Identification, Cloning, and Characterization of a Novel Soluble Receptor That Binds IL-22 and Neutralizes Its Activity1,2

Sergei V. Kotenko,3,4* Lara S. Izotova,* Olga V. Mirochnitchenko,* Elena Esterova,* Harold Dickensheets,† Raymond P. Donnelly,† and Sidney Pestka3*

With the use of a partial sequence of the , we identified a encoding a novel soluble receptor belonging to the class II receptor family. This gene is positioned on 6 in the vicinity of the IFNGR1 gene in a head-to-tail orientation. The gene consists of six exons and encodes a 231-aa with a 21-aa leader sequence. The secreted mature protein demonstrates 34% amino acid identity to the extracellular domain of the IL-22R1 chain. Cross-linking experiments demonstrate that the protein binds IL-22 and prevents binding of IL-22 to the functional cell surface IL-22R complex, which consists of two subunits, the IL-22R1 and the IL-10R2c chains. Moreover, this soluble receptor, designated IL-22-binding protein (BP), is capable of neutralizing IL-22 activity. In the presence of the IL-22BP, IL-22 is unable to induce Stat activation in IL-22-responsive human carcinoma A549 cells. IL-22BP also blocked induction of the suppressors of cytokine signaling-3 (SOCS-3) gene expression by IL-22 in HepG2 cells. To further evaluate IL-22BP action, we used hamster cells expressing a modified IL-22R complex ␥ consisting of the intact IL-10R2c and the chimeric IL-22R1/ R1 receptor in which the IL-22R1 intracellular domain was replaced with the IFN-␥R1 intracellular domain. In these cells, IL-22 activates biological activities specific for IFN-␥, such as up-regulation of MHC class I Ag expression. The addition of IL-22BP neutralizes the ability of IL-22 to induce Stat activation and MHC class I Ag expression in these cells. Thus, the soluble receptor designated IL-22BP inhibits IL-22 activity by binding IL-22 and blocking its interaction with the cell surface IL-22R complex. The Journal of Immunology, 2001, 166: 7096–7103.

ytokines elicit biological activities via binding to specific cytokine receptor family (CRF)5 using structurally homologous membrane-bound receptors. Ligand-induced oligomer- but distinct receptor complexes. Functional receptor complexes for C ization of the receptor extracellular domains causes the both IL-1 and IL-18 consist of the unique ligand-binding chains interaction of the receptor intracellular domains and receptor-as- (IL-1RI and IL-18R, respectively) and accessory chains (IL-1R sociated cytoplasmic , participants of the signal transduc- accessory protein (AcP) and IL-18 AcP-like, respectively), which tion cascade. In this way, the extracellular signal is transmitted are required to initiate signaling and increase cytokine binding through the cellular membrane to the cytoplasm and subsequently affinity of the receptors. The type II IL-1R (IL-1RII) plays the role to the nucleus (1–4). However, functions of several are of decoy receptor for IL-1. IL-1RII has a short intracellular domain also negatively regulated by membrane-bound decoy receptors or 1and, although it is able to bind the ligand and interact with IL-1 by soluble receptors. For example, the role of such cytokine in- AcP, it is unable to transduce signaling. The IL-1RII binds IL-1, hibitors is well characterized for IL-1 and IL-18 signaling (5–10). particularly IL-1␤, preventing ligand binding to the signal-trans- These cytokines signal through receptors belonging to the class I ducing IL-1RI. It may also act by sequestering the IL-1 AcP and, thus, reducing its availability for the functional IL-1R complex. In contrast, the IL-18-binding protein (BP) does not possess a trans- *Department of Molecular and Microbiology, University of Medicine and membrane domain and functions as a soluble inhibitor of IL-18 Dentistry, Robert Wood Johnson Medical School, Piscataway, NJ 08854; and †Divi- sion of Therapeutic Proteins, Center for Biologics Evaluation and Research, Food and actions. The use of decoy or soluble receptors for cytokines using Drug Administration, Bethesda, MD 20892 members of the class II CRF for signaling (3) has been demon- Received for publication January 24, 2001. Accepted for publication April 2, 2001. strated only for IFN-␣. The human IFN-␣R2c, one of the func- The costs of publication of this article were defrayed in part by the payment of page tional chains of the IFN-␣R complex, has two splice variants, the charges. This article must therefore be hereby marked advertisement in accordance membrane bound IFN-␣R2b with a short intracellular domain, with 18 U.S.C. Section 1734 solely to indicate this fact. which appears unable to transduce the signal, and the soluble IFN- 1 This study was supported in part by American Heart Association Grant AHA ␣ 9730247N, State of New Jersey Commission on Research Grant 799-021 (to R2a form (6, 11–16). However, the functions of these receptors S.V.K.), U.S. Public Health Services Grants RO1-CA46465 and 1P30-CA72720 from have not been well characterized. the National Cancer Institute, RO1 AI36450 and RO1 AI43369 from the National IL-22 (or IL-10-related -derived inducible factor) is a Institute of Allergy and Infectious Diseases, and an award from the Milstein Family Foundation (to S.P.). member of the family of IL-10 homologues and requires two re- 2 The cDNA and deduced amino acid sequences of CRF2-10 and its splice variants ceptor chains to assemble the functional IL-22R complex, the were submitted to the GenBank. unique IL-22R1 chain and the IL-10R2c chain (17, 18), which also 3 Address correspondence and reprint requests to Dr. Sergei V. Kotenko or Dr. Sidney Pestka, Department of Molecular Genetics and Microbiology, University of Medicine and Dentistry, Robert Wood Johnson Medical School, Piscataway, NJ 08854. E-mail 5 Abbreviations used in this paper: CRF, cytokine receptor family; AcP, accessory addresses: [email protected] and [email protected] protein; IL-22BP, IL-22-binding protein; F, forward; R, reverse; FL, tagged at N 4 Current address: Department of Biochemistry and Molecular Biology, University of terminus with the FLAG epitope; AP, adapter primer; BAC, bacterial artificial chro- Medicine and Dentistry of New Jersey, New Jersey Medical School, 185 South Or- mosome; CRF, cytokine receptor family; EST, expressed sequence tag; UTR, un- ange Avenue, Newark, NJ 07103. translated region; SOCS, suppressors of cytokine signaling; P, phosphorylatable.

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 7097 serves as a second chain of the IL-10R complex (19). Both chains Cells, transfection, and cytofluorographic analysis belongs to the class II CRF (3). The other family members are two The 16-9 hamster ϫ human somatic cell hybrid line is the Chinese hamster receptor chains for type I IFNs, two receptor chains for type II ovary cell K1 hybrid containing a translocation of the long arm of human IFNs, another chain of the IL-10R complex, and tissue factor. Sev- chromosome 6 encoding the human IFNGR1 gene and a transfected human eral orphan receptors from this family have also been identified HLA-B7 gene (21). The cells were maintained in F12 (Ham) medium (3). The IL-22R complex demonstrates unique features compared (Sigma, St. Louis, MO) containing 5% heat-inactivated FBS (Sigma). COS-1 cells, an SV40-transformed fibroblast-like simian CV-1 cell line, with other receptor complexes in that both chains are able to bind were maintained in DMEM (Life Technologies, Rockville, MD) with 10% IL-22 independently, whereas other ligands (type I and II IFNs and heat-inactivated FBS. Cells were transfected as previously described (19, IL-10) bind with high affinity to only one chain of the correspond- 20). COS-1 cell supernatants were collected at 72 h as a source of the ing receptor complexes, with the second chain only slightly mod- expressed proteins. To detect cytokine-induced MHC class I Ag (HLA-B7) expression, cells ulating affinity of the entire receptor complex (reviewed in Ref. 3). were treated with COS-1 cell supernatants or purified recombinant proteins In a search of novel receptors from the class II CRF, we identified as indicated in the text for 72 h and analyzed by flow cytometry. Cell and cloned a soluble receptor designated CRF class II member 10 surface expression of the HLA-B7 Ag was detected by treatment with (CRF2-10) or IL-22BP, which is the topic of this report. We show mouse anti-HLA (W6/32) (22) mAb followed by FITC-conjugated goat that CRF2-10 binds to IL-22, prevents its interaction with the func- anti-mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA). The cells then were analyzed by cytofluorography as previously described (13). tional IL-22R complex, and, thus, blocks the activity of IL-22. EMSAs and Western and Northern blotting Cells were starved overnight in serum-free medium and then treated with Materials and Methods IL-10 or IL-22 for 15 min at 37°C and used for EMSA experiments to PCR, primers, cDNAs, and expression vectors detect activation of Stat1, Stat3, and Stat5 as previously described (19). EMSAs were performed with a 22-bp sequence containing a Stat1-␣ bind- Primers 5Ј-CAATGGAAAAATAAAGAAGACTGTTGG-3Ј (R10-1 for- ing site corresponding to the IFN-␥ activation sequence element in the Ј Ј Ј ward (F) primer, R10-1F), 5 -GGTACTCAAGAACTCTCTTGTGACC-3 promoter region of the human IFN regulatory factor-1 gene (5 -GATC Ј Ј Ј (R10-2F), 5 -TACTCTTAATTCATCGCCCTCTCCAC-3 (R10-5 reverse GATTTCCCCGAAATCATG-3 ) as previously described (12, 23). Ј Ј (R) primer, R10-5R), 5 -CTTCAGGACTACTGAATTGCATTCAC-3 Three days after transfection, conditioned medium from COS-1 cells (R10-6R) and a library containing cDNA isolated from human placenta transiently transfected with expression plasmids was collected. FLAG- (catalog number HL4025AH, Clontech Laboratories, Palo Alto, CA) were tagged proteins in the conditioned medium were identified by Western used for nested PCR. The first round was performed with R10-1F and blotting with anti-FLAG epitope-specific M2 mAb (Sigma) as previously R10-6R primers followed by the second round with R10-2F and R10-5R described (20). FLAG-tagged proteins were purified from conditioned me- primers and the PCR product of the first round as a template. The resultant dium by immunoaffinity chromatography with the anti-FLAG M2 gel (Sig- PCR product was cloned and sequenced. Three additional primers, 5Ј- ma) according to the manufacturer’s suggested protocols. Ј Ј GGTCACAAGAGAGTTCTTGAGTACC-3 (R10-9R), 5 -TCTGAGTAG Northern blotting was performed as described previously (17) with sup- Ј Ј ␤ CTCCCAGCCGAGGCCG-3 (R10-10R), and 5 -GTAATAAGGTTCCT pressors of cytokine signaling (SOCS)-3 or -actin probes. Ј Ј GTATGTCTGAGG-3 (R10-11R), were designed to clone the 5 end of the CRF2-10 cDNA. Nested PCR was performed with these three primers and Cross-linking adapter primers (AP)1 and 2 and placental cDNA provided with the Mar- The FLAG-tagged P human IL-22 (FL-IL-22-P) was labeled with athon cDNA amplification kit (catalog number K1802-1, Clontech Labo- [32P]ATP and used for cross-linking to cells as previously described (17, ratories) following the manufacturer’s protocol. The first round was per- 23–25). [32P]FL-IL-22-P and soluble receptors were mixed in 200 ␮l PBS formed with the R10-10R primer and AP1 and was followed by the second and incubated at 22°C for 1 h followed by the addition of bis(sulfosuccin- round with either the R10-11R or R10-9R primer and AP2 and the PCR imidyl)suberate to the final concentration 0.5 mM. The reaction mixture product of the first round as a template. Several PCR products were ob- was incubated at 22°C for 10 min. and Tris-HCl buffer (pH 8.0) was added tained and sequenced. to the final concentration at 50 mM for 10 min. The complexes were later CRF2-10-specific primers 5Ј-AGGAACACTGGTTGCCTGAACAGT Ј Ј analyzed on SDS-PAGE. C-3 (R10-22F), 5 -TGGGGATCCAGGAACTCAGTCAACGCATGAGT Ј Ј Ј C-3 (R10-25F), 5 -ACGAGTCATCCTGTTCTCAGGGAGC-3 (R10-16R), Ј Ј Results and 5 -CAGAATTCATGGAATTTCCACACATC-3 (R10-18R) were de- signed to clone a fragment of the CRF2-10 cDNA encoding the mature protein Cloning of the CRF2-10 cDNA into the pEF-SPFL vector (20). These primers and either the library containing A search of the GenBank database with the TBLASTN program cDNA isolated from human placenta or cDNAs synthesized with total RNA isolated from LPS-stimulated PBMCs obtained from a healthy donor were for possible IL-22R1 (CRF2-9) homologues revealed a genomic fragment from human chromosome 6 contained within a bacterial used for nested PCR. The first round was performed with R10-22F and R10- 16R primers followed by the second round with R10-25F and R10-18R primers artificial chromosome (BAC) (GenBank accession number and the PCR product of the first round as a template. Several PCR products AL050337) potentially encoding a fragment of a protein with ho- were purified and sequenced. The PCR product corresponding to the intact mology to IL-22R1. We hypothesized that this genomic fragment CRF2-10 protein was digested with BamHI and EcoRI restriction endonucle- ases and cloned into corresponding sites of the pEF-SPFL vector (20), result- represented an exon of a novel receptor with homology to IL- ing in plasmid pEF-SPFL-CRF2-10. This plasmid encodes CRF2-10 tagged at 22R1. Further analysis of the BAC sequence revealed that several its N terminus with the FLAG epitope (FL-CRF2-10). expressed sequence tags (ESTs) were positioned in proximity to To clone the extracellular domain of the CRF2-8 protein (or ZCYTOR7, this hypothetical exon. These ESTs represented the 3Ј untranslated GenBank accession number XM004438) (3), primers 5Ј-GGGACTGAG Ј Ј region (UTR) of a cDNA with the UniGene designation Hs.126891 CAGTCTGCTGCCC-3 (R8-1F), 5 -GCCGGATCCCTGTGTCTCTGGT Ј Ј Ј Ј consisting of a group of related ESTs. We speculated that the exon GG-3 (R8-2F), 5 -ACACGGTAATAGATATGGGC-3 (R8-3R), and 5 - Ј CCGAATTCTATTTAGCCTTGAACTCT-3 (R8-4R) and the library identified in the homology search is a part of the Hs.126891 gene. containing cDNA isolated from human placenta were used for nested PCR. Two sets of primers were designed for PCR: the sequences of The first round was performed with R8-1 and R8-3 primers followed by F R primers R10-1 and R10-2 were derived from the identified hypo- the second round with R8-2F and R8-3R primers and the PCR product of the first round as a template. The resulting PCR product was digested with thetical exon sequence, and the sequences of primers R10-5 and BamHI and EcoRI restriction endonucleases and cloned into corresponding R10-6 were derived from the 3Ј UTR sequence (see Materials and sites of the pEF-SPFL vector (20), resulting in plasmid pEF-SPFL- Methods). The primers and the human placental cDNA library CRF2-8. This plasmid encodes CRF2-8 tagged at its N terminus with the were used for nested PCR. The resultant PCR product was cloned FLAG epitope (FL-CRF2-8). The construction of the FLAG-tagged phosphorylatable (P) IL-22 was and sequenced. The analysis of the sequence revealed the presence described previously (17), and the nucleotide sequences of the modified of an additional exon (exon 5; Fig. 1; exons are numbered based on regions of all constructs were verified in their entirety by DNA sequencing. the structure of the CRF2-10 gene) between exon 4 (primers R10-1 7098 IL-22-BINDING PROTEIN

IL-22R1 chain (3, 17, 18) and to the extracellular domain of CRF2-8 (3) (Fig. 2D) The function of CRF2-8 was recently char- acterized as a receptor subunit for the IL-20R complex (26). The comparison reveals that the mature CRF2-10 protein demonstrates 34 and 33% amino acid identity to the extracellular domains of IL-22R1 and CRF2-8, respectively.

Expression and purification of CRF2-10 and cross-linking

FIGURE 1. Structure of the CRF2-10 gene. Schematic structure of the To obtain the CRF2-10 protein, an expression vector was created DNA sequence (GeneBank accession number AL050337) containing a encoding FL-CRF2-10. COS-1 cells were transiently transfected fragment of human chromosome 6 (6q24.1–25.2). This DNA fragment with the expression vector and, 3 days later, conditioned medium contains the IFNGR1 gene and the CRF2-10 gene. Analysis of the DNA containing FL-CRF2-10 was collected and tested for protein ex- segment demonstrates that the CRF2-10 gene is positioned near the IFNGR1 pression. Western blotting with anti-FLAG Ab revealed that FL- gene and transcribed in the same direction as the IFNGR1 gene. Exon CRF2-10 was produced and secreted from COS-1 cells (data not positions (exons 1–6) are shown. The CRF2-10 mRNA and two splice shown). FL-CRF2-10 was purified from conditioned medium by variants are also schematically presented. Coding regions of exons are affinity column chromatography and analyzed by Western blotting shaded, and the segments corresponding to 5Ј and 3Ј UTRs are left open. with anti-FLAG Ab (Fig. 3A, lane 2). The purified protein ap- peared on SDS-PAGE as a broad band in the region of ϳ35–45 kDa, suggesting possible glycosylation of the protein. Indeed, and R10-2) and exon 6 (primers R10-5 and R10-6) containing the there are five potential sites for N-linked glycosylation (Asn-X- entire 3Ј UTR. Thr/Ser) in CRF2-10. The FL-CRF2-8 protein was also expressed Three additional primers (R10-9, R10-10, and R10-11) were in COS-1 cells and was purified following similar protocols and designed based on the sequence of exon 4 to clone the 5Ј end of the used in experiments as a control (Fig. 3A, lane 3). CRF2-10 cDNA. Nested PCR was performed with these three FL-IL-22-P was described previously (17). This protein contains primers and APs and placental cDNA provided with the Marathon the FLAG epitope at its N terminus and the consensus amino acid cDNA amplification kit. Several PCR products were cloned and sequence Arg-Arg-Ala-Ser-Val-Ala (P site), recognizable by the sequenced. Different sizes of the PCR products reflected the vari- catalytic subunit of the cAMP-dependent protein kinase (24, 25) ability of the 5Ј UTR of the CRF2-10 cDNA. The CRF2-10 cDNA fused to its COOH terminus. Purified FL-IL-22-P is a glycosylated sequence (Fig. 2) represents the sequence with the longest 5Ј UTR. protein migrating on SDS-PAGE as several bands in the region of The comparison of the sequences with the genomic sequence ϳ25–40 kDa (Fig. 3A, lane 4). The protein can be labeled with (GenBank accession number AL050337) revealed exons 1–3 of [32P]phosphate (Fig. 3A, lane 5) and cross-linked to the IL-22R the CRF2-10 gene (Figs. 1 and 2). One of the PCR products rep- resented a splice variant of the CRF2-10 cDNA, revealing the ex- chains expressed on the cell surface (17). istence of an additional exon 4a (Fig. 1, B and C, and 2). This long To determine whether CRF2-10 is capable of binding IL-22, cross-linking experiments were performed in solution. The FL-IL- splice variant was designated CRF2-10L. When the fragment of the 32 CRF2-10 cDNA encoding mature protein was amplified by PCR 22-P protein was labeled, and the P-labeled FL-IL-22-P was in- either from the placental cDNA library or cDNAs synthesized with cubated with the conditioned medium of COS-1 cells expressing total RNA isolated from LPS-stimulated PBMCs, the existence of FL-CRF2-10 (Fig. 3B, lane 8). After 1 h incubation, bis(sulfosuc- another splice variant lacking exon 5 was revealed (Figs. 1 and cinimidyl)suberate was added and cross-linking was performed. 32 2C). Joining exons 4 and 6 causes a frame-shifting event, resulting As a control, [ P]FL-IL-22-P was incubated with the conditioned in premature termination and, thus, the generation of a short pro- medium of COS-1 cells transfected with the empty vector and tein, which was designated CRF2-10 (Figs. 1 and 2C). Thus, the either left untreated (Fig. 3B, lane 6) or cross-linked after1hof S 32 entire CRF2-10 gene is composed of 6 exons (Fig. 1). The entire incubation (Fig. 3B, lane 7). In addition, [ P]FL-IL-22-P was in- first exon and a part of the second exon encode the 5Ј UTR. The cubated in solution with either purified FL-CRF2-10 (Fig. 3B,1 ␮ ␮ rest of the second exon encodes the signal peptide of the protein. g, lane 9; 0.1 g, lane 10) or with purified FL-CRF2-8 (Fig. 3B, Exons 3–5 and a portion of exon 6 encode the secreted CRF2-10. 1 ␮g, lane 11). After a 1-h incubation, cross-linking was per- The rest of the exon 6 encodes the long 3Ј UTR with the presence formed, and cross-linked complexes were resolved on the gel and of multiple polyadenylation signals. In addition, there are alterna- autoradiographed (Fig. 3B). Radiolabeled IL-22 migrates as a tive splicing events that either insert the exon 4a between exons 4 broad band in the region of 25–40 kDa (Fig. 3, A and B). Cross- and 5, creating a longer CRF2-10 splice variant, or eliminate the linking of the IL-22 in solution resulted in the appearance of an exon 5, creating prematurely terminated short protein (Figs. 1 additional broad band in the region of 100–140 kDa, likely rep- and 2). resenting an IL-22 tetramer. In the presence of the CRF2-10 pro- This BAC (GeneBank accession number AL050337) was tein, cross-linking produced an additional complex migrating as an mapped to human chromosome 6 (6q24.1–25.2). In addition to intense broad band in the region of 55–80 kDa. Moreover, the containing the entire CRF2-10 gene, the BAC also contains the intensity of bands corresponding to the IL-22 monomer was entire IFNGR1 gene (Fig. 2). Analysis of the DNA segment dem- greatly reduced, and cross-linking bands corresponding to the pos- onstrates that the CRF2-10 gene is positioned ϳ24 kb apart from sible tetramer of IL-22 were depleted, indicating that IL-22 tightly the IFNGR1 gene and transcribed in the same direction as the binds CRF2-10, interfering with the formation of IL-22 oligomers. IFNGR1 gene (Fig. 2). All exon/intron and intron/exon splice sites The pattern of cross-linking in the presence of the CRF2-8 soluble conform well to the consensus motifs (exon/GT-intron-AG/exon). extracellular domain in the mixture looks identical with the pattern Comparison of the sequence of the CRF2-10 protein with those of complexes generated by cross-linking of IL-22 to itself (Fig. 3B, of other members of this family of receptors revealed that lanes 11 and 7, respectively) indicating the lack of interaction be- CRF2-10 is mostly homologous to the extracellular domain of the tween CRF2-8 and IL-22. The Journal of Immunology 7099

FIGURE 2. The cDNA and predicted amino acid sequence of CRF2-10. A, The cDNA and the deduced amino acid sequence of CRF2-10. Amino acid residues of the putative signal peptide of CRF2-10 are boxed. Potential glycosylation sites are underlined. Positions of introns and an alternative exon 4a are indicated by arrows. B, The nucleotide sequence of the alternative exon 4a and the encoded amino acid sequence. C, CRF2-10 and its two splice variants, CRF2-10L and ␣ CRF2-10S, are aligned. D, Alignment of amino acid sequences of CRF2-10 (present study), IL-22R1 (3, 17, 18), and CRF2-8 or IL-20R (3, 26). A consensus sequence is shown on the bottom. Identical amino acids corresponding to the consensus sequence are shown in black outline with white lettering. Similar amino acids are shown in gray outline with white lettering. Amino acid residues are numbered starting from the first Met residue (signal peptide amino acids are included). The program PILEUP of the Wisconsin Package Version 9.1 from the Genetics Computer Group (Madison, WI) was used with the following parameters: the gap creation penalty 1, the gap extension penalty 1. The BOXSHADE 3.21 program was used for shading of the alignment file. 7100 IL-22-BINDING PROTEIN

FIGURE 3. CRF2-10 binds IL-22. A, Analysis of soluble proteins used in the study. CRF2-10 was tagged at the N terminus with the FLAG epitope (FL-CRF2-10). FL-CRF2-8 is the soluble extracellular domain of CRF2-8 (3) tagged at the N terminus with the FLAG epitope. IL-22 was tagged at the N terminus with the FLAG epitope and at the C terminus with the Arg-Arg-Ala-Ser-Val-Ala sequence that contains the consensus amino acid motif recognizable by the catalytic subunit of the cAMP-dependent protein kinase (FL-IL-22-P) (17). The proteins were expressed in COS-1 cells, purified from conditioned medium by affinity chromatography, and evaluated by Western blotting with anti-FLAG Ab (lanes 2–4). The 32P-labeled FL-IL-22-P was also resolved on the gel, and the gel was autoradiographed (lane 5). The molecular mass markers are shown on the left. B, Cross-linking in solution was performed to demonstrate the interaction between IL-22 and CRF2-10. [32P]FL-IL-22-P was incubated alone (lanes 6 and 7) or in the presence of COS-1 cell-conditioned medium containing the FL-CRF2-10 protein (lane 8) or either purified FL-CRF2-10 (1 ␮g, lane 9; 0.1 ␮g, lane 10) or FL-CRF2-8 (1 ␮g, lane 11), and cross-linking was performed (lanes 7–11). The cross-linked complexes were analyzed on 10% SDS-PAGE. Untreated [32P]FL-IL-22-P was also loaded as a control (lane 6). The molecular mass markers are shown on the right.

Biological activity Human lung carcinoma A549 cells are responsive to IL-22 as The functional IL-22R complex is composed of two subunits, the measured by the ability of IL-22 to induce Stat activation in these cells (17). In the presence of CRF2-10, the formation of Stat1/ IL-22R1 chain and the second IL-10R2c chain (17–19). Both chains are required for signaling; however, each chain alone is Stat3 DNA-binding complexes in response to IL-22 as measured by EMSA was blocked (Fig. 5, right panel). capable of binding IL-22 (17, 18). The IL-10R2c chain also plays the role of the second chain of the IL-10R complex (19). To de- Many cytokines induce the expression of the SOCS . In termine whether CRF2-10 can neutralize activity of IL-22, we used IL-22-responsive HepG2 human hepatoma cells (27), the expres- hamster cells expressing a chimeric IL-22R complex with the IL- sion of the SOCS-3 gene was induced within 4 h after treatment 22R1 intracellular domain replaced with the IFN-␥R1 intracellular with IL-22 (Fig. 6). IL-22-driven induction of the SOCS-3 gene domain (17). The native receptor complex was modified to facil- expression was inhibited by the addition of CRF2-10 (Fig. 6). itate detection of IL-22-induced biological activities. With this ex- change, IL-22 can activate IFN-␥-like biological responses, such as MHC class I Ag induction and Stat1 activation, in hamster cells expressing the chimeric IL-22R1/␥R1 chain and the intact second chain, IL-10R2c (17). COS-1 cell-conditioned medium containing IL-22 was left untreated or incubated with purified FL-CRF2-10 for 1 h. The medium was then added to the cells expressing the ␥ IL-22R1/ R1 and IL-10R2c chains, and the ability of IL-22 to induce IFN-␥-like biological activities in these cells was tested. IL-22 up-regulated MHC class I Ag expression in these cells (Fig. 4, thin line, open area) (17). Incubation of IL-22 with CRF2-10 before addition to the cells resulted in complete neutralization of the ability of IL-22 to induce MHC class I Ag expression in these cells (Fig. 4, thin line, shaded area). FIGURE 4. MHC class I Ag induction. Chinese hamster cells express- ␥ EMSA was also performed in these cells. IL-22 activates Stat1 ing the intact IL-10R2c chain and the chimeric IL-22R1/ R1 chain were ␥ in these cells as demonstrated by the formation of the Stat1 DNA- used (17). In these cells, IL-22 is able to induce IFN- -specific biological activities such as MHC class I Ag expression as demonstrated here by flow binding complexes after IL-22 treatment (Fig. 5, left panel) (17). cytometry. The cells were left untreated (open area, thick line) or treated Pretreatment for1hofIL-22 with the FL-CRF2-10 protein before with conditioned medium (30 ␮l) from COS-1 cells containing FL-IL-22 addition to the cells eliminated formation of the IL-22-induced that was untreated (open area, thin line) or incubated with the purified Stat1 DNA-binding complexes in a dose-dependent manner (Fig. FL-CRF2-10 protein (5 ␮g) for 1 h before addition to the cells (shaded 5, left panel). The FL-CRF2-8 protein was used as a control and area, thin line). The ordinate represents relative cell number, and the ab- had no effect on IL-22 activity in this assay (Fig. 5, left panel). scissa relative fluorescence. The Journal of Immunology 7101

FIGURE 6. SOCS-3 induction. IL-22-induced SOCS-3 expression in HepG2 cells is inhibited by the CRF2-10. IL-22-responsive HepG2 human hepatoma cells (27) were left untreated or incubated for 4 h with condi- tioned medium from COS-1 cells expressing FL-IL-22 (100 ␮l/106 cells in 1 ml, lanes 2 and 3;10␮l/106 cells in 1 ml, lanes 4–6) that was untreated (lanes 2 and 4) or incubated with purified FL-CRF2-10 (1 ␮g, lanes 3 and 5;5␮g, lane 6) for 1 h before addition to cells. At the end of the incubation period, the cells were harvested, RNA was isolated, and expression of SOCS-3 mRNA was analyzed by Northern blotting. Arrows point to SOCS-3 and ␤-actin transcripts. Equal RNA loading was assessed by eval- uating the expression of the ␤-actin gene.

cross-linking experiments demonstrated that CRF2-10 binds IL-22 and prevents its binding to the cell surface IL-22R complex.

Discussion IL-22 (18, 27, 28) is one of six recently identified IL-10 homo- logues of human and viral origin. In addition to IL-22, the list includes differentiation-associated-7 gene (29), IL-19 (30), ak155 (31), IL-20 (26), and CMV-encoded IL-10 (20). IL-22 FIGURE 5. EMSA. EMSA was used to demonstrate the ability of acts through the IL-22R complex consisting of the specific IL- CRF2-10 to inhibit formation of Stat DNA-binding complexes activated by 22R1 chain and the IL-10R2 chain, which also functions as the IL-22 treatment in intact A549 cells (right panel) or the hamster cells c ␥ second chain of the IL-10R complex (17–19). Both chains of the expressing the intact IL-10R2c chain and the chimeric IL-22R1/ R1 chain (17) (left panel). Cells were incubated with conditioned medium from IL-22R complex belong to the class II CRF, which, in addition, COS-1 cells expressing FL-IL-22 (10 ␮l/106 cells in 1 ml) that was un- includes the first chain of the IL-10R complex, both chains of type treated (lane 2) or incubated with either purified FL-CRF2-10 (1 ␮g, lanes I and type II IFNs, tissue factor, and several orphan receptors (3, 4 and 11; 0.1 ␮g, lane 5) or FL-CRF2-8 (1 ␮g, lane 6) for 1 h before 17). Searching the available sequence of the human genome, we addition to cells. Cellular lysates were prepared and assayed for Stat acti- vation by EMSA as described previously (17). Positions of Stat DNA- binding complexes are indicated by arrows. Abs against Stat1 and Stat3 were added as indicated to reduce the mobility of complexes containing these proteins.

Ligand-binding competition To further evaluate interaction between IL-22 and CRF2-10, ham- ster cells expressing the modified functional IL-22R complex de- scribed above were used for cross-linking. Binding and subsequent cross-linking of [32P]FL-IL-22-P to the cell surface results in for- mation of a number of complexes with molecular masses in the range between 60 and 240 kDa (Fig. 7, lane 1) (17). These com- plexes can be competed out by the addition of an excess of unla- beled IL-22 (Fig. 7, lane 2) (17). In addition, CRF2-10 interferes with binding of IL-22 to the cells. When [32P]FL-IL-22-P and FIGURE 7. Cross-linking. The hamster cells described in the legend to CRF2-10 were added to the cells at the same time, the appearance Fig. 3 were incubated only with [32P]FL-IL-22-P (lane 1) or with [32P]FL- of cross-linking complexes was greatly reduced (Fig. 7, lane 5). A IL-22-P with the addition of either a 100-fold excess of unlabeled IL-22 ␮ 1-h incubation of [32P]FL-IL-22-P with CRF2-10 before addition (competitor, lane 2), purified FL-CRF2-10 (5 g, lanes 3 and 5), or puri- fied FL-CRF2-8 (5 ␮g, lanes 4 and 6). [32P]FL-IL-22-P was incubated with to the cells almost completely eliminated the formation of the each of soluble receptors before the addition to the cells (lanes 3 and 4), or cross-linked complexes (Fig. 7, lane 3). As in all previous exper- the proteins were added at the same time to the cells without prior incu- iments, the presence of the CRF2-8 soluble extracellular domain bation (lanes 5 and 6). Cells were incubated with the proteins, washed, 32 with [ P]FL-IL-22-P, whether the proteins were added to the cells harvested, and cross-linked. The extracted cross-linked complexes were at the same time (Fig. 7, lane 6) or after a 1-h incubation (Fig. 7, analyzed on 7.5% SDS-PAGE. The molecular mass markers are shown on lane 4), did not have any effect on the cross-linking pattern. Thus, the right. 7102 IL-22-BINDING PROTEIN

the ability of IL-22 to induce the expression of SOCS-3 gene in HepG2 human hepatoma cells (Fig. 6). The results demonstrate that the CRF2-10 protein binds IL-22 and, thus, can be designated the IL-22BP. By binding IL-22, CRF2-10 blocks the activities of IL-22 (Figs. 4–6). Although both chains of the IL-22R complex are required to assemble the functional receptor, each chain alone is able to bind IL-22 (17, 18). Thus, it is likely that the IL-22 activity can be negatively regulated by the expression on the cell surface of the

R2c chain unpaired by the IL-22R1 chain. Because IL-22 binding to the R2c chain expressed alone does not lead to signaling, it may prevent shedding of IL-22 into the circulation (local suppression). In addition, the secretion of the soluble IL-22BP into the circula- tion can provide systemic inhibition of IL-22 action. Because in- cubation of cells expressing the functional IL-22R complex with IL-22 in the presence of IL-22BP inhibited binding of IL-22 to the cellular receptors (Fig. 7, lane 5), it seems likely that IL-22BP has higher affinity for IL-22 binding than the membrane-bound IL-22R complex. The fact that the complex of IL-22BP and IL-22, present for 3 days in conditioned medium of cells expressing modified FIGURE 8. Model of the IL-22R complex and . The functional IL-22R complex, was still unable to induce biological functional IL-22 (IL-10-related T cell-derived inducible factor) receptor complex consists of two receptor chains, the unique IL-22R1 chain and the activities (Fig. 4) indicates that the complexes are stable, and little or no dissociation of the complexes occurs. IL-10R2 (R2c) chain, which is a common chain for the IL-10R complex and the IL-22R complex (3, 17–19). Both chains can independently bind IL-22 has been demonstrated to induce production of acute- IL-22, but both chains must be present in the receptor complex to induce phase proteins in (27). The production of IL-22BP may be the signal transduction events. The IL-22 activity can be negatively regu- one of the mechanisms to precisely regulate IL-22 function (Fig. lated by the expression of the R2c chain alone because IL-22 binding to the 8). It is interesting to note that all ESTs for the UniGene R2c chain does not lead to signaling, preventing shedding of IL-22 into the Hs.126891 (the CRF2-10 or IL-22BP gene) were derived from circulation (local suppression). In addition, the secretion of the soluble lung tissue, suggesting that IL-22BP is normally expressed in this IL-22BP into the circulation can provide systemic inhibition of IL-22 tissue and perhaps functions in local inflamation. It would be of action. interest to test whether the profile of the expression of this protein differs between asthmatics and healthy individuals. The homology between IL-22BP and the IL-22R1 extracellular domain (34% have identified the gene and subsequently cloned the cDNA of a identity) is comparable to that between IL-22BP and the CRF2-8 novel receptor (Figs. 1 and 2) belonging to this family of receptors extracellular domain (33% identity). It was recently demonstrated that was first designated CRF2-10. that CRF2-8 is a receptor subunit for IL-20, which may play a role Comparison of the sequence of the CRF2-10 protein with those in (26). Thus, it will be of a great interest to determine of other members of this family of receptors revealed that whether IL-22BP can bind IL-20 (and/or other IL-10 homologues) CRF2-10 is most homologous to the IL-22R1 chain and to the and whether it plays role in psoriasis. Moreover, although it is extracellular domain of CRF2-8. The function of CRF2-8 was re- unlikely that the short splice variant (CRF2-10S) would bind IL- cently characterized as a receptor subunit for the IL-20R complex 22, it is possible that the long splice variant (CRF2-10L) may still (26). However, unlike IL-22R1 or CRF2-8, CRF2-10 is a soluble bind IL-22 and perhaps other IL-10 homologues. It is noteworthy secreted protein lacking the transmembrane and cytoplasmic do- that there is a mouse EST (GeneBank accession number mains. Based on the homology of CRF2-10 to the IL-22R1 extra- BB222214) and a bovine EST (GeneBank accession number cellular domain, we hypothesized that it would bind IL-22 and act BE809214) that encode a mouse and bovine protein, respectively, as an IL-22 antagonist. Results of several experiments confirm this homologous to human CRF2-10, indicating that the CRF2-10 gene hypothesis. By cross-linking, we demonstrated that CRF2-10 binds is conserved between the species. radiolabeled IL-22 in solution (Fig. 3B). Also, in the presence of In conclusion, we have identified, cloned, and characterized a the CRF2-10 protein, IL-22 was unable to interact with the mem- novel soluble receptor belonging to the class II CRF. The gene brane-bound IL-22R complex (Fig. 7). In addition, CRF2-10 neu- encoding this receptor maps to human chromosome 6 in the vi- tralized IL-22 activity. CRF2-10 inhibited the ability of IL-22 to cinity of the IFNGR1 gene. The protein was designated IL-22BP induce Stat activation in intact IL-22-responsive cells such as based on its ability to bind IL-22. Moreover, IL-22BP inhibits A549 human lung carcinoma cells (Fig. 5, right panel). Moreover, IL-22 activity by binding IL-22 and preventing its interaction with to further evaluate the function of the CRF2-10 protein, we used the functional IL-22R complex. Thus, IL-22BP is a naturally oc- IL-22-responsive hamster cells (17). These cells express the hu- curring IL-22 antagonist, but its physiological role remains to be man functional chimeric IL-22R complex composed of the intact determined. ␥ second chain the IL-10R2c and the chimeric IL-22R1/ R1 chain with the IL-22R1 intracellular domain replaced by the IFN-␥R1 Acknowledgments intracellular domain (17). In these cells, IL-22 induced Stat1 ac- We thank Jerome Langer and Michael Newlon for the critical review of the tivation and up-regulated MHC class I Ag expression (Figs. 4 and text and Eleanor Kells for assistance in the preparation of the manuscript. 5, left panels), activities characteristic of IFN-␥ signaling (17). The addition of CRF2-10 inhibited the ability of IL-22 to induce Stat1 References 1. Darnell, J. E., Jr., I. M. Kerr, and G. R. Stark. 1994. Jak-STAT pathways and activation and MHC class I Ag expression in these cells (Figs. 4 transcriptional activation in response to IFNs and other extracellular signaling and 5, left panels). We also demonstrated that CRF2-10 inhibited proteins. Science 264:1415. The Journal of Immunology 7103

2. Ihle, J. N., B. A. Witthuhn, F. W. Quelle, K. Yamamoto, and O. Silvennoinen. 18. Xie, M. H., S. Aggarwal, W. H. Ho, J. Foster, Z. Zhang, J. Stinson, W. I. Wood, 1995. Signaling through the hematopoietic cytokine receptors. Annu. Rev. Im- A. D. Goddard, and A. L. Gurney. 2000. (IL)-22, a novel human munol. 13:369. cytokine that signals through the receptor-related proteins CRF2-4 and 3. Kotenko, S. V., and S. Pestka. 2000. Jak-Stat signal transduction pathway IL-22R. J. Biol. Chem. 275:31335. through the eyes of cytokine class II receptor complexes. Oncogene 19:2557. 19. Kotenko, S. V., C. D. Krause, L. S. Izotova, B. P. Pollack, W. Wu, and S. Pestka. 4. Pestka, S., S. V. Kotenko, G. Muthukumaran, L. S. Izotova, J. R. Cook, and 1997. Identification and functional characterization of a second chain of the in- G. Garotta. 1997. The interferon ␥ (IFN-␥) receptor: a paradigm for the mul- terleukin-10 receptor complex. EMBO J. 16:5894. tichain cytokine receptor. Cytokine Rev. 8:189. 20. Kotenko, S. V., S. Saccani, L. S. Izotova, O. V. Mirochnitchenko, and S. Pestka. 5. McMahan, C. J., J. L. Slack, B. Mosley, D. Cosman, S. D. Lupton, L. L. Brunton, 2000. Human cytomegalovirus harbors its own unique IL-10 homolog (cmvIL- C. E. Grubin, J. M. Wignall, N. A. Jenkins, and C. I. Brannan. 1991. A novel IL-1 10). Proc. Natl. Acad. Sci. USA 97:1695. receptor, cloned from B cells by mammalian expression, is expressed in many 21. Soh, J., R. J. Donnelly, T. M. Mariano, J. R. Cook, B. Schwartz, and S. Pestka. cell types. EMBO J. 10:2821. 1993. Identification of a yeast artificial chromosome clone encoding an accessory ␥ 6. Novick, D., B. Cohen, and M. Rubinstein. 1994. The human interferon ␣/␤ re- factor for the human interferon receptor: evidence for multiple accessory fac- ceptor: characterization and molecular cloning. Cell 77:391. tors. Proc. Natl. Acad. Sci. USA 90:8737. 7. Dinarello, C. A. 1999. Interleukin-18. Methods 19:121. 22. Barnstable, C. J., W. F. Bodmer, G. Brown, G. Galfre, C. Milstein, 8. Dinarello, C. A. 2000. Proinflammatory cytokines. Chest 118:503. A. F. Williams, and A. Ziegler. 1978. Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens–new tools for 9. Akira, S. 2000. The role of IL-18 in innate immunity. Curr. Opin. Immunol. genetic analysis. Cell 14:9. 12:59. 23. Kotenko, S. V., L. S. Izotova, B. P. Pollack, T. M. Mariano, R. J. Donnelly, 10. Daun, J. M., and M. J. Fenton. 2000. Interleukin-1/Toll receptor family members: G. Muthukumaran, J. R. Cook, G. Garotta, O. Silvennoinen, J. N. Ihle, and receptor structure and signal transduction pathways. J. Interferon Cytokine Res. S. Pestka. 1995. Interaction between the components of the interferon ␥ receptor 20:843. complex. J. Biol. Chem. 270:20915. 11. Domanski, P., M. Witte, M. Kellum, M. Rubinstein, R. Hackett, P. Pitha, and 24. Li, B. L., J. A. Langer, B. Schwartz, and S. Pestka. 1989. Creation of phosphor- O. R. Colamonici. 1995. Cloning and expression of a long form of the ␤ subunit ␣ ␣␤ ylation sites in proteins: construction of a phosphorylatable human interferon . of the interferon receptor that is required for signaling. J. Biol. Chem. 270: Proc. Natl. Acad. Sci. USA 86:558. 21606. 25. Pestka, S., L. Lin, W. Wu, and L. Izotova. 1999. Introduction of protein kinase 12. Kotenko, S. V., L. S. Izotova, B. P. Pollack, G. Muthukumaran, K. Paukku, recognition sites into proteins: a review of their preparation, advantages, and O. Silvennoinen, J. N. Ihle, and S. Pestka. 1996. Other kinases can substitute for applications. Protein Expression Purif. 17:203. ␥ Jak2 in signal transduction by interferon- . J. Biol. Chem. 271:17174. 26. Blumberg, H., D. Conklin, W. F. Xu, A. Grossmann, T. Brender, S. Carollo, 13. Kotenko, S. V., L. S. Izotova, O. V. Mirochnitchenko, C. Lee, and S. Pestka. M. Eagan, D. Foster, B. A. Haldeman, A. Hammond, et al. 2001. : ␣ ␣ 1999. The intracellular domain of interferon- receptor 2c (IFN- R2c) chain is discovery, receptor identification, and role in epidermal function. Cell 104:9. responsible for Stat activation. Proc. Natl. Acad. Sci. USA 96:5007. 27. Dumoutier, L., E. Van Roost, D. Colau, and J. C. Renauld. 2000. Human inter- 14. Lutfalla, G., S. J. Holland, E. Cinato, D. Monneron, J. Reboul, N. C. Rogers, leukin-10-related T cell-derived inducible factor: molecular cloning and func- J. M. Smith, G. R. Stark, K. Gardiner, I. M. Kerr, and G. Uze´. 1995. Mutant U5A tional characterization as an hepatocyte-stimulating factor. Proc. Natl. Acad. Sci. cells are complemented by an interferon-␣␤ receptor subunit generated by alter- USA 97:10144. native processing of a new member of a cytokine receptor gene cluster. EMBO J. 28. Dumoutier, L., J. Louahed, and J. C. Renauld. 2000. Cloning and characterization 14:5100. of IL-10-related T cell-derived inducible factor (IL-TIF), a novel cytokine struc- 15. Pfeffer, L. M., L. Basu, S. R. Pfeffer, C. H. Yang, A. Murti, D. Russell-Harde, and turally related to IL-10 and inducible by IL-9. J. Immunol. 164:1814. E. Croze. 1997. The short form of the interferon ␣/␤ receptor chain 2 acts as a 29. Jiang, H., J. J. Lin, Z. Z. Su, N. I. Goldstein, and P. B. Fisher. 1995. Subtraction dominant negative for type I interferon action. J. Biol. Chem. 272:11002. hybridization identifies a novel melanoma differentiation associated gene, mda-7, 16. Hardy, M. P., C. M. Owczarek, S. Trajanovska, X. Liu, I. Kola, and P. J. Hertzog. modulated during human melanoma differentiation, growth and progression. On- 2001. The soluble murine type I interferon receptor Ifnar-2 is present in serum, cogene 11:2477. is independently regulated, and has both agonistic and antagonistic properties. 30. Gallagher, G., H. Dickensheets, J. Eskdale, L. S. Izotova, O. V. Mirochnitchenko, Blood 97:473. J. D. Peat, S. Pestka, N. Vazquez, R. P. Donnelly, and S. V. Kotenko. 2000. 17. Kotenko, S. V., L. S. Izotova, O. V. Mirochnitchenko, E. Esterova, Cloning, chracterization and expression of interleukin-19 (IL-19) a novel homo- H. Dickensheets, R. P. Donnelly, and S. Pestka. 2001. Identification of the func- logue of human interleukin-10 (IL-10). Genes Immun. 1:442. tional interleukin-22 (IL-22) receptor complex: the IL10R2 chain (IL-10R␤)isa 31. Knappe, A., S. Hor, S. Wittmann, and H. Fickenscher. 2000. Induction of a novel common chain of both IL10 and IL22 (IL-10-related T cell-derived inducible cellular homolog of interleukin-10, AK155, by transformation of T lymphocytes factor (IL-TIF)) receptor complexes. J. Biol. Chem. 276:2725. with herpesvirus saimiri. J. Virol. 74:3881.