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Originally published online as doi:10.1189/jlb.0203054 on November 21, 2003

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(Journal of Leukocyte Biology. 2004;75:523-528.)
© 2004 by Society for Leukocyte Biology

RhoA activation promotes transendothelial migration of monocytes via ROCK

Henk Honing*, Timo K. van den Berg*, Susanne M. A. van der Pol*, Christine D. Dijkstra*, Rob A. van der Kammen{dagger}, John G. Collard{dagger} and Helga E. de Vries*,1

* Department of Molecular Cell Biology, VU Medical Center, Amsterdam, The Netherlands; and
{dagger} The Netherlands Cancer Institute, Antoni van Leeuwenhoekziekenhuis, Amsterdam

1 Correspondence: Department of Molecular Cell Biology, VU Medical Center, Postbus 7057, 1007 MB Amsterdam, The Netherlands. E-mail: HE.de_Vries.cell{at}med.vu.nl


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ABSTRACT
 
Monocyte infiltration into inflamed tissue requires the initial arrest of the cells on the endothelium followed by firm adhesion and their subsequent migration. Migration of monocytes and other leukocytes is believed to involve a coordinated remodeling of the actin cytoskeleton. The small GTPases RhoA, Rac1, and Cdc42 are critical regulators of actin reorganization. In this study, we have investigated the role of Rho-like GTPases RhoA, Rac1, and Cdc42 in the adhesion and migration of monocytes across brain endothelial cells by expressing their constitutively active or dominant-negative constructs in NR8383 rat monocytic cells. Monocytes expressing the active form of Cdc42 show a reduced migration, whereas Rac1 expression did not affect adhesion or migration. In contrast, expression of the active form of RhoA in monocytes leads to a dramatic increase in their adhesion and migration across endothelial cells. The effect of RhoA was found to be mediated by its down-stream effector Rho kinase (ROCK), as pretreatment with the selective ROCK inhibitor Y-27632 prevented this enhanced adhesion and migration. These results demonstrate that RhoA activation in monocytes is sufficient to enhance adhesion and migration across monolayers of endothelial cells.

Key Words: GTPase • brain endothelium • cytoskeleton


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INTRODUCTION
 
The extravasation of monocytes into inflamed tissue is a well-orchestrated process, closely regulated by integrins and members of the immunoglobulin (Ig) superfamily. It seems clear that endothelial cells (EC) actively participate in the migration of leukocytes by the activation of signaling pathways that induce disruption of the endothelial monolayer, allowing the passage of infiltrating cells [1 ]. Conversely, signaling events in monocytes that enable them to cross the endothelium also appear to be essential [2 ].

For cells to migrate, a coordinated remodeling of the actin cytoskeleton is required. Studies primarily performed in fibroblasts and other nonhemopoietic cells have shown that the small GTPases RhoA, Rac, and Cdc42 are essential mediators of actin reorganization [3 , 4 ]. This has also revealed that the different GTPases have distinct effects on cellular morphology and behavior. In particular, Rac1 has a part in the formation of lamellipodia and membrane ruffling, and Cdc42 induces the assembly of filopodia. Additionally, RhoA regulates the assembly of actin stress fibers and focal adhesion sites [3 , 4 ]. In monocytes, inhibition of RhoA activation using a C3 exoenzyme has been shown to reduce transmigration across endothelial monolayers [5 ], suggesting that RhoA activation is required in this process. In the same study, no effect of RhoA inhibition on monocyte adhesion was observed, whereas others found such effects [6 ]. Moreover, Cdc42 has been reported to play a role in monocyte transendothelial migration [7 ]. In the present study, we have studied the role of the small GTPases in monocyte transendothelial migration by using monocytic cells that overexpress constitutively activated or dominant-negative forms of RhoA, Rac1, or Cdc42. Our results demonstrate that activation of RhoA promotes monocyte adhesion as well as transmigration and therefore show that RhoA activation is not only required but is also sufficient as a regulator of these processes. Moreover, our results show that these effects are mediated by p160 ROCK, a serine/threonine kinase effector of RhoA.


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MATERIALS AND METHODS
 
Generation of Rho-like GTPases overexpressing monocytes
Myc-tagged V12Rac and V12Cdc42 were cloned into a LZRS-internal ribosome entry site (IRES)-Neo retroviral vector, a modified LZRS vector conferring neomycin resistance. Myc-tagged V14Rho, N19Rho, N17Rac, and N17Cdc42 were cloned as an EcoRI fragment in LZRS-IRES-Zeo that confers zeocin resistance. To produce retroviruses, Phoenix packaging cells were transfected with the retroviral constructs encoding GTPase proteins as described [8 , 9 ]. Subsequently, the rat monocytic NR8383 cell line was transduced with the retroviral constructs, and transductants were selected in Dulbecco’s modified Eagle’s medium containing neomycin (1 mg/ml) or zeocin (0.2 mg/ml). Expression of proteins in the pool of transduced cells was analyzed by Western blot, as described by Sander et al. [8 , 9 ]. For F-actin staining, transduced cells were fixed in 3.7% paraformaldehyde in phosphate-buffered saline (PBS) and were stained with 0.1 µg/ml rhodamine phalloidin (Molecular Probes, Eugene, OR) in PBS as described previously [10 ]. Control NR8383 cells were also loaded with 50 nM Tat-V14RhoA fusion protein (a kind gift Dr. Peter Hordijk, Department of Experimental Immunohematology, CLB, Amsterdam, The Netherlands) to assess their migratory and adhesive capacity (described below).

Monocyte migration assay
The migratory capacity of transduced NR8383 cells to cross a monolayer of brain EC was assessed using time-lapse video microscopy as described previously [10 ]. The well-characterized, immortalized rat brain EC line GP8.3 [11 ] was used as an in vitro model for brain endothelium and was cultured on collagen-coated multiwell plates as described [10 , 11 ]. Briefly, transduced NR8383 cells (3.5x105/ml) were added to 96-well plates containing control or stimulated brain EC monolayers [with 100 ng/ml interleukin (IL)-1ß and 200 U/ml interferon-{gamma} (IFN-{gamma}) for 48 h to up-regulate vascular cell adhesion molecule 1 (VCAM-1) expression on the EC as assessed by fluorescein-activated cell sorter (FACS) analysis]. Monocytes were allowed to settle and migrate over a 4-h period. Migrated monocytes (phase-dark) could be readily distinguished from those remaining on the EC cell surface by their highly refractive (phase-bright) morphology. The level of migration was calculated as the percentage of migrated monocytes of the total monocytes within the field. To identify the role of the adhesion molecules, intercellular adhesion molecule (ICAM) and VCAM, migration of empty vector (EV) Zeo and V14RhoA cells across control and cytokine-stimulated EC [48 h with IL-1ß (100 ng/ml) and IFN-{gamma} (200 U/ml)] was also assessed in the presence of blocking antibodies (10 µg/ml) against the adhesion molecules VCAM and ICAM and isotype-matched antibodies (as described before in ref. [12 ]).

Adhesion assay
The adhesion of monocytes was performed as described previously [10 ]. Briefly, transduced NR8383 were fluorescently labeled with 1 µM 2',7'-biscarboxyethyl-5(6) carboxyfluorescein acetoxymethylester (Molecular Probes), and 1x106 cells/ml were added to nonstimulated and stimulated EC monolayers (100 ng/ml IL-1ß and 200 U/ml IFN-{gamma} for 48 h), which were allowed to adhere for 1 h. After the incubation, nonadherent cells were removed by gently washing the monolayers with prewarmed medium, and the percentage of adhered cells was determined by lysing the cells with 0.1 M NaOH and by measuring the fluorescence intensity in a Tecan X-Fluorscan (excitation wavelength, 485 nm; emission wavelength, 535 nm).

Flow cytometry
For flow cytometry, single-cell suspensions of the rat monocytic cell line NR8383 were washed three times in ice-cold PBS/0.1% bovine serum albumin (BSA) and where indicated, incubated for 30 min at 4°C with primary monoclonal antibodies (mAb), WT-1 [anti lymphocyte function-associated antigen-1 (LFA-1); IgG2a isotype; gift from Dr. T. Tamatani, Department of Immunology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan] or TA-2 [anti very late antigen (VLA)-4; IgG1 isotype; Serotec, Oxford, UK] at 5 µg/ml. After three washes in ice-cold PBS/0.1% BSA, primary antibody binding was detected using rabbit {alpha} mouse F(ab')2 conjugated with phycoerythrin (Dako, Carpinteria, CA) for 30 min at 4°C. After three washes, cells were immediately analyzed on a flow cytometer (FACScan, Becton Dickinson, Oxnard, CA).

Beads assay
Carboxylate-modified TransFluorSpheres (488/645 nm, 1.0 µm; Molecular Probes) were coated with VCAM-Fc as described previously [13 ]. The fluorescent bead-adhesion assay was performed as described in ref. [13 ]. In brief, cells were resuspended in adhesion buffer [20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM CaCl2, 2 mM MgCl2, and 0.5% BSA] at a final concentration of 5 x 106 cells/ml. Ligand-coated fluorescent beads (20 beads/cell) were added, and a suspension of 50 thousand cells was incubated for 30 min at 37°C. Adhesion was determined by measuring the percentage of cells, which have bound, fluorescent beads, by flow cytometry using FACScan (Becton Dickinson).

Statistics
Data are expressed as mean and SD. Significant differences between groups were determined by two-way ANOVA analysis.


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RESULTS
 
Rat monocytic NR8383 cells were transduced with retroviral constructs (using the LZRS-IRES-Zeo and LZRS-IRES-Neo vector) containing myc epitope-tagged, dominant-negative N17Cdc42, N17Rac1, and N19RhoA and constitutively active V12Cdc42, V14RhoA, and V12Rac1. Protein levels of the transduced constructs were determined by Western blotting using anti-myc antibodies, revealing similar levels of expression in all transduced cell lines (Fig. 1A ). Cellular staining for F-actin, using rhodamine phalloidin, revealed cytoskeletal changes in NR8383 cells after transduction with constitutively active and dominant-negative, small GTPases (data not shown), similar as reported previously for macrophages [3 , 14 ]. A typical example of the cytoskeletal organization of RhoV14-overexpressing cells compared with its corresponding EV Zeo is shown in Figure 1B . These observations indicate that transduced constructs of the GTPases are active and indeed influence the arrangement of the cytoskeleton.



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  		Figure 1. Expression of constitutively active V14RhoA, V12Rac1, and V12Cdc42 and dominant-negative N19RhoA, N17Rac1, and N17Cdc42 in the monocytic cell line NR8383. Western blot analysis of total protein lysates (100 µg protein/lane) of the single-cell lines. Blots were immunoprobed with {alpha}Myc antibody to determine the expression of the Myc-tagged proteins. (B) Expression of constitutively active V14RhoA influences cytoskeletal organization. A typical example of the cytoskeletal organization, as stained for actin by rhodamine phalloidin, of monocytes expressing constitutive, active V14RhoA compared their corresponding EV Zeo.

The obtained cells were then used to study the role of the small GTPases in a well-established assay for transendothelial migration [10 ], using rat GP-8 cerebral endothelial cells. After 4 h of migration, NR8383 cells transduced with the active form of RhoA showed a twofold higher migration (P<0.05) compared with EV-transduced NR8383 cells (EV Zeo; n=12; Fig. 2A ). Monocytic cells for 1 h loaded with the RhoV14-Tat protein (50 nM) also showed a twofold increase in their migration across brain endothelium (data not shown). Cells containing N19RhoA had a slight but significant decrease (P<0.05) in their migratory capacity to 81 ± 7.8% (n=16) compared with its control (EV Zeo), suggesting that endogenous RhoA also influences the migratory capacity of monocytes. In contrast, cells with constitutively active Cdc42 had an impaired migration (P<0.05), only 24 ± 2.3% (n=12) of the cells migrated compared with its EV Neo-transduced control, whereas the percentage of migrated cells containing N17Cdc42 was 91 ± 9.6% (n=12) compared with its control (EV Zeo). NR8383 cells containing the active or the dominant-negative form of Rac1 (n=12) had no different migration kinetics compared with EV Neo- and EVZeo-transduced cells. Adhesion of these cells to monolayers to control and cytokine-stimulated EC was also studied (Fig. 2B) . V14RhoA cells showed a twofold increase in their adhesion to control and cytokine-stimulated EC (P<0.05; n=24), whereas the adhesion of N19RhoA cells to control and cytokine-stimulated EC was significantly reduced by 35 ± 4% and 40 ± 5% (P<0.05; n=24), respectively. Again, no significant change in the adhesion of NR8383 cells containing V12Rac1 or N17Rac1 or V12Cdc42 or N17Cdc42 to EC was observed (n=24).



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  		Figure 2. Migration and adhesion of monocytes expressing small GTPases on brain endothelial monolayers. (A) Migration of monocytes expressing constitutively active V14RhoA, V12Rac1, and V12Cdc42 and dominant-negative N19RhoA, N17Rac1, and N17Cdc42 across confluent monolayers of brain EC. Migration is expressed as the percentage of their corresponding EV (100% values for EV Zeo and EV Neo were 12±2.6% and 10±2.8%, respectively). Data are the mean and SD of four independent experiments each containing at least 12 individual wells. *, P < 0.05, indicates significant differences compared with their respective EV control. (B) Adhesion of monocytes expressing V12Cdc42, N17Cdc42, V14RhoA, N19RhoA, V12Rac1, and N17Rac1 and their corresponding vector (EV Zeo and EV Neo) to control EC monolayers (ctrl) and cytokine-stimulated EC monolayers (stim) was determined. Adhesion of EV-transduced cells to unstimulated EC monolayers was regarded as 100% (29±3.6% for EV Zeo and 32±3.9% for EV Neo of the total number of cells added). Data are the mean ± SD of four independent experiments each containing six individual wells. *, P < 0.05, indicates significant differences compared with their respective EV control. (C) Involvement of adhesion molecules VCAM and ICAM in the migration of monocytes expressing small GTPases across control and cytokine-stimulated brain endothelial monolayers. Cellular migration is expressed as the percentage of the migration of EV Zeo-transduced cells across unstimulated EC. Isotype-matched control antibodies showed no effect on the migration (data not shown). Data are the mean ± SD of three independent experiments, each containing eight individual wells. *, P < 0.01, indicates significant differences compared with their migration in the absence of blocking antibodies.

It was important to establish which possible downstream effectors mediated the observed changes. The Rho kinases, ROCKI and -II, have been identified as downstream effectors of RhoA and are involved in Rho-induced formation of stress fibers and focal adhesions [15 16 17 18 ]. Additionally, phosphatidylinositol-3 kinase (PI-3K) is also known to be involved in cellular migration and actin reorganization [19 , 20 ]. Recently, a selective inhibitor of Rho-associated kinases, Y-27632, was pharmacologically characterized [21 ].

We tested this ROCK inhibitor Y-27632 (1, 10, and 100 µM) and wortmannin (WM; 5 and 100 nM) and LY294002 (1.4 and 28 µM), inhibitors of PI-3K, in our in vitro adhesion and migration assays (Fig. 3 ). In the presence of Y-27632 (10 µM), the enhanced migration of V14RhoA-expressing cells was significantly (P<0.05) reduced by 42% to the level of migration of EV Zeo (control). At a concentration of 100 µM Y27632, migration of the control cells (EV Zeo) and V14RhoA and N19RhoA cells was significantly (P<0.05) reduced by 79% (n=16). However, at this concentration, it has been described that Y 27632 µM also influences other signaling cascades [21 ]. Preincubation of the endothelium with Y-27632 (10 µM) does not influence the migration of monocytes across the endothelial cell layer. PI-3K inhibitors WM (5 nM) and LY294002 (1.4 µM) only induced a reduction (P<0.01; n=16) in the migration of RhoV14-expressing cells (Fig. 3A) , which is in agreement with Fine et al. [2 ]. No significant effect of Y-27632 (10 µM) or WM (5 and 100 nM) or LY294002 (1.4 and 28 µM) was observed on the migration of NR8383 cells expressing N19RhoA or the EV Zeo. The enhanced adhesion of V14RhoA cells was also significantly reduced (P<0.01; n=24) to control levels in the presence of the inhibitor of ROCK (Fig. 3B) , whereas this inhibitor did not affect adhesion of the EV Zeo-transduced cells and the N19RhoA cells.



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  		Figure 3. Effects of inhibition of ROCK and PI-3K on the adhesion and migratory capacity of monocytes expressing constitutively active V14RhoA and dominant-negative N19RhoA. (A) Transendothelial migration of monocytes expressing V14RhoA and N19RhoA and of EV-transduced monocytes was measured in the presence of the ROCK inhibitor Y27632 (Y 1, 10, 100 µM; a kind gift of Welfide Corp., Osaka, Japan) and the PI-3K inhibitors WM (5 nM) and LY294002 (LY; 1.4 µM). Data here are expressed as a percentage of migration of the EV Zeo-transduced cells (100% values were 8.7±2.6%). Data are the mean ± SD of four independent experiments of at least four individual wells. *, P < 0.01, indicates significant inhibitory effects to its respective control. (B) Adhesion of monocytes expressing V14RhoA, N19RhoA, and EV Zeo to EC was measured in the absence or presence of the ROCK inhibitor Y27632 (10 µM). Data are expressed as the percentage of the values obtained with EV Zeo-transduced monocytes. Adhesion of EV Zeo-transduced cells to unstimulated EC was regarded as 100%, which was 31 ± 4.1% of the total number of cells added. Data are expressed as the mean ± SD of 12 individual wells. *, P < 0.01, indicates a significant inhibitory effect of Y27632 compared with its respective control.

Rho-like GTPases are known to influence integrin-mediated adhesion by changing their cell-surface expression or by inducing changes in their affinity state [22 , 23 ]. To determine whether observed changes in the adhesion and migration profile of transduced monocytes were a result of differences in their integrin expression, FACS analysis for the surface expression of LFA-1 and VLA-4 on V14RhoA- and V12Cdc42-transduced cells was performed. However, no significant changes in the surface expression of integrins were found compared with their respective controls (data not shown).

To elucidate the functional involvement of integrins in the diapedesis process, migration of V14RhoA-expressing cells and the EV Zeo cells (serving as control) was performed in the presence of mAb against VCAM and ICAM-1, which block interactions with their specific ligands VLA-4 and LFA-1. It was found that only anti-VCAM mAb resulted in a significant inhibition of V14RhoA-expressing cells and control cells (P<0.05, n=24; Fig. 2C ). This relatively important role for VLA-4/VCAM interaction in our system has been reported previously [12 ] and may be related to the typical nature of brain endothelial cells [24 ]. As the relative extent of inhibition did not differ significantly between V14RhoA and control cells, the contribution of the VLA-4/VCAM interaction was apparently not altered after RhoA activation. To investigate whether the observed increased migration of V14RhoA cells was mediated by an altered affinity/avidity status of VLA-4 for VCAM, we performed a binding assay using fluorescent beads coated with VCAM-Fc fusion protein as described before [13 ]. However, there was no difference in the binding of V14RhoA cells compared with the control cells (not shown), suggesting that VLA-4 on V14RhoA cells has no altered affinity and/or avidity for VCAM. Taken together, these data demonstrate that the enhanced migration and adhesion observed with constitutively activated RhoA were not a result of an enhanced expression and/or affinity of VLA-4.


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DISCUSSION
 
These results demonstrate for the first time that active RhoA is not only necessary but is also sufficient to promote migration of monocytes across brain EC. Probably, endogenous RhoA also contributes to this process, as the expression of the dominant-negative form of RhoA (N19RhoA) in these cells already leads to a significant decrease of their migratory capacity. So far, studies have focused on the blocking of RhoA, showing that RhoA is required for monocyte transendothelial migration [5 ]. Moreover, it has been reported that blockade of RhoA and ROCK by C3 exoenzyme or Y-27632, respectively, prevented monocyte tail retraction, and this was considered to explain the role of RhoA in migration [5 ]. Our investigation shows that viral overexpression of dominant-negative RhoA has an inhibitory effect on monocyte migration, which is in line with the above. It should be mentioned that the effects that we found with the dominant-negative GTPases may well be an underestimation of the actual contribution of these molecules, as effective blocking by dominant-negative proteins generally requires relatively high levels of expression to neutralize all endogenous RhoA activity. It is important that we found that constitutively active RhoA but also the loading of monocytes with active RhoV14-Tat fusion protein are sufficient to increase transendothelial migration. In addition, blocking ROCK with Y-27632 abolished the enhanced migration induced by active RhoA, suggesting that ROCK is the major effector involved. Inhibitors of PI-3K only partially prohibited the enhanced migration of constitutive, active RhoA monocytes, implying that PI-3K has a slight part in this process and may act down-stream of RhoA on the organization of the cytoskeleton. Other, yet-unidentified signaling pathways may also contribute to the migration process. These results indicate that the RhoA/ROCK pathway is not only essential but is also sufficient in regulating monocyte transendothelial migration.

Cdc42 has been suggested to play a role in transendothelial migration as well. Overexpression of dominant-negative and active forms of Cdc42 in human monocytes has been shown to inhibit their chemokine-induced migration across human umbilical vein EC (HUVEC) [7 ]. In line with these observations, we indeed observed inhibition of migration with the active form of Cdc42. Cdc42 activity is needed for the dynamic organization of the cytoskeleton, predominantly in the formation of filopodia in macrophages. It is suggested that the active form of Cdc42 and probably filopodia are required for gradient sensing and cell polarization [14 ] rather than cellular migration, a process that requires Rho activation as shown in this study. Surprisingly, no significant effects of the constitutively active and dominant-negative Rac1 were observed in our assays. So far, no reports exist on the exact role of Rac1 in the transendothelial migration of monocytes, but it has been reported that Rac plays a role in the macrophage chemotaxis [14 ]. Taken together, our data show that Rac1 does not affect the spontaneous transendothelial migration of monocytes but that RhoA and Cdc42 have distinct roles in this process.

The role of the small GTPases in regulation of adhesion is controversial. We have shown that expression of active RhoA increases the adhesion of monocytes to the endothelium, which can be completely blocked by inhibition of ROCK with Y-27632. This is in line with Yoshida et al. [6 ] who showed that C3 exoenzyme-treated U937 cells have a reduced adhesion to HUVEC. However, other reports have shown that RhoA is not required for initial adhesion of monocytes but is important for the tail retraction of monocytes and de-adhesion of leukocytes [5 , 25 ]. Differences in results might be a result of differences in the monocytic cell populations used and/or the conditions of the adhesion assay, for instance, the stringency of washing. We show that overexpression of constitutively active RhoA increases adhesion, which can be completely blocked by inhibiting ROCK. However, inhibition of ROCK has no effect on the adhesion of control cells, suggesting that there might be a threshold for RhoA activation before ROCK activation can occur.

As it was important to reveal the mechanism by which activation of the RhoA/ROCK pathway was promoting adhesion and migration, we investigated the involvement of integrins, in particular, VLA-4, which by interacting with endothelial VCAM, plays a role in these processes (Fig. 2C , ref. [23 ]). Our results show that RhoA activation did not affect VLA-4 expression, its affinity or avidity for VCAM, nor the contribution of the VLA-4/VCAM interaction in monocyte migration. This essentially excludes VLA-4 as an (major) effector of the RhoA activation-induced promotion of adhesion and migration. Clearly, future work is necessary to resolve this issue.

In conclusion, the small GTPases RhoA and Cdc42 have distinct effects on monocyte adhesion and migration across brain endothelium in vitro. This study is the first to show that expression of constitutively active RhoA in monocytes is sufficient to enhance adhesion and migration across monolayers of endothelial cells, a process that is mediated via ROCK. Inhibition of RhoA/ROCK signaling in monocytes may reduce monocytic recruitment into inflamed tissue and may thereby have beneficial effects in various conditions of inflammation, such as autoimmunity and allergies.

Received February 3, 2003; revised September 16, 2003; accepted October 7, 2003.


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