Platelets, After Exposure to a High Shear Stress, Induce IL-10-Producing, Mature Dendritic Cells in Vitro

Platelets, after Exposure to a High Shear Stress, Induce IL-10-Producing, Mature Dendritic Cells In Vitro

This information is current as Masao Hagihara, Ayako Higuchi, Noriko Tamura, Yoko of September 23, 2021. Ueda, Kaori Hirabayashi, Yasuo Ikeda, Shunichi Kato, Shinji Sakamoto, Tomomitsu Hotta, Shunnosuke Handa and Shinya Goto J Immunol 2004; 172:5297-5303; ;

doi: 10.4049/jimmunol.172.9.5297 Downloaded from http://www.jimmunol.org/content/172/9/5297

References This article cites 45 articles, 29 of which you can access for free at: http://www.jimmunol.org/content/172/9/5297.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists by guest on September 23, 2021 • Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Platelets, after Exposure to a High Shear Stress, Induce IL-10-Producing, Mature Dendritic Cells In Vitro1

Masao Hagihara,2* Ayako Higuchi,§ Noriko Tamura,† Yoko Ueda,* Kaori Hirabayashi,‡ Yasuo Ikeda,¶ Shunichi Kato,§ Shinji Sakamoto,ʈ Tomomitsu Hotta,* Shunnosuke Handa,† and Shinya Goto†

There is evidence for immune system involvement in atherogenesis. In the present study the effect of platelets on dendritic cells (DC), an important immunologic regulator, was examined in vitro. Platelet-rich plasma, after exposure to shear stress, was added to human monocyte-derived immature DC, which were then examined for surface Ag expression, allogeneic T lymphocyte stim- ulatory activity, and cytokine production. After exposure, the number of anti-CD40 ligand (anti-CD40L) and anti-P-selectin IgG molecules bound per platelet was increased. These activated platelets induced DC maturation, as revealed by signiﬁcant up-

regulation of CD83, CD80, and CD86 Ags. The addition of platelets in the presence of IFN-␥ plus LPS signiﬁcantly enhanced IL-10 Downloaded from production from immature DC. After platelet addition, mature DC provoked a signiﬁcant proliferation of allogeneic naive T lymphocytes. These activated T cells showed lower IFN-␥ production than those stimulated by LPS- and IFN-␥-treated DC. CD40L on the platelet surface was not involved in maturation of DC, as mAb to CD40L failed to block maturation. The effect of platelets was observed even if platelets and DC were separated using large pore-sized membranes or when platelets were depleted from plasma by centrifugation. Furthermore, it was abrogated after the depletion of protein fraction. Thus, soluble protein factors excreted from activated platelets contribute to IL-10-producing DC maturation. The Journal of Immunology, 2004, 172: 5297– http://www.jimmunol.org/ 5303.

he role of cross-talk between platelets and leukocytes in IL-12 and IL-18, both of which are expressed in atherosclerosis the local inflammatory response, particularly with regard lesions, which augment IFN-␥ production (7, 8). Although their T to involvement in the chronic inflammatory aspect of ath- developmental pathways are quite complex, one DC population erosclerosis, has long been discussed (1). Several murine studies differentiates directly from monocytes (monocyte-derived DC) and revealed that immune systems were tightly involved in the course migrates into endothelial cells using specific chemokine receptors of atherogenesis (2). Among them, recruitment of monocytes was (9), which suppose its tight involvement in the plaque formation by guest on September 23, 2021 shown to be important for the formation of atherosclerosis (3). similar to monocytes or macrophages. Recent investigations have Ϫ/Ϫ Experiments using IFN-␥ receptor-deficient, Apoe mice revealed the presence of DCs at atherosclerosis-related lesions, proved that Th1-skewed immune reactions participate in the for- especially at sites prone to rupture (10, 11). mation of atherosclerosis (4). Th1 cells were dominant in early Whether activated platelets attached to an atherosclerotic lesion 3 phases of atherosclerosis (5). Dendritic cells (DC), the most po- influence plaque formation has not yet been elucidated (12). By tent APC, initiate innate and acquired immune responses (6). After releasing adhesive ligands, platelet-derived growth factor, or treatment with LPS plus cytokines, DC secrete large amounts of ␤-amyloid precursor proteins, platelets can provoke activation of APC (1, 13). Platelets express CD40 ligand (CD40L) after stim- Departments of *Hematology and Oncology, †Cardiology, ‡Dermatology, and ¤Cell ulation by thrombin (14). The role of CD40L in the progression of Transplantation and Regenerative Medicine, Tokai University School of Medicine, atheromatous plaque is of particular interest, as many recent re- Kanagawa, Japan; ¶Department of Hematology and Rheumatology, Keio University ʈ ports indicate the presence of CD40 on the surface of atheroma- School of Medicine, Tokyo, Japan; and Pharmaceutical Frontier Research Labora- tories, J.T. Incorporated, Yokohama, Kanagawa, Japan related cells, such as endothelial cells, macrophages, and smooth Received for publication March 18, 2003. Accepted for publication February muscle cells (15). As the CD40-CD40L interaction is important in 24, 2004. the progression of atherosclerosis (16) as well as plaque rupture The costs of publication of this article were defrayed in part by the payment of page mediated by increased matrix metalloprotease production from charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. macrophages and T cells (17), interruption of CD40L binding to CD40 receptors or inhibition of CD40L expression on platelets 1 This work was supported in part by Health and Labor Sciences research grants, Research on Pharmaceutical and Medical Safety; the Japanese Health Sciences Foun- may be a new therapeutic strategy to prevent progression of ath- dation, Research on Health Sciences focusing on Drug Innovation; a Grant-in-Aid for erosclerosis and atheroma rupture. The function of DC is mediated Scientific Research in Japan (15590771), a grant from the Mochida Memorial Foun- dation for Medical and Pharmaceutical Research, a grant for Advanced Medicine by CD40 ligation by CD40L (18). Recently, Hilf et al. (19) re- Supported by the Ministry of Health, Labor, and Welfare; support from Department ported that human platelets inhibited DC activation in the presence of Oriental Medicine (Tsumura) 2003, a grant from the Takeda Scientific Foundation, and a grant from the Novartis Foundation (Japan) for the Promotion of Science (all to of heat shock protein and suggested that a low concentration of S.G.). platelets was a reason for failed DC activation. 2 Address correspondence and reprint requests to Dr. Masao Hagihara, Department of We have focused on the direct interaction between platelets and Hematology and Oncology, Tokai University School of Medicine, Bohseidai, Isehara, DC. Several groups have proposed that the mechanism of in vivo Kanagawa 259-1193, Japan. E-mail address: [email protected] platelet activation might not be the same as that of platelet acti- 3 Abbreviations used in this paper: DC, dendritic cell; CBA, cytometric bead array; CD40L, CD40 ligand; MNC, mononuclear cell; UCB, umbilical cord blood; VWF, vation induced by chemical agonists such as thrombin (20). In- von Willebrand factor. deed, multiple receptor-ligand interactions, including many plasma

and matrix proteins with their corresponding platelet surface re- Effects of platelets after shearing on production of cytokines ceptors, are involved in the process of platelet activation after in- from DC teraction with subendothelial matrix exposed at sites of endothelial Immature DC were washed extensively and replated at a density of 105/ damages (21, 22). Von Willebrand factor (VWF) and its interac- 96-well, flat-bottom plates with 10% FCS containing RPMI 1640. These tion with platelet receptors, gpIb/IX and gpIIb/IIIa, are particularly DC were cultured for 48 h with or without the addition of platelets exposed important, especially at sites exposed to high shear stress (12, 20, to a high shear rate in the presence or the absence of LPS (10 ␮g/ml) plus ␥ 21, 23). Previous reports clearly demonstrated the appearance of IFN- (1000 U/ml). The culture supernatant was harvested and examined for the quantity of IL-10 and IL-12p70 by ELISA (Immunotech) immune-related proteins, such as CD40L and P-selectin, on the surface of platelets after exposure to a high shear stress (23, 24). In Effects of sheared and nonsheared platelets on the proliferation the present study we clarified the role of activated platelets after of allogeneic naive T lymphocytes in response to DC and on exposure to high shear stress on maturation and cytokine produc- cytokine production tion of monocyte-derived DC. DC are currently recognized not UCB-MNCs, depleted of CD14ϩ cells by MACS column separation, were only as immunogenic but also as tolerogenic when they are in added at 6 ϫ 104/well to 96-well, round-bottom plates as responders. Im- immature or semimature stages (25). These opposite paths are de- mature DC, DC matured by LPS and IFN-␥, and DC incubated with plate- termined by the release of proinflammatory cytokines, such as lets exposed or not exposed to a high shear stress were serially diluted after radiation (15 Gy) and added as stimulators. The cells were cultured for 6 IL-12 or IL-10. Mature DC in general, through the production of days in RPMI 1640 with 10% pooled human sera, then counted for their IL-12, are inducers of Th1 immune effectors, whereas immature or proliferative activity by the [3H]thymidine uptake test. Simultaneously, the semimature DC induce Th2 immune effectors (26). Thus, key cy- culture supernatant was harvested from the cultured wells of immature,

tokines (IFN-␥ for Th1, IL-5 for Th2) which are produced from LPS- and IFN-␥-treated DC, or DC incubated with 100 ␮l of platelets as Downloaded from ␥ naive T lymphocytes after stimulation with platelet-activated DC stimulators and examined for the content of cytokines (IFN- and IL-5) using a cytometric bead array (CBA) kit (BD Biosciences). were examined. Naive T cells after incubation with DC were cultured for another 4 days in the presence of IL-2 (20 U/ml; Shionogi), incubated with 20 ␮g/ml PMA (Sigma-Aldrich) and 10 ␮g/ml ionomycin (Sigma-Aldrich) for5hinthe Materials and Methods presence of brefeldin A (Sigma-Aldrich) and permeabilized, and the intra- Blood sample preparation and shear-induced platelet activation cellular expression of IFN-␥ and IL-4 was examined using FITC-IFN-␥ http://www.jimmunol.org/ Blood was collected from 10 healthy adult donors who abstained from the and PE-IL-4 (BD Biosciences) by flow cytometry. Also, the cultured cells 5 ␮ use of drugs known to interfere with platelet function, such as NSAIDs. were extensively washed and replated at a density of 10 /200 linthe presence of PMA/ionomycin, and their supernatant was harvested 16 h Blood was immediately treated with trisodium citrate or with the specific ␥ thrombin inhibitor PPACK to keep physiologic concentrations of divalent later to examine the cytokine (IFN- , IL-4, IL-5, and IL-10) concentrations cations. Platelet-rich plasma was separated by centrifugation at 100 ϫ g for using a CBA kit. ␮ 15 min. Platelets in 400 l of platelet-rich plasma were exposed to a high Statistical analysis shear rate of 10,800 sϪ1 for 6 min in an optically modified cone-plate viscometer (20), the shear rate previously shown to induce VWF-gpIb/IX- All numerical data are expressed as the mean Ϯ SD unless otherwise spec- mediated platelet activation (20, 23, 24). Shear-induced platelet aggrega- ified. The difference between two groups of data was tested by Student’s tion was blocked by the specific anti-gpIIb/IIIa agent tirofiban, enabling paired t test or unpaired t test. A value of p Ͻ 0.05 was considered sta- detection of CD40L and P-selectin molecules expressed on single platelets tistically significant. by guest on September 23, 2021 as we previously reported (24). Umbilical cord blood (UCB) was obtained with written informed consent after approval by the human subjects com- Results mittee of Tokai University Hospital. Platelet surface expression of CD40L and P-selectin after exposure to a hgh shear rate Effects on DC maturation The numbers of platelet-expressing CD40L and P-selectin as well Mononuclear cells (MNCs) were separated from blood samples by Ficoll- Hypaque density (1.077 g/dl) gradient. CD14ϩ cells were isolated from as the mean number of CD40L and P-selectin molecules expressed MNCs using MACS CD14 immunomagnetic beads (Miltenyi Biotec, on the platelet surface were calculated according to a previously Gladbach, Germany), followed by a MACS-positive selection column described procedure (20, 24). Quantitative analysis revealed that ϩ (Miltenyi Biotec). DC were induced from CD14 cells by culture for 7 the mean number of anti-CD40L and anti-P-selectin IgG mole- days in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) containing Ϯ Ϯ 10% FCS (Life Technologies, Gaithersburg, MD) and supplemented with cules bound per platelet increased from 17 156 and 34 148/ GM-CSF (100 ng/ml; a gift from Kirin Brewery, Maebashi, Japan), and platelet before to 388 Ϯ 331 and 1122 Ϯ 554/platelet after shear- IL-4 (10 ng/ml; a gift from Ono Pharmaceutical, Osaka, Japan) ing (both p Ͻ 0.01). These results represent the appearance of DC were then incubated with platelet samples. In all experiments plate- CD40L and P-selectin, not representing the nonspecific increase in lets were prepared from 10 ml of heparinized human blood and adjusted to nonspecific IgG binding to activated platelets, because the number a 200-␮l final volume. Addition of 100 and 10 ␮l corresponded to 1000 and 100 for the platelet to DC ratio, respectively. Positive and negative controls of isotype-matched negative control Ab against thyroglobulin for DC maturation were obtained by culture in the presence or the absence (T-␥) did not increase even after exposure to the same shear stress of LPS (100 ng/ml; Sigma-Aldrich) and IFN-␥ (1000 U/ml; Shionogi, (24). Shear-induced CD40L and P-selectin expression was medi- Osaka, Japan). The level of DC maturation was checked by double-im- ated by VWF-gpIb/IX interaction because mAb blocking VWF- munofluorescence staining using FITC-anti-CD1a mAb (DAKO, Glostrup, Denmark)/PE-anti-CD83 mAb (Immunotech, Marseilles, France), and gpIb/IX interaction, both anti-gpIb/IX and VWF, completely in- FITC-anti-CD80 mAb (Immunotech)/PE-anti-CD86 mAb (Immunotech). hibited shear-induced platelet surface translocation of those In additional experiments, anti-CD40L mAb (Ancell, Bayport, MN) was molecules (24). added together with activated platelets to test the role of CD40L on DC maturation. The efficacy of mAb was confirmed by inhibition of the mat- Effects of platelets on DC maturation uration effect by CD40L transfectant (CD40L; provided from Dr. Schultze, Dana-Farber Cancer Institute, Boston, MA). In the next experiment, plate- As shown in Fig. 1, immature DC started to express cell surface lets were placed in the chamber of a Transwell insert with high density- markers specific for matured DC, CD80, CD83, and CD86 after sized pores (BD Biosciences, San Jose, CA), which prevented direct con- being cultured with platelets exposed to a high shear rate. Further- tact between platelets and DC. Plasma, after depletion of shear-stressed more, the enhancing effect was dose dependent. However, no mat- platelets by centrifugation (10,800 sϪ1, 6 min), or plasma without any treatment was added. In the next experiment, protein components among uration was observed when DCs were incubated with platelets not these plasma samples, after depletion of shear-stressed platelets, were de- exposed to shear. Stimulation with LPS and IFN-␥ significantly pleted by ethanol precipitation, then added to immature DC. enhanced surface expression of CD83, CD80, and CD86 compared The Journal of Immunology 5299 Downloaded from

FIGURE 1. Profiles of DC maturation markers. A, DC were induced ϩ from CD14 monocytes using GM-CSF and IL-4, then cultured as imma- http://www.jimmunol.org/ ture DC (imDC), matured with LPS and IFN-␥, or added with platelets not FIGURE 2. Difference in frequencies in surface markers specific for DC exposed (shear (Ϫ) plt) or exposed to high shear stress in serial dilutions maturation and effect of anti-CD40L mAb. A, DC were generated as de- (plt/DC ratio, 1000 and 100) for 48 h. Maturation of DC was detected by scribed in Fig. 1. Differences in each marker (CD80, CD83, and CD86) CD1a, CD83, CD80, and CD86 expression by double-immunofluorescence among immature DC, LPS- and IFN-␥-treated DC, and DC incubated with staining. B, Immature DC were incubated with platelets (plt/DC ratio, platelets (platelet (plt)/DC ratio, 1000) after exposure to high shear stress 1000) or CD40L transfectant in the absence or the presence of anti-CD40L were compared. B, Anti-CD40L mAb (20 ␮g/ml) was added together with mAb (20 ␮g/ml) for 48 h. Maturation of DC was detected by CD1a, CD83, plt (plt/DC ratio, 1000). The mean Ϯ SD of 10 experiments (samples) are CD80, and CD86 expression by double-immunofluorescence staining. p Ͻ ,ء) presented, and statistically significant differences are indicated

p Ͻ 0.01). by guest on September 23, 2021 ,ءء ;0.05 with no stimulation (Figs. 1 and 2; p Ͻ 0.01). Also, a statistically significant difference ( p Ͻ 0.05) in Ag (CD83, CD80, and CD86) expression was found between DC without and with platelet addition (platelet/DC ratio, 1000). Unexpectedly, mAb to CD40L did by LPS and IFN-␥. Although the magnitude was not as prominent, not significantly neutralize the enhancing effect of platelet-induced DC after platelet addition (platelet/DC ratio, 1000) significantly Ags (Fig. 1B), whereas it prevented an up-regulation of CD83, promoted allo-naive T cell proliferation. No significant prolifera- CD80, and CD86 Ags by CD40L expressed feeder cells. As shown tion was observed when T cells were stimulated with DC cocul- in Fig. 3A, platelets could still up-regulate the expression of CD86 tured with nonsheared platelets or immature DC. Control platelets Ag, even if cell-to-cell contact was avoided using the Transwell did not show any effect of proliferation (data not shown). As insert. Furthermore, plasma, after depletion of shear-stressed plate- shown in Fig. 6, none of the DC samples provoked syngeneic T lets, could still show maturation-enhancing effects, as shown in lymphocyte proliferation. Fig. 3B. Plasma without shearing did not have such a maturation As shown in Fig. 7, T lymphocytes after incubation with LPS- effect. As shown in Fig. 4, these DC maturation effects were com- and IFN-␥-matured DC produced IFN-␥, whereas no significant pletely abrogated after depletion of the protein fraction. production was obtained when they were cocultured with platelet- matured DC or immature DC. IL-5, one of the main Th2 cytokines, Effects of platelets on IL-10 and IL-12 production was produced in low concentrations from mixed cultures with im- Untreated DC did not significantly produce either IL-10 or IL-12, mature DC, LPS and IFN-␥, or platelet-matured DC. As shown in ϩ as revealed by ODs showing almost the lowest level of detection. Fig. 8, CD4 naive T lymphocytes after incubation with platelet- As shown in Fig. 5A, addition of platelets slightly induced IL-10 matured DC exhibited much lower intracellular expression of production, whereas no IL-12 production was observed. In the IFN-␥ than those stimulated with LPS and IFN-␥/DC. However, presence of LPS and IFN-␥, platelet addition did not influence the levels of the other cytokines (IL-4, IL-5, and IL-10) were IL-12 production, but significantly enhanced IL-10 production equivalent in T cells preactivated with LPS and IFN-␥/DC and (Fig. 5B). As a result, DC exposed with LPS and IFN-␥ plus plate- those with shear-stressed platelets. lets showed comparable levels of IL-12 and IL-10 production. CD40L mAb did not affect the enhancement of IL-10 production Discussion by activated platelets (data not shown). The roles of platelet-derived molecules, including platelet-derived growth factor (1), integrins (1), P-selectin (27), and platelet-de- Effects of platelets on proliferation of allo-T lymphocytes and rived microparticles (28), in regulating local inflammatory re- cytokine production from T lymphocytes sponse have been investigated. We have demonstrated that the As shown in Fig. 6, the magnitude of allogeneic naive T cell pro- proteins known to interact with leukocytes, P-selectin and CD40L, liferation was highest when they were stimulated with DC matured were surface translocated by high shear rate conditions, even in the 5300 PLATELETS INDUCE IL-10-PRODUCING DC

FIGURE 4. Effect of protein depletion. Immature DC were left untreated or were incubated with LPS and IFN-␥, with plasma after depletion of shear-stressed platelets (plasma without plt), or with protein-deprived solution from these treated plasma (protein dep.) Maturation of DC was detected by CD1a, CD83, CD80, and CD86 expression by double-immun-

oﬂuorescence staining. Downloaded from

production was promoted by platelet addition in the absence and the presence of LPS and IFN-␥. As a result, naive T cells after stimulation with platelet-matured DC did not acquire the ability to produce the Th1 cytokine, IFN-␥, whereas they maintained IL-5 FIGURE 3. Indirect contact between DC and platelets after high shear production. Gatti et al. (34) applied ﬁxed activated platelets to http://www.jimmunol.org/ stress. A, Platelets after high shear stress were placed in the chamber of a Langerhans cells, which were activated by CD40L on platelets. In Transwell insert with high density-sized pores, which prevented direct con- contrast, our results have shown that CD40-CD40L interaction did tact between platelets (plt) and DC. Under the chamber, DC were cultured not play an important role in maturation of DC, as revealed by for 48 h, and their surface marker expression was compared before and after the addition of platelets. B, Plasma before and after depletion of mAb blocking tests. Henn et al. (31) reported that CD40L ex- shear-stressed platelets or plasma without any treatment was added to im- pressed on the platelet surface is transient and is cleaved to a sol- mature DC, and their surface marker expression was analyzed. uble form after stimulation by soluble agonists, although no soluble CD40L Ag was detected in culture supernatant of DC after

exposure to high shear stress by ELISA (data not shown). Hilf et by guest on September 23, 2021 al. (19) recently reported that thrombin-activated platelets ex- absence of any known platelet-activating agent, such as ADP or pressed heat shock protein (gp96) receptors and down-regulated thrombin. P-selectin, but not CD40L, plays a role in the physical Gp96-mediated DC maturation. They also observed an effect of interaction between flowing leukocytes and collagen-adherent platelets on DC without Gp96 maturation, but in contrast to our platelets (29). By contrast, CD40L plays functionally important results, neither a change in surface Ags nor enhanced IL-10 was roles in the maturation of DC, resulting in specific Ag expression, observed. The ratio of platelets to DC (20:1) was far less than that allo-T cell proliferation, and cytokine production (30). These re- in our experiment (100:1 to 1000:1), which might be a reason why sults suggested that platelets play important roles in the regulation of a local inflammatory response at sites exposed to high shear stress by trapping monocytes that can be transformed to DC and by inducing their maturation through stimulation of the CD40 receptor by surface-translocated CD40L. Although CD40L surface translocation can be induced by platelet activation by soluble agonists such as thrombin (14, 31), these are not likely to play important roles in vivo, because high concentrations of soluble ago- nist are not likely to exist in vivo, especially in the presence of blood flow. VWF-gpIb/IX-mediated platelet CD40L expression is more likely to occur at sites exposed to arterial blood flow conditions generating high shear rates, as animal experiments clearly demonstrate the crucial role of this interaction in arterial throm- bosis (32). Similar VWF-mediated CD40L expression is likely to occur on platelets interacting with exposed subendothelial matrix, such as collagen, because VWF has specific collagen binding sites, FIGURE 5. Production of cytokines from DC. Immature DC were 5 and the VWF-gpIb/IX interaction plays a crucial role in platelet washed extensively and replated at a density of 10 /96-well, flat-bottom thrombus formation on collagen (21, 33). plate with 10% FCS containing RPMI 1640. These DC were cultured for 48 h with or without the addition of platelets exposed to high shear in the We clearly demonstrate that immature DC become mature when absence (A) or the presence (B) of LPS (10 ␮g/ml) plus IFN-␥ (1000 U/ml). they are incubated with platelets after exposure to high shear The culture supernatant was harvested and examined for the quantities of stress. DC matured with platelets expressed all CD83, CD80, and IL-10 and IL-12p70 by ELISA. The mean Ϯ SD of 10 experiments are .(p Ͻ 0.05 ,ء) CD86 Ags at a significantly high level compared with those with- shown, and statistically significant differences are indicated out maturation. IL-12 production from immature DC was not en- Although not shown in this figure, DC with no treatment were not capable hanced by the addition of shear stress-activated platelets. IL-10 of cytokine production. The Journal of Immunology 5301

FIGURE 6. Allogenic and syngeneic T cell stimulation. Allogeneic UCB-MNCs (Allo-MLR) or syngeneic adult-MNCs (AutoMLR), depleted of CD14ϩ cells by MACS column separation, were added at 6 ϫ 104/well to a 96-well, round-bottom plate as responders. Immature DC, DC matured by LPS and IFN-␥, and DC incubated with platelets exposed or not exposed to high shear were serially diluted after radiation (15 Gy) and added Downloaded from as stimulators. The cells were cultured for 6 days in RPMI 1640 with 10% pooled human sera, then proliferative activity was determined by the [3H]thymidine uptake test. Representative data are shown as the mean Ϯ .p Ͻ 0.01 compared with immature DC ,ءء .SD of triplicate wells

discrepant results were obtained. The functionally important roles http://www.jimmunol.org/ of platelet-expressed CD40L in local inﬂammation and progres- FIGURE 8. Flow cytometric analysis of intracellular cytokine expression of atherosclerosis suggest that drugs inhibiting platelet sion (A) and measurement of cytokine production (B) in allogeneic naive T CD40L translocation might prevent atherosclerosis. However, cells after short term culture with DC and IL-2. Naive T cells were incu- contrary to our initial hypothesis, our results clearly prove that bated with DC treated as described in Fig. 6 and were further cultured for soluble factors, other than CD40L, excreted from platelets after 4 days in the presence of IL-2 (20 U/ml). A, Intracellular expression of shear stress, including many of the candidate materials released IFN-␥ and IL-4 was examined by ﬂow cytometry. B, Cytokine production from ␣ granule, such as soluble P-selectin (27), and those released was measured using a CBA kit.

from dense granule, such as ADP (35), might contribute to matu- by guest on September 23, 2021 ration of DC. To differentiate whether the soluble factor made by platelets is lipid or protein, protein fraction was depleted by simple methods of ethanol precipitation. This resulted in the total disap- The roles of macrophages and T cells in the progression of ath- pearance of maturation effects or promotion of IL-10 production in erosclerosis and the onset of atheroma rupture have previously ␥ the presence of LPS and IFN- (data not shown). Activated plate- been established (1). Many immune-related cells, such as chroni- lets make abundant lysophosphatidic acid (36Ð38), a bioactive cally activated CD4ϩ T cells, exist in atheroma (40). There are lipid mediator that can enhance the secretion of IL-10 from DC several candidate Ags that mediate specific expansion of T lym- (39). The addition of diacylglycerol pyrophosphate (8:0), which is phocytes, such as viruses or bacteria and endogenous altered Ags, a selective antagonist of lysophosphatidic acid (37), did not pro- heat shock proteins, or oxidized low density lipoproteins (41). duce any neutralization effect. Those Ags, if presented to professional APC such as DC, can guide naive T cells to Th1-type immune effectors. Actually, CD4ϩ T cell clones in human atherosclerotic lesions specifically respond to oxidized low density lipoproteins in an HLA class II-restricted manner (41). In the present study we focused on DC, which are demonstrated to be in atheroma (10, 11); their role in atherosclerosis was clarified in chronic inflammation related to infection by Chlamydia pneumoniae (42), whereas their important roles in immune regulation need to be studied (6). In the case of Chlamydia infection, host APCs, especially DC, accumulate at infected endothelial cells, take up bacterial Ags, and produce inflammatory cytokines such as IL-6, TNF-␥, or IL-12/18, which accelerate atherosclerosis (42). IL-12 and IL-18 cause Ag-specific CD4ϩ T cells to secrete IFN-␥, which is proinflammatory and proatherogenic (7, FIGURE 7. Effects of sheared platelets on the cytokine production from 8). Platelets accumulate at damaged endothelial cells and are then naive T lymphocytes. UCB-naive T cells were stimulated with immature activated through a gpIb/IX-VWF-mediated interaction, which can DC, LPS- and IFN-␥-treated DC, or DC incubated with sheared platelets result in IL-10-producing DC maturation, as shown in the present for 6 days, and the supernatant of mixed culture wells was examined for ␥ cytokine concentrations using a CBA kit. The mean Ϯ SD of 10 experi- study. Those DC may antagonize IFN- production and thereby con- ments are shown, and statistically significant differences are indicated fer atheroprotection. In vitro and in vivo studies have shown that .(p Ͻ 0.01). rIL-10 suppresses progressive generation of atherosclerosis (43 ,ءء) 5302 PLATELETS INDUCE IL-10-PRODUCING DC

Our study has methodology limitations, particularly in applying 12. Huo, Y., A. Schober, S. B. Forlow, D. F. Smith, M. C. Hyman, S. Jung, the experimental results to understand the role of platelets in reg- D. R. Littman, C. Weber, and K. Ley. 2003. Circulating activated platelets ex- acerbate atherosclerosis in mice deficient in apolipoprotein E. Nat. Med. 9:61. ulating local inflammatory responses in vivo. First, we demon- 13. De Meyer, G. R., D. M. De Cleen, S. Cooper, M. W. Knaapen, D. M. Jans, strated DC maturation in the presence of platelets after exposure to W. Martinet, A. G. Herman, H. Bult, and M. M. Kockx. 2002. Platelet phago- high shear only in a pure culture system, excluding the effects of cytosis and processing of ␤-amyloid precursor protein as a mechanism of macrophage activation in atherosclerosis. Circ. Res. 90:1197. various cellular and protein components in vivo, such as erythro- 14. Henn, V., J. R. Slupsky, M. Grafe, I. Anagnostopoulos, R. Forster, cytes. Other important contributors, such as endothelial cells, ex- G. Muller-Berghaus, and R. A. Kroczek. 1998. CD40 ligand on activated platelets tracellular matrix, and complex blood flow conditions, were not triggers an inflammatory reaction of endothelial cells. Nature 391:591. 15. Mach, F., U. Schonbeck, G. K. Sukhova, T. Bourcier, J. Y. Bonnefoy, considered in our ex vivo system. Second, we demonstrated plate- J. S. Pober, and P. Libby. 1997. Functional CD40 ligand is expressed on let surface translocation of CD40L and P-selectin only after expo- human vascular endothelial cells, smooth muscle cells, and macrophages: sure to homogeneous shear and then could not reproduce the com- implications for CD40-CD40 ligand signaling in atherosclerosis. Proc. Natl. Acad. Sci. USA 94:1931. plex shear stress to which platelets are exposed in vivo. Thus, the 16. Mach, F., U. Schonbeck, G. K. Sukhova, E. Atkinson, and P. Libby. 1998. Re- amounts of CD40L and P-selectin expression may not be physio- duction of atherosclerosis in mice by inhibition of CD40 signalling. Nature logic. Although no clinical data regarding platelet surface CD40L 394:200. are available to date, previous studies have shown P-selectin ex- 17. Schonbeck, U., F. Mach, G. K. Sukhova, E. Atkinson, E. Levesque, M. Herman, P. Graber, P. Basset, and P. Libby. 1999. Expression of stromelysin-3 in athero- pression to be induced in a similar manner when platelets pass over sclerotic lesions: regulation via CD40-CD40 ligand signaling in vitro and in vivo. the damaged surfaces, i.e., coronary intervention (44). Thus, we J. Exp. Med. 189:843. believe that platelet activation, with P-selectin and CD40L surface 18. Caux, C., C. Massacrier, B. Vanbervliet, B. Dubois, C. Van Kooten, I. Durand, and J. Banchereau. 1994. Activation of human dendritic cells through CD40 translocation, can be induced by interaction with damaged vascular cross-linking. J. Exp. Med. 180:1263. Downloaded from surfaces under high shear stress conditions in vivo. 19. Hilf, N., H. Singh-Jasuja, P. Schwarzmaier, C. Gouttefangeas, H. G. Rammensee, In conclusion, it was demonstrated that platelets, after exposure and H. Schild. 2002. Human platelets express heat shock protein receptors and regulate dendritic cell maturation. Blood 99:3676. to high shear stress, play an important role in DC maturation and 20. Goto, S., D. R. Salomon, Y. Ikeda, and Z. M. Ruggeri. 1995. Characterization of subsequent reactions, including allo-T cell proliferation and cyto- the unique mechanism mediating the shear-dependent binding of soluble von kine (IL-10) production. Also, the soluble protein factors secreted Willebrand factor to platelets. J. Biol. Chem. 270:23352. 21. Ruggeri, Z. M., J. A. Dent, and E. Saldivar. 1999. Contribution of distinct ad- from these activated platelets contributed to such DC maturation or http://www.jimmunol.org/ hesive interactions to platelet aggregation in flowing blood. Blood 94:172. IL-10 production. Recent in vivo works have shown that plate- 22. Savage, B., F. Almus-Jacobs, and Z. M. Ruggeri. 1998. Specific synergy of mul- lets were involved in increased risk of atherosclerosis (12, 45). tiple substrate-receptor interactions in platelet thrombus formation under flow. To the contrary, our experimental results suggest that platelets Cell 94:657. 23. Goto, S., K. Eto, Y. Ikeda, and S. Handa. 1999. Abciximab not RGD peptide suppress the progression of atherosclerosis through regulating inhibits von Willebrand factor-dependent platelet activation under shear. Lancet the immunological function of DC. These results show unique, 353:809. dual functions of platelets and their important roles to maintain 24. Tamura, N., M. Yoshida, N. Ichikawa, M. Handa, Y. Ikeda, T. Tanabe, S. Handa, equilibrium in the context of local inflammatory responses at S. Goto. 2002. Shear-induced von Willebrand factor-mediated platelet surface translocation of the CD40 ligand. Thromb. Res. 108:311. sites of vascular injury. 25. Lutz, M. B., and G. Schuler. 2002. Immature, semi-mature and fully mature dendritic cells: which signals induce tolerance or immunity? Trends Immunol. by guest on September 23, 2021 23:445. Acknowledgments 26. Jonuleit, H., E. Schmitt, K. Steinbrink, and A. H. Enk. 2001. Dendritic cells as a We especially thank Hideo Tsukamoto, Ph.D., at Education and Research tool to induce anergic and regulatory T cells. Trends Immunol. 22:394. Support Center, Tokai University School of Medicine, for experimental 27. McEver, R. P., K. L. Moore, and R. D. Cummings. 1995. Leukocyte trafficking mediated by selectin-carbohydrate interactions. J. Biol. Chem. 270:11025. support. 28. Forlow, S. B., R. P. McEver, and M. U. Nollert. 2000. Leukocyte-leukocyte interactions mediated by platelet microparticles under flow. Blood 95:1317. References 29. Burger, P. C., and D. D. Wagner. 2003. Platelet P-selectin facilitates atherosclerotic lesion development. Blood 101:2661. 1. Ross, R. 1993. The pathogenesis of atherosclerosis: a perspective for the 1990s. 30. Morel, Y., A. Truneh, R. W. Sweet, D. Olive, and R. T. Costello. 2001. The Nature 362:801. TNF superfamily members LIGHT and CD154 (CD40 ligand) costimulate 2. Reardon, C. A., L. Blachowicz, T. White, V. Cabana, Y. Wang, J. Lukens, induction of dendritic cell maturation and elicit specific CTL activity. J. Im- J. Bluestone, and G. S. Getz. 2001. Effect of immune deficiency on lipoproteins munol. 167:2479. and atherosclerosis in male apolipoprotein E-deficient mice. Arterioscler. Thromb. Vasc. Biol. 21:1011. 31. Henn, V., S. Steinbach, K. Buchner, P. Presek, and R. A. Kroczek. 2001. The inflammatory action of CD40 ligand (CD154) expressed on activated human 3. Smith, J. D., E. Trogan, M. Ginsberg, C. Grigaux, J. Tian, and M. Miyata. 1995. platelets is temporally limited by coexpressed CD40. Blood 98:1047. Decreased atherosclerosis in mice deficient in both macrophage colony-stimulating factor (op) and apolipoprotein E. Proc. Natl. Acad. Sci. USA 92:8264. 32. Yao, S. K., J. C. Ober, L. I. Garfinkel, Y. Hagay, N. Ezov, J. J. Ferguson, 4. Whitman, S. C., P. Ravisankar, H. Elam, and A. Daugherty. 2000. Exogenous in- H. V. Anderson, A. Panet, M. Gorecki, L. M. Buja, et al. 1994. Blockade of terferon-␥ enhances atherosclerosis in apolipoprotein EϪ/Ϫ mice. Am. J. Pathol. platelet membrane glycoprotein Ib receptors delays intracoronary thrombogen- 157:1819. esis, enhances thrombolysis, and delays coronary artery reocclusion in dogs. Cir- culation 89:2822. 5. Hansson, G. K. 2001. Immune mechanisms in atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 21:1876. 33. Goto, S., Y. Ikeda, E. Saldivar, and Z. M. Ruggeri. 1998. Distinct mechanisms of 6. Palucka, K., and J. Banchereau. 1999. Dendritic cells: a link between innate and platelet aggregation as a consequence of different shearing flow conditions. adaptive immunity. J. Clin. Immunol. 19:12. J. Clin. Invest. 101:479. 7. Gerdes, N., G. K. Sukhova, P. Libby, R. S. Reynolds, J. L. Young, and 34. Gatti, E., M. A. Velleca, B. C. Biedermann, W. Ma, J. Unternaehrer, U. Schonbeck. 2002. Expression of interleukin (IL)-18 and functional IL-18 re- M. W. Ebersold, R. Medzhitov, J. S. Pober, and I. Mellman. 2000. Large Scale culture and selective maturation of human Langerhans cells from gran- ceptor on human vascular endothelial cells, smooth muscle cells, and macro- ϩ phages: implications for atherogenesis. J. Exp. Med. 195:245. ulocyte colony-stimulating factor-mobilized CD34 progenitors. J. Immunol. 8. Uyemura, K., L. L. Demer, S. C. Castle, D. Jullien, J. A. Berliner, M. K. Gately, 164:3600. R. R. Warrier, N. Pham, A. M. Fogelman, and R. L. Modlin. 1996. Cross-regu- 35. Goto, S., N. Tamura, K. Eto, Y. Ikeda, and S. Handa. 2002. Functional signifi- latory roles of interleukin (IL)-12 and IL-10 in atherosclerosis. J. Clin. Invest. cance of adenosine 5Ј-diphosphate recetptor (P2Y12) in platelet activation initi- 97:2130. ated by binding of von Willebrand factor to platelet GP Ib␣ induced by condition 9. Cyster, J. G. 1999. Chemokines and the homing of dendritic cells to the T cell of high shear rate. Circulation 105:2531. areas of lymphoid organs. J. Exp. Med. 189:447. 36. Smyth, S. S., V. A. Sciorra, Y. J. Sigal, Z. Pamuklar, Z. Wang, Y. Xu, 10. Bobryshev, Y. V., and R. S. Lord. 1998. Mapping of vascular dendritic cells in G. D. Prestwich, and A. J. Morris. 2003. Lipid phosphate phosphatases reg- atherosclerotic arteries suggests their involvement in local immune-inflammatory ulate lysophosphatidic acid production and signaling in platelets: studies us- reactions. Cardiovasc. Res. 37:799. ing chemical inhibitors of lipid phosphate phosphatase activity. J. Biol. Chem. 11. Bobryshev, Y. V. 2000. Dendritic cells and their involvement in atherosclerosis. 278:43214. Curr. Opin. Lipidol. 11:511. 37. Rother E., R. Brandl, D. L. Baker, P. Goyal, H. Gebhard, G. Tigyi, and W. Siess. The Journal of Immunology 5303

2003. Subtype-selective antagonists of lysophosphatidic Acid receptors inhibit 42. Miyashita, N., E. Toyota, T. Sawayama, A. Matsumoto, Y. Mikami, N. Kawai, platelet activation triggered by the lipid core of atherosclerotic plaques. Circu- K. Takada, Y. Niki, and T. Matsushima. 1998. Association of chronic infection lation 108:741. of Chlamydia pneumoniae and coronary heart disease in the Japanese. Intern. 38. Aoki, J., A. Taira, Y. Takanezawa, Y. Kishi, K. Hama, T. Kishimoto, K. Mizuno, Med. 37:913. K. Saku, R. Taguchi, and H. Arai. 2002. Serum lysophosphatidic acid is produced 43. Pinderski, L. J., M. P. Fischbein, G. Subbanagounder, M. C. Fishbein, N. Kubo, through diverse phospholipase pathways. J. Biol. Chem. 277:48737. H. Cheroutre, L. K. Curtiss, J. A. Berliner, and W. A. Boisvert. 2002. Overex- 39. Panther, E., M. Idzko, S. Corinti, D. Ferrari, Y. Herouy, M. Mockenhaupt, pression of interleukin-10 by activated T lymphocytes inhibits atherosclerosis in S. Dichmann, P. Gebicke-Haerter, F. Di Virgilio, G. Girolomoni, et al. 2002. The LDL receptor-deficient mice by altering lymphocyte and macrophage pheno- influence of lysophosphatidic acid on the functions of human dendritic cells. types. Circ. Res. 90:1064. J. Immunol. 169:4129. 44. Scharf, R. E., A. Tomer, U. M. Marzec, P. S. Teirstein, Z. M. Ruggeri, and 40. Jonasson, L., J. Holm, O. Skalli, G. Bondjers, and G. K. Hansson. 1986. Regional L. A. Harker. 1992. Activation of platelets in blood perfusing angioplasty- accumulations of T cells, macrophages, and smooth muscle cells in the human damaged coronary arteries: flow cytometric detection. Arterioscler. Thromb. atherosclerotic plaque. Arteriosclerosis 6:131. 12:1475. 41. Stemme, S., B. Faber, J. Holm, O. Wiklund, J. L. Witztum, and G. K. Hansson. 45. Masseberg, S., M. Gawaz, S. Gruner, V. Schulte, I. Konrad, D. Zohlnhofer, 1995. T lymphocytes from human atherosclerotic plaques recognize oxidized low U. Heinzmann, and B. Nieswandt. 2003. A crucial role of glycoprotein VI for density lipoprotein. Proc. Natl. Acad. Sci. USA 92:3893. platelet recruitment to the injured arterial wall in vivo. J. Exp. Med. 197:41. Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021