Endothelial Cadherin Endothelium Is Regulated by Vascular Progenitor

Migration of Human Hematopoietic Progenitor Cells Across Bone Marrow Endothelium Is Regulated by Vascular Endothelial Cadherin This information is current as of October 1, 2021. Jaap D. van Buul, Carlijn Voermans, Veronique van den Berg, Eloise C. Anthony, Frederik P. J. Mul, Sandra van Wetering, C. Ellen van der Schoot and Peter L. Hordijk J Immunol 2002; 168:588-596; ; doi: 10.4049/jimmunol.168.2.588 http://www.jimmunol.org/content/168/2/588 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Migration of Human Hematopoietic Progenitor Cells Across Bone Marrow Endothelium Is Regulated by Vascular Endothelial Cadherin1

Jaap D. van Buul,* Carlijn Voermans,* Veronique van den Berg,* Eloise C. Anthony,* Frederik P. J. Mul,* Sandra van Wetering,* C. Ellen van der Schoot,*† and Peter L. Hordijk2*

The success of stem cell transplantation depends on the ability of i.v. infused stem cells to engraft the bone marrow, a process referred to as homing. Efficient homing requires migration of CD34؉ cells across the bone marrow endothelium, most likely through the intercellular junctions. In this study, we show that loss of vascular endothelial (VE)-cadherin-mediated endothelial cell-cell adhesion increases the permeability of monolayers of human bone marrow endothelial cells (HBMECs) and stimulates the ؉ transendothelial migration of CD34 cells in response to stromal cell-derived factor-1␣. Stromal cell-derived factor-1␣-induced Downloaded from migration was dependent on VCAM-1 and ICAM-1, even in the absence of VE-cadherin function. Cross-linking of ICAM-1 to mimic the leukocyte-endothelium interaction induced actin stress fiber formation but did not induce loss of endothelial integrity, whereas cross-linking of VCAM-1 increased the HBMEC permeability and induced gaps in the monolayer. In addition, VCAM- 1-mediated gap formation in HBMEC was accompanied by and dependent on the production of reactive oxygen species. These data suggest that modulation of VE-cadherin function directly affects the efficiency of transendothelial migration of CD34؉ cells and that activation of ICAM-1 and, in particular, VCAM-1 plays an important role in this process through reorganization of the http://www.jimmunol.org/ endothelial actin cytoskeleton and by modulating the integrity of the bone marrow endothelium through the production of reactive oxygen species. The Journal of Immunology, 2002, 168: 588–596.

ematopoietic stem cell transplantation is applied to re- Fusin, leukocyte-derived seven-transmembrane domain receptor, store hematopoiesis in cancer patients after myelo-abla- or CXCR-4 (6Ð9). SDF-1␣-driven homing of CD34ϩ cells has H tive chemotherapy and/or after irradiation. The success been suggested to be a multistep process similar to the extravasa- of the transplantation depends on the ability of the hematopoietic tion process of leukocytes at inflammatory sites (10) and is medi- stem cells to engraft the bone marrow, a process referred to as ated by adhesion molecules both on CD34ϩ cells (11) and on bone by guest on October 1, 2021 homing (1). An important step in homing is the actual transmigra- marrow endothelial cells. In the final stage of homing, CD34ϩ tion of reinfused stem cells across the bone marrow endothelium to cells migrate across the bone marrow endothelium, presumably via the bone marrow stroma. Although much is known about the mi- the intercellular junctions. Therefore, endothelial cell-cell adhe- gration of granulocytes and T cells, the factors that control the sion is most likely an important regulatory factor in the homing of transendothelial migration of hematopoietic stem cells are still CD34ϩ cells. poorly understood. Endothelial cell-cell adhesion is largely dependent on the ho- Recently, the first powerful chemoattractant for hematopoietic motypic cell-cell adhesion molecule vascular endothelial (VE)- ϩ stem cells (CD34 cells) has been described and identified as stro- cadherin (cadherin-5, CD144). VE-cadherin is a transmembrane mal cell-derived factor-1␣ (SDF-1␣).3 SDF-1␣ is produced by protein that, like other members of the cadherin family (12), as- several types of stromal cell, including those of the bone marrow sociates via its cytoplasmic tail with various cytosolic proteins, (2Ð5), and signals through a G protein-coupled receptor called including ␣-, ␤-, and ␥-catenin (plakoglobin) and p120/p100. These proteins link VE-cadherin to the cortical actin cytoskeleton *Department of Experimental Immunohematology, CLB and Laboratory for Exper- (13Ð15). The role of VE-cadherin in leukocyte transendothelial imental and Clinical Immunology, and †Department of Hematology, Academic Med- migration was first described by Gotsch et al. (16), who showed an ical Center, University of Amsterdam, Amsterdam, The Netherlands accelerated extravasation of neutrophils in a mouse peritonitis Received for publication July 23, 2001. Accepted for publication November 16, 2001. model in vivo upon i.v. injection of a mAb against mouse VE- The costs of publication of this article were defrayed in part by the payment of page cadherin. Transfection experiments and gene inactivation studies charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. have shown that VE-cadherin expression reduces monolayer per- 1 This work was supported by Dutch Cancer Society Grants CLB99-2000 (to J.D.v.B. meability and promotes cell aggregation, motility, and growth, and and E.C.A.) and CLB2001-2544 (to C.V.) and by Landsteiner Foundation for Blood that VE-cadherin is required for the organization of vascular-like Transfusion Research Grant 9910 (to F.P.J.M. and S.v.W.). structures in embryoid bodies (17Ð19). Moreover, VE-cadherin, 2 Address correspondence and reprint requests to Dr. Peter L. Hordijk, Department of together with ␤-catenin, seems to be involved in cell survival (20). Experimental Immunohematology, CLB, Academic Medical Center, University of Amsterdam, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands. E-mail ad- Regulation of VE-cadherin, and thereby of endothelial cell-cell dress: [email protected] adhesion, may occur through tyrosine or serine phosphorylation 3 Abbreviations used in this paper: SDF-1␣, stromal cell-derived factor-1␣; VE, vas- (21Ð24), association with regulatory proteins (20), and modulation cular endothelial; HBMEC, human bone marrow endothelial cell; CB, cord blood; PB, of the endothelial actin cytoskeleton (17, 25). In addition, several peripheral blood; ROS, reactive oxygen species; G␣M-Ig, goat anti-mouse Ig; FN, fibronectin; N-AC, N-acetyl-cysteine; F-actin, filamentous actin; DHR, di-hydro-rho- studies have proposed a role for leukocyte adhesion-induced sig- damine-1,2,3; VLA, very late Ag. naling, e.g., through activation of myosin light chain kinase in the

endothelial cells, which may indirectly regulate VE-cadherin func- cells from CB and Ͼ90% of the cells from PB expressed CD34 as deter- tion in the process of transendothelial migration (26Ð29). mined by FACS analysis with a CD34 Ab (no. 581; Immunotech). A role for specific adhesion molecules in endothelial cell signaling has been suggested by studies on VCAM-1 (CD106) and Cell cultures ICAM-1 (CD54). Lorenzon et al. (30) concluded that VCAM-1 The HBMEC line has been described previously (33). The cells were cul- and endothelial selectins, in addition to their role as adhesion re- tured in FN-coated culture flasks (Nunc, Roskilde, Denmark; Life Tech- ceptors, mediate endothelial stimulation by adherent leukocytes. nologies) in Medium 199 (Life Technologies) supplemented with 10% Moreover, activation of ICAM-1 on endothelial cells after binding (v/v) pooled, heat-inactivated human serum, 10% (v/v) heat-inactivated ␮ of T cells has been reported to induce tyrosine phosphorylation of FCS, 1 ng/ml basic fibroblast factor, 5 U/ml heparin, 300 g/ml glutamine, 100 U/ml penicillin, and 100 ␮g/ml streptomycin. After reaching conflu- the actin-binding protein cortactin (31). In line with this observa- ency, the cells were passaged by treatment with trypsin/EDTA (Life Tech- tion, it was suggested that ICAM-1 mediates cell shape changes nologies). In all experiments, HBMEC monolayers were pretreated with through coupling to the p21Rho GTPase and by inducing phos- IL-1␤ for 4 h. HL-60 and KG-1a cell lines were obtained from the Amer- phorylation of cytoskeletal proteins and transcription factors (32). ican Type Culture Collection (Manassas, VA) and were maintained in IMDM (BioWhittaker, Brussels, Belgium) containing L-glutamine, 100 In the present study, the role of VE-cadherin in the control of U/ml penicillin, 100 ␮g/ml streptomycin, and 10% FCS. All cell lines were permeability of bone marrow endothelium and of the transmigra- cultured at 37¡Cat5%CO2. tion of primary CD34ϩ cells was investigated. For this purpose, we used immortalized human bone marrow endothelial cells Permeability assay (HBMECs) (33) and primary CD34ϩ cells isolated from cord blood (CB) or from peripheral blood (PB) of healthy untreated Permeability of HBMEC monolayers, cultured on 5-␮m-pore, 6.5-mm Transwell filters (Costar, Cambridge, MA), was assayed using FITC-la- Downloaded from volunteers. In this study, we show that transendothelial migration ϩ beled 3000 Dextran as described (17). The permeability response to throm- of primary CD34 cells is promoted by reduced VE-cadherin- bin (1 U/ml) after 30 min was set to 100%. In some experiments, mono- mediated cell-cell adhesion and is accompanied by a focal loss of layers were pretreated with Abs (10 ␮g/ml) for 30 min. Unbound blocking VE-cadherin at sites of transmigration. Cross-linking of VCAM-1, Abs against VCAM-1 and ICAM-1 were washed away before the start of but not ICAM-1, was found to induce loss of endothelial cell-cell the permeability or transmigration assays. Cross-linking Abs and Abs to VE-cadherin were present during the permeability or transmigration assay. adhesion and increased permeability. VCAM-1-mediated gap for- Botulinum C3 toxin was added to the HBMEC monolayers 6 h before the http://www.jimmunol.org/ mation was accompanied by and dependent on the generation of assay. In our previous publication (17), the incubation time for C3 was reactive oxygen species (ROS). These data indicate that activation 18 h. However, we found that a 6-h incubation of HBMEC with C3 was of Ig-like adhesion molecules, in particular VCAM-1, regulate already sufficient to inhibit actin polymerization. The 6-h incubation with C3 did not affect the VE-cadherin localization, whereas the 18-h incubation VE-cadherin function, which might facilitate SDF-1␣-driven ϩ with C3 resulted in diffuse staining of VE-cadherin (17). Kinetics of transendothelial migration of primary CD34 cells across the bone VCAM-1-mediated increase in permeability was performed as described marrow endothelium. by Corada et al. (34). In brief, at indicated time points, 50 ␮l were taken from the lower compartment and fluorescence was measured. After the assay, filters were washed with ice-cold Ca2ϩ- and Mg2ϩ-containing PBS Materials and Methods and then fixed with 2% paraformaldehyde and 1% Triton X-100-containing

Materials PBS and stained with Texas-Red phalloidin to inspect the HBMEC mono- by guest on October 1, 2021 layer by confocal laser scanning microscopy. mAbs against VE-cadherin (cl75) and ␤-catenin were from BD Transduc- tion Laboratories (Amsterdam, The Netherlands). VE-cadherin Ab 7H1 was from BD PharMingen (San Diego, CA). Hybridoma supernatant Transendothelial migration assay TEA1.31 was a kind gift from Dr. E. Dejana (Instituto di Ricerche Farma- cologiche Mario Negri, Milan, Italy). Polyclonal Ab against VE-cadherin Migration assays were performed in Transwell plates of 6.5-mm diameter ␮ (C19) was from SanverTech (Heerhugowaard, The Netherlands). Recom- with 5- m pore filters. Endothelial cells were plated at 20,000Ð30,000 binant human IL-1␤ was from PeproTech (Rocky Hill, NJ); calcein-ace- cells/Transwell on FN-coated filters. Nonadherent cells were removed after toxymethyl, Texas-Red phalloidin, FITC-Dextran 3000, ALEXA 488-la- 18 h. The adherent cells were cultured for 2Ð3 days to obtain confluent beled goat anti-mouse Ig (G␣M-Ig), and ALEXA 488-labeled goat anti- endothelial monolayers. Monolayers of endothelial cells were pretreated ␤ ϩ rabbit Ig secondary Abs were from Molecular Probes (Leiden, The for 4 h with IL-1 . Before adding CD34 cells to the upper compartment, Netherlands). PE-labeled secondary Abs and botulinum C3 toxin were the endothelial monolayers were washed three times with assay medium (IMDM with 0.25% (w/v) BSA (fraction V; Sigma-Aldrich)). Freshly iso- from DAKO (Glostrup, Denmark). Pooled human serum, human serum ϩ albumin, fibronectin (FN), and control Abs IgG1 and IgG2a were obtained lated CD34 cells (50,000Ð100,000) were added to the upper compartment from the CLB (Amsterdam, The Netherlands). FCS was from Life Tech- in 0.1 ml of assay medium, and 0.6 ml of assay medium with or without the ␣ nologies (Paisley, U.K.). Basic fibroblast factor was from Boehringer indicated concentrations of recombinant human SDF-1 (Strathmann Bio- Mannheim (Mannheim, Germany). CXCR-4 expression was quantitated tech, Hannover, Germany) was added to the lower compartment. A 0.1-ml with PE-labeled anti-human Fusin (12G5; BD PharMingen). mAbs against sample containing cells in assay medium was diluted in 0.5 ml of assay ICAM-1 (84H10) and VCAM-1 (1G11) were purchased from Immunotech medium and was kept as input control for quantitation of the number of (Marseille, France). Additional mAbs against ICAM-1 (15.2; CLB) and migrated cells. The Transwell plates were incubated at 37¡C, 5% CO2, for 4 h. Preliminary experiments showed that after 4 h, a substantial fraction of VCAM-1 (4B2; PeproTech) were also used to exclude epitope dependence ϩ Ј ␣ the CD34 cells had migrated. Cells that had migrated to the lower com- of the effects. Cross-linking studies were performed with F(ab )2 of G M- IgG from Jackson ImmunoResearch Laboratories (Baltimore, MD). partment were collected in a FACS tube to which a fixed number of control Thrombin and N-acetyl-cysteine (N-AC) were from Sigma-Aldrich (St. cell line cells (kG-1a) labeled with calcein-acetoxymethyl was added. Louis, MO). FACS analysis was used to determine the ratio between labeled and unla- beled cells, with characteristic light scatter parameters, in the migrated Isolation of CD34ϩ hematopoietic progenitor cells fraction as described before (2). By comparison of this ratio to that of the input control, the number of migrated cells was quantitated. Using this CB was collected after delivery, according to the guidelines of Eurocord, method, we were able to determine reliably a minimum number of 200 and PB from healthy volunteers was obtained from the local blood bank. migrated cells. In blocking experiments, HBMECs were preincubated for These volunteers were normal donors that were not treated with G-CSF or 30 min at 37¡C with mAbs (10 ␮g/ml), followed by washing. As controls, with chemotherapy. Mononuclear leukocytes from PB (500 ml) and from IgG1 and IgG2a isotypes were used. Blocking Abs against VCAM-1 and CB were enriched by density gradient centrifugation over Ficoll-Paque ICAM-1 were not present during the transendothelial migration assay. (1.077 g/ml; Pharmacia Biotech, Uppsala, Sweden). Then the PB mono- However, blocking Abs against VE-cadherin (cl75) were present during nuclear fraction was purified from thrombocytes by elutriation and further the transendothelial migration assay. After the assay, the filters were processed, similar to CB CD34ϩ cell isolation, with the VarioMacs system fixed and stained with Texas-Red phalloidin to inspect HBMEC mono- (Miltenyi Biotec, Gladbach, Germany) as described (2). At least 95% of the layers by confocal laser scanning microscopy. 590 VE-CADHERIN MODULATES MIGRATION OF CD34ϩ CELLS

Immunocytochemistry HBMECs were cultured on FN-coated glass coverslips and were fixed and immunostained as described (17) with mAb against VE-cadherin (7H1, 10 ␮g/ml) or anti-␤-catenin (10 ␮g/ml) followed by staining with fluores- cently labeled secondary Abs (10 ␮g/ml). Filamentous actin (F-actin) was visualized by Texas-Red phalloidin (1 U/ml). In some experiments, cells were pretreated with mAb against VE-cadherin (blocking cl75, 10 ␮g/ml; partially blocking TEA1.31; hybridoma supernatant 1/2 dilution; or the nonblocking 7H1, 10 ␮g/ml) for 30 min. In the analysis after the permeability experiments, a polyclonal Ab against VE-cadherin was used for immunostaining. Images were recorded with a confocal microscope with appropriate filter settings (LSM510; Zeiss, Oberkochen, Germany). Cross- talk between the green and red channels was avoided by use of sequential scanning.

Analysis of ROS production To measure intracellular ROS production, HBMECs were cultured on FN- coated six-well plates and loaded with di-hydro-rhodamine-1,2,3 (DHR; 30 ␮M; Molecular Probes) for 60 min in the presence of catalase (60 ␮g/ml)

and NaN3 (2 mM) at 37¡C. Adhesion molecules were subsequently cross- linked at 37¡C as described in Permeability assay. After 30 min, cells were incubated for 1 min with trypsin at 37¡C, collected on ice, and washed with Downloaded from ice-cold Ca2ϩ- and Mg2ϩ-containing PBS, and DHR ﬂuorescence was ␭ ␭ quantitated by FACS in the FL-2 channel ( EX, 488 nm; EM, 585 nm) for 10,000 counted cells per incubation. For the kinetics of the production of ROS, the endothelial cells were cultured on FN-coated glass coverslips and loaded as described above. Then ROS production was recorded and quantitated by time-lapse confocal microscopy of DHR-loaded HBMECs. In-

tensity values are shown as the percentage of increase relative to the values http://www.jimmunol.org/ at the start of the experiment. In some experiments, cells were pretreated for 18 h with 5 mM of the oxygen radical scavenger N-AC to prevent ROS-mediated signaling. FACS analysis showed that N-AC preincubation had no effect on the IL-1␤-mediated up-regulation of adhesion molecules such as VCAM-1 and ICAM-1.

Statistics All results that were performed at least three times were expressed as the mean Ϯ SD or SEM, as indicated in the legend. Differences were tested by FIGURE 1. a, VE-cadherin-mediated permeability of HBMEC mono- using the Student t test. A value of p Ͻ 0.05 was considered significant. layers. Cells were grown to confluency on FN-coated Transwell filters and by guest on October 1, 2021 prestimulated with IL-1␤, followed by pretreatment for 30 min with Abs Results (10 ␮g/ml) or hybridoma supernatant TEA1.31 (tissue culture supernatant diluted 1/2 in medium). The Transwells were incubated for 3 h with FITC- Role of VE-cadherin in permeability and integrity of HBMEC Dextran 3000 in the upper compartment in the presence of the Abs. Next, monolayers fluorescence in the lower compartment was measured in a fluorometer ␭ ␭ In the initial series of experiments, the role of VE-cadherin in the ( EX, 485 nm; EM, 525 nm) and expressed as described in Materials and control of monolayer integrity of HBMECs was examined. Ab- Methods. The blocking c175 and the partially blocking TEA1.31 Abs to mediated inhibition of VE-cadherin function using two indepen- VE-cadherin, but not the isotype control Ab to VE-cadherin 7H1, increased dent blocking Abs resulted in increased permeability of HBMEC monolayer permeability significantly. Thrombin-induced permeability was set to 100%, as described in Materials and Methods. Data are mean Ϯ monolayers, whereas a nonblocking, isotype-matched VE-cad- SEM of at least three independent experiments (aA, p Ͻ 0.001). b, Block- herin Ab and an irrelevant IgG1 did not have any effect on the ing Abs to VE-cadherin alter VE-cadherin distribution and induce cy- permeability of HBMECs (Fig. 1a). Immunofluorescent staining of toskeletal reorganization. HBMECs were grown to confluency on FN- IL-1␤-prestimulated HBMECs after treatment with the nonblock- coated glass coverslips, pretreated with IL-1␤, and incubated with the ing VE-cadherin Ab showed a jagged distribution of VE-cadherin different anti-VE-cadherin Abs for 1 h. VE-cadherin and F-actin were vi- (Fig. 1bA) and normal F-actin stress fiber formation (Fig. 1bB). At sualized as described in Materials and Methods. The overlays show VE- the ends of the F-actin stress fibers, colocalization of VE-cadherin cadherin in green (A, D, and G) and F-actin in red (B, E, and H); colocal- with actin was observed (Fig. 1bC), as previously described for ization appears in yellow (C, F, and I). The nonblocking 7H1 Ab (10 ␮ primary HUVECs (17, 34, 35). Pretreatment of HBMEC mono- g/ml) did not affect VE-cadherin distribution, whereas the blocking cl75 ␮ layers with a blocking Ab against VE-cadherin (cl75) resulted in a Ab (10 g/ml) caused loss of junctional localization of VE-cadherin (D) and a reorganization of the F-actin cytoskeleton as revealed by the loss of redistribution of VE-cadherin over the cell surface (Fig. 1bD) and stress fibers (E). The TEA1.31 Ab induced a partial loss of cell-cell con- a marked reorganization of the actin cytoskeleton (Fig. 1bE). Al- tacts (H) which, however, was not as prominent as with the blocking cl75 though the partially blocking TEA1.31 Ab to VE-cadherin did in- Ab. VE-cadherin remained localized to cell-cell junctions (G). Bar, 50 ␮m. crease the HBMEC permeability (Fig. 1a), it did not dramatically affect the localization of VE-cadherin (Fig. 1bG) or the actin cy-

toskeleton of the HBMECs (Fig. 1bH), in agreement with pub- ϩ lished results obtained with primary HUVECs (35). These ﬁndings Role of VE-cadherin in transmigration of CD34 cells across underscore the essential role of VE-cadherin in the regulation of HBMECs the integrity of HBMEC monolayers and show that its function Puriﬁed primary CD34ϩ cells from CB and PB and the human (i.e., control of permeability) and localization can be modulated leukemic cell line HL-60, which all express CXCR-4, were tested differentially by different blocking Abs. for their ability to migrate across FN or HBMECs to a gradient of The Journal of Immunology 591

SDF-1␣. HL-60 cells migrated across FN with a similar efficiency across IL-1␤-prestimulated HBMECs. The results show a bell- as primary PB CD34ϩ cells to 30 ng/ml SDF-1␣, whereas primary shaped response with optimal migration at 70 ng/ml SDF-1␣ (Fig. CB CD34ϩ cells showed a higher migration efficiency (Fig. 2a). 2b). When VE-cadherin was blocked with the cl75 Ab on IL-1␤- However, CXCR-4 expression of PB and CB CD34ϩ was com- prestimulated HBMECs, a shift of the optimal concentration for parable (data not shown). Because of the limited supply of primary migration from 70 to 30 ng/ml SDF-1␣ and a significant increase CD34ϩ cells, CXCR-4-expressing HL-60 cells were used as a in migration across HBMECs were observed (Fig. 2b). Moreover, model in dose response studies of SDF-1␣-induced migration migration of primary CB CD34ϩ cells to 30 ng/ml SDF-1␣ across HBMECs pretreated with either the partially blocking TEA1.31 or the blocking cl75 Ab resulted in a significant increase in transmigration efficiency (Fig. 2c). These findings show that VE-cadherin function is an important regulator of efficient migration of primary CD34ϩ cells across bone marrow endothelium. A more detailed analysis of VE-cadherin distribution revealed a focal loss of VE-cadherin immunostaining at sites of transmigration of CD34ϩ cells (Fig. 3a) where stress fibers seemed to con- verge at the periphery of the transmigrating CD34ϩ cell (Fig. 3, b and c). The cell is fixed during its passage through the filter and the endothelial junctions (Fig. 3d). Similarly, also ␤-catenin was re- distributed over the surface of HBMECs at such sites (data not Downloaded from shown). These observations suggest a coordinated interaction between the actin cytoskeleton and VE-cadherin at sites of transmigrating CD34ϩ cells.

Role of p21Rho in VE-cadherin-mediated transmigration of CD34ϩ cells and in the permeability and integrity of HBMEC

monolayers http://www.jimmunol.org/ Blocking VE-cadherin function by the cl75 Ab was accompanied by a reorganization of the actin cytoskeleton (Fig. 1b). Together with the results shown in Fig. 3, these ﬁndings indicate that the actin cytoskeleton, in concert with VE-cadherin, regulates barrier function in HBMECs. Regulation of cytoskeletal contractility in endothelial cells can be mediated by changes in cAMP levels (36) and by the small GTPase p21Rho (37, 38). p21Rho is required for

actin stress fiber formation in response to extracellular stimuli in by guest on October 1, 2021 many cell types and has also been implicated in the organization of cadherin-based cell-cell adhesion in epithelial cells (39, 40). More- over, inactivation of p21Rho by pretreatment of primary HUVECs with the botulinum C3 toxin causes cytoskeletal reorganization, mainly reflected in a loss of actin stress fibers (17). To investigate the role of p21Rho in the control of transendothelial migration of CD34ϩ cells and endothelial permeability, HBMECs were pretreated with the C3 toxin. As a result, a loss of actin stress fibers (Fig. 4a), but no loss of VE-cadherin localization (Fig. 4b), was

FIGURE 2. a, Migration of primary CD34ϩ cells across FN. SDF-1␣ (30 ng/ml)-induced migration of CB CD34ϩ cells (hatched bars) across FN-coated filters was significantly higher than the migration of HL-60 cells (filled bars) or PB CD34ϩ cells from healthy untreated volunteers (open bars). b, SDF-1␣-induced transendothelial migration of HL-60 cells. Dose response of SDF-1␣-induced migration of HL-60 cells across HBMECs showed a bell-shaped curve with an optimal migration at 70 ng/ml SDF-1␣ (filled bars). Open bars represent HBMECs that were pretreated for 30 min FIGURE 3. Transmigration of CB CD34ϩ cells induces focal loss of and subsequently incubated during the assay with blocking Ab cl75 against VE-cadherin. Primary CB CD34ϩ cells and HBMECs were stained and VE-cadherin (10 ␮g/ml), resulting in a significantly increased migration at fixed after3hoftransmigration. Loss of VE-cadherin localization (green) 30 ng/ml SDF-1␣ and a shift of the dose for optimal migration from 70 to was observed at the site of migration of a CB CD34ϩ cell, indicated by the 30 ng/ml SDF-1␣. c, Effect of Ab-mediated loss of VE-cadherin function arrowhead (a). The migrating cell is visualized by F-actin staining in red on SDF-1␣-induced transendothelial migration of primary CB CD34ϩ (b, arrowhead). Yellow indicates colocalization of F-actin and VE-cadherin cells. Pretreatment and incubation of HBMECs with the blocking cl75 Ab at the periphery of the migrating cell (c, arrowhead). The X-Z section (10 ␮g/ml) or the partially blocking TEA1.31 hybridoma supernatant to shows the same migrating CD34ϩ cell in red, protruding into a filter pore VE-cadherin showed a significant increase in transmigration to SDF-1␣. (d). The dashed gray arrow in d corresponds to the gray arrow in the Data are mean Ϯ SD of at least three independent experiments. bA and cA, schematic. The drawing represents a cell (red) protruding into the endo- p Ͻ 0.05; cB, p Ͻ 0.01; aC, p Ͻ 0.001. thelial cells (gray). Bar, 10 ␮m. 592 VE-CADHERIN MODULATES MIGRATION OF CD34ϩ CELLS Downloaded from

FIGURE 4. Role of p21Rho in the transmigration of CB CD34ϩ cells and in permeability and integrity of HBMEC monolayers. HBMECs were treated for 6 h with 10 ␮g/ml C3 toxin to inactivate p21Rho and were immunostained for F-actin and VE-cadherin. The treatment with C3 caused a loss of stress

ﬁbers (a), but staining of VE-cadherin remained localized to cell-cell junctions (b). Colocalization appears in yellow (c). In addition to the C3 treatment, http://www.jimmunol.org/ the monolayer was pretreated for 30 min with the blocking c175 Ab (10 ␮g/ml), which caused a loss of HBMEC cell-cell adhesion (d) and diffuse staining of VE-cadherin over the surface (e). Colocalization appears in yellow (f). Inactivation of p21Rho had no effect on the SDF-1␣-induced migration of CB CD34ϩ cells across HBMECs with or without treatment with the blocking c175 Ab (10 ␮g/ml) (g). Similarly, inactivation of p21Rho had no signiﬁcant effect on the permeability of HBMECs to FITC-Dextran 3000 (h). The VE-cadherin Ab remained present during the assays. Data are mean Ϯ SD of three independent experiments. ␣-VE, VE-cadherin Ab cl75. Bar, 50 ␮m. observed. When, in addition to C3 pretreatment, the blocking cl75 Role of ICAM-1 and VCAM-1 in permeability and integrity of

Ab was used (Fig. 4d), VE-cadherin was diffusely distributed over HBMEC monolayers by guest on October 1, 2021 the cell surface (Fig. 4e) and the cells had lost cell-cell contact as As was shown in Figs. 1 and 2, loss of VE-cadherin function a consequence of reduced VE-cadherin function. This result also increased the permeability of HBMEC monolayers and promoted indicates that cellular contractility is not an absolute requirement transendothelial migration of CD34ϩ cells. To establish a role for for the Ab-mediated loss of VE-cadherin-mediated cell-cell adhe- intracellular signaling in the control of VE-cadherin-mediated cell- sion. Inactivation of p21Rho by C3 did not significantly affect the ϩ cell adhesion, induced by specific adhesion molecules, permeabil- migration of primary CD34 cells across HBMECs (Fig. 4g), in- ity assays in combination with Ab-mediated cross-linking were dicating a minor role for p21Rho in the control of transmigration ϩ performed. Cross-linking of ICAM-1 resulted in a significant de- of primary CD34 cells. Moreover, inhibition of active p21Rho by crease in the HBMEC permeability (Fig. 6a). In contrast, cross- C3 did not affect the permeability of HBMECs either (Fig. 4h). linking of VCAM-1 induced a significant increase in the perme- Role of ICAM-1 and VCAM-1 in VE-cadherin-mediated ability of HBMEC monolayers (Fig. 6a). Cross-linking of ICAM-1 transmigration of CD34ϩ cells induced stress fiber formation (Fig. 6bD), which was not accompanied, however, by altered VE-cadherin distribution at cell-cell ␤ It was previously shown by our group and others that the 1 in- junctions (Fig. 6bE) or discernible gaps in the HBMEC monolayer tegrins very late Ag (VLA)-4 and VLA-5 are required for efficient ϩ (Fig. 6bF). In contrast, cross-linking of VCAM-1 did induce loss transendothelial migration of CD34 cells (2, 11, 41, 42). There- of cell-cell contacts of endothelial cells (Figs. 6bG and 7d) and fore, we investigated which adhesion molecules that are known ϩ concomitant loss of VE-cadherin localization at sites of gap for- ligands for VLA-4 and VLA-5 mediate the migration of CD34 mation (Fig. 6, bH and bI). Kinetics of VCAM-1-induced perme- ␤ cells across HBMECs. ICAM-1 is highly expressed on IL-1 -pre- ability showed maximal increase in permeability already after 90 stimulated HBMECs, whereas VCAM-1 is expressed at a lower min (Fig. 6a, inset). level. Pretreatment of the HBMEC monolayers with blocking Abs to ICAM-1 inhibited the transmigration of PB and CB CD34ϩ cells across HBMECs. Blocking Abs against VCAM-1 also inhib- Role of ROS in VCAM-1-mediated gap formation ited the transmigration of PB and CB CD34ϩ cells. The combi- Cross-linking of VCAM-1 on activated HUVECs has been re- nation of Abs against ICAM-1 and VCAM-1 inhibited the trans- ported to be accompanied by the production of low levels of ROS migration of PB and CB CD34ϩ cells significantly (Fig. 5, a and (43). Moreover, ROS have been described to mediate intercellular b, respectively). After pretreatment of the HBMECs with the gap formation, cell shape change, and F-actin cytoskeletal reorga- blocking cl75 Ab to VE-cadherin, the transmigration of untreated nization in endothelial cells (44). In line with these data, we found PB CD34ϩ (Fig. 5a) and CB CD34ϩ cells (Fig. 5b) remained that incubation with the oxygen radical scavenger N-AC signifi- dependent on ICAM-1 and VCAM-1. Other blocking mAbs to cantly inhibited the VCAM-1-mediated increase in permeability VCAM-1 and ICAM-1 showed similar results. (Fig. 6a). In addition, cross-linking of VCAM-1, but not ICAM-1, The Journal of Immunology 593 Downloaded from http://www.jimmunol.org/

FIGURE 5. Role for endothelial adhesion molecules in SDF-1␣-induced transmigration of primary CD34ϩ cells across HBMECs. a, HBMECs were cultured on Transwell ﬁlters and prestimulated with IL-1␤ as described in Materials and Methods. Before the addition of the CD34ϩ cells from PB of healthy untreated volunteers to the upper compartment, the monolayers on the ﬁlter were incubated for 30 min with blocking Abs against adhesion molecules, followed by washing. mAbs against ICAM-1 (10 ␮g/ml;

␣-ICAM-1) and to VCAM-1 (10 ␮g/ml; ␣-VCAM-1) inhibited the SDF- by guest on October 1, 2021 1␣-induced migration of PB CD34ϩ cells (30 ng/ml SDF-1␣). The combination of Abs against ICAM-1 and VCAM-1 (␣-I-1 ϩ ␣-V-1) showed a significant inhibition of the transmigration. Pretreatment of HBMECs with FIGURE 6. a, Effect of cross-linking of adhesion molecules on the per- blocking Ab c175 against VE-cadherin (10 ␮g/ml, filled bars; VE-cadherin meability of HBMEC monolayers. HBMEC monolayer permeability was ϩ Ab present during the assay) increased basal migration of PB CD34 cells. measured after pretreatment of the endothelial cells with mAbs against Under these conditions, ICAM-1 was still required for efficient migration. ICAM-1, VCAM-1, or IgG1 (30 min, 10 ␮g/ml). Together with the addi- b, mAbs against ICAM-1 (10 ␮g/ml) and VCAM-1 (10 ␮g/ml) also in- tion of FITC-Dextran 3000, the Abs were cross-linked with G␣M-IgG, ϩ hibited the migration of CB CD34 cells. Blocking Ab cl75 against VE- which remained present during the 3-h assay. Cross-linking of ICAM-1 ␮ ␣ cadherin (10 g/ml, filled bars) increased basal SDF-1 -induced migration decreased the permeability of HBMECs (p Ͻ 0.001, compared with con- ϩ of CB CD34 cells, but mAbs against ICAM-1 and VCAM-1 were still trol), whereas cross-linking of VCAM-1 showed an increase in permeabil- Ϯ able to inhibit. Data are mean SD of at least three independent experi- ity of HBMECs (p Ͻ 0.001, compared with control). The oxygen radical Ͻ Ͻ Ͻ ments. bA, p 0.05; aB and bB, p 0.01; aC, p 0.001. scavenger N-AC significantly inhibited the permeability induced by VCAM-1 cross-linking (V1 ϩ N-AC) (p Ͻ 0.05, compared with cross- linked VCAM-1-induced permeability). Thrombin-induced permeability after 30 min was set to 100%. Data are mean Ϯ SD of at least three on HBMECs induced ROS production (Fig. 7a). Kinetic analysis, independent experiments. Inset, Kinetics of VCAM-1-induced permeabil- E f performed by time-lapse confocal microscopy, revealed a rapid ity, measured as described in Materials and Methods. , Control; , VCAM-1 cross-linking. b, Effect of cross-linking of adhesion molecules on increase in ROS production in HBMECs within 5Ð15 min after the morphology of HBMECs. HBMECs were grown to confluency on FN- VCAM-1 cross-linking (Fig. 7b). Moreover, transmigration of pri- ϩ coated glass coverslips, treated as indicated, fixed, and stained for VE- mary CB CD34 cells across HBMECs was significantly dimin- cadherin and F-actin. The images show F-actin in red (A, D, and G) and ished when HBMECs were pretreated with N-AC, indicating a role VE-cadherin (VE-cad) in green, immunostained with a polyclonal Ab (B, for oxygen radicals during transendothelial migration (Fig. 7c). In E, and H). Colocalization appears in yellow (C, F, and I). HBMEC mono- addition, pretreatment of HBMECs with N-AC, which by itself did layers were incubated for 30 min with mAbs against ICAM-1 (X-ICAM-1), not affect the actin cytoskeleton or VE-cadherin distribution, pre- followed by 30 min of cross-linking with G␣M-IgG. F-actin staining vented VCAM-1-induced gap formation (Fig. 7d). showed induction of stress fibers, but this was not accompanied by the ϩ formation of gaps between the cells (D). VE-cadherin was localized nor- Together, these results suggest that, upon adhesion of CD34 mally to cell-cell junctions (E). Cross-linking of VCAM-1 (X-VCAM-1) cells, activation of VCAM-1, probably in conjunction with induced stress fibers and gap formation in the HBMEC monolayer (G), ICAM-1, induces signaling in the endothelial cells that leads to with loss of discrete VE-cadherin localization at sites of absent cell-cell increased stress fiber formation and reduced endothelial cell-cell contacts (H). Staining of endothelial nuclei was due to nonspecific binding adhesion. of the ALEXA 488-labeled goat anti-rabbit Ig secondary Ab. Bar, 20 ␮m. 594 VE-CADHERIN MODULATES MIGRATION OF CD34ϩ CELLS Downloaded from

FIGURE 7. VCAM-1 activation induces generation of ROS in HBMECs. a, VCAM-1 and ICAM-1 were cross-linked as described in Materials and Methods on DHR-loaded and IL-1␤-pretreated HBMECs. After 30 min, the production of ROS was analyzed by FACS as described in Materials and Methods. Cross-linking of VCAM-1 showed a significant 20% increase in ROS production compared with control levels (A, p Ͻ 0.05), whereas ICAM-1 Ϯ cross-linking had no effect. Ten millimolar H2O2 was used as a positive control and increased the DHR response by 44% (data not shown). Data are mean SD of three independent experiments. b, Kinetics of ROS production were measured by time-lapse confocal microscopy during 30 min of VCAM-1 cross-linking as described in Materials and Methods. Data are shown as percentage increase relative to the values at the start of the experiment. f, Control; http://www.jimmunol.org/ Ⅺ, VCAM-1 cross-linking. Data represent the mean of two independent experiments. c, Role for ROS in SDF-1␣-induced transendothelial migration of primary CB CD34ϩ cells. Pretreatment and incubation of HBMECs with the oxygen radical scavenger N-AC (5 mM) showed a significant decrease in transmigration to SDF-1␣. Data represent the mean Ϯ SD of at least three independent experiments (cA, p Ͻ 0.05). d, Phase-contrast images show that gap formation, indicated by the arrowheads, occurred already after 15 min of VCAM-1 cross-linking. To prevent ROS-mediated signaling, HBMECs were pretreated for 18 h with 5 mM N-AC. This treatment prevented VCAM-1-mediated gap formation. Data are representative of three independent experiments. Bar, 100 ␮m. by guest on October 1, 2021 Discussion duced migration of lymphocytes across endothelium. Therefore, The present study demonstrates that VE-cadherin is a crucial reg- shear stress might also have its effect on stem cell homing and on ulator of the permeability of HBMECs and that its adhesive prop- the role of specific adhesion molecules in this process. erties control the efficiency of migration of hematopoietic progen- Conflicting results on the role of VCAM-1 and ICAM-1 in the ϩ itor cells (CD34ϩ cells) across HBMECs. In addition, endothelial transmigration of CD34 cells have been published (46, 47). adhesion molecules such as ICAM-1 and, in particular, VCAM-1 Therefore, we tested the hypothesis that leukocyte-endothelium were found to be important for CD34ϩ cell transmigration and, interactions, mediated by VCAM-1 and ICAM-1, induce endothe- upon activation, for the modulation of endothelial integrity. lial signaling that might control endothelial cell-cell adhesion. Primary CD34ϩ cells induced a focal loss of VE-cadherin and of Cross-linking experiments supported a role for VCAM-1 in the ␤-catenin at sites of transmigration. This focal loss of VE-cadherin regulation of endothelial cell-cell junctions. In contrast, ICAM-1 was reversible. The normal jagged pattern of VE-cadherin distri- failed to induce changes in endothelial cell-cell junctions or for- bution was restored after the transmigration assays (data not mation of gaps between the cells after cross-linking, despite the shown). A similar phenomenon was also reported by Allport et al. induction of actin stress fibers. Similar findings have been reported (35), who studied monocyte migration across HUVECs under by Del Maschio et al. (28). Moreover, activation of the small flow, albeit in the absence of a chemotactic gradient. One of the GTPase p21Rho by ICAM-1 cross-linking in HUVECs has been proposed mechanisms for the loss of VE-cadherin function during suggested (32). Our studies also indicate that in HBMECs, leukocyte passage is the trapdoor mechanism: the VE-cadherin- ICAM-1 may activate Rho, as deduced from the induction of stress catenin complex is mechanically pushed aside by the migrating fibers. However, this response was not sufficient to induce loss of leukocyte and simply re-adheres after leukocyte passage (35). HBMEC integrity. In line with this notion, inhibition of p21Rho by ϩ However, despite the fact that the VE-cadherin distribution was the C3 toxin did not affect the transmigration of CD34 cells or the lost at sites of CD34ϩ cell transmigration, the migration across integrity of HBMEC monolayers, suggesting only a minor role for HBMECs remained dependent on VCAM-1- and ICAM-1-medi- the p21Rho GTPase in the control of CD34ϩ cell transmigration. ated adhesion in the absence of VE-cadherin function. It is impor- Although several reports suggest a signaling role for ICAM-1 dur- tant to note that the transendothelial migration assay is performed ing leukocyte transmigration, e.g., through activation of p21Rho under static conditions. Although the VE-cadherin distribution (36, 48, 49), these findings indicate a differential, possibly cell- during transmigration under static conditions was similar to what type-specific role for a p21Rho signaling pathway in the control of Allport and colleagues (35) found using shear stress, the effect of endothelial monolayer integrity during leukocyte transmigration. blocking Abs to VCAM-1 and ICAM-1 on the transendothelial Moreover, in the absence of p21Rho activity, the endothelial cell- migration of CD34ϩ cells under flow remains to be investigated. cell contacts are still reduced upon Ab-mediated loss of VE-cad- Recently, Cinamon et al. (45) showed that shear stress itself in- herin function. This finding shows that gap formation, induced by The Journal of Immunology 595 anti-VE-cadherin Ab or cross-linking of VCAM-1, might occur also for our insight in endothelial cell function, which is important independently of cellular contractility. in a wide range of (patho)physiological conditions. The increased HBMEC permeability after VCAM-1 cross-linking and the accompanying formation of endothelial gaps suggest Acknowledgments that this Ig-like cell adhesion molecule can act as a bona fide sig- We thank Dr. D. Roos for critically reading the manuscript and Dr. E. naling receptor. Lorenzon et al. (30) already described a prominent Dejana for the TEA1.31 Ab. role for VCAM-1 in the induction of cytoskeletal changes in endothelial cells, although these authors did not analyze changes in References endothelial permeability or VE-cadherin localization. Our present 1. To, L. S., D. N. Haylock, P. J. Simmons, and C. A. Juttner. 1996. The biology and work adds new data to this issue in that the production of ROS clinical uses of blood stem cells. Blood 89:2233. 2. Voermans, C., W. R. Gerritsen, A. E. von dem Borne, and C. E. van der Schoot. appears to be a crucial signaling event in VCAM-1-induced gap ϩ 1999. Increased migration of cord blood-derived CD34 cells across uncoated or formation. The fact that VCAM-1-induced loss of cell-cell adhe- fibronectin-coated filters. Exp. Hematol. 27:1806. sion is dependent on the production of ROS represents a new 3. Aiuti, A., I. J. Webb, C. Bleul, T. Springer, and J. C. Gutierrez-Ramos. 1997. The chemokine SDF-1 is a chemoattractant for human CD34ϩ hematopoietic progen- mechanism by which cell surface molecules can modulate cad- itor cells and provides a new mechanism to explain the mobilization of CD34ϩ herin-mediated cell-cell adhesion. However, activation of ICAM-1 progenitors to peripheral blood. J. Exp. 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Nature 382:829. 7. Oberlin, E., A. Amara, F. Bachelerie, C. Bessia, J. L. Virelizier, endothelium, a function as a signaling receptor that modulates F. Arenzana-Seisdedos, O. Schwartz, J. M. Heard, I. Clark-Lewis, D. F. Legler, HBMEC cell-cell contact, presumably through the production of et al. 1996. The CXC chemokine SDF-1 is the ligand for LESTR/fusin and pre- ROS. Interestingly, ICAM-1-mediated production of ROS in vents infection by T-cell-line-adapted HIV-1. Nature 382:833. 8. Loetscher, M., T. Geisser, T. O’Reilly, R. Zwahlen, M. Baggiolini, and B. Moser.

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