Mature Dendritic Cells Express Functional Thrombin Receptors Triggering Chemotaxis and CCL18/Pulmonary and Activation-Regulated Chemokine Induction This information is current as of September 28, 2021. Xuehua Li, Tatiana Syrovets, Svetlana Paskas, Yves Laumonnier and Thomas Simmet J Immunol 2008; 181:1215-1223; ; doi: 10.4049/jimmunol.181.2.1215 http://www.jimmunol.org/content/181/2/1215 Downloaded from References This article cites 55 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/181/2/1215.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 • Fast Publication! 4 weeks from acceptance to publication by guest on September 28, 2021 *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 © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Mature Dendritic Cells Express Functional Thrombin Receptors Triggering Chemotaxis and CCL18/Pulmonary and Activation-Regulated Chemokine Induction1 Xuehua Li,2 Tatiana Syrovets,2 Svetlana Paskas, Yves Laumonnier, and Thomas Simmet3 Protease-activated receptors (PARs) are a family of G protein-coupled receptors that are activated by serine protease-mediated proteolytic cleavage of their extracellular domain. We have previously characterized the expression and function of PARs in human monocytes and macrophages, yet information about PARs in dendritic cells (DC) is scarce. Monocyte-derived immature DC do not express PARs. Upon maturation with LPS, but not with TNF-␣ or CD40 ligand, DC express PAR1 and PAR3, but not PAR2 or PAR4. Stimulation of DC with the serine protease thrombin or PAR1-activating peptide elicits actin polymerization and concentration-dependent chemotactic responses in LPS-, but not in TNF-␣-matured DC. The thrombin-induced migration is a Downloaded from true chemotaxis with only negligible chemokinesis. Stimulation of PARs with thrombin or the respective receptor-activating peptides activates ERK1/2 and Rho kinase as well as subsequent phosphorylation of the regulatory myosin L chain 2. The ERK1/2- and Rho kinase 1-mediated phosphorylation of myosin L chain 2 was indispensable for the PAR-mediated chemotaxis as shown by pharmaco- logical inhibitors. Additionally, thrombin stimulated the Rho-dependent release of the CC chemokine CCL18/pulmonary and activation- regulated chemokine, which induces chemotaxis of lymphocytes and immature DC as well as fibroblast proliferation. The colocalization ؉ of CD83 DC with CCL18 in human atherosclerotic plaques revealed by immunofluorescence microscopy combined with the presence http://www.jimmunol.org/ of functionally active thrombin receptors on mature DC point to a previously unrecognized functional role of thrombin in DC biology. The thrombin-induced stimulation of mature DC may be of particular relevance in atherosclerotic lesions, which harbor all components of this novel mechanism. The Journal of Immunology, 2008, 181: 1215–1223. hrombin is generated at sites of vascular injury and during Dendritic cells (DC) are essential for the induction of the adaptive inflammation (1, 2). When generated in close proximity to immune response (6, 7). On the basis of their phenotype and their T its cellular receptors (3, 4), thrombin triggers not only in- ability to prime naive T cells, they are commonly subdivided into im- creased fibrin deposition, but also activation of cells expressing pro- mature and mature DC (mDC) (8). Ags, pathogens, LPS, TNF-␣, and tease-activated receptors (PARs),4 which include endothelial and vas- other factors induce functional changes culminating in the transition by guest on September 28, 2021 cular smooth muscle cells as well as various blood cells (1, 2, 5). from the Ag-capturing immature to the Ag-presenting mDC (7, 8). Binding of thrombin to its respective PARs leads to cleavage of Small numbers of DC are localized in the intima of apparently the N-terminal exodomain, unmasking a new N terminus that acts normal, nondiseased arteries (9–11). Interestingly, atherosclerosis- as tethered ligand. Receptor-activating peptides (AP) mimic the prone regions of the murine vessel wall contain abundant DC, newly formed N-terminal sequence of the receptor and activate suggesting a role of these cells in the process of atherogenesis (12). PARs independent of protease activity and receptor cleavage (1, In atherosclerotic arteries, the number of DC further increases, 2). Among the four members of PARs, PAR1, PAR3, and PAR4 indicating that some of them do not migrate to lymph nodes, but are activated by thrombin, whereas PAR2 is activated, by trypsin, remain in the vessel wall, where they could activate T cells directly factor VIIa or Xa, as well as mast cell tryptase (1, 2). within the intima (10, 11, 13). DC-T cell interactions were shown to be frequent in rupture-prone plaque regions, suggesting that this could contribute to plaque destabilization (14). LPS and endoge- nous ligands such as minimally modified low density lipoprotein Institute of Pharmacology of Natural Products and Clinical Pharmacology, Ulm Uni- activate TLR4, whereas genetic deficiency of TLR4 or MyD88 versity, Ulm, Germany involved in TLR signaling reduces atherosclerosis as well as the Received for publication November 7, 2007. Accepted for publication May 13, 2008. Ϫ Ϫ plaque areas in apoE / mice (15, 16). Further evidence for a link The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance between adaptive immune responses and atherogenesis comes from with 18 U.S.C. Section 1734 solely to indicate this fact. the enhanced TLR expression in murine and human atherosclerotic 1 This work was supported by grants from the Deutsche Forschungsgemeinschaft (to lesions. Although pivotal roles for DC and TLR signaling have been T.S and Th.S.). claimed in the development of atherosclerosis (13, 17), little is known 2 X.L. and Ta.S. contributed equally to this work. about the induction of DC stimulation and accumulation. 3 Address correspondence and reprint requests to Dr. Thomas Simmet, Institute of Immature DC (iDC) express only small amounts of PAR1 and Pharmacology of Natural Products and Clinical Pharmacology, Ulm University, PAR3 mRNA, but no detectable PAR proteins on their surface (5). On Helmholtzstrasse 20, D-89081 Ulm, Germany. E-mail address: thomas.simmet@ uni-ulm.de the other hand, a subset of DC may express PAR2. Thus, an endog- 4 Abbreviations used in this paper: PAR, protease-activated receptor; DC, dendritic enous serine protease acting on PAR2 seems to stimulate DC devel- cell; iDC, immature DC; mDC, mature DC; ROCK, Rho kinase; MLC2, myosin L opment from murine bone marrow progenitor cells (18). So far, little chain 2; AP, activating peptide; PARC, pulmonary and activation-regulated chemo- is known about the expression and function of PARs in DC. kine; CD40L, CD40 ligand. In this study, we report expression and function of thrombin re- Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 ceptors in DC matured by different stimuli. We demonstrate for the www.jimmunol.org 1216 ACTIVATION OF mDC BY THROMBIN first time that in LPS-matured DC thrombin triggers chemotactic re- sponses as well as an increased release of the CC chemokine CCL18, also known as pulmonary and activation-regulated chemokine (PARC), for which no murine homolog exists (19). Our data provide insight into novel aspects of DC function linking the coagulation sys- tem to the adaptive immune response. The findings might be of par- ticular significance in atherosclerotic lesions, where all necessary components of this new activation mechanism are colocalized. Sup- port for such a mechanism comes from immunofluorescence micros- copy of plaque material from human carotid endarterectomies show- ing colocalization of matured CD83ϩ DC, thrombin receptors, and CCL18. Materials and Methods Differentiation of DC DC were differentiated from human monocytes obtained from buffy coats with 1000 U/ml GM-CSF (Berlex Laboratories) and 25 ng/ml IL-4 (Pierce) as described previously (5). The differentiation was confirmed by flow cytometric analysis of CD14 (mouse anti-CD14; Dianova), CD1a, HLA- Downloaded from DR, CD83, and CD86 (BD Biosciences). As secondary Abs served PE- Ј conjugated donkey anti-mouse and donkey anti-rabbit F(ab )2 (Dianova). iDC were used after 6 days. Maturation was induced by incubation for an additional 48 h using either 0.5 ␮g/ml LPS (Escherichia coli serotype 055:B5; Sigma-Aldrich), 50 ng/ml TNF-␣ (PeproTech), or 0.5 ␮g/ml CD40 ligand (CD40L; PJK) (20) Cells were kept in FCS-free RPMI 1640 for 6 h before each experiment. We excluded platelet contamination by analysis of CD41 that remained undetectable both by RT-PCR and flow http://www.jimmunol.org/ cytometry (data not shown). mRNA detection Total RNA was extracted using