The CC Chemokine MCP-1 Stimulates Surface Expression of CX3CR1 and Enhances the Adhesion of Monocytes to Fractalkine/CX3CL1 via p38 MAPK This information is current as of September 25, 2021. Simone R. Green, Ki Hoon Han, Yiming Chen, Felicidad Almazan, Israel F. Charo, Yury I. Miller and Oswald Quehenberger J Immunol 2006; 176:7412-7420; ; doi: 10.4049/jimmunol.176.12.7412 Downloaded from http://www.jimmunol.org/content/176/12/7412 References This article cites 52 articles, 29 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/176/12/7412.full#ref-list-1 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 25, 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 © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology The CC Chemokine MCP-1 Stimulates Surface Expression of CX3CR1 and Enhances the Adhesion of Monocytes to Fractalkine/CX3CL1 via p38 MAPK1 Simone R. Green,2* Ki Hoon Han,2,3* Yiming Chen,* Felicidad Almazan,* Israel F. Charo,† Yury I. Miller,* and Oswald Quehenberger4* The membrane-anchored form of CX3CL1 has been proposed as a novel adhesion protein for leukocytes. This functional property of CX3CL1 is mediated through CX3CR1, a chemokine receptor expressed predominantly on circulating white blood cells. Thus far, it is still uncertain at what stage of the trafficking process CX3CR1 becomes importantly involved and how the CX3CR1- dependent adhesion of leukocytes is regulated during inflammation. The objective of this study was to examine the functional effects of chemokine stimulation on CX3CR1-mediated adhesion of human monocytes. Consistent with previous reports, our data Downloaded from indicate that the activity of CX3CR1 on resting monocytes is sufficient to mediate cell adhesion to CX3CL1. However, the basal, nonstimulated adhesion activity is low, and we hypothesized that like the integrins, CX3CR1 may require a preceding activation step to trigger firm leukocyte adhesion. Compatible with this hypothesis, stimulation of monocytes with MCP-1 significantly increased their adhesion to immobilized CX3CL1, under both static and physiological flow conditions. The increase of the adhesion activity was mediated through CCR2-dependent signaling and obligatory activation of the p38 MAPK pathway. Stimulation with MCP-1 also induced a rapid increase of CX3CR1 protein on the cell surface. Inhibition of the p38 MAPK pathway prevented this http://www.jimmunol.org/ increase of CX3CR1 surface expression and blunted the effect of MCP-1 on cell adhesion, indicating a causal link between receptor surface density and adhesion activity. Together, our data suggest that a chemokine signal is required for firm CX3CR1-dependent adhesion and demonstrate that CCR2 is an important regulator of CX3CL1-dependent leukocyte adhesion. The Journal of Immunology, 2006, 176: 7412–7420. he interaction of leukocytes with the vascular wall and the adhesion of leukocytes (8, 9). Chemokine signals also stimulate subsequent migration across endothelial junctions are the subsequent diapedesis of the adherent cells through the vascu- central to both immune surveillance and host defense. The lar wall and their migration to sites of inflammation. T by guest on September 25, 2021 molecular control of these trafficking processes is exquisitely reg- Recently, a novel integrin-independent pathway for leukocyte ulated and, in accordance with the classic multistep model of leu- capture has been described that involves the chemokine fractalkine kocyte recruitment, requires the coordinated actions of adhesion (or CX3CL15 in the standard chemokine nomenclature) (5). molecules and chemotactic factors (1–3). At least three distinct CX3CL1, the sole member of the CX3C family of chemokines families of adhesion molecules orchestrate leukocyte adhesion in- established on the basis of the arrangement of N-terminal con- cluding selectins, integrins, and members of the Ig superfamily (4). served cysteine residues, is structurally distinct from other chemo- Each is involved at different stages of leukocyte emigration, and kines and is encoded as a type I transmembrane protein containing the synchronization of their action is essential for functional im- multiple domains (10, 11). The chemokine domain is anchored to mune response. Immune surveillance under quiescent conditions the plasma membrane through an extended mucin-rich stalk, fused entails the continuous interaction of the circulating leukocytes with to a transmembrane helix and an intracellular domain (12, 13). the endothelium. However, in the absence of chemokines this in- CX3CL1 is highly expressed on vascular endothelial cells but only teraction is reversible and is mediated primarily by selectins (5–7). upon activation with inflammatory cytokines (10). Other cellular In the inflammatory event, locally produced chemokines lead to sources for CX3CL1 include neurons (14), epithelial cells (15, 16), the activation of integrins, which then allows shear-resistant firm smooth muscle cells (17), dendritic cells (18), and macrophages (19). The extended mucin-rich stalk of the endothelial-bound *Department of Medicine, University of California, San Diego, La Jolla, California 92093; and †Gladstone Institute of Cardiovascular Disease, San Francisco, California CX3CL1 allows the chemokine domain to be effectively presented 94141 to circulating leukocytes. Based on in vitro studies, the membrane- Received for publication September 27, 2005. Accepted for publication March bound configuration of CX3CL1 induces cell adhesion through 30, 2006. interaction with its cognate receptor CX3CR1, expressed on leu- The costs of publication of this article were defrayed in part by the payment of page kocytes including monocytes, T cells, and NK cells (20, 21). Like charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. other chemokine receptors, CX3CR1 is a seven-transmembrane 1 This work was supported by National Institutes of Health Grant HL56989 (La Jolla domain G protein-coupled receptor, but the adhesion does not re- Specialized Center of Research in Molecular Medicine and Atherosclerosis). quire transmembrane signaling and is independent of calcium 2 S.R.G. and K.H.H. contributed equally to this study. fluxes (22, 23). In addition to its function as an adhesion molecule, 3 Current address: College of Medicine, University of Ulsan, Seoul, South Korea. 4 Address correspondence and reprint requests to Dr. Oswald Quehenberger, Depart- 5 Abbreviations used in this paper: CX3CL1, fractalkine according to the new che- ment of Medicine, 0682, University of California, San Diego, 9500 Gilman Drive, La mokine nomenclature; his-CX3CL1, histidine-tagged secreted form of CX3CL1; Jolla, CA 92093-0682. E-mail address: [email protected] PTX, pertussis toxin. Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 7413 CX3CL1 can be released from the cell surface through either stim- 100%, and any changes in the mean fluorescence induced by the various ulated or constitutive proteolytic cleavage to produce a soluble treatments were normalized to that baseline. Flow cytometry was per- molecule (24, 25). In this form, CX3CL1 acts like a conventional formed using a FACSCalibur instrument with CellQuest software (BD Bioscience). chemokine exerting potent chemotactic activity (10, 11). In this study, we examined the mechanism by which the Static adhesion assay CX3CR1-dependent adhesion of monocytes is regulated under Wells of a 96-well plate (Corning) were coated overnight at 4°C with conditions of inflammation in the presence of chemokines. We ascitic fluid containing monoclonal anti-polyhistidine Ab (1/1000 dilution found that MCP-1 markedly enhanced the CX3CR1 expression on in PBS). After two washes with PBS, the wells were blocked with adhesion freshly isolated human peripheral blood monocytes or monocytic buffer (RPMI 1640, 1 mg/ml BSA, 10 mM HEPES, pH 7.4) for1hatroom temperature. The buffer was removed, the his-CX3CL1 was added in 50 ␮l cell lines and stimulated the adhesion of these cells to immobilized of adhesion buffer at a final concentration of 30 nM and incubated for 1 h CX3CL1. In contrast to the basal adhesion, the chemokine-stim- at 37°C. The wells were washed three times, and static adhesion assays ulated adhesion activity required the activation of CCR2, was were performed essentially as described (20). blocked by pertussis toxin (PTX) and involved MAPK-mediated Before the adhesion assay, THP-1 cells or freshly isolated human and murine monocytes were washed once with PBS, resuspended in adhesion signaling events. From our data, we concluded that chemokines buffer at 1 ϫ 106 cells/ml, and stimulated with various concentrations