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Competitive Glycosaminoglycan BindingChemokine Cooperativity Is Caused by

J. R. Zaman and Martine J. SmitVolkman, Amanda E. I. Proudfoot, Henry F. Vischer, GuidoZiarek, Johan van der Vlag, Tracy M. Handel, Brian F. Angelique L. W. M. M. Rops, David C. Aguilar, Joshua J.der Lee, Lambertus H. C. J. van Lith, Anne O. Watts, Folkert Verkaar, Jody van Offenbeek, Miranda M. C. van

ol.1302159http://www.jimmunol.org/content/early/2014/03/17/jimmun

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The Journal of Immunology

Chemokine Cooperativity Is Caused by CompetitiveGlycosaminoglycan 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 proteins that activate G protein–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 chemokines to membrane-tethered glycosaminoglycans (GAGs), which establishes a chemo-

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-

mation, in which multiple chemokines are secreted simultaneously. The Journal of Immunology, 2014, 192: 000–000.

Chemokines are 8- to 12-kDa–sized secreted proteins thatmediate the directed migration (chemotaxis) of leukocytes.The chemokine family encompasses nearly 50 members,

which are classified based on the relative position of their conservedN-terminal cysteine residues (CC, CXC, CX3C, and C). Chemokineselicit intracellular responses via G protein–coupled receptors. Uponligand binding, chemokine receptors activate G proteins of the Gai

family, leading to inhibition of adenylyl cyclases and mobilization

of Ca2+ from intracellular stores. Furthermore, activated chemokinereceptors bind to the scaffolding protein b-arrestin (1–3). Chemokinereceptor activation mediates leukocyte chemotaxis toward lym-phoid organs or sites of inflammation along a chemokine gradientthat is established by binding of chemokines to membrane-tetheredand extracellular matrix–associated glycosaminoglycans (GAGs)(4). GAGs represent a heterogeneous population of unbranchedpolysaccharides, with heparin, heparan sulfate, dermatan sulfate,chondroitin sulfate, and hyaluronic acid comprising the largestgroups.An interesting feature of chemokine biology is the ability of

distantly related chemokines to enhance each other’s function (5–12). This behavior is referred to as chemokine cooperativity orchemokine synergy, but a clear mechanistic understanding ofthis phenomenon is lacking. We became interested in chemokinecooperativity while testing the ability of several chemokines toactivate the chemokine receptor CCX-CKR. CCX-CKR is anatypical chemokine receptor in that it does not activate conven-tional G protein–mediated signaling pathways (3, 13, 14), but itdoes recruit the scaffolding protein b-arrestin2 upon activationby CCL19, CCL21, and CCL25 (3). We noted that several che-mokines that did not activate or bind CCX-CKR directly enhancedthe potency of CCL19 and CCL21 to activate CCX-CKR. We sub-sequently observed that multiple chemokine combinations cansynergistically enhance the activation of typical (G protein–cou-pled) chemokine receptors, such as CCR7 and CXCR5. Throughanalysis of chemokine mutants that are deficient in GAG binding,we discovered that chemokine cooperativity is mediated by com-petitive binding of chemokines to GAGs.

Materials and MethodsReagents

All wild-type chemokines were obtained from PeproTech (London, U.K.).[125I]-labeled CCL19 ([125I]-CCL19) was from Perkin Elmer (Boston,

*Division of Medicinal Chemistry, Faculty of Sciences, VU University Amsterdam,1081 HVAmsterdam The Netherlands; †Molecular Pharmacology and Drug Metab-olism and Pharmacokinetics, Merck Research Laboratories, 5340 BH Oss, The Neth-erlands; ‡Department of Nephrology, Radboud University Medical Centre, 6500 HCNijmegen, The Netherlands; xSkaggs School of Pharmacy and Pharmaceutical Sci-ences, University of California, La Jolla, CA 92093; {Department of Biochemistry,Medical College of Wisconsin, Milwaukee, WI 53226; ‖Merck Serono Geneva Re-search Centre, 1202 Geneva, Switzerland; and #Netherlands Translational ResearchCenter B.V., 4340 AG Oss, The Netherlands

1Current address: Department of Biological Chemistry and Molecular Pharmacology,Harvard Medical School, Boston, MA.

2G.J.R.Z. and M.J.S. contributed equally to this work.

Received for publication August 14, 2013. Accepted for publication February 11,2014.

This work was supported by National Institutes of Health/National Institute of Al-lergy and Infectious Diseases Grant R01 AI37113 (to T.M.H.), National Institutesof Health/National Institute of General Medical Sciences Grant K12 GM068524IRACDA (to D.C.A.), Dutch Kidney Foundation Grant KJPB 09.01 and GLYCORENConsortium Grant CP09.03 (to A.L.W.M.M.R. and J.v.d.V.), and Dutch Top InstitutePharma Initiative Program D1-105 (to F.V. and J.v.O.).

Address correspondence and reprint requests to Dr. Folkert Verkaar, VU UniversityAmsterdam, De Boelelaan 1083, Amsterdam, 1081 HV, The Netherlands. E-mailaddress: folkert.verkaar@glpg.com

The online version of this article contains supplemental material.

Abbreviations used in this article: BCS, bovine calf serum; CHO, Chinese hamsterovary; EFC, enzyme fragment complementation; GAG, glycosaminoglycan; [125I]-CCL19, [125I]-labeled CCL19; mtCXCL, mutant CXCL; wtCXCL12, wild-type CXCL12.

Copyright� 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00

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MA). Forskolin was from Sigma-Aldrich (Steinheim, Germany). Produc-tion of mutant CXCL (mtCXCL)12 (15), mtCXCL11 (16), CXCL121 (17),and CXCL122 (18) has been described previously.

Cell culture

The generation of the Chinese hamster ovary (CHO)-CCX-CKR cells hasbeen described previously (3). These cells were maintained in DMEM F12(PAA Laboratories, Colbe, Germany) supplemented with 10% v/v bovinecalf serum (BCS; Hyclone, Logan, UT), 250 mg/ml hygromycin, 800 mg/mlgeneticin, 100 U/ml penicillin, and 100 mg/ml streptomycin (all from Invi-trogen, Carlsbad, CA). CHO cells stably expressing the b-arrestin2-EA fu-sion protein and G protein–coupled receptors C-terminally extended witha complementary b-galactosidase mutant (Prolink) (i.e., CHO-CCR7,CHO-CXCR5, and CHO-CCR5 cells) were purchased from DiscoveRx(Fremont, CA). CHO-CXCR3 cells were provided by T. van der Doelen(Merck, Sharp, and Dohme [Merck Worldwide], Oss, The Netherlands).These cell lines were cultured in the medium mentioned above. HEK293Tand CHO cells were purchased from the American Type Culture Collectionand maintained in DMEM F12 supplemented with 10% v/v BCS, 100 U/mlpenicillin, and 100 mg/ml streptomycin. L1.2 cells (American Type CultureCollection) were cultured in RPMI 1640 (Sigma-Aldrich) containing 10% v/vheat-inactivated BCS, 100 U/ml penicillin, 100 mg/ml streptomycin, 25 mMHEPES, 2 mM L-glutamine, 1% nonessential amino acids, 1 mM sodiumpyruvate (all from PAA Laboratories), and 50 mM 2-ME (Invitrogen).

Enzyme fragment complementation assays

EFC assays were performed, as described previously (3). Briefly, cells weredispensed in a 384-well white CulturPlate (Perkin Elmer) with 13 104 cells in15ml OPTImem (Invitrogen) containing 1%BCS, 100U/ml penicillin, and 100mg/ml streptomycin. Following overnight culturing in a humidified incubator(37˚C, 5% CO2, 95% humidity), cooperative and receptor-engaging chemo-kines were added in 5 ml OPTImem each, followed by 90-min incubation at37˚C. Subsequently, cells were disrupted by addition of 12.5 ml PathHunterDetection Reagent (DiscoveRx) in the formulation specified by the manufac-turer. Following 1-h incubation at room temperature in the dark, luminescencewas measured using a Victor3 multilabel plate reader (Perkin Elmer).

Radioligand binding

Radioligand-binding experiments were performed, as described previously(3). For saturation-binding experiments, 253 104 CHO cells in 100 ml culturemedium were dispensed in a poly-L-lysine–coated 96-well culture plate(Greiner) and incubated overnight in a humidified incubator. Medium wasreplaced by 50 ml nonradiolabeled chemokine in 20 mM HEPES, 5 mMMgCl2, 1 mM CaCl2, and 100 mM NaCl (pH 7.4) at 4˚C (assay medium),followed by addition of 50 ml [125I]-CCL19 in assay medium. Plates wereincubated 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 mlradioimmunoprecipitation assay buffer (Sigma-Aldrich), and radioactivity inlysates 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) containing0.1% BSA (assay buffer) was added to the lower compartment of a 96-wellChemoTx plate (5 mm pore size; Neuroprobe, Gaithersburg, MD). Aquantity amounting to 25 3 104 L1.2 cells in 20 ml assay buffer wasadministered to the upper compartment of the plate. Cells were thenallowed to migrate through the filter for 5 h in a humidified incubator(37˚C). Twenty-five microliter assay buffer from the lower well was thentransferred to a white 96-well CulturPlate Plate (Perkin Elmer), followedby addition of 25 ml calcein AM (1 mg/ml final concentration) in assaybuffer. Fluorescence intensities were measured on a Victor3 plate reader.

Data analysis

Sigmoidal dose-response curves were plotted using Graphpad Prism 6.0software. All data are presented as averages6 SEM. Statistical significanceof observed differences was determined using Student t test or two-wayANOVA, as indicated in the figure legend and indicated in the figures withasterisks (*). A p value , 0.05 was regarded as statistically significant.

ResultsCXCL13 enhances the ability of CCL19, CCL21, and CCL25 toactivate CCX-CKR

Previous studies have suggested that CXCL13 is a ligand for CCX-CKR (13, 19). We therefore tested the ability of this chemokine to

activate or inhibit CCX-CKR in CHO cells genetically engineeredto express complementary b-galactosidase mutants coupled toCCX-CKR and the scaffolding protein b-arrestin2 (CHO-CCX-CKR cells). The recruitment of b-arrestin2 to activated CCX-CKR enables enzyme fragment complementation (EFC) betweenthe two b-galactosidase mutants, thus allowing measurement ofreceptor activation by assessing b-galactosidase activity (3, 20). Inaccordance, when CHO-CCX-CKR cells were treated with CCL19,CCL21, and CCL25, they responded with a concentration-dependentincrease in b-galactosidase activity (3) (Fig. 1A). In contrast,CXCL13 did not activate CCX-CKR in these cells (Fig. 1A,filled circles). CXCL13 was also unable to displace radiolabeledCCL19 ([125I]-CCL19) from CHO cells expressing CCX-CKR(Supplemental Fig. 1A, filled circles). Thus, CXCL13 is unlikelyto be a ligand for CCX-CKR. Nonetheless, we found that CXCL13

FIGURE 1. CXCL13 increases the ability of CCL19 to activate CCX-

CKR. (A) CHO cells stably expressing CCX-CKR fused to a peptide frag-

ment of b-galactosidase and b-arrestin2 coupled to a complementary

b-galactosidase mutant (CHO-CCX-CKR cells) were treated with increas-

ing concentrations of CCL19 (N) or CXCL13 (d) before measurement of

b-galactosidase activity. In addition, CHO-CCX-CKR cells were treated

with increasing concentrations of CXCL13 in the presence of 2 nM CCL19

(;). Asterisks (*) indicate statistically significant differences (two-way

ANOVA, p , 0.05) between cells treated with 2 nM CCL19 in the presence

and absence of CXCL13. (B) CHO-CCX-CKR 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. pEC50 values were as follows: vehicle: 8.6 6 0.2

(4), 100 nM CXCL13: 9.1 6 0.2 (4), and 1000 nM CXCL13: 9.4 6 0.1 (4)

(average 6 SD [n]). Differences in pEC50 values between vehicle-treated

cells and cells treated with either 100 or 1000 nM CXCL13 reached

statistical significance (Student t test, p , 0.05).

2 CHEMOKINE COOPERATIVITY IS GAG DEPENDENT

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concentration dependently enhanced the potency with whichCCL19 activated CCX-CKR (Fig. 1). This effect was not limited toactivation of CCX-CKR by CCL19, as similar results wereobserved in experiments using CCL21 and CCL25 (SupplementalFig. 1B, 1C).Secondly, we measured b-arrestin2 recruitment to CCX-CKR

using a bioluminescence resonance energy transfer approach (3).For this, we transiently transfected HEK293T cells with vectorsencoding CCX-CKR genetically extended with Renilla luciferaseand b-arrestin2 fused to yellow fluorescent protein. Similar toresults for the b-galactosidase complementation assay, CXCL13increased the potency with which CCL19 activated CCX-CKR(Supplemental Fig. 1D). Thus, CXCL13 cooperates with CCL19,CCL21, and CCL25 in activating CCX-CKR.

Chemokine cooperativity is specific for certain chemokinecombinations

We then tested whether the cooperative effect of CXCL13 wasspecific for certain chemokine pairs or chemokine receptors. Tothis end, we used CHO cells that complement b-galactosidase

following recruitment of b-arrestin2 to the Gai-coupled chemokinereceptors CCR7 (CHO-CCR7), CCR5 (CHO-CCR5), and CXCR5(CHO-CXCR5). CXCL13 enhanced the potency with which CCL19induced b-arrestin recruitment and Gai activation by CCR7 in CHO-CCR7 cells (Fig. 2A, 2B). Reciprocally, CCL19 enhanced the potencyof CXCL13 toward activation of CXCR5 (Fig. 2C). However, bothCCL19 and CXCL13 were incapable of potentiating CCL3-inducedactivation of CCR5 (Fig. 2D).Next, a panel of chemokines was tested for their ability to co-

operate with CCL19 in activation of CCX-CKR. Several chemo-kines other than CXCL13, including CXCL12 and CXCL11, werefound to cooperate with CCL19, whereas several others (CCL3,CCL4, and CCL23) did not (Fig. 2E). Essentially the same patternwas observed when CCX-CKR was activated with CCL21, asfollows: CXCL13, CXCL12, and CXCL11 cooperated with CCL21,whereas CCL3 and CCL4 did not (Supplemental Fig. 2).

CXCL13 enhances CCL19-mediated chemotaxis

Next, we used a transwell migration assay to test whether the en-hanced receptor activity translated to a more potent chemotactic

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).

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response. We used murine L1.2 cells, a pre-B lymphoma cell line,which migrated toward a gradient of CCL19 (Fig. 3A), implying thatthese cells endogenously express CCR7. However, the L1.2 cellsdid not migrate toward CXCL13 (Fig. 3A). In accordance with ourfunctional assays, 1 mM CXCL13 significantly potentiated CCL19-mediated chemotaxis in these cells (Fig. 3B).

Chemokine cooperativity is dependent on GAG binding

Our analysis revealed that CCL3 and CCL4 did not induce che-mokine cooperativity (Fig. 2E). These chemokines bind to severalGAGs with relatively low affinity compared with other chemo-kines (e.g., CXCL8, CCL2, and CCL5) (21, 22). To investigate theimportance of GAG binding for chemokine cooperativity, wetested the ability of GAG-binding–deficient chemokine mutants tocooperate in CCX-CKR activation. Such mutants have been de-scribed for CXCL12 and CXCL11 (15, 16, 23). In sharp contrastto wildtype CXCL12 (wtCXCL12), a GAG binding-deficientCXCL12 mutant (CXCL12K24S,H25S,K27S, hereafter referred to asmtCXCL12) did not cooperate with CCL21 in the EFC assay(Fig. 4A). Likewise, we found that mtCXCL12 did not cooperatewith CCL21 in activating CCX-CKR in the bioluminescenceresonance energy transfer assay (Supplemental Fig. 3). Simi-larly, a GAG-binding–deficient mtCXCL11 (CXCL11 50s orCXCL11K57A,K59A,R62A) was impaired in its ability to cooperatewith CCL21 in activating CCX-CKR (Fig. 4B). Both mtCXCL12and mtCXCL11 were capable of activating their cognate receptorswith potencies and efficacies comparable to wild-type chemokines(Supplemental Fig. 4).

Chemokine cooperativity is mediated by both chemokinemonomers and dimers

Chemokine heterodimerization has previously been suggested tounderlie chemokine cooperativity (10). GAG binding and dimer-ization of chemokines are intimately linked processes (18, 22).Indeed, the CXCL12-GAG binding interface significantly overlapswith the CXCL12 (homo)dimerization surface, and mtCXCL12 isnot only impaired in its ability to bind to GAGs but also in itsability to dimerize (18). To differentiate between dimerization andGAG binding as potential mechanisms for chemokine coopera-tivity, we used a CXCL12 mutant that forms obligate monomers(termed CXCL121) and a constitutively dimeric mutant ofCXCL12 (CXCL122) (17, 18, 24, 25). CXCL121 and CXCL122are strictly monomeric and dimeric at concentrations up to 10 mM(17, 24). Both mutants bound to heparin, although CXCL121 didso with a ∼10-fold lower potency than wtCXCL12 (17–18). Whentested in the EFC assay on CHO-CCX-CKR cells, both CXCL121 andCXCL122 cooperated with CCL21 in the activation of CCX-CKR(Fig. 4C). In conclusion, both monomeric and dimeric CXCL12proteins can mediate chemokine cooperativity.

Chemokine cooperativity is caused by competitive GAGbinding

The simplest model that explains the necessity for GAG bind-ing in chemokine cooperativity is one in which the cooperativechemokines displace the receptor-engaging chemokine frombinding to GAGs. This scenario would result in elevated con-centrations of soluble receptor-engaging chemokines that areavailable to bind and activate receptors. To test whether CXCL12and CXCL13 compete with CCL19 for binding to GAGs on the cellsurface, we monitored the binding of [125I]-CCL19 to CHO cells.The CHO cells did not express CCX-CKR or CCR7, as determinedby RT-PCR (data not shown), whereas they abundantly expressGAGs (23, 26). Incubation of CHO cells with [125I]-CCL19 led to aconcentration-dependent increase in cell-associated radioactivity, alarge portion of which could be competed for by adding excess(1 mM) nonlabeled CCL19 (Fig. 5A). wtCXCL12 was also capableof displacing [125I]-CCL19 from CHO cells, whereas mtCXCL12was 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 bindingto these cells (Fig. 5B and data not shown). These data indicatethat cooperative chemokines compete with CCL19 for GAGbinding on cells, whereas noncooperative chemokines do not(Fig. 6).

DiscussionChemokine cooperativity has previously been suggested to becaused by convergence of intracellular signaling pathways down-stream of receptor activation (5–7, 11, 12). Our data indicate thatchemokine cooperativity is not strictly dependent on the receptorof the cooperative chemokine. For example, mtCXCL12 andmtCXCL11 fully activate their respective G protein–coupledreceptors (Supplemental Fig. 4) but do not cooperate withCCL21 (Fig. 4). Furthermore, RT-PCR analyses and cAMPmeasurements indicated that CHO-CCX-CKR cells do not ex-press CXCR5 or CXCR4, the cognate Gai-coupled receptors forCXCL13 and CXCL12, respectively (data not shown). Our dataconcur with previous evidence suggesting that activation of thereceptor for the cooperative chemokine is not necessary forcooperativity (8, 9).Alternatively, chemokine cooperativity has been suggested to

originate from chemokine heterodimerization (9, 10). Our obser-

FIGURE 3. CXCL13 enhances the ability of CCL19 to induce chemo-

taxis. (A) Murine pre-B lymphoma (L1.2) cells were tested for their ability

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

migrate toward ascending concentrations of CCL19 in the absence or

presence of 1 mM CXCL13. Asterisks (*) indicate statistically significant

differences (two-way ANOVA, p , 0.05) between vehicle- and CXCL13-

treated cells.

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vation that both monomeric and dimeric CXCL12 species caninduce 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 raisingthe effective free concentration of chemokine that can engage itsreceptor (Fig. 6). Other researchers performed experiments onheparinase-treated cells to test whether GAGs influence chemo-kine cooperativity and observed no effect of heparinase treatmenton chemokine cooperativity (8, 10). Similarly, pretreatment ofCHO-CCX-CKR cells with a mixture of heparinases did not in-fluence the ability of CXCL13 to cooperate with CCL19 (data notshown). A flaw in this approach is that GAGs form a large het-erogeneous population of molecules, and it is experimentallychallenging to ascertain that all of the GAGs relevant to chemo-kine cooperativity are removed by treatment with heparinase andchondroitinase mixtures. Our analysis with GAG-binding–defi-cient mutants represents a method of analysis that lacks this pit-fall. A remaining challenge will be to deduce which GAGsubtypes are actually involved in chemokine cooperativity. Such

FIGURE 4. Mutant chemokines that do not bind to GAGs do not display

chemokine cooperativity. (A) CHO-CCX-CKR cells were treated with

wtCXCL12 and a CXCL12 mutant that is impaired in its ability to bind to

GAGs (mtCXCL12) in the presence of increasing concentrations of CCL21.

Subsequently, b-galactosidase activity was determined. pEC50 values were as

follows: vehicle: 7.7 6 0.3 (5), wtCXCL12: 8.7 6 0.4 (5), and mtCXCL12:

7.76 0.2 (5) (average6 SD [n]). Values for vehicle-treated and mtCXCL12-

treated cells were not statistically different, whereas wtCXCL12 signifi-

cantly differed from the other treatments (Student t test, p , 0.01).

(B) CHO-CCX-CKR cells were treated with CCL21 and 1 mM wild-type

CXCL11 (wtCXCL11) or 1 mM mtCXCL11 that is impaired in its ability

to bind GAGs, followed by measurement of b-galactosidase activity.

pEC50 values were as follows: vehicle: 7.8 6 0.2 (3), wtCXCL11: 9.6 60.3 (3), and mtCXCL11: 8.3 6 0.4 (3) (average 6 SD [n]). Values for

vehicle-treated and mtCXCL11-treated cells were not statistically dif-

ferent, whereas wtCXCL11 significantly differed from the other treat-

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

of CCL21, followed by b-galactosidase measurement. pEC50 values were

as follows: vehicle: 7.7 6 0.2 (6), wtCXCL12: 8.5 6 0.2 (6), CXCL121:

8.36 0.3 (6), and CXCL122: 8.56 0.5 (6) (average6 SD [n]). Differences

between values for vehicle-treated cells and cells treated with all three

CXCL12 proteins reached statistical significance (Student t test, p, 0.01).

pEC50 values for wtCXCL12, CXCL121, and CXCL122 were not statis-

tically different.

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

determination of cell-associated radioactivity. Asterisks (*) indicate sta-

tistically significant differences (Student t test, p , 0.01).

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GAGs may be identified by studying the ability of chemokines tocompete for binding on arrays or columns containing immobilizedsynthetic or purified GAGs (21, 27).Another outstanding question is whether chemokine coopera-

tivity occurs in vivo. Although systemic, homeostatic levels ofCCL19, CCL21, CXCL13, and CXCL12 are generally much lowerthan the chemokine concentrations required to induce chemokinecooperativity in our functional assays, local concentrations ofchemokine may well exceed the concentrations necessary for coop-erativity to occur. Of note, CCL19, CCL21, CXCL12, and CXCL13have exceptionally broad and overlapping expression patterns,which would provide many potential opportunities for synergisticinteractions. For instance, CCL19, CCL21, and CXCL12 areexpressed on the luminal side of high endothelial venules (28), andCCL21, CXCL12, and CXCL13 are expressed in lymph nodes(29–31). Indeed, CXCL12 has been shown to enhance the CCR7-mediated recruitment of naive T cells from high endothelialvenules into the underlying lymphoid tissue (5). Furthermore,CCL19, CCL21, CXCL12, and CXCL13 are expressed locallyduring chronic inflammation (32–34), and individual chemo-kine concentrations within these inflamed sites may very wellexceed the threshold for cooperativity to occur. Additionally,previous work has suggested that chemokine mixtures can con-certedly induce cooperativity at low individual chemokineconcentrations (10). Because multiple chemokines are expressedconcomitantly during inflammation and homeostatic trafficking(35–37), the concentration of several cooperative chemokinescombined may be sufficient for chemokine cooperativity to occur.How would chemokine cooperativity influence chemokine

function, and how will chemokine cooperativity be altered by(patho)physiological processes? Because chemokine cooperativitywould allow chemokines to activate their cognate receptors at lowerchemokine concentrations, it is likely to extend the range fromwhich chemokines can induce recruitment of leukocytes. Anotherimplication from our data is that alterations in GAG composition orabundance, as have been observed to occur in several pathologies

(38–41), might alter chemokine cooperativity. Lastly, our datapresent a new perspective on therapeutic targeting of the chemo-kine system. Through modulation of cooperativity, therapeuticstrategies aimed at inhibiting chemokine–GAG interactions mightestablish a broader effect on leukocyte trafficking than targetingchemokine receptors. For instance, chemokine cooperativity islikely to be modulated by administration of heparin or GAGderivatives, which have previously been shown to affect chemo-kine gradient formation (42, 43). Furthermore, small molecules orAbs 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.

AcknowledgmentsWe thankMarloes 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,

Danny Scholten, and Sabrina de Munnik (VU University Amsterdam) for

expert technical assistance. We thank Danny Scholten, Saskia Nijmeijer

(VU University Amsterdam), and Laura Heitman (Leiden University, Lei-

den, The Netherlands) for helpful discussions.

DisclosuresThe authors have no financial conflicts of interest.

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FIGURE 6. A model for chemokine cooperativity. (A) In the absence of

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activation.

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