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CXCL8 Direct Interaction with the Chemokine TSG-6 Inhibits

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CXCL8 Direct Interaction with the Chemokine TSG-6 Inhibits TSG-6 Inhibits Neutrophil Migration via Direct Interaction with the Chemokine CXCL8 This information is current as Douglas P. Dyer, Jennifer M. Thomson, Aurelie Hermant, of September 23, 2021. Thomas A. Jowitt, Tracy M. Handel, Amanda E. I. Proudfoot, Anthony J. Day and Caroline M. Milner J Immunol 2014; 192:2177-2185; Prepublished online 5 February 2014; doi: 10.4049/jimmunol.1300194 Downloaded from http://www.jimmunol.org/content/192/5/2177 Supplementary http://www.jimmunol.org/content/suppl/2014/02/05/jimmunol.130019 Material 4.DCSupplemental http://www.jimmunol.org/ References This article cites 56 articles, 27 of which you can access for free at: http://www.jimmunol.org/content/192/5/2177.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 23, 2021 • No Triage! Every submission reviewed by practicing scientists • 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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology TSG-6 Inhibits Neutrophil Migration via Direct Interaction with the Chemokine CXCL8 Douglas P. Dyer,*,† Jennifer M. Thomson,* Aurelie Hermant,‡ Thomas A. Jowitt,*,x Tracy M. Handel,† Amanda E. I. Proudfoot,‡ Anthony J. Day,*,x and Caroline M. Milner* TNF-stimulated gene/protein-6 (TSG-6) is expressed by many different cell types in response to proinflammatory cytokines and plays an important role in the protection of tissues from the damaging consequences of acute inflammation. Recently, TSG-6 was identified as being largely responsible for the beneficial effects of multipotent mesenchymal stem cells, for example in the treatment of animal models of myocardial infarction and corneal injury/allogenic transplant. The protective effect of TSG-6 is due in part to its inhibition of neutrophil migration, but the mechanisms underlying this activity remain unknown. In this study, we have shown that TSG-6 inhibits chemokine-stimulated transendothelial migration of neutrophils via a direct interaction (KD, ∼25 nM) between TSG-6 and the glycosaminoglycan binding site of CXCL8, which antagonizes the association of CXCL8 with heparin. Further- Downloaded from more, we found that TSG-6 impairs the binding of CXCL8 to cell surface glycosaminoglycans and the transport of CXCL8 across an endothelial cell monolayer. In vivo this could limit the formation of haptotactic gradients on endothelial heparan sulfate proteoglycans and, hence, integrin-mediated tight adhesion and migration. We further observed that TSG-6 suppresses CXCL8-mediated chemotaxis of neutrophils; this lower potency effect might be important at sites where there is high local expression of TSG-6. Thus, we have identified TSG-6 as a CXCL8-binding protein, making it, to our knowledge, the first soluble mammalian chemokine-binding protein to be described to date. We have also revealed a potential mechanism whereby TSG-6 http://www.jimmunol.org/ mediates its anti-inflammatory and protective effects. This could inform the development of new treatments for inflammation in the context of disease or following transplantation. The Journal of Immunology, 2014, 192: 2177–2185. eutrophils play an essential role in the inflammatory in neutrophil activation and transmigration (2). It is produced in response to infection and injury, which is dependent response to proinflammatory stimuli, for example, by endothelial N on their rapid recruitment from the vasculature. This oc- cells (3), monocytes/macrophages, and mast cells (4), and it in- curs via a multistep process that involves sequential tethering teracts with glycosaminoglycans (GAGs), such as heparan sulfate and rolling, activation by chemoattractants (e.g., chemokines), (HS) and chondroitin sulfate (5), on the vascular endothelium (6–8). by guest on September 23, 2021 tight adhesion, and transendothelial migration (1). However, the GAG binding mediates presentation of CXCL8 to its G protein– destructive potential of neutrophils requires that their extravasa- coupled receptors on neutrophils (i.e., CXCR1 and CXCR2) (9), tion be tightly regulated, otherwise tissue damage occurs, as seen enables chemokine oligomerization (where the monomeric and in conditions such as ischemia/reperfusion injury, rheumatoid ar- dimeric forms of CXCL8 have differential functions in vivo) (10), thritis, and asthma. The chemokine CXCL8 (IL-8) plays a key role and generates haptotactic gradients that guide neutrophil re- cruitment (11, 12). There is evidence that tissue-specific variations in the content, concentration, and distribution of GAGs can mod- *Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United ulate the CXCL8 monomer/dimer equilibrium and, thereby, regu- † Kingdom; Skaggs School of Pharmacy and Pharmaceutical Sciences, University of late neutrophil extravasation (13). California, San Diego, La Jolla, CA 92093; ‡Merck Serono Geneva Research Center, 1202 Geneva, Switzerland; and xWellcome Trust Centre for Cell-Matrix Research, TNF-stimulated gene/protein-6 (TSG-6), an ∼35-kDa secreted Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United glycoprotein (14), is expressed at sites of inflammation and in- Kingdom jury; for example, it is abundant in the synovial fluids of patients Received for publication January 18, 2013. Accepted for publication January 5, 2014. with inflammatory arthritis (15), and serum concentrations of TSG-6 D.P.D. was supported by a Biotechnology and Biological Sciences Research Council correlate with the progression of murine proteoglycan-induced Collaborative Award in Science and Engineering Doctoral Training Award in con- junction with Merck Serono (Geneva, Switzerland). J.M.T. was the recipient of arthritis (16). There is growing evidence that TSG-6 acts to in- a Biotechnology and Biological Sciences Research Council Collaborative Award in hibit the extravasation of neutrophils in vivo (17–23), affecting both Science and Engineering Doctoral Training in conjunction with Eden Biodesign their rolling and transendothelial migration (17, 19). For example, (Liverpool, U.K.). This work was also supported by Arthritis Research U.K. Grants 16539 and 18472 (to A.J.D. and C.M.M.). TSG-6–deficient mice develop much more severe proteoglycan- Address correspondence to Dr. Caroline M. Milner and Prof. Anthony J. Day, Faculty induced arthritis than do controls, with elevated neutrophil infil- of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, tration into the paw joints (20). Furthermore, secretion of TSG-6 Manchester M13 9PT, U.K. E-mail addresses: [email protected] by multipotent mesenchymal stromal cells (MSCs) has been shown (C.M.M.) and [email protected] (A.J.D.) to reduce inflammatory damage in rodent models of myocardial The online version of this article contains supplemental material. infarction (21), corneal wounding (22), and corneal transplanta- Abbreviations used in this article: b-, biotinylated; CKBP, chemokine-binding pro- tein; GAG, glycosaminoglycan; HA, hyaluronan; HS, heparan sulfate; HSPG, hep- tion (24). These protective properties of TSG-6 and the fact that it aran sulfate proteoglycan; MSC, mesenchymal stromal cell; rh, recombinant human; is produced primarily in the context of inflammation suggest that SPR, surface plasmon resonance; TSG-6, TNF-stimulated gene/protein-6; WT, wild- it forms part of an endogenous pathway to limit tissue damage type. during acute inflammatory episodes: TSG-6 is released from the Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 secretory granules of neutrophils (25) and mast cells (16) as well www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300194 2178 TSG-6 INHIBITS CXCL8-MEDIATED NEUTROPHIL MIGRATION as being expressed by macrophages (14, 26) and a wide variety of neutrophils) with CXCL8, without or with TSG-6, in the lower chamber. stromal cell types (18). Although it is evident that the inhibitory Migrated neutrophils were recovered from the media below each mem- 3 effect of TSG-6 on neutrophil transendothelial migration (17, 19) brane by centrifugation (10 min, 400 g) and counted. The transport of CXCL8 across endothelial cell monolayers was in- contributes to its protective effects in inflammatory models, the vestigated using a modification of the method described in Pruenster et al. molecular basis of this activity has not yet been determined. (37). Biotinylated (b-)CXCL8 (prepared as in Ref. 38 and shown to signal TSG-6 consists mainly of contiguous Link and CUB modules through CXCR2 with similar efficiency to unmodified CXCL8) was added (14, 18). It interacts with protein ligands, including inter-a-in- (3 nM) below HUVEC monolayers, in the absence/presence of Link_TSG6 (5-fold molar excess), and Transwells were incubated at 37˚C for 2 h. hibitor (17, 27) and thrombospondin-1 (28), as well as with various Media were collected from the upper and lower
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