Biology of monocyte interactions with the endothelium : the platelet factor - Chapter 4 "Platelet binding to monocytes increases the adhesive properties of monocytes by upregulating the expression and functionality o

UvA-DARE (Digital Academic Repository)

Biology of monocyte interactions with the endothelium : the platelet factor

da Costa Martins, P.A.

Publication date

2005

Link to publication

Citation for published version (APA):

da Costa Martins, P. A. (2005). Biology of monocyte interactions with the endothelium : the

platelet factor.

General rights

It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons).

Disclaimer/Complaints regulations

If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible.

"Platelett binding to monocytes increases

thee adhesive properties of monocytes by

upregulatingg the expression and

functionalityy of pi and fe integrins"

11

Paula A. da Costa Martins,

Janine M. van Gils,

Anita Mol,

11

Peter L Hordijk and

Jaap J. Zwaginga

departmentt of Immunohematology, Sanquin Research, Location CLB, Amsterdam departmentt of Hematology, Academical Medical Center, Amsterdam, Thee Netherlands Submittedd for publication

Abstract t

Humann monocytes adhere to activated platelets that display P-selectin, an adhesionn molecule that recognizes P-selectin glycoprotein-1 (PSGL-1), which is a specificc ligand for P-selectin on leukocytes. We have recently shown that platelet bindingg to monocytes increases the adhesive capacity of monocytes to activated endothelium.. To better understand the effect of platelet binding on the capacity of monocytess to adhere to activated endothelium the PselectinPSGL1 interaction -inducedd changes in integrin functionality were studied. The binding of platelets to monocytess via P-selectin-PSGL-1 interactions was shown to increase expression andd activity of a4(3i- and OMp2- integrin, with a concomitant decrease in L-selectin

expression.. Furthermore, the binding of platelets to monocytes resulted in increasedd monocyte adhesion to ICAM-1, VCAM-1 and fibronectin. Platelet bindingg was also responsible for an increase in monocyte transendothelial migration.. Similar effects were observed after engagement of PSGL-1 with specific antibodiess or with P-selectin-lg protein.

Ourr data suggest that platelets by binding via P-selectin to PSGL-1 on monocytess induce PSGL-1 signaling, leading to upregulation and activation of (3i-andd p2- integrins and increased adhesion of monocytes to activated endothelium.

Hence,, monocytes within platelet-monocyte complexes are in a higher state of activationn and have an increased atherogenic capacity.

Introduction n

Whilee monocyte adhesion to the damaged endothelium is essential for atherogenesis,, platelet recruitment to atherosclerotic lesions is responsible for acute thrombo-embolicc events causing myocardial infarction (1-5). The interactions betweenn platelets and monocytes might therefore modulate both thrombosis and atherogenesis.. In this respect, platelet-leukocyte complexes in the circulation are knownn markers of platelet activation associated with vascular damage caused by atheroscleroticc lesions (6,7).

Activatedd platelets express P-selectin, a member of the selectin family, which uponn activation is translocated from the a-granules to the platelet surface (8,9). The mainn ligand for P-selectin is P-selectin Glycoprotein Ligand-1 (PSGL-1) - a disulfide-linkedd homodimer with a molecular weight of -220 kDa on platelets and most leukocytess (10-12). P-selectin and PSGL-1 are considered to be the main players in platelet-monocytee interactions, which not only mediate the binding of leukocytes to activatedd platelets or thrombi localized at the injured vessel wall but also the formationn of platelet-leukocyte complexes (mainly platelet-monocyte complexes) in thee circulation (2,13,14).

Monocytes,, as other leukocytes, are recruited to cytokine-activated endotheliumm in a multistep process. Initially, monocytes in the blood stream have to bee slowed down by a capturing mechanism and roll over the endothelial layer. Activationn of the monocytes during the rolling phase will result in firm adhesion and transmigrationn (15,17). While the latter process is mediated by interactions between thee leukocyte integrins and their endothelial ligands, capture and rolling are mainly mediatedd by selectins and their respective receptors (17-19). By comparing monocytess and PMC regarding their capacity to adhere to the endothelium, we have previouslyy shown that PMC are more adhesive. This increased adhesion is to a large extentt dependent on enhanced monocyte-PMC interactions leading to the formation off flow-oriented monocyte and PMC clusters. P-selectin - PSGL-1 interactions are involvedd in the formation of these secondary tethers (14).

P-selectinn has been shown to increase B2 integrin functionality on neutrophils

(20-22)) and to enhance the nuclear transcription of NF-KB that is required for the

productionn of cytokines such as MCP-1 and TNF-a (23). The idea of PSGL-1 ligation -- mediated signaling has been emphasized by the observation that cross-linking of PSGL-11 on neutrophils induced protein-tyrosine phosphorylation, activated MAP kinases,, and stimulated IL-8 secretion. Moreover, PSGL-1 engagement on mouse neutrophilss induces LFA-1 - and Mac-1 - dependent adhesion to ICAM-1 (24). Furthermore,, P-selectin binding to its counterreceptor PSGL-1 promotes a46i

-dependentt adhesion of monocytes to vascular cell adhesion molecule 1 (VCAM-1) (25). .

Altogetherr these observations suggest a radical change, both quantitative and qualitative,, in the leukocyte repertoire of surface-expressed adhesion molecules uponn platelet binding. To better characterize the effect of platelet binding on the adhesivee capacity of monocytes to adhere to activated endothelium we focused on thee P-selectin-PSGL-1 interaction. The present study demonstrates that the binding off platelets to monocytes via P-selectin-PSGL-1 interactions results in stronger adhesionn of monocytes to ICAM-1, VCAM-1 and fibronectin. Similar results were obtainedd after engagement of PSGL-1 with a specific antibody or with P-selectin-lg. Furthermore,, PSGL-1 engagement not only caused changes in integrin activation but itt also changed the integrin expression pattern on monocytes.

Ourr data suggest that platelets by binding, via P-selectin, to PSGL-1 on monocytes,, induce PSGL-1 signaling leading to translocation of pi and B2 integrins to

thee monocyte surface as well as integrin activation. Hence, monocytes within platelet-monocytee complexes are in a higher activation state and therefore, by adheringg more to the endothelium, their atherogenic capacity will be increased.

Materiall and Methods

Reagents.Reagents. Human serum albumin (HSA) was purchased from Sanquin

Immunoreagentss (Amsterdam, The Netherlands). Bovine serum albumin and PMA weree from Sigma-Aldrich (St. Louis, MO, USA). Recombinant TNF-a was from Boehringerr Mannheim (Germany) and recombinant MCP-1 was from Strathman Biotechh (Hannover, Germany). Alexa Fluor 488 phalloidin and Hoechst were from Molecularr Probes (Eugene, OR, USA). Washing buffer contained PBS supplemented withh 0.5% human serum albumin and 13 mM trisodium citrate. Incubation buffer containedd 20 mM HEPES, 132 mM NaCI, 6 mM KCI, 1 mM MgS04, 1.2 mM KH2P04

supplementedd with 5 mM glucose, 1.0 mM CaCI2 and 0.5% (w/v) HSA. IMDM

mediumm was from BioWhittaker (Verviers, Belgium) and tissue culture supplies (media,, antibiotics and trypsin) were from Gibco, Life Technologies Inc (Paisley, UK).

ProteinsProteins and Monoclonal antibodies. Human P-selectin-Fc chimera,

ICAM-1-lgGG and VCAM-1-IgG were purchased from R&D Systems (Minneapolis, MN, USA).. Human plasma fibronectin was from Sigma-Aldrich. Monoclonal antibodies (mAbs)) WASP 12.2 (CD62P, anti P-selectin), DREG 56 (CD62L, anti L-selectin), IB4 (CD18,, anti B2-integrin) and 44A (CD11b) were isolated from the supernatant of

hybridomashybridomas obtained from the American Type Culture Collection (Rockville, MD, USA).. The mAbs as mentioned above are functionally blocking antibodies. Control

antibodyy W6/32 (anti-HLA-A,B,C) was isolated from the supernatant of a hybridoma obtainedd from the American Type Culture Collection. Specific mAbs against human

PSGL-1,, PL-1 (blocking of PSGL-1 binding) and PL-2 (non-blocking) were kindly providedd by Dr. Kevin L. Moore (University of Oklahoma, OK, USA). MAb KPL-1 (blocking-PSGL-11 antibody) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA).. MAbs against human CD49d (HP2/1, a4piintegrin), VCAM-1 (1G11, CD106)

andd ICAM-1 (84H10, CD54) were purchased from Immunotech (Marseille, France). Thee CD11b conformational-dependent mAb CBRM1/5 was a kind gift of Dr. Kevin L. Mooree (University of Oklahoma, OK, USA). The pHntegrin conformation-dependent mAbb HUTS21 was kindly provided by Dr. F. Sanchez-Madrid (Hospital de la Princesa,, Madrid, Spain). The following directly labeled mAbs were purchased from Sanquinn Immunoreagents (Amsterdam, The Netherlands): IgGi/FITC, lgG2a/FITC, CD14/FITCC (clone CLB-mon/1, 8G3), CD62P/PE (clone CLB thromb/6), CD42b/PE (clonee CLB-MB45), CD29/FITC (clone 2A4), CD18/FITC (clone CLB-LFA-1/1), CD11b/FITCC (clone CLB-mon-gran/1,B2) and mouse I g d (FITC- or PE- labeled). FITC-labeledd 44H6 mAb (anti human CD49d) was from Chemicon International (Temecula,, CA) and FITC-labeled CD62L (anti L-selectin) was from Becton Dickinsonn (San Jose, CA). Human and mouse IgG were purchased from Sigma (St. Louis,, MO).

MonocyteMonocyte isolation. Whole blood, anticoagulated with 0.4% trisodium citrate

(pHH 7.4) was obtained from healthy volunteers from the Sanquin Blood Bank (Amsterdam,, The Netherlands). Monocytes were negatively selected from human peripherall blood by means of a MACS monocyte isolation kit according to the manufacturer'ss instructions (Miltenyi Biotech GMBH, Bergisch Gladbach, Germany). Thiss procedure resulted in monocyte fractions containing more than 90% monocytes (CD14-positivee cells in FacScan), the viability exceeding 95% (as determined by Trypann blue exclusion). To obtain PMC-poor monocyte suspensions, the monocytes weree incubated with a mouse IgG mAb against GPIIIa for 20 min at . After one washingg step, the cells were incubated with goat-anti-mouse-IgG microbeads (Dynabeads,, Dynal A.S., Oslo, Norway), at a ratio of two beads per platelet, for 20 minn at . After magnetic extraction of the beads, the number of PMC was less than 5%% of the total number of monocytes. After isolation, the cells were resuspended in incubationn buffer. For blocking experiments, monocyte suspensions with or without PMCC were incubated with mAbs for 10 min at C prior to the perfusion experiments.. In some instances, washed platelets were added to the monocyte suspensionn just before perfusion (addition of 1 or 3 platelets per monocyte resulted in thee formation of 10-20 or 20-40% of PMC, respectively).

PlateletPlatelet isolation. Whole blood was centrifuged at 150 *g for 10 min to obtain

platelet-richh plasma (PRP), which was diluted 1:1 in Krebs-Ringer solution (4 mM KCI,, 107 mM NaCI, 20 mM NaHC03, 2 mM Na2S04, 19 mM tri-sodium citrate, 0.5%

(w/v)) glucose in H20, pH 6.1). The mixture was centrifuged at 500 *g for 10 min and

thee supernatant was removed. The platelets in the pellet were resuspended in 2 ml of Krebs-Ringerr solution and centrifuged at 500 *g for 10 min. This process was repeatedd two times, the final suspension being made up in Krebs-Ringer solution to a concentrationn of 300,000 platelets/ul.

CellCell culture. Human umbilical vein endothelial cells (HUVEC) were isolated

fromm human umbilical cord veins as described (26,27). Cells were cultured in RPMI 16400 containing 20% (v/v) human serum, 200 ug/ml penicillin and streptomycin (Gibco)) and were grown to confluence in 5-7 days. Endothelial cells from the third passagee were used in the experiments. TNF-a (100 U/ml) was added directly to the mediumm 6 hours prior to the experiments. U937 cells (a monocytic cell line derived fromm human histiocytic lymphoma) were purchased from the American Type Culture Collectionn (Manassas, VA, USA). The cells were cultured in RPMI 1640 (Gibco) containingg 10% (v/v) heat-inactivated fetal calf serum (Gibco), 2 mM L-glutamine, 50 lU/mll penicillin and 50 ug/ml streptomycin (complete medium).

AdhesionAdhesion assay. Adhesion assays were performed as previously described

(28,29)) with some modifications. Briefly, 96-well microtiter plates (Costar No. 3596, Cambridge,, MA) were coated by incubation with fibronectin (10 ug/ml), VCAM-1 or ICAM-1/Fcc chimera (1 ug/ml) for 1h at C or 4% HSA as control for 1h at . Afterr incubation, the wells were washed with PBS and then blocked with 4% HSA at CC for 30 minutes. Control wells were filled with 4% HSA in PBS. Monocytes were labeledd with calcein AM (Molecular Probes, Eugene, OR, USA) at a final concentrationn of 5 ug/1 *107 cells. In some instances, monocytes were incubated withh platelets or P-selectin after calcein labeling. For PMA stimulation, cells were addedd to wells containing 10 ng/ml of PMA. For blocking experiments, cells were addedd to wells containing the function-blocking monoclonal antibodies. Plates were thenn incubated at C and cells were allowed to settle for 30 minutes. After incubation,, non-adherent cells were removed by washing twice with PBS and adherentt cells were lysed in 0.5% Triton X-100 for 10 min at room temperature. Adhesionn was quantified with a microplate fluorescence plate reader (Tecan GENios plus,, Tecan Group Ltd, Mannedorf, Switzerland). Fluorescence was measured at excitationn wavelength 485 nm and emission wavelength 525 nm. The adhesion ratio (%)) was calculated as follows: [fluorescence from experimental sample

-fluorescencefluorescence from negative control sample) / total fluorescence added to well * 100%. 100%.

MonocyteMonocyte perfusion and evaluation of cell adhesion. Monocytes in

suspensionn (2 x 106 cells/ml in incubation buffer) were aspirated from a reservoir throughh plastic tubing and perfused through a chamber with a Harvard syringe pump (Harvardd Apparatus, South Natic, MA, USA). The shear stress through the chamber wass precisely controlled and was kept at 0.8 dyn/cm2. During perfusions the flow chamberr (27,30) was mounted on a microscope stage (Axiovert 25, Zeiss, Germany),, equipped with a B/W CCD video camera (Sanyo, Osaka, Japan), and coupledd to a VHS video recorder. Video images were evaluated for the number of adherentt monocytes and the rolling velocity per cell with dedicated routines made in thee image analysis software Optimas 6.1 (Media Cybernetics Systems, Silverspring, MD,, USA). The monocytes that were in contact with the surface appeared as bright white-centeredd cells after proper adjustment of the microscope during recording. The numberr of surface-adherent monocytes was measured after 5 minutes of perfusion att a minimum of 25 randomized high-power fields. To automatically determine the velocityy of rolling cells, custom-made software was developed in Optimas 6.1. A sequencee of 50 frames representing an adjustable time interval (5t, with a minimal intervall of 80 milliseconds) was digitally captured. The position of every cell was detectedd in each frame, and for all subsequent frames the distance traveled by each celll and the number of images in which a cell appears in focus was measured. The cut-offf value to distinguish between rolling and static adherent cells was set at 1 nm/sec.. With this method, static adherent, rolling and free flowing cells (which were nott in focus) could be clearly distinguished.

FlowcytometryFlowcytometry and confocal microscopy to determine cell adhesion moleculesmolecules expression upon PSGL-1 engagement on monocytes. Expression of

adhesionn molecules on the monocyte surface was investigated by flowcytometry (FACSS Vantage, Becton Dickinson, San Jose, CA) with cells that were incubated, or not,, with platelets (see Material and Methods) or with a P-selectin-lg. By distinguishingg between monocytes with no platelets bound to their surface (CD14-positive/CD42b-negativee events) and platelet-bound monocytes (CD14/CD42b-positivee events), two different populations of monocytes were characterized, regardingg the expression of different adhesion molecules. The expression of CD62L, CD18,, CD11a, CD11b, CD29 and CD49d was determined by incubating monocytes withh specific, directly labeled antibodies. Isotype-matched control antibodies IgGi andd IgGza were taken along. Integrin activation was investigated by incubating monocytess with the antibodies CBRM1/5 (activation-dependent epitope on aM

subunit)) or HUTS 21 (activation-dependent epitope on pi integrins).

Too further investigate the integrin expression and activation state induced by PSGL-11 engagement by P-selectin, the distribution of aMB2 (CD11b) and a4pi

(CD49d)) was characterized by confocal microscopy. Monocytes, untreated or treated withh P-selectin-lg or PMA (positive control) were fixed with 3.7% formaldehyde in

PBSS containing 1 mM Ca2+ and 1 mM Mg2+ for 10 minutes at room temperature. After blockingg with PBS containing 0.5% (w/v) bovine serum albumin, 1 mM Ca2+ and 1 mMM Mg2+: CD11a and CD49d were detected with FITC-labeled antibodies. Images

weree recorded with a Zeiss LSM 510 confocal laser scanning microscope.

TransmigrationTransmigration assay. Monocyte transmigration was studied under flow and

staticc conditions. Monocyte suspensions (<5%, 10-20% and 20-40% PMC) were perfusedd over 6-hour - TNF-a - activated EC. After 5 minutes of perfusion, non-adherentt cells were washed away and incubation medium was further perfused for 100 minutes. The adherent cells that migrated through the endothelial cell layer were manuallyy counted every two minutes.

Transmigrationn assays under static conditions were performed in 6.5-mm, 5 um-poree Transwell plates (Corning Costar, Cambridge, MA), coated with fibronectin. Freshlyy isolated monocytes or PMC (1 *105) were added to the upper compartment inn 0.1 ml of assay medium (IMDM medium with 0.25% (w/v) BSA) and 0.6 ml of assayy medium with 10 ng/ml recombinant human MCP-1 were added to the lower compartment.. The Transwell plates were then incubated at , 5% C02 l for

differentt time periods (30, 60, 90 and 120 minutes). Cells that migrated to the lower compartmentt were collected in a tube to which a fixed number of control U937 cells, labeledd with calcein, were added. Flowcytometry analysis was used to determine the ratioo between labeled and unlabeled cells. By comparing this ratio with that of the inputt control, the number of migrated cells was quantified. After the assay cells from thee upper side of the filter were removed with a cotton swab. The filters were then fixedd and stained with Hoechst. The migrated cells on the bottom side of the filters weree counted with a microscope equipped with a UV filter in different fields of the cell filter. .

StatisticalStatistical analysis. Data are represented as the mean S.E.M. of at least 3

independentt experiments and were compared with a two-tailed Student's t-test or a one-wayy ANOVA with Bonferroni correction. P values < 0.05 were considered to be significant. .

Results s

PSGL-1PSGL-1 ligation by P-selectin increases monocyte adhesion to immobilizedimmobilized fibronectin, VCAM-1 and ICAM-1. To test the effect of P-selectin in

monocytee adhesiveness, we analyzed changes in monocyte adhesion to fibronectin. Freshlyy isolated monocytes were treated with various concentrations of P-selectin-lg andd added to fibronectin-coated wells of tissue culture plates. After incubation at 37 CC for 30 minutes, unbound cells were washed with PBS, and bound monocytes were quantified.. P-selectin-lg enhanced monocyte adhesion to fibronectin in a concentration-dependentt manner. A maximal effect was obtained at 10ug/ml (data nott shown).

Wee further characterized the adhesive capacity of monocytes after PSGL-1 engagement,, by platelets or P-selectin-lg, to various immobilized proteins (fibronectin,, VCAM-1, and ICAM-1). Monocytes were incubated with different concentrationss of platelets (allowing the formation of PMC, see Material and Methods)) or with P-selectin-lg (10ug/ml). As shown in figure 1A, platelets enhance monocytee adhesion to the different protein surfaces in a concentration-dependent manner.. The strongest effect was obtained when 3 platelets per monocyte were addedd (20-40% PMC). Similarly, binding of P-selectin also enhanced monocyte adhesionn to the different surfaces.

Too verify the specificity of this effect, i.e., the role of platelets and integrins, the physicall interaction of platelets with monocytes was inhibited by antibodies to cell-surfacee receptors. A blocking antibody against P-selectin blocked the increment in monocytee adhesion to all immobilized proteins (Figure 1B). When monocytes were incubatedd with HP2/1 (a mAb to integrin a4(3i that blocks leukocyte adhesion) the

incrementt in monocyte adhesion to fibronectin or VCAM-1 was completely abrogated.. Similarly, when monocytes were preincubated with IB4 (a mAb to the integrinn (32 subunit that blocks leukocyte adhesion) or 44a (a blocking mAb to integrin

aM)) the monocyte adhesion to ICAM-1 was very low (Figure 1B). A mouse IgGi

antibodyy had no detectable effect. These data indicate that P-selectin specifically increasess monocyte adhesion and suggests that the increased adhesion is mediated byy both pi - and p2 -integrins.

AdhesionAdhesion of monocytes to ECM and endothelial substrates under flow conditions.conditions. We next examined the effects of PSGL-1 engagement on monocyte

adhesivenesss to fibronectin, VCAM-1, and ICAM-1 under flow conditions. Similar to thee results obtained under static conditions, also under flow conditions we observed aa significant increase in monocyte adhesion to the different protein surfaces (Figure 2,, upper panel).

11 D<5%PMC 10-20% PMC cc 7SJ CS 20-40% PMC P-sel Ig 50 0 88 25

£ÈÈ £ÈÈ

Ctrll FN VCAM-1 ICAM-1 PMA

B B

^ ^

FN N

l - . l l

controll control WASP12.2 HP2H mlgG1

VCAM-1 1

40 0 20 0 0 0

controll control WASP12.2 HP2/1 mlgG1

ICAM-1 1

a a

controll control WASP12.2 IB4 44a mlgG1

Figuree 1. P-selectin/platelet binding induces monocyte adhesion to fibronectin, VCAM-1 andd ICAM-1. Freshly isolated monocytes labeled with calcein, untreated or incubated with platelets

(10-20%% or 20-40% PMC, see Material and Methods) or P-selectin-lg (10ug/ml), were added to 96-welll tissue culture plates coated without (control) or with fibronectin, VCAM-1-lg or ICAM-1-lg (A). For antibodyy inhibition experiments (B) 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 CD11bb - blocking antibody) was performed after binding of P-selectin Ig to the monocytes and before addingg them to the wells on the tissue culture plate. As a control, monocytes were also incubated with mousee IgG (mlgG!), prior to addition to the coated wells. After incubation at C the plates were washedd and the bound cells were lysed with 0.5% w/v Triton X-100 for 10 minutes at room temperature.. Plates were then read on a microplate fluorescence plate reader at excitation wavelengthh 485 nm and emission wavelength 525 nm. All results are expressed as the mean S.E.M. valuess of adherent cells / mm2 _{of three independent experiments (**p<0.01, *p<0.05).}

Too determine whether integrin activation, modulated by PSGL-1 - P-selectin binding,, changes the monocyte capacity to adhere to a model of inflamed endotheliumm we perfused freshly isolated monocytes, incubated or not with platelets orr with P-selectin-lg, over TNF-a - activated HUVEC (Figure 2, lower panel). When thee PMC content in the monocyte suspension was less than <5%, blocking antibodiess to VLA-4 or Mac-1 (on monocytes) and VCAM-1 or ICAM-1 (on HUVEC) reducedd monocyte adhesion by 30%. As expected and as shown before 14, a P-selectinn blocking antibody (WASP12.2) did not have an effect on monocyte adhesion underr these conditions. With a PMC content of 20-40%, the same integrin - blocking antibodiess also inhibited monocyte adhesion to HUVEC. However, a stronger inhibitoryy effect (50%) was obtained by blocking P-selectin on platelets.

1000 0 800 0 600 0

—..

3-3- albumin FN VCAM-1 ICAM-1

c c .22 1000 to o <B <B =00 800 (0 0 gg 600 400 0 200 0

Figuree 2. P-selectin/platelet binding induces monocyte adhesion to fibronectin, VCAM-1 andd ICAM-1 under flow conditions. Freshly isolated monocytes, untreated or incubated with plateletss (10-20% or 20-40% PMC, see Material and Methods) or P-selectin-lg (10ug/ml), were perfusedd over glass coverslips coated with albumin, fibronectin, VCAM-1-Ig or ICAM-1-Ig (A) or with TNF-aa - activated HUVEC (B). For antibody inhibition experiments, platelets or P-selectin Ig were treatedd with WASP12.2 (P-selectin - blocking antibody) prior to incubation with monocytes. Incubation off 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 , just before startingg the perfusion. Results are expressed as the mean S.E.M. values of adherent cells / mm2 of threee independent experiments (**p<0.01, *p<0.05).

monocytes 010-20%% PMC

20-40% PMC P-sel Ig

FlowFlow cytometric analysis of fa and fa integrin expression on P-selectin-oror platelet-bound monocytes. We analyzed the expression of cupN and 0MP2

integrinss on monocytes and determined whether P-selectin binding enhanced the levell of integrins expressed on the monocyte surface. PSGL-1 ligation on monocytes wass induced by incubation of monocytes with P-selectin-lg or with different amounts off platelets (see Material and Methods). Platelet- or P-selectin - bound monocytes showedd increased expression of both cupi and aMp2 integrins (CD49d and CD11b,

respectively,, Table I, * p<0.05) while a decrease in L-selectin expression was observed,, suggesting monocyte activation upon P-selectin binding. When P-selectin orr platelet binding to monocytes was blocked by incubation of the cells with the anti-P-selectinn mAb WASP12.2, no increase in integrin expression was observed, as previouslyy shown (14). Although the increase in integrin expression on platelet-bound monocytess was stronger, an increase in P2 integrin expression was also observed on thee naked monocytes within the suspensions to which platelets were added.

Tablee 1. Expression of integrins on monocytes is increased upon platelet/P-selectin binding.

adhesion n molecule e CD62L L CD18 8 CDllb b CDD 29 CD49d d monocytes s <5%% 10-20% 20-40% PMCC PMC PMC 44 0 4 00 0 4 44 4 7 1411344 4 1 44 1 1 PMC C <5%% 10-20% 20-40% PMCC PMC PMC n.d.. 4 * n.d.. * * n.d.. * * n.d.. * * n.d.. 8 * P-selectin n (10(ig/ml) ) * * * * 7 7 * * * *

Freshlyy isolated monocytes (<5% PMC), were incubated with platelets for 30 minutes at , to alloww the formation of platelet-monocyte complexes (PMC, see Material and Methods) or with P-selectin-lg,, (10ug/ml). Incubation of monocytes with a CD42b/PE antibody was used to distinguish two populationss of monocytes: monocytes with no platelets bound to their surface and platelet-monocyte complexess (PMC, CD42b positive events). Expression of adhesion molecules was analysed and describedd in Material and Methods. Specific isotype-matched control <lgG<i and lgG2a) antibodies were takenn along. All results are expressed as the mean S.E.M. values of mean fluorescence intensity (MFI)) of five independent experiments (*p<0.05).

Integrinn activation was assessed by the use of specific antibodies such as CBRM1/55 and HUTS 21. CBRM1/5 antibody reacts with an activation-dependent epitopee on the aM subunit (Mac-1) while HUTS 21 antibody reacts with an

activation-dependentt epitope on Pi integrins. Incubation of monocytes with platelets or with P-selectin-lgg resulted in 3-5 fold increase in pi and P2 integrin activation (Figure 3). In PMC-richh suspensions the naked monocytes did not show an increase in integrin activity. .

Besidess the effect of P-selectin binding to PSGL-1, also the influence of specificc PSGL-1 antibodies (KPL-1, PL-1 and PL-2) on integrin upregulation on monocytess was investigated. Addition of PL-2 (a non-blocking PSGL-1 antibody), KPL-11 or PL-1 (two blocking mAbs to PSGL-1) to monocytes (no platelets present) alsoo resulted in increased integrin expression and activation (Figure 4). These results indicatee that mAb interaction with PSGL-1 on monocytes is sufficient to induce functionall up-regulation of Pi and p2 integrins.

£ 5 5 2 i i B B Dmonocytess «PMC 88 2 400 oo m So o o O O 33 ?

U U

—ii Jfl I

I I

Figuree 3. Platelet/P-selectin binding to monocytes induces integrin activation.

Monocytess were incubated with platelets (see Material and Methods), P-selectin-lg (10ug/ml) or PMA (100 ng/ml). Within the monocyte suspensions to which platelets were added, incubation of monocytes withh a CD42b/PE antibody was used to distinguish two populations of monocytes: monocytes with no plateletss bound to their surface (open bars) and platelet-monocytes complexes (CD42b positive events,, black bars). The expression of integrin activation-dependent epitopes was determined as describedd in Material and Methods. HUTS21 is a PHntegrin conformational-dependent mAb (A) and CBRM1/55 is a CD11b conformational-dependent mAb (B). All results are expressed as the mean S.E.M.. values of mean fluorescent intensity (MFI) of three independent experiments (** p<0.01,* p<0.05,, **)

ConfocalConfocal microscopy analysis of integrin expression on P-selectin-boundbound monocytes. To further characterize the integrin expression and activation

integrinss on the monocyte surface was investigated by confocal microscopy (Figure 5).. Non-treated cells (control) stained for CD11b or CD49d showed only a weak and puntuactedd staining for both antibodies. As a positive control, PMA stimulation stronglyy induced a bright staining pattern. Stimulation by P-selectin-lg chimera resultedd in an intermediate staining pattern with the patches being larger and more abundantt than in the control conditions. These data indicate induction of variable degreess of avidity of both ciMp2 and a4Pi integrins by P-selectin and PMA.

DlgG11 «PL-2 HKPL-1 «PL-1

Figuree 4. PSGL-1 antibodies enhance integrin expression and activation on monocytes. Washedd monocytes were incubated with PL-1 or KPL-1 (blocking mAbs to PSGL-1) or PL-2 (a non-blockingg mAb to PSGL-1) antibody. Expression of CD11a, CD11b and CD49d was determined by flowcytometry.. Isotype-matched control antibodies (IgG! and lgG2a) were taken as controls. Integrin

activationn was analysed by flowcytometry after incubation of cells with antibodies specific for activation-dependentt epitopes of CD11b (CBRM1/5) and pyintegrins (HUTS21). Data are expressed ass the mean S.E.M. values of mean fluorescence intensity (MFI) of three different experiments (**p<0.01,*p<0.05). .

EffectEffect of platelet binding on monocyte transmigration. We further

investigatedd a possible correlation between the observed increase in monocyte adhesionn to HUVEC upon PSGL-1 ligation and an increase in monocyte transmigration.. An increase in monocyte transmigration was observed when 20-40% PMCC were present. Under static conditions the percentage of migration towards MCP-11 was 25% higher when PMC were present (Figure 6). PMC presence also influencedd monocyte transmigration under flow conditions because 50% of the cells migratedd within the first 6 minutes of perfusion while in the absence of PMC this processs took 10 minutes (data not shown).

control l P-sell Ig PMA A

Figuree 5. P-selectin induces p^ and p2-integrin expression and clustering. Monocytes

weree treated without (control) or with P-selectin-lg and PMA. Cells were then fixed with 3.7% formaldehydee in PBS containing 1 mM Ca2+ and 1 mM Mg2+ for 10 minutes at room temperature. After blockingg with PBS containing 0.5% w/v bovine serum albumin, 1 mM Ca2+ and 1 mM Mg2+, cells were incubatedd with a IgG^ CD11b or CD49d directly-labeled mAbs. Images were taken with a confocal laserr scanning microscope. The presented data are representative images of three independent experimentss (bar=10 urn).

16 6 12 2

I I

Figuree 6. Platelet binding to monocytes induces monocyte transendothelial migration.

Transmigrationn of freshly isolated monocytes, untreated (monocytes) or incubated with platelets (PMC,, see Material and Methods) was studied under static conditions and under flow. For studies underr static conditions, monocytes were added to the upper compartment of a Transwell plate coated withh fibronectin and 10 ng/ml of recombinant human MCP-1 was added to the lower compartment. Afterr incubation of the Transwell plates at C for different time periods (30, 60, 90 and 120 minutes),, the percentage of migration was quantified. All results are expressed as the mean S.E.M. valuess of percentage of migration or number of migrated cells / mm2 of three independendent experimentss (*p<0.05)

Discussion n

Interactionss of PSGL-1 with P-selectin mediate the initial tethering of leukocytess to activated platelets or endothelial cells at sites of infection or tissue injuryy (13, 31). We recently showed that platelet-monocyte complexes support monocytee adhesion by enhancing secondary tethering (14). In the present study, we extendd our previous work by investigating the consequences of platelet binding on thee monocyte phenotype regarding expression of (3i and 02 integrins and the adhesivee capacity to an inflamed endothelium model. We demonstrated that platelet bindingg to monocytes induces a high monocyte-activation state, characterized by down-regulationn of L-selectin and rapid activation of cuPr and OMp2- integrins. Similar effectss on monocyte activation were observed after ligation of PSGL-1 by P-selectin-Igg chimera or by specific antibodies to PSGL-1. This P-selectin - triggered integrin activationn was completely blocked by a blocking antibody to P-selectin, indicating thatt physical binding of P-selectin to PSGL-1 on monocytes is essential for this process. .

P-selectinn - triggered signaling and its stimulatory effects on human leukocytes havee been described in several previous reports. P-selectin binding to PSGL-1 has beenn shown to promote p2-integrin - dependent homotypic neutrophil aggregation andd neutrophil-platelet conjugation (22,32). Hidari et al. (24) demonstrated that ligationn of PSGL-1 on human neutrophils with mAbs or with P-selectin increased proteinn tyrosine phosphorylation, activated the ERK MAP kinases and induced secretionn of IL-8. Monocytes, upon binding of activated platelets, were shown to secretee monocyte chemotactic protein-1 and IL-8 (33) and express tissue factor (34-36). .

Variouss mechanisms for a role of P-selectin in influencing the activity of pi and P22 integrins have been suggested. However, most studies have shown that P-selectinn cannot directly stimulate integrin activation on human leukocytes (20, 33, 36).. Instead, P-selectin was described as an anchoring molecule, allowing monocytess to bind to activated endothelium thus facilitating the binding of EC surface-boundd PAF to its receptor on the leukocyte surface. Subsequently, immobilizedd chemoattractant PAF induced integrin activation (20). Our data indicate thatt the increase in integrin expression and activation occurs upon PSGL-1 ligation byy platelets, P-selectin or by PSGL-1 - specific antibodies. We observed an increase inn monocyte adhesion to immobilized fibronectin, VCAM-1 and ICAM-1, indicative of integrinn conformational changes. Furthermore, upon platelet binding to monocytes theree was an increase in aM$2 and a43i - dependent adhesion of monocytes to

activatedd endothelial cells under flow conditions. This is in agreement with previous studiess showing induction of QMp2 integrin upon platelet binding to human neutrophils

(20,, 21, 37) and an increased affinity of monocytes to VCAM-1 by P-selectin binding underr flow conditions (4, 25, 38).

Thee presence of additional platelet-released synergistic factors such as the cytokiness PAF and RANTES (4) seem to be required to induce optimal leukocyte activation.. This might explain the observed monocyte adhesion upon P-selectin-lg bindingg or PSGL-1 antibodies which was, in some instances, lower than the one obtainedd by adding freshly isolated platelets to monocytes. A very recent study (20) onn neutrophils suggested an intermediate state of integrin activation induced by engagementt of PSGL-1 by either P-selectin Ig or antibodies to PSGL-1. This intermediatee integrin activation state is compatible with a moderate increase in monocytee adhesion to immobilized proteins or activated endothelial cells. Furthermore,, depending on the leukocyte type, PSGL-1 structure and subsequent affinityy for its main ligand, P-selectin, seems to differ. When compared to neutrophils, PSGL-11 on eosinophils has been shown to bind 10-fold stronger to P-selectin (39). Recently,, platelets were shown to preferentially bind monocytes over neutrophils underr flow (40), suggesting differences in structure/affinity of PSGL-1 on the two differentt cell types. These differences might result in the induction of different

PSGL-1-mediatedd integrin-signaling pathways and explain the effect of platelet binding on bothh integrin expression and activation observed by us, in contrast to others (20).

Inn conclusion, our data show an increase in expression and adhesive capacity off a4- and aw integrins upon PSGL-1 ligation by P-selectin on human monocytes.

PSGL-11 ligation by platelet binding results in increased integrin activation and subsequentlyy increased cell adhesion to fibronectin, VCAM-1, ICAM-1 and activated endotheliall cells. The concerted action of a variety of stimuli such as chemoattractants,, and P-selectin, provided by platelet binding, seems to modulate thee activation of monocyte integrins relevant for the monocyte extravasation process andd thus for their atherogenic capacity.

Aknowledgements s

Wee thank Dr. F. Sanchez-Madrid for kindly providing the HUTS21 antibody andd Dr. Kevin L. Moore for providing the PL-1, PL-2 and CBRM1/5 antibodies. We alsoo would like to thank Dr. A. Tool for technical help with the adhesion assays and Dr.. M. Femandez-Borja for the helpful discussions and suggestions.

References s

1.. van Zanten, G.H., de Graaf S., Slootweg P.J. , Heijnen H.F., Connolly T.M., de Groot P.G., Sixmaa J.J. (1994) Increased platelet deposition on atherosclerotic coronary arteries. J. Clin. Invest. 93:615-632. .

2.. Huo, Y., Schober A., Forlow S.B., Hyman M.C., Jung S., Liftman D.R., Weber C , Ley K. (2003) Circulatingg activated platelets exacerbate atherosclerosis in mice deficient in apolipoprotein E. Nat. Med.. 9:61-67.

3.. Sarma, J., Laan C.A., Alam S., Jha A., Fos K.A., Dransfield I. (2002) Increased platelet binding too circulating monocytes in acute coronary syndromes. Circulation. 105:2166-2171.

4.. Furman, M.I., Benoit S.E., Barnard M.E., Valeri CR., Borbone M.L., Becker R.C., Hechtman H.B.,, Michelson A.D. (1998) Increased platelet reactivity and circulating monocyte-platelet aggregates inn patients with stable coronary artery disease. J. Am. Coll. Cardio. 31:352-358.

5.. Broijersen, A., Hamsten A., Eriksson M., Angelin B., Hjemdahl P. (1998) Platelet activity in vivo inn hyperlipoproteinemia - importance of combined hyperlipidemia. Thromb. Haemost. 79:268-275. 6.. Consigny P.M. Pathogenesis of atherosclerosis. (1995) Am. J. Roentgenol. 164:553-558. 7.. Munro, J.M., Cotran R.S. (1998) The pathogenesis of atherosclerosis: atherosclerosis and inflammation.. Lab. Invest. 58:249-261.

8.. Stenberg, P.E., McEver R.P., Schuman M.A., Jacques Y.V., Bainton D.F. (1985) A platelet alpha-granulee membrane protein (GMP-140) is expressed on the plasma membrane after activation. J.. Cell Biol. 101:880-886.

9.. Bonfanti, T.G., Furie B.C., Furie B., Wagner D.D. (1989) PADGEM (GMP-140) is a component off Weibel-Palade bodies of human endothelial cells. Blood. 73:1109-1112.

10.. Moore, K.L., Stults N.L., Diaz D., Smith D.F., Cummings R.D., Varki A., McEver R.P. (1992) Identificationn of a specific glycoprotein ligand for P-selectin (CD62) on myeloid cells. J. Cell Biol. 118:445-456. .

11.. Sako, D., Chang X.J., Barone K.M., Vachino G., White H.M., Shaw G., Veldman G.M., Bean K.M.,, Ahem T.J., Furie J., Cumming DA., Larsen G.A. (1993) Expression cloning of a functional glycoproteinn ligand for P-selectin. Cell. 75:1179-1186.

12.. Ma, L-, Raycroft L., Asa D., Anderson D.C., Geng J.G. (1994) A sialoglycoprotein from human leukocytess functions as a ligand for P-selectin. J. Biol. Chem. 169:27739-27746.

13.. Walcheck, B., Moore K.L., McEver R.P., Kishimoto T.K. (1996) Neutrophil-neutrophil interactionss under hydrodynamic shear stress involve L-selectin and PSGL-1 - a mechanism that amplifiess initial leukocyte accumulation on P-selectin in vitro. J. Clin. Invest. 98:1081-1087.

14.. da Costa Martins, P.A., van den Berk N., Ulfman L.H., Koenderman L, Hordijk P.L., Zwaginga J.J.. (2004) Platelet-monocyte complexes support monocyte adhesion to endothelium by enhancing secondaryy tethering and cluster formation. Art. Thromb. Vase. Biol. 24:193-199.

15.. Ley, K., Tedder T.F. (1995) Leukocyte interactions with vascular endothelium. New insights into selectin-mediatedd attachment and rolling. J. Immunol. 155:525-528.

16.. Beekhuizen, H., van Furth R. (1993) Monocyte adherence to human vascular endothelium. J. Leuk.. Biol. 54:363-378.

17.. Luscinskas, F.W., Kansas G.S., Ding H., Pizcueta P., Schleiffenbaum B.E., Tedder T.F., Gimbronee M.A.Jr. (1994) Monocyte rolling, arrest and spreading on IL-4 activated vascular endotheliumm under flow is mediated via sequential action of L-selectin, beta 1-integrins and beta 2-integrins.. J. Cell Biol. 125:1417-1427.

18.. Tedder, T.F., Steeber D A , Chen A., Engel P. (1995). The selectins: vascular adhesion molecules.. FASEB J. 9:866-873.

19.. McEver, R.P., Moore K.L., Cummings R.D. (1995) Leukocyte trafficking mediated by selectin carbohydratee interactions. J. Biol. Chem. 270:11025-11028.

20.. Ma, Y.Q., Plow E.F., Geng J.G. (2004) P-selectin binding to P-selectin glycoprotein ligand 1 inducess an intermediate state of aM(32 activation and acts cooperatively with extracellular stimuli to

supportt maximal adhesion of human neutrophils. Blood. 104:2549-2556.

21.. Blanks, J.E., Moll T., Eytner R., Vestweber D. (1998) Stimulation of P-selectin glycoprotein ligand-11 on mouse neutrophils activates (32 integrin - mediated cell attachment to ICAM-1. Eur. J. Immunol.. 28: 433-443.

22.. Evangelista, V., Manarini S., Sideri R., Rotondo S., Martelli N., Piccoli A., Totani L , Piccardoni R.,, Vestweber D., de Gaetano G., Cerletti C. (1999) Platelet/polymorphonuclear leukocyte interaction: P-selectinn triggers protein-tyrosine phosphorylation-dependent CD11b/CD18 adhesion: role of PSGL-11 as a signaling molecule. Blood. 93:876-885.

23.. Weyrich, A.S., Mclntyre T.M., McEver R.P., Prescott S.M., Zimmerman G.A. (1995) Monocyte tetheringg by P-selectin regulates monocyte chemotactic protein-1 and tumor necrosis factor-alpha secretion.. Signal integration and NF-kappa B translocation. J. Clin. Invest. 95:2297-2303.

24.. Hidari, K.I., Weyrich A.S., Zimmerman G.A., McEver R.P. (1997). Engagement of P-selectin glycoproteinn ligand-1 enhances tyrosine phosphorylation and activates mitogen-activated protein kinasess in human neutrophils. J. Biol. Chem. 272:28750-28756.

25.. Yago, T., Tsukuda M., Minami M. (1999) P-selectin binding promotes the adhesion of monocytess to VCAM-1 under flow conditions. J. Immunol. 163:367-373.

26.. Jaffe, E.A., Nachman R.L., Becker C.G., Minick C.R. (1973) Culture of human endothelial cells derivedd from umbilical veins. Identification by morphologic and immunologic criteria. J. Clin. Invest. 52:2745-2756. .

27.. van Zanten, G.H., Saelman E.U., Schut Hese K.M., Wu Y.P., Slootweg P.J., Nieuwenhuis H.K., dee Groot P.G., Sixma J.J. (1996) Platelet adhesion to collagen type IV under flow conditions. Blood 88:3862-3871. .

28.. Weber C , Alon R., Moser B., Springer T.A. (1996) Sequential regulation of a46i and a5pi

integrinn activity by CC chemokines in monocytes: implications for transendothelial chemotaxis. J Cell Biol.. 134:1063-1073.

29.. Weber C , Kitayama J., Springer T.A. (1996) Differential regulation of Br and B2-integrin avidity

byy chemoattractants in eosinophils. Proc. Natl. Acad. Sci. 93:10939-10944.

30.. Sakariassen, K.S., Aarts P.A., de Groot P.G., Houdijk W.P., Sixma J.J. (1983) A perfusion chamberr developed to investigate platelet interaction in flowing blood with human vessel wall cells, theirr extracellular matrix, and purified components. J. Lab. Clin. Med. 102:522-535.

31.. Mayadas, T.N., Johnson R.C, Rayburn H., Hynes R.O., Wagner D.D. (1993) Leukocyte rolling andd extravasation are severely compromised in P selectin-deficient mice. Cell. 74:541-554.

32.. Evangelista, V., Manarini S., Rotondo S., Martelli N., Polischuk R., McGregor J.L., de Gaetano G.,, Cerletti C. (1996) Platelet/ polymorphonuclear leukocyte interaction in dynamic conditions: evidencee of adhesion cascade and cross talk between P-selectin and the beta 2 integrin CD11b/CD18.. Blood. 88:4183-4194.

33.. Weyrich, A.S., Elstad M.R., McEver R.P., Mclntyre T.M., Moore K.L., Morrissey J.H., Prescott S.M.,, Zimmerman G.A. (1996) Activated platelets signal chemokine synthesis by human monocytes. J.Clin.. Invest. 97:1525-1534.

34.. Celi, A., Pellegrini G., Lorenzet R., De Blasi A., Ready N., Furie B.C., Furie B. (1994) P-selectin inducess the expression of tissue factor on monocytes. Proc. Natl. Acad. Sci. USA. 91:8767-8771. 35.. Pellegrini, G., Malandra R., Celi A., Furie B.C., Furie B., Lorenzet R. (1998) 12-Hydroxyeicosatetraenoicc acid upregulates P-selectin-induced tissue factor activity on monocytes. FEBSS Lett. 441:463-466.

36.. Elstad, M.R., La Pine T.R., Cowley F.S., McEver R.P., Mclntyre T.M., Prescott S.M., Zimmermann G.A. (1995) P-selectin regulates platelet-activating factor synthesis and phagocytosis by monocytes.. J. Immunol. 155:2109.

37.. Piccardoni, P., Sideri R., Manarini S., Piccoli A., Martelli N., de Gaetano G., Cerletti C , Evangelistaa V. (2001) Platelet/polymorphonuclear leukocyte adhesion: a new role for SRC kinases in Mac-11 adhesive function triggered by P-selectin. Blood.98:108-116.

38.. Huo, Y., Moghadam A.H., Ley K. (2000) Role of vascular cell adhesion molecule-1 and fibronectinn connecting segment-1 in monocyte rolling and adhesion on early atherosclerotic lesions. Circ.. Res. 87:153-159

39.. Symon F.A., Lawrence M.B., Williamson M.L., Walsh G.M., Watson S.R., Wardlaw A.J. (1996) Functionall and structural characterization of the eosinophil P-selectin ligand. J. Immunol. 157:1711-1719. .

40.. Ahn K.C., Jun A.J., Pawar P., Jadhav S., Napier S., McCarty O.J., Konstantopoulos K. (2005) Preferentiall binding of platelets to monocytes over neutrophils under flow. Biochem. Biophys. Res. Commun.. 329:345-355.