Intercellular Adhesion Molecule (ICAM)-1, But Not ICAM-2, Activates RhoA and Stimulates c-fos and rhoA Transcription in Endothelial Cells This information is current as of September 25, 2021. Paul W. Thompson, Anna M. Randi and Anne J. Ridley J Immunol 2002; 169:1007-1013; ; doi: 10.4049/jimmunol.169.2.1007 http://www.jimmunol.org/content/169/2/1007 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. The Journal of Immunology

Intercellular Adhesion Molecule (ICAM)-1, But Not ICAM-2, Activates RhoA and Stimulates c-fos and rhoA Transcription in Endothelial Cells1

Paul W. Thompson,*† Anna M. Randi,‡ and Anne J. Ridley2*†

ICAM-1 and -2 are integrin-binding Ig superfamily adhesion molecules that are important for leukocyte transmigration across endothelial monolayers. ICAM-1 cross-linking is known to activate the small GTPase RhoA and induce stress fiber formation in endothelial cells, but ICAM-2 signaling has not been investigated. In this study, we compare ICAM-1 and ICAM-2 signaling and localization in HUVECs. Although ICAM-1 and ICAM-2 both localize with the -binding moesin in apical microvilli, only ICAM-1 colocalizes with moesin after cross-linking. Unlike ICAM-1, ICAM-2 does not activate RhoA or alter actin cytoskel-

etal organization. Interestingly, ICAM-1 stimulates transcription of c-fos, a known early response . In addition, it up-regulates Downloaded from rhoA expression, suggesting that it activates a positive feedback pathway after RhoA activation. These results indicate that in endothelial cells, ICAM-1, but not ICAM-2, rapidly stimulates signaling responses involving RhoA. The Journal of Immunology, 2002, 169: 1007–1013.

he vascular endothelium serves as a selective blood/tissue sponses (7). In contrast, in vitro studies indicate that ICAM-2 barrier, controlling the extravasation of solutes and mac-

contributes to leukocyte transendothelial migration under non- http://www.jimmunol.org/ T romolecules and the passage of leukocytes into surround- inflammatory conditions and therefore could play a role in ing tissues. Leukocyte transmigration is dependent upon the ex- leukocyte recirculation (8, 9). Although lymphocyte homing to pression of adhesion receptors on the apical surface of the lymph nodes was apparently unimpaired in ICAM-2-deficient endothelium, including Ig superfamily members such as VCAM-1 mice (10), it has recently been reported that ICAM-2 plays an and ICAM-1 (1, 2). ICAM-2 is closely related to ICAM-1, but only important role in mediating DC transmigration across resting has two Ig-like extracellular domains, compared with the five Ig endothelium (5), and thus it may contribute to the tissue domains of ICAM-1 (3). Both were identified through their ability recruitment of a subset of hemopoietic cell types. ␤ to bind the same ligand, the 2 integrin LFA-1 (CD11a/CD18), As well as being critical for leukocyte adhesion, ICAM-1 can which is expressed on all leukocytes (3, 4). Recently, ICAM-2 has activate signaling pathways. Ab-induced cross-linking of ICAM-1 by guest on September 25, 2021 also been reported to bind to another ligand, dendritic cell (DC)3- on rat brain-derived endothelial cell lines has been reported to specific ICAM-grabbing nonintegrin, a C-type lectin expressed induce the activation of the Src tyrosine kinase and phosphoryla- only on DCs (5). tion of the cytoskeletally associated focal adhesion kinase, In resting endothelial cells, ICAM-1 is expressed at very low paxillin, p130Cas and cortactin, as well as the activation of the levels whereas ICAM-2 is expressed abundantly. Proinflamma- small GTPase Rho (11Ð13). In the same cell lines, ICAM-1 cross- tory cytokines such as TNF-␣ induce a large increase in ICAM-1 levels (1) and a concomitant decrease in ICAM-2 linking induced actin cytoskeletal reorganization, which was levels on the surface of endothelial cells (6). ICAM-1 is shown to involve Rho, intracellular calcium signaling, and p38 essential for stable adhesion and transmigration of leukocytes in mitogen-activated protein kinase activation (12, 14). Rho mediates nearly all types of inflammatory response (1), and ICAM-1- the assembly of stress fibers and associated focal contacts in many deficient mice have impaired inflammatory and immune re- cell types, including endothelial cells (12, 15, 16), and induces contractility in HUVECs leading to the formation of intercellular gaps which could aid the transmigration of leukocytes (16). In *Ludwig Institute for Cancer Research, Royal Free and University College School of Medicine, and †Department of Biochemistry and Molecular Biology, University Col- addition, Rho is required for monocyte adhesion to HUVECs, and lege London, London, United Kingdom; and ‡Experimental Medicine, GlaxoSmith- mediates clustering of ICAM-1 underneath the monocytes (17). Kline, Addenbrooke’s Center for Clinical Investigation, Addenbrooke’s Hospital, Furthermore, Rho can activate in coordination Cambridge, United Kingdom with Ras activation, by activating the serum response element Received for publication February 13, 2002. Accepted for publication May 7, 2002. (SRE) promoter sequence (18). In contrast to ICAM-1, a possible The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance signaling function of ICAM-2 in endothelial cells has not been with 18 U.S.C. Section 1734 solely to indicate this fact. investigated. However, in T cells, it has recently been reported that 1 P.W.T. was funded by a Medical Research Council studentship (U.K.). ICAM-2 can activate the serine/threonine kinase Akt/protein 2 Address correspondence and reprint requests to Dr. Anne J. Ridley, Ludwig Institute kinase B (19). for Cancer Research, 91 Riding House Street, London W1W 7PS, U.K. E-mail ad- The cytoplasmic tails of ICAM-1 and ICAM-2 could be respon- dress: [email protected] sible for transducing signals in the cell. Despite their short length 3 Abbreviations used in this paper: DC, dendritic cell; SRE, serum response element; ERM, , , and moesin; N-ERMAD, N-terminal domain of ERM proteins; (ICAM-1 has 28 aa and ICAM-2 has 26 aa), both ICAM-1 and F-actin, filamentous actin; CPERM, C-terminally phosphorylated ERM protein; ICAM-2 cytoplasmic tails can interact with ezrin in vitro (20). TRITC, tetramethylrhodamine isothiocyanate; EGM, endothelial growth medium; TRBD, rhotekin Rho-binding domain; GST-TRBD, TRBD bound to GST; SHP-2, Src Ezrin is a member of the ERM (ezrin, radixin, and moesin) family, homology 2 domain-containing tyrosine phosphatase-2. which can function as a linker between the plasma membrane and

Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00 1008 ICAM-1, ICAM-2, AND RHO

the actin (21), and play an important role in lamelli- with human fibronectin until confluent. To obtain quiescent-starved cells, podium extension (22), cell-cell adhesion (23), microvillus assem- the culture medium was replaced by medium containing 10% FCS, but no bly, and cytokinesis (23). Ab-cross-linked ICAM-1 coclusters with heparin or other growth factors. Cells were incubated in this medium for no longer than 24 h. Cells grown in EGM-2 were starved in 1% FCS. ERM proteins in endothelial cells (17). The N-terminal domain of No differences between HUVEC responses were observed between cells ERM proteins (N-ERMAD) can interact with both phosphoino- grown in different media. sitides and a number of proteins, while the C-terminal domain of ERM proteins binds to filamentous actin (F-actin). There is Receptor clustering and immunofluorescence evidence that Rho activation occurs downstream of ERMs as N-ERMADs can interact with RhoGDI, which normally holds Rho To induce receptor clustering, either mouse monoclonal anti-ICAM-2 Ab in an inactive GDP-bound complex in the cytoplasm. N-ERMAD was added to starved cells at a final concentration of 10 ␮g/ml for 60 min, or mouse monoclonal anti-ICAM-1/ICAM-2 were added to cells that had binding to RhoGDI facilitates exchange of GDP for GTP on Rho, been stimulated for 24 h with 10 ng/ml TNF-␣. After incubation with presumably because Rho is released from RhoGDI (24). Also, primary Abs, TNF-␣ and the primary Abs were removed from the cell more recently, the ICAM-1 cytosolic tail has been shown to inter- medium and 10 ␮g/ml of Alexa 488-labeled goat anti-mouse Ab was added act with the Src homology 2 domain containing tyrosine phospha- to the cells for between 15 and 60 min. Cells were then washed three times tase-2 (SHP-2) in a phosphotyrosine-dependent manner (25). in TBS (10 mM Tris, pH 7.5, 150 mM NaCl) containing 0.25% BSA, fixed in 4% paraformaldehyde for 20 min at room temperature, permeabilized for In this study, we compare the effects of ICAM-1 and -2 cross- 6 min with 0.2% Triton X-100, and then incubated with 1 ␮g/ml TRITC- linking in primary human umbilical cord endothelial cells upon phalloidin for 45 min to stain actin filaments, or for 45 min with rabbit cytoskeletal arrangements and gene expression. We show that al- polyclonal anti-moesin Ab diluted 1/200, or rabbit polyclonal anti-myosin though ICAM-1, ICAM-2, and moesin all localize in microvilli in II Ab diluted 1/25, followed by the appropriate TRITC-labeled secondary Downloaded from Ab. The specimens were mounted in moviol. To observe responses in cells unstimulated cells, only ICAM-1 cross-linking coclusters moesin, without receptor clustering, cells were incubated with primary Abs as be- activates RhoA, and causes stress fiber formation, whereas fore for 60 min. These cells were then washed, fixed, and stained with the ICAM-2 cross-linking does not affect the actin cytoskeleton. Fur- secondary Ab for 45 min. Staining for intracellular epitopes was then con- thermore, ICAM-1 cross-linking induces rhoA and c-fos gene ex- ducted as described above. All incubations were conducted in TBS con- pression. Thus, we provide a mechanistic basis for the difference taining 0.25% BSA. To use the phosphospecific anti-CPERM Ab, a different fixation proce- between the roles of ICAM-1 and -2 in leukocyte transmigration. dure was used as previously described (38). In brief, after receptor clus- http://www.jimmunol.org/ tering, cells were fixed in 10% TCA in distilled water at 4¡C for 20 min. Materials and Methods All further staining was conducted in TBS containing 0.25% BSA. Cells Materials were incubated with rat monoclonal anti-CPERM Ab (297S) for 45 min, followed by Cy5-conjugated goat anti-rat Ab for 45 min. Reagents were obtained from the following sources: medium 199 modified Confocal laser scanning microscopy was conducted with an LSM 510 Earle’s salt solution and HBSS (Life Technologies, Paisley, U.K.); Nutri- (Zeiss, Welwyn Garden City, U.K.) mounted over an affinity corrected doma NS (Boehringer Mannheim, Lewes, U.K.); pooled HUVECs and Axioplan microscope (Zeiss). Image files were collected as a matrix of endothelial growth medium (EGM)-2 (Clonetics, San Diego, CA); human 1024 ϫ 1024 pixels describing the average of eight frames scanned at fibronectin, heparin, endothelial cell growth supplement, tetramethylrho- 0.062 Hz where FITC, TRITC, and Cy5 were excited at 488, 543, and 633

damine isothiocyanate (TRITC)-phalloidin, HEPES, collagenase type II, nm and visualized with 540 Ϯ 25, 608Ϯ 32, and 690 Ϯ 30 nm bandpass by guest on September 25, 2021 aprotinin, leupeptin, and other lysis buffer components, unless otherwise filters, respectively, where the levels of interchannel cross-talk were stated (Sigma-Aldrich, Gillingham, U.K.); TNF-␣ (Insight Biotechnology, insignificant. Wembley, U.K.); mouse monoclonal anti-ICAM-1 Ab (clone BBIG-I1; R&D Systems, Abingdon, U.K.); mouse monoclonal anti-ICAM-2 Ab (clone CBR-IC2/2; Alexis Biochemicals, Nottingham, U.K.); goat poly- DNA constructs and transfection of HUVECs clonal anti-moesin Ab (C-15; Sigma-Aldrich); Alexa 488-labeled goat anti- mouse IgG and Alexa 546-labeled goat anti-mouse IgG Abs (Molecular pCDM8-ICAM-1 (human) has been previously described (26). The cDNA Probes, Leiden, The Netherlands); FITC- and TRITC-labeled goat anti- encoding human ICAM-2 (3) was subcloned into pcDNA3 using EcoRV mouse and goat anti-rabbit Abs (Southern Biotechnology Associates, Bir- and NotI restriction sites. HUVECs were transfected using a mixture of P6 mingham, AL); Cy5-conjugated goat anti-rat Ab (Jackson ImmunoRe- integrin peptide and Lipofectin (Life Technologies) as previously de- search Laboratories, West Grove, PA); rabbit polyclonal anti-myosin II Ab scribed (27). Briefly, P6 integrin-targeting peptide, lipofectin, and plasmid (Biogenesis, Poole, U.K.); mouse monoclonal anti-RhoA Ab (Santa Cruz DNA were allowed to form a complex, which was incubated with subcon- Biotechnology, Santa Cruz, CA); HRP-conjugated goat anti-mouse Ab fluent cells in Optimem for 4 h. Cells were then washed in Optimem and (Bio-Rad, Hemel Hempstead, U.K.); and Fugene 6 (Boehringer Mann- grown to confluence in their normal growth medium (medium 199 with heim). Rabbit polyclonal anti-moesin Ab was generously provided by Dr. supplements). P. Mangeat (Universite« Montpellier II, Montpellier, France); rat monoclo- nal anti-COOH (Thr-558) C-terminally phosphorylated ERM proteins (anti- CPERM; 2975) were generously provided by Dr. S. Tsukita (Kyoto Uni- RhoA activity assay versity Faculty of Medicine, Kyoto, Japan). Integrin peptide P6 was GST-rhotekin Rho-binding domain (TRBD; a gift from Dr. M. Schwartz, generously provided by Dr. S. Hart (Institute of Child Health, London, Scripps Institute, San Diego, CA) was expressed in Escherichia coli and U.K.). Lightcycler apparatus and PCR reagents were obtained from Roche purified as previously described (28). TNF-␣-activated HUVECs (on (Basel, Switzerland), primers for rhoA gene expression analysis were ob- 10-cm dishes) were subject to Ab-induced ICAM-1/ICAM-2 clustering as tained from Life Technologies, and primers for c-fos gene expression anal- described above. Control dishes were treated with primary or secondary ysis were obtained from Sigma-Genosys (Pampisford, U.K.). Abs alone, or were given fresh medium before lysis. Cells were lysed in Cell culture lysis buffer (50 mM Tris, pH 7.3, 1% Triton X-100, 0.5% sodium deoxy- ␮ cholate, 0.1% SDS, 500 mM NaCl, 10 mM MgCl2,10 g/ml each of HUVECs were isolated from umbilical cords using 0.1% collagenase type leupeptin and aprotinin, 1 mM PMSF). Lysates were incubated with 20 ␮g II. The cells were cultured in TC Nunclon flasks in medium 199 modified of GST-TRBD beads for1hat4¡C. Beads were washed in buffer B (50

Earle’s salt solution containing 1.25 g/L NaHCO3 and Glutamax and sup- mM Tris pH 7.3, 1% Triton X-100, 150 mM NaCl, 10 mM MgCl2,10 plemented with 20% FCS, 100 ␮g/ml endothelial cell growth supplement, ␮g/ml each of aprotinin and leupeptin, 100 ␮M PMSF). Proteins retained 1% Nutridoma NS, and 100 ␮g/ml heparin. Alternatively, HUVECs from on the GST-TRBD beads were resolved by 13% SDS-PAGE. Bound RhoA Clonetics were grown in EGM-2. Cells were cultured at 37¡C in humidified was detected by Western blotting using mouse monoclonal anti-RhoA Ab.

air containing 5% CO2. For experiments, cells were used between one and Quantification of relative RhoA activity was performed using the photo- three passages. densitometry program Quantity One (Bio-Rad). The amount of activated For immunofluorescence experiments, cells were grown on glass cov- RhoA was normalized to the amount of total RhoA in the corresponding erslips coated with 10 ␮g/ml human fibronectin until confluent. For bio- lysate for each treatment. The significance of any difference in RhoA ac- chemical experiments, cells were grown on 6- and 10-cm dishes coated tivity after ICAM clustering was determined by Student’s t test. The Journal of Immunology 1009

Gene expression analysis localization of ICAMs and ERM proteins in primary endothelial TNF-␣-activated HUVECs (10 ng/ml, 24 h) were stimulated with either cells. In resting HUVECs, ICAM-2 colocalized with moesin at ICAM-1 or -2 cross-linking Abs for either 60 or 90 min. Total RNA was intercellular junctions and at the apical surface (Fig. 1, aÐc). In- isolated using the Qiagen RNeasy kit (Valencia, CA). First strand-cDNA terestingly, colocalization also occurred in distinct microvillus-like synthesis was performed on the total RNA preparations to provide a cDNA structures on the apical surface. These structures also contained template for gene expression analysis. F-actin (Fig. 1, dÐf) and are therefore likely to be microvilli. Co- Qualitative gene expression was performed using the Lightcycler sys- ␣ tem. Primers for real-time PCR were designed close to the 3Ј end of rhoA localization of moesin and ICAM-2 was also observed in TNF- - and c-fos and were as follows: rhoA 5Ј-ATGTGCCCACAGTGTTTGA activated HUVECs (data not shown). ICAM-1 was not expressed GAAC; rhoA 3Ј-CGTTGGGACAGAAATGCTTGACT; c-fos 5Ј-TCAC significantly in resting, unstimulated HUVECs (data not shown; CCTGCCTCTCCTCAAT; c-fos 3Ј-GCTGCATAGAAGGACCCAGATAG. Ref. 1). After 24 h of TNF-␣ stimulation (10 ng/ml), ICAM-1 Standard curves were generated using rhoA cDNA as a template and the rhoA primers. Therefore, samples were analyzed for gene copy numbers as expression was highly induced and colocalized with moesin at in- compared with the rhoA standard curve. In the case of samples analyzed for tercellular junctions and in microvillus-like structures with moesin c-fos, the standard curve was still made from rhoA transcripts so this anal- (Fig. 1, gÐi), similar to the distribution of ICAM-2. These results ysis could only be qualitative. Normalization of the samples was conducted indicate that leukocyte-binding receptors ICAM-1 and -2 are op- by assessing the GAPDH mRNA content in each sample against a GAPDH timally positioned on endothelial cell microvilli for interaction standard curve. For qualitative analysis, all 0-min timepoint samples were ␤ taken as the reference point, with changes induced by stimulation repre- with 2 integrins. sented as changes relative to the 0-min timepoint. Results were obtained from two separate stimulations. ICAM-1, but not ICAM-2, cross-linking induces stress fiber

RhoA protein levels were also determined after ICAM-1 and -2 cross- formation and moesin coclustering Downloaded from linking. After cross-linking, cells were lysed in 2ϫ Laemmli sample buffer. Proteins were resolved by SDS-PAGE and transferred to polyvinylidene It has previously been shown that Ab-induced ICAM-1 clustering difluoride membrane followed by immunoblotting for RhoA. Blots shown induces the activation of Rho and the formation of stress fibers in are representative of three separate experiments. rat brain-derived microvascular endothelial cell lines (13). There- fore, we investigated the effects of ICAM-1 and -2 Ab-induced Results

Both ICAM-1 and -2 colocalize with moesin and F-actin in http://www.jimmunol.org/ HUVECs ICAMs have been shown to interact with ERM proteins in vitro and using exogenously expressed proteins. Endothelial cells ex- press ICAM-2 constitutively and can be induced to express high levels of ICAM-1 by inflammatory stimuli. Therefore, immuno- fluorescence techniques were used to investigate the subcellular by guest on September 25, 2021

FIGURE 2. ICAM-1 cross-linking induces actin stress fiber formation in HUVECs. Starved monolayers were either treated with 10 ng/ml TNF-␣ FIGURE 1. ICAMs colocalize with moesin in endothelial cell mi- for 24 h (aÐf) or left untreated (g and h). Untreated cells (g and h) had been crovilli. Confluent HUVECs were starved for 24 h in medium 199 con- transfected 5 days previously with pCDM8-ICAM-1(human). Cells were taining 10% FCS. Starved monolayers were either untreated (aÐf) or treated incubated with 10 ␮g/ml mouse monoclonal anti-ICAM-1 Ab for 60 min. with 10 ng/ml TNF-␣ for 24 h (gÐi), fixed in 4% paraformaldehyde, and Cells were either washed, fixed, and stained with Alexa 488-goat anti- stained for ICAM-2 (a, c, d, and f) or ICAM-1(g and i) with mouse mAbs. mouse Ab for 60 min (a and b), or washed in conditioned medium and then Cells were then refixed, permeabilized in 0.2% Triton X-100, and stained incubated with the secondary Ab for 30 min (e and f)or60min(cÐe and for moesin (b, c, h, and i) using a rabbit polyclonal Ab, and for F-actin (e h). All cells were then fixed, permeabilized, and stained for either F-actin and f) using TRITC-phalloidin. In merged images, ICAMs are shown in (b, d, and h) using TRITC-phalloidin, or moesin (f) using a rabbit poly- green, while F-actin and moesin are shown in red. Bars, 10 ␮m. clonal Ab. Bars, 10 ␮m. 1010 ICAM-1, ICAM-2, AND RHO cross-linking on the actin cytoskeleton of HUVECs. Ab-cross- In contrast to ICAM-1, Ab cross-linking of ICAM-2 for 60 min linking of ICAM-1 for 60 min on HUVECs induced the formation on resting HUVECs only induced the formation of small clusters of F-actin bundles which aligned parallel to the elongated axis of of ICAM-2 and had no effect on the arrangement of F-actin (Fig. the cell (compare Fig. 2, b and d). ICAM-1 cross-linking also 3, aÐd). Myosin II staining also confirmed that no increase in induced the assembly of myosin II-containing filaments (data not stress fibers occurred in response to ICAM-2 cross-linking (data shown), confirming that the newly formed F-actin structures were not shown). TNF-␣ treatment for 24 h induced a small increase in stress fibers, and this was accompanied by the appearance of in- stress fibers relative to quiescent HUVECs (compare Fig. 3, b and tercellular gaps, indicative of increased contractility. f), but ICAM-2 cross-linking after TNF-␣ stimulation had no effect Ab-induced cross-linking led to the accumulation of large upon the number or organization of F-actin cables (Fig. 3, eÐh). ICAM-1 clusters (Fig. 2c), as previously described (17). The ma- Surprisingly, moesin did not cocluster with ICAM-2 (Fig. 3, i and jority of these large ICAM-1 clusters were on the cell surface and j), although it colocalized with moesin in microvilli before clus- not internalized (data not shown). As we showed that ICAM-1 tering (Fig. 1, aÐc). colocalized with moesin in microvilli (Fig. 1), we investigated the effects of Ab-induced ICAM-1 cross-linking on the localization of ICAM-2 does not activate RhoA moesin. ICAM-1 cross-linking caused a dramatic redistribution of As stress fiber formation is dependent on RhoA (15), we assayed moesin, so that moesin colocalized with ICAM-1 clusters (Fig. 2, the effects of ICAM-1 and -2 cross-linking upon RhoA activity e and f), consistent with our previous report of ERM colocalization using the TRBD to affinity-precipitate endogenous GTP-bound with cross-linked ICAM-1 (17). RhoA from endothelial cell lysates (28). RhoA was activated be- To determine whether TNF-␣ signaling is required for ICAM-1 tween 15 and 30 min after ICAM-1 clustering and remained acti- Downloaded from to induce stress fiber assembly, we transfected HUVECs with ex- vated at 1 h (Fig. 4). RhoA activity showed a 2.5-fold increase as ogenous ICAM-1 so that they could be cross-linked on resting, compared with resting levels at this time point. ICAM-2 clustering, unstimulated cells. Under these conditions, ICAM-1 cross-linking however, had no effect on RhoA activity. This data confirms re- was able to stimulate the formation of stress fibers (Fig. 2, g and sults from immunofluorescence experiments in Figs. 2 and 3, and h), indicating that TNF-␣ prestimulation is not required for this response. http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. ICAM-1, but not ICAM-2, cross-linking induces RhoA ac- tivation. a, Rho activity was assayed using GST-TRBD to pull-down GTP- bound Rho only. Confluent 10-cm dishes of starved HUVECs were treated with TNF-␣ (10 ng/ml) for 24 h. Following ICAM-1 or -2 cross-linking, cells were lysed at the indicated timepoints. Lysates were then incubated with GST-TRBD on glutathione beads for 60 min and bound proteins were resolved by 13% SDS-PAGE (upper panel). Proteins were transferred to polyvinylidene difluoride membranes and immunoblotted for RhoA using a mouse monoclonal anti-RhoA Ab. A fraction of each lysate was retained to determine the relative level of Rho (total Rho) in each lysate by immu- noblot analysis (lower panel). 1Ј: primary mouse anti-ICAM-1 or -2 Ab FIGURE 3. ICAM-2 cross-linking does not induce actin cytoskeleton alone; 2Ј: secondary goat anti-mouse Ab alone. Blots shown are represen- rearrangement. Starved monolayers were either left untreated (aÐd, i, and tative of three separate experiments. b, Relative RhoA activation was quan- j) or treated with 10 ng/ml TNF-␣ for 24 h (eÐh). Cells were incubated with tified using photodensitometry. The data represent means Ϯ SD of three 10 ␮g/ml mouse monoclonal anti-ICAM-2 for 60 min. Cells were either independent experiments. Densitometry readings for activation were nor- washed, fixed, and stained with Alexa 488-goat anti-mouse Ab (a, b, e, and malized to “Total Rho” densitometry readings for each individual treat- f), or washed in conditioned medium and then incubated with the secondary ment. GAM: secondary goat anti-mouse Ab alone; MoMo: primary mouse Ab for 60 min (c, d, and gÐj). All cells were then fixed, permeabilized, and anti-ICAM-1 or -2 Ab alone. RhoA activation induced by ICAM-1 clus- stained for either F-actin (b, d, f, and h) using TRITC-phalloidin, or moesin tering was significantly different from that produced by ICAM-2 clustering (i and j) using a rabbit polyclonal Ab. Bar, 10 ␮m. (p Ͻ 0.005) as determined by the paired Student’s t test. The Journal of Immunology 1011 indicates that the effects of ICAM-1 and -2 cross-linking on the min of ICAM-2 cross-linking induced a relatively small 1.4-fold endothelial cytoskeleton are dramatically different. increase in c-fos mRNA levels. It is possible that this small in- crease was caused by addition of Abs alone, rather than a specific ICAM-1 cross-linking induces rhoA and c-fos gene expression consequence of ICAM-2-induced signaling pathways. RhoA has been shown to stimulate transcription of the c-fos gene Interestingly, ICAM-1 cross-linking also induced a small in- through activation of the SRE (29). As ICAM-1 cross-linking ac- crease in rhoA mRNA expression levels (Fig. 5b). At 90 min after tivated RhoA, we tested whether c-fos expression was affected by ICAM-1 cross-linking, a 1.5-fold increase in rhoA mRNA levels ICAM-1 or -2 cross-linking. ICAM-1 cross-linking induced a dra- was observed as compared with resting levels. This increase in matic 6-fold increase in c-fos mRNA levels (Fig. 5a), returning to mRNA was paralleled by an increase in RhoA protein levels after resting levels after 90 min of ICAM-1 cross-linking. In contrast, 60 ICAM-1 cross-linking (Fig. 5c). Quantification of the increase in protein levels by photodensitometry showed that at 135 min of ICAM-1 cross-linking, the level of RhoA protein levels was in- creased by 1.5-fold compared with resting levels. In contrast, ICAM-2 cross-linking did not induce an increase in rhoA mRNA or protein levels. In fact, at 90 min of ICAM-2 cross-linking, there was a small decrease in rhoA mRNA levels (Fig. 5b). There results suggest that activation of RhoA may feed back to stimulate an increase in RhoA protein levels. Downloaded from Discussion ICAM-1 and ICAM-2 are related leukocyte-binding receptors on endothelial cells involved in mediating leukocyte transmigration across the endothelium. As leukocyte-endothelial interaction can trigger responses in endothelial cells (13, 17), we have used Ab- induced cross-linking to compare responses induced by ICAM-1 http://www.jimmunol.org/ and -2. We have shown that ICAM-1 and -2 both colocalize with the ERM family protein moesin and F-actin in endothelial cell microvilli. However, the responses to ICAM-1 and ICAM-2 are strikingly different. Ab cross-linked ICAM-1 coclusters moesin, activates RhoA to induce the formation of stress fibers in HUVECs, and induces expression of rhoA and c-fos , whereas cross-linked ICAM-2 does not colocalize with moesin or

alter RhoA activity. by guest on September 25, 2021

ICAM localization and ERM proteins We have shown that both ICAM-2 in resting cells and ICAM-1 on activated cells localize with moesin in apical microvilli. This is consistent with previous reports showing the presence of CD44 and exogenously expressed ICAM-2 at microvilli in fibroblasts, although fibroblasts do not express endogenous ICAM-2. Position- ing of leukocyte-binding receptors on endothelial cell microvilli is likely to facilitate leukocyte capture. We have observed a high level of C-terminal phosphorylation of ERM proteins in HUVECs (data not shown), indicating that they are predominantly in an ac- tive conformation and are therefore likely to link ICAMs to F-actin at microvilli.

ICAM-1 and RhoA activation

FIGURE 5. ICAM-1, but not ICAM-2, cross-linking induces rhoA and We have found that ICAM-1 cross-linking on primary endothelial c-fos transcription. a, TNF-␣-activated HUVECs (10 ng/ml, 24 h) were cells induces the formation of stress fibers and the activation of treated with ICAM-1 or -2 cross-linking Abs for 60 and 90 min. Total RNA RhoA, in accordance with previous results with a rat brain-derived was isolated and analyzed for expression of c-fos using real-time PCR and endothelial cell line (12). Although ICAM-1 is only normally ex- specifically designed 5Ј and 3Ј c-fos primers. Results are shown as a quan- pressed in HUVECs following activation with cytokines such as titative measure of fold increase from c-fos mRNA levels in untreated cells. TNF-␣, cytokine stimulation is not required for ICAM-1 responses The results are from two separate cross-linking treatments. b, HUVECs as cross-linking of exogenously expressed ICAM-1 on unstimu- were treated as in a and rhoA mRNA expression was analyzed using 5Ј and lated HUVECs can still induce stress fiber formation. The level of Ј 3 rhoA primers. Results are from two separate treatments. Results are ICAM-1 engaged by Ab in transfected cells is considerably lower shown as a qualitative measure of fold increase from rhoA mRNA levels in than that on TNF-␣-stimulated cells, and may be similar to the untreated cells. The results are from two separate cross-linking treatments. c, HUVECs were treated with cross-linking Abs for either 60 or 135 min amount of ICAM-1 normally engaged by monocyte binding. and then lysed in 2ϫ Laemmli sample buffer. RhoA was analyzed by In contrast to ICAM-1, ICAM-2 does not activate RhoA, either Western blotting (upper panel) and differences were quantitated by pho- in resting HUVECs or in TNF-␣-stimulated HUVECs. Ab-induced todensitometry (figures in parentheses). The blot was reprobed with an ICAM-2 clusters are very small and do not aggregate into larger anti-␤-tubulin Ab (lower panel) to show equal protein loading. clusters unlike those formed by ICAM-1. Both ICAMs can bind to 1012 ICAM-1, ICAM-2, AND RHO

␤ the same ligand, the 2 integrin, LFA-1, but their short cytoplas- uously survey the tissues for incoming foreign Ags. Immature DCs mic tails are markedly different in sequence and may therefore have to migrate from the blood into the periphery and this is not interact with different signaling proteins (1, 3, 30). Several proteins dependent upon inflammatory signals. Therefore, constitutive ex- have been reported to interact with the ICAM-1 cytosolic tail (20, pression of ICAM-2 on endothelial cells may be important for the 25, 31), and of these, the SHP-2 and ERM proteins could be in- transmigration of immature DCs. volved in ICAM-1 signal transduction. SHP-2 has recently been Our results showing that ICAM-1, but not ICAM-2, activates shown to interact with a poorly conserved immunoreceptor ty- RhoA, induces actin reorganization, and stimulates gene expres- rosine-based inhibitory motif contained within the cytosolic tail of sion fit with their different roles in leukocyte transmigration. Under ICAM-1, after ICAM-1-mediated adhesion of cells to fibrinogen inflammatory conditions, where ICAM-1 is involved, RhoA acti- (25). SHP-2 has been reported to down-regulate RhoA activity in vation would contribute to the inflammatory response of endothe- fibroblasts and epithelial cells (32, 33). ICAM-2 would not be lial cells through its effects in disrupting intercellular junctions, expected to bind to SHP-2 as the sequence in the ICAM-1 tail that enhancing endothelial permeability (16, 37). Increased transcrip- binds SHP-2 is not conserved in ICAM-2. Alternatively, ERM tion of selected genes is likely to contribute to the progression and proteins could mediate RhoA activation downstream of ICAM-1 eventual resolution of the endothelial response to inflammation. (34). Rho-GDI has been shown to interact with moesin, and this During routine leukocyte trafficking, such as immature DC trans- interaction with ERM proteins causes a decrease in the ability of migration, these responses to RhoA would be detrimental as they Rho-GDI to inhibit nucleotide exchange on Rho (24), leading to would promote vascular leakage, and thus it makes sense that Rho activation. Results from this study show that moesin coclus- ICAM-2 does not activate Rho. Indeed, it may actively inhibit tered with ICAM-1 rather than ICAM-2 and therefore it is possible Rho signaling, as indicated by the decrease in rhoA mRNA Downloaded from that an ICAM-1/moesin interaction is required for signaling to levels following ICAM-2 cross-linking. Therefore, the role of RhoA. However, it is also likely that the function of ERM inter- endothelial cell signaling in leukocyte transmigration is likely action with ICAMs is to stabilize interaction with the actin to be very different in inflammatory compared with noninflam- cytoskeleton. matory conditions.

ICAM-1 induces gene expression http://www.jimmunol.org/ In this study, we show that ICAM-1 cross-linking in TNF-␣-acti- Acknowledgments vated HUVECs stimulates expression of c-fos and rhoA. ICAM-1 We thank the staff in the maternity ward of University College Hospital for supplying umbilical cords; Dr. Martin Schwartz for the plasmid-encoding cross-linking has previously been reported to stimulate expression GST-TRBD; Dr. Paul Mangeat (Montpellier, France) for the anti-moesin of the leukocyte adhesion molecule VCAM-1 and to activate the Ab; Dr. S. Tsukita (Kyoto, Japan) for the anti-phospho-ERM Ab; Dr. S. transcription factor activator protein (AP)-1 in resting endothelial Hart (Institute of Child Health, London, U.K.) for advice and P6 integrin cells (35), but ICAM-1-mediated changes in transcription in TNF- peptide for HUVEC transfections; and Dr. M. Goulden for assistance with ␣-stimulated cells, which are more physiologically relevant as they Lightcycler studies. express high levels of ICAM-1, have not been investigated before. by guest on September 25, 2021 Active RhoA has been shown to induce gene expression through References activation of the SRE (29). In particular, RhoA can enhance tran- 1. Springer, T. A. 1994. Traffic signals for lymphocyte recirculation and leukocyte scriptional activation of c-fos, egr-1, and cox-2. Therefore, the emigration: the multistep paradigm. Cell 76:301. ICAM-1-induced stimulation of c-fos transcription correlates well 2. Ley, K., and T. F. Tedder. 1995. Leukocyte interactions with vascular endothe- with the observed activation of RhoA. It is extremely interesting lium. J. Immunol. 155:525. 3. Staunton, D. E., M. L. Dustin, and T. A. Springer. 1989. Functional cloning of that ICAM-1 cross-linking induced up-regulation of both rhoA ICAM-2, a cell adhesion ligand for LFA-1 homologous to ICAM-1. 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