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Blockade of Chemokine Activity by a Soluble Chemokine Binding Protein from Vaccinia Virus Antonio Alcamí, Julian A. Symons, Paul D. Collins, Timothy J. Williams and Geoffrey L. Smith This information is current as of October 3, 2021. J Immunol 1998; 160:624-633; ; http://www.jimmunol.org/content/160/2/624

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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 © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Blockade of Chemokine Activity by a Soluble Chemokine Binding Protein from Vaccinia Virus1

Antonio Alcamı´,* Julian A. Symons,* Paul D. Collins,† Timothy J. Williams,† and Geoffrey L. Smith2*

Chemokines direct migration of immune cells into sites of inflammation and infection. Chemokine receptors are seven-transmembrane domain proteins that, in contrast to other cytokine receptors, cannot be easily engineered as soluble chemokine inhibitors. Poxviruses encode several soluble cytokine receptors to evade immune surveillance, providing new strategies for immune modulation. Here we show that vaccinia virus and other orthopoxviruses (cowpox and camelpox) express a secreted 35-kDa chemokine binding protein (vCKBP) with no sequence similarity to known cellular chemokine receptors. The vCKBP ؍ binds CC, but not CXC or C, chemokines with high affinity (Kd 0.1–15 nM for different CC chemokines), blocks the interaction of chemokines with cellular receptors, and inhibits chemokine-induced elevation of intracellular calcium levels and cell mi- Downloaded from gration in vitro, thus representing a soluble inhibitor that binds and sequesters chemokines. The potential of vCKBP as a therapeutic agent in vivo was illustrated in a guinea pig skin model by the blockade of eotaxin-induced eosinophil infiltration, a feature of allergic inflammatory reactions. Furthermore, vCKBP may enable the rational design of antagonists to neutralize pathogens that use chemokine receptors to initiate infection, such as HIV or the malarial parasite. The Journal of Immunology, 1998, 160: 624–633. http://www.jimmunol.org/ central element in host immune surveillance is the mi- that the binding site of chemokines for proteoglycans and specific gration of immune cells into areas of injury or infection chemokine receptors (CKRs) are distinct (8, 9). A during the inflammatory response. Chemokines are che- The activity of chemokines is tightly regulated to prevent ex- moattractant cytokines that regulate trafficking and effector func- cessive inflammation that can cause disease, and these molecules tions of leukocytes, and thus play a key role in inflammation and represent potential targets for therapeutic intervention in a wide host defense against pathogens (1–5). Chemokines are divided into range of diseases. The production of soluble versions of cytokine at least three structural groups based on the number and arrange- receptors containing only the extracellular binding domain repre- ment of conserved cysteines: CC (␤) chemokines such as sents a physiologic and therapeutic strategy to block the activity of

3 by guest on October 3, 2021 RANTES, macrophage inflammatory protein-1␣ (MIP-1␣), and some cytokines (10, 11). However, the seven-transmembrane do- eotaxin; CXC (␣) chemokines such as IL-8 and growth-related main structure of CKRs makes the construction of soluble, inhib- ␣ oncogene (GRO)- (alternatively known as melanoma growth itory CKRs difficult, and thus antagonists based on mutated che- stimulating activity); and the C chemokine lymphotactin. mokines, blocking peptides, or Abs are alternative inhibitors of the Chemokines exert their activity by interacting with seven-trans- chemokines under evaluation (1, 3). membrane domain receptors expressed in different cell subsets, Poxviruses, a family of complex DNA viruses (12), have thus determining the leukocyte subtype that predominates in dif- evolved unique strategies for evasion of the host immune re- ferent types of inflammation. Chemokines are thought to form a sponse (13–16) and are the only virus family known to produce chemical gradient in an immobilized phase via electrostatic inter- secreted versions of receptors for cytokines such as TNF, IL- actions with negatively charged proteoglycans, which may in- 1␤, IFN-␣/␤, and IFN-␥. These poxvirus proteins bind cyto- crease the specificity of chemokine action (6, 7). There is evidence kines with high affinity and block their activity by preventing interaction with receptors on the target cell. Inactivation of virus genes encoding these cytokine inhibitors has profound ef- *Sir William Dunn School of Pathology, University of Oxford, Oxford; and †Im- perial College School of Medicine at the National Heart and Lung Institute, fects on viral pathogenesis. Poxvirus cytokine receptors were London, United Kingdom initially identified from sequence similarity to the extracellular Received for publication August 1, 1997. Accepted for publication September binding domain of cellular cytokine receptors, but binding and 30, 1997. functional analysis led to the identification of the IFN-␣/␤R The costs of publication of this article were defrayed in part by the payment of encoded by vaccinia virus (VV), which has very limited simi- page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. larity to known cellular counterparts (17, 18). 1 This work was supported by grants from The Wellcome Trust, the Royal Soci- Here we report that a soluble 35-kDa protein encoded by VV ety, and the National Asthma Campaign. strain Lister has chemokine binding activity, and similar proteins 2 Address correspondence and reprint requests to Prof. Geoffrey L. Smith, Sir are produced by 11 orthopoxviruses representing three species: William Dunn School of Pathology, University of Oxford, South Parks Rd., Ox- VV, cowpox, and camelpox viruses. During the preparation of this ford, United Kingdom OX1 3RE. manuscript, Graham et al. (19) reported that the T1/35-kDa family 3 Abbreviations used in this paper: MIP-1␣, macrophage inflammatory protein- 1␣; GRO-␣, growth-related oncogene ␣; CKR, chemokine receptor; VV, vaccinia of poxvirus-secreted proteins binds CC and CXC chemokines and virus; vCKBP, virus chemokine binding protein; MCP, monocyte chemoattrac- modulates the influx of inflammatory cells into virus-infected tis- tant protein; NAP-2, neutrophil activating peptide-2; LTB4, leukotriene B4; WR, Western Reserve; EDC, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide; EGS, sues. However, the mechanism by which the T1/35-kDa proteins ethylene glycol-bis-succinamidyl succinate. modulate chemokine activity was not demonstrated. Here we show

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 The Journal of Immunology 625 that the virus chemokine binding protein (vCKBP) binds CC che- MIP-1␣ or as binding to 200 pg of purified B8R-Fc, was subtracted and mokines with high affinity and inhibits their ability to induce signal represented 3.8 to 7.5% of the total counts per minute bound. Binding data transduction and cell migration in vitro. However, vCKBP does were analyzed by the LIGAND program (26). The number of binding sites for MIP-1␣ per molecule of vCKBP was calculated from the number of not bind or inhibit CXC or C chemokines. We demonstrate that the binding sites deduced from the Scatchard analysis and from the concen- vCKBP inhibitory mechanism is the blockade of the interaction of tration and m.w. of recombinant 35K-Fc. chemokines with cellular receptors, thus representing the first soluble protein identified that sequesters chemokines and blocks their Construction of recombinant baculovirus activity. Furthermore, the therapeutic application of this novel sol- The 35-kDa gene was obtained by PCR using VV Lister DNA and oligo- uble chemokine inhibitor is illustrated in a guinea pig skin model nucleotides 5Ј-ATCGGTACCAATTATGAAACAATATATCGTCC-3Ј, by the ability of vCKBP to block eotaxin-induced eosinophil in- which inserts a KpnI site, and 5Ј-GTTGGATCCTCAGACACACGCTTT Ј filtration in vivo, a feature of allergic inflammatory reactions. GAGTTTTG-3 , containing a BamHI site. The 782-bp fragment was se- quenced and cloned into pAcCL29-1 (27), generating pAc35K. The sequence contained His148 instead of Arg148 (21), but both sequences are Materials and Methods natural variants of the VV Lister 35-kDa protein and bind chemokines with Viruses the same specificity. Recombinant 35-kDa protein containing a C-terminal six-histidine tag (35K.His) was produced in the baculovirus system. The Ϫ The growth conditions of human U937 and TK 143B cells, and the 35-kDa gene was obtained by PCR using pCOS35K-Fc DNA (see Con- sources of VV strains have been described previously (20). The Evans and struction of 35K-Fc fusion and expression below) and oligonucleotides Lister strains used in this study and a Lister virus lacking the gene encoding 5Ј-TCAGAATTCATTATGAAACAATATATCGTCC-3Ј, containing an the 35-kDa protein (Lister⌬35K or V357) (21) were provided by A. H. EcoRI site, and 5Ј-ATCCTCGAGGACACACGCTTTGAGTTTTG-3Ј, Patel (Institute of Virology, Glasgow, U.K.). containing an XhoI site. The 778-bp fragment was cloned into pBAC-1 Downloaded from (Novagen, Madison, WI), generating pAc35K.His. The sequence was con- Reagents firmed and contained the Arg148 variant of the Lister 35-kDa protein. The Radioiodinated recombinant human IFN-␥ (90 ␮Ci/␮g) and RANTES, recombinant baculoviruses Ac35K and Ac35K.His were produced from MIP-1␣, IL-8, and GRO-␣ (2200 Ci/mmol) were obtained from DuPont- pAc35K and pAc35K.His, respectively, as described previously (20). The New England Nuclear (Boston, MA). Recombinant human [125I]MIP-1␣ 35K.His protein was purified by metal chelate affinity chromatography (Ni- (2000 Ci/mmol) used for determination of affinity constants was obtained NTA resin; Qiagen, Inc., Chatsworth, CA). The recombinant baculoviruses from Amersham (Little Chalfont, U.K.). Recombinant human RANTES, AcB15R and AcB8R have been described (20, 22). MIP-1␣, monocyte chemoattractant protein-1 (MCP-1), MCP-2, MCP-4, http://www.jimmunol.org/ eotaxin, IL-8, GRO-␣, 78-amino acid epithelial cell-derived neutrophil ac- Construction of 35K-Fc fusion and expression ␥ tivator, IFN- -inducible protein-10, platelet factor 4, neutrophil-activating The VV Lister 35-kDa gene fused to the Fc region of human IgG1 was ␣ peptide-2 (NAP-2), stromal cell-derived factor-1 , and lymphotactin, constructed in pCOSFCLINK (a gift from Dr. P. R. Young, SmithKline ␣ mouse eotaxin, and rat RANTES and MIP-1 were purchased from Pep- Beecham Pharmaceuticals, King of Prussia, PA). The insert was obtained ␣ roTech (Rocky Hill, NJ). Recombinant mouse MIP-1 and human eotaxin, by PCR using VV Lister DNA and the oligonucleotides 5Ј-TCAGAATT ␥ GRO- , and I309 were obtained from R&D Systems (Minneapolis, MN). CATTATGAAACAATATATCGTCC-3Ј, which introduces an EcoRI site, ␥ Recombinant human IFN- was purchased from Genzyme (Cambridge, and 5Ј-ATCGGTACCGACACACGCTTTGAGTTTTG-3Ј, which inserts MA). Leukotriene B4 (LTB4) was purchased from Cascade Biochem Ltd Gly and Thr via a KpnI site after amino acid 258 of the 35-kDa protein (Reading, U.K.). The following were gifts: guinea pig eotaxin from Drs. G. before the hinge region of human IgG1, which was provided by pCOSF- Andrews and H. J. Showell (Pfizer Central Research, Groton, CT), human CLINK, generating pCos35K-Fc. The sequence was confirmed and con- by guest on October 3, 2021 IL-5 from Dr. T. N. C. Wells (Glaxo-Wellcome Molecular, Geneva, Swit- tained the Arg148 variant of the Lister 35-kDa protein. The 35K-Fc fusion zerland), and human C5a from Dr. J. Van Oostrum (Ciba Geigy, Summit, protein was purified from supernatants of CHO cells stably transfected with NJ). Heparin (6 kDa) and heparan sulfate from porcine or bovine intestinal pCOS35K-Fc on a protein A-Sepharose column (Pharmacia, Piscataway, mucosa, respectively, were purchased from Sigma Chemical Co. (St. NJ). The B8R-Fc fusion protein will be described elsewhere (J. A. Symons Louis, MO). Rabbit antiserum to purified VV Lister 35-kDa protein, pro- and G. L. Smith, unpublished). vided by A. H. Patel (Institute of Virology), or VV Western Reserve (WR) B15R protein expressed in baculovirus have been described previously (21, Measurement of calcium mobilization in human eosinophils 22). and neutrophils Preparation of medium for binding and biologic assays Changes in intracellular calcium flux were determined in purified human Supernatants from orthopoxvirus-infected TKϪ143B cells or baculovirus- neutrophils and eosinophils as described previously (28). Briefly, granu- infected Sf cells were harvested 1 or 3 days postinfection, respectively, and locytes were separated from mononuclear leukocytes, RBCs, and platelets prepared as previously described (22). Baculovirus supernatants were con- using a combination of dextran sedimentation and density centrifugation centrated and dialyzed against PBS as described previously (18). VV su- over Percoll-plasma gradients. For neutrophil experiments, granulocytes pernatants were inactivated with 4,5Ј,8-trimethylpsoralen and UV light for comprised Ͼ95% neutrophils, and the remaining cells were a mixture of cell migration assays (23). eosinophils and mononuclear cells. For eosinophil experiments, blood was obtained from healthy atopic volunteers, and eosinophils were purified Binding assays (Ͼ98%) from the isolated granulocytes by immunomagnetic separation using anti-human CD16 microbeads (Midimacs, Miltenyl Biotec, Tedding- The binding medium was RPMI 1640 containing 20 mM HEPES (pH 7.4) ton, U.K.) (29). Purified cells were loaded with fura-2/AM (neutrophils, 1 and 0.1% BSA. Cross-linking experiments with 1-ethyl-3-(3-dimethyl- ␮M; eosinophils, 2.5 ␮M) and, after two washes, resuspended at 106 aminopropyl)-carbodiimide (EDC) or ethylene glycol-bis-succinamidyl 2ϩ 2ϩ 125 cells/ml in Ca /Mg -free PBS containing 10 mM HEPES (pH 7.4), 10 succinate (EGS; 1 mg/ml) to I-labeled chemokines (0.4 nM) or mM glucose, and 0.25% BSA. Aliquots of cells were taken, and the ex- 125 ␥ ␮ [ I]IFN- (2 nM) were performed in a volume of 25 l as previously ternal calcium concentration was adjusted to 1 mM. Changes in fluores- described (20, 24). Samples were analyzed by SDS-PAGE in 16- or 20- cence were monitored at 37°C using a spectrometer (LS50, Perkin-Elmer cm-long gels with 12 or 14% acrylamide. In the competition assays with 125 Corp., Norwalk, CT) at excitation wavelengths of 340 and 380 nm and an U937 cells, supernatants were preincubated with 100 pM [ I]chemokine emission wavelength of 510 nm. in 100 ␮lfor1hat4°C. Subsequently, 2.5 ϫ 106 U937 cells were added ␮ 125 in 50 l and incubated for2hat4°C. Bound [ I]chemokine was deter- Chemotaxis assay mined by phthalate oil centrifugation as previously described (22). A scintillation proximity assay (Amersham) (25) was used for determination of Cell migration was evaluated in 10-mm tissue culture inserts (Life Tech- the affinity constant. Purified 35K-Fc (200 pg) was incubated with nologies, Grand Island, NY) placed in 24-well plates. A 0.5-ml aliquot of [125I]MIP-1␣, with or without unlabeled chemokines, in 100 ␮lfor2hat test reagents diluted in RPMI 1640 with 1 mg/ml BSA was placed in the room temperature. Protein A-fluoromicrospheres containing scintillant lower compartment, and 0.5 ml of U937 cell suspension (4 ϫ 106 cells/ml) were added in 50 ␮l and incubated for 2 h, and bound [125I]MIP-1␣ was was placed in the upper chamber, separated by a polycarbonate filter (8-␮m determined by scintillation proximity assay in a beta counter. Nonspecific pore size). After incubation at 37°C for 2 h, the filter was washed, fixed, binding, determined in the presence of a 1000-fold excess of unlabeled and stained with 4Ј6,-diamidino-2-phenylindole (DAPI) (30). The number 626 VACCINIA CHEMOKINE BINDING PROTEIN Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 1. Soluble chemokine binding activity produced by orthopoxviruses. a, Media from cultures uninfected (mock) or infected with the indicated orthopoxviruses were incubated with [125I]RANTES and treated with the cross-linker EDC. b, Media from infected cultures were either harvested 24 h postinfection (L) or treated with the inhibitor of DNA synthesis cytosine arabinoside (40 ␮g/ml) and harvested 7 h postinfection (E). Media from cultures uninfected (mock) or infected were incubated with [125I]RANTES and cross-linked with EDC. c, Media from cultures uninfected (mock) or infected were incubated with the CC chemokines [125I]RANTES or [125I]MIP-1␣ or with the CXC chemokine [125I]GRO-␣ and treated with EDC or EGS. The amount of medium used was equivalent to 2 to 7 ϫ 104 cells. An autoradiograph of the SDS-PAGE analysis, with molecular masses in kilodaltons, is shown. The positions of RANTES (R), MIP-1␣ (M), GRO-␣ (G), and ligand-receptor complexes (square brackets) are indicated. of migrating cells in five high powered fields was counted using a fluo- to induce blood eosinophilia (31). Guinea pig eotaxin, human C5a, or rescence microscope. LTB4, with or without purified 35K.His protein, was then injected intradermally (50 ␮l/site) into individual sites in the shaved dorsal skin of the Bioassay of eosinophil accumulation in guinea pig skin animals. After 2 h, the animals were killed, and the skin sites were excised Accumulation of endogenous peripheral blood eosinophils. Sedated guinea (11 mm in diameter) for quantification of eosinophil accumulation by mea- pigs were pretreated for 1 h with human IL-5 (18 pmol/kg; i.v. injection) surement of eosinophil peroxidase as previously described (31). The Journal of Immunology 627 Downloaded from

FIGURE 2. Identiﬁcation of the VV Lister 35-kDa protein as the vCKBP. a, Cross-linking of [125I]RANTES with EDC to supernatants from cultures infected with the indicated VVs, recombinant baculoviruses, or 20 ng of puriﬁed Fc fusion proteins in the absence or the presence of a 1000-fold http://www.jimmunol.org/ excess of RANTES (RAN) or IFN-␥. b, Cross-linking of [125I]RANTES with EDC to supernatants from cells infected with VV Lister or Ac35K in the presence of 5 ␮l of preimmune, anti-35K, or anti-B15R rabbit serum. c, Cross-linking of [125I]IFN-␥ with EDC to supernatants from cultures infected with the indicated viruses. The amount of supernatant used was equivalent to 2 ϫ 104 cells (VV) or 4 ϫ 103 cells (baculovirus). An autoradiograph of the SDS-PAGE analysis, with molecular masses in kilodaltons, is shown. The positions of RANTES (R), IFN-␥ monomers (M), IFN-␥ dimers (D), and ligand-receptor complexes (triangles) are indicated.

Accumulation of 111In-labeled eosinophils pox and United Soviet Socialists Republic (USSR) (Fig. 1b). The

Eosinophils were purified from the peritoneal cavity of horse serum-treated RANTES-vCKBP complex was 37 to 44 kDa when the cross- by guest on October 3, 2021 111 linker EGS was used (Fig. 1b), suggesting that two RANTES mol- donor animals and radiolabeled with InCl3 as previously described (28). 111In-labeled eosinophils (5 ϫ 106 cells/animal) were injected i.v. into ecules could form a complex with vCKBP. Similar results were recipient animals, and after 10 min, guinea pig eotaxin and human C5a, observed with [125I]MIP-1␣, another CC chemokine (Fig. 1c). with or without purified 35K.His protein, were injected intradermally (100 MIP-1␣ can dimerize, but there is controversy about binding of CC ␮l/skin site; two sites per treatment) into individual sites in the shaved dorsal skin. After 2 h, the animals were killed, and the skin sites were chemokines as monomers or dimers to cellular CKRs (4). Cross- 125 excised (17 mm in diameter) for quantification of eosinophil accumulation linking of media from all viruses shown in Figure 1a to [ I]IL-8 by measurement of 111In counts using a gamma counter (28). (not shown) or [125I]GRO-␣ (Fig. 1c and data not shown) was negative or very weak, suggesting vCKBP specificity for CC, but Results not CXC, chemokines. VV, cowpox, and camelpox viruses encode soluble chemokine binding activity The VV Lister 35-kDa secreted protein encodes vCKBP Although no CKR homologues were found in the VV genome Although the predicted vCKBP size was 25 to 29 kDa, its expres- sequence (32), we searched for the expression of soluble chemo- sion profile in orthopoxviruses (Fig. 1, a and c) correlated with the kine binding proteins by orthopoxviruses. Binding assays with expression of a 35-kDa major secretory protein in VV Lister, [125I]RANTES were performed with media from cultures infected Evans, and rabbitpox, but not in WR, Tian-Tan, or Wyeth (21, 33). with 13 strains of VV, rabbitpox, or buffalopox viruses (considered The 35-kDa gene is present in cowpox (34), but is truncated by a VV strains), cowpox virus (Brighton Red strain), elephantpox vi- frameshift mutation within the signal peptide in VV Copenhagen, rus (considered a cowpox virus strain), or camelpox virus, fol- where it is called B29R or C23L (32), and in VV WR, where it lowed by chemical cross-linking with EDC. A [125I]RANTES- produces a 7.5-kDa protein (21, 35). The expression of vCKBP vCKBP complex was observed with several viruses, including VV before (early) and after (late) viral DNA synthesis (Fig. 1b) was Evans (Fig. 1c), but not with the best characterized VV strains WR also consistent with the transcriptional regulation of the 35-kDa and Copenhagen (Fig. 1a). The size of the ligand-vCKBP complex gene from the strong early/late promoter p7.5, widely used in VV ranged from 33 to 37 kDa, suggesting a vCKBP size of 25 to 29 expression vectors (36). kDa after subtraction of the 8-kDa monomeric RANTES. The vari- The absence of RANTES-binding activity in supernatants ation in vCKBP size may reflect different polypeptide lengths or from cultures infected with a VV Lister mutant lacking the 35- degrees of glycosylation among viruses. The different size pre- kDa gene (Lister⌬35K) (21) indicated that the 35-kDa protein is dicted for vCKBP from orthopoxviruses by Graham et al. (19) in the vCKBP encoded by VV Lister (Fig. 2a). This was confirmed similar experiments (41 kDa) might be due to the use of higher by showing specific binding of [125I]RANTES to the 35-kDa resolution gels in this study. The soluble vCKBP was expressed protein produced from recombinant baculovirus (Ac35K) and before (early) and after (late) viral DNA synthesis by VV rabbit- when fused to the Fc region of human IgG1 (35K-Fc), but not 628 VACCINIA CHEMOKINE BINDING PROTEIN to the controls AcB15R (VV IL-1␤R) (22), AcB8R (VV IFN- ␥R) (20), or B8R-Fc (VV IFN-␥R fused to human IgG1; J. A. Symons and G. L. Smith, unpublished; Fig. 2a). Additional evidence was the neutralization of the RANTES binding activity of VV Lister and Ac35K supernatants by antiserum against purified 35-kDa protein (21), but not by antiserum against the VV IL-1␤R B15R (22) (Fig. 2b). Recently, the myxoma virus-soluble IFN-␥R (24), but not the VV IFN-␥R (B8R) (20), was demonstrated to bind a wide range of chemokines by interaction with their proteoglycan binding domain (19, 37). Here we demonstrate that VV expresses a 35-kDa vCKBP that does not bind IFN-␥ and is not B8R by showing that 1) the binding of [125I]RANTES to natural (VV Lister) or recombinant (Ac35K and 35K-Fc) 35-kDa protein was inhibited by unlabeled RANTES, but not IFN-␥ (Fig. 2a); 2) [125I]IFN-␥ was cross-linked to the natural IFN-␥R expressed from all three viruses, WR, Lister, and Lister⌬35K, or the recombinant IFN-␥R (AcB8R), but not to the 35-kDa protein (Ac35K; Fig. 2c); 3) the

molecular sizes of monomeric IFN-␥ complexed to IFN-␥R were Downloaded from 60 and 52 kDa for the natural and recombinant IFN-␥R, respectively (20), which were higher than the 35-kDa size of the RAN- TES-vCKBP complex and could not be explained by the size difference between IFN-␥ (17 kDa) and RANTES (8 kDa; Fig. 2, a and c); 4) the 35-kDa size of the RANTES-vCKBP complex was ␥ smaller than the 50-kDa size predicted for the RANTES-IFN- R http://www.jimmunol.org/ complex, as reported for the myxoma IFN-␥R/M7T (37); and 5) the binding of [125I]IFN-␥ to the natural (VV Lister) or recombinant (AcB8R) VV IFN-␥R was inhibited by unlabeled IFN-␥ but not by RANTES (Fig. 2c). It is also demonstrated that VV IFN-␥R does not bind RANTES by the inability of RANTES to bind rIFN-␥R (AcB8R and B8R-Fc) (Fig. 2a) and the lack of binding of RANTES to supernatants from cells infected with Lister⌬35K, which still express IFN-␥Rs (Fig. 2, a and c). In addition, a higher FIGURE 3. Binding specificity of vCKBP encoded by VV, cowpox virus, and camelpox virus. Cross-linking of 0.4 nM human [125I]RAN- molecular size of the soluble IFN-␥R, known to be encoded by all by guest on October 3, 2021 TES with EDC to medium from infected cultures in the absence (NC) or the orthopoxviruses included in this study (20), complexed with the presence of increasing concentrations of unlabeled chemokines 125 125 ␣ 125 [ I]RANTES, [ I]GRO- ,or[ I]IL-8 was not observed (Fig. from different species. The amount of medium was equivalent to 1 ϫ 1, a and c, and data not shown), indicating that the IFN-␥R from 104 cells for orthopoxviruses and 2 ϫ 103 cells for Ac35K. The con- other strains of VV, cowpox, or camelpox viruses does not bind centrations of unlabeled chemokines were 50-, 100-, 500-, or 2000- CC or CXC chemokines. fold excesses for VV Lister and Ac35K, and 100- or 2000-fold excesses for cowpox and camelpox. An autoradiograph of the SDS-PAGE anal- Binding specificity and affinity of vCKBP for chemokines ysis showing the ligand-receptor complexes is presented. Lptn, The binding specificity of vCKBP was further investigated in lymphotactin. cross-linking experiments (Fig. 3). The vCKBP expressed from recombinant baculovirus, VV Lister, cowpox virus, or camelpox 125 virus bound human [ I]RANTES, and this binding was effec- 35K-Fc was saturable and of high affinity: in two experiments Kd tively competed in a dose-dependent manner by all human CC values of 103 Ϯ 4 pM (Fig. 4a) and 128 Ϯ 9 pM (not shown) were chemokines tested, including eotaxin, a specific chemoattractant of obtained. The high affinity interaction of vCKBP for CC chemo- ϭ eosinophils. In contrast, human CXC chemokines or the C che- kines (Kd 115 pM) was similar or 100-fold higher than the mokine lymphotactin were not bound by vCKBP. The failure of affinity of chemokines for cellular CKRs (1, 4, 5). Scatchard anal- high doses of GRO-␣ or IL-8 to block [125I]RANTES binding was ysis of the binding data predicted 4.2 binding sites for MIP-1␣ in consistent with the negative or very weak cross-linking of vCKBP the 35K-Fc protein in both experiments, suggesting 2.1 binding from orthopoxviruses to [125I]GRO-␣ or [125I]IL-8 (Fig. 1c and sites for chemokines in the 35-kDa protein. This was consistent data not shown), suggesting a very low, and probably physiolog- with the binding of two chemokine molecules to vCKBP, as sug- ically insignificant, affinity for CXC chemokines. The vCKBP also gested by cross-linking studies (Fig. 1c). The interaction of bound CC chemokines from mouse and rat. This vCKBP binding [125I]MIP-1␣ with 16.5 pM 35K-Fc was inhibited by a similar specificity was confirmed in experiments cross-linking [125I]MIP- dose of purified recombinant 35-kDa protein, which was produced 1␣ to VV Lister supernatants in the presence of unlabeled chemo- in baculovirus and containing a C-terminal six-histidine tag kines (not shown). These results demonstrate that vCKBP from (35K.His), suggesting that both proteins bind MIP-1␣ with similar VV, cowpox, and camelpox viruses bind CC, but not CXC, affinities (Fig. 4b). chemokines. The affinity of vCKBP for other chemokines was determined in The affinity of vCKBP for CC chemokines was determined in binding assays of 35K-Fc to [125I]MIP-1␣ in the presence of in- studies of binding of purified recombinant 35K-Fc to [125I]MIP- creasing doses of unlabeled chemokine competitors (Fig. 4c). 1␣, and quantification of bound MIP-1␣ was achieved using a These data showed that vCKBP binds CC chemokines with dif- scintillation proximity assay (25). The interaction of MIP-1␣ to ferent affinities: MIP-1␣ Ͼ eotaxin Ͼ RANTES Ͼ MCP-1 Ͼ I309. The Journal of Immunology 629

FIGURE 4. Affinity constant of vCKBP binding to chemokines. a, Saturation curve and Scatchard analysis of [125I]MIP-1␣ binding to 35K-Fc. Puri- fied 35K-Fc protein was incubated with the indicated doses of [125I]MIP-1␣ for 2 h, and the radioactivity bound was determined by scintillation proximity assay with protein A-fluoromicrospheres containing scintillant. The mean (ϮSEM) specific binding of triplicate samples is shown. b, Competitive inhibition with purified 35K.His. Pu- rified 35K-Fc protein was incubated in triplicate with 50 pM [125I]MIP-1␣ in the presence of increasing doses of purified 35K.His produced in the baculovirus system. The percentage of specific binding (mean Ϯ SEM) refers to binding in the absence of competitor, which was 4,235 cpm. c, Competitive inhibition with various doses of CC chemokines. Purified 35K-Fc protein was incubated in triplicate with 50 pM [125I]MIP- 1␣ in the presence of increasing doses of unlabeled human CC chemokines. The percentage of Downloaded from specific binding (mean Ϯ SEM) refers to binding in the absence of competitor, which was 4,049 or

4,561 cpm. The Kd values calculated from the data are indicated. d, Competitive inhibition with CC, CXC, and C chemokines. Puriﬁed 35K-Fc protein was incubated in triplicate with 50 pM [125I]MIP-1␣ in the presence of a 10,000-fold ex- http://www.jimmunol.org/ cess of unlabeled human (h), mouse (m), or rat (r) chemokines. The percentage of speciﬁc binding (mean Ϯ SEM) refers to binding in the absence of competitor, which was 4,340 cpm. e, MIP-1␣ binding in the presence of glycosaminoglycans. Increasing doses of heparin or heparan sulfate were incubated in triplicate with 50 pM [125I]MIP-1␣ for 30 min before the addition of

puriﬁed 35K-Fc protein. The percentage of spe- by guest on October 3, 2021 ciﬁc binding (mean Ϯ SEM) refers to binding in the absence of competitor, which was 3,014 cpm. Lptn, lymphotactin; ENA78, 78 amino acid epithelial cell-derived neutrophil activator; IP-10, IFN-␥ inducible protein 10; PF-4, platelet factor 4; SDF-1␣, stromal cell derived factor-1␣.

The lower affinity for I309 probably reflects the amino acid se- The vCKBP blocks binding of CC chemokines to cell surface quence of this chemokine being the most divergent of the CC receptors chemokine group (4). Competition experiments with other chemo- Biologic activity of vCKBP for CC chemokines was shown by the kines corroborated, in a different quantitative binding assay, the ability of supernatants from orthopoxviruses and recombinant bac- specificity of vCKBP for CC, but not CXC or C, chemokines and ulovirus Ac35K to inhibit the binding of radioiodinated RANTES extended these studies to 19 chemokines (Fig. 4d). and MIP-1␣, but not GRO-␣, to cellular receptors (Fig. 5a). This again demonstrated the specificity of vCKBP for CC chemokines. The vCKBP does not bind to the proteoglycan binding site of As previously reported (38, 39), excess unlabeled RANTES failed chemokines to block the binding of [125I]RANTES to cells (Fig. 5a). The bind- Chemokines interact with glucosaminoglycans such as heparin or ing of [125I]MIP-1␣, but not [125I]GRO-␣, to U937 cells was in- heparan sulfate through their carboxyl terminus (6, 9). This inter- hibited in a dose-dependent manner by supernatants containing the action is thought to facilitate chemokine localization to the endo- 35-kDa protein (Fig. 5b). The potent inhibitory activity of vCKBP thelial cells and does not interfere with chemokines binding to was illustrated by the complete blockade of MIP-1␣ binding to their receptors. To ascertain whether vCKBP bound to the proteo- cells in the presence of 0.1 to 1 ␮l of medium, equivalent to the glycan binding site of chemokines, [125I]MIP-1␣ was preincubated amount of vCKBP synthesized by only 200 to 2000 cells. Further- with various doses of heparin or heparan sulfate before incubation with the 35K-Fc protein. Figure 4e shows that heparin or heparan more, 10 or 50 pM purified recombinant 35K.His protein produced 125 ␣ sulfate did not interfere with MIP-1␣ binding to vCKBP at doses in baculovirus blocked the binding of 100 pM [ I]MIP-1 to up to 10 ␮g/ml and had a minor effect at higher doses up to 200 U937 cells by 50 and 95%, respectively (Fig. 5c). These results ␮g/ml, equivalent to a 6 ϫ 105-fold molar excess of heparin over showed that the soluble vCKBP blocks the binding of CC chemo- MIP-1␣. kines to their cellular receptors and were consistent with the high 630 VACCINIA CHEMOKINE BINDING PROTEIN Downloaded from http://www.jimmunol.org/

FIGURE 5. Inhibition of chemokine binding activity by vCKBP. a, Binding of 100 pM [125I]RANTES, [125I]MIP-1␣,or[125I]GRO-␣ to U937 cells in the presence of supernatants from 5 ϫ 104 infected cells or a 100-fold excess of unlabeled chemokine. The mean Ϯ SEM from duplicate samples are expressed as the percentage of counts bound in the presence of competitor compared with that bound without competitor (6,601, 10,620, and 2,351 cpm for RANTES, MIP-1␣, and GRO-␣, respectively). b, Binding of [125I]MIP-1␣ or [125I]GRO-␣ to U937 cells in the presence of different amounts of culture supernatants or a 100-fold excess of unlabeled MIP-1␣ or GRO-␣. The mean Ϯ SEM from duplicate samples and total counts per minute bound to cells are expressed as described in a. c, Binding of [125I]MIP-1␣ to U937 cells in the presence of different amounts of purified 35K.His. The mean Ϯ SEM from duplicate samples is expressed as described in a. The total radioactivity bound was 4,518 cpm. by guest on October 3, 2021 affinity interaction of MIP-1␣ and other CC chemokines with re- model using eosinophil peroxidase as an index of eosinophil num- combinant 35K-Fc. bers (Fig. 7a). The inhibitory effect of various doses of vCKBP on eosinophil accumulation in the skin in response to eotaxin was The vCKBP blocks the in vitro biologic activity of CC determined in guinea pigs injected i.v. with 111In-labeled eosino- chemokines phils (Fig. 7b). The potency of this inhibitor in vivo was illustrated The blockade of CC chemokine binding to cell receptors suggested by the complete blockade of eotaxin activity by a threefold molar that vCKBP would inhibit their biologic activity. This was dem- excess of vCKBP and 50% inhibition by an equal molar concen- onstrated by the ability of vCKBP to inhibit the rapid and transient tration (Fig. 7, a and b). This emphasizes the potential therapeutic increase in intracellular calcium in eosinophils by the CC chemo- application of vCKBP in inflammatory diseases. kines MCP-4 and eotaxin (Fig. 6a). This inhibitory effect was se- lective for these agonists, since vCKBP had no effect on the cal- Discussion cium response induced by C5a in eosinophils (not shown). In In this study we have identified and characterized a soluble contrast, vCKBP had no effect on the elevation of intracellular vCKBP from poxviruses. We demonstrate that vCKBP is the VV calcium levels in human neutrophils in response to the CXC che- Lister 35-kDa secreted protein, and similar proteins are expressed mokines IL-8, GRO-␣, and NAP-2 (Fig. 6b). This demonstrated by other orthopoxviruses. This protein is encoded by gene B29R or specificity of vCKBP for CC, but not CXC, chemokines in a bi- C23L in the VV strain Copenhagen (32), where it is truncated and ologic assay. inactive, and is transcribed from the strong, early-late promoter The vCKBP from VV Lister or recombinant baculovirus Ac35K p7.5, frequently used in VV expression vectors (36). also blocked the migration of U937 cells in vitro in response to the In contrast to known cellular CKRs, which are hydrophobic pro- CC chemokine MIP-1␣ (Fig. 6c). In contrast, Lister⌬35K and teins with seven transmembrane domains, the 35-kDa protein is a AcB8R supernatants did not. cysteine-rich, acidic (pI 4.2), soluble protein with no sequence similarity to cellular counterparts. Many of the poxvirus-encoded Blockade of eotaxin chemotactic activity by vCKBP in vivo cytokine receptors mimic the extracellular binding domain of The blockade of chemokine activity by vCKBP was demonstrated known cellular counterparts; thus, vCKBP may represent a viral in vivo in a guinea pig skin model of eotaxin-induced eosinophil version of an unidentified cellular molecule, as has been proposed infiltration (31). Purified recombinant 35K.His bound guinea pig for the VV IFN-␣/␤R (18). eotaxin, as determined by cross-linking assay (not shown). The Chemokine binding proteins closely related to the VV Lister vCKBP inhibited local eosinophil infiltration induced by intrader- 35-kDa protein are found in other VV strains, the leporipoxviruses mal injection of eotaxin, but not other agents, in a guinea pig Shope fibroma virus (S-T1) and myxoma virus (M-T1), racoonpox The Journal of Immunology 631 Downloaded from http://www.jimmunol.org/

FIGURE 7. Inhibition of eotaxin-induced eosinophil accumulation in guinea pig skin by vCKBP. a, Guinea pigs were pretreated for 1 h with human IL-5 to induce blood eosinophilia. Guinea pig eotaxin,

human C5a, or LTB4 was injected intradermally in the presence of by guest on October 3, 2021 purified 35K.His (30 pmol/skin site) or control medium, and eosinophil accumulation was determined after 2 h. Results represent the number of eosinophils per skin site Ϯ SEM from four or five animals. A statis- tically significant difference between groups that did or did not receive purified vCKBP is indicated by an asterisk ( pϽ0.02). The detection limit of the assay is indicated with a dashed line. No eosinophil accumulation was detected in response to the injection vehicle (HBSS containing 0.1% low endotoxin BSA), purified 35K.His, or control medium alone. b, 111In-labeled eosinophils were injected i.v. into guinea pigs, and after 10 min, guinea pig eotaxin or human C5a, with or without 35K.His, was injected intradermally into individual sites. Ac- cumulation of 111In-labeled eosinophils was determined after 2 h. Re- sults represent the mean Ϯ SEM from four animals. Eosinophil accumulation in response to the injection vehicle is indicated with a dashed line and was similar to that obtained with purified 35K.His or FIGURE 6. Blockade of chemokine biologic activity by vCKBP in control medium alone. vitro. a, Elevation of intracellular calcium levels in human eosinophils by CC chemokines. MCP-4 or eotaxin was preincubated with the indicated doses of purified 35K.His or control supernatant for 5 min virus, cowpox virus, and camelpox virus (Ref. 19 and this report), before its addition to human eosinophils and measurement of calcium and are predicted to be produced by strains of variola virus, the mobilization. The maximum elevation of intracellular calcium cause of smallpox (G3R) (40, 41). Interestingly, VV WR gene (mean Ϯ SEM) in three experiments with eosinophils from different A41L is predicted to encode a secretory protein more distantly donors is shown. Representative traces from one experiment are rep- related to the VV Lister 35-kDa protein (33, 42) and may represent resented in the insets. b, Elevation of intracellular calcium levels in another vCKBP that binds chemokines not included in this study. ␣ human neutrophils by CXC chemokines. IL-8, GRO- , or NAP-2 was Concerning the specificity of vCKBP for different chemokines, preincubated with purified 35K.His or control supernatant for 5 min we demonstrate unequivocally that it is a specific inhibitor of CC, before its addition to human neutrophils and measurement of calcium but not CXC or C, chemokines. This is shown by cross-linking mobilization. The maximum elevation of intracellular calcium experiments with cold competitors, in more quantitative binding (mean Ϯ SEM) in four experiments with neutrophils from different donors is shown. c, Chemotactic activity of MIP-1␣ on U937 cells. The experiments, and by determination of affinity constants in studies mean (ϮSEM) number of migrated cells per field under high power in which 19 chemokines were analyzed. Most importantly, the CC magnification (n ϭ 5) in the presence of MIP-1␣ (50 ng/ml), with or specificity was confirmed in biologic assays showing that vCKBP without competitor, or 10Ϫ7 M FMLP (line) is shown. does not block the interaction of CXC to cellular receptors or their 632 VACCINIA CHEMOKINE BINDING PROTEIN capacity to transduce signals. This contradicts the conclusion of this or alternative models of poxvirus infection are required to Graham et al. (19), who reported that it had a broad specificity for fully understand the role of vCKBP in poxvirus pathogenesis. CC and CXC chemokines. However, consistent with our results, The important role of chemokines and CKRs in the pathogenesis these researchers found that excess IL-8 could not compete the of virus infections is emphasized by the expression of 1) chemo- cross-linking of RANTES to vCKBP, but the effect on the biologic kine-like proteins by molluscum contagiosum virus (46), Kaposi’s activity of CXC chemokines was not investigated. sarcoma-associated herpes virus (47, 48), and murine CMV (49), The mechanism by which vCKBP inhibits chemokine activity representing potential alternative ways to modulate chemokine ac- might be its interaction with the proteoglycan binding domain of tivity; and 2) seven-transmembrane domain CKRs by large DNA chemokines, which is thought to facilitate chemokine localization viruses such as herpesviruses and poxviruses (14). Moreover, the to endothelial cells, as suggested by Graham et al. (19). Alterna- MIP-1␣ knockout mouse is less able to clear influenza virus in- tively, vCKBP might interact with the receptor binding domain of fection (50), and RANTES, MIP-1␣, and MIP-1␤ increase resis- chemokines and thus block binding to cellular receptors, as re- tance to HIV infection (2, 3). ported for poxvirus soluble receptors (13, 14). The data in this Poxvirus proteins that counteract the immune system have been paper show that vCKBP blocks CC chemokine activity by binding optimized during the evolution of viruses with their hosts; are pro- chemokines with high affinity, at a site different from the proteo- viding insights into the physiologic role of immune regulatory glycan binding domain, and preventing their interaction with cel- molecules such as cytokines (51) or chemokines; and represent lular receptors. Consistent with this, the affinity of vCKBP for potential sources of immunomodulatory proteins and new strate- ␣ ϭ human MIP-1 (Kd 115 pM) is similar or 10- to 100-fold higher gies of immune modulation. In this report a novel soluble chemo- than the affinity of most chemokines for cellular CKRs, which kine inhibitor from poxviruses with therapeutic potential is de- Downloaded from normally ranges from 1 to 10 nM (1, 4, 5). The vCKBP also binds scribed. Eosinophil accumulation in response to eotaxin is a ϭ eotaxin, RANTES, and MCP-1 with high affinity (Kd 1–15 nM). feature of allergic inflammatory reactions, such as those occurring The lower affinity of RANTES for myxoma M-T1 protein reported in allergic asthma. The potent eotaxin inhibitory effect of vCKBP ϭ by Graham et al. (19) (Kd 73 nM) might be due to the use of in vivo emphasizes its potential therapeutic application in inflam- human chemokines with the M-T1 protein from myxoma virus, matory diseases. The vCKBP has binding specificity for CC che-

whose natural host is the rabbit. There is precedent for myxoma mokines, but the soluble inhibitor or variants from other poxvi- http://www.jimmunol.org/ virus-soluble cytokine receptors binding human cytokines with ruses may provide the structural scaffolding needed to design much lower affinity than rabbit cytokines (43, 44). specific soluble inhibitors of other chemokines. Evidence for the binding of vCKBP to two chemokine mole- The finding of a soluble vCKBP also has implications for in- cules was found in cross-linking experiments using EGS and in fectious agents such as HIV, in which CKRs play an important role Scatchard analysis of the binding of MIP-1␣ to 35K-Fc. MIP-1␣ in determining transmission and disease progression (2, 3). Inhi- and IL-8 can dimerize, but there is controversy about binding to bition of the interaction of the HIV envelope with the chemokine cellular CKRs and the biologic activity of chemokines as mono- binding domain of CKRs has recently emerged as a new target for mers or dimers (4). If vCKBP does bind to dimeric chemokines, intervention. The vCKBP might be engineered to create soluble this might enhance the inhibitory activity of vCKBP. molecules that bind to the domain of gp120, which interacts with by guest on October 3, 2021 Poxviruses have evolved two different mechanisms to block the CKR to block HIV infection at an early stage (2, 3). A similar chemokine activity. First, the soluble IFN-␥R from myxoma virus approach might yield therapeutic agents to block attachment of the (M-T7), but not VV strain WR, binds the heparin-binding domain malarial parasite to the Duffy Ag on erythrocytes and initiation of of a wide range of chemokines (19, 37), and this additional func- the infection (52). tion of M-T7 is consistent with an increased leukocyte infiltration The poxvirus chemokine inhibitor reported here represents a to sites of myxoma virus replication in rabbits infected with a new and exciting virus immune evasion mechanism that provides deletion mutant lacking this protein (45). The myxoma M-T7 pro- insights into virus pathogenesis, the function of key immune reg- tein may prevent the correct localization of chemokines in vivo, ulatory molecules, and new strategies for therapeutic intervention which form a gradient by interacting with proteoglycans at the in immune responses and disease. surface of endothelial cells. We show here that the soluble IFN-␥R from 19 orthopoxviruses (20), including strains of VV, cowpox, Acknowledgments and camelpox viruses, does not bind CC or CXC chemokines. We thank A. H. Patel (Institute of Virology, Glasgow, U.K.) for the VV Second, we show that orthopoxviruses encode a vCKBP that binds Lister lacking the 35-kDa protein and Abs against this protein. chemokines with high affinity at a site different from the proteoglycan binding domain and blocks their interaction with cellular CKRs. References The expression of a potent, soluble, CC-specific vCKBP by pox- 1. Howard, O. M. Z., A. Ben-Baruch, and J. J. Oppenheim. 1996. Chemokines: progress toward identifying molecular targets for therapeutic agents. Trends Bio- viruses suggests an important role for CC chemokines in anti-viral technol. 14:46. defense. Nonetheless, deletion of the 35-kDa protein in VV rab- 2. Fauci, A. S. 1996. 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