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(Journal of Leukocyte Biology. 2001;70:601-609.)
© 2001 by Society for Leukocyte Biology

Differential regulation of transendothelial migration of THP-1 cells by ICAM-1/LFA-1 and VCAM-1/VLA-4

John A. Ronald, Carmen V. Ionescu, Kem A. Rogers and Martin Sandig

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The adhesion molecules intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expressed in atherogenic lesions are thought to regulate monocyte diapedesis. To better understand their specific roles we used function-blocking antibodies and examined in a culture model the morphology, motility, and diapedesis of THP-1 cells interacting with human coronary artery endothelial cells. The number of motile THP-1 cells was reduced only when VCAM-1 or both ICAM-1 and VCAM-1 were blocked. Blockade of ICAM-1 and VCAM-1, either separately or together, reduced to the same degree the distance that THP-1 cells traveled. Diapedesis was reduced only during the simultaneous blockade of both adhesion molecules. Blockade of either ICAM-1 or VCAM-1 inhibited pseudopodia formation, but ICAM-1 blockade induced the formation of filopodia. We suggest that the interactions of endothelial ICAM-1 and VCAM-1 with their ligands differentially regulate distinct steps of diapedesis by modulating the ratio of active and inactive forms of small GTPases such as Rho, Rac, and Cdc42.

Key Words: adhesion molecules • integrins • endothelial cells • microfilaments • cell motility


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Transendothelial migration (diapedesis) of leukocytes plays a key role in the pathogenesis of inflammatory diseases, including the development of atherosclerosis [1 ]. In vivo studies have shown that one of the earliest events in diet-induced atherosclerosis is the migration of monocytes from the blood into the subendothelial space [2 3 4 ]. Although the exact mechanisms that control the recruitment of monocytes in atherogenesis are not completely understood, it is well documented that this process is mediated, in part, by the interaction between endothelial adhesion molecules and ligands present on monocytes [5 6 7 ]. Intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) are two of the endothelial adhesion molecules that belong to the immunoglobulin (Ig) superfamily of adhesion receptors believed to mediate monocyte migration across the endothelium [8 9 10 ]. It is interesting that a significant correlation has been found between the degree of macrophage infiltration and endothelial ICAM-1 and VCAM-1 expression in atherosclerotic lesions [11 ].

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 {alpha}, 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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells
Human coronary artery ECs (HCAECs) (Clonetics Corp., San Diego, CA) were routinely cultured in EGM-2-MV purchased from Clonetics Corp. This medium consists of endothelial basal medium 2 supplemented with 5% fetal bovine serum, human recombinant epidermal growth factor, hydrocortisone, vascular endothelial growth factor, human fibroblast growth factor-basic with heparin, Long R3-human recombinant insulin-like growth factor 1, ascorbic acid, heparin, gentamicyn 1000 (Clonetics Corp.), penicillin G (10,000 U/100 mL), and streptomycin sulfate (10,000 µg/100 mL) (GIBCO/BRL, Burlington, Canada). HCAECs in the fourth through ninth passages were used for experiments. THP-1 cells (human acute monocytic leukemia cells), a gift from M. Huff (The J. P. Robarts Research Institute, London, ON, Canada), were routinely cultured in RPMI 1640 (GIBCO/BRL) supplemented with 10% fetal bovine serum (Hyclone), 50 µM 2-mercapthoethanol (Sigma Biosciences), penicillin G (10,000 U/100 mL), and streptomycin sulfate (10,000 µg/100 mL) (GIBCO/BRL).

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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Blockade of ICAM-1 or VCAM-1 differentially affects monocyte morphology
Our previous studies showed that during diapedesis the distribution of LFA-1 and F-actin in THP-1 cells and catenins in the endothelium in our culture model is similar to that in PBMs migrating through rat aortas in situ, indicating that THP-1 cells are a good model for analyzing molecular events during monocyte diapedesis. Because spreading of THP-1 cells on the apical surface is a component of diapedesis and depends on the presence of ICAM-1 and VCAM-1, we first ascertained by immunocytochemistry that, on Matrigel, endothelial monolayers express both adhesion molecules after stimulation with IL-1ß (data not shown). The effects of endothelial treatment with anti-ICAM-1 and anti-VCAM-1 were analyzed and quantified with respect to monocyte spreading. Phase-contrast images revealed that THP-1 cells normally extend long pseudopodia and lamellipodia once they are spread on top of the endothelium (Fig. 1a ). ICAM-1 and VCAM-1 blockade produced THP-1 cells that remained somewhat round (Fig. 1b 1c) , but ICAM-1 blockade alone induced the formation of filopodia by THP-1 cells (Fig. 1b) . Blockade of both ICAM-1 and VCAM-1 simultaneously resulted in THP-1 cells with a similar round morphology to those under VCAM-1 blockade (Fig. 1d) .



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  		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.

 
To examine the changes in morphology in more detail and to ascertain that PBMs behave similarly to THP-1 cells, we labeled cocultures for F-actin and analyzed them using LSCM. On control endothelia and those pretreated with control IgG, THP-1 cells and PBMs formed lamellipodia and pseudopodia, whereas pseudopodia formation was largely reduced on endothelia pretreated with anti-ICAM-1 or anti-VCAM-1. Prominent extensions of filopodia were seen when ICAM-1/LFA-1 interactions were blocked (Fig. 2c , d ). These filopodia appeared as thin protrusions and were often tapered with a broad base and thinner tip pointing outward. They had variable lengths (sometimes longer than 10 µm) and contained high concentrations of F-actin. Anti-VCAM-1 pretreatment inhibited the formation of pseudopodia or lamellipodia (Fig. 2d 2h) . However, in contrast to the effects of pretreatment with anti-ICAM-1, monocytes interacting with anti-VCAM-1 pretreated HCAECs did not extend filopodia but remained rather compact (Fig. 2d 2h) . Pretreatment with control IgG did not appear to affect the morphology of THP-1 cells or PBMs, when they interacted with HCAECs (Fig. 2b) . When we scored THP-1 cells according to the shape of their surface extensions, we found a significant fourfold reduction in the percentage of cells with long pseudopodia or lamellipodia after either anti-ICAM-1 or anti-VCAM-1 pretreatment of HCAECs (Fig. 3 ). After VCAM-1 blockade, a twofold increase in the number of THP-1 cells that remained rather compact was observed as compared with THP-1 cells interacting with untreated ECs. These cells, in addition, did not form any filopodia. In contrast to the effect of anti-VCAM-1, anti-ICAM-1 pretreatment of HCAECs resulted in a dramatic 10-fold increase in the number of THP-cells that formed prominent filopodia (Fig. 3) .



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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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  		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).

 
ICAM-1 and VCAM-1 differentially regulate the migration of THP-1 cells along HCAECs
After their adhesion and spreading on the apical surface of ECs, monocytes migrate along the endothelium [20 ]. Because pretreatment of HCAECs with antibodies to ICAM-1 or VCAM-1 greatly affected the ability of THP-1 cells to form normal pseudopodia or lamellipodia, it was of interest to examine how this would affect the lateral migration of THP-1 cells in our assay system (Fig. 4 ). Using time-lapse video microscopy, we counted the number of cells that translocated along the endothelial surface over 30 min. In control cultures, 84% of monocytes migrated laterally. ICAM-1 blockade had no effect on the ability of THP-1 cells to translocate on the endothelium. In contrast, blockade of VCAM-1 inhibited by 30% the ability of THP-1 cells to migrate laterally (Fig. 4a) . These results suggest that VCAM-1/VLA-4 interactions alone are involved in the lateral migration of THP-1 cells across the apical surface of ECs. However, the simultaneous blockade of both ICAM-1 and VCAM-1 resulted in a 55% decrease in the percentage of monocytes translocating on the EC surface suggesting that ICAM-1/LFA-1 interactions are also involved in this process (Fig. 4a) . It is conceivable that ICAM-1 and/or VCAM-1 blockade differentially affect the distance that THP-1 cells travel along the EC surface over a 30-min period. We therefore used the movies produced to measure the distances traveled under various conditions (Fig. 4b) . In control cultures, THP-1 cells translocated on average a distance of 33 µm. Blockade of ICAM-1, VCAM-1, and both adhesion molecules together reduced, to the same degree, the distance an individual THP-1 cell traveled. Compared with the control, an ~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.



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  		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).

 
ICAM-1 and VCAM-1 together regulate diapedesis of THP-1 cells
Because it was apparent that ICAM-1/LFA-1 and VCAM-1/VLA-4 interactions regulated the trafficking of monocytes along the endothelial surface, we questioned to what extent these interactions were involved in the trafficking of monocytes through the endothelium. To evaluate the effects of function-blocking antibodies on diapedesis, we used LSCM after labeling for F-actin [35 ]. The process of diapedesis can be divided into four recognizable stages based on the location and morphology of the monocytes, as illustrated in Figure 5 . These four categories are then used to quantify diapedesis during a period of 30 min. THP-1 cells with a spherical morphology and with HCAEC stress fibers present in optical sections below the plane of the THP-1 cells (Fig. 5A III , solid arrow) were called "round on top." THP-1 cells that had acquired a flattened morphology and extended pseudopodia (Fig. 5B 5II , open arrow) on the EC surface and with endothelial stress fibers present in optical sections below the plane of the THP-1 cells (Fig. 5B III , solid arrow) were categorized as being "spread on top" of the endothelial monolayer. "Transmigrating" THP-1 cells (Fig. 5C 5I –III) were identified by having three morphologically distinct regions: a spherical part situated above the endothelial monolayer (Fig. 5C 5I) ; a spread part underneath the endothelial monolayer (Fig. 5C III ), and a narrow circular transmigration passage between these two (Fig. 5C 5II , arrowhead), through which the THP-1 cell nucleus translocates during diapedesis. In most of the cases, the transmigration passage was located at the contact region between two or more adjacent ECs. THP-1 cells that were located completely "underneath" the endothelium (Fig. 5D 5I –III) had several pseudopodia (open arrow in Fig. 5D III ) and were identified by the presence of intact inter-EC junctions or endothelial microfilament bundles (solid arrow in Fig. 5D 5I ) in optical planes above the THP-1 cells. In Figure 6 , the numbers of transmigrating THP-1 cells and monocytes are expressed as the summed data of the monocytes scored as transmigrating and those underneath the endothelium (i.e., those that had transmigrated). No significant decrease in the percentage of transmigrating THP-1 cells was noted after either VCAM-1 or ICAM-1 blockade (Fig. 6a) , which suggests that neither ICAM-1 nor VCAM-1 alone are involved in the transendothelial migration of monocytes. However, the blockade of both ICAM-1 and VCAM-1 significantly reduced the percentage of transmigrating monocytes, by 45% (Fig. 6a) . Therefore, it appears that both VCAM-1/VLA-4 and ICAM-1/LFA-1 interactions in concert regulate monocyte transendothelial migration. Similarly, preliminary results indicated that diapedesis of PBMs is also affected by antibodies to both adhesion molecules (Fig. 6c) , whereas pretreatment of HCAEC monolayers with an isotype-matched control IgG had no effect on THP-1 cell diapedesis (Fig. 6b) .



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  		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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  		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).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A considerable number of studies have explored the mechanisms responsible for mediating monocyte diapedesis. In the last decade it became clear that endothelial ICAM-1 and VCAM-1 interactions with their monocytic counter receptors LFA-1 and VLA-4, respectively, are involved in mediating diapedesis [6 , 9 , 15 ]. In the present study we used a novel culture model of diapedesis combined with time-lapse video microscopy and confocal microscopy to further dissect the specific roles of LFA-1/ICAM-1 and VLA-4/VCAM-1 interactions with function-blocking antibodies to ICAM-1 and VCAM-1. Most of our experiments were performed with THP-1 cells. These cells have characteristics of monocytes and transmigrate through cultured endothelial monolayers [35 ] in a manner similar to PBMs in situ [34 ], and, in contrast to PBMs, they can be genetically engineered to dissect molecular events during diapedesis.

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.


   ACKNOWLEDGEMENTS
 
J. Ronald and V. Ionescu were supported by studentships from the government of Ontario. M. Sandig is a Heart and Stroke Foundation of Ontario Scholar. This work was supported by the Heart and Stroke Foundation of Ontario, grant T3723. We thank Ms. Nicole Bechard for help with imaging. The THP-1 cells were a gift from Dr. M. Huff, The Robarts Research Institute, London, ON. J. Ronald and C. Ionescu contributed equally to this work.

Received December 7, 2000; revised April 25, 2001; accepted April 26, 2001.


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 DISCUSSION
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