Cooperativity Is Caused by Competitive Glycosaminoglycan Binding Folkert Verkaar, Jody van Offenbeek, Miranda M. C. van der Lee, Lambertus H. C. J. van Lith, Anne O. Watts, This information is current as Angelique L. W. M. M. Rops, David C. Aguilar, Joshua J. of September 27, 2021. Ziarek, Johan van der Vlag, Tracy M. Handel, Brian F. Volkman, Amanda E. I. Proudfoot, Henry F. Vischer, Guido J. R. Zaman and Martine J. Smit

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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. Published March 17, 2014, doi:10.4049/jimmunol.1302159 The Journal of Immunology

Chemokine Cooperativity Is Caused by Competitive Glycosaminoglycan Binding

Folkert Verkaar,*,† Jody van Offenbeek,*,† Miranda M. C. van der Lee,† Lambertus H. C. J. van Lith,† Anne O. Watts,* Angelique L. W. M. M. Rops,‡ David C. Aguilar,x Joshua J. Ziarek,{,1 Johan van der Vlag,‡ Tracy M. Handel,x Brian F. Volkman,{ Amanda E. I. Proudfoot,‖ Henry F. Vischer,* Guido J. R. Zaman,#,2 and Martine J. Smit*,2

Chemokines comprise a family of secreted that activate G –coupled chemokine receptors and thereby control the migration of leukocytes during inflammation or immune surveillance. The positional information required for such migratory

behavior is governed by the binding of to membrane-tethered glycosaminoglycans (GAGs), which establishes a chemo- Downloaded from kine concentration gradient. An often observed but incompletely understood behavior of chemokines is the ability of unrelated chemokines to enhance the potency with which another chemokine subtype can activate its cognate receptor. This phenomenon has been demonstrated to occur between many chemokine combinations and across several model systems and has been dubbed chemokine cooperativity. In this study, we have used GAG binding-deficient chemokine mutants and cell-based functional (mi- gration) assays to demonstrate that chemokine cooperativity is caused by competitive binding of chemokines to GAGs. This

mechanistic explanation of chemokine cooperativity provides insight into chemokine gradient formation in the context of inflam- http://www.jimmunol.org/ mation, in which multiple chemokines are secreted simultaneously. The Journal of Immunology, 2014, 192: 000–000.

hemokines are 8- to 12-kDa–sized secreted proteins that of Ca2+ from intracellular stores. Furthermore, activated chemokine mediate the directed migration () of leukocytes. receptorsbindtothescaffolding protein b-arrestin (1–3). Chemokine C The chemokine family encompasses nearly 50 members, receptor activation mediates leukocyte chemotaxis toward lym- which are classified based on the relative position of their conserved phoid organs or sites of inflammation along a chemokine gradient N-terminal cysteine residues (CC, CXC, CX3C, and C). Chemokines that is established by binding of chemokines to membrane-tethered

elicit intracellular responses via G protein–coupled receptors. Upon and extracellular matrix–associated glycosaminoglycans (GAGs) by guest on September 27, 2021 ligand binding, chemokine receptors activate G proteins of the Gai (4). GAGs represent a heterogeneous population of unbranched family, leading to inhibition of adenylyl cyclases and mobilization polysaccharides, with heparin, heparan sulfate, dermatan sulfate, chondroitin sulfate, and hyaluronic acid comprising the largest groups. *Division of Medicinal Chemistry, Faculty of Sciences, VU University Amsterdam, 1081 HV Amsterdam The Netherlands; †Molecular Pharmacology and Drug Metab- An interesting feature of chemokine biology is the ability of olism and Pharmacokinetics, Merck Research Laboratories, 5340 BH Oss, The Neth- distantly related chemokines to enhance each other’s function (5– erlands; ‡Department of Nephrology, Radboud University Medical Centre, 6500 HC 12). This behavior is referred to as chemokine cooperativity or Nijmegen, The Netherlands; xSkaggs School of Pharmacy and Pharmaceutical Sci- ences, University of California, La Jolla, CA 92093; {Department of Biochemistry, chemokine synergy, but a clear mechanistic understanding of Medical College of Wisconsin, Milwaukee, WI 53226; ‖Merck Serono Geneva Re- this phenomenon is lacking. We became interested in chemokine # search Centre, 1202 Geneva, Switzerland; and Netherlands Translational Research cooperativity while testing the ability of several chemokines to Center B.V., 4340 AG Oss, The Netherlands activate the CCX-CKR. CCX-CKR is an 1Current address: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA. atypical chemokine receptor in that it does not activate conven- 2G.J.R.Z. and M.J.S. contributed equally to this work. tional G protein–mediated signaling pathways (3, 13, 14), but it does recruit the scaffolding protein b-arrestin2 upon activation Received for publication August 14, 2013. Accepted for publication February 11, 2014. by CCL19, CCL21, and CCL25 (3). We noted that several che- This work was supported by National Institutes of Health/National Institute of Al- mokines that did not activate or bind CCX-CKR directly enhanced lergy and Infectious Diseases Grant R01 AI37113 (to T.M.H.), National Institutes the potency of CCL19 and CCL21 to activate CCX-CKR. We sub- of Health/National Institute of General Medical Sciences Grant K12 GM068524 sequently observed that multiple chemokine combinations can IRACDA (to D.C.A.), Dutch Kidney Foundation Grant KJPB 09.01 and GLYCOREN Consortium Grant CP09.03 (to A.L.W.M.M.R. and J.v.d.V.), and Dutch Top Institute synergistically enhance the activation of typical (G protein–cou- Pharma Initiative Program D1-105 (to F.V. and J.v.O.). pled) chemokine receptors, such as CCR7 and CXCR5. Through Address correspondence and reprint requests to Dr. Folkert Verkaar, VU University analysis of chemokine mutants that are deficient in GAG binding, Amsterdam, De Boelelaan 1083, Amsterdam, 1081 HV, The Netherlands. E-mail we discovered that chemokine cooperativity is mediated by com- address: [email protected] petitive binding of chemokines to GAGs. The online version of this article contains supplemental material. Abbreviations used in this article: BCS, bovine calf serum; CHO, Chinese hamster ovary; EFC, enzyme fragment complementation; GAG, glycosaminoglycan; [125I]- Materials and Methods CCL19, [125I]-labeled CCL19; mtCXCL, mutant CXCL; wtCXCL12, wild- Reagents type CXCL12. All wild-type chemokines were obtained from PeproTech (London, U.K.). 125 125 Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 [ I]-labeled CCL19 ([ I]-CCL19) was from Perkin Elmer (Boston,

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302159 2 CHEMOKINE COOPERATIVITY IS GAG DEPENDENT

MA). Forskolin was from Sigma-Aldrich (Steinheim, Germany). Produc- activate or inhibit CCX-CKR in CHO cells genetically engineered tion of mutant CXCL (mtCXCL)12 (15), mtCXCL11 (16), CXCL121 (17), to express complementary b-galactosidase mutants coupled to and CXCL122 (18) has been described previously. CCX-CKR and the scaffolding protein b-arrestin2 (CHO-CCX- Cell culture CKR cells). The recruitment of b-arrestin2 to activated CCX- CKR enables enzyme fragment complementation (EFC) between The generation of the Chinese hamster ovary (CHO)-CCX-CKR cells has b been described previously (3). These cells were maintained in DMEM F12 the two -galactosidase mutants, thus allowing measurement of (PAA Laboratories, Co¨lbe, Germany) supplemented with 10% v/v bovine receptor activation by assessing b-galactosidase activity (3, 20). In calf serum (BCS; Hyclone, Logan, UT), 250 mg/ml hygromycin, 800 mg/ml accordance, when CHO-CCX-CKR cells were treated with CCL19, geneticin, 100 U/ml penicillin, and 100 mg/ml streptomycin (all from Invi- b CCL21, and CCL25, they responded with a concentration-dependent trogen, Carlsbad, CA). CHO cells stably expressing the -arrestin2-EA fu- b sion protein and G protein–coupled receptors C-terminally extended with increase in -galactosidase activity (3) (Fig. 1A). In contrast, a complementary b-galactosidase mutant (Prolink) (i.e., CHO-CCR7, CXCL13 did not activate CCX-CKR in these cells (Fig. 1A, CHO-CXCR5, and CHO-CCR5 cells) were purchased from DiscoveRx filled circles). CXCL13 was also unable to displace radiolabeled (Fremont, CA). CHO-CXCR3 cells were provided by T. van der Doelen CCL19 ([125I]-CCL19) from CHO cells expressing CCX-CKR (Merck, Sharp, and Dohme [Merck Worldwide], Oss, The Netherlands). (Supplemental Fig. 1A, filled circles). Thus, CXCL13 is unlikely These cell lines were cultured in the medium mentioned above. HEK293T and CHO cells were purchased from the American Type Culture Collection to be a ligand for CCX-CKR. Nonetheless, we found that CXCL13 and maintained in DMEM F12 supplemented with 10% v/v BCS, 100 U/ml penicillin, and 100 mg/ml streptomycin. L1.2 cells (American Type Culture Collection) were cultured in RPMI 1640 (Sigma-Aldrich) containing 10% v/v heat-inactivated BCS, 100 U/ml penicillin, 100 mg/ml streptomycin, 25 mM Downloaded from HEPES, 2 mM L-glutamine, 1% nonessential amino acids, 1 mM sodium pyruvate (all from PAA Laboratories), and 50 mM 2-ME (Invitrogen). Enzyme fragment complementation assays EFC assays were performed, as described previously (3). Briefly, cells were dispensed in a 384-well white CulturPlate (Perkin Elmer) with 1 3 104 cells in 15 ml OPTImem (Invitrogen) containing 1% BCS, 100 U/ml penicillin, and 100 mg/ml streptomycin. Following overnight culturing in a humidified incubator http://www.jimmunol.org/ (37˚C, 5% CO2, 95% humidity), cooperative and receptor-engaging chemo- kines were added in 5 ml OPTImem each, followed by 90-min incubation at 37˚C. Subsequently, cells were disrupted by addition of 12.5 ml PathHunter Detection Reagent (DiscoveRx) in the formulation specified by the manufac- turer. Following 1-h incubation at room temperature in the dark, luminescence was measured using a Victor3 multilabel plate reader (Perkin Elmer). Radioligand binding Radioligand-binding experiments were performed, as described previously (3). For saturation-binding experiments, 25 3 104 CHO cells in 100 mlculture by guest on September 27, 2021 medium were dispensed in a poly-L-lysine–coated 96-well culture plate (Greiner) and incubated overnight in a humidified incubator. Medium was replaced by 50 ml nonradiolabeled chemokine in 20 mM HEPES, 5 mM MgCl2,1mMCaCl2, and 100 mM NaCl (pH 7.4) at 4˚C (assay medium), followed by addition of 50 ml[125I]-CCL19 in assay medium. Plates were incubated for 3 h at 4˚C and then washed with ice-cold assay medium sup- plemented with 500 mM NaCl. Cells were disrupted by addition of 150 ml radioimmunoprecipitation assay buffer (Sigma-Aldrich), and radioactivity in lysates was measured with a Compugamma gamma counter (Perkin Elmer). Bare-filter migration (chemotaxis) assays A total of 30 ml chemokine in RPMI 1640 (Sigma-Aldrich) containing 0.1% BSA (assay buffer) was added to the lower compartment of a 96-well ChemoTx plate (5 mm pore size; Neuroprobe, Gaithersburg, MD). A quantity amounting to 25 3 104 L1.2 cells in 20 ml assay buffer was administered to the upper compartment of the plate. Cells were then allowed to migrate through the filter for 5 h in a humidified incubator (37˚C). Twenty-five microliter assay buffer from the lower well was then FIGURE 1. CXCL13 increases the ability of CCL19 to activate CCX- transferred to a white 96-well CulturPlate Plate (Perkin Elmer), followed CKR. (A) CHO cells stably expressing CCX-CKR fused to a peptide frag- by addition of 25 ml calcein AM (1 mg/ml final concentration) in assay ment of b-galactosidase and b-arrestin2 coupled to a complementary buffer. Fluorescence intensities were measured on a Victor3 plate reader. b-galactosidase mutant (CHO-CCX-CKR cells) were treated with increas- Data analysis ing concentrations of CCL19 (N)orCXCL13(d) before measurement of b-galactosidase activity. In addition, CHO-CCX-CKR cells were treated Sigmoidal dose-response curves were plotted using Graphpad Prism 6.0 6 with increasing concentrations of CXCL13 in the presence of 2 nM CCL19 software. All data are presented as averages SEM. Statistical significance ; of observed differences was determined using Student t test or two-way ( ). Asterisks (*) indicate statistically significant differences (two-way , ANOVA, as indicated in the figure legend and indicated in the figures with ANOVA, p 0.05) between cells treated with 2 nM CCL19 in the presence asterisks (*). A p value , 0.05 was regarded as statistically significant. and absence of CXCL13. (B) CHO-CCX-CKR cells were treated with increasing concentrations of CCL19 in the presence of 1000 nM CXCL13, Results 100 nM CXCL13, or corresponding vehicle, followed by measurement of b 6 CXCL13 enhances the ability of CCL19, CCL21, and CCL25 to -galactosidase activity. pEC50 values were as follows: vehicle: 8.6 0.2 6 6 activate CCX-CKR (4), 100 nM CXCL13: 9.1 0.2 (4), and 1000 nM CXCL13: 9.4 0.1 (4) (average 6 SD [n]). Differences in pEC50 values between vehicle-treated Previous studies have suggested that CXCL13 is a ligand for CCX- cells and cells treated with either 100 or 1000 nM CXCL13 reached CKR (13, 19). We therefore tested the ability of this chemokine to statistical significance (Student t test, p , 0.05). The Journal of Immunology 3

concentration dependently enhanced the potency with which following recruitment of b-arrestin2 to the Gai-coupled chemokine CCL19 activated CCX-CKR (Fig. 1). This effect was not limited to receptors CCR7 (CHO-CCR7), CCR5 (CHO-CCR5), and CXCR5 activation of CCX-CKR by CCL19, as similar results were (CHO-CXCR5). CXCL13 enhanced the potency with which CCL19 observed in experiments using CCL21 and CCL25 (Supplemental induced b-arrestin recruitment and Gai activation by CCR7 in CHO- Fig. 1B, 1C). CCR7 cells (Fig. 2A, 2B). Reciprocally, CCL19 enhanced the potency Secondly, we measured b-arrestin2 recruitment to CCX-CKR of CXCL13 toward activation of CXCR5 (Fig. 2C). However, both using a bioluminescence resonance energy transfer approach (3). CCL19 and CXCL13 were incapable of potentiating CCL3-induced For this, we transiently transfected HEK293T cells with vectors activation of CCR5 (Fig. 2D). encoding CCX-CKR genetically extended with Renilla luciferase Next, a panel of chemokines was tested for their ability to co- and b-arrestin2 fused to yellow fluorescent protein. Similar to operate with CCL19 in activation of CCX-CKR. Several chemo- results for the b-galactosidase complementation assay, CXCL13 kines other than CXCL13, including CXCL12 and CXCL11, were increased the potency with which CCL19 activated CCX-CKR found to cooperate with CCL19, whereas several others (CCL3, (Supplemental Fig. 1D). Thus, CXCL13 cooperates with CCL19, CCL4, and CCL23) did not (Fig. 2E). Essentially the same pattern CCL21, and CCL25 in activating CCX-CKR. was observed when CCX-CKR was activated with CCL21, as follows: CXCL13, CXCL12, and CXCL11 cooperated with CCL21, Chemokine cooperativity is specific for certain chemokine whereas CCL3 and CCL4 did not (Supplemental Fig. 2). combinations We then tested whether the cooperative effect of CXCL13 was CXCL13 enhances CCL19-mediated chemotaxis specific for certain chemokine pairs or chemokine receptors. To Next, we used a transwell migration assay to test whether the en- Downloaded from this end, we used CHO cells that complement b-galactosidase hanced receptor activity translated to a more potent chemotactic http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 2. Distinct chemokine combinations display cooperativity. (A) CHO-CCR7 cells were treated with increasing concentrations of CCL19 in the presence of 1000 nM CXCL13, 100 nM CXCL13, or corresponding vehicle, followed by measurement of b-galactosidase activity. (B) CHO-CCR7 cells were treated with 0.5 mM forskolin and increasing concentrations of CCL19 in the presence of 1000 or 100 nM CXCL13, followed by measurement of cAMP levels. (C) CHO-CXCR5 cells were treated with increasing concentrations of CXCL13 in the presence of 1000 nM CCL19, 100 nM CCL19, or corresponding vehicle, followed by measurement of b-galactosidase activity. (D) CHO-CCR5 cells were treated with ascending concentrations of CCL3 and 1000 nM CCL19, 1000 nM CXCL13, or corresponding vehicle, followed by measurement of b-galactosidase activity. (E) CHO-CCX-CKR cells were treated with 2 nM CCL19 in the absence or presence of 1000 nM of the indicated chemokines (x-axis), followed by measurement of b-galactosidase activity. Asterisks (*) indicate statistically significant differences (Student t test, p , 0.05). 4 CHEMOKINE COOPERATIVITY IS GAG DEPENDENT response. We used murine L1.2 cells, a pre-B lymphoma cell line, Chemokine cooperativity is mediated by both chemokine which migrated toward a gradient of CCL19 (Fig. 3A), implying that monomers and dimers these cells endogenously express CCR7. However, the L1.2 cells Chemokine heterodimerization has previously been suggested to did not migrate toward CXCL13 (Fig. 3A). In accordance with our underlie chemokine cooperativity (10). GAG binding and dimer- m functional assays, 1 M CXCL13 significantly potentiated CCL19- ization of chemokines are intimately linked processes (18, 22). mediated chemotaxis in these cells (Fig. 3B). Indeed, the CXCL12-GAG binding interface significantly overlaps Chemokine cooperativity is dependent on GAG binding with the CXCL12 (homo)dimerization surface, and mtCXCL12 is not only impaired in its ability to bind to GAGs but also in its Our analysis revealed that CCL3 and CCL4 did not induce che- ability to dimerize (18). To differentiate between dimerization and mokine cooperativity (Fig. 2E). These chemokines bind to several GAG binding as potential mechanisms for chemokine coopera- GAGs with relatively low affinity compared with other chemo- tivity, we used a CXCL12 mutant that forms obligate monomers kines (e.g., CXCL8, CCL2, and CCL5) (21, 22). To investigate the (termed CXCL12 ) and a constitutively dimeric mutant of importance of GAG binding for chemokine cooperativity, we 1 CXCL12 (CXCL12 ) (17, 18, 24, 25). CXCL12 and CXCL12 tested the ability of GAG-binding–deficient chemokine mutants to 2 1 2 are strictly monomeric and dimeric at concentrations up to 10 mM cooperate in CCX-CKR activation. Such mutants have been de- (17, 24). Both mutants bound to heparin, although CXCL12 did scribed for CXCL12 and CXCL11 (15, 16, 23). In sharp contrast 1 so with a ∼10-fold lower potency than wtCXCL12 (17–18). When to wildtype CXCL12 (wtCXCL12), a GAG binding-deficient tested in the EFC assay on CHO-CCX-CKR cells, both CXCL121 and CXCL12 mutant (CXCL12K24S,H25S,K27S, hereafter referred to as CXCL122 cooperated with CCL21 in the activation of CCX-CKR mtCXCL12) did not cooperate with CCL21 in the EFC assay Downloaded from (Fig. 4C). In conclusion, both monomeric and dimeric CXCL12 (Fig. 4A). Likewise, we found that mtCXCL12 did not cooperate proteins can mediate chemokine cooperativity. with CCL21 in activating CCX-CKR in the bioluminescence resonance energy transfer assay (Supplemental Fig. 3). Simi- Chemokine cooperativity is caused by competitive GAG larly, a GAG-binding–deficient mtCXCL11 (CXCL11 50s or binding CXCL11K57A,K59A,R62A) was impaired in its ability to cooperate The simplest model that explains the necessity for GAG bind-

with CCL21 in activating CCX-CKR (Fig. 4B). Both mtCXCL12 http://www.jimmunol.org/ and mtCXCL11 were capable of activating their cognate receptors ing in chemokine cooperativity is one in which the cooperative with potencies and efficacies comparable to wild-type chemokines chemokines displace the receptor-engaging chemokine from (Supplemental Fig. 4). binding to GAGs. This scenario would result in elevated con- centrations of soluble receptor-engaging chemokines that are available to bind and activate receptors. To test whether CXCL12 and CXCL13 compete with CCL19 for binding to GAGs on the cell surface, we monitored the binding of [125I]-CCL19 to CHO cells. The CHO cells did not express CCX-CKR or CCR7, as determined

by RT-PCR (data not shown), whereas they abundantly express by guest on September 27, 2021 GAGs (23, 26). Incubation of CHO cells with [125I]-CCL19 led to a concentration-dependent increase in cell-associated radioactivity, a large portion of which could be competed for by adding excess (1 mM) nonlabeled CCL19 (Fig. 5A). wtCXCL12 was also capable of displacing [125I]-CCL19 from CHO cells, whereas mtCXCL12 was not (Fig. 5A). Similarly, CXCL13 displaced 0.5 nM [125I]- CCL19 from both CHO and HEK293T cells, whereas the nonco- operative chemokine CCL3 did not affect [125I]-CCL19 binding to these cells (Fig. 5B and data not shown). These data indicate that cooperative chemokines compete with CCL19 for GAG binding on cells, whereas noncooperative chemokines do not (Fig. 6).

Discussion Chemokine cooperativity has previously been suggested to be caused by convergence of intracellular signaling pathways down- stream of receptor activation (5–7, 11, 12). Our data indicate that chemokine cooperativity is not strictly dependent on the receptor of the cooperative chemokine. For example, mtCXCL12 and mtCXCL11 fully activate their respective G protein–coupled receptors (Supplemental Fig. 4) but do not cooperate with CCL21 (Fig. 4). Furthermore, RT-PCR analyses and cAMP measurements indicated that CHO-CCX-CKR cells do not ex- FIGURE 3. CXCL13 enhances the ability of CCL19 to induce chemo- press CXCR5 or CXCR4, the cognate Ga -coupled receptors for taxis. (A) Murine pre-B lymphoma (L1.2) cells were tested for their ability i CXCL13 and CXCL12, respectively (data not shown). Our data to migrate toward ascending concentrations of CCL19 or CXCL13 in a bare-filter migration assay. (B) L1.2 cells were tested for their ability to concur with previous evidence suggesting that activation of the migrate toward ascending concentrations of CCL19 in the absence or receptor for the cooperative chemokine is not necessary for presence of 1 mM CXCL13. Asterisks (*) indicate statistically significant cooperativity (8, 9). differences (two-way ANOVA, p , 0.05) between vehicle- and CXCL13- Alternatively, chemokine cooperativity has been suggested to treated cells. originate from chemokine heterodimerization (9, 10). Our obser- The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/

FIGURE 5. Cooperative chemokines displace membrane-associated CCL19. (A) CHO cells were treated with increasing concentrations of radiolabeled CCL19 ([125I]-CCL19) in the presence of 1 mM CCL19, wtCXCL12, mtCXCL12, or vehicle, followed by determination of cell- associated radioactivity. (B) CHO cells were incubated with 0.5 nM [125I]-

CCL19 in the presence of 1 mM CCL19, CXCL13, and CCL3, followed by by guest on September 27, 2021 determination of cell-associated radioactivity. Asterisks (*) indicate sta- tistically significant differences (Student t test, p , 0.01).

vation that both monomeric and dimeric CXCL12 species can induce chemokine cooperativity argues against this mode of ac- tion. In this study, we demonstrate a determining role for GAG binding in chemokine cooperativity. Our data suggest that coop- erative chemokines compete for binding to GAGs, thereby raising the effective free concentration of chemokine that can engage its FIGURE 4. Mutant chemokines that do not bind to GAGs do not display receptor (Fig. 6). Other researchers performed experiments on chemokine cooperativity. (A) CHO-CCX-CKR cells were treated with wtCXCL12 and a CXCL12 mutant that is impaired in its ability to bind to heparinase-treated cells to test whether GAGs influence chemo- GAGs (mtCXCL12) in the presence of increasing concentrations of CCL21. kine cooperativity and observed no effect of heparinase treatment

Subsequently, b-galactosidase activity was determined. pEC50 values were as on chemokine cooperativity (8, 10). Similarly, pretreatment of follows: vehicle: 7.7 6 0.3 (5), wtCXCL12: 8.7 6 0.4 (5), and mtCXCL12: CHO-CCX-CKR cells with a mixture of heparinases did not in- 7.7 6 0.2 (5) (average 6 SD [n]). Values for vehicle-treated and mtCXCL12- fluence the ability of CXCL13 to cooperate with CCL19 (data not treated cells were not statistically different, whereas wtCXCL12 signifi- shown). A flaw in this approach is that GAGs form a large het- cantly differed from the other treatments (Student t test, p , 0.01). erogeneous population of molecules, and it is experimentally B m ( ) CHO-CCX-CKR cells were treated with CCL21 and 1 M wild-type challenging to ascertain that all of the GAGs relevant to chemo- m CXCL11 (wtCXCL11) or 1 M mtCXCL11 that is impaired in its ability kine cooperativity are removed by treatment with heparinase and to bind GAGs, followed by measurement of b-galactosidase activity. chondroitinase mixtures. Our analysis with GAG-binding–defi- pEC50 values were as follows: vehicle: 7.8 6 0.2 (3), wtCXCL11: 9.6 6 0.3 (3), and mtCXCL11: 8.3 6 0.4 (3) (average 6 SD [n]). Values for cient mutants represents a method of analysis that lacks this pit- vehicle-treated and mtCXCL11-treated cells were not statistically dif- fall. A remaining challenge will be to deduce which GAG ferent, whereas wtCXCL11 significantly differed from the other treat- subtypes are actually involved in chemokine cooperativity. Such ments (Student t test, p , 0.01). (C) CHO-CCX-CKR cells were treated with either 1 mM wtCXCL12, constitutively monomeric (CXCL121), or dimeric CXCL12 (CXCL122) in the presence of increasing concentrations between values for vehicle-treated cells and cells treated with all three of CCL21, followed by b-galactosidase measurement. pEC50 values were CXCL12 proteins reached statistical significance (Student t test, p , 0.01). as follows: vehicle: 7.7 6 0.2 (6), wtCXCL12: 8.5 6 0.2 (6), CXCL121: pEC50 values for wtCXCL12, CXCL121, and CXCL122 were not statis- 8.3 6 0.3 (6), and CXCL122: 8.5 6 0.5 (6) (average 6 SD [n]). Differences tically different. 6 CHEMOKINE COOPERATIVITY IS GAG DEPENDENT

(38–41), might alter chemokine cooperativity. Lastly, our data present a new perspective on therapeutic targeting of the chemo- kine system. Through modulation of cooperativity, therapeutic strategies aimed at inhibiting chemokine–GAG interactions might establish a broader effect on leukocyte trafficking than targeting chemokine receptors. For instance, chemokine cooperativity is likely to be modulated by administration of heparin or GAG derivatives, which have previously been shown to affect chemo- kine gradient formation (42, 43). Furthermore, small molecules or Abs targeting chemokines, such as those described for CXCL12 (44–46), may have applicability as cooperativity-disrupting drugs. Future endeavors will have to elucidate how chemokine cooper- ativity is modulated in such instances.

Acknowledgments We thank Marloes van der Zwam (University of Groningen, Groningen, The Netherlands), and Winfried Mulder and Toon van der Doelen (Merck, Sharp, and Dohme [Merck Worldwide]) for providing reagents. We grate-

fully acknowledge Azra Mujic-Delic, Maarten Bebelman, Quinte Braster, Downloaded from Danny Scholten, and Sabrina de Munnik (VU University Amsterdam) for FIGURE 6. A model for chemokine cooperativity. (A) In the absence of expert technical assistance. We thank Danny Scholten, Saskia Nijmeijer cooperative chemokines, CCL19/CCL21 binds to its seven-transmembrane (VU University Amsterdam), and Laura Heitman (Leiden University, Lei- receptors (CCX-CKR or CCR7) or to membrane-tethered GAGs. (B) In the den, The Netherlands) for helpful discussions. presence of cooperative chemokines, CCL19/CCL21 is competed from the GAGs, raising the free concentration of CCL19, which results in enhanced Disclosures association of CCL19/CCL21 with its receptors and consequent receptor http://www.jimmunol.org/ activation. The authors have no financial conflicts of interest.

GAGs may be identified by studying the ability of chemokines to References compete for binding on arrays or columns containing immobilized 1. Lohse, M. J., J. L. Benovic, J. Codina, M. G. Caron, and R. J. Lefkowitz. 1990. beta-Arrestin: a protein that regulates beta-adrenergic receptor function. Science synthetic or purified GAGs (21, 27). 248: 1547–1550. Another outstanding question is whether chemokine coopera- 2. Canals, M., D. J. Scholten, S. de Munnik, M. K. Han, M. J. Smit, and R. Leurs. tivity occurs in vivo. Although systemic, homeostatic levels of 2012. Ubiquitination of CXCR7 controls receptor trafficking. PLoS One 7: e34192. CCL19, CCL21, CXCL13, and CXCL12 are generally much lower

3. Watts, A. O., F. Verkaar, M. M. van der Lee, C. A. Timmerman, M. Kuijer, J. van by guest on September 27, 2021 than the chemokine concentrations required to induce chemokine Offenbeek, L. H. van Lith, M. J. Smit, R. Leurs, G. J. Zaman, and H. F. Vischer. b cooperativity in our functional assays, local concentrations of 2013. -Arrestin recruitment and G protein signaling by the atypical chemokine decoy receptor CCX-CKR. J. Biol. Chem. 288: 7169–7181. chemokine may well exceed the concentrations necessary for coop- 4. Proudfoot, A. E., T. M. Handel, Z. Johnson, E. K. Lau, P. LiWang, I. Clark- erativity to occur. Of note, CCL19, CCL21, CXCL12, and CXCL13 Lewis, F. Borlat, T. N. Wells, and M. H. Kosco-Vilbois. 2003. Glycosamino- glycan binding and oligomerization are essential for the in vivo activity of have exceptionally broad and overlapping expression patterns, certain chemokines. Proc. Natl. Acad. Sci. USA 100: 1885–1890. which would provide many potential opportunities for synergistic 5. Bai, Z., H. Hayasaka, M. Kobayashi, W. Li, Z. Guo, M. H. Jang, A. Kondo, interactions. For instance, CCL19, CCL21, and CXCL12 are B. I. Choi, Y. Iwakura, and M. Miyasaka. 2009. CXC chemokine ligand 12 promotes CCR7-dependent naive T cell trafficking to lymph nodes and Peyer’s expressed on the luminal side of high endothelial venules (28), and patches. J. Immunol. 182: 1287–1295. CCL21, CXCL12, and CXCL13 are expressed in lymph nodes 6. Gouwy, M., S. Struyf, J. Catusse, P. Proost, and J. Van Damme. 2004. Synergy (29–31). Indeed, CXCL12 has been shown to enhance the CCR7- between proinflammatory ligands of G protein-coupled receptors in neutrophil activation and migration. J. Leukoc. Biol. 76: 185–194. mediated recruitment of naive T cells from high endothelial 7. Gouwy, M., S. Struyf, S. Noppen, E. Schutyser, J. Y. Springael, M. Parmentier, venules into the underlying lymphoid tissue (5). Furthermore, P. Proost, and J. Van Damme. 2008. Synergy between coproduced CC and CXC chemokines in monocyte chemotaxis through receptor-mediated events. Mol. CCL19, CCL21, CXCL12, and CXCL13 are expressed locally Pharmacol. 74: 485–495. during chronic inflammation (32–34), and individual chemo- 8. Kuscher, K., G. Danelon, S. Paoletti, L. Stefano, M. Schiraldi, V. Petkovic, kine concentrations within these inflamed sites may very well M. Locati, B. O. Gerber, and M. Uguccioni. 2009. Synergy-inducing chemokines enhance CCR2 ligand activities on monocytes. Eur. J. Immunol. 39: 1118–1128. exceed the threshold for cooperativity to occur. Additionally, 9. Sebastiani, S., G. Danelon, B. Gerber, and M. Uguccioni. 2005. CCL22-induced previous work has suggested that chemokine mixtures can con- responses are powerfully enhanced by synergy inducing chemokines via CCR4: certedly induce cooperativity at low individual chemokine evidence for the involvement of first beta-strand of chemokine. Eur. J. Immunol. 35: 746–756. concentrations (10). Because multiple chemokines are expressed 10. Paoletti, S., V. Petkovic, S. Sebastiani, M. G. Danelon, M. Uguccioni, and concomitantly during inflammation and homeostatic trafficking B. O. Gerber. 2005. A rich chemokine environment strongly enhances leukocyte migration and activities. Blood 105: 3405–3412. (35–37), the concentration of several cooperative chemokines 11. Krug, A., R. Uppaluri, F. Facchetti, B. G. Dorner, K. C. Sheehan, combined may be sufficient for chemokine cooperativity to occur. R. D. Schreiber, M. Cella, and M. Colonna. 2002. IFN-producing cells respond How would chemokine cooperativity influence chemokine to CXCR3 ligands in the presence of CXCL12 and secrete inflammatory che- mokines upon activation. J. Immunol. 169: 6079–6083. function, and how will chemokine cooperativity be altered by 12. Vanbervliet, B., N. Bendriss-Vermare, C. Massacrier, B. Homey, O. de (patho)physiological processes? Because chemokine cooperativity Bouteiller, F. Brie`re, G. Trinchieri, and C. Caux. 2003. The inducible CXCR3 would allow chemokines to activate their cognate receptors at lower ligands control plasmacytoid responsiveness to the constitutive chemokine stromal cell-derived factor 1 (SDF-1)/CXCL12. J. Exp. Med. 198: chemokine concentrations, it is likely to extend the range from 823–830. which chemokines can induce recruitment of leukocytes. Another 13. Gosling, J., D. J. Dairaghi, Y. Wang, M. Hanley, D. Talbot, Z. Miao, and T. J. Schall. 2000. Cutting edge: identification of a novel chemokine receptor that implication from our data is that alterations in GAG composition or binds dendritic cell- and T cell-active chemokines including ELC, SLC, and abundance, as have been observed to occur in several pathologies TECK. J. Immunol. 164: 2851–2856. The Journal of Immunology 7

14. Townson, J. R., and R. J. Nibbs. 2002. Characterization of mouse CCX-CKR, venules promotes the adhesion and chemotaxis of naive T lymphocytes. Proc. a receptor for the lymphocyte-attracting chemokines TECK/mCCL25, SLC/ Natl. Acad. Sci. USA 95: 258–263. mCCL21 and MIP-3beta/mCCL19: comparison to human CCX-CKR. Eur. J. 30. Ansel, K. M., V. N. Ngo, P. L. Hyman, S. A. Luther, R. Fo¨rster, J. D. Sedgwick, Immunol. 32: 1230–1241. J. L. Browning, M. Lipp, and J. G. Cyster. 2000. A chemokine-driven positive 15. O’Boyle, G., P. Mellor, J. A. Kirby, and S. Ali. 2009. Anti-inflammatory therapy feedback loop organizes lymphoid follicles. Nature 406: 309–314. by intravenous delivery of non-heparan sulfate-binding CXCL12. FASEB J. 23: 31. Hargreaves, D. C., P. L. Hyman, T. T. Lu, V. N. Ngo, A. Bidgol, G. Suzuki, 3906–3916. Y. R. Zou, D. R. Littman, and J. G. Cyster. 2001. A coordinated change in 16. Severin, I. C., J. P. Gaudry, Z. Johnson, A. Kungl, A. Jansma, B. Gesslbauer, chemokine responsiveness guides plasma cell movements. J. Exp. Med. 194: 45– B. Mulloy, C. Power, A. E. Proudfoot, and T. Handel. 2010. Characterization of 56. the chemokine CXCL11-heparin interaction suggests two different affinities for 32. Mazzucchelli, L., A. Blaser, A. Kappeler, P. Scha¨rli, J. A. Laissue, glycosaminoglycans. J. Biol. Chem. 285: 17713–17724. M. Baggiolini, and M. Uguccioni. 1999. BCA-1 is highly expressed in Heli- 17. Ziarek, J. J., A. E. Getschman, S. J. Butler, D. Taleski, B. Stephens, I. Kufareva, cobacter pylori-induced mucosa-associated lymphoid tissue and gastric lym- T. M. Handel, R. J. Payne, and B. F. Volkman. 2013. Sulfopeptide probes of the phoma. J. Clin. Invest. 104: R49–R54. CXCR4/CXCL12 interface reveal oligomer-specific contacts and chemokine 33. Hjelmstro¨m, P., J. Fjell, T. Nakagawa, R. Sacca, C. A. Cuff, and N. H. Ruddle. allostery. ACS Chem. Biol. 8: 1955–1963. 2000. Lymphoid tissue homing chemokines are expressed in chronic inflam- 18. Ziarek, J. J., C. T. Veldkamp, F. Zhang, N. J. Murray, G. A. Kartz, X. Liang, mation. Am. J. Pathol. 156: 1133–1138. J. Su, J. E. Baker, R. J. Linhardt, and B. F. Volkman. 2013. Heparin oligo- 34. Pablos, J. L., A. Amara, A. Bouloc, B. Santiago, A. Caruz, M. Galindo, saccharides inhibit chemokine (CXC motif) ligand 12 (CXCL12) cardioprotection T. Delaunay, J. L. Virelizier, and F. Arenzana-Seisdedos. 1999. Stromal-cell by binding orthogonal to the dimerization interface, promoting oligomerization, derived factor is expressed by dendritic cells and endothelium in human skin. and competing with the chemokine (CXC motif) receptor 4 (CXCR4) N terminus. Am. J. Pathol. 155: 1577–1586. J. Biol. Chem. 288: 737–746. 35. Adams, D. H., and A. R. Lloyd. 1997. Chemokines: leucocyte recruitment and 19. Feng, L. Y., Z. L. Ou, F. Y. Wu, Z. Z. Shen, and Z. M. Shao. 2009. Involvement activation . Lancet 349: 490–495. of a novel chemokine decoy receptor CCX-CKR in breast cancer growth, me- 36. Mahalingam, S., and G. Karupiah. 1999. Chemokines and chemokine receptors tastasis and patient survival. Clin. Cancer Res. 15: 2962–2970. in infectious diseases. Immunol. Cell Biol. 77: 469–475. 20. Verkaar, F., J. W. van Rosmalen, M. Blomenro¨hr, C. J. van Koppen, 37. Luster, A. D. 1998. Chemokines—chemotactic cytokines that mediate inflam- W. M. Blankesteijn, J. F. Smits, and G. J. Zaman. 2008. G protein-independent mation. N. Engl. J. Med. 338: 436–445. Downloaded from cell-based assays for drug discovery on seven-transmembrane receptors. Bio- 38. Taylor, K. R., and R. L. Gallo. 2006. Glycosaminoglycans and their proteo- technol. Annu. Rev. 14: 253–274. glycans: host-associated molecular patterns for initiation and modulation of in- 21. Kuschert, G. S., F. Coulin, C. A. Power, A. E. Proudfoot, R. E. Hubbard, flammation. FASEB J. 20: 9–22. A. J. Hoogewerf, and T. N. Wells. 1999. Glycosaminoglycans interact selectively 39. Sasisekharan, R., Z. Shriver, G. Venkataraman, and U. Narayanasami. 2002. with chemokines and modulate receptor binding and cellular responses. Bio- Roles of heparan-sulphate glycosaminoglycans in cancer. Nat. Rev. Cancer 2: chemistry 38: 12959–12968. 521–528. 22. Hoogewerf, A. J., G. S. Kuschert, A. E. Proudfoot, F. Borlat, I. Clark-Lewis, 40. Lortat-Jacob, H. 2009. The molecular basis and functional implications of

C. A. Power, and T. N. Wells. 1997. Glycosaminoglycans mediate cell surface chemokine interactions with heparan sulphate. Curr. Opin. Struct. Biol. 19: 543– http://www.jimmunol.org/ oligomerization of chemokines. Biochemistry 36: 13570–13578. 548. 23. Amara, A., O. Lorthioir, A. Valenzuela, A. Magerus, M. Thelen, M. Montes, 41. Rops, A. L., J. van der Vlag, J. F. Lensen, T. J. Wijnhoven, L. P. van den Heuvel, J. L. Virelizier, M. Delepierre, F. Baleux, H. Lortat-Jacob, and F. Arenzana- T. H. van Kuppevelt, and J. H. Berden. 2004. Heparan sulfate proteoglycans in Seisdedos. 1999. Stromal cell-derived factor-1alpha associates with heparan glomerular inflammation. Kidney Int. 65: 768–785. sulfates through the first beta-strand of the chemokine. J. Biol. Chem. 274: 42. Johnson, Z., M. H. Kosco-Vilbois, S. Herren, R. Cirillo, V. Muzio, P. Zaratin, 23916–23925. M. Carbonatto, M. Mack, A. Smailbegovic, M. Rose, et al. 2004. Interference 24.Veldkamp,C.T.,C.Seibert,F.C.Peterson,N.B.DelaCruz,J.C.Haugner, with heparin binding and oligomerization creates a novel anti-inflammatory III,H.Basnet,T.P.Sakmar,andB.F.Volkman. 2008. Structural basis of strategy targeting the chemokine system. J. Immunol. 173: 5776–5785. CXCR4 sulfotyrosine recognition by the chemokine SDF-1/CXCL12. Sci. 43. Severin, I. C., A. Soares, J. Hantson, M. Teixeira, D. Sachs, D. Valognes, Signal. 1: ra4. A. Scheer, M. K. Schwarz, T. N. Wells, A. E. Proudfoot, and J. Shaw. 2012. 25. Drury, L. J., J. J. Ziarek, S. Gravel, C. T. Veldkamp, T. Takekoshi, S. T. Hwang, Glycosaminoglycan analogs as a novel anti-inflammatory strategy. Front.

N. Heveker, B. F. Volkman, and M. B. Dwinell. 2011. Monomeric and dimeric Immunol. 3: 293. by guest on September 27, 2021 CXCL12 inhibit metastasis through distinct CXCR4 interactions and signaling 44. Hachet-Haas, M., K. Balabanian, F. Rohmer, F. Pons, C. Franchet, S. Lecat, pathways. Proc. Natl. Acad. Sci. USA 108: 17655–17660. K. Y. Chow, R. Dagher, P. Gizzi, B. Didier, et al. 2008. Small neutralizing 26. Zhang, L., R. Lawrence, B. A. Frazier, and J. D. Esko. 2006. CHO glycosylation molecules to inhibit actions of the chemokine CXCL12. J. Biol. Chem. 283: mutants: proteoglycans. Methods Enzymol. 416: 205–221. 23189–23199. 27. de Paz, J. L., E. A. Moseman, C. Noti, L. Polito, U. H. von Andrian, and 45. Blanchetot, C., D. Verzijl, A. Mujic´-Delic´, L. Bosch, L. Rem, R. Leurs, P. H. Seeberger. 2007. Profiling heparin-chemokine interactions using synthetic C. T. Verrips, M. Saunders, H. de Haard, and M. J. Smit. 2013. Neutralizing tools. ACS Chem. Biol. 2: 735–744. nanobodies targeting diverse chemokines effectively inhibit chemokine function. 28. Okada, T., V. N. Ngo, E. H. Ekland, R. Fo¨rster, M. Lipp, D. R. Littman, and J. Biol. Chem. 288: 25173–25182. J. G. Cyster. 2002. Chemokine requirements for entry to lymph nodes and 46. Gonzalo, J. A., C. M. Lloyd, A. Peled, T. Delaney, A. J. Coyle, and Peyer’s patches. J. Exp. Med. 196: 65–75. J. C. Gutierrez-Ramos. 2000. Critical involvement of the chemotactic axis 29. Gunn, M. D., K. Tangemann, C. Tam, J. G. Cyster, S. D. Rosen, and CXCR4/stromal cell-derived factor-1 alpha in the inflammatory component of L. T. Williams. 1998. A chemokine expressed in lymphoid high endothelial allergic airway disease. J. Immunol. 165: 499–508.