P-Selectin Glycoprotein Ligand-1 Is Expressed on Endothelial Cells and Mediates Monocyte Adhesion to Activated Endothelium
Objective— The purpose of this study was to investigate the presence and functionality of P-selectin glycoprotein ligand-1 (PSGL-1) on activated endothelial cells (ECs).
Methods and Results— We show here that PSGL-1 is expressed at the mRNA and protein levels in umbilical vein and microvascular ECs. Furthermore, this endothelial PSGL-1 (ePSGL-1) is functional and mediates adhesion of monocytes or platelet-monocyte complexes (PMCs) to the activated endothelium in a flow model. ePSGL-1 expression was not affected by treating ECs with inflammatory stimuli (tumor necrosis factor α, interleukin-1β, thrombin, or histamine). However, the functional binding capacity of ePSGL-1 to monocytes or P-selectin/Fc chimera significantly increased by stimulation of the ECs with TNFα. By means of a siRNA approach to specifically knock-down the genes involved in the glycosylation of PSGL-1 we could show that tumor necrosis factor α–induced glycosylation of ePSGL-1 is critical for its binding capacity.
Conclusion— Our results show that ECs express functional PSGL-1 which mediates tethering and firm adhesion of monocytes and platelets to inflamed endothelium.
- P-selectin glycoprotein ligand-1
- monocyte adhesion
- platelet-monocyte complexes
PSGL-1 is one of the best characterized selectin ligands known to date. PSGL-1 is expressed as a homodimer of two 120-kDa subunits that binds all three selectins, with the highest affinity for P-selectin,1 and is known to be constitutively expressed on the surface of platelets2 and most types of leukocytes.3 PSGL-1, besides playing a critical role in the inflammatory response by mediating leukocyte–leukocyte and leukocyte–endothelium interactions it also participates in the hemostatic process by mediating leukocyte–platelet interactions.4 In vivo studies showed that leukocyte PSGL-1 mediates rolling of leukocytes over E-selectin and P-selectin on activated ECs5 whereas PSGL-1 – L-selectin interactions mediate leukocyte secondary tethering at the activated endothelium.6
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PSGL-1–dependent interactions appear to enable the presence of inflammatory cells at the hemostatic thrombus by binding to P-selectin on activated platelets localized at the injured vessel wall.7 These platelet–leukocyte interactions, however, also occur when activated platelets are present in the circulation giving rise to circulating platelet–leukocyte complexes, mainly platelet–monocyte complexes (PMCs). PMCs are currently regarded not just as markers of vessel wall disease8,9 but also as thromboatherogenic particles with high adhesive capacity to activated endothelium.10,11
Only few studies have assessed the presence of PSGL-1 on the endothelium. Laszik et al3 described the presence of PSGL-1 in the small venules and capillaries of benign hyperplasia samples, although no vascular-associated staining could be detected in normal tissues or tissues undergoing acute inflammation. Also Sperandio et al6 failed to show PSGL-1 expression on resting or inflamed endothelium and platelets in mice. For many years the presence of PSGL-1 on ECs has not been considered to be important and therefore not further investigated. Recently, Ley et al12 demonstrated the presence of PSGL-1 in venules of the mesenteric lymph node and small intestine of mice. We show in this report that PSGL-1 is expressed at the mRNA and protein levels in human vein and foreskin microvascular ECs (HUVECs and FMVECs, respectively). Importantly, we also show that endothelial PSGL-1 plays an important role in mediating the rolling and adhesion of monocytes, platelets, and PMCs over activated endothelium. Further, PSGL-1 expression was demonstrated on the endothelial lining of atherosclerotic coronary arteries, suggesting a role in the formation of the inflammatory infiltrate in this type of lesions. These findings reveal a new mechanism by which selectins and their ligands participate in the onset of inflammation and/or atherosclerosis.
Materials and Methods
HUVECs were isolated from human umbilical cord veins as described.13 Immortalized HUVECs, EC-RF2414 cells, were kindly provided by Prof H. Pannekoek (Academic Medical Center, Amsterdam, The Netherlands). FMVECs15,16 were kindly provided by Prof V.W.M. van Hinsbergh (VU Medical Center, Amsterdam, The Netherlands). Cells were cultured in RPMI 1640 containing 20% (v/v) human serum, 200 μg/mL penicillin, and streptomycin (Life Technologies) and grown to confluence in 5 to 7 days.
The plasmids pSUPER/β4GalT-7, pSUPER/GST-1, pSUPER/GST-2, pSUPER/PSGL-1, and pSUPER/FX were generated and transfected into HUVECs as described in supplemental Methods (available online at http://atvb.ahajournals.org) to induce silencing of the genes β4GALT-7, GST-1, GST-2, PSGL-1, and FX.
Isolation of Blood Cells
Whole blood, anticoagulated with 0.4% trisodium citrate (pH 7.4), was obtained from healthy volunteers from the Sanquin Blood Bank (Amsterdam, The Netherlands). Monocytes were isolated by negative selection from human peripheral blood by means of a MACS monocyte isolation kit according to the manufacturer’s instructions (Miltenyi Biotech GMBH). This procedure resulted in more than 90% pure monocyte suspensions (measured as CD14-positive cells by flowcytometry).
Reverse Transcriptase Polymerase Chain Reaction
Total RNA was prepared from freshly isolated monocytes and untreated or IL-1β (4 hours) treated HUVECs or EC-RF24 cells with the Absolutely RNA kit (Stratagene). Total RNA (2 μg) was converted to cDNA using 0.5 μg of dT12–18 primer (Invitrogen), Superscript II (Invitrogen), and 20 U of RNAsin (Promega).
Monocytes and HUVECs were lysed in 1.5% Triton X-100, 0.1% SDS, 0.1% NP-40, 100 mmol/L Tris-HCl pH 7.4, 150 mmol/L NaCl, and 1 mmol/L CaCl2 buffer. Proteins from the cell lysates (1×106 monocytes and 2×106 HUVECs) were separated on 7% SDS-PAGE, transferred to a polyvinylidene fluoride (PVDF) membrane, and blotted with PL-1 antibody. The bound antibody was detected by using HRP-conjugated secondary antibody.
Flow Cytometry and Confocal Microscopy
PSGL-1 surface expression on ECs was investigated by flow cytometry (FACS; Vantage, Becton Dickinson) with cells from different passages, stimulated or not with TNF-α (10 and 30 minutes, 2, 6, 12, and 24 hours), IL-1β (6 hours), thrombin (5 and 10 minutes), or histamine (5 and 10 minutes). After stimulation, ECs were resuspended in washing buffer and incubated with a control antibody (fluorescein isothiocyanate [FITC]-labeled goat anti-mouse IgG), or an antibody against PSGL-1, P-selectin, E-selectin, vascular cell adhesion molecule (VCAM)-1, or PECAM-1 for 45 minutes at 4°C.
Monocyte/Platelet Perfusion and Evaluation of Adhesion and Rolling Velocity
Monocytes (2×106 cells/mL) were perfused over ECs seeded on glass slides as previously described.11 The perfusion chamber was mounted on a microscope stage (Axiovert 25, Zeiss), equipped with a B/W charge-coupled device (CCD) video camera (Sanyo). The flow rate through the chamber was precisely controlled and the monocytes were perfused at 0.8 dyn/cm2. The cut-off value to distinguish between rolling and static adherent cells was set at 1 μm/s.
Tissue Specimens: Immunohistochemistry and Immunofluorescence
Portions of coronary arteries were obtained from autopsy specimens at the Academic Medical Centre (Amsterdam, The Netherlands) according to institutional guidelines. Coronary arteries undergoing atherosclerosis were snap-frozen and sectioned using conventional techniques.
Data are represented as the mean±SEM of at least 3 independent experiments and were compared with a two-tailed Student t test or a one-way ANOVA with Bonferroni correction. Probability values <0.05 were considered to be significant.
For detailed Methods please see the supplemental materials, available online at http://atvb.ahajournals.org.
Expression of PSGL-1 in Endothelial Cells
Because PSGL-1 is involved in leukocyte–endothelium interactions and there is controversy concerning the presence of PSGL-1 on inflamed endothelium, we investigated whether PSGL-1 is indeed expressed on ECs. Immunofluorescence analysis (flow cytometry and microscopy) showed that PSGL-1 is expressed on the surface of ECs (Figures 1A, 1B, and 2⇓C). In contrast to E-selectin and VCAM-1, there was no increase in surface levels of PSGL-1 after EC activation using TNF-α (6 hours, Figures 1A, 1B, and 2⇓C) or IL-1β (data not shown), at different incubation times (10 or 30 minutes, 2, 6, 12, or 24 hours; data not shown). PSGL-1 expression levels on FMVECs were similar to those obtained on HUVECs (data not shown). PSGL-1 was not upregulated by recruitment from intracellular stores as shown by stimulation with thrombin or histamine for 5 or 10 minutes (Figure 1C), in contrast to P-selectin (used as positive control).
PSGL-1 transcripts were shown by RT-PCR in untreated and TNF-α or IL-1β–treated primary HUVECs (Figure 2A), EC-RF24 cells (data not shown), and monocytes (positive control). No detectable differences between stimulated and unstimulated cells were observed. As a positive control for TNFα or IL-1β stimulation we analyzed intercellular adhesion molecule-1 (ICAM-1) mRNA which showed a dramatic increase in expression in response to these cytokines (data not shown). Quantitative real-time PCR did not show significant differences in the expression levels of PSGL-1 transcripts in both HUVECs and FMVECs (shown in supplemental Figure I). The expression of PSGL-1 was also confirmed by Western blot analysis (Figure 2B). Although the level of PSGL-1 protein in ECs was much lower than in monocytes, a protein of similar apparent molecular weight was observed in both cell types (≈120 kDa).
Platelet Adhesion to Endothelial PSGL-1
To determine the functionality of endothelial PSGL-1, platelets were perfused over ECs and adhesion was quantified. Washed and labeled platelets were incubated with a control (W6/32) or a blocking P-selectin antibody (WASP 12.2) and perfused at high shear over untreated or TNF-α–treated (6 hours) ECs. Platelet adhesion was strongly increased after activation of ECs (Figure 3A). This effect was strongly inhibited when PSGL-1 on activated ECs or P-selectin on platelets was blocked with PL-1 or WASP12.2 antibodies, respectively.
Although similar amounts of PSGL-1 are present on the surface of unstimulated and stimulated ECs, only stimulated cells are able to support platelet adhesion. The P-Selectin/Fc chimera was used to test whether there is an increase in PSGL-1 affinity for its receptor on stimulation. Analysis by flow cytometry (Figure 3B) and immunofluorescence microscopy (Figure 3C) indeed showed that the P-selectin/Fc protein bound significantly more to stimulated than to unstimulated ECs. The binding of the P-selectin/Fc chimera to PSGL-1 was inhibited by a blocking antibody to PSGL-1, which underscored the specificity of the interaction between the P-selectin/Fc chimera and PSGL-1. These results indicate that, despite PSGL-1 being constitutively expressed on ECs, the affinity for its receptor is increased by cytokine stimulation of ECs.
PSGL-1 Expression in Atherosclerotic Coronary Arteries
Sections of coronary arteries undergoing acute inflammation such as atherosclerosis were examined for the expression of PSGL-1 (Figure 4). Expression of PECAM-1 was used as a marker for endothelial cells and a strong and regular staining was observed. Although not as regular, the sections also exhibited luminal staining with the anti–PSGL-1 antibody indicating clear PSGL-1 expression on the vascular endothelium of these arteries. In contrast, staining of the endothelium with an irrelevant mouse IgG1 MAb was not detected (Figure 4A). Simultaneous detection of PSGL-1 and PECAM-1, as an endothelial marker, show that these two molecules colocalize on the surface of activated endothelium (Figure 4B). Detection of an IgG-control antibody was at background levels.
Rolling/Adhesion of Monocytes and PMCs to ECs via Endothelial PSGL-1
To investigate whether endothelial PSGL-1 is also functional in mediating monocyte and platelet-monocyte complex (PMC) interactions with the endothelium under flow, monocytes were perfused over HUVECs (untreated or TNF-α–treated for 6 hours). Video recordings were analyzed for the number of adhered monocytes and for rolling velocity. Perfusions of monocytes or PMCs only resulted in rolling when the ECs had been treated with TNFα. In the presence of PMCs, blocking PSGL-1 on ECs significantly inhibited monocyte adhesion by 30% (P<0.05, Figure 5A, black bars) and strongly increased monocyte rolling velocity (Figure 5B, black bars). Simultaneous inhibition of PSGL-1 on ECs and on monocytes caused a synergistic reduction of monocyte adhesion (data not shown). To test the role of endothelial PSGL-1 in the adhesion of monocytes in the absence of platelets, PMCs were removed from the cell suspension by immunodepletion. As previously reported,11 low levels of PMCs resulted in reduced monocyte adhesion to the endothelium. By blocking PSGL-1 on ECs, monocyte adhesion was further decreased 30% (Figure 5A, empty bars), whereas rolling velocity was significantly increased (Figure 5B, empty bars). As was shown before,11 blocking of P-selectin on the endothelium did not have any effect in cell adhesion.
To investigate whether endothelial PSGL-1 can interact with L-selectin on monocytes, an L-selectin–blocking antibody was used on a monocyte suspension containing <5% PMCs. To rule out a possible contribution of remaining platelets, the monocytes were, where indicated, incubated with an antibody to P-selectin to prevent PMC formation. When the cells were incubated with the DREG 56 antibody to L-selectin, adhesion to the endothelium was inhibited by 35% (P<0.05, supplemental Figure IIIA). This effect was similar to that obtained by blocking PSGL-1 on ECs. Although not statistically significant, when both L-selectin on monocytes and PSGL-1 on ECs were blocked, monocyte adhesion was further inhibited. As a control we used a nonblocking antibody against PSGL-1 (PL-2) which did not affect monocyte adhesion to ECs (data not shown).
Previously it has been shown that the expression of L-selectin ligands in endothelial cells is modulated by sulfation,17 and that TNF-α upregulates the expression of two sulfotransferases implicated in the sulfation of L-selectin ligands.18,19 To investigate whether the mechanism of increase in monocyte adhesion described here is dependent on the sulfation of PSGL-1 an RNA interference approach was designed. The genes targeted were GST-1 and -2, implicated in the sulfation of N- and O-linked glycans, β4GalT-7, involved in the initiation of the glycosaminoglycan chains, and FX, which controls the synthesis of GDP-Fucose (supplemental Figure I). Additionally, a knock-down for PSGL-1 and a sequence without homology in the human genome were used as a positive and negative control, respectively. The silencing of PSGL-1 results in a decrease in monocyte adhesion (supplemental Figure IIIB) and an increase in rolling velocity (data not shown), which are comparable to the effect of blocking with PL-1. Furthermore, the silencing of GST-1 was able to mimic the effects of silencing PSGL-1, whereas any of the other treatments were ineffective (supplemental Figure IIIB). In agreement, the binding of P-selectin/Fc to activated endothelial cells was also decreased when cells were transfected with pSUPER/PSGL-1, pSUPER/GST-1, and, to a lesser extent, with pSUPER/GST2 (shown in supplemental Figure II).
The molecular mechanisms by which leukocyte recruitment to inflamed tissues occurs have been extensively studied over the past years. The initial tethering and rolling of monocytes along the vessel wall is generally accepted to be mediated by selectins and their ligands that are expressed on ECs, platelets, and leukocytes. PSGL-1, one of the primary selectin ligands, is known to be expressed on leukocytes and platelets. In this study we show that functional PSGL-1 is expressed on ECs on treatment with proinflammatory cytokines.
PSGL-1 expression was shown at the mRNA and protein level on primary microvascular and umbilical ECs and on an endothelial cell line. PSGL-1 is restricted to the surface of ECs and is not increased by stimulation with inflammatory cytokines such as TNFα or IL-1β, in contrast to other cytokine-induced adhesion molecules such as ICAM-1, VCAM-1, and E-selectin. In addition, activators such as thrombin or histamine, which induce elevated surface expression of P-selectin on ECs,20 had no effect on the expression levels of endothelial PSGL-1. Thus, PSGL-1 is constitutively expressed in primary ECs and in immortalized endothelial cells, and is not stored in P-selectin–containing vesicles within ECs.
Endothelial PSGL-1, at higher shear stress, was able to interact with platelets and recruit them to the endothelium. This effect was inhibited by blocking P-selectin on platelets or PSGL-1 on ECs. The increase in affinity of endothelial PSGL-1 to P-selectin was further demonstrated by the strong binding of a P-selectin/Fc protein to stimulated HUVECs, which was abrogated by incubating the cells with a blocking antibody to PSGL-1.
Our flow system enabled us to show functionality of endothelial PSGL-1 as a ligand for selectins also on monocytes. When 10 to 20% PMCs were present in the monocyte suspension, we found a significant reduction (30%) in monocyte adhesive interactions with the endothelium, accompanied by an increase in cell rolling velocity when TNF-α–stimulated ECs were preincubated with a PSGL-1–blocking antibody. Under low shear conditions, platelet interactions with the endothelium are mainly characterized by transient tethering and rolling, whereas firm adhesion rarely occurs.11,21 However, it is important to discern whether PSGL-1 on ECs interacts mainly with L-selectin on monocytes or might also interact with P-selectin on platelets. To investigate this, we used a PMC-free monocyte suspension. Blocking of endothelial PSGL-1 or L-selectin on monocytes increased monocyte rolling velocity and inhibited monocyte adhesion to ECs by 30%, indicating that monocyte L-selectin is a primary receptor for endothelial PSGL-1. However, the contribution of molecular interactions, other than selectin-dependent, cannot be completely ruled out. P-selectin on platelets or platelet microparticles has been implicated in triggering monocyte arrest by deposition of chemokines, namely RANTES, on activated endothelium.22,23 Such mechanism could contribute to a more pronounced recruitment with PMC-rich monocyte suspensions. However, because preincubation of monocytes or platelets with a P-selectin–specific blocking antibody was able to block rolling to at least 50%, it is possible to conclude that P-selectin–PSGL-1 interactions are involved in monocyte/platelet rolling over activated endothelial cells.
Interestingly, PSGL-1 was only functional after cytokine treatment of ECs. Although most lymphocytes express PSGL-1, only 10 to 20% of cells are able to bind P-selectin,24 which shows that PSGL-1 expression does not necessarily imply functional relevance. The glycosylation of PSGL-1 is essential for functionality,25 and dramatic changes in endothelial cell glycosylation have been reported on TNFα treatment.19 Here we show that silencing of the sulfotransferase GST-1, and partially GST-2, mimics the effect of silencing PSGL-1 or using the blocking antibodies PL-1 or DREG 56. Altogether, these data indicate that TNFα-induced expression of functional PSGL-1 is dependent on the expression of GST-1, and partially GST-2, whereas fucosylation, or the expression of glycosaminoglycans do not contribute. These findings are in line with those of Li et al,18 demonstrating the critical role of GST-1 and -2 in shear-resistant leukocyte rolling via L-selectin.
Additionally, we show the expression of PSGL-1 on the ECs of atherosclerotic lesions, suggesting a potential role in the recruitment of inflammatory cells to the lesion. Although expressed at low levels, PSGL-1 on activated ECs is able to functionally bind P- and L-selectin on platelets and monocytes, respectively, mediating monocyte initial tethering and platelet recruitment to the endothelium. Our results strongly suggest that PSGL-1 has a crucial role in monocyte/PMCs and platelet recruitment to the vascular endothelium and should be considered as an important participant in the onset of inflammation and/or atherosclerosis.
Sources of Funding
This work was supported by grants from the Dutch Heart Foundation (1999B059 and M93.007).
P.d.C.M. and J.-J.G.-V. contributed equally to this study.
Original received July 17, 2006; final version accepted February 6, 2007.
Tedder TF, Steeber DA, Chen A, Engel P. The selectins: vascular adhesion molecules. FASEB J. 1995; 9: 866–873.
Frenette PS, Denis CV, Weiss L, Jurk K, Subbarao S, Kehrel B, Hartwig JH, Vestweber D, Wagner DD. P-Selectin glycoprotein ligand 1 (PSGL-1) is expressed on platelets and can mediate platelet-endothelial interactions in vivo. J Exp Med. 2000; 191: 1413–1422.
Laszik Z, Jansen PJ, Cummings RD, Tedder TF, McEver RP, Moore KL. P-selectin glycoprotein ligand-1 is broadly expressed in cells of myeloid, lymphoid, and dendritic lineage and in some nonhematopoietic cells. Blood. 1996; 88: 3010–3021.
Yang J, Hirata T, Croce K, Merrill-Skoloff G, Tchernychev B, Williams E, Flaumenhaft R, Furie BC, Furie B. Targeted gene disruption demonstrates that P-selectin glycoprotein ligand 1 (PSGL-1) is required for P-selectin-mediated but not E-selectin-mediated neutrophil rolling and migration. J Exp Med. 1999; 190: 1769–1782.
Sperandio M, Smith ML, Forlow SB, Olson TS, Xia L, McEver RP, Ley K. P-selectin glycoprotein ligand-1 mediates L-selectin-dependent leukocyte rolling in venules. J Exp Med. 2003; 197: 1355–1363.
da Costa Martins P, van den Berk N, Ulfman LH, Koenderman L, Hordijk PL, Zwaginga JJ. Platelet-monocyte complexes support monocyte adhesion to endothelium by enhancing secondary tethering and cluster formation. Arterioscler Thromb Vasc Biol. 2004; 24: 193–199.
Rivera-Nieves J, Burcin TL, Olson TS, Morris MA, McDuffie M, Cominelli F, Ley K. Critical role of endothelial P-selectin glycoprotein ligand 1 in chronic murine ileitis. J Exp Med. 2006; 203: 907–917.
Fontijn R, Hop C, Brinkman HJ, Slater R, Westerveld A, van Mourik JA, Pannekoek H. Maintenance of vascular endothelial cell-specific properties after immortalization with an amphotrophic replication-deficient retrovirus containing human papilloma virus 16 E6/E7 DNA. Exp Cell Res. 1995; 216: 199–207.
Li X, Tu L, Murphy PG, Kadono T, Steeber DA, Tedder TF. CHST1 and CHST2 sulfotransferase expression by vascular endothelial cells regulates shear-resistant leukocyte rolling via L-selectin. J Leukoc Biol. 2001; 69: 565–574.
García Vallejo JJ, van Dijk W, van het Hof B, van Die I, Engelse ME, van Hinsbergh VWM, Gringhuis SI. Activation of human endothelial cells by tumor necrosis factor-α results in profound changes in the expression of glycosylation-related genes. J Cell Physiol. 2006; 206: 203–210.
Theilmeier G, Lenaerts T, Remacle C, Collen D, Vermylen J, Hoylaerts MF. Circulating activated platelets assist THP-1 monocytoid/endothelial cell interaction under shear stress. Blood. 1999; 94: 2725–2734.
Schober A, Manka D, von Hundelshausen P, Huo Y, Hanrath P, Sarembock IJ, Ley K, Weber C. Deposition of platelet RANTES triggering monocyte recruitment requires P-selectin and is involved in neointima formation after arterial injury. Circulation. 2002; 106: 1523–1529.
Mause SF, von Hundelshausen P, Zernecke R, Koenen RR, Weber C. Platelet microparticles: a transcellular delivery system for RANTES promoting monocyte recruitment on endothelium. Arterioscler Thromb Vasc Biol. 2005; 25: 1512–1518.
Vachino G, Chang XJ, Veldman GM, Kumar R, Sako D, Fouser LA, Berndt MC, Cumming DA. P-selectin glycoprotein ligand-1 is the major counter-receptor for P-selectin on stimulated T cells and is widely distributed in non-functional form on many lymphocytic cells. J Biol Chem. 1995; 270: 21966–21974.
Leppanen A, Mehta P, Ouyang YB, Ju T, Helin J, Moore KL, van Die I, Canfield WM, McEver RP, Cummings RD. A novel glycosulfopeptide binds to P-selectin and inhibits leukocyte adhesion to P-selectin. J Biol Chem. 1999; 274: 24838–24848.