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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.
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Chapterr 2
"Platelet-monocytee complexes support
monocytee adhesion to endothelium by
enhancingg secondary tethering and
clusterr formation"
P.P. da Costa Martins1, N. Van den Berk1, L. H. Ulfman2, LL Koenderman2, P.L. Hordijk1 and J.J. Zwaginga13
departmentt of Immunohematology, Sanquin Research, Location CLB, Amsterdam departmentt of Lung Diseases, University Hospital Utrecht, Utrecht departmentt of Hematology, Academical Medical Center, Amsterdam, Thee Netherlands Publishedd in Atherioscler. Thromb. Vase. Biol. 2004;24:193-199
Abstract t
Adhesionn of monocytes to endothelium can be supported by monocyte-monocytee interactions resulting in the formation of cell aggregates at the vessel wall (clusters).. Since platelets, bound to the injured vessel wall, support monocyte adhesion,, and platelet activation in the circulation leads to formation of platelet-monocytee complexes (PMC), we examined whether adhesion of PMC to the vessel walll enhances monocyte clustering. The effect of PMC formation in monocyte adhesionn and clustering on HUVEC was studied in vitro with a perfusion system. In thee presence of 10-20% PMC, monocyte adhesion and cluster formation to stimulatedd HUVEC increased twofold above levels obtained with pure monocytes. Whilee the observed effects increased with higher PMC levels, blocking-monoclonal antibodiess directed against platelet-associated P-selectin or monocyte PSGL-1 reversedd adhesion and clustering to control values. In the presence of PMC, blocking L-selectinn decreased adhesion by 25%. When PMC were present, clustering was onlyy supported by L-selectin at higher shear. These data indicate that monocyte adhesionn to the vessel wall is enhanced by PMC-mediated monocyte secondary tethering.. These interactions are mainly mediated by P-selectin and PSGL-1. Therefore,, PMC in the circulation might be proatherogenic, and prevention of their formationn is a possible therapeutic goal.
Introduction n
Thee recruitment of peripheral monocytes to the site of vascular damage is one off the first steps in atherogenesis and inflammation 1|2. However, the interactions betweenn the inflammatory and hemostatic response, as indicated by the colocalisationn of leukocytes, platelet and fibrin depositions are also considered essentiall in this process.
Thee powerful adhesive interactions that are required for monocytes to withstand local floww at the vessel wall can be described as a multistep process, mediated by different adhesionss molecules3. The initial tethering and rolling of monocytes over the vascularr endothelium are mediated by reversible binding of selectins to their cognate cell-surfacee glycoconjugates. Selectins are expressed on activated endothelial cells (E-- and P-selectin), activated platelets (P-selectin) and peripheral leukocytes (L-selectin)4,5. .
Selectinss also mediate an additional mechanism for leukocyte adhesion, the so-calledd secondary tethering 6J. This process involves the rolling of circulating leukocytess over ligands that are present on surface-adherent leukocytes. The formationn of strings or clusters of adhered cells is the result and accounts for the majorityy of cell accumulation at higher shear stresses 6. Although this mechanism hass been studied more extensively for neutrophil adhesion to endothelial cells 8, fibrinogenn 9, endothelial cell matrix and vascular glycoproteins 7,1 , secondary tetheringg of monocytes to endothelial cells was also described to be L-selectin-dependent11. .
Althoughh platelets adhered to injured vessel wall form strong adhesive substrates for leukocytes,, activated platelets in the circulation also bind leukocytes 12. These platelet-leukocytee complexes are mostly considered as markers of platelet-activating conditionss and vessel wall disease such as unstable atherosclerosis 13,14, stable coronaryy disease 15 and hypercholesterolemia 16. Platelet activation was recently reportedd to induce the development of atherosclerotic lesions of carotid arteries of ApoEApoEAA mice by increasing the number of vessel wall-adhered leukocytes 17. Furthermore,, an earlier report suggested that platelets bound to monocytoid cells mediatee increased adhesion to endothelial cells 18.
Soo far, the influence of platelet binding to monocytes on monocyte-monocyte interactionss or cluster formation was not described. We hypothesised that platelets onn the monocyte surface enhance monocyte adhesion and clustering at the vessel walll by "bridging" interactions with other monocytes. To test this hypothesis, we perfusedd isolated monocytes or PMC in a transparent perfusion chamber. Real-time imagee analysis was used to show that platelets on circulating monocytes strongly enhancee secondary tethering and, subsequently, monocyte adhesion to stimulated endotheliall cells. Our findings suggest the existence of a new proatherogenic
pathwayy in which platelet contribution to leukocyte-leukocyte capture may increase thee kinetics of leukocyte recruitment as well as exacerbate inflammation.
Materiall and methods
Reagents.Reagents. Human serum albumin (HSA) was purchased from the CLB
(Amsterdam,, The Netherlands). Recombinant TNF-a was purchased from Boehringerr Mannheim (Germany). Washing buffer contained phosphate-buffered salinee (PBS) supplemented with 0.5% HSA and 13 mM trisodium citrate. Incubation bufferr contained 20 mM HEPES, 132 mM NaCI, 6 mM KCI, 1 mM MgS04) 1.2 mM
KH2P044 supplemented with 5 mM glucose, 1.0 mM CaCI2 and 0.5% (w/v) HSA.
Tissuee culture supplies (media, antibiotics and trypsin) were purchased from Gibco, Lifee Technologies Inc (Paisley, UK).
MonoclonalMonoclonal antibodies. MoAbs WASP 12.2 (CD62P, anti P-selectin) and
DREGG 56 (CD62L, anti L-selectin) and W6/32 (anti HLA-A, B and C) were isolated fromm the supernatant of hybridomas obtained from the American Type Culture Collectionn (Rockville, MD, USA). MoAb ENA2 (anti E-selectin, CD62e) was kindly
providedd by Dr. W.A. Buurman (University Hospital, Maastricht). MoAbs C17 (CD61, GPIIIa),, 6C9 (CD41, GPIIb) and MB45 (CD41b, anti-GPIba) were purchased from CLBB Immunoreagentia (Amsterdam, The Netherlands). The above-mentioned MoAbs aree functionally blocking antibodies. MoAbs PL-1 (blocking of PSGL-1 binding) and PL-22 (non-functional blocking) were provided by Dr. Kevin L. Moore (University of Oklahoma,, Oklahoma, USA). The CD11a conformation-dependent MoAb CBRM 1/5 wass a kind gift of Dr. T.A.Springer (Harvard Medical School, Boston, MA). All other antibodiess were directly FITC-labeled: CD18 (CLB-LFA1/1), CD11a (CLB-LFA1/2), CD111 b (CLB-T11.2/1), CD62L (Leu-8, Becton & Dickinson).
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 isolated from human peripheral bloodd by means of MACS monocyte isolation kit according to the manufacturer's instructionss (Miltenyi Biotech GMBH, Bergisch Gladbach, Germany). This procedure resultedd in monocyte fractions containing more than 90% monocytes (CD14-positive cellss in FacScan, 15-20 * 106 monocytes were isolated from 50 ml of whole blood), thee viability exceeding 95% (Trypan blue exclusion). In order to obtain PMC-poor monocytee suspensions the monocytes were incubated with a mouse IgG MoAb againstt GPIIIa for 20 min at . After one washing step, the cells were incubated withh goat-anti-mouse-IgG microbeads (ratio platelets: beads = 1:2; Dynabeads, Dynall A.S., Oslo, Norway) for 20 min at . Magnetic extraction of the beads
resultedd in a 30-40% toss of the initial amount of monocytes and in a PMC presence off less than 5% of the total amount of monocytes. After isolation, the celts were resuspendedd in HEPES buffer. For blocking experiments, the cells were incubated withh MoAbs (10 jag/ml) for 10 min at C prior to the perfusion experiments. In some instances,, washed platelets were added to the monocyte suspension just before perfusion. .
PlateletPlatelet isolation. Whole blood was centrifuged at 150 g for 10 min to obtain
platelet-richh plasma (PRP), which was diluted in 1:1 of Krebs-Ringer solution (4 mM KCI,, 107 mM NaCI, 20 mM NaHC03, 2mM NaS04, 19 mM tri-sodium citrate, 0.5%
(w/v)) glucose in h-feO, pH 6.1). The mixture was centrifuged at 500 g for 10 min and thee supernatant was removed. The pelleted platelets 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 thee concentration of 300 000 platelets/pl.
EndothelialEndothelial cells. Human umbilical vein endothelial cells (HUVEC) were
isolatedd from human umbilical cord veins as described 19. The cells were cultured in RPMII 1640 containing 20% (v/v) human serum, 200 |ug/ml penicillin and streptomycinn {Life Technologies). Cells were grown to confluence in 5-7 days. Endotheliall cells of the second or third passage were used in perfusion assays. HUVECC monolayers were activated by TNF-a (100U/ml, 6 hrs, ) prior to the perfusionn experiments.
MonocyteMonocyte perfusion and evaluation of adhesion and cluster formation.
Duringg perfusions, the flow chamber 20,21 was mounted on a microscope stage (Axiovertt 25, Zeiss, Germany), which was equipped with a B/W CCD-video camera (Sanyo,, Osaka, Japan) and coupled to a VHS video recorder. Video images were evaluatedd for the number of adherent cells and the cluster index, with dedicated routiness made in the image analysis software Optimas 6.1 (Media Cybernetics Systems,, Silverspring, MD, USA). The monocytes that were in contact with the surfacee appeared as bright white-centred cells after proper adjustment of the microscopee during recording. The cluster index was measured as previously describedd 9 . In short, for each adherent cell the number of cells in a surrounding areaa of approximately 1750 jim2 was measured. The cluster index per cell was set to bee the difference between the measured and the expected number of cells inside an arbitraryy area around the cell. For each experiment, the mean cluster index of a minimall of 500 cells was calculated.
PMCPMC quantification and cell adhesion molecule expression. The
percentagee of monocytes positive for platelet-specific markers was determined in monocytee suspensions by flowcytometry (FACS Vantage, Becton Dickinson & Co., Mountainn View, USA) and also directly in whole blood after lysing the red blood cells. Monocytess from whole blood or bead-isolation were incubated with antibodies againstt surface markers (GPIb, GPIIbllla and CD14). The percentage of double positivee events represents the percentage of monocytes that have at least bound one platelet.. The mean number of platelets associated per monocyte was determined manuallyy by light and confocal microscopy. Per experiment at least 100 monocytes weree evaluated, whereas larger aggregates (>3 monocytes) were omitted (n=3). In somee instances a platelet/monocyte ratio was determined. The expression of differentt adhesion molecules was determined by incubating monocytes from lysed bloodd or bead-isolation with specific directly labeled antibodies (CD62L, CD18, CD11a,, CD11b, CD15, CD162).
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
QuantificationQuantification and analysis of platelet-monocyte complexes (PMC). The
presencee The presence of monocyte-bound platelets was investigated by flowcytometryy and light microscopy. Ten to 20% of the bead-isolated monocytes weree associated with platelets with a mean number of 1 0.3 platelets per monocyte (Tablee 1). To confirm that PMC formation also occurs in whole blood, FACS analysis wass performed on lysed whole blood samples. Five to 10% of the monocytes were positivee for the platelet-specific markers GPIb and GPIIbllla. By adding platelets to thee monocyte suspension (platelets:monocyte; 3:1) the mean number of monocyte-boundd platelets increased to 2.2 + 0.5 per monocyte, and the percentage of PMC increasedd to 40-60%. In contrast, after PMC removal, less than 5% of the bead-isolatedd monocytes showed binding of platelets (Table 1).
Tablee 1. Platelet binding to monocytes and recetor exression on monocytes in whole bloodd and after isolation.
I s o l a t i o n n p r o c e d u r e e Monocyte» » Inn w h o l e M o o d d Bead-isolated d m o n o c y t e * * Treatment t c o n t r o l l c o n t r o l l «Herr P M C extraction n a d d i t i o nn o f 3:1 1 platelets s Platelet** per m o n o c y t e e n . d . . 1 1 0 3 3 n . d . . 2222 %% P M C 1 0 . 2 0 0 2 0 - 4 0 0 < 5 5 5 0 - 7 0 0 CD18 8 1155 1 2 6 x 1 1 1 2 0 * 6 6 1 3 7 * 2 2 R e c e p t o rr expression (MFU) CD11aa C O I I b s L e x L-sel PSGL-1 7 4 * 0 0 7 3 * 6 6 8 4 * 5 5 8 7 * 5 5 3 8 * 7 7 5 3 * 5 5 5 6 * 3 3 6 8 * 5 5 3 6 6 * 7 7 3 2 5 ** 1 n.d d n.d d 1 4 6 * 8 8 9 6 * 4 4 9 8 * 6 6 6 1 * 7 7 1 1 1 * 1 1 1 2 8 * 2 2 n.d d n.d d
Monocytess isolated by immunobeads were compared to monocytes in RBC-lysed whole blood, in respectt to the number of PMCs in suspension and also the number of platelets per monocyte was determinedd by light microscopy (n=3). The percentage of monocytes positive for platelet-specific markerss (FITC-labeled GPIb and GPIIbllla) was determined by immunofluorescence flowcytometry. Monocytess from RBC-lused whole blood were also compared to monocytes isolated with immunobeads,, considering their expression of various adhesion molecules. The surface marker expressionn was determined by flowcytometry. N.D. indicates not determined. * The data are representedd as the mean fluorescence intensity (MFI) SEM.
Surfacee marker expression of monocytes in whole blood and of isolated monocytess was determined by flowcytometry (Table 1). When compared to monocytess from lysed blood, beads isolated monocytes showed some loss of L-selectinn suggesting a slightly stimulated phenotype. This, however, was not significant.. Moreover, upregulation of p2 -integrins, which indicates that the isolation
proceduree has triggered cell activation, was not observed. In addition, expression of activatedd p2 -integrins was similar for all monocyte populations (data not shown). Also
PSGL-11 and sLex expression levels were similar after both isolation procedures. Additionn of platelets did not induce a significant increase in integrin expression on monocytes.. However, a decrease in L-selectin expression (from 96 4 to 61 2, p<< 0.01) on PMC compared to single monocytes was observed suggesting that additionall platelets activate monocytes to some degree (Figure 1). PMC-associated P-selectinn mediates binding of monocytes to HUVEC. Monocytes in the presence of PMCC showed rolling and firm adhesion to the endothelium. Secondary tethering or celll cluster formation also occurred. By the latter mechanism, flow-oriented trails of celll clusters (Figure 2 and 3) were formed.
Bothh monocytes without platelets on their surface and PMC were involved in all differentt interactions. However, the influence of PMC on adhesion and cluster formationn became clear from experiments in which PMC were removed from the monocytee suspension (<5% PMC in suspension). This resulted in a clear decrease in adhesionn and cluster formation (Figure 4). PMC-associated P-selectin accounted
mainlyy for this result as the P-selectin-blocking MoAb WASP 12.2 similarly inhibited adhesionn by about 60% (711 125 to 304 139 cells/mm2; p<0.001) and clustering byy 65% when PMC were present in the monocyte suspension (Figure 4, panel A). In agreement,, under PMC-poor conditions, WASP 12.2 could only reduce adhesion by 30%,, while clustering decreased (1.23 0.15 to 0.93 0.05). The presence of P-selectinn on the endothelial surface was excluded because preincubation of endotheliall cells with WASP 12.2 failed to influence adhesion and clustering (data nott shown)
CBRM1/5 5 CD62L L
Figuree 1. Expression of activation markers on monocytes derived from different isolationn procedures. Monocytes in lysed whole blood and bead-isolated monocytes were incubated
withh an antibody against L-selectin or an antibody specific for the activated conformation of CD11a (CBRM1/5).. Mean fluorescence was determined by flowcytometry.
Thee contribution of E-selectin to monocyte adhesion and/or clustering was also investigated.. The blockade of E-selectin on activated endothelial cells inhibited monocytee adhesion by 13% however, no effect on clustering (data not shown) was observed.. Finally, and apart from transient interactions, individual platelets in the monocytee suspensions rarely show firm adhesion to endothelium and therefore did nott support monocyte adhesion.
Figuree 2. Platelets promote secondary tethering of monocytes on stimulated endothelial cells.. Confocal microscopy detail of a monocyte cluster, formed in the direction of flow, in which
plateletss (arrows) show bridging interactions with monocytes. Platelets were labeled with FITC GPIb. Bar:: 10 urn.
Figuree 3. Monocyte cluster formation on stimulated endothelial cells. Monocyte
suspensionss containing 10-20% PMCs were perfused over 6 hours TNF-a - activated endothelium at aa shear stress of 0.8 dyn/cm2. Images were recorded on video during 5 minutes. The images depicted weree taken after 30 seconds of cell perfusion in a time framed as indicated. The arrow indicates the floww direction. Bar: 30 urn.
L-selectinL-selectin dependent adhesion and clustering. Although we show that in
thee presence of PMC P-selectin plays an important role in secondary tethering, L-selectinn was first identified to mediate this process11,12. Therefore the role of L-selectinn was investigated via a L-selectin blocking antibody (DREG 56). In the presencee of PMC, DREG 56 decreased adhesion by 25% (701 126 to 533 132 cells/mm2,, p<0.05) (Figure 4, panel B). Clustering, in contrast, was not inhibited by
DREGG 56 (2.94 0.43 to 3.37 0.09). Under PMC-poor conditions DREG 56
inhibitedd adhesion by 57% (338 13 to 148 18 cells/mm2; p<0.001) and cluster formationn by 42% (1.2 0.1 to 0.7 0.2; p<0.05) also at low shear.
Ass clustering is more important for supporting adhesion at higher shears we repeated thesee experiments also at higher shear stresses (Figure 5). Although lower adhesion wass observed at higher shear, clustering increased up to shear forces of 2.4
dyn/cm2.. At 3.6 dyn/cm2, the amount of cells that adhere was so low that cluster formationn was also hampered. L-selectin did mediate clustering at all higher shear stresses. .
1000-, ,
+PMCC -PMC +PMCC -PMC
Figuree 4. Effect of blocking P- or L-selectin on monocyte interactions with stimulated endotheliall cells. PMC-rich (+PMC) or PMC-poor (-PMC) monocyte suspensions were incubated
withh a control (A and B, empty bars), a blocking P-selectin antibody (A, filled bars), or a blocking L-selectinn antibody (B, filled bars) prior to perfusion over stimulated EC ata shear stress of 0.8 dyn/cm2.
Thee adhesion and clustering behaviors of monocytes are expressed as absolute adhesion (cells/mm2) andd clustering index, respectively. Means SEM of 3 to 5 experiments are given. Statistically significantt effects are indicated (*P<0.05; **P<0.001).
SecondarySecondary tethering is mainly mediated by platelet P-selectin on PMC.
Too better understand the initial events in cluster formation at low shear and the way inn which P- and L-selectin are involved, we analysed images of monocyte perfusions overr stimulated endothelial cells and counted the number of PMC that contributed to clusterss and to single adhering monocytes. To estimate this contribution, we counted thee total number of platelets bound to single adherent monocytes and the total numberr of platelets bound to monocytes within the clusters. When monocytes were pre-treatedd with a control antibody we observed a higher amount of platelets bound perr monocyte within the clusters compared to the amount of platelets bound to single
adheringg monocytes (data not shown). This indicates preferential contribution of PMCss in the clusters but also that both PMC and monocytes without platelets form clusters.. The blocking of L-selectin did not affect the p/m ratio within the clusters, but thee p/m ratio in single adhering cells increased significantly when compared to the controll situation (data not shown).
900 0 cc ,_, OO OJ ii I 600 "22 « CDD = %% 8 300 oo — X X ®® 4 . T3 3 | | in in o o 0.8 8 1.6 6 24 4 3.6 6 0.8 8 1.6 6 2.4 4 36 6 shearr stress (dyn/cm2)
Figuree 5. Effect of increasing shear stress on monocyte adhesion and clustering to activatedd endothelium after blockade of P- or L-selectin. PMC-rich monocyte suspensions were
perfusedd over stimulated endothelium at different shear stresses (0.8, 1.6, 2.4 and 3.6 dyn/cm2). Monocytess were treated with a control (0), anti P-selectin ( ) or anti L-selectin (V) antibody for 5 minutess just before perfusion. Adhesion and clustering behavior of monocytes are expressed in absolutee numbers (cells/mm2) and clustering index, respectively. Means SEM of 3 experiments are given. .
Too better define the role of platelets in secondary tethering, cross-over design typee of experiments were performed. Monocytes from a PMC-rich (10-20% PMC) or aa PMC-poor (<5% PMC) suspension were allowed to adhere statically to glass coverslipss for 10 minutes at . Mounted in the perfusion chamber, the coverslips weree then washed with incubation buffer and exposed to PMC-rich or PMC-poor monocytee suspensions at a shear of 0.8 dyn/cm2. When a PMC-poor monocyte suspensionn was perfused over PMC-poor or PMC-rich monocyte surfaces, the flowingg monocytes did not seem to interact with monocytes/PMC on the surface sincee the presence of PMC on the surface did not affect monocyte adhesion or clusteringg (Figure 6). However, when PMC are present in the perfusate an increase inn monocyte adhesion and clustering was observed. This effect was increased when PMCC were present on the surface and in the perfusate, suggesting that platelets withinn the PMC enhance monocyte adhesion to the endothelium by promoting monocytee secondary tethering.
perfusate: : i<5%% PMC • 10-20% PMC 310-20%% PMC + anti P-selectin || E | EE 100 == ü 400 X X
l u l l l
perfusate:: <5% PMC 10-20%% PMC T3 3 C C 5 5 3 3 Ö ÖI I
perfusate:: <5% PMC 10-20%% PMCFiguree 6. Role of platelets in monocyte secondary tethering. Monocytes from PMC-poor
andd PMC-rich suspensions were allowed to adhere statistically to glass coverslips for 10 minutes at 37 .. PMC-poor or PMC-rich monocyte suspensions were then perfused over these monocyte surfaces att a shear stress of 0.8 dyn/cm2. The cells from the perfusate that adheres appeared as bright white-centeredd cells while the surface cells were spread over the glass and not in focus. The adhesion and clusteringg behaviors of monocytes are expressed as absolute adhesion (cells/mm2) and clustering
index,, respectively. Means SEM of 3 experiments are given. Statistically significant effects are indicatedd (*P<0.05, **P<0.01)
P-selectinP-selectin on platelets binds to PSGL-1 expressed on monocytes. To
investigatee the monocyte ligand for P-selectin on platelets, we incubated a PMC-rich monocytee suspension with a PSGL-1-blocking or non-blocking antibody (PL-1 and PL-2,, respectively). Incubation of cells with PL-1 antibody decreased adhesion by 45%% (709 71 to 389 40, p<0.001) and cell clustering by 57% (2.9 0.4 to 1.3 0.1,, p<0.01) (Figure 7). An isotype-matched control antibody (W6/32) or PL-2 showedd no effect.
PlateletsPlatelets augment monocyte adhesion and clustering. To investigate
whetherr we could correlate the amount of PMC in suspension with the number of firmlyy adhering monocytes, we compared a monocyte suspension with no PMC with monocytee suspensions with different amounts of PMC. Unstimulated and washed
plateletss (ratio platelet:monocyte used: 1:1, 3:1, 5:1) were added to monocyte suspensionss containing 10-20% PMC. Addition of platelets to monocytes in all ratios resultedd in an increase in PMC formation. An increase in PMC content from led to an increasee in both monocyte and cluster formation. However, when a ratio of 5:1 was used,, and the percentage of formed PMC increased to 70-90%, a decrease in adhesionn was observed. This is in agreement with previous results 18, which showed aa steep, bell-shaped dose-response curve for leukocyte adhesion to stimulated HUVEC,, at very low platelet-leukocyte ratios, with a peak at 3 platelets per leukocyte. Thee blockade of P-selectin decreased adhesion and clustering to basal levels (data nott shown).
1D00n n
controll PL-1 PL-2 controll PL-1 PL-2
Figuree 7. Effect of PSGL-1 blockade on the adhesion and clustering behavior of monocytes.. Monocytes were treated with a specific antibody for PSGL-1 and perfused over
stimulatedd HUVECs at a shear stress of 0.8 dyn/cm2. PL-1 is a functional blocking antibody, and PL-2
iss a non-blocking antibody. Monocyte adhesion and clustering are expressed in absolute numbers (cells/mm2)) and clustering index, respectively. Means SEM of 3 to 5 experiments are given. Statisticallyy significant effects are indicated (*P<0.01)
PossiblePossible platelet-platelet - mediated interactions in clustering. The
potentiall role of platelet-platelet interactions in bridging PMC within the clusters was investigated.. No influence on monocyte adhesion or clustering by antibody-mediated blockadee of the main receptors that mediate platelet-platelet interactions (GPIb, and GPIIbllla)) was observed. Also addition of the plasma proteins (von Willebrand factor andd fibrinogen) that bridge the mentioned receptors had no effect (data not shown).
Discussion n
Thee recruitment of monocytes to the vessel wall plays a key role in the pathophysiologyy of atherosclerosis. Often observed as rolling interactions on the vessell wall, two different types of cell tethering can be discriminated: 1. Primary tetheringg directly to the endothelial surface and 2. Secondary tethering to already adheredd cells. Both mechanisms precede firm adhesion of flowing monocytes but onlyy secondary tethering is associated with cluster formation 14 since secondary tetheringg reduces monocyte velocity, thus facilitating adhesion, downstream of adherentt cells (Figure 2 and 3). Interactions between L-selectin and syalomucin-like ligandss were reported to be responsible for inter-leukocytic cluster formation 7. In this respect,, human monocytes express P-selectin glycoprotein ligand-1 (PSGL-1), a heavilyy glycosylated sialomucin that can serve as a ligand for L-selectin 8'22. We showw here that monocyte complexes (PMC), which are markers of platelet-activatingg conditions, promote monocyte clustering and adhesion to stimulated endothelium.. Our experiments show that the formation of monocyte clusters is mediatedd by platelet-expressed P-selectin and monocyte-expressed PSGL-1. The factt that L-selectin did not mediate monocyte clustering at low shear stress indicates thatt PSGL-1 is not the main ligand for L-selectin under these conditions. A possible interactionn of endothelial P-selectin with the PSGL-1 on monocytes was excluded becausee preincubation of the endothelial cells with an antibody to P-selectin did not
influenceinfluence monocyte/PMC adhesion or clustering. Additionally, monocytes themselves doo not express P-selectin 3. Furthermore, blocking the main platelet receptors, GPIb
andd GPIIbllla, which could possibly interact with fibrinogen or vWF in the plasma, alsoo did not affect monocyte adhesion or clustering.
Previouss reports have indicated that P-selectin expressed on activated platelets is responsiblee for an increase in leukocyte adhesion by direct binding to endothelial cellss in vitro 23 and in vivo 17. These interactions were claimed to assist leukocyte adhesionn to the vessel wall, at shear rates that normally do not allow leukocyte adhesionn to activated endothelium 18. However, intravital microscopy, and also our ownn observations, characterised interactions of free platelets with endothelial cells as rapidlyy reversible, leaving doubts about platelet role in leukocyte tethering when boundd to their membrane. Theilmeier et al.1 8 suggested a role for PMC in enhancing monocytee adhesion to the endothelium but used highly diluted PMC concentrations whichh did not result in cluster formation. Our observations show, for the first time, non-randomm homotypic monocyte/PMC clustering in the direction of flow suggesting aa previously unidentified role for platelet in mediating secondary tethering of monocytes. .
Inn addition, PSGL-1 has also been described as being important for leukocyte adhesionn to endothelium 24,25 and as a ligand for E-selectin 22. However, only a slight decreasee (about 13%) in adhesion and no effect on clustering of monocytes/PMC
wass observed by the blockade of E-selectin. This indicates that PSGL-1, by mediatingg monocyte-platelet interactions, plays a major rote in secondary monocyte tetheringg (Figure 8). However, we can not exclude that other adhesion molecules suchh as fibrinogen 9 and GPIba 26 on platelets and p2-integrins on leukocytes also
facilitatee PMC formation.
Thee role of L-selectin on monocyte clustering and adhesion, in the presence of PMC wass also investigated. In a PMC-rich monocyte suspension, at 0.8 dyn/cm2, L-selectinn blockade decreased monocyte/PMC adhesion by 25% but did not effect cell clustering.. At higher shear rates, however, the role of L-selectin in clustering became clearer.. The observation that L-selectin mediates adhesion at all shear rates, irrespectivee of PMC presence, indicates that monocyte-expressed L-selectin is functionallyy mediating primary tethering of monocytes to carbohydrate ligands on endotheliall cells. L-selectin blockade in the absence of PMC had a strong inhibitory effectt on monocyte adhesion. The latter might be due to the fact that in this situation onlyy E-selectin remains as mediator of initial tethering. In contrast, when PMC are present,, the additional clustering capacity of PMC as initially adhering monocytes mightt compensate the effect of L-selectin blockade on primary tethering and total adhesion.. Monocytes with no platelets (and thus no P-selectin expression) on their membranee will in case of L-selectin blockade miss the most prominent selectins for primaryy interactions with endothelial cells. In contrast, monocytes with platelets boundd on their cell membrane are able to use P-selectin or even GPIb and GP llbllla onn platelets for primary tethering to endothelial cells. Therefore, the p/m ratio in the singlee adhering cells increases. The selection and presence of these PMC on the endotheliall surface makes the blockade or L-selectin not critical for monocyte secondaryy tethering at low shear stresses. This study provides evidence for an importantt contribution of circulating activated platelets in the secondary tethering of monocytess at the site of inflammation. Cluster formation can, theoretically, start by thee adhesion of circulating PMC to the already-adhered monocytes, but can also followw PMC adhesion to the endothelium, which form a better platform for secondary tetheringg of free-flowing monocytes.
Ourr in vitro experiments show that the clustering mechanism seems capable of clearingg the circulation from the free activated platelets. The in vivo fate of platelet/leukocytee complexes in vivo is still unknown. In fact, for a possible acceleratingg effect of PMC on atherogenesis, clustered monocytes will have to efficientlyy penetrate further into the vessel wall. Equally important is the question if plateletss bound to monocytes transmigrate with these monocytes into the subendotheliall tissue. These questions are currently under investigation.
Inn conclusion, our results suggest that prevention of platelet activation or platelet interactionss with monocytes, and thus formation of PMC, might become an interestingg therapeutic approach to modulate atherogenesis.
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