Lymphocytes emigrate from the circulation to target tissues through the microvascular endothelial cell (EC) barrier. During paracellular transmigration cell-cell junctions have been proposed to disengage and provide homophilic and heterophilic interaction surfaces in a zip-like process. However, it is not known whether ECs modulate junction proteins during this process. Here we show that tyrosine phosphorylation of adherens junction vascular endothelial cadherin (VEC) is required for successful transendothelial lymphocyte migration. We found that adhesion of lymphocytes or activation of the endothelial intercellular adhesion molecule 1 (ICAM1) led to tyrosine phosphorylation of VEC. Substitution of tyrosine for phenylalanine in VEC at positions 645, 731 or 733 produced ECs that were significantly less permissive to lymphocyte migration. We also found that these same tyrosine residues are involved in ICAM1-dependent changes of VEC phosphorylation. ICAM1 activation enhanced transendothelial permeability, suggesting the occurrence of junction disassembly. In agreement, the expression of VEC mutated at Y645F, Y731F or Y733F predominantly affected lymphocyte transmigration in paracellular areas. Taken together, these results demonstrate that phosphorylation of adherens junctions constitutes a molecular endpoint of lymphocyte-induced vascular EC signaling and may be exploited as a new target of anti-inflammatory therapies.
Intercellular adhesion molecule 1 (ICAM1) on the luminal surface membrane of vascular endothelial cells (ECs) plays a crucial role during the egress of lymphocytes from the vascular compartment. It binds to β2 integrins, such as leucocyte-function-associated antigen 1 (LFA-1), a heteromer of αL (ITGAL, also known as CD11a) and ITGB2 (CD18), on lymphocytes and promotes their stable arrest (Butcher, 1991). However, endothelial ICAM1 is not only a docking receptor for lymphocyte adhesion and migration but its engagement also triggers endothelial signaling cascades that contribute fundamentally to vascular compliance to lymphocyte diapedesis and the endothelial inflammatory response (Turowski et al., 2005). ICAM1-induced signaling comprises changes in levels of intracellular Ca2+ and dynamic actin as well as activation of the small GTPase Rho, and Src and C protein kinases. Despite the identification of an increasing number of key players involved in ICAM1-mediated endothelial signaling, downstream effectors and molecular endpoints that ultimately modulate lymphocyte migration remain elusive.
Both tight and adherens junction (AJ) proteins form attachments between ECs, and are major sites of regulation in the transport of molecules and cells across vascular barriers (Bazzoni and Dejana, 2004). At least in the case of paracellular leukocyte extravasation, various junction molecules disengage and, in turn, form homo- and heterophilic interactions with leukocyte adhesion proteins (Imhof and Aurrand-Lions, 2004; Muller, 2003; Rao et al., 2007). In this process the EC has been ascribed a rather passive role, where junctions are forced open by the migrating leukocyte (Imhof and Aurrand-Lions, 2004; Shaw et al., 2001). We investigated whether intercellular junction modulation is part of the endothelial response to lymphocyte migration.
Results and Discussion
Migration of antigen-activated lymphocytes across brain microvascular ECs in vitro does not require stimulation of inflammatory cytokines, occurs in the absence of chemokine gradients and is predominantly dependent on ICAM1 but not vascular cell adhesion molecule 1 (VCAM1) (Greenwood et al., 1995). ICAM1-mediated signaling pathways involve Rho GTPases and MAP kinase cascades and have generally been studied by antibody-mediated crosslinking of serum-starved cells (Turowski et al., 2005). In the rat brain microvascular EC cell line (GPNT) ICAM1 activation led to a rapid and transient increase of tyrosine phosphorylation of a number of proteins which was maximal within 10-15 minutes of ICAM1 stimulation (Fig. 1A). When analyzed by immunocytochemistry (Fig. 1B) the increase in phosphorylated tyrosine was most marked at the intercellular junction area and preceded major actin re-arrangements typically seen following ICAM1 crosslinking (Adamson et al., 1999). Immunoprecipitation and subsequent analysis of phosphorylated tyrosine of junction proteins revealed that VEC (Fig. 1C,E), but not the tight junction protein 1 (ZO-1), occludin, or any of the catenins (Fig. 2), displayed altered tyrosine phosphorylation following ICAM1 crosslinking. Phosphorylation of VEC was rapid and transient, reaching a maximum after 15 minutes of ICAM1 crosslinking (Fig. 1D). It depended on upstream signaling by Rho GTPase, dynamic actin and also intracellular Ca2+ (Fig. 1F), all of which are essential for successful lymphocyte migration (Turowski et al., 2005). No involvement of Src family protein kinases was observed in our experimental set up. This is in contrast to a recent report by Allingham et al. (Allingham et al., 2007) and may reflect that different signaling modules are activated and used by different subsets of ECs and/or leukocytes. Indeed, T-cell adhesion to GPNT cells also induced a significant rise in VEC tyrosine phosphorylation (Fig. 1G,H), albeit with a slightly delayed time course (presumably reflecting settling and adhesion times). Taken together these data suggest that interendothelial junctions can be molecular endpoints of ICAM1-mediated signaling during leukocyte adhesion and migration.
To study the functional significance of ICAM1-mediated VEC phosphorylation, the structure of the cytoplasmic domain of VEC was further analyzed in silico and by mutagenesis. The cytoplasmic tail of VEC contains eight conserved tyrosine residues (Fig. 3A), three of which are predicted to constitute phospho-acceptor sites for insulin receptor family (Y645, Y731) or Src family (Y685) protein kinases (http://kinasephos.mbc.nctu.edu.tw, data not shown). A structural computer model of the VEC intracellular domain bound to β-catenin suggested that Y725, Y731 and Y733 are exposed and accessible to phosphorylation, whereas Y685, Y757 and Y774 are oriented towards the bound β-catenin and, possibly, inaccessible (Fig. 3B). Y645 and Y658 are located outside of this area in a domain predicted to mediate binding to p120 (Lampugnani et al., 2002).
Recently, Y658, Y685 and Y731 have been described as target phosphorylation sites (Potter et al., 2005; Wallez et al., 2006), and we therefore asked whether phosphorylation of any of these three tyrosines residues is important for lymphocyte migration. For this we derived EC lines from VEC-null mouse endotheliomas that re-expressed wild-type (wt) VEC, Y658F-VEC, Y685F-VEC or Y731F-VEC. The VEC expression levels and localization were similar in all four cell lines (Fig. 4A,B), suggesting that Y to F mutations do not interfere with junctional targeting. Migration of activated T cells across VEC-null ECs was very low but significantly enhanced when wt VEC was re-expressed (Fig. 4C). A similar, twofold increase was observed when either a clonal cell line that stably re-expressed wt VEC or a pool of cells transiently expressing EGFP-VEC was analyzed. Absolute migration rates measured after 4 hours of lymphocyte co-culture increased from ∼10% to 20-25% when VEC was reconstituted in VEC-null endothelioma cells and were then in the same range as those usually observed for GPNT cells (ca. 30%) indicating that the expression of VEC is vital to the functional integrity of the vascular endothelium. This was somewhat unexpected because VEC disruption using antibodies has been reported to enhance neutrophil migration in vivo (Gotsch et al., 1997). However, VEC elimination leads to significant changes in the composition of AJs (Zanetta et al., 2005) and might, in this way, inhibit leukocyte migration indirectly. Alternatively, VEC might have opposing effects on the migration of lymphocytes and neutrophils. When cells that express VEC mutants were analyzed we found that the re-expression of VECs carrying single mutations in Y658 or Y685 did not have any effect on lymphocyte transmigration (Fig. 4D). However, mutation of Y731 led to an approximately twofold reduction in lymphocyte migration. This indicated that VEC phosphorylation may be important for leukocyte migration.
To test the role of intracellular tyrosines of VEC more systematically and in an EC system more relevant to inflammation, VEC mutants containing single Y to F substitutions were transiently expressed as N-terminal fusion proteins of EGFP in GPNT cells. Expression levels and overall localization of the individual VEC mutants were similar and mostly restricted to the periphery of the cell (Fig. 5A). Some cells expressed the protein at very high levels throughout the cell but these appeared non-viable and were never integrated into endothelial monolayers. In all other expressing cells, EGFP was almost exclusively found at cell-cell junctions once EC monolayers were formed (Fig. 5B). Again we observed that the introduction of Y731F-VEC significantly reduced lymphocyte migration. In addition we also observed impaired lymphocyte migration across GPNT cells that expressed Y645F-VEC or Y733F-VEC (Fig. 5C). By contrast, the introduction of wt VEC or any other Y to F mutant did not affect migration. The inhibitory effect of Y645F-VEC and Y733F-VEC was also corroborated in a VEC-null background. Transient expression of Y645F-VEC–GFP or Y733F-VEC–GFP but not any other VEC-GFP mutants, produced endothelioma cells with a similar propensity to reduced lymphocyte migration (Fig. 5D). In all cases the mutant VEC clearly interfered on the level of lymphocyte diapedesis because adhesion rates were comparable.
Commercially available antibodies against VEC phosphorylated at Y658 or Y731 (Potter et al., 2005) were not reactive with VEC immunoprecipitates from ICAM1-crosslinked cells (data not shown). They also appeared to recognize a protein of the same size whether full-length or truncated VEC (Lampugnani et al., 2002) was expressed (data not shown). To examine whether ICAM1-induced tyrosine phosphorylation involved the tyrosine at positions 645, 731 or 733 the phosphorylation of wt and mutant GFP-VEC was analyzed in CHO expressing ICAM1 cells (CHO-ICAM1). Crosslinking of ectopic human ICAM1 on CHO cells induced tyrosine phosphorylation of co-expressed GFP-VEC in a time dependent manner (Fig. 6A), similar to that observed in GPNT cells. When the phosphorylation status of the Y to F mutants of GPF-VEC was tested, only mutant Y733F-VEC displayed significantly reduced tyrosine phosphorylation in response to ICAM1 ligation (Fig. 6B), suggesting that this mutation interfered with tyrosine phosphorylation of VEC. Next, GFP-VEC was also isolated and analyzed from 32P-labelled cells. Significantly, GFP-VEC was immunoprecipitated as a strongly phosphorylated protein, with the majority of phosphate present as phosphorylated serine and only very little phosphorylated tyrosine (data not shown). This observation is also in agreement with the recently reported phosphorylation of S665 by PAK (Gavard and Gutkind, 2006). Tryptic phosphopeptide mapping revealed at least five groups of peptides with apparently variable phosphorylation levels (Fig. 6C,D; supplementary material Fig. S2). When ICAM1 was crosslinked most phosphopeptides remained unchanged but two peptides displayed significantly reduced chromatographic mobility, indicative of additional phosphorylation (arrows in Fig. 6D). The Y645F mutation suppressed phosphorylation of one peptide but enhanced that of the other, indicating that this mutation interferes with the recognition of the phosphorylation site. Mutation Y731F induced a perfect reversal to VEC from non-crosslinked cells suggesting that this site was directly phosphorylated. Overall phosphorylation was low in Y733F-VEC and the migration of most phosphopeptides was altered, which suggests that this mutation renders most sites unphosphorylatable. More refined analysis of phosphopeptides by mass spectroscopy will be required to delineate the exact phosphorylation changes that occur on tyrosine and serine residues when ECs are stimulated. In summary, VEC mutations of Y645, Y731 or Y733, which dominantly inhibit lymphocyte migration, also affect ICAM1-mediated VEC phosphorylation, suggesting that these events are mechanistically linked.
VEC has been reported to be phosphorylated on tyrosine under various conditions (Andriopoulou et al., 1999; Esser et al., 1998) and, indeed, we found that tyrosine phosphorylation was also increased when GPNT cells were treated with a variety of vasoactive compounds (Fig. 7A). Generally it is thought that such phosphorylation induces the disruption of AJs, which can be measured by concomitant rises in transendothelial permeability. We found that ICAM1 crosslinking is likely to affect AJ organization because it also induced an increase in paracellular permeability (Fig. 7B) and monolayer impedance (data not shown). However, in contrast to growth factor stimulation, where this is frequently associated with altered catenin binding (Lampugnani et al., 1997; Potter et al., 2005; Weis et al., 2004), ICAM1 ligation did not affect the levels of VEC-associated α-, β- or γ- catenin (Fig. 7C,D), suggesting that the molecular mechanism may be different. Intuitively, ICAM1-mediated VEC phosphorylation might lead to the disengagement of AJs and thus facilitate the path during paracellular migration. In support of such a mechanism we found that the expression of Y645F-VEC, Y731F-VEC or Y733F-VEC led to a significant reduction of migration in paracellular areas of the EC monolayer (Fig. 7E) and this correlated well with the general rate of inhibition (see Fig. 5C). Therefore, paracellular but not transcellular migration might be affected by dysfunctional VEC. However, more careful analyses using dynamic confocal microscopy of stable transfectants are required to draw a definite conclusion, in particular because transcellular migration also relies on juxta-junctional positioning of the lymphocyte and, subsequently, on a transcytotic pathway (Carman et al., 2007; Millan et al., 2006) – in which VEC may also be involved (Gavard and Gutkind, 2006; Xiao et al., 2005).
Leukocyte adhesion and subsequent ICAM1 engagement on EC induces an endothelial response and a complex network of signaling pathways regulating the inflammatory response of the endothelium (Turowski et al., 2005). From the results presented here, tyrosine phosphorylation of VEC meets all the criteria for a molecular endpoint of an endothelial signaling cascade that regulates lymphocyte migration: it is induced by surface engagement of ICAM1, it can be placed downstream of pathways that have previously been identified to affect lymphocyte migration and it appears to regulate paracellular migration directly. The importance of this mechanism and its general validity is underscored by recent findings by Allingham et al. (Allingham et al., 2007) who reported similar VEC phosphorylation to operate when neutrophils migrate across human umbilical vein endothelial cells. Collectively, these results demonstrate that VEC plays a more active role during leukocyte migration than previously anticipated. Single amino acid mutations, namely at Y645, Y731 or Y733, suppressed this function and, surprisingly, did so in a dominant fashion. Since dominant effects are rarely observed with mutations introducing non-phosphorylatable amino acids (as opposed to phospho-mimetic substitutions), it will be interesting to elucidate how these mutations interfere with the function of endogenous VEC during leukocyte migration. Currently, anti-adhesion therapies are amongst the most specific and advanced strategies to treat inflammation. The identification of a dominant molecular endpoint of adhesion-induced signaling may constitute a valid target for novel anti-inflammatory therapies.
Materials and Methods
Cell culture and treatment
The rat brain microvascular EC line GPNT (Romero et al., 2003) and mouse brain endothelioma cells bEND5 (Lyck et al., 2003) were grown as previously described. Mouse ECs with a homozygous null mutation of the VEC gene and the cell line re-expressing wild-type VEC derived by retroviral gene transfer have been described in detail (Lampugnani et al., 2002). Additionally, cell lines that stably express Y658F-VEC, Y685F-VEC, or Y731F-VEC have been generated in the same manner. Wild-type Chinese hamster ovary (CHO) cells and stable transfectants expressing human ICAM1 (CHO-ICAM1; a kind gift from Jeremy Pearson, King's College London, UK) were cultured in DMEM/F12 supplemented with 10% FCS.
Antibody-mediated ICAM1 crosslinking was performed as previously described in serum-starved cells (Adamson et al., 1999) using monoclonal antibodies against rat (clone 1A29), mouse (clones YNI-1 and 25ZC7) or human (clone 15.2) ICAM1.
Lymphocyte-EC co-cultures were carried out with peripheral lymph node cells (PLNCs) isolated from Lewis rats (Harlan Olac) as previously described (Adamson et al., 1999). For biochemical analysis of the ECs, PLNCs were washed and fixed using 3.7% formaldehyde before being added to the ECs, thus avoiding analysis of lymphocyte proteins after cell lysis. For adhesion assays, PLNCs were fluorescently labeled with 1 μM calcein-AM (Molecular Probes) before addition to EC monolayers (approximately ten labeled PLNCs per EC). Co-cultures were left at 37°C for 60 minutes and adherent T cells quantified in a fluorescent plate reader. Migration assays were performed using a myelin basic protein (MBP) Ag-specific T-cell line and time-lapse video microscopy as previously reported (Adamson et al., 1999). Migration was considered paracellular when it clearly occurred in defined areas of the periphery of the EC that appeared bright by phase-contrast microscopy. Only T cells that had completed transmigration were counted as migrated cells.
Permeability assays on GPNT cells were conducted in transwell inserts using FITC-dextrans (Sigma) of varying molecular weight (1 mg/ml) (Esser et al., 1998).
Cell lysis, immunoprecipitations and western blots
Total cell extracts were prepared in 50 mM Tris-Cl pH 7.5, 1% SDS, 1 mM orthovanadate. For immunoprecipitations cells were lysed in a buffer containing 10 mM HEPES-NaOH pH 7.4, 100 mM NaCl, 50 mM β-glycerophosphate, 2 mM MgCl2, 5 mM EGTA, 5 mM EDTA, 1% Igepal (Sigma), 1 mM orthovanadate, 1 mM NaF and protease inhibitors (Roche). Clarified cell extracts were incubated with 1 μg of antibodies against VEC (Santa Cruz, 6458), α- or β-catenin (Sigma), γ-catenin or p120 catenin (BD Biosciences) or 5 μg of anti-GFP antibody (Abcam 290). Antigen-antibody complexes were collected using protein A- or protein G-sepharose beads (GE Healthcare). SDS-PAGE and immunoblotting was performed as previously described (Turowski et al., 1999) using antibodies against phosphorylated tyrosine (4G10, PY20 or RC20H), VEC or catenin. For quantitative analyses immunoblots were scanned and quantified using the NIH imaging software ImageJ. The signal intensity of the phosphorylated tyrosine residues was normalized to the corresponding amount of VEC, and expressed as fold-increase of untreated controls.
GPNT cells were fixed in 3.7% formaldehyde and extracted with acetone (–20°C). For the detection of phosphorylated tyrosine the monoclonal antibody 4G10 (Upstate) was biotinylated and used at 2 μg/ml. Cells were then stained using streptavidin-coupled Texas Red (GE Healthcare) and phalloidin conjugated to Oregon Green (Molecular Probes; 1:50). VEC was revealed using a polyclonal goat antibody (sc-6458, Santa Cruz). Cells were mounted using Moviol 4-88 and analyzed by standard epifluorescent or confocal microscopy (Turowski et al., 1999).
Plasmids, mutagenesis and transfections
Mouse VEC was mutagenized using the Quickchange mutagenesis (Stratagene). Subsequently, wt and mutant VEC were cloned into pEGFP-N1 (BD Biosciences) to result in expression of the open reading frame of mouse VEC (aa 1- 784), followed by the linker sequence LVDPPVAT and the entire sequence of EGFP. Resulting plasmids (pEGFP-N′-VEC) were verified by DNA sequencing and purified using endotoxin-free preparation methods (Qiagen). Subsequently, GPNT cells or mouse VEC-null endothelioma cells were nucleofected using 20 μg plasmid per 2×106 cells according to the manufacturer's instructions (Amaxa). CHO cells were transfected using Lipofectamine 2000 (Invitrogen).
32P labeling and phosphoamino acid and phosphopeptide analysis
Transfected CHO-ICAM1 cells were labeled with 32P (PBS 13; GE Healthcare; 20 MBq/ml) in phosphate-free DMEM for 3 hours, ICAM1 crosslinked, lysed and VEC-GFP was precipitated using 5 μg of a polyclonal anti-GFP antibody (Abcam 290). Immunoprecipitates were resolved by SDS-PAGE and transferred to nitrocellulose or PVDF. Radiolabelled VEC was excised and subjected to acid hydrolysis for phosphoamino acid analysis or to digestion with trypsin using standard procedures (Boyle et al., 1991). Amino acids or phosphopeptides were subsequently resolved on cellulose TLC plates by electrophoresis and chromatography and detected by autoradiography.
Computer model of the VEC cytoplasmic domain
A homology-based model was obtained using the coordinates of E-cadherin-β-catenin complex (pdb: 1i7x, 1i7w). Cytoplamic domains of VEC from various species and E-cadherin were aligned using ClustalW. The optimal alignment was used by PyMol to build the VEC model.
We thank David Barford for help with deriving the structure model of the VEC–β-catenin complex. This work was supported by a Programme Grant from the Wellcome Trust (062403, awarded to J.G. and P.A.) and in part by the European Community (NoE MAIN 502935, to E.D.).
Supplementary material available online at http://jcs.biologists.org/cgi/content/full/121/1/29/DC1
↵‡ Present address: Kennedy Institute of Rheumatology, Imperial College, London, W6 8LH, UK
↵§ Present address: Faculty of Life Sciences, University of Manchester, Manchester, UK
↵¶ Present address: Ophthaltec Limited, London, SW1Y 4QU, UK
- Accepted September 25, 2007.
- © The Company of Biologists Limited 2008