematography and LSCM to investigate the effects of function-blocking antibodies to ICAM-1 and VCAM-1 on different stages of diapedesis, and we show that inhibiting ICAM-1/LFA-1 and VCAM-1/VLA-4 interactions differentially affected monocyte morphology, lateral migration, and transendothelial migration. In line with reports implicating Rho, Rac, and Cdc42 in leukocyte locomotion, we suggest that ICAM-1/LFA-1 and VCAM-1/VLA-4 interactions illicit signaling mechanisms mediated by specific small GTPases.
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Human peripheral blood monocytes (PBMs) were obtained from healthy adult donors and isolated by gradient centrifugation according to manufacturer’s protocols using OptiPrep leukocyte isolation medium (Accurate Chemicals, Westbury, NY). This isolation method yields monocytes with an average purity of 88–96%, and >95% viability [36 ]. PBMs (2.5x106 cells/mL) were resuspended in EGM-2-MV before they were cocultured with endothelial-cell monolayers.
Antibodies
Monoclonal mouse antihuman ICAM-1/CD54 IgG1 isotype, clone K562,
was obtained from Serotec Ltd., Kidlington, United Kingdom. Anti-ICAM-1
recognizes a functional epitope on ICAM-1, localized at the level of
the first Ig-like domain (NH terminal), specifically blocking the
interaction of ICAM-1 with LFA-1 on leukocytes. Anti-ICAM-1 inhibits
lymphocyte adhesion to ECs [37
]. Monoclonal mouse
anti-human VCAM-1, IgG1 isotype, clone P3C4 was obtained from Chemicon
International Inc., Temecula, CA. Previous studies confirmed that
monoclonal antibody P3C4 inhibits leukocyte adherence to bone marrow
stromal cells that express VCAM-1 [38
]. For control
experiments, human IgG pooled from normal serum was obtained from Sigma
Biosciences (catalog no. I4506). Fluorescein isothiocyanate
(FITC)-conjugated goat anti-mouse IgG was obtained from GIBCO/BRL, and
AlexaTM 488 goat anti-mouse IgG (H+L) from Molecular Probes, Eugene,
OR.
Transendothelial migration assay
Diapedesis was assayed as previously described
[35
]. Briefly, to provide a substrate for monocyte
transendothelial migration, sterile round glass coverslips (12 mm in
diameter) were coated with Matrigel (Becton Dickinson, Franklin Lakes,
NJ) at a dilution of 1:8. The Matrigel was air-dried at room
temperature for 1 h, followed by rehydration in Hank’s balanced
salt solution (GIBCO/BRL). HCAECs were removed from culture dishes by
trypsinization with a solution containing 0.025% trypsin and 0.1 mM
EDTA in Hank’s balanced salt solution lacking Ca2+ and
Mg2+ (GIBCO/BRL), resuspended in EGM-2-MV, and seeded onto
Matrigel at a concentration of about 2.5–2.8 x 105
cells/mL in a 200-µL drop of medium. This number of cells was
sufficient and necessary to obtain a confluent monolayer without
additional cell proliferation. Cells were allowed to attach and spread
in the drop on the Matrigel by incubating for 2 h at 37°C in a
humidified atmosphere containing 5% CO2. Coverslips were
then flooded with EGM-2-MV and incubated for at least 24 h before
the experiments. Confluent HCAEC monolayers on Matrigel were incubated
for 12–18 h in EGM-2-MV containing 10 ng/mL of recombinant human
IL-1ß (Endogen, Cambridge, MA), which has been shown to
up-regulate the expression of ICAM-1 and VCAM-1 [39
],
and was washed with EGM-2-MV containing IL-1ß before addition of
leukocytes. To analyze diapedesis, THP-1 cells or PBMs were resuspended
in EGM-2-MV and added to IL-1ß-stimulated HCAECs at a ratio of 
1
monocyte to 6 HCAECs [35
]. The cocultures were incubated
at 37°C for 30 min before fixation and immunostaining.
Time-lapse cinematography
To record cellular behavior and shape changes with an inverted
microscope, delta T dishes (Bioptechs, Inc., Butler, PA), each
containing a heatable glass coverslip as the bottom surface, were
coated with Matrigel. HCAEC monolayers were established and activated
with IL-1ß as previously described. After stimulation with IL-1ß,
the medium in cultures was replaced with sterile filtered 15 µM
Hanks’ HEPES-buffered medium containing IL-1ß. To analyze their
lateral migration on HCAECs, THP-1 cells were resuspended in
sterile-filtered 15 µM Hanks’-HEPES-buffered medium and added to
IL-1ß-stimulated HCAECs at a ratio of 1 THP-1 cell to 3 HCAECs.
The cocultures were then placed on a heated stage (37°C; Bioptechs, Inc.) mounted on a Leica DM-IRB microscope (Leica, Deerfield, IL). Floating mineral oil on the top of the culture medium prevented evaporation of the medium. Digital-phase contrast images of cocultures magnified 400-fold were then taken every 14 s for 30 min using a computer-controlled digital camera (Sony, Tokyo, Japan). Illumination of cultures by the microscope and timing of the digital camera were controlled using Northern Eclipse and Cell Time software (Empix Image, Inc., Mississauga, Canada). Images were arranged sequentially using Adobe Premiere 5.0 software (Adobe Systems, Inc., Mountain View, CA) to create a cinematization of THP-1 monocytes interacting with HCAEC monolayers.
Quantification of lateral migration
To analyze lateral migration of THP-1 cells along the apical
surface of HCAECs, monolayer plots of the positions of THP-1 cells on
top of the underlying endothelium within the last frame of the
cinematization were made. Individual cells were then visualized
throughout the entire length of the cinematization, and their lateral
movement with respect to the underlying endothelium was recorded.
Lateral migration was assessed as occurring only if the nucleus of a
spread THP-1 cell or the center of a round THP-1 cell moved relative to
the underlying endothelium. Endothelial monolayer shifting, which could
result in the movement of THP-1 cells within the frame of reference,
was not taken as evidence of lateral migration. Results are expressed
as mean percentages of control values ± SE. Two
groups were compared by Student’s t test, and differences
at P < 0.05 were considered to be significant. To
record the distance that individual THP-1 cells move in relation to the
underlying endothelium, a stage micrometer was photographed using the
same magnification as that during the recording of cocultures. This
image was transferred to Adobe Premiere 5.0, and a ruler was
constructed from the image on the computer screen. The ruler was then
used on the computer screen images of the cinematizations to determine
the distance migrated along the endothelium. The distances of only
those THP-1 cells that remained in every frame throughout the
cinematization were recorded. Results are expressed as mean percentages
of control values ± SE of two separate experiments
done in triplicate. Data from a minimum of 40 cells per treatment were
collected. Two groups were compared by a nonparametric signed rank test
according to Wilcoxon [40], and differences at P <
0.05 were considered to be significant.
Use of function-blocking antibodies
To examine the effect of function-blocking antibodies on
monocyte motility and diapedesis, confluent monolayers of
IL-1ß-stimulated HCAECs on Matrigel were preincubated for 30 min with
EGM-2-MV containing 10 or 20 µg/mL of monoclonal mouse anti-human
ICAM-1 or 5, 10, or 100 µg/mL of monoclonal mouse anti-human VCAM-1,
before addition of THP-1 cells or PBMs. EC monolayers were then washed
with EGM-2-MV containing 10 ng/mL of IL-1ß except in experiments
involving video microscopy, in which recording took place in the
presence of function-blocking antibodies. The diapedesis assays were
performed as described above. In control experiments, HCAECs were
incubated with an equivalent volume of medium lacking antibodies or
containing 10 µg/mL of an unrelated isotype-matched control IgG.
Immunofluorescence labeling
Confluent HCAEC monolayers were fixed at room temperature for 10
min in 2% paraformaldehyde in phosphate-buffered saline (PBS).
Cultures were washed three times for 5 min in PBS and then
labeled for F-actin, DNA, and either ICAM-1 or VCAM-1 as follows:
cultures were incubated for 1 h at room temperature with
monoclonal mouse anti-human ICAM-1/CD54 clone K562 or with monoclonal
mouse anti-human VCAM-1 clone P3C4 at a dilution of 1:50 in PBS
containing 1% bovine serum albumin (BSA). Either of two different
fluorescently labeled secondary antibodies was used: FITC-conjugated
goat anti-mouse IgG (GIBCO/BRL) or AlexaTM 488 goat anti mouse IgG
(H+L) at a dilution of 1:300 in PBS containing 1% BSA. To visualize
F-actin-fixed HCAECs were permeabilized for 5 min with a buffer
containing 15 mM Tris, 120 mM NaCl, 25 mM KCl, 2 mM EDTA, 2 mM EGTA,
and 0.5% Triton X-100 at pH 7.4 and were incubated for an hour at room
temperature with either Texas Red-X phalloidin or with fluorescein
phalloidin, at a dilution of 1:10 in PBS containing 1% BSA. Control
monolayers were treated by omitting the primary antibodies from the
procedures. Between incubation periods, cultures were rinsed three
times for 5 min in PBS. Nuclei were labeled for 5 min with
Bisbenzimide (Hoechst 33342; Sigma) at a concentration of 10 nM in PBS
before mounting. To prevent the contact of cells with the microscope
slide, coverslips were placed on plastic spacers and mounted in
Vectashield (Vector Laboratories, Burlington, Canada). Coverslips were
then sealed with nail enamel.
Microscopic analysis
Samples were analyzed by fluorescence microscopy, using a Zeiss
Axiophot microscope equipped with epifluorescence and appropriate
filters, or by LSCM. Serial optical sections (0.8–1.0 µm thick) of
the samples were obtained with a Zeiss LSM 410 laser scan confocal
microscope (Carl Zeiss Canada Ltd., Toronto, Canada). The following
excitation wavelengths were used: 488 nm for FITC, 568 nm for Texas
Red, and 351–364 nm for Hoechst dye. The dichroic beam splitters
(DBSP) used were DBSP 488/568 for excitation by 488 and 568 nm, and FT
395 for excitation by UV.
Quantification of diapedesis
To score monocytes at different stages of diapedesis, their
positions with regard to ECs were evaluated on samples double labeled
for F-actin and DNA [35
]. Monocytes were divided into
the following categories: a) round on top (spherical monocytes on top
of ECs with stress fibers located underneath the monocytes); b) spread
on top (monocytes located on the apical surface of ECs but showing
cytoplasmic protrusions); c) transmigrating (monocytes located
partially above and partially below EC stress fibers; d) underneath
(monocytes located underneath ECs [stress fibers were situated above
the monocyte]). Each experiment was done in triplicate and repeated at
least twice. Unless otherwise specified, 300 cells were scored for each
coverslip. Results are expressed as mean percentages of control
values ± SE. Groups were compared by performing a
one-way analysis of variance followed by Scheffe’s multiple-contrast
test, and differences at P < 0.05 were considered to
be significant.
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 View larger version (147K): [in a new window] |
Figure 1. Effects of function blocking antibodies to ICAM-1 and VCAM-1 on the
morphology of THP-1 cells adhered to HCAEC monolayers. THP-1 cells were
cultured for 30 min with stimulated HCAECs that were untreated (a) or
pretreated with anti-ICAM-1 (b), anti-VCAM-1 (c), or both anti-ICAM-1
and anti-VCAM-1 simultaneously (d). Phase contrast micrographs of
cocultures revealed that THP-1 cells formed prominent long pseudopodia
and lamellipodia (arrows in panel a). ICAM-1 or VCAM-1 blockade (b, c)
resulted in THP-1 cells remaining more compact (arrowheads) but with
slender filopodia extensions (arrows in b) in ICAM-1 blockade. The
extension of pseudopodia was lacking in cocultures with HCAECs that
were pretreated with both antibodies (d). Scale bar, 10µm.
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Figure 2. Effects of ICAM-1 (c, g) or VCAM-1 (d, h) blockade on F-actin
distribution in THP-1 cells (a–d) and PBMs (e–h) adhered to
HCAEC monolayers. Monocytes were cultured for 30 min with stimulated
HCAECs that were untreated (a, e) or pretreated with an unrelated
isotype-matched control antibody (b, f), anti-ICAM-1 (c, g), or anti
VCAM-1 (d, h). Cultures were fixed, stained with phalloidin, and
examined by confocal microscopy. Monocytes spreading on control
cultures (a, e, b, f) formed F-actin-rich lamellipodia (arrowheads in
panels a and e) and pseudopodia (open arrows in panels b and f). In
contrast, monocytes extended prominent F-actin rich filopodia (solid
arrows in panels c and g) on the surface of anti-ICAM-1-pretreated
HCAECs. Anti-VCAM-1 pretreatment of HCAECs inhibited the formation of
pseudopodia and filopodia (d, h). Scale bar, 10µm.
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 View larger version (27K): [in a new window] |
Figure 3. Quantification of the morphologies of THP-1 cells after the blockade of
ICAM-1 or VCAM-1 on HCAECs. THP-1 cells were cocultured for 30 min with
untreated (black bars), anti-ICAM-1-pretreated (hatched bars), or
anti-VCAM-1 pretreated (white bars) HCAEC monolayers and scored
according to their morphology. ICAM-1 and VCAM-1 blockade resulted in a
fourfold reduction in the percentage of THP-1 cells extending normal
pseudopodia. A twofold increase in the percentages of cells with
compact morphology was observed after VCAM-1 blockade. In addition,
ICAM-1 blockade resulted in a 10-fold increase in the percentage of
cells that formed filopodia. Results are expressed as the mean
percentage ± SD of three separate experiments done in
triplicate. *Significantly different compared with controls
(P<0.05).
|
15-µm decrease or 40% reduction in the distance traveled was
recorded for all treatments (Fig. 4b)
. This suggests that ICAM-1 and
VCAM-1 might affect the same pathway that is used to maintain the
migration of monocytes along HCAECs.
 View larger version (17K): [in a new window] |
Figure 4. Effects of ICAM-1 and VCAM-1 blockade on THP-1 cell migration along
HCAECs. THP-1 cells were cocultured with HCAEC monolayers pretreated
with anti-ICAM-1, anti-VCAM-1, or both anti-ICAM-1 and anti-VCAM-1
together. THP-1 cells were recorded for 30 min by time-lapse video
microscopy, and the number of translocating cells (a) and the distances
traveled (b) were determined. VCAM-1 blockade resulted in a 30%
decrease in the percentage of THP-1 cells migrating along HCAECs
compared with control cells, whereas simultaneous blockade of ICAM-1
and VCAM-1 inhibited lateral migration by 55%. No significant
effect was seen when ICAM-1 was blocked. The blockade of ICAM-1,
VCAM-1, or both molecules together reduced the distance that cells
traveled by approximately 40% (b). Results are expressed as mean
percentage ± SE of two separate experiments performed
in triplicate. *, significantly different compared with controls
(P<0.05).
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 View larger version (66K): [in a new window] |
Figure 5. Morphological changes during THP-1 cell diapedesis. Optical sections
obtained by LSCM illustrate the four stages of monocyte (M) diapedesis
observable in HCAEC cocultures. THP-1 cells were cocultured for 30 min
with IL-1ß-stimulated HCAECs, fixed, and stained for F-actin. In
series A, cells were, in addition, labeled for LFA-1 to identify
monocytes, whereas in series B, cultures were labeled for ICAM-1 to
demonstrate the EC surface. The levels at which each optical section
was taken are indicated in the illustrations at left. The location of
the monocyte with respect to endothelium was determined by judging its
location relative to endothelial nuclei and/or microfilament bundles
(solid arrows in panels A, III; B, III; C, III; and D, I). Note that
monocytes were seen to either spread on top (B, II) or underneath (C,
III; D, III) the endothelium by extending pseudopodia (open arrows).
Transmigrating cells contained an F-actin-lined circular transmigration
passage at the level of the endothelium (arrowhead in panel C, II).
Nuclei are labeled blue with Hoechst dye. Scale bars,
10µm.
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 View larger version (19K): [in a new window] |
Figure 6. Effects of function-blocking antibodies to ICAM-1 or VCAM-1 on monocyte
diapedesis through HCAEC monolayers. THP-1 cells (a, b) or PBMs (c)
were cocultured with HCAEC monolayers pretreated with anti-ICAM-1 (a),
anti VCAM-1 (a), both antibodies together (a, c), or an unrelated
isotype-matched control IgG (b). The numbers of transmigrating or
transmigrated (i.e., underneath) monocytes with a morphology as
illustrated in Figure 5C
and 5D
, respectively, were scored at different
time points and are expressed as percentages of control values.
Blockade of either ICAM-1 or VCAM-1 alone had no significant effect on
the percentage of monocyte diapedesis. In contrast, the simultaneous
blockade of ICAM-1 and VCAM-1 significantly reduced the percentage of
transmigrating monocytes by 45%. In panels a and b, results are
expressed as mean percentages ± SE of three separate
experiments performed in triplicate. Panel c represents data from one
experiment performed in triplicate. *, Significantly different compared
with controls (P<0.05).
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Monocyte shape changes
Previous studies have referred to monocytes that are spread on ECs
as "stretched monocytes" [29
, 30
]. Our
study demonstrates that most of the THP-1 cells and PBMs that are
spread on the apical surface of IL-1ß-stimulated HCAECs extended
lamellipodia or long pseudopodia. It is not surprising that
concentrations of F-actin are observed at the leading front of
pseudopodia or lamellipodia, suggesting that these monocytes are in a
motile state at the time of fixation. Using function-blocking
antibodies to ICAM-1 and VCAM-1, we present evidence that the formation
of pseudopodia and lamellipodia is differentially regulated by
endothelial ICAM-1 and VCAM-1. The antibodies used have previously been
shown to specifically block ICAM-1/LFA-1 interactions and
VCAM-1-mediated binding of leukocytes to bone marrow stromal cells
[37
]. We obtained similar results with antibody
concentrations up to 100µg/mL, indicating that the 10-µg/mL
concentration used throughout the study was sufficient to saturate the
appropriate interaction sites. The fact that these antibodies have
different effects on the shape and motility of monocytes indicates that
these effects are not caused by nonspecific interactions. In addition,
treatment with isotype-matched control IgG did not affect monocyte
morphology, indicating that our results are unlikely to be caused by Fc
receptor blockade or activation.
The spreading of monocytes on human umbilical vein ECs has previously been reported to be mediated by ICAM-1 but to be independent of VCAM-1/VLA-4 interactions [29 , 30 ]. Our finding that VCAM-1 blockade prevented the formation of pseudopodia and lamellipodia by monocytes contradicts this report and suggests that both ICAM-1/LFA-1 and VCAM-1/VLA-4 interactions play a role in a monocyte’s ability to form normal pseudopodia and lamellipodia on HCAEC monolayers.
Recent studies suggest cross talk between different integrins, leading to a modulation of leukocyte diapedesis [32 ]. Our results show that the blockade of ICAM-1, VCAM-1 or both adhesion molecules together increases the number of monocytes that maintain a round morphology. Therefore, it is possible that downstream members of the Rho family of GTP-binding proteins [24 , 41 ] are regulated by both ICAM-1/LFA-1 and VCAM-1/VLA-4 interactions. Rho inhibition has been shown to reduce the cortical tension in macrophages and cause cell spreading [26 ]. We suggest that both ICAM-1/LFA-1 and VCAM-1/VLA-4 interactions are required to deactivate Rho, thereby decreasing cortical tension and allowing monocytes to spread on top of the endothelium.
The observation that ICAM-1 but not VCAM-1 blockade resulted in the extension of filopodia by monocytes suggests a specific response linking ICAM-1/LFA-1 interactions with downstream regulation of Cdc42. Injection of active Cdc42 has been shown to induce the formation of filopodia in macrophages [26 ]. It is conceivable that VCAM-1 might be responsible for the induction of filopodia formation through the activation of Cdc42 but that this formation is tightly regulated by ICAM-1. ICAM-1 could possibly inhibit Cdc42 so that filopodia are not normally formed when monocytes interact with the apical surface of the endothelium. In an alternative scenario, VCAM-1 could promote the activation of Cdc42 and ICAM-1 the activation of Rac, resulting in the formation of pseudopodia and lamellipodia. The role of Rac in lamellipodia formation has been shown in macrophages after microinjection with active Rac [26 ]. Because Rho may be regulated by ICAM-1, an increased cortical tension might also inhibit the formation of normal pseudopodia when VCAM-1 and ICAM-1 are blocked. Thus the ratios of active and inactive forms of Rho, Rac, and Cdc42 determine the proper cytoskeletal rearrangements needed to produce functional lamellipodia and pseudopodia. The absence of either ICAM-1/LFA-1 or VCAM-1/VLA-4 interactions disrupts these ratios and affects monocyte morphology.
Lateral migration of monocytes
The mechanisms by which a leukocyte moves along a cellular surface
are poorly understood, but they appear to depend on signaling events
that result in changes in cytoskeletal organization, which provide both
the protrusive and contractile forces necessary for cell migration
[24
, 26
, 27
,
42
43
44
]. Our results show that blockade of VCAM-1 but not
ICAM-1 reduced the number of monocytes migrating along the endothelium.
It is interesting that ICAM-1 blockade had an inhibitory effect on the
number of monocytes migrating but only when VCAM-1 was simultaneously
blocked. In addition, blockade of ICAM-1, VCAM-1, and both adhesion
molecules together reduced, to the same degree, the distance that
monocytes traveled. Therefore, it appears that independent mechanisms
regulate which monocytes respond by migrating and to what extent
migration proceeds.
Monocyte diapedesis
Our results showed that the blockade of either ICAM-1 or VCAM-1
has no significant effect on the number of THP-1 cells that
transmigrate through the endothelial monolayer. However, the
simultaneous blockade of both ICAM-1 and VCAM-1 resulted in a
significant reduction in THP-1 cell and PBM diapedesis. Perhaps
ICAM-1/LFA-1 interactions alone are able to initiate the cytoskeletal
rearrangements sufficient to allow monocytes to migrate through the
endothelium when VCAM-1 is blocked. During ICAM-1 blockade, the
opposite may be true. This compensatory ability to support diapedesis
might be caused by shared downstream signaling events involving members
of the Rho family of GTPases. It has been suggested that ICAM-1 and
VCAM-1 activations are transient and that this may affect the kinetics
of diapedesis [31
]. Lateral migration may require
specific cytoskeletal rearrangements initiated by a specific ratio of
inactive and active forms of Rho, Rac, and Cdc42. This ratio might
exist for only a short period, until the avidity of ICAM-1 and VCAM-1
for their respective ligands changes. A change in avidity could affect
the ratio of active and inactive Rho family members, resulting in the
formation of appropriate pseudopodial extensions through intercellular
junctions.
The observed involvement of VCAM-1 in transendothelial migration is not in agreement with previous reports [31 ] that show that monocyte migration through human umbilical-vein EC monolayers on polycarbonate membranes is independent of VCAM-1. One possibility for this discrepancy could be the different types of ECs analyzed. Differences in the expression of certain cell adhesion molecules within different EC populations have been reported in studies comparing the expression levels of these molecules in human arterial and venous ECs. For example, cell-type-specific differences in VCAM-1 expression and regulation by cytokines have been observed [45 , 46 ]. The differential expression of these cell adhesion molecules might therefore be functionally correlated, not only to the number of monocytes transmigrating at particular vascular sites but also to the different mechanisms that may regulate diapedesis.
Taken together, we demonstrated that blocking ICAM-1, VCAM-1, or both adhesion molecules on ECs prevents the formation of pseudopodia and lamellipodia by monocytes spreading on the monolayer. This blockade reduced the ability of monocytes to migrate along the endothelium. Therefore, a correlation appears to exist between the types of protrusions monocytes make while spreading on top of ECs and the rate at which they migrate along the monolayer. It also appears that the morphological changes necessary for lateral migration are different from those for transendothelial migration, but both are regulated in part by ICAM-1/LFA-1 and VCAM-1/VLA-4 interactions.
Based on the results of our study, we propose that during monocyte diapedesis, ICAM-1/LFA-1 and VCAM-1/VLA-4 interactions differentially modulate the ratio between active and inactive forms of Rho, Rac, and Cdc42. Future studies should aim at dissecting the precise roles of these GTPases in linking ICAM-1- and VCAM-1-mediated adhesion to changes in monocyte motility.
Received December 7, 2000; revised April 25, 2001; accepted April 26, 2001.
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A. H. Wagner, B. Guldenzoph, B. Lienenluke, and M. Hecker CD154/CD40-Mediated Expression of CD154 in Endothelial Cells: Consequences for Endothelial Cell-Monocyte Interaction Arterioscler. Thromb. Vasc. Biol., April 1, 2004; 24(4): 715 - 720. [Abstract] [Full Text] [PDF]
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