Published online before print January 13, 2006
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* Departments of Experimental Immunohematology and
Molecular Cell Biology, Sanquin Research at CLB and Landsteiner Laboratory, Academic Medical Center, Amsterdam, The Netherlands; and
Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, The Netherlands
1 Correspondence: Sanquin Research (CLB), Dept. of Experimental Immunohematology (P112), Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands. E-mail: p.martinsdacosta{at}sanquin.nl
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4ß1 and Mß2integrin, with a concomitant decrease in L-selectin expression. Furthermore, the binding of platelets to monocytes resulted in increased monocyte adhesion to intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and fibronectin. Platelet binding was also responsible for an increase in monocyte transendothelial migration. Similar effects were observed after engagement of PSGL-1 with specific antibodies or with P-selectin immunoglobulin protein. Our data suggest that platelets, by binding via P-selectin to PSGL-1 on monocytes, induce up-regulation and activation of ß1 and ß2integrins and increased adhesion of monocytes to activated endothelium. Hence, monocytes within PMC are in a higher state of activation and may have, therefore, an increased atherogenic capacity.
Key Words: endothelial cells • platelet-monocyte complexes • cell surface adhesion molecules • cell activation
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Activated platelets express P-selectin, a member of the selectin family, which upon activation, is translocated from the -granules to the platelet surface [8
, 9
]. The main ligand for P-selectin is P-selectin glycoprotein ligand-1 (PSGL-1), a disulfide-linked homodimer with a molecular weight of 
220 kDa on platelets and most leukocytes [10
11
12
]. P-selectin and PSGL-1 are considered to mediate platelet-monocyte interactions, which not only mediate the binding of leukocytes to activated platelets or thrombi localized at the injured vessel wall but also the formation of platelet-leukocyte complexes [mainly platelet-monocyte complexes (PMC)] in the circulation [2
, 13
, 14
].
Monocytes, as other leukocytes, are recruited to cytokine-activated endothelium in a multistep process. Initially, monocytes in the bloodstream have to be slowed down by a capturing mechanism and roll over the endothelial layer. Activation of the monocytes during the rolling phase will result in firm adhesion and transmigration [15 16 17 ]. Although the latter process is mediated by interactions between the leukocyte integrins and their endothelial ligands, capture and rolling are mainly mediated by selectins and their respective receptors [17 18 19 ]. By comparing monocytes and PMC regarding their capacity to adhere to the endothelium, we have previously shown that PMC are more adhesive. This increased adhesion is to a large extent dependent on enhanced monocyte-PMC interactions leading to the formation of flow-oriented monocyte and PMC clusters. P-selectin-PSGL-1 interactions are involved in the formation of these secondary tethers [14 ].
P-selectin has been shown to increase ß2 integrin functionality on neutrophils [20
21
22
] and to enhance the nuclear transcription of nuclear factor-
B, which is required for the production of cytokines such as monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor (TNF-) [23
]. The idea of PSGL-1 ligation-mediated signaling has been emphasized by the observation that cross-linking of PSGL-1 on neutrophils induced protein-tyrosine phosphorylation, activated mitogen-activated protein kinases (MAPK), and stimulated interleukin (IL)-8 secretion. Moreover, PSGL-1 engagement on mouse neutrophils induces lymphocyte function-associated antigen-1 (LFA-1)- and membrane-activated complex-1 (Mac-1)-dependent adhesion to intercellular adhesion molecule-1 (ICAM-1) [24
]. Furthermore, P-selectin binding to its counter-receptor PSGL-1 promotes 4ß1-dependent adhesion of monocytes to vascular cell adhesion molecule-1 (VCAM-1) [25
].
Altogether, these observations suggest a radical change, quantitatively and qualitatively, in the leukocyte repertoire of surface-expressed adhesion molecules upon platelet binding. In the present study, we focused on the P-selectin-PSGL-1 interaction to better characterize the effect of platelet binding on the adhesive capacity of monocytes to activated endothelium.
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was from Boehringer Mannheim (Germany), and recombinant MCP-1 was from Strathman Biotech (Hannover, Germany). Alexa Fluor-488 phalloidin and Hoechst 33258 were from Molecular Probes (Eugene, OR). Washing buffer contained phosphate-buffered saline (PBS) supplemented with 0.5% HSA and 13 mM trisodium citrate. Incubation buffer contained 20 mM HEPES, 132 mM NaCl, 6 mM KCl, 1 mM MgSO4, and 1.2 mM KH2PO4 supplemented with 5 mM glucose, 1.0 mM CaCl2, and 0.5% (w/v) HSA. Iscove’s modified Dulbecco’s medium (IMDM) was from BioWhittaker (Verviers, Belgium), and other tissue-culture supplies (media, antibiotics, and trypsin) were from Gibco, Life Technologies Inc. (Paisley, UK).
Cell culture
Human umbilical vein endothelial cells (HUVEC) were isolated from human umbilical cord veins as described previously [26
, 27
]. Cells were cultured in RPMI 1640 containing 20% (v/v) human serum and 200 µg/ml penicillin and streptomycin (Gibco, Life Technologies Inc.) and were grown to confluence in 5–7 days. EC from the third passage were used in the experiments. TNF- (100 U/ml) was added to the medium 6 h prior to the experiments. U937 cells (a monocytic cell line derived from human histiocytic lymphoma) were purchased from the American Type Culture Collection (ATCC; Manassas, VA). The cells were cultured in RPMI 1640 (Gibco, Life Technologies Inc.) containing 10% (v/v) heat-inactivated fetal calf serum (Gibco, Life Technologies Inc.), 2 mM L-glutamine, 50 IU/ml penicillin, and 50 µg/ml streptomycin (complete medium).
Proteins and monoclonal antibodies (mAb)
Human P-selectin-Fc chimera, ICAM-1-immunoglobulin G (IgG), and VCAM-1-IgG were purchased from R&D Systems (Minneapolis, MN). Human plasma fibronectin was from Sigma-Aldrich. The antibodies used in the present study are described in Table 1
. The hybridomas for mAb WASP12.2, DREG56, IB4, 44a, and W6/32 were from the ATCC. The CD11b conformation-dependent antibody CBRM1/5 and the antibodies against PSGL-1, PL-1 and PL-2, were kindly provided by Dr. Kevin L. Moore (University of Oklahoma, Norman). KPL-1 was from Santa Cruz Biotechnology (CA). Antibodies 1G11 and 84H10 were from Immunotech (Marseille, France). The ß1integrin conformation-dependent mAb HUTS21 was a kind gift of Dr. Francisco Sanchez-Madrid (Hospital de La Princesa, Madrid, Spain). The FITC-labeled 44H6 mAb was from Chemicon International (Temecula, CA), and the FITC-labeled DREG56 antibody was from Becton Dickinson (San Jose, CA). The Alexa-488-labeled goat anti-mouse (GAM) Ig antibody was from Molecular Probes. All the other antibodies were purchased from Sanquin Immunoreagents. Human and mouse IgG were purchased from Sigma-Aldrich.
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Table 1. Name and Characteristics of All Antibodies Used in This Study
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Platelet isolation
Whole blood was centrifuged at 150 g for 10 min to obtain platelet-rich plasma, which was diluted 1:1 in Krebs-Ringer solution [4 mM KCl, 107 mM NaCl, 20 mM NaHCO3, 2 mM Na2SO4, 19 mM tri-sodium citrate, 0.5% (w/v) glucose in H2O, pH 5.0]. The mixture was centrifuged at 500 g for 10 min, and the supernatant was removed. The platelets in the pellet were resuspended in 2 ml Krebs-Ringer solution and centrifuged at 500 g for 10 min. This process was repeated two times, and the final suspension was made up in Krebs-Ringer (pH 6.1) solution to a concentration of 300,000 platelets/µl.
Adhesion assay
Adhesion assays were performed as described previously [28
29
30
31
32
] with some modifications. Briefly, 96-well microtiter plates (No. 3596, Corning Costar, Cambridge, MA) were coated by incubation with fibronectin (10 µg/ml), VCAM-1, or ICAM-1/Fc chimera (1 µg/ml) for 1 h at 37°C or 4% HSA as control for 1 h at 37°C. After incubation, the wells were washed with PBS and then blocked with 4% HSA at 37°C for 30 min. Control wells were filled with 4% HSA in PBS. Monocytes were labeled with calcein alveolar macrophage (Molecular Probes) at a final concentration of 5 µg/1 x 107 cells. In some instances, monocytes were incubated with platelets or P-selectin after calcein labeling. For PMA stimulation, cells were added to wells containing 10 ng/ml PMA. For blocking experiments, cells were added to wells containing the function-blocking mAb. Plates were then incubated at 37°C, and the monocytes were allowed to settle for 30 min. After incubation, nonadherent cells were removed by washing twice with PBS, and adherent cells were lysed in 0.5% Triton X-100 for 10 min at room temperature. Adhesion was quantified with a microplate fluorescence plate reader (GENios Plus, Tecan Group Ltd., Männedorf, Switzerland). Fluorescence was measured at excitation wavelength 485 nm and emission wavelength 525 nm. The adhesion ratio (%) was calculated as follows: (fluorescence from experimental sample–fluorescence from negative control sample)/total fluorescence added to well x 100%.
Monocyte perfusion and evaluation of cell adhesion
Monocytes in suspension (2x106 cells/ml in incubation buffer at 37°C) were aspirated from a reservoir through plastic tubing, a valve, and the flow chamber [27
, 33
] with a Harvard syringe pump (Harvard Apparatus, South Natic, MA). The flow rate through the chamber was controlled precisely (according to the manufacturer’s instructions), and the shear stress was kept at 0.8 dyn/cm2. The wall shear stress was calculated according to the Navier Stokes equation: t = (6Q.
)/(w.h2). In this equation, Q is the flow rate, is the suspending-medium viscosity, w is the slit width, and h is the slit height of the flow chamber [33
]. The shear stress is proportional to the rate of flow of the cells and can be calculated as dyn/cm2. During perfusions, the flow chamber was mounted on a microscope stage (Axiovert 25, Zeiss, Germany), equipped with a black and white charged-coupled device video camera (Sanyo, Osaka, Japan) and coupled to a VHS video recorder. Video images were evaluated for the number of adherent monocytes and the rolling velocity per cell with dedicated routines made in the image analysis software Optimas 6.1 (Media Cybernetics Systems, Silver Spring, MD). The monocytes, which were in contact with the surface, appeared as bright, white-centered cells after proper adjustment of the microscope during recording. The number of surface-adherent monocytes was measured after 5 min of perfusion at a minimum of 25 randomized, high-power fields. The rolling velocity of cells was measured as described previously [34
]. In short, a sequence of 50 frames representing an adjustable time interval (
t, with a minimal interval of 80 ms) was captured digitally. The position of every cell was detected in each frame, and for all subsequent frames, the distance traveled by each cell and the number of images in which a cell appears in focus were measured. The cut-off value to distinguish between rolling and static adherent cells was set at 1 µm/s. With this method, static adherent, rolling, and free-flowing cells (which were not in focus) could be clearly distinguished.
Flow cytometry and confocal microscopy to determine cell adhesion molecules expression upon PSGL-1 engagement on monocytes
Calibration of the flow cytometer was performed according to a standard procedure, which has been set and approved by the Dutch Foundation for Quality Assessment in Clinical Laboratories (SKML; Nijmegen, The Netherlands). Expression of adhesion molecules on the monocyte surface was investigated by flow cytometry (FACSVantage, Becton Dickinson) with cells that were incubated, or not, with platelets (see Monocyte isolation in Materials and Methods) or with a P-selectin Ig. By distinguishing between monocytes with no platelets bound to their surface (CD42b–) and platelet-bound monocytes (CD42b+), two different populations of monocytes were characterized, regarding the expression of different adhesion molecules. The expression of CD62L, CD18, CD11a, CD11b, CD29, and CD49d was determined by incubating monocytes with specific, directly labeled antibodies for 1 h at 4°C (according to the manufacturer’s instructions). Samples were also incubated with isotype-matched control antibodies (IgG1 and IgG2a, considering the antibody of interest, see Table 1 ). Integrin activation was investigated by incubating monocytes with the purified antibodies CBRM1/5 (activation-dependent epitope on M subunit) or HUTS21 (activation-dependent epitope on ß1 integrins) or the respective isotype control antibodies (unlabeled IgG1 and IgG2a, see Table 1
) for 1 h at 4°C. After washing one time with washing buffer, cells were further incubated with Alexa-488-labeled goat anti-mouse Ig antibody.
To further investigate the integrin expression and activation state induced by PSGL-1 engagement by P-selectin, the distribution of Mß2 (CD11b) and 4ß1 (CD49d) was characterized by confocal microscopy. Monocytes, untreated or treated with P-selectin Ig or PMA (positive control), were fixed with 3.7% formaldehyde in PBS containing 1 mM Ca2+ and 1 mM Mg2+ for 10 min at room temperature. After blocking with PBS containing 0.5% (w/v) BSA, 1 mM Ca2+, and 1 mM Mg2+, the expression of CD11b and CD49d was detected with the FITC-labeled antibodies. Images were recorded with a Zeiss LSM 510 confocal laser-scanning microscope.
Transmigration assay
Monocyte transmigration was studied under flow and static conditions. Monocyte suspensions (containing <5%, 10–20%, or 20–40% PMC) were perfused over 6 h—TNF--activated EC. After 5 min of perfusion, nonadherent cells were washed away, and incubation medium was perfused further for 10 min. The adherent cells that migrated through the EC layer were manually counted every 2 min.
Transmigration assays under static conditions were performed in 6.5 mm, 5 µm pore Transwell plates (Corning Costar), coated with fibronectin. Freshly isolated monocytes or PMC (1x105) were added to the upper compartment in 0.1 ml assay medium [IMDM medium with 0.25% (w/v) BSA], and 0.6 ml assay medium with 10 ng/ml recombinant human MCP-1 were added to the lower compartment. The Transwell plates were then incubated at 37°C, 5% CO2, for different time periods (30, 60, 90, and 120 min). Cells that migrated to the lower compartment were collected in a tube to which a fixed number of control U937 cells, labeled with calcein, were added. Flow cytometry analysis was used to determine the ratio between labeled and unlabeled cells. By comparing this ratio with that of the input control, the number of migrated cells was quantified. After the assay, cells from the upper side of the filter were removed with a cotton swab. The filters were then fixed and stained with Hoechst 33258. The migrated cells on the bottom side of the filters were counted with a microscope equipped with an ultraviolet filter in different fields of the cell filter.
Statistical analysis
Data are represented as the mean ± SD of three to five independent experiments, and comparisons were analyzed using the Mann-Whitney U test. All effects were assessed at the 0.05 level of significance.
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We further characterized the adhesive capacity of monocytes after PSGL-1 engagement by platelets or P-selectin Ig to various immobilized proteins (fibronectin, VCAM-1, and ICAM-1). Monocytes were incubated with different concentrations of platelets (allowing the formation of PMC, see Monocyte isolation in Materials and Methods) or with P-selectin Ig (10 µg/ml). As shown in Figure 1A , platelets enhance monocyte adhesion to the different protein surfaces in a concentration-dependent manner. The strongest effect was obtained when three platelets per monocyte were added (20–40% PMC). Similarly, the binding of P-selectin also enhanced monocyte adhesion to the different surfaces (Fig. 1B) .
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Figure 1. P-selectin/platelet binding induces monocyte adhesion to fibronectin, VCAM-1, and ICAM-1. Freshly isolated monocytes labeled with calcein, untreated (A, open bars) or incubated with platelets (10–20% or 20–40% PMC; shaded and solid bars, respectively, A), or P-selectin Ig (10 µg/ml, B) were added to 96-well tissue-culture plates coated with HSA (control), fibronectin (FN), VCAM-1 Ig, or ICAM-1 Ig. As a positive control, monocytes were treated with PMA before addition to the wells. This is represented by the line at 100%. All results are expressed as the mean ± SD values of adherent cells/mm2 of four independent experiments (**, P<0.01; *, P<0.05).
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4ß1, which blocks leukocyte adhesion), the increment in monocyte adhesion to fibronectin or VCAM-1 was abrogated completely. Similarly, when monocytes were preincubated with IB4 (a mAb to the integrin ß2 subunit, which blocks leukocyte adhesion) or 44a (a blocking mAb to integrin M), the monocyte adhesion to ICAM-1 was low. A mouse IgG1 antibody had no detectable effect. These data indicate that P-selectin specifically increases monocyte adhesion and suggests that the increased adhesion is mediated by ß1 and ß2 integrins.
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Figure 2. Antibodies against integrins or P-selectin block the platelet-dependent effect on monocyte adhesion. Monocytes were left untreated (control, open bars) or were treated with P-selectin Ig (–). For antibody inhibition experiments, P-selectin Ig was treated with WASP12.2 (a P-selectin-blocking antibody) prior to incubation with monocytes. Incubation with W6/32 (anti-HLA-A, -B, and -C, control antibody), HP2/1 (a CD49d-blocking antibody), and IB4 (a CD18-blocking antibody) or 44A (a CD11b-blocking antibody) was performed after binding of P-selectin Ig to the monocytes and before adding them to the wells on the tissue-culture plate. As a control, monocytes were also incubated with mouse IgG (mIgG1), prior to addition to the coated wells. After incubation at 37°C, the plates were washed, and the bound cells were lysed with 0.5% w/v Triton X-100 for 10 min at room temperature. Plates were then read on a microplate fluorescence plate reader at excitation wavelength 485 nm and emission wavelength 525 nm. All results are expressed as the mean ± SD values of adherent cells/mm2 of three independent experiments (**, P<0.01; *, P<0.05).
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Figure 3. P-selectin/platelet binding induces monocyte adhesion to fibronectin, VCAM-1, and ICAM-1 under flow conditions. Freshly isolated monocytes, untreated or incubated with platelets (10–20% or 20–40% PMC, see Materials and Methods) or P-selectin Ig (10 µg/ml), were perfused over glass coverslips coated with albumin, fibronectin, VCAM-1 Ig, or ICAM-1 Ig (upper graphic) or with TNF--activated HUVEC (lower graphic). For antibody inhibition experiments, platelets or P-selectin Ig were treated with WASP12.2 (P-selectin-blocking antibody) prior to incubation with monocytes. For all other conditions, incubation of monocytes with W6/32 (anti-HLA-A, -B, and -C, control antibody), HP2/1 (CD49d-blocking antibody), or 44A (CD11b-blocking antibody) and of HUVEC with 1G11 (VCAM-1-blocking antibody) or 84H10 (ICAM-1-blocking antibody) was performed for 10 min at 37°C just before starting the perfusion. Results are expressed as the mean ± SD values of adherent cells/mm2 of three independent experiments (**, P<0.01; *, P<0.05). VLA-4, Very late antigen-4.
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-activated HUVEC (Fig. 3
, lower graphic). We have previously shown [14
] that monocytes do not adhere to unstimulated endothelium under flow conditions. We have performed similar control experiments, which confirmed this (data not shown). Therefore, monocyte adhesion to the endothelium was used as control for proper stimulation of the EC by TNF-. When the PMC content in the monocyte suspension was less than 5%, blocking antibodies to VLA-4 or Mac-1 (on monocytes) and VCAM-1 or ICAM-1 (on HUVEC) reduced monocyte adhesion by 30%. As expected and as shown before [14
], a P-selectin-blocking antibody (WASP12.2) did not have an effect on monocyte adhesion under these conditions. With a PMC content of 20–40%, the same integrin-blocking antibodies also inhibited monocyte adhesion to HUVEC. However, a stronger inhibitory effect (50%) was obtained by blocking P-selectin on platelets.
Flow cytometric analysis of ß1 and ß2 integrin expression on P-selectin- or platelet-bound monocytes
We analyzed the expression of 4ß1 and Mß2 integrins on monocytes and determined whether P-selectin binding enhanced the level of integrins expressed on the monocyte surface. PSGL-1 ligation on monocytes was induced by incubation of monocytes with P-selectin Ig or with different amounts of platelets (see Materials and Methods). In Figure 4
, a histogram representative of the expression pattern of adhesion molecules on the surface of cells from the two distinct monocyte subpopulations (naked monocytes and monocytes with platelets on their surface, PMC) is shown. Clear differences regarding the expression of CD11a and CD62L on monocytes from the two subpopulations can be observed. After analyzing the expression of several other adhesion molecules, the differences between the two subpopulations were quantified and are indicated in Table 2
. Platelet- or P-selectin-bound monocytes showed increased expression of 4ß1 and Mß2 integrins (CD49d and CD11b, respectively, Table 2
, *P<0.05), and a decrease in L-selectin expression was observed, suggesting monocyte activation upon P-selectin binding. When P-selectin or platelet binding to monocytes was blocked by incubation of the cells with the anti-P-selectin mAb WASP12.2, no increase in integrin expression was observed, as shown previously [14
]. Although the increase in integrin expression on platelet-bound monocytes was stronger, an increase in ß2 integrin expression was also observed on the naked monocytes within the suspensions to which platelets were added, suggesting some degree of cell activation.
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Figure 4. Monocytes show a different pattern in the expression of adhesion molecules depending on the binding or absence of platelets on their surface. Monocytes were incubated with platelets to allow the formation of PMC (three platelets per monocyte, resulting in the formation of 20–40% PMC in the monocyte suspension; see Materials and Methods). Incubation of this cell suspension with a CD42b/PE antibody was used to distinguish two populations of monocytes: monocytes (no platelets bound to their surface and thus, CD42b–) and PMC (monocytes with platelets bound to their surface and thus, CD42b+). To investigate whether monocytes belonging to the two subpopulations show differences in an integrin-expression pattern, monocytes belonging to each subpopulation were analyzed regarding the expression of LFA-1 (CD11a) and L-selectin (CD62L) as described in Materials and Methods. The presented data are representative images of three independent experiments.
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Table 2. Influence of Platelet Binding on the Expression of Various Adhesion Molecules on the Monocyte Surface
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M subunit (Mac-1), and HUTS21 antibody reacts with an activation-dependent epitope on ß1 integrins. Incubation of monocytes with platelets or with P-selectin Ig resulted in a three- to fivefold increase in ß1 and ß2 integrin activation (Fig. 5
). Although the naked monocytes in PMC-rich suspensions did show a partial increase in integrin expression, these integrins were not activated.
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Figure 5. Platelet/P-selectin binding to monocytes induces integrin activation. Monocytes were incubated with platelets (10–20% or 20–40% PMC, see Materials and Methods), P-selectin Ig (10 µg/ml), or PMA (10 ng/ml). Within the monocyte suspensions to which platelets were added, incubation of monocytes with a CD42b/PE antibody was used to distinguish two populations of monocytes: monocytes with no platelets bound to their surface (open bars) and platelet-monocytes complexes (solid bars). The expression of integrin activation-dependent epitopes was determined as described in Materials and Methods. HUTS21 is a ß1integrin conformational-dependent mAb (upper graphic), and CBRM1/5 is a CD11b conformational-dependent mAb (lower graphic). All results are expressed as the mean ± SD values of MFI of three independent experiments (**, P<0.01; *, P<0.05).
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Figure 6. PSGL-1 antibodies enhance integrin expression and activation on monocytes. Washed monocytes were incubated with PL-1 or KPL-1 (blocking mAb to PSGL-1) or PL-2 (a nonblocking mAb to PSGL-1) antibody. Expression of CD11a, CD11b, and CD49d was determined by flow cytometry. Isotype-matched control antibodies (IgG1 and IgG2a) were taken as controls. Integrin activation was analyzed by flow cytometry after incubation of cells with antibodies specific for activation-dependent epitopes of CD11b (CBRM1/5) and ß1integrins (HUTS21) and subsequent staining with an Alexa-488-labeled goat anti-mouse Ig antibody. Data are expressed as the mean ± SD values of MFI of three different experiments (**, P<0.01; *, P<0.05).
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4ß1 and Mß2 integrins on the monocyte surface was investigated by confocal microscopy (Fig. 7
). Nontreated cells (control) stained for CD11b or CD49d showed only a weak and punctuated staining for both antibodies. As a positive control, PMA stimulation strongly induced a bright staining pattern. Stimulation by P-selectin Ig chimera resulted in an intermediate staining pattern, and the patches were larger and more abundant than in the control conditions. These data indicate induction of variable degrees of avidity of Mß2 and 4ß1 integrins by P-selectin and PMA. As the effect of platelet binding to monocytes on integrin up-regulation was so clear, the results were not quantitated.
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Figure 7. P-selectin induces ß1 and ß2 integrin expression and clustering. Monocytes, untreated (control) or treated with P-selectin Ig and PMA, were fixed with 3.7% formaldehyde in PBS containing 1 mM Ca2+ and 1 mM Mg2+ for 10 min at room temperature. After blocking with PBS containing 0.5% w/v BSA, 1 mM Ca2+, and 1 mM Mg2+, cells were incubated directly with FITC-labeled IgG1, CD11b, or CD49d mAb. Images were taken with a confocal laser-scanning microscope. The presented data are representative images of three independent experiments (original bar, 10 µm).
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Figure 8. Platelet binding to monocytes induces monocyte transendothelial migration. Transmigration of freshly isolated monocytes, untreated (monocytes) or incubated with platelets (PMC, see Materials and Methods), was studied under static conditions and under flow. For studies under static conditions, monocytes were added to the upper compartment of a Transwell plate coated with fibronectin, and 10 ng/ml recombinant human MCP-1 was added to the lower compartment. After incubation of the Transwell plates at 37°C for different time periods (30, 60, 90, and 120 min), the percentage of migration was quantified. All results are expressed as the mean ± SD values of percentage of migration or number of migrated cells/mm2 of three independent experiments (*, P<0.05).
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4ß1 and Mß2 integrins. Similar effects on monocyte activation were observed after ligation of PSGL-1 by P-selectin Ig chimera or by specific antibodies to PSGL-1. This P-selectin-triggered integrin activation was blocked completely by a blocking antibody to P-selectin, indicating that physical binding of P-selectin to PSGL-1 on monocytes is essential for this process. In fact and although the naked monocytes within the monocyte suspensions to which platelets have been added show a slight increase in integrin expression as a result of possible reversible platelet binding or platelet-released effectors, their integrin functionality remains unchanged. P-selectin-triggered signaling and its stimulatory effects on human leukocytes have been described in several previous reports. P-selectin binding to PSGL-1 has been shown to promote ß2integrin-dependent homotypic neutrophil aggregation and neutrophil-platelet conjugation [22 , 36 ]. Hidari et al. [24 ] demonstrated that ligation of PSGL-1 on human neutrophils with mAb or with P-selectin increased protein tyrosine phosphorylation, activated the extracellular signal-regulated kinase and MAPK, and induced secretion of IL-8. Monocytes, upon binding of activated platelets, were shown to secrete MCP-1 and IL-8 [37 ] and to express tissue factor [38 39 40 ].
Various mechanisms for a role of P-selectin in influencing the activity of ß1 and ß2 integrins have been suggested. However, most studies have shown that P-selectin cannot stimulate integrin activation directly on human leukocytes [20
, 37
, 40
]. Instead, P-selectin was described as an anchoring molecule, allowing monocytes to bind to activated endothelium, thus facilitating the binding of EC surface-bound platelet-activating factor (PAF) to its receptor on the leukocyte surface. Subsequently, immobilized chemoattractant PAF induced integrin activation [20
]. Our data indicate that the increase in integrin expression and activation occurs upon PSGL-1 ligation by platelets, P-selectin, or PSGL-1-specific antibodies. We observed an increase in monocyte adhesion to immobilized fibronectin, VCAM-1, and ICAM-1, indicative of integrin conformational changes. Furthermore, upon platelet binding to monocytes, there was an increase in Mß2- and 4ß1-dependent adhesion of monocytes to activated EC under flow conditions. This is in agreement with previous studies showing induction of Mß2 integrin upon platelet binding to human neutrophils [20
, 21
, 41
] and an increased affinity of monocytes to VCAM-1 by P-selectin binding under flow conditions [4
, 25
, 42
].
The presence of additional platelet-released, synergistic factors, such as the cytokines PAF and regulated on activation, normal T expressed and secreted [4 ], seems to be required to induce optimal leukocyte activation. This might explain the observed monocyte adhesion upon P-selectin-Ig binding or PSGL-1 antibodies, which was in some instances lower than the one obtained by adding freshly isolated platelets to monocytes. A recent study [20 ] about neutrophils suggested an intermediate state of integrin activation induced by engagement of PSGL-1 by P-selectin Ig or antibodies to PSGL-1. This intermediate integrin activation state is compatible with a moderate increase in monocyte adhesion to immobilized proteins or activated EC. Furthermore, depending on the leukocyte type, PSGL-1 structure and subsequent affinity for its main ligand, P-selectin, seem to differ. When compared with neutrophils, PSGL-1 on eosinophils has been shown to bind tenfold stronger to P-selectin [43 ]. Recently, platelets were shown to preferentially bind monocytes over neutrophils under flow [44 ], suggesting differences in structure/affinity of PSGL-1 on the two different cell types. These differences might result in the induction of different PSGL-1-mediated integrin-signaling pathways and explain the effect of platelet binding on integrin expression and activation observed by us, in contrast to others [20 ].
In conclusion, our data show an increase in expression and adhesive capacity of 4 and M integrins upon PSGL-1 ligation by P-selectin on human monocytes. PSGL-1 ligation by platelet binding results in increased integrin activation and subsequently, increased cell adhesion to fibronectin, VCAM-1, ICAM-1, and activated EC. Although an increase in monocyte transmigration was also observed, the exact role of platelets in this phenomenon needs further investigation.
The concerted action of a variety of stimuli such as chemoattractants and P-selectin, provided by platelet binding, seems to modulate the activation of monocyte integrins relevant for the monocyte extravasation process and thus, for their atherogenic capacity.
Received June 15, 2005; revised November 18, 2005; accepted November 21, 2005.
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-granule membrane protein (GMP-140) is expressed on the plasma membrane after activation J. Cell Biol. 101,880-886Mß2 activation and acts cooperatively with extracellular stimuli to support maximal adhesion of human neutrophils Blood 104,2549-2556 secretion. Signal integration and NF- B translocation J. Clin. Invest. 95,2297-2303[Medline]4ß1 and 5ß1 integrin activity by CC chemokines in monocytes: implications for transendothelial chemotaxis J. Cell Biol. 134,1063-1073 4 ß 1 (VLA-4) and 4 ß 7 on the human B cell line JY J. Biol. Chem. 267,8366-8370This article has been cited by other articles:
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P. Haselmayer, L. Grosse-Hovest, P. von Landenberg, H. Schild, and M. P. Radsak TREM-1 ligand expression on platelets enhances neutrophil activation Blood, August 1, 2007; 110(3): 1029 - 1035. [Abstract] [Full Text] [PDF]
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R. R. Koenen, P. von Hundelshausen, and C. Weber Inflammatory Blues Turns Velvet Skin Into Rawhide: Monocyte Rolling on Modified Endothelial PSGL-1 Arterioscler. Thromb. Vasc. Biol., May 1, 2007; 27(5): 990 - 992. [Full Text] [PDF]
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