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

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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 recruitment (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 conjunction 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 protein; 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 chambers, followed by GAGs, for example, heparin, HS, chondroitin-4-sulfate, dermatan incubation with 103 PBS for 3 min to recover any CXCL8 bound to cell sulfate, and hyaluronan (HA) (27, 29, 30). All of these ligands bind surface GAGs; cells were then lysed with RIPA buffer (10 mM Tris [pH to the Link module domain of TSG-6, which is also responsible 7.4], 150 mM NaCl, 1% (v/v) Triton X-100, 1% (v/v) sodium deoxy- for the inhibition of neutrophil migration (17, 19); however, it is cholate, 0.1% (w/v) SDS, 5 mM EDTA). All samples were subject to SDS- PAGE and CXCL8 was detected and quantified by Western blot analysis not clear whether any of these interactions contributes to the anti– with streptavidin conjugated to IRDye 800CW (LI-COR Biosciences) using migratory activity of TSG-6 (17). an Odyssey imaging system (LI-COR Biosciences). In this study, we have demonstrated that TSG-6 acts to inhibit Analysis of the TSG-6 interaction by surface plasmon CXCL8-induced transendothelial migration of human neutrophils resonance via a direct interaction between the TSG-6 Link module and CXCL8, which antagonizes the binding of CXCL8 to heparin/HS. Surface plasmon resonance (SPR) analysis was carried out using a Biacore

Our data indicate that TSG-6 can impair the transport of CXCL8 3000 (GE Healthcare), where ligands (i.e., Link_TSG6, rhTSG-6, or Downloaded from ∼ across the endothelium and its presentation by cell surface GAGs. CXCL8) were immobilized onto a C1 Biacore chip ( 500 response units) as follows. The flow rate was set at 40 ml/min and the surface was equili- At high concentrations TSG-6 was also seen to inhibit neutrophil brated with SPR running buffer, that is, SPR 6.0 (10 mM NaOAc, 150 mM chemotaxis. To our knowledge, this work identifies TSG-6 as the NaCl, 0.05% [v/v] Tween 20 [pH 6]) or SPR 7.2 (10 mM HEPES, 150 mM first soluble, mammalian chemokine-binding protein and reveals NaCl, 0.05% [v/v] Tween 20 [pH 7.2]), for 1 h (39). Paired cells (on the m a molecular mechanism for its tissue-protective effects during in- SPR chip) were then activated by injecting 70 l of a 1:1 mixture of 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.1 M N-hydrox- flammation. ysuccinimide (39, 40). Protein ligands (rhTSG-6, Link_TSG6, or CXCL8 http://www.jimmunol.org/ at 20 mg/ml) were immobilized by passing them over one of the activated cells, using buffer conditions found to be optimal in initial scouting Materials and Methods experiments (rhTSG-6 in 10 mM sodium acetate [pH 6], Link_TSG6 and Protein and GAG preparation CXCL8in10mMHEPES[pH7.4]),until the signal increased by 500 response units. Any remaining active sites in the reference and ligand-containing Full-length, wild-type (WT) recombinant human (rh)TSG-6, its isolated cells were then blocked by injecting 1 M ethanolamine (70 ml) (39, 40). Link module (Link_TSG6), and biotinylated Link_TSG6 were produced Analytes (rhTSG-6, CXCL8, or other chemokines), at a range of con- as described previously (29, 31); the Link_TSG6_D (K34A/K54A) and centrations, were passed over the immobilized CXCL8, rhTSG-6, or Link_TSG6_T (K20A/K34A/K41A) mutants were prepared/characterized Link_TSG6 in SPR running buffer at either pH 6 (SPR 6.0) or pH 7.2 as in Mahoney et al. (27). WT CXCL8 and the CXCL8_S (R68A) and

(SPR 7.2); the resulting sensograms were analyzed using the 1:1 by guest on September 23, 2021 CXCL8_T (K64A/K67A/R68A) mutants, expressed and purified as de- Langmuir interaction model with BIAevaluation software (GE Health- scribed in Tanino et al. (32), and CCL3, CCL5, and CXCL11 were pro- care). vided by the Pharmaceutical Research Laboratory (Amanda E. I. Proudfoot, Geneva, Switzerland). Heparin (Fourth International Standard) was bio- Characterization of the CXCL8/TSG-6 interaction using tinylated as described in Clark et al. (33). plate-based binding assays Cell culture Mictrotiter plate assays to compare the interaction of CXCL8 with full- length TSG-6 and its isolated Link module were carried out essentially All cell cultures were incubated at 37˚C and 5% (v/v) CO . EA.hy 926 cells 2 as described previously (27, 41). rhTSG-6 or Link_TSG6 (50 pmol/well) in and HUVECs were cultured in DMEM with 10% (v/v) FBS and endo- coating buffer (20 mM Na CO [pH 9.6]) was immobilized onto 96-well thelial basal medium (EBM-2; Lonza), respectively. HL-60 cells (Ameri- 2 3 Nunc MaxiSorp plates (Thermo Fisher Scientific) by incubation overnight can Type Culture Collection, Manassas, VA) were maintained in IMDM at room temperature. Plates were washed three times and blocked with 1% with 20% (v/v) FBS; differentiation to neutrophil-like cells, as assessed by (w/v) BSA for 90 min at 37˚C. After further washing, CXCL8 (0–500 nM) CD11b upregulation and morphological changes, was induced by incu- was added to each well and plates were incubated for 2 h at room tem- bation with 1.5% (v/v) DMSO for 120 h (34). The murine pre–B cell line perature and then for 90 min at room temperature with biotinylated anti- 300-19 (35) and stable transfectants expressing CXCR1 (clone 1B4) or human CXCL8 Ab (PeproTech; 0.3 mg/ml). Bound CXCL8 was detected CXCR2 (clone 1D5) were cultured in RPMI 1640 with 10% (v/v) FBS, 1% 2 using extravidin–alkaline phosphatase (1:10,000; Sigma-Aldrich), fol- (w/v) glutamine, and 5 3 10 5 M 2-ME, under puromycin (1.5 mg/ml) lowed by SigmaFast p-nitrophenyl phosphate solution (200 ml/well; selection (35). Sigma-Aldrich), with absorbance measurements at 405 nm being taken after 10 min. To determine the specificity of the CXCL8-Link_TSG6 in- CXCL8 transport and chemokine-induced neutrophil teraction, CXCL8 (50 pmol/well) was immobilized onto MaxiSorp plates transmigration across endothelial cell monolayers overnight and biotinylated Link_TSG6 (10 nM) was added in the fluid phase, in combination with unlabeled Link_TSG6 (0–1000 nM). Binding EA.hy 926 cells were seeded on top of 6.5-mm Transwell filters in 24-well was detected as described above. plates (Corning permeable supports, 3-mm polyester membrane), and HUVECs were seeded in the same way following coating of the filters with fibro- CXCL8/heparin binding assays nection (10 mg/ml in PBS for 1 h at 37˚C). Cells (5 3 104 cells/well in 100 ml media) were incubated at 37˚C overnight, with 600 ml media below the Microtiter plate assays were carried out essentially as described previously membrane. Monolayer formation was confirmed by eye using a light mi- (27, 33). Briefly, CXCL8, CXCL8_S, or CXCL8_T (50 pmol/well in 20 croscope, cells were washed with PBS, and Transwells were then transferred mM Na2CO3 [pH 9.6]) was immobilized onto 96-well Nunc MaxiSorp to fresh wells containing serum-free medium with or without chemokine plates (Thermo Fisher Scientific) by incubation at room temperature over- in the absence/presence of rhTSG-6 or Link_TSG6 (WT or mutant) at the night. All subsequent washes (three times after each incubation), dilutions, concentrations indicated. and incubations were performed in SAB6 (10 mM NaOAc, 150 mM NaCl, For transmigration assays, differentiated HL-60 cells (as a model of 2% [v/v] Tween 20 [pH 6]). After blocking with 5% (w/v) BSA, for human neutrophils) (34) or human neutrophils isolated from buffy coats 90 min at 37˚C, biotinylated heparin (b-heparin) was added alone (0–100 ng/ (36) were washed, resuspended (3.75 3 106 cells/ml) in fresh DMEM, and well) or (at 25 ng/well) in combination with TSG-6 proteins (0–1000 nM) added (750,000 cells/well) to the top of endothelial monolayers. Trans- for 4 h at room temperature. Bound b-heparin was detected by addition of wells were then incubated at 37˚C for 24 h (HL-60 cells) or 2 h (primary extravidin–alkaline phosphatase (1:10,000; Sigma-Aldrich) followed by The Journal of Immunology 2179

SigmaFast p-nitrophenyl phosphate solution (200 ml/well; Sigma-Aldrich). Results Absorbance measurements at 405 nm were taken after 10 min and cor- TSG-6 inhibits CXCL8-mediated transendothelial migration of rected against blank wells. neutrophils via its Link module domain Interaction of CXCL8 with cell surface receptors TSG-6 is a potent inhibitor of neutrophil extravasation in vivo Binding of CXCL8 to murine 300-19 cells (35) stably expressing CXCR1 (17, 20), and because CXCL8 is an important chemoattractant (clone 1B4) or CXCR2 (clone 1D5) was determined by flow cytometry and activator for neutrophils, we chose to investigate the effects of using a biotinylated human CXCL8 Fluorokine kit (R&D Systems) ac- cording to the supplier’s instructions. Cells (1 3 105 in PBS) were incu- TSG-6 on CXCL8 using a Transwell assay. In this system CXCL8 bated at 4˚C for 1 h with b-CXCL8 that had been preincubated (1 h at 37˚C) upregulated the transendothelial migration of both differentiated in the absence or presence of Link_TSG6. Cell-associated CXCL8 was HL-60 cells (Fig. 1A) and primary human neutrophils (Fig. 1B) in detected, following addition of avidin-fluorescein, using a CyAn ADP a dose-dependent manner; 3.6 nM CXCL8 increased the numbers flow cytometer (Beckman Coulter) with excitation at 488 nm. Gating ∼ ∼ was applied to select live cells on the basis of forward scatter versus side of migrated cells by 2- and 6-fold, respectively, which is con- scatter. sistent with previous studies (19, 42). Under these conditions, we showed that a 5-fold molar excess (i.e., 18 nM) of Link_TSG6 CXCL8-mediated chemotaxis of human neutrophils completely ablated the CXCL8-induced migration of differenti- Neutrophils were purified from fresh human blood and their chemotaxis ated HL-60 cells (Fig. 1C) and significantly reduced the migration through 3-mm pores was assayed as reported previously (36). CXCL8 of primary human neutrophils (Fig. 1D). Furthermore, Link_TSG6 (1 nM) and Link_TSG6 (0–10 mM) were placed in the lower chambers of 96-well ChemoTx plates (Neuroprobe, Cabin John, MD), neutrophils (in and full-length rhTSG-6 had equivalent neutrophil inhibitory effects (Fig. 1E), confirming that this activity resides within the Link RPMI 1640 without red phenol, 2% [v/v] heat-inactivated FBS, 1% Downloaded from penicillin/streptomycin, 1% [w/v] L-glutamine) were added to the upper module domain of TSG-6. Given that the inhibition of neutrophil chambers, and migrated cells were counted (using a CyQUANT kit; migration was seen when both the CXCL8 and TSG-6 proteins Molecular Probes) after 45 min at 37˚C. were added to Transwells below the endothelial cell layer, we Statistical analysis hypothesized that this activity of TSG-6 was due to its direct in- Statistically significant differences between groups were identified by re- teraction with CXCL8. peated measures ANOVA, and a Bonferroni post hoc test was used to compare each condition with controls (GraphPad Prism, version 5.0). A TSG-6 binds to CXCL8 via its Link module http://www.jimmunol.org/ two-tailed Student t test was used for statistical analyses involving pairwise We investigated the binding ofTSG-6toCXCL8usingSPR, comparisons of data sets. A p value ,0.05 was considered statistically significant. When data are presented as mean values 6 SEM, the number which revealed that these proteins interact with high affinity of independent experiments (n) is indicated, with all conditions being set (see Table I). For example, when CXCL8 was flowed over im- up at least in triplicate within each experiment. mobilized rhTSG-6, at pH 6 (Supplemental Fig. 1A) or pH 7.2 by guest on September 23, 2021

FIGURE 1. TSG-6 inhibits transendothelial migration of differentiated HL-60 cells via interaction of its Link module domain with CXCL8. Migration of differentiated HL-60 cells across a monolayer of EA.hy 926 cells (A) or primary human neutrophils across a monolayer of HUVECs (B) was measured in response to a range of concentrations of WT CXCL8 (1.2, 3.6, 6, 9, and 12 nM) (n = 3). Migration of differentiated HL-60 cells across a monolayer of EA. hy 926 cells was measured in response to (C) CXCL8 (3.6 nM [+]) alone or in combination with Link_TSG6 (at 1:1, 2:1, 5:1, and 10:1 molar ratios) (n =3– 7) or (E) CXCL8 (3.6 nM [+]) alone or in combination with a 5-fold molar excess of Link_TSG6 or rhTSG-6 (n = 3). Migration of primary human neutrophils across a monolayer of HUVECs was determined in response to (D) WT CXCL8 (3.6 nM [+]) alone or in combination with Link_TSG6 (at 1:1, 2:1, 5:1, and 10:1 molar ratios) (n = 3). Data are plotted as mean values (6SEM) relative to nonstimulated controls (2). *p , 0.05, **p , 0.01, ***p , 0.001 compared with non stimulated controls (A, B) or to CXCL8 alone (C–E), as determined using repeated measures ANOVA analysis with a Bonferroni post hoc test. In this and subsequent ﬁgures, the dotted line indicates baseline neutrophil migration in the absence of CXCL8 stimulus. 2180 TSG-6 INHIBITS CXCL8-MEDIATED NEUTROPHIL MIGRATION

Table I. Dissociation constants for the interactions of rhTSG-6 and Link_TSG6 with CXCL8 and other chemokines

a 2 21 21 21 Immobilized Ligand Fluid-Phase Analyte KD (nM) x kon (M s ) koff (s ) rhTSG-6 CXCL8 (pH 6) 26 6.5 2.9 3 104 7.5 3 1024 rhTSG-6 CXCL8 (pH 7.2) 19 16.7 3.4 3 104 6.5 3 1024 Link_TSG6 CXCL8 (pH 6) 6 44.3 5.3 3 104 3.2 3 1024 Link_TSG6 CXCL8 (pH 7.2) 21 18.1 1.9 3 104 1.1 3 1023 rhTSG-6 CXCL8_Sc (pH 6) 581 2.2 3.1 3 103 1.8 3 1023 rhTSG-6 CXCL8_Td (pH 6) 2,109 1.4 640 1.4 3 1023 Link_TSG6 CXCL8_S (pH 6) 203 2.0 8.4 3 103 1.7 3 1023 CXCL8b rhTSG-6 (pH 6) 74 8.9 5.7 3 103 4.2 3 1024 CXCL8 rhTSG-6 (pH 7.2) 20 15.3 2.3 3 104 1.1 3 1023 Link_TSG6 CCL5 (pH 7.2) 2 7.3 5.7 3 104 1.1 3 1024 rhTSG-6 CXCL11 (pH 6) 16 7.2 6.4 3 103 1.1 3 1024 rhTSG-6 CCL3 (pH 6) 15,100 7.4 4.4 3 103 6.6 3 1023

a KD values determined by full kinetic analysis of SPR data. bNo fittable data were generated when Link_TSG6 was used as the analyte, owing to nonspecific interactions between this protein and the biosensor chip. cCXCL8 R68A mutant. dCXCL8 K64A/K67A/R68A mutant. Downloaded from (Supplemental Fig. 1D), analysis of the resultant sensograms To determine whether this inhibitory effect was due to TSG-6 (a (using the 1:1 Langmuir model) revealed affinities (KD)of26 known heparin-binding protein) interacting with heparin, we used and 19 nM, respectively. When rhTSG-6 was used as the analyte the Link_TSG6 mutants K34A/K54A (Link_TSG6_D) and K20A/ (and CXCL8 immobilized), KD values within the same order of K34A/K41A (Link_TSG6_T), which have ∼50 and ∼10% of WT magnitude were obtained, that is, 74 nM at pH 6 (Supplemental heparin-binding activity, respectively (27). Both mutants antago- Fig. 1C) and 20 nM at pH 7.2 (Supplemental Fig. 1F). Sim- nized the CXCL8/heparin interaction; Link_TSG6_D had similar ilar affinities were also observed with immobilized Link_TSG6 activity to WT Link_TSG6, whereas Link_TSG6_T showed en- http://www.jimmunol.org/ (6 nM at pH 6 [Supplemental Fig. 1B] and 21 nM at pH 7.2 hanced activity with maximal inhibition at 0.125 mM (Fig. 3C). [Supplemental Fig. 1E]), demonstrating that the interaction with CXCL8 is mediated via the Link module of TSG-6; this was further confirmed in plate-based assays where CXCL8 bound to immobilized rhTSG-6 and Link_TSG6 (Fig. 2A). Furthermore, unlabeled Link_TSG6 competed for the binding of biotinylated Link_TSG6 to immobilized CXCL8 with an IC50 of 15 6 3 nM (Fig. 2B), which is

comparable to the KD value determined above by SPR. by guest on September 23, 2021 Taken together, these results indicate that there is a specific, high-affinity interaction between the Link module of TSG-6 and CXCL8 (with KD values in the range ∼10–70 nM), which could contribute to the inhibition of chemokine-induced neutrophil migration. Overall, there was little difference between the binding affinities determined by SPR at pH 6 and pH 7.2; this is in contrast to other interactions of TSG-6, for example, with HA (43), heparin (27, 28), and thrombospondin-1 (28), which are all sensitive to pH in the range pH 6 to pH 7.5.

The Link module of TSG-6 inhibits CXCL8/GAG interactions Chemokine/GAG interactions play essential roles in neutrophil migration through enabling the sequestration of chemokines by HS proteoglycans (HSPGs), and hence the formation of haptotactic gradients, on the lumen of the endothelium (5, 11, 12); they have also been implicated in the transport of chemokines (produced in interstitial tissues) across the endothelium (11). For example, mutants of CXCL8 with impaired heparin-binding activities showed deficient transcytosis and reduced accumulation on the apical surface of the endothelium in skin (7). Therefore, FIGURE 2. TSG-6 binds specifically to CXCL8 via its Link module do- we investigated whether binding of TSG-6 to CXCL8 could main. (A) rhTSG-6 or Link_TSG6 (50 pmol/well) was immobilized overnight antagonize the interaction between CXCL8 and heparin, a GAG onto a MaxiSorp microtiter plate. CXCL8 (0–500 nM) was added in the fluid that is often used as a model for HS owing to its similar structure phase and binding was detected using a biotinylated anti-CXCL8 Ab (n =4). B and greater availability. In plate-based assays, where immobi- ( ) CXCL8 (50 pmol/well) was immobilized onto a MaxiSorp plate overnight and biotinylated Link_TSG6 (10 nM) was added in fluid phase in combination lized WT CXCL8 bound to heparin in a dose-dependent manner with unlabeled Link_TSG6 (0–1000 nM) (n = 4). Binding was detected using (Fig. 3A), we found that Link_TSG6 and rhTSG-6 were simi- extravidin–alkaline phosphatase followed by disodium p-nitrophenyl phos- ∼ larly effective as competitors for this interaction (IC50sof 70 phate; absorbance at 405 nm was determined after 10 min. Data were plotted and ∼80 nM, respectively); CXCL8/heparin binding was com- as mean values 6 SEM using OriginPro version 8. In (B)anIC50 of 15 6 3nM pletely abolished in the presence of 0.5–1 mM TSG-6 protein was obtained for the inhibition of biotinylated Link_TSG6 binding to immo- (Fig. 3B). bilized CXCL8 by unlabeled Link_TSG6. The Journal of Immunology 2181 Downloaded from

FIGURE 3. Interaction between the Link module domain of T6G-6 and the GAG-binding site of CXCL8 inhibits CXCL8/heparin binding and CXCL8- mediated neutrophil transmigration. WT or mutant CXCL8 (250 nM) was immobilized onto MaxiSorp plates and b-heparin (0–100 ng/well) (A)or http://www.jimmunol.org/ b-heparin (25 ng/well) in combination with a range of concentrations of rhTSG-6 or Link_TSG6 (0-1000 nM) (B), or in combination with a range of concentrations of WT Link_TSG6, Link_TSG6_D, or Link_TSG6_T (0–2000 nM) (C), was added in the ﬂuid phase. Binding of b-heparin was detected using extravidin–alkaline phosphatase followed by disodium p-nitrophenyl phosphate; absorbance at 405 nm was determined after 5 (A) or 10 min (B, C).

Data are plotted as mean values (n =4)6 SEM and ﬁtted using OriginPro version 8. KD values of 5 6 12 and 26 6 11 nM were determined for the binding of b-heparin to WT CXCL8 and CXCL8_D, respectively (A). IC50 values of 81 6 18 and 71 6 14 nM for the inhibition of b-heparin binding to CXCL8 by rhTSG-6 and Link_TSG6, respectively (B), and 105 6 5, 172 6 3, and 45 6 5 nM for the inhibition of this interaction by WT Link_TSG6, Link_TSG6_D, and Link_TSG6_T, respectively (C), were determined. In (D), migration of differentiated HL-60 cells across an EA.hy 926 cell monolayer was determined in response to CXCL8 (3.6 nM [+]) alone or in combination with 5:1 molar ratios of WT Link_TSG6, Link_TSG6_D, or Link_TSG6_T (n = 4). Data are

plotted as mean values (6SEM) relative to nonstimulated controls (2). **p , 0.01, ***p , 0.001 compared with CXCL8 alone, as determined using by guest on September 23, 2021 repeated measures ANOVA analysis with a Bonferroni post hoc test.

Furthermore, in the Transwell system, both Link_TSG6_D and TSG-6 inhibits CXCL8-mediated neutrophil transmigration by Link_TSG6_T inhibited the transendothelial migration of differ- interacting with the GAG-binding surface of CXCL8 and entiated HL-60 cells with potencies similar to WT Link_TSG6 blocking CXCL8/GAG binding (Fig. 3D). Overall, these data indicate that both the inhibitory To further investigate the mechanism of the anti-migratory activ- effect of TSG-6 on CXCL8-induced neutrophil transmigration and ity of TSG-6, we used the CXCL8 mutants R68A (CXCL8_S) and its impairment of the CXCL8/heparin interaction are independent K64A/K67A/R68A (CXCL8_T) (5, 8, 32), which we found to of the heparin-binding activity of TSG-6. have reduced heparin-binding activity (∼20–60% of WT) and no Using the Transwell system with b-CXCL8 and Link_TSG6 in measurable activity, respectively (Fig. 3A). In SPR experiments the lower chamber, we went on to show that Link_TSG6 (at the TSG-6 showed weak binding to both mutants (Supplemental Fig. same concentration seen to inhibit neutrophil transmigration) caused 1G–I, Table I); the affinities for the CXCL8_S and CXCL8_T significant reductions in 1) the binding of b-CXCL8 to GAGs on interactions with rhTSG-6 were reduced by 20- and 80-fold, ∼ the basolateral surface of HUVECs ( 4-fold), 2) the movement of respectively, compared with WT CXCL8 (due to slower rates b-CXCL8 out of the lower Transwell chamber, and 3) the subsequent of association and faster rates of dissociation). These data ∼ association of chemokine with GAGs on the apical cell surface ( 5- suggest that the heparin- and TSG-6–binding sites on CXCL8 fold) (Fig. 4). We observed no significant effect of Link_TSG6 on the overlap. amount of intracellular b-CXCL8. This could, at least in part, reflect Despite its somewhat impaired heparin-binding function, that Link_TSG6 inhibits the GAG-mediated uptake of CXCL8 by en- CXCL8_S induced transendothelial migration of differentiated dothelial cells, but has no effect on other mechanisms, for example, HL-60 cells (Fig. 5A) with similar efficiency to WT protein; in via the Duffy Ag receptor for chemokines. Additionally, Link_TSG6 contrast, CXCL8_T had no such activity (Fig. 5B), reflecting the did not alter the amount of b-CXCL8 in the media of the upper essential role of GAG-binding in CXCL8-mediated neutrophil Transwell chamber. This is likely because most CXCL8 present here migration. However, Link_TSG6 had no inhibitory effect on the is due to nonspecific paracellular diffusion, as has been previously CXCL8_S-mediated migration of either differentiated HL-60 cells demonstrated (11), and is therefore independent of the transcytosis (Fig. 5C) or primary human neutrophils (Fig. 5D), even when mechanism inhibited by Link_TSG6. Taken together, these data present at 10- or 20- fold molar excess. Because the affinity of suggest that TSG-6 might inhibit both GAG-mediated transcytosis of TSG-6 for CXCL8_S is ∼20-fold weaker than for WT CXCL8, CXCL8 and the presentation of CXCL8 on the lumen of the vascular these data indicate that a direct interaction between TSG-6 and endothelium. CXCL8 is required to inhibit CXCL8-mediated transendothelial 2182 TSG-6 INHIBITS CXCL8-MEDIATED NEUTROPHIL MIGRATION

inhibitory effects on the interaction of b-CXCL8 with a HUVEC monolayer (data not shown). TSG-6 inhibits CXCL8-mediated neutrophil chemotaxis In the absence of an endothelial cell monolayer, we observed dose-dependent inhibition by Link_TSG6 of CXCL8-induced neutrophil chemotaxis with an IC50 of 2.4 6 0.3 mM (Fig. 6A). To promote neutrophil migration, CXCL8 must bind to CXCR1 and/or CXCR2. We therefore used ﬂow cytometry to directly test the effect of TSG-6 on the interactions of CXCL8 with its receptors. Preincubation with Link_TSG6 gave rise to a dose-dependent reduction in the binding of b-CXCL8 to cell surface CXCR2, with an IC50 of ∼5 mM (Fig. 6B); however, we did not detect any effect on the CXCL8/CXCR1 interaction even at molar excesses of Link_TSG6 as high of 600:1 (not shown). The similar potencies with which Link_TSG6 inhibited CXCL8/CXCR2 binding and CXCL8-induced chemotaxis indicate that TSG-6 can operate via an alternative mechanism to modulate CXCL8 activity, whereby its

binding to the chemokine weakly inhibits subsequent interaction Downloaded from with CXCR2 (Fig. 7). However, this activity is only seen with micromolar concentrations of TSG-6, in contrast to its inhibition of neutrophil transmigration (∼10 nM; Fig. 1C) and CXCL8/heparin FIGURE 4. TSG-6 inhibits the binding of CXCL8 to endothelial surface binding (IC50 of ∼70 nM; Fig. 3B). GAGs and the transport of CXCL8 across an endothelial cell monolayer. HUVEC monolayers were incubated in Transwells with b-CXCL8 (3 nM),

Discussion http://www.jimmunol.org/ in the absence or presence of Link_TSG6 (15 nM), for 2 h. b-CXCL8, in In this study, we have determined that TSG-6 is a novel ligand the media from the upper and lower chambers, bound to GAGs on the apical and basolateral surfaces of the endothelial cells (recovered in 103 for the neutrophil chemoattractant CXCL8. This high-afﬁnity in- ∼ PBS) and in the HUVEC lysates (intracellular), was quantiﬁed by Western teraction (KD of 25 nM; an average of the values obtained by blot analysis using an Odyssey system. Data, represented as a percentage SPR) occurs via the Link module domain of TSG-6 and directly of the total b-CXCL8 recovered, are plotted as mean values (n =6)6 inhibits CXCL8-induced neutrophil transendothelial migration SEM. *p , 0.05 as determined by pairwise comparison of samples with and, to a lesser extent, chemotaxis. Both TSG-6 and CXCL8 are and without Link_TSG6 using the Student t test. GAG-binding proteins, where associations with GAGs are important in regulating their functions (27, 44). We have demonstrated in migration. These data indicate that TSG-6 might inhibit neutrophil the present study that binding of TSG-6 to CXCL8 inhibits the by guest on September 23, 2021 migration by impairment of CXCL8 binding to endothelial cell interaction of CXCL8 with heparin and, therefore, likely blocks GAGs, and this is supported by our observation that Link_TSG6 binding to HS and other GAGs; consistent with this, TSG-6 was and a heparin oligosaccharide (dp8) have essentially identical found to impair the presentation of CXCL8 on endothelial GAGs.

FIGURE 5. The mutant CXCL8_S, with impaired binding to heparin and TSG-6, mediates transendothelial migration of neutrophils, but TSG-6 does not inhibit this activity. Migration of differentiated HL-60 cells across EA.hy 926 monolayers was measured in response to WT CXCL8 (3.6 nM) (A and B), CXCL8_S (3.6, 7.2, 12, and 24 nM) (n =4)(A), or CXCL8_T (3.6, 7.2, 12, and 24 nM) (n =4)(B) alone, or in response to (C) CXCL8_S (3.6 nM [+]) alone or in combination with Link_TSG6 (at 1:1, 5:1, 10:1 and 20:1 molar ratios) (n = 6). Migration of primary human neutrophils across HUVEC monolayers (D) was measured in response to CXCL8_S (3.6 nM [+]) alone or in combination with Link_TSG6 (at 5:1 and 10:1 molar ratios) (n = 3). Data are plotted as mean values 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001 compared with nonstimulated controls (A, B)orto CXCL8_S alone (C, D), as determined using repeated measures ANOVA analysis with a Bonferroni post hoc test. The Journal of Immunology 2183

the formation of haptotactic gradients, where CXCL8 is associated with proteoglycans on the lumenal surface of the vascular endothelium, is critical for the presentation of CXCL8 to its receptors on neutrophils (5, 11, 12). Our data indicate that TSG-6 can inhibit the immobilization of CXCL8 on endothelial GAGs, for example, by blocking CXCL8/HS binding ((a) in Fig. 7). This would result in reduced concentrations of cell-associated versus fluid-phase chemokine (where the latter would then be washed away by venular flow) and/or upset the equilibrium between monomeric and dimeric CXCL8, thereby reducing receptor activation and/or increasing receptor desensitization and internalization (see Ref. 46); GAG binding is directly coupled to the dimerization of CXCL8 (8), where the dimer and monomer have distinct roles in the formation of chemokine gradients in vivo (10). In turn, this would limit integrin- mediated attachment of neutrophils to the endothelium and their subsequent transmigration. CXCL8 that is produced (e.g., by macrophages) in inflamed or damaged extravascular tissue is transported to the lumenal surface

of the endothelium via pericellular and transcellular mechanisms Downloaded from (11, 37, 47). The Duffy Ag receptor for chemokines plays an important role in trancytosis (37, 48, 49), but this process is also dependent on the interaction of CXCL8 with HSPGs on the abluminal surface of endothelial cells (11). Our data indicate that TSG-6, expressed at an inﬂammatory site, could potentially limit

the transport of CXCL8 across the endothelium by antagonizing http://www.jimmunol.org/ CXCL8/HSPG interactions ((b) in Fig. 7). Human neutrophils carry the receptor CXCR1 that binds with FIGURE 6. Link_TSG6 can inhibit the chemotaxis of human neu- high affinity to CXCL8 (and less tightly to CXCL6) and CXCR2, trophils and the interaction of CXCL8 with its receptor CXCR2. (A) Pu- which binds to CXCL1–3 and CXCL5–8 (9). There is evidence rified human neutrophils were added to the upper chambers of ChemoTx that CXCR1 and CXCR2 act in a coordinated manner, with CXCR2 plates, where the lower chambers contained CXCL8 (1 nM) alone or in being most important for early stage CXCL8-induced neutrophil combination with increasing concentrations of Link_TSG6 (0–20 mM). Migrated neutrophils were counted after 2 h; data were plotted as mean values (n =3)6 SEM. (B) Biotinylated CXCL8 (120 nM), alone or fol- by guest on September 23, 2021 lowing preincubation with Link_TSG6 (at 10:1 to 200:1 molar excess), was added to cells expressing CXCR2. Cell-associated CXCL8 was detected by flow cytometry following addition of avidin-conjugated fluorescein, with gating applied to select live cells. An overlay of histograms (incidents of absorbance against fluorescence), each representative of three independent experiments, is shown as an inset. Data, as a percentage of the maximal signal (i.e., CXCL8 alone), were plotted as mean values (n =3)6 SEM. *p , 0.05, ***p , 0.001 relative to cells incubated with CXCL8 alone. Graphs were generated and data fitted using OriginPro version 8 giving rise to an IC50 of 2.4 6 0.3 mM for the inhibition of neutrophil chemotaxis by Link_TSG6 (A), and an IC50 of 4.9 mM (i.e., ∼40-fold molar excess) for the inhibition of CXCL8 binding to CXCR2 by Link_TSG6 (B).

The use of mutants revealed that suppression of CXCL8/GAG binding is independent of the heparin-binding properties of TSG- 6 and that the TSG-6– and GAG–binding surfaces on CXCL8 are, at least, partially overlapping. We have also shown that at high concentrations, TSG-6 antagonizes the binding of CXCL8 to its receptor CXCR2 and that this correlates with an inhibition of neutrophil chemotaxis. CXCL8 has distinct GAG- and receptor-binding sites, in contrast to some other chemokines such as CCL3 and CCL4 (36). However, the structure of CXCL8 (45) reveals that these are close enough together that by binding to a surface that primarily overlaps the GAG-binding site FIGURE 7. Potential mechanisms for the modulation of CXCL8 func- TSG-6 could partially occlude/perturb the receptor-binding site. tion by TSG-6. Inhibition by TSG-6 of CXCL8/GAG interactions could antagonize (a) binding of CXCL8 to HSPGs on the lumenal surface of the This would be consistent with our observations that TSG-6 inhibits endothelium, thereby preventing the formation of haptotactic gradients the CXCL8/heparin interaction more effectively than the CXCL8/ and/or (b) binding of CXCL8 to GAGs on the ablumenal surface, thus ∼ ∼ m CXCR2 interaction (i.e., with IC50 values of 70–80 nM and 5 M, impairing transcytosis of CXCL8. Very high local concentrations of TSG-6 respectively). might inhibit the CXCL8/CXCR2 interaction (c, d), thereby limiting the The sites at which TSG-6 might act to regulate CXCL8-mediated movement of neutrophils in response to a chemotactic gradient of CXCL8. neutrophil extravasation are summarized in Fig. 7. As noted above, d, CXCL8; s, TSG-6. 2184 TSG-6 INHIBITS CXCL8-MEDIATED NEUTROPHIL MIGRATION recruitment, whereas CXCR1 triggers cytotoxic effects, i.e., at through neutralization of chemokine activities. The soluble CKBP sites of injury/infection (10, 46). Our observation that TSG-6 from S. mansoni binds to various chemokines including CXCL8 inhibits the interaction of CXCL8 with cell-associated CXCR2 and inhibits CXCL8-mediated neutrophil migration in models of and also CXCL8-induced chemotaxis, with similar potencies, inflammatory disease (54), whereas the viral CKBPs block chemo- suggests that TSG-6 obstructs the receptor-binding surface of kine functions through interacting with their GAG-binding and/or CXCL8. Antagonism of the CXCL8/CXCR2 interaction in vivo their receptor-binding domains (see Refs. 53, 56). Evasin-3, in the could prevent tight adhesion/diapedesis of neutrophils (Fig. 7c) saliva of ticks, binds selectively to CXCL8 (KD of ∼1 nM) and and/or their response to chemotactic gradients in the extravascular suppresses the CXCL8/CXCR1 interaction; it is a potent inhibitor tissue (Fig. 7d). The effect of TSG-6 on chemotaxis (IC50 of ∼2.5 of neutrophil recruitment (55, 57), reducing myocardial infarct mM) is ∼250-fold less potent than its inhibition of neutrophil size in a mouse model of ischemia/reperfusion injury and also transmigration (IC50 of ∼10 nM), suggesting that TSG-6 may only decreases the local production of TNF in the synovial joints of regulate chemotaxis in situations where it is present at a high local mice with Ag-induced arthritis. Thus, there are many parallels concentration, for example, when secreted by neutrophils in re- between the effects of these various CKBPs and the activities of sponse to inflammatory cytokines (25). This could reflect the TSG-6 described in the present study and previously, for example, greater affinity of the interaction between CXCL8 and CXCR2 in murine models of inflammation (17), arthritis (20), and myo- (KD of ∼1 nM) (50) than between CXCL8 and heparin (low mi- cardial infarction (21). Although TSG-6 does not show any ob- cromolar IC50 value) (5, 32) and between CXCL8 and TSG-6 (KD vious relationship to the parasitic or viral CKBPs in either sequence of ∼25 nM). We did not detect any effect of TSG-6 on CXCL8 or tertiary structure, there appear to be similarities in the mechanisms binding to CXCR1; however, this does not exclude the possibility through which these proteins act to neutralize chemokine func- Downloaded from that the inhibition of neutrophil chemotaxis by TSG-6 might in- tion; i.e., CKBPs from pathogenic organisms might mimic the ac- volve perturbation of both CXCR1- and CXCR2-mediated pro- tivity of TSG-6. cesses. In summary, this study has identified an endogenous mech- It is becoming well established that TSG-6, e.g., when expressed anism whereby TSG-6 might regulate neutrophil extravasation by tissue-resident cells in response to inflammation, represents an in vivo and thus prevent/limit tissue damage during acute inflam-

endogenous pathway that is anti-inflammatory and protects tissues mation. Our discovery of a soluble mammalian CKBP could inform http://www.jimmunol.org/ from damage, for instance through the inhibition of neutrophil the development of new anti-inflammatory therapeutics, for diseases migration (17–23). Previously reported mechanisms that might of unresolved inflammation (e.g., rheumatoid arthritis and cardio- contribute to its regulation of neutrophil recruitment in vivo in- vascular disease), where the regulation of chemokine activity is clude 1) the formation of TSG-6/HA complexes that can modulate a potential target, or indeed lead to improved methods for trans- CD44-mediated leukocyte attachment to the vascular endothelium plantation. (see Ref. 51), and 2) attenuation by TSG-6 of TLR2/NF-kB signaling in resident macrophages resulting in reduced production of Acknowledgments proinflammatory mediators (23), again perhaps by regulating HA/ We thank Ann Canfield (University of Manchester, Manchester, U.K.), CD44 engagement. The results described in the present study, Mark Fuster (University of California San Diego, La Jolla, CA), and Bernhard by guest on September 23, 2021 where the binding of TSG-6 to CXCL8 directly suppresses the Moser (University of Cardiff, Cardiff, U.K.) for providing EA.hy 926 cells, promigratory effects of CXCL8, are clearly independent of the HUVECs, and the 300-19 cell line, respectively, and Barbara Mulloy HA-binding properties of TSG-6. Thus, we have identified a new (National Institute for Biological Standards and Control, U.K.) for provision of the Fourth International Standard of Heparin. We are grateful to Catherina mechanism whereby TSG-6 contributes to a negative feedback Salanga (Skaggs School of Pharmacy and Pharmaceutical Sciences, University loop to limit excessive neutrophil recruitment at sites of inflam- of California, San Diego) for assistance with the chemokine transport assay. mation. The CXCL8-binding activity of TSG-6 could largely ex- plain its protective effects in in vivo models of inflammatory Disclosures disease, regardless of the source of the TSG-6, i.e., endogenous, The authors have no financial conflicts of interest. exogenously administered (see Ref. 18), or MSC expressed (21– 24). This could also provide the mechanism whereby MSCs can promote the survival of tissue transplants in animal models given References the recent finding that TSG-6 can suppress rejection of corneal 1. Ley, K., C. Laudanna, M. I. Cybulsky, and S. Nourshargh. 2007. Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat. Rev. Immu- allografts in mice (24) and that transplantation can give rise to nol. 7: 678–689. high levels of CXCL8 expression (52); TSG-6 might limit in- 2. Smith, W. B., J. R. Gamble, I. Clark-Lewis, and M. A. Vadas. 1991. Interleukin-8 flammation after surgery and thus prevent the onset of an adaptive induces neutrophil transendothelial migration. Immunology 72: 65–72. 3. Hillyer, P., E. Mordelet, G. Flynn, and D. Male. 2003. Chemokines, chemokine immune response. receptors and adhesion molecules on different human endothelia: discriminating In this study we have identified TSG-6 as a soluble chemokine- the tissue-specific functions that affect leucocyte migration. Clin. Exp. Immunol. binding protein (CKBP) that, to our knowledge, is the first to be 134: 431–441. 4. Lisignoli, G., S. Toneguzzi, C. Pozzi, A. Piacentini, M. Riccio, A. Ferruzzi, described in mammals. In addition to CXCL8, we have shown G. Gualtieri, and A. Facchini. 1999. Proinflammatory cytokines and chemokine (using SPR) that TSG-6 interacts with some, but not all, of the production and expression by human osteoblasts isolated from patients with rheumatoid arthritis and osteoarthritis. J. Rheumatol. 26: 791–799. CC and CXC chemokines tested. For example, it binds with high 5. Kuschert, G. S., A. J. Hoogewerf, A. E. Proudfoot, C. W. Chung, R. M. Cooke, affinity to CCL5 (KD of 2 nM), CXCL11 (KD of 16.4 nM) (see R. E. Hubbard, T. N. Wells, and P. N. Sanderson. 1998. Identification of a glycos- Table I), and CCL2 (not shown), but does not bind to CCL3 aminoglycan binding surface on human interleukin-8. Biochemistry 37: 11193–11201. 6. Webb, L. M., M. U. Ehrengruber, I. Clark-Lewis, M. Baggiolini, and A. Rot. (Table I) or to the CXCR2 ligand CXCL1 and has no effect on 1993. Binding to heparan sulfate or heparin enhances neutrophil responses to CXCL1-mediated neutrophil transmigration (data not shown). Thus, interleukin 8. Proc. Natl. Acad. Sci. USA 90: 7158–7162. TSG-6 binds to at least two members of each of the CC and 7. Middleton, J., S. Neil, J. Wintle, I. Clark-Lewis, H. Moore, C. Lam, M. Auer, E. Hub, and A. Rot. 1997. Transcytosis and surface presentation of IL-8 by CXC chemokine families. Soluble CKBPs are known to be pro- venular endothelial cells. Cell 91: 385–395. duced by viruses (reviewed in Ref. 53) and also by parasites, such 8. Frevert, C. W., M. G. Kinsella, C. Vathanaprida, R. B. Goodman, D. G. Baskin, A. Proudfoot, T. N. Wells, T. N. Wight, and T. R. Martin. 2003. Binding of as the helminth Shistosoma mansoni (54) and blood-sucking ticks interleukin-8 to heparan sulfate and chondroitin sulfate in lung tissue. Am. J. (55), enabling these organisms to evade the host immune response Respir. Cell Mol. Biol. 28: 464–472. The Journal of Immunology 2185

9. Lee, J., R. Horuk, G. C. Rice, G. L. Bennett, T. Camerato, and W. I. Wood. 1992. Kinetics of chemokine-glycosaminoglycan interactions control neutrophil mi- Characterization of two high affinity human interleukin-8 receptors. J. Biol. gration into the airspaces of the lungs. J. Immunol. 184: 2677–2685. Chem. 267: 16283–16287. 33. Clark, S. J., V. A. Higman, B. Mulloy, S. J. Perkins, S. M. Lea, R. B. Sim, and 10. Das, S. T., L. Rajagopalan, A. Guerrero-Plata, J. Sai, A. Richmond, R. P. Garofalo, A. J. Day. 2006. His-384 allotypic variant of factor H associated with age-related and K. Rajarathnam. 2010. Monomeric and dimeric CXCL8 are both essential for macular degeneration has different heparin binding properties from the non- in vivo neutrophil recruitment. PLoS ONE 5: e11754. disease-associated form. J. Biol. Chem. 281: 24713–24720. 11. Wang, L., M. Fuster, P. Sriramarao, and J. D. Esko. 2005. Endothelial heparan 34. Jacob, C., M. Leport, C. Szilagyi, J. M. Allen, C. Bertrand, and V. Lagente. 2002. sulfate deficiency impairs L-selectin- and chemokine-mediated neutrophil traf- DMSO-treated HL60 cells: a model of neutrophil-like cells mainly expressing ficking during inflammatory responses. Nat. Immunol. 6: 902–910. PDE4B subtype. Int. Immunopharmacol. 2: 1647–1656. 12. Colditz, I. G., M. A. Schneider, M. Pruenster, and A. Rot. 2007. Chemokines at 35. Loetscher, M., B. Gerber, P. Loetscher, S. A. Jones, L. Piali, I. Clark-Lewis, large: in-vivo mechanisms of their transport, presentation and clearance. Thromb. M. Baggiolini, and B. Moser. 1996. Chemokine receptor specific for IP10 and Haemost. 97: 688–693. mig: structure, function, and expression in activated T-lymphocytes. J. Exp. Med. 13. Gangavarapu, P., L. Rajagopalan, D. Kolli, A. Guerrero-Plata, R. P. Garofalo, 184: 963–969. and K. Rajarathnam. 2012. The monomer-dimer equilibrium and glycosamino- 36. Proudfoot, A. E., T. M. Handel, Z. Johnson, E. K. Lau, P. LiWang, I. Clark- glycan interactions of chemokine CXCL8 regulate tissue-specific neutrophil Lewis, F. Borlat, T. N. Wells, and M. H. Kosco-Vilbois. 2003. Glycosamino- recruitment. J. Leukoc. Biol. 91: 259–265. glycan binding and oligomerization are essential for the in vivo activity of 14. Lee, T. H., H. G. Wisniewski, and J. Vilcek. 1992. A novel secretory tumor ne- certain chemokines. Proc. Natl. Acad. Sci. USA 100: 1885–1890. crosis factor-inducible protein (TSG-6) is a member of the family of hyaluronate 37. Pruenster, M., L. Mudde, P. Bombosi, S. Dimitrova, M. Zsak, J. Middleton, binding proteins, closely related to the adhesion receptor CD44. J. Cell Biol. 116: A. Richmond, G. J. Graham, S. Segerer, R. J. Nibbs, and A. Rot. 2009. The 545–557. Duffy antigen receptor for chemokines transports chemokines and supports their 15. Mahoney, D. J., C. Swales, N. A. Athanasou, M. Bombardieri, C. Pitzalis, promigratory activity. Nat. Immunol. 10: 101–108. K. Kliskey, M. Sharif, A. J. Day, C. M. Milner, and A. Sabokbar. 2011. TSG-6 38. Allen, S. J., D. J. Hamel, and T. M. Handel. 2011. A rapid and efficient way to inhibits osteoclast activity via an autocrine mechanism and is functionally obtain modified chemokines for functional and biophysical studies. Cytokine 55: synergistic with osteoprotegerin. Arthritis Rheum. 63: 1034–1043. 168–173. 16. Nagyeri, G., M. Radacs, S. Ghassemi-Nejad, B. Tryniszewska, K. Olasz, 39. Biacore. 1997. BIAevaluation Software Handbook Version 3.0. Biacore AB, G. Hutas, Z. Gyorfy, V. C. Hascall, T. T. Glant, and K. Mikecz. 2011. TSG-6 Uppsala, Sweden. Downloaded from protein, a negative regulator of inflammatory arthritis, forms a ternary complex 40. O’Shannessy, D. J., M. Brigham-Burke, and K. Peck. 1992. Immobilization with murine mast cell tryptases and heparin. J. Biol. Chem. 286: 23559–23569. chemistries suitable for use in the BIAcore surface plasmon resonance detector. 17. Getting, S. J., D. J. Mahoney, T. Cao, M. S. Rugg, E. Fries, C. M. Milner, Anal. Biochem. 205: 132–136. M. Perretti, and A. J. Day. 2002. The link module from human TSG-6 inhibits 41. Mahoney, D. J., C. D. Blundell, and A. J. Day. 2001. Mapping the hyaluronan- neutrophil migration in a hyaluronan- and inter-a-inhibitor-independent manner. binding site on the link module from human tumor necrosis factor-stimulated J. Biol. Chem. 277: 51068–51076. gene-6 by site-directed mutagenesis. J. Biol. Chem. 276: 22764–22771. 18. Milner, C. M., and A. J. Day. 2003. TSG-6: a multifunctional protein associated 42. Park, C. J., N. P. Gabrielson, D. W. Pack, R. D. Jamison, and A. J. Wagoner

with inflammation. J. Cell Sci. 116: 1863–1873. Johnson. 2009. The effect of chitosan on the migration of neutrophil-like HL60 http://www.jimmunol.org/ 19. Cao, T. V., M. La, S. J. Getting, A. J. Day, and M. Perretti. 2004. Inhibitory cells, mediated by IL-8. Biomaterials 30: 436–444. effects of TSG-6 Link module on leukocyte-endothelial cell interactions in vitro 43. Blundell, C. D., D. J. Mahoney, M. R. Cordell, A. Almond, J. D. Kahmann, and in vivo. Microcirculation 11: 615–624. A. Perczel, J. D. Taylor, I. D. Campbell, and A. J. Day. 2007. Determining the 20. Szańto´,S.,T.Ba´rdos, I. Ga´l, T. T. Glant, and K. Mikecz. 2004. Enhanced molecular basis for the pH-dependent interaction between the link module of neutrophil extravasation and rapid progression of proteoglycan-induced arthritis human TSG-6 and hyaluronan. J. Biol. Chem. 282: 12976–12988. in TSG-6-knockout mice. Arthritis Rheum. 50: 3012–3022. 44. Handel, T. M., Z. Johnson, S. E. Crown, E. K. Lau, and A. E. Proudfoot. 2005. 21. Lee, R. H., A. A. Pulin, M. J. Seo, D. J. Kota, J. Ylostalo, B. L. Larson, Regulation of protein function by glycosaminoglycans: as exemplified by che- L. Semprun-Prieto, P. Delafontaine, and D. J. Prockop. 2009. Intravenous hMSCs mokines. Annu. Rev. Biochem. 74: 385–410. improve myocardial infarction in mice because cells embolized in lung are ac- 45. Clore, G. M., E. Appella, M. Yamada, K. Matsushima, and A. M. Gronenborn. tivated to secrete the anti-inflammatory protein TSG-6. Cell Stem Cell 5: 54–63. 1990. Three-dimensional structure of interleukin 8 in solution. Biochemistry 29: 22. Oh, J. Y., G. W. Roddy, H. Choi, R. H. Lee, J. H. Ylo¨stalo, R. H. Rosa, Jr., and 1689–1696.

D. J. Prockop. 2010. Anti-inflammatory protein TSG-6 reduces inflammatory 46. Richardson, R. M., R. J. Marjoram, L. S. Barak, and R. Snyderman. 2003. Role by guest on September 23, 2021 damage to the cornea following chemical and mechanical injury. Proc. Natl. of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migra- Acad. Sci. USA 107: 16875–16880. tion, activation, and regulation. J. Immunol. 170: 2904–2911. 23. Choi, H., R. H. Lee, N. Bazhanov, J. Y. Oh, and D. J. Prockop. 2011. Anti- 47. Middleton, J., A. M. Patterson, L. Gardner, C. Schmutz, and B. A. Ashton. 2002. inflammatory protein TSG-6 secreted by activated MSCs attenuates zymosan- Leukocyte extravasation: chemokine transport and presentation by the endo- induced mouse peritonitis by decreasing TLR2/NF-kB signaling in resident thelium. Blood 100: 3853–3860. macrophages. Blood 118: 330–338. 48. Lee, J. S., C. W. Frevert, M. M. Wurfel, S. C. Peiper, V. A. Wong, K. K. Ballman, 24. Oh, J. Y., R. H. Lee, J. M. Yu, J. H. Ko, H. J. Lee, A. Y. Ko, G. W. Roddy, and J. T. Ruzinski, J. S. Rhim, T. R. Martin, and R. B. Goodman. 2003. Duffy antigen D. J. Prockop. 2012. Intravenous mesenchymal stem cells prevented rejection of facilitates movement of chemokine across the endothelium in vitro and promotes allogeneic corneal transplants by aborting the early inflammatory response. Mol. neutrophil transmigration in vitro and in vivo. J. Immunol. 170: 5244–5251. Ther. 20: 2143–2152. 49. Lee, J. S., M. M. Wurfel, G. Matute-Bello, C. W. Frevert, M. R. Rosengart, 25. Maina, V., A. Cotena, A. Doni, M. Nebuloni, F. Pasqualini, C. M. Milner, M. Ranganathan, V. W. Wong, T. Holden, S. Sutlief, A. Richmond, et al. 2006. A. J. Day, A. Mantovani, and C. Garlanda. 2009. Coregulation in human leu- The Duffy antigen modifies systemic and local tissue chemokine responses kocytes of the long pentraxin PTX3 and TSG-6. J. Leukoc. Biol. 86: 123–132. following lipopolysaccharide stimulation. J. Immunol. 177: 8086–8094. 26. Chang, M. Y., C. K. Chan, K. R. Braun, P. S. Green, K. D. O’Brien, A. Chait, 50. Paolini, J. F., D. Willard, T. Consler, M. Luther, and M. S. Krangel. 1994. The A. J. Day, and T. N. Wight. 2012. Monocyte-to-macrophage differentiation: chemokines IL-8, monocyte chemoattractant protein-1, and I-309 are monomers synthesis and secretion of a complex extracellular matrix. J. Biol. Chem. 287: at physiologically relevant concentrations. J. Immunol. 153: 2704–2717. 14122–14135. 51. Baranova, N. S., E. Nileba¨ck, F. M. Haller, D. C. Briggs, S. Svedhem, A. J. Day, 27. Mahoney, D. J., B. Mulloy, M. J. Forster, C. D. Blundell, E. Fries, C. M. Milner, and R. P. Richter. 2011. The inflammation-associated protein TSG-6 cross-links and A. J. Day. 2005. Characterization of the interaction between tumor necrosis hyaluronan via hyaluronan-induced TSG-6 oligomers. J. Biol. Chem. 286: factor-stimulated gene-6 and heparin: implications for the inhibition of plasmin 25675–25686. in extracellular matrix microenvironments. J. Biol. Chem. 280: 27044–27055. 52. Citro, A., E. Cantarelli, P. Maffi, R. Nano, R. Melzi, A. Mercalli, E. Dugnani, 28. Kuznetsova, S. A., A. J. Day, D. J. Mahoney, M. S. Rugg, D. F. Mosher, and V. Sordi, P. Magistretti, L. Daffonchio, et al. 2012. CXCR1/2 inhibition enhances D. D. Roberts. 2005. The N-terminal module of thrombospondin-1 interacts with pancreatic islet survival after transplantation. J. Clin. Invest. 122: 3647–3651. the link domain of TSG-6 and enhances its covalent association with the heavy 53. Alcami, A. 2003. Viral mimicry of cytokines, chemokines and their receptors. chains of inter-a-trypsin inhibitor. J. Biol. Chem. 280: 30899–30908. Nat. Rev. Immunol. 3: 36–50. 29. Parkar, A. A., and A. J. Day. 1997. Overlapping sites on the Link module of 54. Smith, P., R. E. Fallon, N. E. Mangan, C. M. Walsh, M. Saraiva, J. R. Sayers, human TSG-6 mediate binding to hyaluronan and chrondroitin-4-sulphate. FEBS A. N. McKenzie, A. Alcami, and P. G. Fallon. 2005. Schistosoma mansoni Lett. 410: 413–417. secretes a chemokine binding protein with antiinflammatory activity. J. Exp. 30. Marson, A., D. E. Robinson, P. N. Brookes, B. Mulloy, M. Wiles, S. J. Clark, Med. 202: 1319–1325. H. L. Fielder, L. J. Collinson, S. A. Cain, C. M. Kielty, et al. 2009. Development 55. De´ruaz, M., A. Frauenschuh, A. L. Alessandri, J. M. Dias, F. M. Coelho, of a microtiter plate-based glycosaminoglycan array for the investigation of R. C. Russo, B. R. Ferreira, G. J. Graham, J. P. Shaw, T. N. Wells, et al. 2008. glycosaminoglycan-protein interactions. Glycobiology 19: 1537–1546. Ticks produce highly selective chemokine binding proteins with antiinflammatory 31. Nentwich, H. A., Z. Mustafa, M. S. Rugg, B. D. Marsden, M. R. Cordell, activity. J. Exp. Med. 205: 2019–2031. D. J. Mahoney, S. C. Jenkins, B. Dowling, E. Fries, C. M. Milner, et al. 2002. A 56. Fallon, P. G., and A. Alcami. 2006. Pathogen-derived immunomodulatory novel allelic variant of the human TSG-6 gene encoding an amino acid differ- molecules: future immunotherapeutics? Trends Immunol. 27: 470–476. ence in the CUB module. Chromosomal localization, frequency analysis, mod- 57. Montecucco, F., S. Lenglet, V. Braunersreuther, G. Pelli, C. Pellieux, C. Montessuit, eling, and expression. J. Biol. Chem. 277: 15354–15362. R. Lerch, M. Deruaz, A. E. Proudfoot, and F. Mach. 2010. Single administration of 32. Tanino, Y., D. R. Coombe, S. E. Gill, W. C. Kett, O. Kajikawa, A. E. Proudfoot, the CXC chemokine-binding protein Evasin-3 during ischemia prevents myocardial T. N. Wells, W. C. Parks, T. N. Wight, T. R. Martin, and C. W. Frevert. 2010. reperfusion injury in mice. Arterioscler. Thromb. Vasc. Biol. 30: 1371–1377.