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Originally published online as doi:10.1189/jlb.0305141 on May 20, 2005 Originally published online as doi:10.1189/jlb.0305141 on May 17, 2005

Published online before print May 17, 2005
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(Journal of Leukocyte Biology. 2005;78:435-441.)
© 2005 by Society for Leukocyte Biology

Differential and additive effects of platelet-derived chemokines on monocyte arrest on inflamed endothelium under flow conditions

Thomas Baltus*,{dagger}, Philipp von Hundelshausen*, Sebastian F. Mause*, Wolfgang Buhre{dagger}, Rolf Rossaint{dagger} and Christian Weber*,1

* Departments of Molecular Cardiovascular Research and
{dagger} Anesthesiology, University Hospital Aachen, Rheinisch-Westfälische Technische Hochschule Aachen, Germany

1 Correspondence: Kardiovaskuläre Molekularbiologie, Universitätsklinikum Aachen, Pauwelstrasse 30, 52074 Aachen, Germany. E-mail: cweber{at}ukaachen.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Platelet-derived chemokines, such as regulated on activation, normal T expressed and secreted (RANTES; CC chemokine ligand 5), platelet factor 4 [PF4; CXC chemokine ligand 4 (CXCL4)], and epithelial neutrophil-activating protein 78 (ENA-78; CXCL5), or precursors, such as ß-thromboglobulin, which can be processed to neutrophil-activating protein-2 (NAP-2; CXCL7), may play an important role in monocyte recruitment during atherogenesis. Platelets can deposit chemokines on inflamed endothelium; however, little is known about differential or additive effects of platelet chemokines on monocyte arrest. Here, we demonstrate that preincubation of activated human microvascular endothelial cells (HMVECs) with RANTES, PF4, or NAP-2 but not ENA-78 dose-dependently increased surface immobilization and subsequent monocyte arrest in flow. RANTES was the most potent and efficient arrest chemokine. Pretreatment of HMVECs with ß-thromboglobulin enhanced monocyte arrest in the presence of cathepsin G generating NAP-2. Combined pretreatment of HMVECs with RANTES and PF4 at suboptimal concentrations synergistically increased arrest, and preincubation with chondroitinase ABC abrogated RANTES- and PF4-induced monocyte arrest. This was associated with reduced expression of chondroitin sulfate, RANTES, and PF4 on the HMVEC surface. Perfusion of HMVECs with platelets known to deposit RANTES and PF4 on the endothelial surface enhanced monocyte arrest, which was inhibited by Met-RANTES, chondroitinase, or a blocking antibody to PF4 but not to ENA-78. The relevance of platelet-derived chemokines was confirmed in adhesion assays with activated whole blood, where Met-RANTES and to a lesser extent, antibodies to PF4 and NAP-2 inhibited arrest of CD14-positive monocytes. Thus, multiple platelet-derived chemokines and processable precursors, which can be presented by specific endothelial proteoglycans, may contribute and cooperate differentially to induce monocyte recruitment.

Key Words: adhesion • inflammation • atherosclerosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Monocytes play an important role during the pathogenesis of atherosclerosis. Different steps of leukocyte recruitment, i.e., rolling, arrest, and transmigration, are influenced by chemokines expressed or immobilized by inflamed endothelium [1 , 2 ]. Chemokines bind to glycosaminoglycans (GAGs) such as heparan or chondroitin sulfate on the endothelial cell surface and are presented to their cognate receptors. Chemokines are produced by a variety of inflammatory cell types, including endothelial cells and leukocytes. Moreover, platelets contain a multitude of proinflammatory chemokines and precursors stored in their {alpha}-granules [3 , 4 ]. For instance, platelets can release the CXC chemokine platelet factor 4 [PF4; CXC chemokine ligand 4 (CXCL4)] or epithelial neutrophil-activating protein 78 (ENA-78; CXCL5), ß-thromboglobulin as a precursor for neutrophil-activating protein-2 (NAP-2; CXCL7), and the CC chemokine regulated on activation, normal T expressed and secreted [RANTES; CC chemokine ligand 5 (CCL5)] or macrophage-inflammatory protein-1{alpha} (CCL3). Although NAP-2 (CXCL7) is generated by proteolytic cleavage of connective tissue-activating protein III (CTAP-III) and ß-thromboglobulin, e.g., by the neutrophil serine protease cathepsin G, mRNA coding for ENA-78 is found in platelets [3 ].

ENA-78 and NAP-2 are ligands for the CXC chemokine receptor 2 (CXCR2), which mediates monocyte adhesion triggered by keratinocyte-derived chemokine or growth-related oncogene-{alpha} (GRO-{alpha}) on activated or atherosclerotic endothelium in shear flow [5 , 6 ]. NAP-2 and CTAP-III, following its proteolytic conversion, induce CXCR2-dependent neutrophil adhesion to endothelial cells [7 ]. Moreover, RANTES bound to activated microvascular or aortic endothelial cells triggers CC chemokine receptor 1 (CCR1)-mediated monocyte arrest in shear flow [8 , 9 ], which requires its oligomerization [10 ]. Conversely, PF4 has been shown to dose-dependently induce firm adhesion of neutrophils on endothelial cells by mechanisms differing from interleukin (IL)-8 stimulation [11 ]. The adhesion triggered by NAP-2 can be further enhanced by substimulatory concentrations of PF4 [7 ].

It has been well documented that activated platelets can deposit chemokines, namely RANTES and PF4, on the surface of inflamed or atherosclerotic endothelium in a process involving platelet P-selectin in shear flow [8 , 12 , 13 ]. This can trigger subsequent monocyte arrest and may explain a prominent role of platelets in atherogenic monocyte recruitment and exacerbation of plaque or neointima formation. Although RANTES has clearly been implicated in neointimal hyperplasia after injury and native atherosclerosis [12 , 14 ], little is known about differential contributions and potentially concerted effects of various platelet chemokines presented on the endothelial surface in monocyte arrest.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and reagents
Human microvascular endothelial cells (HMVECs; Promocell, Heidelberg, Germany) were cultured as described. HMVECs were stimulated with IL-1ß (10 ng/ml) overnight. Whole blood was obtained from healthy volunteers and anticoagulated with heparin. Monocytic Mono Mac 6 cells were cultured as described [5 ]. Platelets were isolated from platelet concentrates from healthy, voluntary donors. After stabilization with prostaglandin E2 (PGE2), the concentrates were purified using ADIAgel® tubes containing Sephadex® to remove plasma proteins. Human RANTES, NAP-2, ENA-78, and IL-1ß were from PeproTech (Rocky Hill, NJ); ß-thromboglobulin was from ICN (Eschwege, Germany). An antibody against chondroitin sulfate was from Santa Cruz Biotechnology (CA); antibody against PF4 was from Chemicon (Temecula, CA). Monoclonal antibodies (mAb) against RANTES, NAP-2, ENA-78, or their biotinylated correlates were from R&D Systems (Minneapolis, MN). Secondary antibodies were from Sigma Chemical Co. (St. Louis, MO). PGE2 was from Calbiochem (Schwalbach, Germany); thrombin receptor-activating peptide 6 (TRAP-6) was from Bachem (Weil am Rhein, Germany). All other reagents were from Sigma Chemical Co.

Leukocyte recruitment under flow conditions
For laminar flow assays, confluent HMVECs activated with IL-1ß overnight were preincubated with platelet chemokines for 30 min at 37°C, or platelets activated with TRAP-6 (2 µmol/l) were preperfused for 20 min. Whole blood from healthy volunteers without medication was anticoagulated with heparin, incubated for 45 min at 37°C with a phycoerythrin (PE)-conjugated CD14 mAb (1:50, R&D Systems), and subsequently, activated with TRAP-6. Whole blood was preincubated with Met-RANTES (1 µg/ml, 30 min, 37°C) or antibodies (10 µg/ml) as indicated. Mono Mac 6 cells (106 cells/ml) were resuspended in a Hepes-buffered Hanks’ balanced salt solution containing 0.5% bovine serum albumin and 1 µmol/l magnesium and calcium (HHMC). For laminar flow assays, Mono Mac 6 cells, pretreated with or without the RANTES peptide antagonist Met-RANTES (kindly provided by Dr. Amanda Proudfoot, Serono Pharmaceuticals, Rockland, MA, 1 µg/ml, 30 min), the small molecule CCR1 antagonist BX471 (kindly provided by Dr. Richard Horuk, Berlex Biosciences, Montville, NJ, 1 µg/ml), pertussis toxin (PTX; Calbiochem, 500 ng/ml, 2 h), or the CXCR2 antagonist 8-73 GRO-{alpha} (kindly provided by Dr. Ian Clark-Lewis, University of British Columbia, Vancouver, Canada; 1 µg/ml, 30 min), were perfused on HMVECs in a parallel flow chamber at 1.5 dyne/cm2 for 5 min and analyzed by video microscopy. For some experiments, HMVECs were pretreated with chondroitinase ABC (0.5 U/ml) or with antibodies (10 µg/ml) to PF4 (Chemicon, polyclonal), ENA-78 (clone 33170.111), or NAP-2 (clone 59418) for 30 min. Whole blood assays were performed using HMVECs grown to confluence on glass slides in a parallel wall flow chamber on the stage of an Olympus IX71 microscope. CD14+ cells were detected after 10 min of perfusion at 1.5 dyne/cm2 by fluorescence microscopy. Images were recorded with an exposure of 1 s. Adherent monocytes were counted in multiple fields.

Flow cytometry
HMVECs activated with IL-1ß (10 ng/ml) overnight were removed from the dish by treatment with EDTA solution and mechanical scraping, washed twice, and resuspended in HHMC. Cells were stained with antibodies against chondroitin, RANTES, and PF4 on ice for 30 min and reacted with fluorescein isothiocyanate-conjugated secondary antibodies. After 30 min, cells were washed and analyzed by flow cytometry, which using NAP-2 mAb after preincubation of HMVEC with ß-thromboglobulin at 500 ng/ml, revealed a slight but distinct binding of ß-thromboglobulin to the surface (not shown).

Enzyme-linked immunosorbent assay (ELISA)
Confluent HMVECs were cultured in 96-well plates and stimulated overnight with IL-1ß (10 ng/ml). Chemokines in phosphate-buffered saline (PBS) were reinsulated at indicated concentrations for 2 h at 37°C. Plates were washed with PBS/Tween 20 (0.05%) and reacted with biotinylated chemokine antibodies (R&D Systems) for 2 h. After washing, antibody binding was detected by treatment with horseradish peroxidase-streptavidin and tetramethylbenzidine substrate; optical density (OD) was measured at 405 nm.

Statistical analysis
Data were presented as mean ± SD of at least three independent experiments. Statistical analysis was performed using one-way ANOVA with Newman-Keuls correction for multiple comparisons. Differences were considered statistically significant when P values were less than 0.05. Data were analyzed using GraphPad Prism (GraphPad Software, San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
RANTES, PF4, and NAP-2 but not ENA-78 bind to inflamed microvascular endothelium
Cell-surface ELISA was used to detect the immobilization of platelet-derived chemokines on the surface of activated endothelium following preincubation with concentrations ranging from 20 to 1000 ng/ml. Preincubation of activated HMVECs with RANTES resulted in a dose-dependent surface binding with an effective concentration (EC)50 of 100 ng/ml and the highest maximal binding of all chemokines tested (Fig. 1A ). Preincubation with PF4 also yielded robust and dose-dependent surface binding with an EC50 of 20 ng/ml (Fig. 1B) . Although NAP-2 showed slight but distinct binding only at the highest concentrations used (Fig. 1C) , ENA-78 did not exhibit any binding above background (Fig. 1D) . Thus, platelet-derived chemokines revealed a differential capacity for surface binding on HMVECs, and RANTES and PF4 were immobilized most efficiently.



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  		Figure 1. Cell-surface ELISA of RANTES (A), PF4 (B), NAP-2 (C), and ENA-78 (D) immobilized on inflamed HMVECs following pretreatment with these chemokines at indicated concentrations for 20 min. Data represent mean ± SD of at least four independent experiments.

 
Platelet chemokines enhance the arrest of monocytes on inflamed HMVECs
We next tested whether differences in surface binding affect the potential of these platelet-derived chemokines to trigger monocyte arrest under flow conditions. As expected, preincubation of HMVECs with RANTES resulted in a dose-dependent and up to fourfold increase in monocyte arrest with an EC50 <20 ng/ml (Fig. 2A ). RANTES-triggered arrest was mediated by CCR1, as shown by inhibition with the antagonist BX471 (Fig. 2A) . In contrast, PF4 triggered a slight increase in monocyte arrest only at the highest concentrations used (Fig. 2B) . PF4-induced arrest was not modulated by pretreatment with PTX, indicating that it was not mediated by PTX-sensitive heptahelical receptors (Fig. 2B) . Moreover, NAP-2 caused a significant and up to twofold enhancement of monocyte arrest with an EC50 of ~250 ng/ml (Fig. 2C) , and preincubation with ENA-78 in accordance with its lack of surface binding did not stimulate monocyte arrest (Fig. 2D) . NAP-2-triggered arrest was mediated by CXCR2, as shown by inhibition with 8-73 GRO-{alpha} (Fig. 2C) . Thus, RANTES was clearly the most efficient platelet-derived arrest chemokine, and NAP-2 showed an intermediate activity. The dissociation of surface binding and its limited arrest function infers differential mechanisms for adhesion induction by PF4.



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  		Figure 2. Monocytic cell arrest triggered by platelet-derived chemokines on inflamed endothelium. Activated HMVECs were reinsulated with RANTES (A) and the small molecule CCR1 antagonist BX471 (A, {circ}), PF4 (B) and PTX (B, {circ}), NAP-2 (C) and the CXCR2 antagonist 8-73 GRO-{alpha} (C, {circ}), and ENA-78 (D) at indicated concentrations for 20 min. Data represent mean ± SD of four independent experiments.

 
ß-Thromboglobulin triggers monocyte arrest when processed by cathepsin G
The precursor CTAP-III has been shown to stimulate adhesion of neutrophils to endothelium, only following its proteolytic conversion to NAP-2 [7 , 15 16 17 ]. Similarly, preincubation of HMVECs with the NAP-2 precursor ß-thromboglobulin failed to enhance monocyte arrest in flow (Fig. 3 ). However, processing of ß-thromboglobulin on inflamed endothelium by simultaneous pretreatment with cathepsin G significantly increased monocyte arrest (Fig. 3) , which could be abrogated by a blocking antibody to NAP-2, indicating that the arrest chemokine NAP-2 has been generated on the HMVEC surface. Although immunoblotting using a NAP-2 mAb detected ß-thromboglobulin but not NAP-2 in activated platelet lysates, indicating that platelets do not contain cathepsin G to process ß-thromboglobulin, flow cytometry revealed that ß-thromboglobulin can be bound to the HMVEC surface (data not shown). Hence, it is conceivable that circulating leukocytes may secrete cathepsin G to process ß-thromboglobulin to NAP-2, which may significantly induce monocyte arrest on endothelium, already at low surface concentrations (see Figs. 1C and 2C ).



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  		Figure 3. Monocytic cell arrest triggered by processing of ß-thromboglobulin on inflamed endothelium. Activated HMVECs were reinsulated with ß-thromboglobulin (500 ng/ml) for 20 min and subsequently, treated with cathepsin G (2.5 µg/ml) for 1 h at 37°C. Data represent mean ± SD (n=4; *, P<0.01, vs. control; **, P<0.01, vs. ß-thrombogobulin+cathepsin G).

 
Synergistic effect of RANTES and PF4 bound to inflamed HMVECs on monocyte arrest
To test the combined influence of platelet-derived chemokines on monocyte arrest, HMVECs were pretreated with suboptimal concentrations of RANTES and PF4, alone or in combination (Fig. 4A ). Although each chemokine alone produced the expected increase in monocyte arrest, the combination resulted in a synergistic enhancement of monocyte arrest on inflamed HMVECs in flow, possibly as a result of concerted signaling or improved receptor activation by either chemokine.



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  		Figure 4. Additive effect of RANTES and PF4 on monocytic cell arrest on inflamed endothelium. HMVECs activated with IL-1ß (10 ng/ml) overnight were reinsulated with RANTES (100 ng/ml) and PF4 (100 ng/ml) and with chondroitinase ABC (0.5 U/ml) for 30 min at 37°C as indicated (A). Data represent mean ± SD (n=4; *, P<0.001, vs. control; **, P<0.01, vs. RANTES+PF4). Surface expression of chondroitin sulfate, RANTES, and PF4 on inflamed endothelium. HMVECs activated with IL-1ß (10 ng/ml) overnight were reinsulated with or without RANTES (C), PF4 (D, 1 µg/ml each), or chondroitinase ABC (B–D) for 30 min at 37°C. HMVECs were stained for chondroitin sulfate (B), RANTES (C), or PF4 (D) and analyzed by flow cytometry. Representative histograms are shown.

 
Role of chondroitin sulfate in RANTES- and PF4-triggered monocyte arrest
We next analyzed the role of chemokine binding to GAGs on the endothelial surface. Pretreatment of HMVECs with chondroitinase ABC significantly inhibited the increase in monocyte arrest induced by RANTES and PF4 (Fig. 4A) , indicating that chondroitin sulfate on the endothelial surface is involved crucially and specifically in platelet chemokine binding and subsequent monocyte arrest. Flow cytometry confirmed the involvement of chondroitin sulfate, revealing that it is detectable on the HMVEC surface and that its expression was markedly reduced by pretreatment with chondroitinase ABC (Fig. 4B) . Concomitantly, the surface binding of RANTES (Fig. 4C) and PF4 (Fig. 4D) to HMVECs was moderately attenuated by pretreatment with chondroitinase ABC. This indicates that the surface binding of platelet chemokines appears to be not exclusively mediated by chrondroitin sulfate; however, chondroitin sulfate appears to be most important and sufficient for triggering their functions in monocyte arrest.

Role of RANTES and PF4 in monocyte arrest on HMVECs after perfusion of platelets
The preperfusion of HMVECs with activated platelets enhanced monocyte arrest in flow (Fig. 5 ), which recently has been attributed to RANTES deposition [8 ]. In line with these data, pretreatment of monocytes with the RANTES antagonist Met-RANTES markedly inhibited arrest after platelet preperfusion, implicating RANTES receptor activation. A blocking antibody to PF4 slightly but significantly reduced monocyte arrest, indicating that PF4 is required to trigger monocyte arrest in concert with RANTES (Fig. 5) . In contrast, monocyte arrest was not affected by a blocking antibody to ENA-78 (Fig. 5) , consistent with its lack of immobilization on HMVECs. RANTES and PF4 appear to play an important and nonredundant role in monocyte arrest on inflamed endothelium after platelet perfusion.



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  		Figure 5. Monocytic cell arrest on inflamed endothelium after platelet preperfusion. Mono Mac 6 cells pretreated with the RANTES antagonist Met-RANTES (1 µg/ml) were perfused with 1.5 dyne/cm2. Activated HMVECs were preincubated with antibodies (Ab; 10 µg/ml) to PF4 or ENA-78, or chondroitinase ABC (0.5 U/ml) for 30 min at 37°C, and were preperfused with activated platelets for 20 min. Data represent mean ± SD (n=4; *, P<0.01, vs. platelet perfusion; **, P<0.05, vs. platelet perfusion).

 
Role of platelet chemokines in monocyte arrest after perfusion of activated whole blood
To more closely simulate the conditions of monocyte recruitment in vivo, we performed adhesion assays with activated whole blood under flow conditions and detection of adherent monocytes with a PE-conjugated CD14 mAb. Without pretreatment, blood monocyte arrest amounted to 48 ± 6 cells/mm2 (Fig. 6A ). The pretreatment of blood monocytes with Met-RANTES significantly inhibited arrest by 40% (Fig. 6B) . Although a control antibody had no effect, a PF4 antibody caused a slight but significant reduction of blood monocyte arrest (Fig. 6B) . These data confirm the involvement of platelet-derived chemokines and in particular, RANTES in the recruitment of peripheral blood monocytes.



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  		Figure 6. Monocyte arrest on inflamed endothelium after perfusion of activated whole blood. Anticoagulated blood was incubated with CD14-PE and activated by TRAP. After perfusion of whole blood for 10 min on IL-1ß-activated endothelium, adherent CD14-positive monocytes were detected by fluorescence microscopy (A). Whole blood was pretreated with Met-RANTES, PF4 antibody, or control immunoglobulin G (IgG) antibody for 30 min at 37°C. Adherent monocytes were counted in multiple high-power fields, and adhesion was expressed as percentage of untreated control (B). Data represent mean ± SD (n=3; *, P<0.01, vs. control).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Platelets contain multiple chemokines, which are released from the {alpha}-granules following activation [4 ]. We have shown that platelet-derived chemokines can be immobilized on inflamed or early atherosclerotic endothelium and can trigger subsequent monocyte arrest under flow conditions [5 ]. Although the dose-dependent impact of various CXC chemokines and precursors (e.g., PF4 and NAP-2 converted from CTAP-III) on neutrophil adhesion to endothelium under static conditions has been characterized [7 ], the differential and combined effects of platelet chemokines on monocyte arrest in flow have not yet been elucidated. Here, we show that RANTES and PF4, immobilized via chondroitin sulfate, act in concert to induce shear-resistant monocyte arrest. Although ENA-78 was ineffective, the ß-thromboglobulin product NAP-2 also efficiently triggered monocytic cell arrest in flow. At least part of the function as an arrest chemokine appears to be related to the capacity for surface binding. Notably, RANTES and PF4 are crucial for monocyte arrest after perfusion of activated platelets or whole blood.

Of the platelet-derived chemokines tested, RANTES bound to activated endothelium was the most efficacious and potent in triggering monocyte arrest under flow conditions. Although RANTES induced arrest at concentrations as low as 20 ng/ml used for preincubation, the enhancement of arrest showed saturation characteristics starting at 100 ng/ml. The fact that RANTES is the most effective chemokine may be related to its ability to form higher order oligomers, which have been shown to be required for its arrest function [10 ]. Not expressed by platelets in its fully processed and active form, the binding of NAP-2 to endothelial cells was slightly less effective in enhancing monocyte arrest. On a quantitative basis, PF4 and ß-thromboglobulin form the most prominent fraction among the platelet-derived chemokines and precursors [4 ]. However, as compared with RANTES or NAP-2, the proteolytic product of ß-thromboglobulin, PF4 bound to HMVECs exhibited only a moderate but dose-dependent enhancement of monocyte arrest in flow. In contrast, ENA-78, which is structurally related to NAP-2, did not trigger monocyte arrest. Although ß-thromboglobulin did not exert any effect, an enhancement of monocyte arrest was observed after treatment of HMVECs with cathepsin G, which is released by neutrophils [15 ]. Thus, it is safe to assume that ß-thromboglobulin bound to endothelial cells is processed to NAP-2 and triggers subsequent monocyte arrest. These data are in good accordance with data showing that neutrophil adhesion to endothelial cells was stimulated by NAP-2 and to a lesser extent, PF4 but not by the NAP-2 precursor CTAP-III [7 ].

The function of arrest chemokines was closely correlated to their ability to bind to the surface of endothelial cells. The binding of chemokines to cell-surface GAGs, e.g., via the BBXB motif in RANTES, has been involved in their arrest function [5 ]. In fact, RANTES, NAP-2, and PF4 were immobilized dose-dependently to the HMVEC surface. Conversely, ENA-78, which did not trigger monocyte arrest, did not bind to the endothelial surface. As ENA-78 shows a 53% analogy to NAP-2 in its amino acid sequence, the binding motif for endothelial GAGs may differ or not be located within the shared amino acid sequence. The finding that PF4 was more readily immobilized than NAP-2 but less efficient in triggering arrest may indicate that the mechanisms for arrest induction may differ for PF4 and NAP-2, as inferred by distinct signaling pathways identified for PF4 and NAP-2 in neutrophils [11 ].

The surface binding of RANTES and PF4 was attenuated, and the arrest function was abrogated by cleavage of chondroitin sulfate with chondroitinase, thereby demonstrating a specific involvement of this GAG subspecies. RANTES and PF4 have been found to bind to various GAGs; for instance, RANTES binding to heparin displayed higher affinity than that to chondroitin sulfate. Specific GAG binding with low or intermediate affinity may be suitable to mediate an arrest function of chemokines, as they may be more permissive in conferring chemokines to their leukocyte receptors. Although the binding of these chemokines to chondroitin sulfate may appear to be moderate, it may be functionally more relevant than that to other receptors or GAGs.

It is important that we could demonstrate for the first time that the coincubation of inflamed endothelium with the platelet chemokines RANTES and PF4 at suboptimal concentrations results in an additive effect on monocyte arrest under flow conditions, exceeding the effect of each individual chemokine. In particular, given the negligible effects of PF4 at low concentrations, the enhancement of RANTES-mediated responses reveals synergistic features. The finding is in accordance with a report showing that the exposure of neutrophils to substimulatory concentrations of PF4 enhances the stimulation of their adhesion to endothelium by NAP-2 [7 ]. Additive or synergistic effects of PF4 and RANTES may be a result of a convergence of distinct signaling pathways activating integrin adhesiveness or attributable to the recently identified heterophilic interaction of RANTES and PF4, which may increase monocyte arrest by supporting the recruitment of additional GAG binding sites or chemokine receptors [18 ]. It is conceivable that such suboptimal concentrations of RANTES and PF4 may occur in vivo [19 ].

The release and deposition of RANTES and PF4 on inflamed and early atherosclerotic endothelium or on monocytes have been demonstrated after perfusion or exposure to activated platelets and have been implicated in RANTES-triggered monocyte recruitment [8 , 12 , 13 ]. In line with the additive or rather synergistic enhancement of monocyte arrest by recombinant RANTES and PF4, monocyte arrest after perfusion of activated platelets was inhibited by RANTES receptor blockade and reduced by a blocking PF4 antibody, demonstrating for the first time that both chemokines contribute to arrest mediated by platelet secretory products in a nonredundant manner. As PF4 is released most abundantly by activated platelets, the concentrations of RANTES achieved on the endothelial surface may be rate-limiting for the induction and modulation of monocyte arrest on inflamed endothelium. Moreover, we have now demonstrated the relevance of RANTES and to a lesser extent, PF4 for the shear-resistant arrest of monocytes in activated whole blood under conditions more closely resembling their physiological recruitment in vivo. In accordance with the comparative analysis of recombinant proteins, RANTES also appeared to be the most important platelet-derived arrest chemokine in monocyte adhesion studies using whole blood. As these chemokines appear to play a crucial role in monocyte recruitment affected by platelets, variable options for therapeutic intervention can be envisioned, e.g., by combined inhibition of platelet degranulation or by selectively targeting individual platelet chemokines to affect their functional consequences [20 ].

In conclusion, platelet-derived chemokines deposited by activated platelets on the surface of inflamed endothelium or present in whole blood differentially modulate monocyte arrest in flow. Although some chemokines appear to be more specialized than others in triggering arrest related to their capacity for immobilization on the endothelial surface, others, in particular, RANTES and PF4, may act in concert to mediate enhanced monocyte arrest.


   ACKNOWLEDGEMENTS
 
This work was supported by Deutsche Forschungsgemeinschaft (Grant We1913/5) and Interdisciplinary Centre for Clinical Research on Biomaterials (BMBF Grant 01KS9503/9). T. B. and P. v. H. contributed equally to this work.

Received March 11, 2005; revised April 25, 2005; accepted April 27, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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