A Disintegrin and Metalloproteinase 10-Mediated Cleavage and Shedding Regulates the Cell Surface Expression of CXC Chemokine Ligand 16 This information is current as of October 2, 2021. Peter J. Gough, Kyle J. Garton, Paul T. Wille, Marcin Rychlewski, Peter J. Dempsey and Elaine W. Raines J Immunol 2004; 172:3678-3685; ; doi: 10.4049/jimmunol.172.6.3678 http://www.jimmunol.org/content/172/6/3678 Downloaded from References This article cites 35 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/172/6/3678.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 October 2, 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 © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology A Disintegrin and Metalloproteinase 10-Mediated Cleavage and Shedding Regulates the Cell Surface Expression of CXC Chemokine Ligand 16 Peter J. Gough,2* Kyle J. Garton,* Paul T. Wille,* Marcin Rychlewski,* Peter J. Dempsey,† and Elaine W. Raines* CXC chemokine ligand (CXCL)16 and scavenger receptor for phosphatidylserine and oxidized low-density lipoprotein were independently identified as a chemokine and a scavenger receptor, respectively, but have since been shown to be identical. CXCL16 is synthesized as a transmembrane protein with its chemokine domain at the end of a mucin-rich stalk. When expressed at the cell surface, CXCL16 functions as a scavenger receptor, binding and internalizing oxidized low-density lipoprotein and ,bacteria. As a soluble form, CXCL16 is a chemoattractant for activated CD4؉ and CD8؉ T cells through binding its receptor CXCR6. In this study, we examined the mechanisms that regulate the conversion between these two functionally distinct forms Downloaded from of CXCL16. We demonstrate that murine CXCL16 is synthesized as an intracellular precursor that is rapidly transported to the cell surface where it undergoes metalloproteinase-dependent cleavage, causing the release of a fragment that constitutes the majority of the CXCL16 extracellular domain. Using a novel retroviral system for the generation of short interfering RNAs, we show that knockdown of a disintegrin and metalloproteinase (ADAM) family protease ADAM10 decreases this constitutive shed- ding of CXCL16. Furthermore, we show that overexpression of ADAM10 increases CXCL16 shedding, whereas overexpression of a dominant-negative form of ADAM10 lowers shedding of CXCL16 in a similar manner to short interfering RNAs. Through http://www.jimmunol.org/ the modulation of ADAM10 function, we demonstrate that ADAM10-mediated constitutive shedding is a key regulator of CXCL16 cell surface expression. The identification of ADAM10 as a major protease responsible for the conversion of CXCL16 from a membrane-bound scavenger receptor to a soluble chemoattractant will provide new information for understanding the physio- logical function of this molecule. The Journal of Immunology, 2004, 172: 3678–3685. he scavenger receptor for phosphatidylserine and oxi- PSOX is expressed by macrophages in vitro and in atherosclerotic dized low-density lipoprotein (SR-PSOX)3 and CXC che- lesions in vivo (1, 5), suggesting that SR-PSOX activity may be mokine ligand (CXCL)16 were independently identified involved in the massive accumulation of cellular cholesterol dur- T by guest on October 2, 2021 as a scavenger receptor and chemokine, respectively, but have ing the generation of macrophage foam cells associated with ath- since been shown to be identical (1–3). SR-PSOX was identified erosclerotic lesion development (6). Similar to other members of through an expression cloning strategy designed to identify recep- the scavenger receptor family, SR-PSOX has recently been shown tors that could mediate cell adhesion to phosphatidylserine-coated to mediate the uptake of Gram-positive and -negative bacteria surfaces (1). It was subsequently shown to bind and internalize when expressed by macrophages and dendritic cells, indicating oxidized low-density lipoprotein (OxLDL) (1), making it a mem- that this receptor may play a role in innate immunity and initiation ber of the structurally diverse scavenger receptor family of cell of the acquired immune response (7, 8). surface receptors that is defined by the ability to recognize mod- CXCL16 was independently identified by two groups as the li- ified low-density lipoprotein (4). Further analysis showed that SR- gand for the orphan G-protein-coupled chemokine receptor Bonzo/ CXCR6 (2, 3). CXCL16 is the second transmembrane chemokine identified to date, and bears significant structural homology to frac- *Department of Pathology, University of Washington, Harborview Medical Center, talkine/CX3C chemokine ligand (CX3CL)1 (9, 10). A combination Seattle, WA 98104; and †Pacific Northwest Research Institute, Seattle, WA 98122 of immunohistochemistry and FACS analysis showed that Received for publication October 8, 2003. Accepted for publication January 13, 2003. CXCL16 is selectively expressed by APCs, including DCs, mac- The costs of publication of this article were defrayed in part by the payment of page rophages, and B cells (2, 3). When expressed by macrophages, charges. This article must therefore be hereby marked advertisement in accordance soluble CXCL16 is released into the medium and has chemoat- with 18 U.S.C. Section 1734 solely to indicate this fact. tractant activity that is mediated solely through the CXCR6 recep- 1 This work was supported by National Institutes of Health Grants HL18645 and tor. CXCR6 is expressed by many cell types including naive CD8ϩ HL67267 (to E.W.R.) and DK59778 and DK63363 (to P.J.D.), a postdoctoral fel- T cells, NK T cells, and a subset of memory CD4ϩ T cells, al- lowship from the American Heart Association (to P.J.G.), and the Paul G. Allen ϩ ϩ Foundation for Medical Research (to K.J.G.). though only activated CD4 and CD8 T cells appear to migrate 2 Address correspondence and reprint requests to Dr. Peter J. Gough at the current ad- strongly to the soluble chemokine (2, 11). Furthermore, CXCL16- ϩ dress: Atherosclerosis Department, Medicines Research Centre, GlaxoSmithKline, Gun- positive cells in the spleen were seen in close opposition to CD8 nels Wood Road, Stevenage SG1 2NY, U.K. E-mail address: [email protected] T cells, suggesting that, similar to CX3CL1, CXCL16 may act as 3 Abbreviations used in this paper: SR-PSOX, scavenger receptor for phosphatidyl- an intercellular adhesion molecule when expressed on the cell sur- serine and oxidized low-density lipoprotein; CXCL, CXC chemokine ligand; OxLDL, face (2, 12, 13). oxidized low-density lipoprotein; CX3CL, CX3C chemokine ligand; ADAM, a dis- integrin and metalloproteinase; ThioM␾, thioglycolate-elicited peritoneal macro- Given the potential distinct functional activities of membrane- phage; BMDM, bone marrow-derived macrophage; m, murine; HA, hemagglutinin; bound CXCL16 as a scavenger receptor, and soluble CXCL16 as siRNA, small interfering RNA; RIPA, radioimmunoprecipitation assay; SIN, self in- activating; LPA, lysophosphatidic acid; GPCR, G-protein-coupled receptor; EGF, a chemokine, the mechanisms that regulate the conversion between epidermal growth factor. these two forms would appear to be important for determining the Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 The Journal of Immunology 3679 role played by this molecule in vivo. We have previously shown transduction, 2 ϫ 105 cells were plated per well of a six-well plate 24 h before a 10-h incubation with retroviral supernatants containing 4 ␮g/ml Polybrene that membrane-bound CX3CL1 can be proteolytically cleaved from the cell surface by at least two distinct metalloproteinases (Sigma-Aldrich). Transduction efficiency was enhanced by centrifuging plates at 1700 ϫ g for2hat37°C at the beginning of the 10-h incubation period. (14). We identified a disintegrin and metalloproteinase (ADAM) After transduction, retroviral supernatant was replaced with fresh medium, and family member ADAM17 as the protease responsible for stimu- cells were allowed to recover for at least 48 h before use in subsequent ex- lated shedding of CX3CL1 (14, 15), whereas constitutive release of periments. For puromycin selection, cells were cultured in the presence of 15 ␮g/ml puromycin for 48 h following recovery from transduction. CX3CL1 has subsequently been shown to be mediated by ADAM10 (16). Given the structural similarity between CX3CL1 and CXCL16, we have examined whether similar proteolytic CXCL16 shedding assays mechanisms are responsible for generating soluble CXCL16. In Macrophages (RAW-264, BMDM, and ThioM␾) were plated at a density this study, we demonstrate that CXCL16 is synthesized as an in- of 2.5 ϫ 106 cells per 60-mm dish, and dermal fibroblasts at a density of tracellular precursor that is rapidly