Platelets Recruit Human Dendritic Cells Via Mac-1/JAM-C Interaction and Modulate Dendritic Cell Function In Vitro Harald F. Langer, Karin Daub, Gregor Braun, Tanja Schönberger, Andreas E. May, Martin Schaller, Gerburg M. Stein, Konstantinos Stellos, Andreas Bueltmann, Dorothea Siegel-Axel, Hans P. Wendel, Hermann Aebert, Martin Roecken, Peter Seizer, Sentot Santoso, Sebastian Wesselborg, Peter Brossart, Meinrad Gawaz Objective—Thrombotic events and immunoinflammatory processes take place next to each other during vascular remodeling in atherosclerotic lesions. In this study we investigated the interaction of platelets with dendritic cells (DCs). Methods and Results—The rolling of DCs on platelets was mediated by PSGL-1. Firm adhesion of DCs was mediated through integrin ␣M␤2 (Mac-1). In vivo, adhesion of DCs to injured carotid arteries in mice was mediated by platelets. Pretreatment with soluble GPVI, which inhibits platelet adhesion to collagen, substantially reduced recruitment of DCs to the injured vessel wall. In addition, preincubation of DCs with sJAM-C significantly reduced their adhesion to platelets. Coincubation of DCs with platelets induced maturation of DCs, as shown by enhanced expression of CD83. In the presence of platelets, DC-induced lymphocyte proliferation was significantly enhanced. Moreover, coincubation of DCs with platelets resulted in platelet phagocytosis by DCs, as verified by different cell phagocytosis assays. Finally, platelet/DC interaction resulted in apoptosis of DCs mediated by a JAM-C– dependent mechanism. Conclusions—Recruitment of DCs by platelets, which is mediated via CD11b/CD18 (Mac-1) and platelet JAM-C, leads to DC activation and platelet phagocytosis. This process may be of importance for progression of atherosclerotic lesions. (Arterioscler Thromb Vasc Biol. 2007;27:1463-1470.) Key Words: adhesion molecules 䡲 cell trafficking 䡲 dendritic cells 䡲 platelets B eyond their role in hemostasis and thrombosis,1 platelets represent an important linkage between thrombosis, inflammation, and atherogenesis.2 Moreover, there is growing evidence regarding the importance of dendritic cells (DCs) in the pathogenesis of atherosclerosis and of vulnerable coronary plaques.3 In atherosclerotic plaques, the number of DCs is substantially enhanced and DCs preferentially accumulate at rupture-prone regions.4,5 Recently, DCs were shown to accumulate in the intima of atherosclerosis-predisposed regions of the aorta of C57BL/6 mice.6 However, the mechanisms involved in the recruitment of circulating DCs at site of vascular lesions are poorly understood so far. It is well recognized that platelets rapidly adhere to the extracellular matrix of the subendothelium at sites of vascular lesions. If this process is controlled, platelets passivate vascular injury and initiate the healing process.1 However, uncontrolled platelet-mediated thrombus formation leads to acute thrombotic occlusion or plaque progression resulting in, eg, acute coronary syndrome.7 Platelet-mediated cell recruitment to the atherosclerotic plaque plays a central role for vascular repair mechanisms. DCs participate both in the innate and adaptive immune system and represent highly specialized antigen-presenting cells.8 Thereby, they are capable of stimulating naive, memory, and effector T-cells, as well as activating natural killer cells.8 Proteins are internalized by phagocytosis, degraded into short peptides, and presented via the MHC II receptors.9 During maturation, DCs express various adhesion receptors, which enable DCs to interact with other cell types and mediate homing of DCs to target tissues.4,8 Original received September 24, 2006; final version accepted February 21, 2007. From Innere Medizin (H.F.L., K.D., G.B., T.S., A.E.M., K.S., A.B., D.S.-A., P.S., M.G.), Abteilung III, Eberhard Karls University Tuebingen, Germany; Department of Dermatology (M.S., M.R.), Eberhard Karls University Tuebingen, Germany; Internal Medicine I (G.M.S., S.W.), Eberhard Karls University Tuebingen, Germany; Department of Thoracic, Cardiac, and Vascular Surgery (H.P.W., H.A.), Eberhard Karls University Tuebingen, Germany; Institute for Clinical Immunology and Transfusion Medicine (S.S.), Justus-Liebig-University Giessen; Internal Medicine II (P.B.), Eberhard Karls University Tuebingen, Germany. P.B. and M.G. contributed equally to this work and both should be considered first authors. Correspondence to Harald F. Langer, MD, Medizinische Klinik III, Eberhard Karls Universität Tübingen, Otfried-Müller Str. 10, 72076 Tübingen, Germany. E-mail harald.langer@med.uni-tuebingen.de; or to Meinrad Gawaz, MD, Medizinische Klinik III, Universitätsklinikum Tübingen, OtfriedMüllerstr.10, 72076 Tübingen, Germany. E-mail meinrad.gawaz@med.uni-tuebingen.de © 2007 American Heart Association, Inc. Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org DOI: 10.1161/ATVBAHA.107.141515 1463 Downloaded from http://atvb.ahajournals.org/ by guest on February 13, 2016 1464 Arterioscler Thromb Vasc Biol. June 2007 The present study evaluates the role of platelets for DC adhesion to vascular lesions and shows that platelets play a critical role for the recruitment and function of DCs. Materials and Methods DCs were generated from buffy coats derived from healthy donors and differentiated to immature monocyte-derived DCs or mature DCs (MDCs) (supplemental Figure I, please see http://atvb.ahajournals. org). For blocking experiments, soluble Fc fusion proteins JAM-AFc, JAM-C-Fc, GPVI-Fc, and Fc-control were generated. Adhesion of DCs to platelets (all experiments were performed with isolated platelets) was evaluated in vitro using a static adhesion assay, as well as a dynamic flow model simulating arterial shear rates with and without blocking fusion proteins or antibodies. Recruitment of DCs by platelets in vivo was evaluated by intravital microscopy in mice. Transmission electron microscopy was used to analyze platelet phagocytosis by DCs and direct interaction between the 2 cells. Phenotyping of DCs and differentiation of DCs by platelets was evaluated by flow cytometry, activation of DCs by platelets using a mixed lymphocyte reaction assay with and without blocking substances. Platelet phagocytosis by DCs further was visualized by phase contrast microscopy, standard and confocal immunofluorescence microscopy and flow cytometry. Platelet-induced DC apoptosis was measured by propidium iodide staining, the method of Nicoletti et al, and terminal deoxynucleotidyl transferase-catalyzed deoxyuridinephosphate-nick end labeling (terminal deoxynucleotidyl transferase-mediated deoxyuridinephosphate nick end-labeling assay). For detailed Material and Methods, please see http://atvb.ahajournals.org Results Dendritic Cells Adhere to Immobilized Platelets Under Static and Dynamic Flow Conditions Platelets play a critical role in the recruitment and adhesion of circulating blood leukocytes toward vascular lesions.2 Recently, we could demonstrate that immobilized platelets are able to interact with and to recruit endothelial progenitor cells.10 Besides endothelial progenitor cells, dendritic cells have been postulated to play a role in vascular repair mechanisms and atherosclerosis.3,4 To test whether DCs bind to platelets, isolated platelets (2⫻108/mL) were allowed to adhere to 96-well plates coated with collagen type I. Subsequently, immature DCs (immature monocyte-derived DCs) or MDCs were added to the wells and adhesion of DCs on platelets was evaluated. Under static conditions, DCs substantially adhered to immobilized platelets compared with immobilized collagen type I alone (n⫽6; P⬍0.05; Figure 1A, 1B). Adhesion of DCs to platelets was dependent on the maturation status, as adhesion of MDC onto platelets was significantly enhanced compared with immature monocytederived DCs (n⫽6; P⬍0.05; Figure 1B). In the control experiment, adhesion of DCs to immobilized fibronectin was run in parallel.11 To further characterize adhesion of DCs to platelets, DCs were coincubated with adherent platelets and evaluated by electron microscopy. We found that platelets attached to DCs via forming pseudopodia, indicating that specific adhesion receptors are involved (Figure 1C). To evaluate, whether DC adhesion to immobilized platelets occurs in vivo, we used a carotid injury model of intravital microscopy as described.12 We found that virtually no adhesion of DCs to the intact carotid vessel wall occurs (Figure 1D). However, after vascular injury adhesion of circulating DCs to the site of injury occurs rapidly (Figure 1D). Both Figure 1. Interaction of DCs with platelets under static conditions. A, 96-well plates precoated with collagen I (10 ␮g/mL) were incubated with or without freshly isolated platelets in order to achieve adherent platelet layers as described in Materials and Methods. DCs (1⫻105/mL) were allowed to adhere to these plates. After 30 minutes, the plates were gently washed twice and adherent DCs were quantified by using a defined frame that was projected to each photograph. Wells coated with fibronectin (10 ␮g/mL) served as positive control. B, The mean and SD of 6 independent experiments are shown. *P⬍0.05 as compared with control. C, DCs (1⫻105/mL) were incubated with freshly isolated platelets (2⫻108/ mL) and transmission electron microscopy was performed as described in Materials and Methods to visualize interaction of DCs with platelets (Plt) (magnification ⫻12.000); Ps indicates pseudopodium. D, To assess DC recruitment by platelets in vivo, we used intravital fluorescence microscopy. Virtually no DCs adhered to the intact vessel wall of mouse carotid arteries; 5 minutes after induction of vascular injury by ligation, the number of transient as well as firmly adherent DCs increased significantly. The mean and SD of 6 independent experiments are shown. *P⬍0.001 as compared with noninjured vessels. Downloaded from http://atvb.ahajournals.org/ by guest on February 13, 2016 Langer et al transient and firm adhesion was evident and reached a maximum at 5 minutes and finally reached plateau (Figure 1D). To further analyze the role of platelets for DC recruitment to vascular lesions, mice were pretreated with the soluble collagen receptor GPVI-Fc, which inhibits adhesion of platelets to the injured carotid artery in vivo.13 Pretreatment of mice with soluble collagen receptor GPVI-Fc significantly reduced adhesion of DCs 5 minutes (P⬍0.005) and 30 minutes (P⬍0.05) after induction of injury (supplemental Figure II). Thus, adhesion of DCs to platelets occurs in vivo. Next, we analyzed adhesion receptors expressed on DCs that are potentially involved in adhesion to immobilized platelets. We found that both subunits of the ␤2-integrin Mac-1, CD11b (␣M-subunit), and CD18 (␤2-subunit), are highly expressed on DCs (supplemental Figure III). Moreover, CD29 (␤1-subunit), CD49d (␣4-subunit), and CD162 (PSGL-1) were substantially surface expressed on DCs. Interestingly, surface expression of the ␤2-chain was further enhanced in DCs cultivated in the presence of MDC compared with immature monocyte-derived DCs (supplemental Figure III). Next, we evaluated the determinants that mediate DC adhesion to platelets under arterial flow conditions. In a parallel plate flow chamber, DCs cultured in the presence of granulocyte-macrophage colony-stimulating factor/IL-4/ CD40L (MDC) were perfused over platelets immobilized on collagen type I at a wall shear rate of 2000 s⫺1 as described.10 Cell rolling was significantly enhanced on platelets compared with the collagen surface alone (Figure 2A). Preincubation with a blocking monoclonal antibody to CD162, but not with a control antibody (2D1), significantly reduced this cell rolling (Figure 2A). Furthermore, DCs showed enhanced firm adhesion to immobilized platelets compared with immobilized collagen alone (Figure 2B, 2C). When DCs were preincubated with a blocking anti-CD18 mAb (5 ␮g/mL), firm adhesion of DCs to immobilized platelets was significantly reduced compared with experiments in which an irrelevant mAb (2D1) was used (Figure 2B, 2C). This indicates that the ␤2-integrin is critically involved in DC/ platelet adhesion. In contrast, a blocking mAb directed against CD49d had no effect on DC adhesion onto platelets (Figure 2B, 2C). To identify the platelet counter receptor/ ligand for DCs, we evaluated the effect of soluble recombinant JAM-C fusion protein (sJAM-C), which is known as the heterophilic counter-receptor of Mac-1 integrin, on DC/platelet adhesion. For the control experiments, sJAM-A, soluble collagen receptor GPVI-Fc, sGPIb, or Fc was applied. In the presence of sJAM-C, but not sJAM-A, sGPIb, soluble collagen receptor GPVI-Fc (not shown), or Fc, DC–platelet interaction was significantly (P⬍0.05, n⫽4 to 8) reduced (Figure 2D, 2E). To identify the distinct ␤2-integrin involved in the adhesion process, further experiments with blocking monoclonal antibodies were performed. Although there was a certain reduction in adhesion after pre-incubation with an anti-CD11c mAb, an obvious decrease in DC adhesion to platelets could be observed after pre-incubation with an anti-CD11b mAb, which showed statistical significance (P⫽0.006) only in this group (Figure 2F). Taken together, these data indicate that PSGL-1 mediates an initial contact Platelets and Dendritic Cells 1465 between DCs and platelets under arterial flow conditions followed by firm adhesion mediated to a substantial part via Mac-1/JAM-C. Platelets Induce Differentiation of DCs and Enhance Their Capacity to Stimulate Lymphocyte Proliferation Coincubation of DCs with platelets over several days suggested an induced differentiation of the DCs as evidenced by enhanced expression of CD83 (Figure 3A), which reached a plateau at day 3 as evaluated by coexpression of CD1a/CD83 (Figure 3B). Furthermore, expression of differentiation markers CD1a/CD83, CD54, CD40, and CCR-7 was evaluated in the presence of blocking mAbs to CD40L, CD18, PF-4, or sJAM-C (Figure 3C). Pre-incubation with a blocking mAb to CD18 or sJAM-C reduced coexpression of CD1a/CD83 and expression of CCR-7, but not expression of CD54 and CD40 in the presence of platelets. Pre-incubation with a blocking mAb to platelet factor 4 (anti-PF-4) reduced coexpression of CD1a/CD83, expression of CD54, CD40, and CCR-7 in the presence of platelets; mAb to CD40L, however, had no effect on the expression of these differentiation markers under platelet influence (Figure 3C). The ability of the generated DC populations to stimulate allogenic T-cell responses was furthermore analyzed in a mixed lymphocyte reaction.14 DCs coincubated with platelets showed an enhanced T-cell stimulatory capacity, dependent on the maturation stimulus used (Figure 3D). Immature DCs cultivated in the presence of granulocyte-macrophage colonystimulating factor/IL-4 showed clearly increased T-cell stimulation after exposure to platelets (n⫽3, P⬍0.05; Figure 3D), similar to DCs additionally treated with CD40L but without platelets. However, DCs cultivated in the presence of granulocyte-macrophage colony-stimulating factor/IL-4/ CD40L (MDC) revealed a further enhanced T-cell stimulatory capacity, when additionally coincubated with platelets (n⫽3, P⬍0.05; Figure 3D). Thus, platelets substantially enhance the capacity of DCs to initiate lymphocyte proliferation, a critical step in the initiation of primary immune responses. To further characterize the influence of platelets on DC activation, we performed additional mixed lymphocyte reaction assays with or without blocking monoclonal antibodies. We found that a blocking mAb to CD40L significantly (n⫽4, P⬍0.05) reduced lymphocyte proliferation induced by DCs that were exposed to platelets (Figure 3E). A blocking mAb to CD18 or sJAM-C, however, had no effect in this setting (Figure 3E). Phagocytosis of Platelets by DCs After Prolonged Interaction To further characterize the interaction of DCs with platelets, we coincubated these 2 cell types for up to 12 days. After 3 to 7 days, platelets started to disappear and after ⬇10 days of coincubation; virtually none of the platelets could be found extracellularly (Figures 4A and 5A). In turn, DCs showed brown intracellular granules, probably representing phagocytosed platelets (Figure 4A). To further analyze phagocytosis of platelets by DCs, platelets were labeled with the Fluorochrome Celltracker orange CMTMR and added to DCs. After Downloaded from http://atvb.ahajournals.org/ by guest on February 13, 2016 1466 Arterioscler Thromb Vasc Biol. June 2007 Figure 2. Adhesion of DCs to immobilized platelets under arterial shear conditions. (A,B,C) Coverslips were precoated with collagen I (10 ␮g/mL) and additionally pre-incubated with or without freshly isolated platelets (2⫻108/mL) to achieve adherent platelet layers as described in Materials and Methods. Resuspended DCs (2.5⫻105/mL) were perfused over these coverslips in a flow chamber using arterial shear rates. In similar fashion, DCs were perfused over immobilized platelets in the presence or absence of blocking mAbs (5 ␮g/mL) as indicated. From minutes 2 to 3, 5 to 6, and 8 to 9, rolling DCs were counted (n⫽4, A). *P⬍0.05 as compared with control antibody. After 10 minutes, firmly adherent DCs were quantified by offline counting (n⫽4; B,C). The mean and SD of 4 independent experiments are shown. *P⬍0.05 as compared with control-IgG. C, Representative offline images of perfusion experiments after 10 minutes. D, To identify the involved counterreceptor for DC adhesion on platelets, isolated platelets were immobilized on collagen and incubated with DCs with or without soluble Fc fusion proteins as described in Materials and Methods. *P⬍0.05 as compared with control, n⫽4 to 8. E, Representative microscopy images of these experiments. F, To further characterize the receptor mediating adhesion to platelets on DCs, isolated platelets were immobilized on collagen and incubated with DCs with or without blocking monoclonal antibodies. *P⬍0.05 as compared with control, n⫽4. Downloaded from http://atvb.ahajournals.org/ by guest on February 13, 2016 Langer et al Platelets and Dendritic Cells 1467 7 days, substantial amounts of fluorescent platelets were found within DCs as verified by confocal fluorescence microscopy (Figure 4B). Using transmission electron microscopy, we further visualized the process of platelet phagocytosis (Figure 4C). Platelets initiate the contact with DCs via protrusions (Figures 4C and 1C). Subsequently, platelets are incorporated and lysed to cell fragments (Figure 4C). To analyze the exact kinetics of platelet uptake by DCs, platelets were labeled with mepacrine and DCs were analyzed by flow cytometry at different days. Hereby, we could show that platelet uptake started after 3 days and reached a maximum at 5 to 7 days (Figure 4D). Similarly, time-lapse experiments showed that platelets are phagocytosed at this time point (supplemental film, please see http://atvb.ahajournals.org). Platelets Induce Apoptosis of DCs Recently, platelets have been described to induce apoptosis of endothelial cells.15 We analyzed the importance of platelet/DC interaction for the induction of apoptosis of DCs using propidium iodide staining as described in Materials and Methods. After coincubation of DCs with isolated platelets, vesicles appeared around DCs (Figure 5A), indicating apoptosis of DCs. Using the same coincubation model and the method of Nicoletti et al, induction of apoptosis was significantly enhanced in DCs (immature monocyte-derived DCs and MDCs) treated with platelets compared with control (P⬍0.05; Figures 5B, IV). Mitomycin C treatment of DCs, which served as positive control, showed similar levels of apoptotic cell death (Figure 5B and supplemental Figure IV). Similarly, using a terminal deoxynucleotidyl transferasemediated deoxyuridinephosphate nick end-labeling assay, we could show, that platelets induced apoptosis of DCs (P⬍0.05, n⫽3; Figure 5C). Analyzing the kinetics of platelet-induced DC apoptosis by propidium iodide staining, we could show that apoptosis starts after 3 to 5 days and reaches a maximum after 7 days (Figure 5D). To further elucidate the mechanisms mediating platelet-induced DC apoptosis, we performed experiments with pre-incubation with blocking antibodies and the method of Nicoletti et al Thereby, we could show, that the presence of sJAM-C or a blocking mAb to CD11b resulted in significantly decreased apoptosis of DCs (Figure 5E), suggesting this to be one of the central responsible mechanisms. Figure 3. Effect of platelets on DC differentiation and activation. A, DCs were coincubated with platelets and analyzed by fluorescence-activated cell sorter flow cytometry. After 24 hours, DCs coincubated with platelets showed enhanced expression of CD83 and CD86 and virtually no expression of CD14. B, Coexpression of CD1a/CD83 was enhanced after 1 day and showed a maximum increase after 2 to 3 days. C, Additionally, expressions of differentiation markers CD1a/CD83, CD54, CD40, and CCR-7 were evaluated with or without blocking mAbs to CD40L, CD18, and PF-4 or sJAM-C (n⫽4). D, After coincubation with platelets, DCs were irradiated and PBMNCs were added. Proliferation was measured in a mixed lymphocyte reaction assay. Platelets were able to activate PBMNCs as shown by a significant increase in proliferation. Proliferation was measured by 3H-thymidin incorporation (CPM indicates counts per minute). *P⬍0.05 compared with control. E, Immature monocyte-derived DCs were exposed to platelets with or without application of blocking mAbs to CD40L, CD18, or sJAM-C. *P⬍0.05 as compared with control, n⫽4. Downloaded from http://atvb.ahajournals.org/ by guest on February 13, 2016 1468 Arterioscler Thromb Vasc Biol. June 2007 Application of a blocking antibody to PDGF-AB showed a slight, yet not significant decrease, whereas inhibition of CD40L revealed virtually no reduction of DC apoptosis (Figure 5E). Discussion In this study, we have shown that platelets regulate adhesion and function of DCs in vitro. The major findings are as follows. DCs adhere to immobilized platelets via the CD11b/ CD18 complex (␣M␤2, Mac-1) after an initial contact has been established by dendritic cell PSGL-1. Blocking experiments further showed that CD11c and JAM-A play a minor, yet not significant, role. Platelets enhance the capacity of DCs to initiate lymphocyte proliferation. DCs phagocyte platelets and undergo apoptosis, which was mediated by JAM-C. The recruitment of DCs by platelets to injured carotid arteries in vivo, as verified by intravital microscopy, emphasizes the (patho-) physiological relevance of the identified mechanism. Atherosclerosis is a chronic disease that involves thrombotic but also immunoinflammatory mechanisms.16 DCs are found in the intima of atherosclerosis-prone vessel areas and form cell clusters.6,17 During atheroprogression, the number of DCs markedly increases preferentially within plaque shoulders, which represent plaque rupture-prone regions4,5 associated with plaque destabilization,18 indicating that DCs might be involved in the process of atherosclerosis. DCs can originate from CD34⫹ progenitor cells and DC precursors, which circulate via the bloodstream to reach their target tissues.4,9 However, the mechanisms that regulate DC recruitment toward the atherosclerotic plaque are not understood. Platelets accumulate within seconds to sites of vascular injury and release a variety of potent chemotactic factors and adhesion receptors onto the platelet surface that induce recruitment of circulating blood cells toward sites of vascular lesions.1 Recently, circulating endothelial progenitor cells have been shown to home at sites of vascular lesions,19 most likely mediated by adherent platelets.10 In the present study, we show that DCs adhere to immobilized platelets under flow conditions similar to arterial shear rates. Our data suggest that PSGL-1, which is surfaceexpressed on DCs, is able to mediate an initial contact between platelets and DCs. We found that both subunits Figure 4. Coincubation of DCs with platelets. A, Platelets were coincubated with DCs in 96-well plates up to 12 days. After 3 to 7 days, platelets increasingly disappeared and in projection to DCs (Š), brown vesicles could be observed. B, To verify platelet phagocytosis by dendritic cells, platelets (2⫻108/mL) were labeled with the Fluorochrome Celltracker orange CMTMR and coincubated with DCs for 7 days in chamber slides. Subsequently, cells were analyzed by standard and confocal fluorescence microscopy. C, Furthermore, transmission electron microscopy was performed as described in Materials and Methods. Hereby, we could clearly visualize an established contact (⬍) between DCs (Š) and platelets (3), internalization, and finally lysis of the platelets resulting in intracellular platelet components within DCs (magnification as indicated in photographs). D, Platelets were stained with mepacrine and after coincubation of DCs with these platelets, phagocytosis was analyzed by assessment of mepacrine-positive DCs using flow cytometry from days 1 to 11. DCs alone served as control. Downloaded from http://atvb.ahajournals.org/ by guest on February 13, 2016 Langer et al Platelets and Dendritic Cells 1469 CD11b/CD18 of the ␣M␤2 integrin (Mac-1) are highly surface-expressed on DCs and that adhesion of DCs onto platelets is mediated by CD11b/CD18 but not CD49d. Previously it was shown that DCs bind to fibronectin, possibly via ␤1-integrins.11 Similarly, in our studies immature DCs bound to fibronectin, but obviously weaker to collagen, which is the major constituent of the extracellular matrix of atherosclerotic plaques. However, when platelets adhere to collagen, they are activated and mediate adhesion of DCs via interaction with ␤2-integrin. In the present study we could show that JAM-C,20 but not GPIb21 or fibrinogen,22 acts as a specific counter receptor, which is required on platelets to mediate DC adhesion under arterial shear rates. Hagihara et al23 demonstrated that activated platelets induce IL-10 –producing MDCs in vitro derived from mononuclear cells. Similar to the study by Hagihara et al, our data indicate that platelets induce a differentiation of DCs, as shown by enhanced coexpression of CD1a/CD83, which started already after 1 day and peaked at days 2 to 3 of coincubation. Furthermore, we could show that platelets enhance the capacity of DCs to initiate lymphocyte proliferation. Thus, once adherent to platelets, DCs are stimulated to regulate immunoinflammatory responses. Activated platelets release a variety of potent inflammatory compounds, including IL-1, CD40 ligand, or growth factors, that might stimulate maturation and function of DCs. Thus, it is tempting to speculate that in the microenvironment of adherent platelets, immature DCs adhere and mature through stimulation of platelet-derived compounds.23 Because of our experiments, CD40L as one of these candidate substances is involved in platelet-mediated DC activation, as a blocking mAb to CD40L could reduce the effect of platelets on DCs in a mixed lymphocyte reaction. Once homed to target tissues, DCs continuously and efficiently sample the antigenic content of their microenvironment by phagocytosis.8 We found that platelets are substantially internalized into DCs. As platelets and DCs were coincubated over several days, the platelets presumably were activated. When platelets were coincubated for up to 12 days with DCs, a complete uptake of platelets was obvious. Because platelet-containing DCs changed their morphology Figure 5. Platelet induced apoptosis of DCs. A, After coincubation of DCs with platelets, platelets were phagocyted by DCs and disappeared. Instead, after 7 to 12 days, around DCs vesicles appeared (arrow), indicating an apoptotic process. B and supplemental Figure IV, DCs were incubated with isolated platelets for 9 days. Mitomycin C-treated (25 ␮g/mL) DCs served as positive control. Subsequently, induction of apoptosis was assessed by propidium iodide staining of hypodiploid apoptotic nuclei and flow cytometry. Compared with untreated cells, DCs incubated with isolated platelets revealed significantly increased levels of apoptosis, similar to the positive control. *P⬍0.05 vs control; n⫽4. C, After incubation with platelets, mitomycin C, or control, a terminal deoxynucleotidyl transferase-mediated deoxyuridinephosphate nick end-labeling assay was performed, as described in Materials and Methods. Compared with control, significantly more apoptosis could be detected in DCs exposed to platelets and in the positive control group. P⬍0.05; n⫽3. D, To evaluate kinetics of platelet-induced apoptosis of DCs, propidium iodide staining of hypodiploid apoptotic nuclei and flow cytometry was performed from days 1 to 11. Platelet induced apoptosis of DCs started at day 3 and reached a maximum at day 7. E, Similarly, on day 7, apoptosis was evaluated with or without pre-incubation with blocking soluble proteins or blocking monoclonal antibodies as indicated. P⫽0.001 compared with DCs and platelets; n⫽3. Downloaded from http://atvb.ahajournals.org/ by guest on February 13, 2016 1470 Arterioscler Thromb Vasc Biol. June 2007 significantly, we asked whether they undergo apoptosis. We found that platelet phagocytosis induces apoptosis of DCs as measured by the generation of hypodiploid apoptotic nuclei and terminal deoxynucleotidyl transferase-mediated deoxyuridinephosphate nick end-labeling assay. Interestingly, platelet phagocytosis and DC apoptosis occurred at parallel time points, possibly implying that the one may be linked to the other process. By experiments with blocking proteins, we could show that JAM-C/CD11b is of importance for this process. Our experimental data are strengthened by recent clinical data, which indicate that DCs may be involved in atherosclerosis.3,5,24 For example, application of statins leads to lower numbers of DCs in atherosclerotic plaques.5 An interaction between platelets and dendritic cells thus may be one of the critical cellular links between atherosclerosis and immunologic processes. Acknowledgments We acknowledge the excellent technical assistance of Sarah Gehring, Iris Schäfer, Alexandra Gau␤, Heike Runge, Sylvia Stephan, Solveig Daecke and Birgit Fehrenbacher. Sources of Funding The study was supported by grants of the Deutsche Forschungsgemeinschaft (GRK1302 to S.W., SFB 685 to P.B. and S.W., We 1801/2-4 to S.W., and Graduiertenkolleg MA2186/3-1 “Zellbiologische Mechanismen immunassoziierter Prozesse,” GK 794, and Nr. 2186/3-1 to M.G.), the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (Center for Interdisciplinary Clinical Research, IZKF; Fö. 01KS9602 to S.W.), SFB 685 (M.R.), and the Karl und Lore Klein Stiftung and the Sandersstiftung (Nr. 2003.0601 to M.G.), the fortüne program of the UKT, and the Novartis foundation (H.F.L. and M.G.). Disclosures None. References 1. Ruggeri ZM. Platelets in atherothrombosis. Nat Med. 2002;8:1227–1234. 2. Gawaz M, Langer H, May AE. Platelets in inflammation and atherogenesis. J Clin Invest. 2005;115:3378 –3384. 3. Bobryshev YV. Dendritic cells in atherosclerosis: current status of the problem and clinical relevance. Eur Heart J. 2005;26:1700 –1704. 4. Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med. 2005;352:1685–1695. 5. Yilmaz A, Lochno M, Traeg F, Cicha I, Reiss C, Stumpf C, Raaz D, Anger T, Amann K, Probst T, Ludwig J, Daniel WG, Garlichs CD. Emergence of dendritic cells in rupture-prone regions of vulnerable carotid plaques. Atherosclerosis. 2004;176:101–110. 6. Jongstra-Bilen J, Haidari M, Zhu SN, Chen M, Guha D, Cybulsky MI. Low-grade chronic inflammation in regions of the normal mouse arterial intima predisposed to atherosclerosis. J Exp Med. 2006;203:2073–2083. 7. Massberg S, Schulz C, Gawaz M. Role of platelets in the pathophysiology of acute coronary syndrome. Semin Vasc Med. 2003;3:147–162. 8. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature. 1998;392:245–252. 9. Lipscomb MF, Masten BJ. Dendritic cells: immune regulators in health and disease. Physiol Rev. 2002;82:97–130. 10. Langer H, May AE, Daub K, Heinzmann U, Lang P, Schumm M, Vestweber D, Massberg S, Schonberger T, Pfisterer I, Hatzopoulos AK, Gawaz M. Adherent platelets recruit and induce differentiation of murine embryonic endothelial progenitor cells to mature endothelial cells in vitro. Circ Res. 2006;98:e2–10. 11. Saint-Vis B, Bouchet C, Gautier G, Valladeau J, Caux C, Garrone P. Human dendritic cells express neuronal Eph receptor tyrosine kinases: role of EphA2 in regulating adhesion to fibronectin. Blood. 2003;102: 4431– 4440. 12. Massberg S, Brand K, Gruner S, Page S, Muller E, Muller I, Bergmeier W, Richter T, Lorenz M, Konrad I, Nieswandt B, Gawaz M. A critical role of platelet adhesion in the initiation of atherosclerotic lesion formation. J Exp Med. 2002;196:887– 896. 13. Massberg S, Konrad I, Bultmann A, Schulz C, Munch G, Peluso M, Lorenz M, Schneider S, Besta F, Muller I, Hu B, Langer H, Kremmer E, Rudelius M, Heinzmann U, Ungerer M, Gawaz M. Soluble glycoprotein VI dimer inhibits platelet adhesion and aggregation to the injured vessel wall in vivo. FASEB J. 2004;18:397–399. 14. Appel S, Mirakaj V, Bringmann A, Weck MM, Grunebach F, Brossart P. PPAR-gamma agonists inhibit toll-like receptor-mediated activation of dendritic cells via the MAP kinase and NF-kappaB pathways. Blood. 2005;106:3888 –3894. 15. Wassmer SC, de Souza JB, Frere C, Candal FJ, Juhan-Vague I, Grau GE. TGF-{beta}1 released from activated platelets can induce TNFstimulated human brain endothelium apoptosis: a new mechanism for microvascular lesion during cerebral malaria. J Immunol. 2006;176: 1180 –1184. 16. Ross R. Atherosclerosis–an inflammatory disease. N Engl J Med. 1999; 340:115–126. 17. Lord RS, Bobryshev YV. Clustering of dendritic cells in athero-prone areas of the aorta. Atherosclerosis. 1999;146:197–198. 18. Bobryshev YV, Lord RS. Co-accumulation of dendritic cells and natural killer T cells within rupture-prone regions in human atherosclerotic plaques. J Histochem Cytochem. 2005;53:781–785. 19. Urbich C, Heeschen C, Aicher A, Dernbach E, Zeiher AM, Dimmeler S. Relevance of monocytic features for neovascularization capacity of circulating endothelial progenitor cells. Circulation. 2003;108:2511–2516. 20. Santoso S, Sachs UJ, Kroll H, Linder M, Ruf A, Preissner KT, Chavakis T. The junctional adhesion molecule 3 (JAM-3) on human platelets is a counterreceptor for the leukocyte integrin Mac-1. J Exp Med. 2002;196:679 – 691. 21. Chavakis T, Santoso S, Clemetson KJ, Sachs UJ, Isordia-Salas I, Pixley RA, Nawroth PP, Colman RW, Preissner KT. High molecular weight kininogen regulates platelet-leukocyte interactions by bridging Mac-1 and glycoprotein Ib. J Biol Chem. 2003;278:45375– 45381. 22. Konstantopoulos K, Neelamegham S, Burns AR, Hentzen E, Kansas GS, Snapp KR, Berg EL, Hellums JD, Smith CW, McIntire LV, Simon SI. Venous levels of shear support neutrophil-platelet adhesion and neutrophil aggregation in blood via P-selectin and beta2-integrin. Circulation. 1998;98:873– 882. 23. Hagihara M, Higuchi A, Tamura N, Ueda Y, Hirabayashi K, Ikeda Y, Kato S, Sakamoto S, Hotta T, Handa S, Goto S. Platelets, after exposure to a high shear stress, induce IL-10-producing, mature dendritic cells in vitro. J Immunol. 2004;172:5297–5303. 24. Ranjit S, Dazhu L, Qiutang Z, Yibo F, Yushu L, Xiang W, Shen CL, Yuan T. Differentiation of dendritic cells in monocyte cultures isolated from patients with unstable angina. Int J Cardiol. 2004;97:551–555. Downloaded from http://atvb.ahajournals.org/ by guest on February 13, 2016 Platelets Recruit Human Dendritic Cells Via Mac-1/JAM-C Interaction and Modulate Dendritic Cell Function In Vitro Harald F. Langer, Karin Daub, Gregor Braun, Tanja Schönberger, Andreas E. May, Martin Schaller, Gerburg M. Stein, Konstantinos Stellos, Andreas Bueltmann, Dorothea Siegel-Axel, Hans P. Wendel, Hermann Aebert, Martin Roecken, Peter Seizer, Sentot Santoso, Sebastian Wesselborg, Peter Brossart and Meinrad Gawaz Arterioscler Thromb Vasc Biol. 2007;27:1463-1470; originally published online March 22, 2007; doi: 10.1161/ATVBAHA.107.141515 Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2007 American Heart Association, Inc. All rights reserved. Print ISSN: 1079-5642. Online ISSN: 1524-4636 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://atvb.ahajournals.org/content/27/6/1463 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Arteriosclerosis, Thrombosis, and Vascular Biology can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Arteriosclerosis, Thrombosis, and Vascular Biology is online at: http://atvb.ahajournals.org//subscriptions/ Downloaded from http://atvb.ahajournals.org/ by guest on February 13, 2016 Data Supplement (unedited) at: http://atvb.ahajournals.org/content/suppl/2007/03/26/ATVBAHA.107.141515.DC1.html Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Arteriosclerosis, Thrombosis, and Vascular Biology can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Arteriosclerosis, Thrombosis, and Vascular Biology is online at: http://atvb.ahajournals.org//subscriptions/ Downloaded from http://atvb.ahajournals.org/ by guest on February 13, 2016 Material and Methods Reagents For cultivation of DCs from human mononuclear cells, human granulocyte macrophage colony stimulating factor (GM-CSF; Leukine Liquid Sargramostin; Berlex Laboratories, Richmond, CA), interleukin-4 (IL-4; R&D Systems, Wiesbaden, Germany) and CD40 ligand (CD40L, Biozol, Eching, Germany) were used. Human fibronectin and collagen I were from Becton Dickinson (BD, Heidelberg, Germany). Blocking monoclonal antibody (mAb) directed against CD49d (α4-integrin, clone 9F10) was purchased from Immunotech (Marseille, France), CD18 (β2-integrin,clone IB4) and CD162 (P-Selectin Glycoprotein Ligand-1 (PSGL-1), clone 3E2.25.5 PL-1) from Ancell (Bayport, USA), CD11a (clone HI111), CD11b (αM integrin, clone ICRF44), CD11c (clone 3.9) and CD40L from Biozol, Eching, Germany. Blocking polyclonal Ab to CD11d (clone A01) was from Abnova, Taipei, Taiwan. The mAb AP1 against GPIb was a kind gift from Dr. P. Newman, Blood Research Institute, Milwaukee, USA. A monoclonal antibody to PDGF-AB was from Promega (Madison, USA). For flow cytometry, fluorescein isothiocyanate (FITC) or phycoerythrin (PE) labeled antibodies specific for CD29 (clone 3S3), CD49b (clone AK7), CD49e (clone SAM-1) and CD49f (clone MCA699) from Serotec (Düsseldorf, Germany), CD49d (clone 9F10), CD162 (clone KPL-1) and CD18 (clone IB4) from BD, CD41 (clone P2), CD51 (clone AMF7), CD61 (clone SZ21) and CD11b (clone Bear 1) from Immunotech, CD62P (clone G1-4) from Ancell and corresponding IgG controls were used. The fluorochrome celltrackertm orange CMTMR (Molecular Probes, Leiden, Netherlandes) and quinacrine dihydrochloride (mepacrine, Sigma, Taufkirchen, Germany) was used to evaluate platelet phagocytosis. Mitomycin C was from Medac (Hamburg, Germany). Glycocalicin, the soluble extracellular region of GPIbα (sGPIbα) 1 was a kind gift from Dr. K. Clemetson, Theodor Kocher Institute, Bern, Switzerland. Isolation of platelets Human platelets were isolated as described before.2 Briefly, venous blood was drawn from the antecubital vein of healthy volunteers and collected in acid citrate dextrose (ACD)-buffer. After centrifugation at 430 x g for 20 min, platelet-rich plasma (PRP) was removed, added to Tyrodes-HEPES (HEPES 2.5 mmol/L, NaCl 150 mmol/L, KCl 2.5 mmol/L, NaHCO3 0.36 mmol/L, glucose 5.5 mmol/L, BSA 1 mg/ml, pH 6.5) and centrifugated at 900 x g for 10 min. After removal of the supernatant, the resulting platelet pellet was resuspended in Tyrodes-HEPES buffer (pH 7.4 supplemented with CaCl2, 1 mmol/L; MgCl2, 1 mmol/L). Isolation of DCs DCs were generated from buffy coats derived from healthy donors.3,4 In brief, peripheral blood mononuclear cells (PBMNCs) were isolated by Ficoll/Paque (Biochrom, Berlin, Germany) density gradient centrifugation of HLA-A02 – positive buffy coat preparations. Cells were resuspended in serum-free X-VIVO 20 medium (Cambrex, Verviers, Belgium) and allowed to adhere (1 x 107 cells/ well) in 6-well plates in a final volume of 2 ml. After 2 hours of incubation at 37°C and 5% CO2, nonadherent cells were removed. Immature monocyte derived dendritic cells (iMDC) were generated by culturing the adherent blood monocytes in medium (RPMI 1640 with GlutaMAX-1 supplemented with 10% heat-inactivated fetal calf serum and 100 IU/ml penicillin/streptomycin [Invitrogen, Karlsruhe, Germany]) in the presence of human recombinant granulocyte macrophage colony - stimulating factor (GM-CSF; 100 ng/ml) and interleukin-4 (IL-4; 20 ng/ml) for 7 days. 5-7 For maturation, iMDC were additionally treated with CD40L (100 ng/ml) for further 24 hours (-> MDC). After generation of dendritic cells, prior to each functional experiments, a washing step was included to remove differentiating substances and DCs were processed with RPMI medium and cells/ substances as described below.To control purity of the cells and to exclude presence of cells of the monocyte/macrophage lineage, DCs were characterized by flow cytometry and verified to be positive for CD1a, CD83, CD80, CD86, CD40, CD54 and for the human leukocyte antigen DR (HLA- DR), while lacking the expression of CD14, as described previously 8,9 (Suppl. Fig 1). Generation of soluble Fc-fusion proteins Cloning of JAM-A-Fc, JAM-C-Fc, GPVI-Fc and Fc-control. Fc fusion proteins or Fc control were generated as described before.10 For human JAM-A a PCR was performed with cgcgggagatctaccaccatggggacaaaggcgcaag-3’ gcgggggcggccgccattccgctccacagcttcc-3’, for the primers and human JAM-C 5’5’- with 5’- gcggggggtaccaccatggcgctgaggcggcc-3’ and 5’-gcgggggcggccgcctccgccaatgttcaggtca -3’. For amplification, the plasmid pcDNA–FRT (BamHI/EcoRV) was used. Generation and harvesting of JAM-A-Fc (soluble JAM-A, sJAM-A), JAM-C-Fc (soluble JAM-A, sJAM-C), GPVI-Fc (soluble GPVI, sGPVI) or Fc was performed as described before.10 Adhesion of DCs to platelets under static and dynamic conditions Static adhesion. To evaluate DC/ platelet adhesion under static conditions, isolated platelets (2x108/ml) were allowed to adhere to 96-well plates coated with collagen type I (10 µg/ml) for 2 hours followed by blocking with BSA (2%). DCs (iMDC, MDC) were added and incubated for 30 min. After two gentle washing steps with PBS, residual adherent DCs were counted by direct phase contrast microscopy. As control, experiments were performed with immobilized fibronectin (10 µg/ml). Where indicated, DCs (MDC) or platelets were pre-treated with sJAM-C, sJAM-A, sGPVI, sGPIbα or Fc alone as negative control (20 µg/ml each) or blocking monoclonal antibodies to CD 11a, CD11b, CD11c, CD11d, CD42 (GPIb). Dynamic adhesion. Adhesion experiments under flow conditions were basically performed as previously described.11 In brief, glass coverslips were coated with collagen type I (10 µg/ml) and used in a flow chamber (Oligene, Berlin, Germany). Isolated platelets (2x108/ml) were allowed to adhere to collagen-coated coverslips. Where indicated, DCs were pre-treated with anti-CD162 mAb (5µg/ml), anti-CD18 mAb (5µg/ml), anti-CD49d mAb (5µg/ml) or with an irrelevant mAb (2D1, 5µg/ml) 10 for 30 min before perfusion was started. Perfusion was performed with DCs (MDC) resuspended in Tyrodes-HEPES buffer (pH 7.4 CaCl2, 1 mmol/L; MgCl2, 1 mmol/L) at shear rates of 2000 s-1 (high shear). All experiments were recorded in real time on video-CD and evaluated off-line. Carotid ligation in mice and assessment of DC adhesion by intravital microscopy To assess DC recruitment by platelets in vivo, we used intravital fluorescence microscopy as described before.12 Wild-type C57BL6/J mice (Charles River Laboratories) were anesthesized by intraperitoneal injection of a solution of midazolame (5 mg/kg body weight; Ratiopharm), medetomidine (0.5 mg/kg body weight; Pfizer) and fentanyl (0.05 mg/kg body weight, CuraMed/Pharma GmbH). Polyethylene catheters (Portex) were implanted into the right jugular vein and fluorescent DCs (5x104 cells/250µl) were injected intraveneously. The right common carotid artery was dissected free and ligated vigorously near the carotid bifurcation for 5 min to induce vascular injury.12 Before and after vascular injury, interaction of the fluorescent DCs with the injured vessel wall was visualized by in situ in vivo video microscopy of the right common carotid artery using a Zeiss Axiotech microscope (20x water immersion objective, W 20x/0,5; Carl Zeiss MicroI maging, Inc.) with a 100-W HBO mercury lamp for epi-illumination. Adherent DCs were quantified as described before.12 Where indicated, mice were pre-treated with soluble GPVI-Fc (4mg/kg injection 12h and 1h prior to each experiment), which inhibits adhesion of platelets to the injured carotic artery in vivo 10 or Fc in equimolar concentration as negative control. Electron microscopy For transmission electron microscopy, DCs (MDC) were grown to 70-80% confluency and coincubated with isolated platelets (2x108/ml) for various time intervals in culture medium. Subsequently, cells were fixed in Karnovsky`s solution, postfixed in osmiumtetroxide and embedded in glycid ether prior to electron microscopy.11 For scanning electron microscopy, DCs were incubated on glass discs. Thereafter, discs were gently rinsed with isotonic saline to remove weakly attached cells, fixed in 2 % (w/v) glutaraldehyde in PBS (1 h), dehydrated through ascending grades of ethanol up to absolute ethanol, mounted, critical point dried, sputtered with gold-palladium, and analyzed by a scanning electron microscope (Cambridge Stereoscan, Cambridge, UK). Flow cytometry Receptor surface expression of DCs (iMDC/ MDC) was evaluated using flow cytometry. DCs were incubated with fluorescence-labelled (FITC or PE) mAbs for 30 min as indicated. As control, PE- or FITC- labeled isotype-matched IgG was used. To evaluate differentiation of DCs (iMDC) under platelet influence, expression of CD1a, CD14, CD83 and co-expression of CD1a/CD83, CD40, CD54 and CCR-7 on DCs was analyzed after coincubation of both cells for up to 3 days. To identify mechanisms involved in platelet mediated DC differentiation, blocking substances were added as indicated in figure legends. Flow cytometric analysis was performed on a FACScalibur (Beckton-Dickinson, Heidelberg, Germany). Mean immunofluorescence (MIF) was used as index of antigen expression. Mixed-Lymphocyte-Reaction (MLR) assay To evaluate the activation of DCs by platelets, a mixed lymphocyte reaction (MLR) assay was carried out.13 Briefly, DCs (iMDC/ MDC, 2.5x105/ml) were coincubated with or without freshly isolated platelets (2x108/ml) for seven days in 48-well plates at 37°C. Subsequently, cells were detached, washed and adjusted to a concentration of 1x105/ml. To inhibit further proliferation of DCs, the cells were exposed to radiation of 30 Gray. Then, DCs were coincubated with PBMNCs (1x106/ml) in a 96 microtiterwell for 5 days at 37°C, to evaluate the stimulating effect of the DCs (with or without platelets) on PBMNC proliferation. This effect was measured by the detection of 3Hthymidin uptake.13 PBMNCs and platelets alone served as an internal control. To identify mechanisms involved in platelet mediated DC activation, blocking substances were added as indicated in figure legends. Coincubation and phagocytosis assays For coincubation experiments, DCs (2.5x105/ml) were added to isolated platelets (2x108/ml) for up to 12 days. Visual microscopic controls were carried out daily and phase contrast images were taken on day 1, 7 and 12. To visualize phagocytosis of platelets by DCs, platelets were labeled with the fluorochrome celltrackertm orange CMTMR and coincubated with DCs (iMDC) for 7 days in chamber slides. Subsequently, cells were analyzed by standard and confocal fluorescence microscopy. To evaluate the kinetics of platelet phagocytosis by DCs, platelets were stained with mepacrine for 4h and after co-incubation of DCs (iMDC) with platelets, mean fluorescence intensity was analyzed in the DC gate on day 1, 2, 5, 7 and 11. Moreover using the staining protocol with celltrackertm orange CMTMR, on day 5 cells (co-cultures) were analyzed by time-lapse fluorescence and phase contrast microscopy using an Olympus IX 81 microscope (Olympus, Hamburg, Germany) and cell^P software (Olympus Soft Imaging Solutions, LeinfeldenEchterdingen, Germany). The analyses were performed at 37°C in an incubator containing an atmosphere of 10% CO2 in air (Incubator S, Pecon, Erbach, Germany). Phase contrast images and celltrackertm orange CMTMR (red) fluorescence were acquired at 30 min intervals over 48 hours. Data are presented as an overlaid sequence of phase contrast and fluorescence images. Apoptosis assay For detection of apoptosis, DCs (2.5x105/ml culture medium) were coincubated with platelets (2x108/ml) in 96-well microtiter plates for 9 days. The leakage of fragmented DNA from apoptotic nuclei was measured by the method of Nicoletti et al.14 Briefly, apoptotic nuclei from DCs (iMDC/ MDC) were prepared by lysing cells in a hypotonic lysis buffer (1% sodium citrate, 0.1% Triton X-100 and 50 µg/mL propidium iodide) and subsequently analyzed by flow cytometry. To identify mechanisms involved in platelet mediated DC apoptosis, blocking substances were added as indicated in figure legends. To evaluate the kinetics of DC (iMDC) apoptosis induced by platelets, apoptosis was analyzed on day 1, 3, 5, 7, 9 and 11 using this method. Furthermore, to detect DNA-fragments the terminal deoxynucleotidyl transferase (TdT)-catalyzed deoxyuridinephosphate (dUTP)-nick end labeling (TUNEL) assay was carried out at day 9 to measure platelet induced DC (iMDC) apoptosis, using the MEBSTAIN Apoptosis kit Direct (Coulter-Immunotech, Krefeld, Germany) generally following the instructions of the manufacturer. Briefly, cells were cultured in 24 well plates for the indicated times, transferred into FACS tubes and washed twice with PBS/0.2%BSA. Cells were fixed in 4% paraformaldehyde (in 0.1M NaH2PO4, pH 7.4) for 30 min at 4°C followed by 2 washing steps and permeabilization with 70% ethanol at -20°C for another 30 min. After washing and incubation with TdT and FITC-dUTP for 1h at 37°C, reaction was blocked with PBS/BSA. Mitomycin C treated cells served as positive control. After washing, cells were measured by flow cytometry. Data presentation and statistics Comparisons between group means were performed using Student t-test or ANOVA analysis. Data are presented as mean + standard deviation. P<0.05 was considered as statistically significant. References Reference List 1. Santoso S, Sachs UJ, Kroll H, Linder M, Ruf A, Preissner KT, Chavakis T. The junctional adhesion molecule 3 (JAM-3) on human platelets is a counterreceptor for the leukocyte integrin Mac-1. J Exp Med. 2002;196:679691. 2. Massberg S, Gawaz M, Gruner S, Schulte V, Konrad I, Zohlnhofer D, Heinzmann U, Nieswandt B. A crucial role of glycoprotein VI for platelet recruitment to the injured arterial wall in vivo. J Exp Med. 2003;197:41-49. 3. Brossart P, Grunebach F, Stuhler G, Reichardt VL, Mohle R, Kanz L, Brugger W. Generation of functional human dendritic cells from adherent peripheral blood monocytes by CD40 ligation in the absence of granulocyte-macrophage colony-stimulating factor. Blood. 1998;92:4238-4247. 4. Appel S, Mirakaj V, Bringmann A, Weck MM, Grunebach F, Brossart P. PPARgamma agonists inhibit toll-like receptor-mediated activation of dendritic cells via the MAP kinase and NF-kappaB pathways. Blood. 2005;106:3888-3894. 5. Dorfel D, Appel S, Grunebach F, Weck MM, Muller MR, Heine A, Brossart P. Processing and presentation of HLA class I and II epitopes by dendritic cells after transfection with in vitro-transcribed MUC1 RNA. Blood. 2005;105:31993205. 6. Grunebach F, Muller MR, Brossart P. New developments in dendritic cellbased vaccinations: RNA translated into clinics. Cancer Immunol Immunother. 2005;54:517-525. 7. Muller MR, Tsakou G, Grunebach F, Schmidt SM, Brossart P. Induction of chronic lymphocytic leukemia (CLL)-specific CD4- and CD8-mediated T-cell responses using RNA-transfected dendritic cells. Blood. 2004;103:1763-1769. 8. Nencioni A, Schwarzenberg K, Brauer KM, Schmidt SM, Ballestrero A, Grunebach F, Brossart P. Proteasome inhibitor bortezomib modulates TLR4induced dendritic cell activation. Blood. 2006;108:551-558. 9. Brossart P, Zobywalski A, Grunebach F, Behnke L, Stuhler G, Reichardt VL, Kanz L, Brugger W. Tumor necrosis factor alpha and CD40 ligand antagonize the inhibitory effects of interleukin 10 on T-cell stimulatory capacity of dendritic cells. Cancer Res. 2000;60:4485-4492. 10. Massberg S, Konrad I, Bultmann A, Schulz C, Munch G, Peluso M, Lorenz M, Schneider S, Besta F, Muller I, Hu B, Langer H, Kremmer E, Rudelius M, Heinzmann U, Ungerer M, Gawaz M. Soluble glycoprotein VI dimer inhibits platelet adhesion and aggregation to the injured vessel wall in vivo. FASEB J. 2004;18:397-399. 11. Langer H, May AE, Daub K, Heinzmann U, Lang P, Schumm M, Vestweber D, Massberg S, Schonberger T, Pfisterer I, Hatzopoulos AK, Gawaz M. Adherent platelets recruit and induce differentiation of murine embryonic endothelial progenitor cells to mature endothelial cells in vitro. Circ Res. 2006;98:e2-10. 12. Massberg S, Brand K, Gruner S, Page S, Muller E, Muller I, Bergmeier W, Richter T, Lorenz M, Konrad I, Nieswandt B, Gawaz M. A critical role of platelet adhesion in the initiation of atherosclerotic lesion formation. J Exp Med. 2002;196:887-896. 13. Nencioni A, Lauber K, Grunebach F, Van Parijs L, Denzlinger C, Wesselborg S, Brossart P. Cyclopentenone prostaglandins induce lymphocyte apoptosis by activating the mitochondrial apoptosis pathway independent of external death receptor signaling. J Immunol. 2003;171:5148-156. 14. Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods. 1991;139:271-279. Langer and coworkers Platelets and Dendritic Cells Figure I (please see www.ahajournals.org) Dendritic cells were generated from PBMNCs using IL-4/GM-CSF with or without CD40L. DC morphology and immunologic properties were characterized by scanning electron microscopy (upper panel) and flow cytometry (lower panel). After culture on glass disks for scanning electron microscopy, DCs showed an adhesive phenotype with numerous cellular processes. Furthermore, DCs are typically positive for CD1a, HLA-DR, CD80, CD86, CD40 and CD54 and negative for CD14. In more mature DCs, expression of CD83, CD40 and CD54 is increased, CD14 stays negative. Negative control depicts isotype matched IgG control. Figure II (please see www.ahajournals.org) To assess DC recruitment by platelets in vivo, we used intravital fluorescence microscopy. Mice were pre-treated with soluble GPVI or Fc control 12 h and 1 h prior to each experiment. Adhesion of DCS was assessed 5 min or 30 min after induction of vessel injury or when no injury was induced. The mean and standard deviation of 4-6 independent experiments is shown. * indicates p<0.005, # p<0.05 as compared to Fc control. Figure III (please see www.ahajournals.org) Expression of adhesion receptors on the surface of DCs was evaluated by FACS flow cytometry with FITC- or PE- labeled mAbs. Corresponding isotype-matched IgG served as control antibody. The analysis of 4 independent experiments (mean+standard deviation of the mean fluorescence) is shown. * indicates p<0.05, p<0.01 vs. IgG control. 1 # Langer and coworkers Platelets and Dendritic Cells Figure IV (please see www.ahajournals.org) DCs were incubated with isolated platelets for 9 days or Mitomycin C (25 µg/ml) as positive control. Induction of apoptosis was assessed by propidium iodide staining of hypodiploid apoptotic nuclei and flow cytometry. Representative dot plots of the flow cytometry experiments are shown. Suppl. film To evaluate kinetics of platelet phagocytosis by DCs, we stained platelets with cell trackerTM as described in Materials and Methods. Phagocytosis reached a maximum between d5 – d7 as evidenced by increased red signal in projection to dendritic cells. 2 Fig. I x 1000 iMDC (IL-4/GM-CSF) MDC (IL-4/GM-CSF/CD40L) 35,45 35,46 101,36 CD86 CD1a 10,00 5,98 27,52 22,24 1,76 20,09 2,30 49,38 HLA DR CD80 38,44 CD14 618,59 CD54 CD86 CD83 CD40 CD14 247,96 CD1a CD80 CD83 82,30 CD40 174,26 HLA DR CD54 Fig. II DC adhesion [cells/mm2] 1500 no injury 5 min 30 min 1000 # * 500 0 Fc GPVI Fc GPVI injury Fig. III CD29 MDC (IL-4/ GM-CSF/ CD40L) iMDC (IL-4/ GM-CSF) * CD49b * CD49d CD49e CD49f # CD11b * CD18 CD41 CD51 CD61 CD62P CD162 * control IgG 0 100 200 Mean Immunofluorescence 300 Propidium iodide positive (MIF) Fig. IV DCs DCs + Mito C DCs + Plts Plts Forward Scatter