Cloning and Characterization of IL-1HY2, a Novel Interleukin-1 Family Member*

The interleukin-1 (IL-1) family members play an important role in the process of inflammation and host defense. We describe here the identification and characterization of a novel member of the IL-1 family, IL-1HY2. The human IL-1HY2 protein shares significant amino acid sequence similarity (37%) with the IL-1 receptor antagonist and has a predicted three-dimensional structure similar to that of the IL-1 receptor antagonist. TheIL-1HY2 gene is located in close proximity to other IL-1 family genes on human chromosome 2, and the genomic organization of theIL-1HY2 gene is highly conserved with other IL-1 family members. IL-1HY2 protein is secreted from mammalian cells, and the purified recombinant IL-1HY2 protein binds soluble IL-1 receptor type I. IL-1HY2 is expressed in human skin, spleen, and tonsil. Immunohistochemical analysis showed that the IL-1HY2 protein is expressed in the basal epithelia of skin and in proliferating B cells of the tonsil. These data suggest that IL-1HY2 is a novel IL-1 family member and that it may participate in a network of IL-1 family members to regulate adapted and innate immune responses.

Cytokines are often expressed at low levels owing to their potent effects. To identify novel cytokine genes expressed at low levels, a unique high throughput cDNA screening approach (10,11) was employed. This approach utilizes a set of oligonucleotide probes to hybridize successively to arrays of human cDNAs from various tissues. Hybridization patterns allow the cDNA clones to be clustered into groups of similar or identical sequences. The number of clones in a particular cluster represents the relative expression level of a specific gene. Representative clones from clusters with low numbers of members were selected for sequencing. Novel cytokine family members were identified using various sequence analysis algorithms. The IL-1HY1 gene was recently identified using this approach (5).
In the current report, we describe the discovery of a novel IL-1 family member referred to as IL-1HY2. The characteristics of IL-1HY2 indicate that it shares a common ancestral precursor gene with other IL-1 family members. We demonstrate that IL-1HY2 is secreted, binds the soluble IL-1 receptor, and is expressed in human skin, spleen, and tonsil, suggesting that it may play a role as a secreted ligand in host defense and other immune responses.

EXPERIMENTAL PROCEDURES
Isolation of the IL-1HY2 cDNA and Sequence Analysis-A cDNA library was constructed from human fetal skin mRNA (Invitrogen) in the pSport1 vector (Life Technologies, Inc.). Inserts of the library were amplified by PCR using primers specific to pSport1 vector sequences that flank the inserts. These samples were then analyzed using the screening by hybridization approach as described previously (10,11). Briefly, the PCR products were spotted onto nylon membranes and hybridized with oligonucleotide probes to give sequence signatures. The clones were clustered into groups of similar or identical sequences, and single representative clones for rarely expressed transcripts were selected for gel sequencing. The 5Ј sequence of the amplified inserts was then deduced using the reverse M13 sequencing primer by ABD Big Dye terminator cycle sequencing (PerkinElmer Life Sciences). The cDNA sequence data base was analyzed using the BLAST and motif search algorithms. A novel cDNA sequence encoding a polypeptide homologous to the IL-1ra protein was identified. The sequence was confirmed in part by sequencing PCR products generated from 5Ј rapid amplification of cDNA ends analysis using a Marathon cDNA amplification kit (CLONTECH). This novel gene sequence was designated IL-1HY2.
Chromosomal Localization and Genomic Organization of the IL1HY2 Gene-PCR primers specific to IL-1HY2 (5Ј-CCGCACCAAGGTCCCC-ATTTTC-3Ј and 5Ј-GAGCCCACAAGGATAACCCAGG-3Ј) were used to screen the NIGMS human/rodent somatic cell hybrid mapping panel 2 (12) and the Stanford G3 Human/Hamster Radiation Hybrid panel (13) (Research Genetics, Huntsville, AL). A PCR product of 824 base pairs was amplified using the following conditions: 94°C for 3 min; 30 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 1 min; and 72°C for 10 min. Linkage analysis and subsequent chromosomal localization were obtained using the Stanford Human Genome Center RH server. To determine the genomic structure of the IL-1HY2 gene, human BAC clones (Research Genetics) were screened by PCR with primers specific to the IL-1HY2 cDNA. The BAC393I6 containing the IL-1HY2 gene was partially digested by Sau3AI enzyme and size-selected restriction fragments were inserted into the BamHI site of pUC18 (Amersham Phar-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AF334755 and AF334756.
‡ Present address: Center for Biomolecular Science and Engineering, University of California, Santa Cruz, CA 95064.
Transfection of Chinese Hamster Ovary (CHO) Cells and Protein Analysis by Western Blot Analysis-The coding region of the IL-1HY2 cDNA was cloned into the pcDNA3.1/V5-His TOPO vector (Invitrogen) using PCR primers (5Ј-GAGCCGCCATGTGTTCCCTCCCCATGGCAA-G-3Ј and 5Ј-GCTACCAGCTCTGTTCAAAGTAAAAC-3Ј). CHO cells obtained from the American Type Culture Collection were cultured in F12K supplemented with 10% fetal bovine serum, 100 units/ml penicillin G, and 100 g/ml streptomycin sulfate at 37°C in 5% CO 2 . CHO cells were transfected with the IL-1HY2 expression vector or a control empty vector using Fugene-6 according to the manufacturer's instructions (Roche Molecular Biochemicals). After the transfection, medium was replaced with serum-free F12K medium, and the cells were incubated for an additional 24 h prior to harvesting. Conditioned medium was centrifuged and then filtered through a 0.2-m filter (Pall Gelman Laboratory) to remove cell debris. The conditioned medium was subsequently concentrated by 10-fold using the Microcon YM-10 according to the manufacturer's instructions (Amicon). Cell lysates and conditioned media were analyzed by Western blot analysis. After SDS-polyacrylamide gel (15%) electrophoresis, protein samples were electroblotted onto an Immobilon-P membrane (Millipore) and probed with the anti-IL-1HY2 antibodies. Immunocomplexes were visualized by SuperSignal West Pico chemiluminescence detection reagents with goat anti-rabbit IgG conjugated with horseradish peroxidase as a secondary antibody (Pierce).
Purification of Recombinant IL-1HY2 Protein and Measurement of Receptor-Ligand Binding Affinities-The coding region of IL-1HY2 cDNA was cloned into pCR II/TOPO vector (Invitrogen) using PCR primers (5Ј-GTCATATGTGTTCCCTCCCCATGGCAAG-3Ј and 5Ј-GAA-GCTTGATCACTACCAGCTCTGTTCAAAGTAAAAC-3Ј). The sequence of the IL-1HY2 coding region was confirmed by sequencing. The NdeI/ HindIII restriction fragment containing IL-1HY2 was isolated and inserted between the NdeI and HindIII sites of the bacterial expression vector pRSET B (Invitrogen). The resulting plasmid, pRSET IL-1HY2, was transformed into Escherichia coli strain BL21(DE3)pLysS (Invitrogen). IL-1HY2 protein expression was induced by isopropyl-1-thio-␤-Dgalactopyranoside, and cells expressing IL-1HY2 were harvested by centrifugation at 10, 000 ϫ g for 30 min. The cells were then lysed in 10 volumes of lysis buffer (20 mM Tris, pH 8.0, 1 mM EDTA) by passing through an Avestin homogenizer. IL-1HY2 present in the supernatant was purified by a combination of Q-Sepharose anion exchange, phenyl-Sepharose hydrophobic interaction, and DEAE-Sepharose anion exchange chromatography (AP Biotech). The IL-1HY2 protein was 95% pure as determined by SDS-polyacrylamide gel electrophoresis and Coomassie staining. The dissociation equilibrium constants (K D ) of IL-1HY2, IL-1ra, and IL-1␤ binding to soluble IL-1 receptor type I (sIL-1RI) were measured using the IAsys affinity sensor (Labsystems Affinity Sensors, Franklin, MA) according to the manufacturer's instructions and as described (14,15). Purified recombinant IL-1HY2 protein, recombinant IL-1ra, and IL-1␤ proteins (R & D Systems) were biotinylated using EZ-Link Sulfo-NHS-LC-Biotin (Pierce) according to the manufacturer's instructions. The biotinylated proteins (1.2 M) were immobilized onto the avidin-layered biotin-coated cuvette surface. After ligand immobilization, sIL-1RI (R & D Systems) at various concentrations (70 -500 nM) was added to the cuvette. Protein binding kinetics was measured, and the K D of each protein to the IL-1 receptor was obtained using the software Fastfit (Labsystems Affinity Sensors) (14,15).
Generation of Antibodies and Immunohistochemistry-Anti-IL-1HY2 polyclonal antibodies were raised in rabbit against a synthetic peptide (CTLPNRGLDRTKVP) specific to the IL-1HY2 protein. The antibodies specific to the peptide were subsequently isolated by affinity purification using standard procedures (16). The anti-IL-1HY2 antibodies did not cross-react with other IL-1 family members, including IL-1ra, IL-1␤, and IL-1HY1, in a Western blot analysis (data not shown). The anti-CD20 monoclonal antibody was purchased from DAKO (Carpenteria, CA), and the anti-Ki67 was obtained from Coulter Immunotech (Miami, FL). Immunohistochemistry was performed at QualTek Molecular Systems, Inc. (Santa Barbara, CA) using a procedure described previously (17). Briefly, sections of adult human tonsil and skin were reacted with the primary anti-IL-1HY2 antibodies that were detected by a biotinylated secondary antibody followed by streptavidin-AP. Fast Red was used as the chromogen (red), and the slides were counter stained with hematoxylin (blue nuclear stain). In the negative control staining, slides were processed without adding the primary antibody. For the double labeling immunohistochemistry, the second primary antibody, the anti-CD20 monoclonal antibody, or the anti-Ki67 monoclonal antibody, was detected using a biotinylated secondary antibody followed by streptavidin-horseradish peroxidase. Diaminobenzidine (DAB) was used as the chromogen (brown).
Three-dimensional Structure Modeling-The GeneAtlas TM software package (Molecular Simulations Inc., San Diego, CA) was used to generate three-dimensional structural models for IL-1HY2. The threedimensional structural models of the IL-1HY2 protein were predicted based on a search of 4250 nonredundant Protein Data Bank structures using the multiple alignment sequence profile-based searching method (PSI-BLAST) (18), an automated sequence and structure searching procedure (High Throughput Modeling) (19), and a fold recognition method (SeqFold) (20). The known structures of IL-1ra (21) and IL-1␤ (22) (1i1b for IL-1␤ and 1irp, 1ilr, and 1ilt for IL-1ra) that were the best fit structures were used as templates.

RESULTS
To identify additional IL-1 family member genes, BLAST and motif algorithms were used to search an expressed human gene sequence data base generated using screening by hybridization technology (10,11). A cDNA sequence from a human fetal skin library was identified that encodes a polypeptide with homology to the IL-1ra protein. Sequencing of the cDNA clone and subsequent 5Ј rapid amplification of cDNA ends analysis led to the identification of a full-length coding se- quence designated as IL-1HY2. The nucleotide and the deduced protein sequences are shown in Fig. 1. Blast search analysis indicated that this nucleotide sequence had not been previously reported in any public data bases. The IL-1HY2 cDNA contains an open reading frame corresponding to a 152-amino acid residue protein with a predicted molecular mass of 17 kDa and a predicted isoelectric point of 4.7.
The genomic organization of the IL-1HY2 gene was determined by sequencing of the human BAC clone containing the IL-1HY2 gene (see "Experimental Procedures"). Like the other IL-1 family members, the IL-1HY2 gene consists of four coding exons (Fig. 3). The structure of the IL-1HY2 gene is remarkably similar to that of the other IL-1 family genes, with a conserved pattern of intron placement (Fig. 2A).
To determine the expression pattern of the IL-1HY2 gene, a panel of cDNA libraries derived from various human tissues was analyzed by a semi-quantitative PCR using primers specific to IL-1HY2. The IL-1HY2 transcripts were expressed in fetal skin and at a lower level in spleen (Fig. 4A). To determine whether IL-1HY2 protein is secreted in mammalian cells, an IL-1HY2 expression construct was used to transfect CHO cells. The recombinant IL-1HY2 protein was detected in both the cell lysate and conditioned medium from transiently transfected CHO cells (Fig. 4B).
Because the IL-1HY2 protein, which belongs to the IL-1 family, is secreted in mammalian cells, the recombinant IL-1HY2 protein was tested for binding to the sIL-1RI using the IAsys affinity sensor. Biotinylated recombinant IL-1HY2, IL-1ra, or IL-1␤ protein was immobilized on the avidin-layered biotin-coated cuvette surface, and their dissociation equilibrium constants (K D ) with sIL-1RI were measured (see "Experimental Procedures"). The recombinant IL-1HY2 protein bound sIL-1RI, although the binding affinity of IL-1HY2 was lower (higher K D ) than those of IL-1ra and IL-1␤ (Table I).
To analyze IL-1HY2 protein expression in the human skin, polyclonal antibodies raised against a peptide specific to IL-1HY2 were used in the immunohistochemistry study (Fig. 5). IL-1HY2 protein was expressed mostly in the basal epithelia of the human skin (Fig. 5B). To identify the cell types in immune tissues that express the IL-1HY2 protein, immunohistochemistry was performed on the tonsil (Fig. 6). From the staining data, the IL-1HY2 protein was expressed in the germinal center of the tonsil (Fig. 6B). Using double labeling immunostaining, the IL-1HY2 protein was detected in a subset of B cells (CD-20-positive) (Fig. 6C), most of which were proliferating (Ki67-positive) cells (Fig. 6D). Furthermore, the IL-1HY2positive cells did not react with the anti-CD45RO (T cell marker) antibody or the anti-CD14 (monocyte marker) antibody (data not shown), suggesting that the IL-1HY2 protein was not expressed in T cells or monocytes in the tonsil. DISCUSSION Recently, five new IL-1 family members, including IL-1HY1 FIL1⑀, FIL1, and FIL1 (7), and IL-1H1 (8), were identified. In this report, we describe the discovery of a novel IL-1 family gene, IL-1HY2, expanding the IL-1 family to include a total of 10 family members (IL-1␣, IL-1␤, IL-1ra, IL-18, IL-1HY1, FIL1⑀, FIL1, FIL1, IL-1H1, and IL-1HY2). It would appear that IL-1HY2 was derived from a duplication event from an ancestral gene common to the other IL-1 family members. This notion is evidenced by significant amino acid sequence homologies (19 -41%) that the IL-1HY2 protein shares with the other IL-1 family members (Fig. 2, A and B) and by the high degree of conservation in the genomic organization of the IL-1HY2 gene with the other family member genes (Figs. 1 and  3). Based on the amino acid sequence homologies, IL-1HY2, IL-1HY1, and IL-1ra appeared to form a subfamily that evolved from a common ancestor (Fig. 2C).
The three-dimensional structural model of the IL-1HY2 pro-tein was predicted based on a search of 4250 nonredundant Protein Data Bank structures. The known crystal structure of IL-1ra (21), which was one of the best fit structures, was used as a template (Fig. 7). The IL-1HY2 structural model displays a 12-␤-stranded trefoil structure and is highly similar to the IL-1ra crystal structure (Fig. 7). The structural model of IL-1HY2 predicted based on the IL-1␤ crystal structure (22) also shares similarity to the IL-1␤ structure (data not shown). The similarity between the IL-1HY2 structural models and the IL-1ra and IL-1␤ structures provides more evidence that IL-1HY2 belongs to the IL-1 family. The recombinant IL-1HY2 proteins expressed in mammalian  and IL-1␤ binding to sIL-1RI The dissociation equilibrium constant (K D ) was measured using the IAsys affinity sensor (Labsystems Affinity Sensors) as described (14,15). Data from a representative experiment are presented.  5. Analysis of IL-1HY2 protein expression in human skin by immunohistochemistry. Sections of the human skin were processed without primary antibodies (A) or with affinity purified polyclonal antibodies specific to the IL-1HY2 polypeptide (B). A biotinylated secondary antibody was used followed by streptavidin-AP. The anti-IL-1HY2 antibodies were detected using Fast Red as the chromogen (red). Slides were counterstained with hematoxylin (blue nuclear stain). Pictures are shown at 150ϫ magnification.
FIG. 6. Cell type-specific expression of the IL-1HY2 protein in the human tonsil. Sections of the human tonsil were reacted without primary antibodies (A) or with affinity purified polyclonal antibodies specific to the IL-1HY2 polypeptide (B) as described in the legend for Fig. 5. For double labeling of the tissues, anti-CD20 antibody was used as a second primary antibody in addition to the anti-IL-1HY2 antibodies (C) or the anti-IL-1HY2 and anti-Ki67 antibodies (D) were used as primary antibodies. The second primary antibody (anti-CD20 antibody or anti-Ki67 antibody) was detected using a biotinylated secondary antibody followed by streptavidin-horseradish peroxidase. Diaminobenzidine (DAB) was used as the chromogen (brown). Sections shown represent the edges of a germinal center within a tonsil. Pictures are shown at 400ϫ magnification.

FIG. 7.
Comparison of the three-dimensional structural model of IL-1HY2 with the crystal structure of IL-1ra. The IL-1HY2 structural model was generated using the GeneAtlas TM software package (Molecular Simulations Inc., San Diego, CA). A and C, the crystal structure of IL-1ra (blue) and the predicted 12-␤-stranded trefoil structural model of IL-1HY2 (yellow) based on the IL-1ra template, respectively. Both the crystal structure and the model are viewed down the ␤-barrel axis. B, the superimposed crystal structure and the IL-1HY2 structural model. molecular mass and the predicted molecular mass of IL-1HY2 may be due to other post-translational modifications, such as phosphorylation.
Although IL-1HY2 lacks an obvious signal peptide, we have demonstrated that the recombinant IL-1HY2 protein is secreted from CHO cells (Fig. 3B), suggesting that IL-1HY2 may act as a secreted ligand. Other IL-1 family proteins, including IL-1␤ and IL-18, also lack a classical signal peptide and are still secreted. The secretion of IL-1␤ and IL-18 involves cleavage by the IL-1␤ converting enzyme (25)(26)(27). The IL-1HY2 protein does not contain an apparent consensus site for the IL-1␤ converting enzyme cleavage. Thus, secretion of the IL-1HY2 protein may involve an alternative secretory mechanism.
IL-1HY2 was shown to bind sIL-1RI, although it does not bind as avidly as IL-1␤ and IL-1ra to sIL-1RI (Table I). The K D values of IL-1ra and IL-1␤ binding to sIL-1RI presented here are higher than those reported by other groups (28,29). This may reflect the different methodology and instrumentation (IAsys affinity sensor) that we used. Thus, the binding affinities of IL-1HY2 can only be compared with those of IL-1ra and IL-1␤ determined using the same conditions. The affinity of IL-1HY2 binding to the IL-1 receptor was determined using the soluble form of the receptor. It has been shown that IL-1 receptor accessory protein plays a role in enhancing IL-1 receptor-ligand binding affinities and signaling activities (30,31). Thus, the IL-1 receptor accessory protein or another accessory protein may affect the binding affinity of IL-1HY2 to the IL-1 receptor. The fact that IL-1HY2 binds to sIL-1RI suggests that IL-1HY2 may play a role in regulating the IL-1 receptor function.
The data presented in the current report indicate that IL-1HY2 is expressed in skin and activated B cells of the human tonsil. The tissue-specific expression pattern of IL-1HY2 suggests that it may be involved in the regulation of normal immune responses and inflammatory pathophysiology.