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Functional Chicken IL-12Identification and Molecular Cloning of

Zuilekom, Joan Burnside and Virgil E. J. C. SchijnsWinfried G. J. Degen, Nancy van Daal, Hanneke I. van

http://www.jimmunol.org/content/172/7/4371doi: 10.4049/jimmunol.172.7.4371

2004; 172:4371-4380; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2004 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Identification and Molecular Cloning of FunctionalChicken IL-121

Winfried G. J. Degen,2* Nancy van Daal,* Hanneke I. van Zuilekom,* Joan Burnside,† andVirgil E. J. C. Schijns*

By a combination of large-scale sequencing, bioinformatics, and traditional molecular biology, we identified the long-searched-forcDNA sequences encoding the homologues of the chicken IL-12p35 and IL-12p40 chains. These molecules are the first discoverednonmammalian IL-12 subunits. The homologies of the chicken IL-12p35 and IL-12p40 proteins to the corresponding knownsubunits of various species, i.e., humans, sheep, horse, cat, bovine, mouse, and woodchuck, ranged between 21 and 42%, respectively.The expression of IL-12 subunits was observed in lymphoid cells and proved to be dependent on the cell type and stimulus, whileexpression was not detected in stimulated primary chicken embryo fibroblast cells. Following transient expression of both molecules inCOS-7 cells, we confirmed the necessity of heterodimerization into IL-12p70 to yield bioactivity as was also shown for its mammaliancounterparts. The chicken IL-12p70 molecule, generated either by transient coexpression of monomeric IL-12p35 and monomericIL-12p40 or as a fusion protein (as in a fusion linker construct), induced IFN-� synthesis and proliferative activity of freshly exposedchicken splenocytes. The high degree of functional similarity between chicken IL-12 and IL-12 of higher mammalian vertebrates,despite their poor sequence homology, illustrates the conservation and vital importance of the IL-12 molecule since the evolu-tionary dichotomy of birds and mammals >300 million years ago. In this article, we describe the first nonmammalian IL-12molecule and show that this chicken IL-12 molecule is bioactive. The Journal of Immunology, 2004, 172: 4371–4380.

T he group of soluble secreted molecules, collectivelytermed cytokines, represents critical communication sig-nals among the cells of the immune system and between

immune and nonimmune system cells. In mammals, a group ofcomposite heterodimeric cytokines has been identified based on com-plexes of a 40-kDa protein subunit (p40). The mammalian p40 sub-unit links covalently, by disulfide binding, with a 35-kDa protein(p35) to form the heterodimeric p40-p35 IL-12 (IL-12p70 or IL-12)molecule (1–3). In addition, p40 may form the composite cytokineIL-23, after combining with a 19-kDa protein (p19) (4), a moleculestructurally related to IL-6, p35, and G-CSF. IL-12, also known as NKcell stimulatory factor (5) or cytotoxic lymphocyte maturation factor(6), is a pleiotropic cytokine that is produced by monocytes (7), mac-rophages (8), and dendritic cells (9) and has been shown to mediatemultiple effects on T cells and NK cells (10, 11). These effects mainlyinclude induction of IFN-� and TNF production by resting and acti-vated T and NK cells and induction of activated T and NK cell pro-liferation (5). Furthermore, IL-12 plays an important and crucial rolein mammals in the regulation of IFN-� characteristic Th1-type im-mune responses (12, 13), while it inhibits the Th2 pathway. Th1-biased immune responses are essential in the control of mostly intra-cellular infections of bacterial, parasitic, fungal, or viral nature (for areview, see Ref.14).

The importance of the Th1/Th2 effector subsets and the key roleof IL-12 in directing Th1-associated cell-mediated immunity ex-plains the necessity to clone and characterize this molecule forspecies of veterinary relevance, where it can be used as a tool forprophylactic/therapeutic vaccination or for tumor therapy. To datebovine (15), cat (16–18), dog (19, 20), goat (J. C. Beyer, W. P.Cheevers, and N. M. Kumpula-McWhirter, unpublished observa-tions), horse (21), pig (22), and sheep (23–25) homologues of IL-12p40 and IL-12p35 subunits have been cloned. Some of them arecurrently being tested in clinical applications.

Unfortunately, no avian IL-12 subunit has been cloned yet. Infact, no nonmammalian IL-12 molecule has been identified or iso-lated so far. It is also very remarkable that there is no evidence fora balanced Th1/Th2 system in nonmammalian species. Yet, theexistence of a Th1-like cytokine network in birds was, in part,evidenced some years ago by the identification of chicken IFN-�(26) and the recent discovery of chicken IL-18 (27). One of themajor reasons for the failure to identify avian IL-12 thus far is thelow sequence homology, usually restricted to �30–50%, betweenavian and mammalian cytokine sequences. This explains the di-saster of classical approaches to identify avian homologues ofmammalian cytokines. The identification by PCR amplification us-ing primers based on mammalian sequences proved to be verydifficult and unpredictable (28, 29). As a result of this poor ho-mology, cloning and sequencing of avian cytokines lags dramati-cally behind similar work achieved in mammals. So far only alimited number of avian cytokines have been identified (for tworecent reviews, see Refs. 28 and 29).

By using a combination of large-scale sequencing, bioinformatics,and traditional molecular biology, we succeeded in the isolation andcharacterization of an avian (chicken (Ch)3) IL-12p40 (ChIL-12p40) and IL-12p35 (ChIL-12p35) subunit. Despite the limited

*Department of Vaccine Technology and Immunology, Intervet International BV,Boxmeer, The Netherlands; and †Department of Animal and Food Sciences DelawareBiotechnology Institute, Delaware Technology Park, Newark, DE 19711

Received for publication June 23, 2003. Accepted for publication January 21, 2004.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported in part by the Center for Agricultural Biotechnology,University of Delaware (to J.B.).2 Address correspondence and reprint requests to Dr. Winfried G. J. Degen, Departmentof Vaccine Technology and Immunology R&D, Intervet International BV, P.O. Box 31,5830 AA Boxmeer, The Netherlands. E-mail address: winfried.degen@Intervet.com

3 Abbreviations used in this paper: Ch, chicken; CEF, chicken embryo fibroblast;EST, expressed sequence tag; Fe, feline; IBV-N, N protein of avian infectious bron-chitis virus; Mu, mouse; ODN, oligodeoxynucleotide; SPF, specific pathogen free;CD40L, CD40 ligand; semi-Q, semiquantitative.

The Journal of Immunology

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sequence homology, they show remarkable functional similarity tothe group of known mammalian IL-12 molecules, underscoring theevolutionary conserved functional importance of this molecule.

Materials and MethodsCell culture

The mammalian cell line COS-7 (African green monkey kidney cells) andthe chicken DT-40 B cell line (a kind gift from P. Staeheli, University ofFreiburg, Freiburg, Germany) were grown in DMEM (supplemented with10% FCS, 2 mM glutamine, 100 �g/ml streptomycin, 100 U/ml penicillin,and 1 mM pyruvate), whereas the chicken HD-11 (macrophage origin) andthe CU91 (T cell origin) cell lines (a kind gift from T. Schat, CornellUniversity, Ithaca, NY) were grown in RPMI 1640 (supplements as forDMEM). Chicken embryo fibroblasts (CEF) were isolated using 3.5 U/mltrypsin treatment and homogenization of 10-day-old SPF (specific patho-gen-free (SPF)) chicken embryos. CEF cells were grown as monolayers inDMEM (supplemented with 5% FCS). All cell lines were maintained in ahumidified atmosphere of 5% CO2 at 37°C. Chicken primary splenocyteswere isolated from 3-wk-old normal White Leghorn SPF chickens as de-scribed before (30) and grown in RPMI 1640 (see above) in a humidifiedatmosphere of 5% CO2 at 41°C.

Chickens

Three-week-old normal White Leghorn SPF chickens were derived from theIntervet Animal Facilities and housed under SPF conditions. The animals re-ceived water and food ad libitum. All experiments were conducted accordingto protocols approved by the Intervet Animal Welfare Committee.

Treatment of cells with various stimulants

Approximately 700.000 cells were incubated overnight in 3-cm dishes with1 ml of cell culture medium and one of the following stimulants: 10 �gCon A (Miles Scientific, Naperville, IL), 10 �g of an immunostimulatingchicken CpG-oligodeoxynucleotide (ODN), 10 �g chicken CD40 ligand(CD40L; a kind gift from J. Young, Institute for Animal Health, Compton,U.K.), 10 �g LPS (Sigma-Aldrich, St. Louis, MO), or 5 �g PMA (Sigma-Aldrich). After incubation, cells were isolated, washed three times withPBS, and stored at �70°C or used immediately for RNA isolation.

RNA isolation

Total RNA was isolated using TRIzol reagent (Invitrogen Life Technolo-gies, Grand Island, NY) as described by the manufacturer. RNA qualitywas evaluated on 1.2% agarose gels. RNA samples were used subsequentlyfor RT-PCR or stored at �70°C.

PCR and sequence reactions

Plasmid cDNA template (10–20 ng) was mixed with 0.5 �l of 1 U/�lSupertaq (HT Biotechnology, Cambridge, U.K.), 1 �l of 10 ng/�l of eachprimer, 1.6 �l of 2 mM dNTPs, and 2 �l of 10� ST PCR buffer (HTBiotechnology) in a final volume of 20 �l. The reaction cycling conditionswere: 5 min 94°C, 30 cycles (30 s 94°C, 1 min 55°C, 1 min 72°C), and 5min 72°C for a final extension. For cloning purposes, PCR products weregel purified using the Qiaquick Gel Extraction kit (Qiagen, Valencia, CA)and ligated into the pDrive (Qiagen PCR Cloning kit; Qiagen) or thepCR2.1-TOPO vector (TA Cloning kit; Invitrogen Life Technologies).Plasmid DNA was purified using the Qiagen Plasmid Midi kit. All cloneswere extensively sequenced in both 5� and 3� directions using a DNAsequencing kit (BigDye Terminator V3.0 Cycle Sequencing Ready Reac-tion; Applied Biosystems, Foster City, CA) and suitable sequencing prim-ers. Sequences were analyzed with the Sequencher 4.0 software (GeneCodes, Ann Arbor, MI).

Semiquantitative RT-PCR (semi-Q-RT-PCR)

Two micrograms of total RNA was reverse-transcribed into cDNA usingthe Superscript II RT protocol (Invitrogen Life Technologies) in a 20-�lreaction. One microliter of cDNA was used as a template for PCR ampli-fication with the conditions as described above. To determine the optimumnumber of PCR cycles required for a near linear relationship between theamount of RNA and amplified DNA band intensity, a variable number ofcycles was performed for each marker. The following internal primers wereused: 5�-TGGGCACTGCCACCTCCTGC-3� and 5�-CATCTCTGCAGTGAGGGCAC-3� for ChIL-12p35 (516-bp fragment; 30 and 35 PCR cycles);5�-GAACGATGAGACACCAGCTA-3� and 5�-TTATCTGCAAAGCGTGGACCACTCACTCCAGGAT-3� for ChIL-12p40 (844-bp fragment; 30 PCRcycles); 5�-GGACATACTGCAAGTAGTCT-3� and 5�-GGCCAGGTCCAT

GATATCTT-3� for ChIFN-� (European Molecular Biology Laboratory(EMBL)/GenBank accession number X99774; 308-bp fragment; 35 PCR cy-cles); and 5�-GGCCGCCTGGTCACCAGGGCTGCC-3� and 5�-GGAGGAGTGGGGGAGACAGAAGGG-3� for ChGAPDH (EMBL/GenBank ac-cession number K01458; 1099-bp fragment; 25 PCR cycles). PCR productswere separated on a 1.5% agarose gel and analyzed.

Sequence analysis

Comprehensive sequence analysis, including BLAST searches, were per-formed using the in-house interBLAST tools from Intervet Innovation (Schwa-benheim, Germany), the Wisconsin Package Version 10.2 (Genetics ComputerGroup, Madison, WI), Sequencher 4.0 (Gene Codes), OMIGA 2.0 (OxfordMolecular, Oxford, U.K.), and GeneDoc 2.6 (www.psc.edu/biomed/genedoc,Pittsburgh Supercomputing Center, Pittsburgh, PA).

ChIL-12p35, ChIL-12p40, ChFlexi-IL-12, and feline (Fe)Flexi-IL-12 eukaryotic expression constructs

ChIL-12p35. Full-length ChIL-12p35 (pat.pk0055.c11), originally clonedin pcDNA3 (Invitrogen Life Technologies) (31), was excised frompcDNA3 using EcoRI and NotI and cloned into the eukaryotic expressionvector pcDNA3.1� (Invitrogen). The EcoRI/NotI ChIL-12p35 fragmentwas also cloned into pcDNA3.1� (Invitrogen Life Technologies) to obtainan antisense control construct.ChIL-12p40. Full-length ChIL-12p40, present in a cDNA library con-structed from pooled T and B cells isolated from vaccinated chickens andrecloned into pDrive (Qiagen) (see also Results), was excised from pDriveusing NotI and HindIII and cloned into the eukaryotic expression vectorpcDNA3.1� (Invitrogen Life Technologies).ChFlexi-IL-12. A single-chain chicken IL-12 molecule was generated bya strategy described by McMonagle et al. (32). The following primers wereused to amplify ChIL-12p35 without the putative 35-aa signal peptide se-quence (as determined by the SPScan program from the Wisconsin PackageVersion 10.2; Genetics Computer Group) and that introduced a 5�-BamHI anda 3�-HindIII restriction site: 5�-TTGGATCCGGTGGCGGCGGATCTCTGCCACCTCCTGCCCA-3� and 5�-CCAAGCTTTTACATCTCTGCAGTGAGGGCACTCAGGTAGC-3�. For ChIL-12p40 the following primers were usedthat introduced a 5�-NotI and a 3�-BamHI restriction site: 5�-TTGCGGCCGCCATGTCTCACCTGCTATTTGCCTTACTTTC-3� and 5�-TGGATCCACCACCGCCCGAGCCACCGCCACCTCTGCAAAGCGTGG-3�. Both PCRfragments were separately cloned into pCR2.1-TOPO (Invitrogen Life Tech-nologies) and extensively sequenced. ChIL-12p40 was excised from pCR2.1-TOPO as a NotI/BamHI fragment and cloned into the pcDNA3.1� vector(Invitrogen Life Technologies). The ChIL-12p35 was excised from pCR2.1-TOPO as a BamHI/HindIII fragment and cloned into the (ChIL-12p40)-(pcDNA3.1�) construct downstream of the p40 fragment. This resulted in asingle-chain p40-p35 heterodimeric construct in which the p40 chain is linkedto the p35 chain by an in-frame (G4S)3-linker; this molecule was designatedChFlexi-IL-12 (for chicken Flexi-IL-12).FeFlexi-IL-12. FeFlexi-IL-12, a construct similar to the ChFlexi-IL-12,was a kind gift from L. Nicolson (University of Glasgow VeterinarySchool, Glasgow, U.K.) and was cloned into the eukaryotic pCI-neo vector(Promega, Madison, WI).

Transient expression of cDNA clones in COS-7 cells

COS-7 cells were transfected with 1.5 �g of each cDNA construct usingthe Invitrogen Life Technologies Lipofectamine reagent (as described bythe manufacturer) and cultured in 3-cm dishes with DMEM (without FCSand penicillin/streptomycin). After 8 h, transfected cells were washed andcultured in DMEM with penicillin/streptomycin and 10% FCS. After a72-h incubation at 37°C in 5% CO2, the cell culture supernatants wereharvested and centrifuged at 13,000 rpm for 10 min at 4°C to remove celldebris. The supernatants were analyzed via Western blotting and used im-mediately or stored at �70°C.

Western blot analysis

Cell culture supernatants from transfected COS-7 cells were size fraction-ated using 4–12% Nu-PAGE (Invitrogen Life Technologies) and blottedonto nitrocellulose filters (Schleicher & Schuell, Keene, NH). Westernblots were blocked in MPBS (3% skimmed milk in PBS) and subsequentlyincubated with a polyclonal Ab that was raised against a FeIL-12p40 pep-tide (32) diluted 1/300 in MPBS. After extensive washing, blots were in-cubated with alkaline phosphatase-conjugated goat anti-rabbit IgG Abs(Sanbio, Uden, The Netherlands) diluted 1/1000 in MPBS. After washing,bound alkaline phosphatase-labeled secondary Abs were visualized viastaining. Blocking and Ab incubations were performed for 1 h at roomtemperature.

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Bioactivity assays for ChIL-12

NO assay for the induction of splenic ChIFN-� by ChIL-12. Chprimary spleen cells (splenocytes) were seeded in triplicate in a96-well plate at a density of 0.5 � 106 cells/well in 100 �l andincubated with 50 �l of serial dilutions of cell culture supernatantsfrom COS-7 cells transfected with cDNA clones encoding ChIL-12p40, ChIL-12p40 mixed with ChIL-12p35, ChFlexi-IL-12, Fe-Flexi-IL-12, or with an empty pcDNA3.1 plasmid (mock). Forty-eight hours after the addition of proteins, supernatants (75 �l) werecollected and analyzed for the presence of biologically activeChIFN-�. For this, 100 �l of 1.5 � 106/ml HD-11 cells wereincubated with 75 �l of the collected supernatants for 24 h at 37°Cin 5% CO2 in 96-well plates. Activation of HD-11 cells byChIFN-� was measured as a function of nitrite accumulation in theculture supernatants using the Griess assay (33, 34).

Assay for spleen cell proliferation by ChIL-12. After removing75 �l of the supernatants (see above) 50 �l of medium and 18.5kBq [methyl-3H]thymidine (25 �l/well) were added to the remain-ing 75 �l in the 96-well plate and incubated for 18–20 h at 41°Cin 5% CO2. After incubation, the incorporated radioactivity wascounted using an LKB Betaplate beta counter (LKB Instruments,Gaithersburg, MD).

Statistical analysis

The significance of the differences between the means of NO production orbetween the means of cell proliferation was analyzed using the Student’s ttest. Differences were considered significant at a confidence level of 95%( p � 0.05).

ResultsIsolation of the putative ChIL-12p35 cDNA clone

Cloning and sequencing of avian cytokines lags behind similarwork done in mammals and so far only a few avian cytokines havebeen identified. Because of the low sequence homology to mam-malian cytokines, classical molecular approaches to characterizeavian cytokine homologues are usually not successful. The iden-tification of these “missing” avian cytokines will therefore mainlycome from 1) large-scale sequencing projects resulting in ex-pressed sequence tags (ESTs), 2) the availability of EST clones indatabases, and 3) sophisticated bioinformatics.

From a high throughput sequencing project, in which a cDNAlibrary was used constructed from an enriched pool of T cellsstimulated with Con A (31), only 2 of the 5500 cDNA clonesisolated showed homology to the IL-12p35 cDNA sequence ofseveral species. One of the two clones (pat.pk0055.c11) appearedto be full length and was used for further studies. Databasesearches with this cDNA clone did not result in the identificationof more copies.

Sequence analysis of the putative ChIL-12p35 subunit

Clone pat.pk0055.c11 is 618 nt long (from start to stop) and en-codes a protein of 205 aa (Fig. 1A). Analysis of the pat.pk0055.c11cDNA sequence containing the open reading frame (nt 1–618)showed an overall nucleotide homology to the IL-12p35 cDNAsequences of human, sheep, horse, cat, bovine, mouse, and wood-chuck of 43, 45, 48, 43, 45, 42, and 45%, respectively (Score TableNucleic Acid PAM 65; results not shown). A multiple alignment ofthe predicted pat.pk0055.c11 protein to human, sheep, horse, cat,bovine, mouse, and woodchuck IL-12p35 proteins yields an over-all amino acid similarity of 27, 25, 30, 24, 25, 29, and 21%, re-spectively (Score Table Blosum 62; Fig. 2A). The differences insimilarity between pat.pk0055.c11 and the IL-12p35 protein ofvarious species are the result of substitutions, insertions, and de-letions of amino acid residues. Striking are the two large deletions

in pat.pk0055.c11 of 13 and 15 aa when comparing this sequenceto the other IL-12p35 sequences. The predicted ChIL-12p35 pro-tein is a precursor protein with a signal sequence of 35 aa (asdetermined by using SPScan from the Wisconsin Package Version10.2), resulting in a mature protein of 170 aa. The precursor formof ChIL-12p35 contains nine cysteines, of which four are con-served between the analyzed species (Fig. 2A). In addition, oneputative N-glycosylation site is present at amino acid position 48.When removing the first 62 aa (nt 1–186) of pat.pk0055.c11, allhomologies increase �7% (data not shown), indicating that theN-terminal fragment of pat.pk0055.c11 is not as highly conservedas the rest of the protein. Based on the homologies, we conclude tohave cloned the putative ChIL-12p35 subunit.

Isolation of the putative ChIL-12p40 cDNA clone

The coding sequence, i.e., nt 35–1042, of the mouse IL-12p40subunit (MuIL-12p40; EMBL/GenBank accession numberM86671) was used to search the Biotechnology and BiologicalSciences Research Council (Manchester, U.K.) Chicken ESTProject Database (http://www.chick.umist.ac.uk/) (35) using thetBlastX program. A chicken EST sequence (clone IDChEST582p2, EST name 603603708F1, derived from chickenadult kidney) was retrieved that showed 51% identity with aa 251–

FIGURE 1. Nucleotide and deduced amino acid sequences of ChIL-12p35 (A) and ChIL-12p40 (B). The chicken nucleotide sequences willappear in the EMBL/GenBank database with the accession numbersAY262751 (ChIL-12p35) and AY262752 (ChIL-12p40).

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279 and 66% identity with aa 310–327 of the MuIL-12p40 se-quence. At that moment, no GenBank accession number was as-signed to this ChEST582p2 clone and the sequence was notannotated. A database search in the same chicken EST databasewith this ChEST582p2 clone did not result in a longer or full-length clone, indicating that this database at that moment containedonly one incomplete copy of this molecule. A similar databasesearch in the UD Chicken EST database (http://www.chick-est.udel.edu/) with the coding sequence, i.e., nt 35–1042, of MuIL-12p40 (EMBL/GenBank accession number M86671) did not resultin a significant hit using the BlastN program nor did a search withthe ChEST582p2 clone result in a longer or full-length clone.

Although the ChEST582p2 clone is 848 nt long, only the first233 nt contain an open reading frame encoding a peptide of 76 aa(including the stop codon). To obtain the full-length ChIL-12p40cDNA clone, a cDNA library was screened that was constructedfrom pooled T and B cells isolated from vaccinated chickens. Inthis library cDNA molecules are unidirectionally cloned betweenthe NotI and EcoRI sites of the eukaryotic expression vectorpBlueScript (Stratagene, La Jolla, CA). In a PCR with a 5� end

pBlueScript vector primer, �120 nt upstream of the NotI restric-tion site, and the 3� end ChEST582p2 primer (5�-TTATCTGCAAAGCGTGGACCACTCACTCCAGGAT-3�; nucleotide posi-tions 915–948 in Fig. 1B), a PCR fragment of �1000 nt wasobtained that was cloned into pDrive (Qiagen). This clone wasdesignated pND89.

Several months after its initial discovery we discovered that theChEST582p2 EST clone was annotated in the above-mentionedBiotechnology and Biology Sciences Research Council (35) data-base as an IL-12p40 subunit with the highest homology (57%) tothe IL-12p40 subunit of Marmota monax (woodchuck; X97019 inEMBL/GenBank). ChEST582p2 was also deposited in the EMBL/GenBank database with accession number BU291084 (releasedate, November 28, 2002) but without the IL-12p40 annotation.

Sequence analysis of the putative ChIL-12p40 subunit

Clone pND89 was extensively sequenced and revealed that thecDNA clone (from start to stop) is 948 nt long and encodes aprotein of 315 aa (Fig. 1B). Analysis of the pND89 cDNA se-quence containing the open reading frame (nt 1–948) showed an

FIGURE 2. Multiple protein alignments of ChIL-12p35 (A) and ChIL-12p40 (B). Multiple alignments of the ChIL-12p35 and ChIL-12p40 proteins tothe IL-12p35 and IL-12p40 proteins of several species was performed using the GeneDoc program (Score Table Blosum 62). Gaps (–) are introduced toallow maximal alignment. Conserved cysteines are indicated with asterisks (�), whereas putative N-glycosylation sites in the ChIL-12 subunits are indicatedwith arrows (2). The proteins used for the multiple alignments have the following EMBL/GenBank accession numbers for p35/p40: human, M65271/M65272; sheep, AF173557/AF004024; horse, Y11130/Y11129; cat, Y07761/Y07762; bovine, U14416/U11815; mouse, M86672/M86671; woodchuck,X970189/X97019. 55.c11, ChIL-12p35 (clone pat.pk0055.c11); pND89, ChIL-12p40.

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overall nucleotide homology to the IL-12p40 cDNA sequences ofhuman, sheep, horse, cat, bovine, mouse, and woodchuck of 57,56, 57, 55, 56, 55, and 57%, respectively (Score Table NucleicAcid PAM 65; results not shown). A multiple alignment of thepredicted pND89 protein to human, sheep, horse, cat, bovine,mouse, and woodchuck IL-12p40 proteins yields an amino acidhomology of 41, 40, 40, 42, 39, 36, and 41%, respectively (ScoreTable Blosum 62; Fig. 2B). The differences in similarity betweenpND89 and the IL-12p40 protein of various species are the resultof substitutions, insertions and deletions of amino acid residues.Sequence analysis further revealed the presence of a signal peptidewith the cleavage site between aa 20 and 21 (as determined byusing SPScan from the Wisconsin Package Version 10.2) resultingin a mature protein of 295 aa acids. The precursor form of ChIL-12p40 contains nine cysteines, of which six are conserved betweenall analyzed species (Fig. 2B). Next to this, five putative N-glyco-sylation sites are present (amino acid positions 84, 134, 215, 239,and 304). The presence of a WSXWS box (aa 305–311), an Ig-likeC2-type domain (aa 41–94), and a fibronectin-type III domain (aa228–308), which are both characteristic for IL-12p40, were con-firmed by similarity. Based on these homologies we conclude tohave cloned the putative chicken IL-12p40 subunit.

Cellular source of ChIL-12p35 and ChIL-12p40 mRNA

In mammals, IL-12 expression is generally restricted to APCs suchas macrophages, dendritic cells, and B cells (for a recent review,see Ref. 36). To examine cell type-associated expression of ChIL-12p35 and ChIL-12p40 mRNA, several available immunorelevantchicken cell types were tested as candidate producers in a prelim-inary study using semi-Q-RT-PCR. mRNA expression was exam-ined in freshly isolated chicken splenocytes and CEF cells and inseveral chicken cell lines including HD-11 (macrophage derived),DT-40 (B cell derived), and CU91 (T cell-derived). Cells wereexposed overnight to Con A, PMA, an immunostimulating chickenCpG-ODN, recombinant ChCD40L, or LPS (Fig. 3). Unstimulatedcells, cultured in medium only, showed minimal or hardly detect-able ChIL-12 subunit expression levels, suggesting a low basalChIL-12p35 and ChILp40 mRNA expression. In general, in stim-ulated cells detection of ChIL-12p40 mRNA proved to be easierthan ChIL-12p35 mRNA, in which case we had to increase thenumber of PCR cycles from 30 to 35 to obtain visible bands.

Up-regulated ChIL-12p40 mRNA expression was noted insplenocytes exposed to Con A, CpG-ODN, ChCD40L, and LPS(Fig. 3, lanes 1 and 3–5), but not following PMA treatment (lane2). ChIL-12p35 mRNA was only detected after 35 cycles; expres-

sion was noted faintly in most cell types (Fig. 3, lanes 1–6). InHD-11 cells, ChIL-12p40 mRNA expression was clearly inducedby CpG-ODN, ChCD40L, and LPS (Fig. 3, lanes 9–11), whileChIL-12p35 mRNA levels were hardly detectable even at 35 PCRcycles. In contrast, for DT-40 cells up-regulated expression ofChIL-12p40 mRNA was noted only after LPS induction (Fig. 3,lane 17). Notably, this stimulus mainly stimulated ChIL-12p35mRNA expression in DT-40 cells. Analysis of the T cell line CU91however showed marked ChIL-12p40 up-regulation after exposureto PMA and to a lesser extent to ChCD40L (Fig. 3, lanes 20 and22). ChIL-12p35 mRNA was also up-regulated by PMA andChCD40L (Fig. 3, lanes 20 and 22). Nonlymphoid primary CEFcells showed no detectable expression levels of ChIL-12p35 andChIL-12p40 mRNA with or without LPS treatment, suggestingthat also ChIL-12 is mainly a product of inflammatory cells (Fig.3, lanes 25 and 26). Collectively, if produced at all, these prelim-inary results show that ChIL-12p40 mRNA was expressed in largeexcess over the ChIL-12p35 mRNA, which is in line with mam-malian IL-12 results (36). The expression of ChIL-12 subunits mayvary depending on the cell type and its stimulus. It should be keptin mind that the available cell lines tested may have altered char-acteristics in terms of stimulus-induced IL-12 expression whencompared with nontransformed cells.

Characterization of recombinant ChIL-12

To detect secreted ChIL-12p40 and ChIL-12p35 subunits after(co-)transfection of COS-7 cells, nondenaturing Western blot anal-ysis was applied. For this, a polyclonal Ab that was raised againsta peptide of the FeIL-12p40 subunit was used. McMonagle et al.(32) reported that this Ab recognized the equine IL-12p40 unitbased upon a homology of �85% between the feline peptide andthe corresponding equine protein sequence. Because the homologybetween this FeIL-12p40 peptide and the corresponding chickenprotein sequence is �80%, we hypothesized that this Ab wouldpossibly also recognize the ChIL-12p40 subunit on Western blot.Indeed, when using this anti-FeIL-12p40 peptide Ab, we were ableto detect ChIL-12p40 and the ChIL-12p40 homodimer ChIL-12p80 (i.e., ChIL-12p40-ChIL-12p40) in the supernatants of trans-fected COS-7 cells (Fig. 4, lane 1). In the supernatants fromCOS-7 cells transfected with both ChIL-12p35 and ChIL-12p40,we detected the heterodimeric ChIL-12p70 (ChIL-12p35-ChIL-12p40) protein (Fig. 4, lane 4). The formation of heterodimericChIL-12p70 was more efficient than the formation of homodimericChIL-12p80 as no or only very small amounts of ChIL-12p80could be detected. Also, formation of ChIL-12p70 is specific as

FIGURE 3. Semi-Q-RT-PCR analysis of ChIL-12p35 and ChIL-12p40 mRNA in several chicken cell types. Chicken cells were treated overnight withseveral stimulants, RNA was isolated and used for semi-Q-RT-PCR analysis. PCR products were separated on agarose gel, stained with ethidium bromide,and visualized under UV light. ChGAPDH was used for RT-PCR control and semiquantitative comparison for ChIL-12p35 and ChIL-12p40 mRNAexpression. A typical result obtained in several experiments is shown. The number of PCR cycles is indicated for each marker in parentheses. Chicken cellsused: freshly isolated splenocytes and CEF cells, macrophage-derived HD-11 cell line, B cell-derived DT-40 cell line, and T cell-derived CU91 cell line.CpG, immunostimulating chicken CpG-ODN; DNA, DNA plasmid encoding p35, p40, or ChGAPDH; H2O, water control; medium, cell culture medium;p35, ChIL-12p35; p40, ChIL-12p40.

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cotransfection of ChIL-12p40 cDNA with antisense ChIL-12p35cDNA or with a cDNA construct encoding an irrelevant viral pro-tein (IBV-N) did not result in heterodimerization (Fig. 4, lanes 5and 6). A double band was detected in the cell culture supernatantrecovered from the FeFlexi-IL-12 transfection (Fig. 4, lane 7).These bands may reflect differential glycosylation states for thesingle-chain FeFlexi-IL-12, a phenomenon also reported for theequine Flexi-IL-12 (32). The high molecular band that was de-tected in all FeFlexi-IL-12 samples possibly represents protein ag-gregates. In conclusion, these results clearly show that the ChIL-12p40 and ChIL-12p35 subunits heterodimerize into the ChIL-12p70 molecule after cotransfection in COS-7 cells.

ChIL-12-dependent induction of ChIFN-�

A hallmark of IL-12 activity in mammals is its induction of IFN-�by T lymphocytes (5). To assess whether ChIL-12 also inducesChIFN-� we incubated freshly isolated primary chicken spleencells with dilutions of various proteins isolated after transient(co-)expression in COS-7 cells. Activation of macrophage-likechicken HD-11 cells by released IFN-� was subsequently mea-sured as a function of nitrite accumulation in the HD-11 cell cul-ture supernatant using the (indirect) Griess assay. Since IFN-� isthe major macrophage-activating factor in chicken (37), release ofNO is a generally accepted indicator for IFN-� production. Asshown in Fig. 5A, only heterodimeric ChIL-12p70 (a cotransfec-tion of ChIL-12p40 with ChIL-12p35) is able to significantly ( p �0.05) induce the production of IFN-� in chicken spleen cells in aconcentration-dependent manner. No other combination of trans-fection, nor mock or ChIL-12p40 alone, could induce ChIFN-�secretion to a level comparable to the heterodimeric ChIL-12p70.Next to this it is clear that only species-specific IL-12 inducessignificant ( p � 0.05) IFN-� in chicken spleen cells as the Fe-Flexi-IL-12, which is bioactive in the mammalian IL-12 bioassay(data not shown), induced no significant amounts of ChIFN-�.Theoretically, beside IFN-�, other factors, e.g., CD40L or TNF-�,may be able to evoke NO production by HD-11 cells. Therefore,we also measured ChIFN-� mRNA expression directly via semi-Q-RT-PCR on primary chicken splenocytes 48 h postincubationwith various proteins isolated after transient (co-)transfection in

COS-7 cells. For this assay we used undiluted cell culture super-natant. The results from this semi-Q-RT-PCR analysis confirmedthat ChIL-12 induces ChIFN-� mRNA expression (Fig. 5C) whichcoincided with NO production by HD-11 cells after exposure tosplenocyte supernatants (data not shown). Taken together, the NOand semi-Q-RT-PCR results demonstrate, as known for its mam-malian counterparts, that ChIL-12 induces ChIFN-�.

ChIL-12-dependent proliferation of chicken spleen cells

Another characteristic of IL-12, shared with several other cyto-kines, is its induction of T cell proliferation. The growth responseof freshly isolated chicken splenocytes to various proteins, isolatedafter transient (co-)expression in COS-7 cells, was measured by acell proliferation assay. Only heterodimeric ChIL-12p70 (a co-transfection of ChIL-12p40 with ChIL-12p35) was able to inducesignificant ( p � 0.05) proliferation of chicken spleen cells (Fig.5B). The relatively low proliferation data observed for the firstdilutions are possibly explained by overdose effects for this pa-rameter. This phenomenon was described before by McMonagle etal. (32). Neither ChIL-12p40 alone nor FeFlexi-IL-12 were able toinduce similar proliferative responses. These results show thatChIL-12 (p70) is able to induce the proliferation of chicken spleencells. We therefore conclude to have cloned the fully bioactivesubunits of ChIL-12.

The use of undiluted ChIL-12 COS-7 cell culture supernatantsin the semi-Q-RT-PCR analysis and the subsequent positive iden-tification of ChIFN-� mRNA expression (Fig. 5C) is in line withthe observed NO induction, but seems to contradict the low pro-liferation data observed at high ChIL-12 protein concentrations(Figs. 5B and 6, B and D). However, cell proliferation and IFN-�production, as measured via NO release, are two distinct physio-logical processes that may both be activated by IL-12. It is clearfrom Figs. 5 and 6 that at high ChIL-12 concentrations prolifera-tion can be repressed, but NO synthesis is not affected. Apparently,IFN-� synthesis is not affected at high concentrations of IL-12(Figs. 5A and 6, A and C). The results from the semi-Q-RT-PCRanalysis for ChIFN-� mRNA expression support this (Fig. 5C).

Bioactivity of single-chain ChFlexi-IL-12

After showing that cotransfection of single-chain ChIL-12p40 withsingle-chain ChIL-12p35 resulted in the formation of a bioactiveChIL-12 heterodimer (Figs. 4 and 5), a single-chain IL-12 mole-cule (ChFlexi-IL-12) was constructed. ChFlexi-IL-12 is a single-chain p40-p35 heterodimeric construct in which the ChIL-12p40chain is linked to the ChIL-12p35 chain by an in-frame (G4S)3-linker. McMonagle and coworkers (32) already showed that thisapproach resulted in a bioactive equine IL-12 molecule. Westernblot analysis showed that the expression profile of the ChFlexi-IL-12 after transfection in COS-7 cells is comparable to FeFlexi-IL-12 (data not shown). Following incubation of freshly isolatedchicken spleen cells with ChFlexi-IL-12, we observed both therelease of ChIFN-�, measured via NO release and expression ofChIFN-� mRNA via semi-Q-RT-PCR, as well as cell proliferation(Figs. 5C and 6, A and B) indicating that the Chflexi-IL-12 con-struct is also bioactive. At high ChFlexi-IL-12 concentrations, theproliferation of splenocytes appeared to be limited, likely as a re-sult of overdosing or contaminating factors, as we also noticed forthe heterodimeric ChIL-12p70 (a cotransfection of ChIL-12p40with ChIL-12p35) molecule (see Fig. 5B).

To directly compare the potency of ChFlexi-IL-12 vs ChIL-12p70 (p35 plus p40), we monitored the bioactivity of both con-structs in one experiment using the splenocytes from the sameindividual chicken. We found no significant difference ( p � 0.05)

FIGURE 4. Western blot analysis of COS-7 cell culture supernatantsafter transfection with ChIL-12. COS-7 cells were transfected with thecDNAs encoding the following molecules: ChIL-12p40 (p40), ChIL-12p35(p35); IBV-N (irrelevant control protein), FeFlexi-IL-12, or with the emptypcDNA3.1 vector (mock). Cell culture supernatants were analyzed by4–12% Nu-PAGE and Western blotting using a polyclonal Ab raisedagainst a feline IL-12p40 peptide. A representative Western blot is shown.The positions of monomeric p40, heterodimeric p40-p35, and homodimericp40-p40 are indicated on the left.

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in bioactivity between the two constructs by measuring NO releaseand cell proliferation (Fig. 6, C and D).

DiscussionIn this study, we described the isolation and characterization of thefirst avian IL-12 molecule, namely, ChIL-12. To the best of ourknowledge, this is also the first report describing a nonmammalianIL-12 molecule. The predicted sequences of ChIL-12p40 andChIL-12p35 show homology to several mammalian IL-12 mole-cules. Depending on the analyzed species and subunit, the proteinhomology for ChIL-12p35 and ChIL-12p40 ranged between 21and 30% and 36 and 42%, respectively. The ChIL-12p40 andChIL-12p35 subunits, similar to their mammalian counterparts,need to heterodimerize to exert IL-12 bioactivity. Based on thehomologies and the bioactivity of ChIL-12, we hereby provideclear evidence that we have cloned the first nonmammalian IL-12molecule.

In mammals, IL-12 is a critical inducer of Th1-type immuneresponses typically associated with IFN-� production. This mole-cule has long been searched for in birds. Although ChIFN-��, alsoknown as IFN-I, was the first discovered cytokine in birds (38), ittook �35 years until ChIFN-� was discovered (26). Only recentlythe characterization of ChIL-18 (27) and ChIL-6 (39) has beenreported. The presently discovered ChIL-12 molecule comple-ments the set of typically Th1-associated cytokines in birds. Thesearch for new chicken cytokines is hampered by the poor homol-ogy between avian and mammalian cytokine sequences. As such,traditional molecular biological approaches based on RT-PCR of-ten fail. For comparison, the feline IL-12 (17) and guinea pig IL-12(40) were cloned by using a rather standardized RT-PCR approachbased on available mammalian IL-12 sequences. As a consequencefor the chicken, the successful discovery of new cytokines dependson large-scale EST sequencing projects combined with sophisti-cated bioinformatic analysis. Using both approaches and tradi-tional molecular biology, we succeeded in the isolation of the p40and p35 subunits of ChIL-12. The homology of the ChIL-12 sub-units to several mammalian counterparts ranges between 21 and30% (p35) and 36 and 42% (p40). These homologies are in a rangesimilar to other recently isolated and characterized chicken cyto-kines, i.e., IL-6 (average 40%) (39) and IL-18 (average 30%) (27).ChIL-6 was isolated using the suppression subtractive hybridiza-tion technology (41), whereas ChIL-18 was isolated using a data-base search combined with a PCR on a chicken cDNA library.

After the identification of the ChIL-12 subunit sequences, wewere able in a preliminary study to monitor cell type-associatedexpression using semi-Q-RT-PCR. Several chicken cell types werestimulated with known mammalian IL-12 inducers such asCD40L, CpG-ODN, which triggers the Toll-like receptor 9 forimmunostimulatory activity, and LPS, triggering Toll-like receptor4 (42). Remarkably, Con A stimulation of freshly isolated spleno-cytes triggered ChIL-12 mRNA expression probably via a T cell-dependent pathway. However, Con A stimulation of the T cell lineCU91 failed to induce ChIL-12p40 and ChIL-12p35 mRNA ex-pression above cell culture medium levels. Strikingly, however,

supernatant from transfected COS-7 cells or Con A (positive control). PCRproducts were separated on agarose gel, stained with ethidium bromide andvisualized under UV light. ChGAPDH was used for RT-PCR control andsemiquantitative comparison for ChIFN-� mRNA expression. A typicalresult obtained in several experiments is shown. The number of PCR cyclesis indicated in parentheses. p40 � p35, cotransfection of ChIL-12p40 andChIL-12p35; mock, empty pcDNA3.1 plasmid; DNA, DNA plasmid en-coding ChIFN-� or ChGAPDH; H2O, water control.

FIGURE 5. A, Chicken heterodimeric IL-12-dependent induction ofIFN-�. Freshly isolated chicken splenocytes were stimulated in triplicatewith serial dilutions of various cell culture supernatants of transfectedCOS-7 cells. After 48 h of incubation, supernatants were collected andtested for ChIFN-� characteristic NO induction. B, Chicken heterodimericIL-12-dependent proliferation of chicken spleen cells. Freshly isolatedchicken splenocytes were treated as described for A but after a 48-h incu-bation proliferation was assessed by [3H]thymidine uptake. For clarity rea-sons, we omitted data below 2000 cpm. A typical response repeatedlyobserved in numerous experiments is shown. Data are expressed as geo-metric mean � SD (vertical bars) and are representative of at least fourindependent experiments. �, Significantly different (p � 0.05) from mocktreatment. C, Semi-Q-RT-PCR analysis of ChIFN-� mRNA expression inchicken splenocytes at 48 h after incubation with undiluted cell culture

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FIGURE 6. A, ChFlexi-IL-12-dependent induction of IFN-�. Freshly isolated chicken splenocytes were stimulated in triplicate with serial dilutions ofvarious cell culture supernatants of transfected COS-7 cells. After 48 h of incubation, supernatants were collected and tested for ChIFN-� characteristic NOinduction. B, ChFlexi-IL-12-dependent proliferation of chicken spleen cells. Freshly isolated chicken splenocytes were treated as described for A but after 48 h ofincubation proliferation was assessed by [3H]thymidine uptake. For clarity reasons, we omitted data below 2000 cpm. C and D, Comparison of the bioactivitybetween ChFlexi-IL-12 and p40 � p35 as measured via (C) the induction of IFN-� and (D) cell proliferation. A typical response repeatedly observed in numerousexperiments is shown. Data are expressed as geometric mean � SD (vertical bars) and are representative of at least four independent experiments. �, Significantlydifferent (p � 0.05) from mock treatment. p40 � p35, cotransfection of ChIL-12p40 and ChIL-12p35; mock, empty pcDNA3.1 plasmid.

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exposure of this cell line to PMA activated the mRNA expressionof both subunits. To the best of our knowledge, mammalian T cellsare normally not identified as IL-12 producers (36). Although in-triguing, this might also be related to the transformed phenotype ofthis cell line and therefore an artifact. In line with mammalian data,we noted ChIL-12 mRNA synthesis following stimulation of Bcells, in particular after stimulation with LPS, while for macro-phage-type HD-11 cells up-regulation was noted for known mam-malian IL-12 inducers, including CpG-ODN, ChCD40L, and LPS.

IL-12 was initially recognized as an inducer of IFN-� synthesis(5) and since all mammalian IL-12 molecules isolated thus fartypically show this activity we tested whether ChIL-12 was alsoable to characteristically induce ChIFN-�. Our data clearly showedthat the activity of ChIL-12 is in line with this mammalian concept.This was surprising because of the limited sequence homology ofthe ChIL-12 subunits with the group of known mammalian IL-12molecules. The high degree of functional similarity betweenChIL-12 and higher mammalian vertebrates illustrates, therefore,the conservation and vital importance of the IL-12 molecule sincethe evolutionary dichotomy of birds and mammals �300 millionyears ago. Species specificity of ChIL-12 function was evidencedby the failure of bioactive FeIL-12 to induce sufficient ChIFN-� infreshly isolated chicken spleen cells. Because ChIL-12p40 aloneexhibits no ChIFN-� inducing capacity, we conclude that biolog-ically active ChIL-12 depends on the interaction of the p40 andp35 subunits covalently linked to each other into the 70-kDa het-erodimer p70. Gubler et al. (1) showed already in 1991 that bio-active IL-12 is a heterodimeric cytokine composed of disulfide-linked p35 and p40 subunits.

Another characteristic feature of IL-12 is its capacity to inducecell proliferation. The growth response of splenocytes to variousforms of ChIL-12 showed that only the ChIL-12p40 with ChIL-12p35 combination (heterodimeric ChIL-12p70) was able to in-duce the proliferation of chicken spleen cells. These resultsshowed that proliferation of freshly isolated chicken spleen cells isreadily induced upon exposure to ChIL-12 (p70) only.

Using a single-chain IL-12 molecule (ChFlexi-IL-12), consist-ing of a single chain ChIL-12p40 linked to the ChIL-12p35 chainby an in-frame (G4S)3-linker, we showed proliferation, NO-induc-ing activity, and ChIFN-� mRNA expression in freshly isolatedchicken splenocytes.

From human and mouse studies, it is known that homodimericp40 (p80) competes with heterodimeric p40-p35 (p70) for bindingto the IL-12R which results in an inhibition of cell proliferationand IFN-� synthesis in vitro (43, 44). To investigate whether ho-modimeric chicken p40 (p80) also has this antagonizing effect, anin vitro experiment was set up in which the 10-fold concentratedsupernatant of COS-7 cells transfected with ChIL-12p40 was pre-incubated for 2 h with splenocytes. After that, serial dilutions ofChFlexi-IL-12 protein were added and IFN-� release was mea-sured. In contrast to Gillessen et al. (43) and Ling et al. (44), wedid not observe a clear decrease in IFN-� synthesis (data notshown). This might be in line with the results found for human p80(45). Human p80, in contrast to mouse, has only a minor ability toantagonize IL-12 and might therefore work differently when com-pared with the mouse system. Our preliminary data suggest thatthis might also be true for chicken p80.

The discovery of ChIL-12 further provides evidence that thechicken Th1 cytokine pathway was already functional before theavian and mammalian lineages separated 300 million years ago(46). The conservation of this pathway in vertebrate evolution il-lustrates its importance for species fitness. Remarkably, within thechicken thus far only Th1-type cytokines have been isolated andclearly characterized. The absence of Th2-associated IgE, eosino-

phils, and allergies suggests no or limited Th2 responses in thechicken and possibly nonmammalian vertebrates in general. How-ever, using Ag delivery through scavenger receptors on avian mac-rophages, Vandaveer et al. (47) were able to manipulate Th1/Th2-associated responses in chickens. Interestingly, within severalchicken EST databases sequences are present which are annotatedas the ChIL-10R, indicating the presence, although only in part, ofa putative Th2 pathway in the chicken. Recently, P. Kaiser andcoworkers presented evidence for the presence of the IL-4, IL-5,and IL-13 genes which have to be functionally analyzed (48). Moreresearch will be necessary to identify components of the avian Th2pathway. Combinations of ongoing large-scale sequencing projects(a draft of the chicken genome sequence is expected to be com-pleted by the end of 2003) and bioinformatic approaches willtherefore be necessary to isolate and characterize novel avian cy-tokines. Both techniques are definitively necessary to explore theexisting cytokine gap between birds and mammals. These inves-tigations may also help to unravel the evolutionary development ofthe immune system of birds and mammals.

In summary, we have isolated and characterized the chickenIL-12 molecule which is to the best of our knowledge the firstnonmammalian IL-12 molecule discovered. We showed that themolecule has functional similarities with mammalian IL-12 homo-logues despite the rather poor homology. Future experiments arenecessary to examine the role of ChIL-12 in vivo.

References1. Gubler, U., A. O. Chua, D. S. Schoenhaut, C. M. Dwyer, W. McComas,

R. Motyka, N. Nabavi, A. G. Wolitzky, P. M. Quinn, and P. C. Familletti. 1991.Coexpression of two distinct genes is required to generate secreted bioactivecytotoxic lymphocyte maturation factor. Proc. Natl. Acad. Sci. USA 88:4143.

2. Wolf, S. F., P. A. Temple, M. Kobayashi, D. Young, M. Dicig, L. Lowe,R. Dzialo, L. Fitz, C. Ferenz, and R. M. Hewick. 1991. Cloning of cDNA fornatural killer cell stimulatory factor, a heterodimeric cytokine with multiple bi-ologic effects on T and natural killer cells. J. Immunol. 146:3074.

3. Trinchieri, G. 1994. Interleukin-12: a cytokine produced by antigen-presentingcells with immunoregulatory functions in the generation of T-helper cells type 1and cytotoxic lymphocytes. Blood 84:4008.

4. Oppmann, B., R. Lesley, B. Blom, J. C. Timans, Y. Xu, B. Hunte, F. Vega,N. Yu, J. Wang, K. Singh, et al. 2000. Novel p19 protein engages IL-12p40 toform a cytokine, IL-23, with biological activities similar as well as distinct fromIL-12. Immunity 13:715.

5. Kobayashi, M., L. Fitz, M. Ryan, R. M. Hewick, S. C. Clark, S. Chan, R. Loudon,F. Sherman, B. Perussia, and G. Trinchieri. 1989. Identification and purificationof natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologiceffects on human lymphocytes. J. Exp. Med. 170:827.

6. Stern, A. S., F. J. Podlaski, J. D. Hulmes, Y. C. Pan, P. M. Quinn, A. G. Wolitzky,P. C. Familletti, D. L. Stremlo, T. Truitt, and R. Chizzonite. 1990. Purification tohomogeneity and partial characterization of cytotoxic lymphocyte maturation fac-tor from human B-lymphoblastoid cells. Proc. Natl. Acad. Sci. USA 87:6808.

7. D’Andrea, A., M. Rengaraju, N. M. Valiante, J. Chehimi, M. Kubin, M. Aste,S. H. Chan, M. Kobayashi, D. Young, and E. Nickbarg. 1992. Production ofnatural killer cell stimulatory factor (interleukin 12) by peripheral blood mono-nuclear cells. J. Exp. Med. 176:1387.

8. Kato, T., R. Hakamada, H. Yamane, and H. Nariuchi. 1996. Induction of IL-12p40 messenger RNA expression and IL-12 production of macrophages via CD40-CD40 ligand interaction. J. Immunol. 156:3932.

9. Sousa, C. R., S. Hieny, T. Scharton-Kersten, D. Jankovic, H. Charest,R. N. Germain, and A. Sher. 1997. In vivo microbial stimulation induces rapidCD40 ligand-independent production of interleukin 12 by dendritic cells and theirredistribution to T cell areas. J. Exp. Med. 186:1819.

10. Trinchieri, G., and P. Scott. 1994. The role of interleukin 12 in the immuneresponse, disease and therapy. Immunol. Today 15:460.

11. Cho, D., W. J. Lee, P. J. Halloran, G. Trinchieri, and Y. B. Kim. 1996. Enhance-ment of porcine natural killer cell activity by recombinant human and murineIL-12. Cell. Immunol. 172:29.

12. Hsieh, C. S., S. E. Macatonia, C. S. Tripp, S. F. Wolf, A. O’Garra, andK. M. Murphy. 1993. Development of TH1 CD4� T cells through IL-12 pro-duced by Listeria-induced macrophages. Science 260:547.

13. Schopf, L. R., J. L. Bliss, L. M. Lavigne, C. L. Chung, S. F. Wolf, andJ. P. Sypek. 1999. Interleukin-12 is capable of generating an antigen-specificTh1-type response in the presence of an ongoing infection-driven Th2-type re-sponse. Infect. Immun. 67:2166.

14. Rengarajan, J., S. J. Szabo, and L. H. Glimcher. 2000. Transcriptional regulationof Th1/Th2 polarization. Immunol. Today 21:479.

4379The Journal of Immunology

by guest on Novem

ber 17, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

15. Zarlenga, D. S., A. Canals, R. A. Aschenbrenner, and L. C. Gasbarre. 1995.Enzymatic amplification and molecular cloning of cDNA encoding the small andlarge subunits of bovine interleukin 12. Biochim. Biophys. Acta 1270:215.

16. Bush, K., N. K. Day, L. A. Kraus, R. A. Good, and W. G. Bradley. 1994. Mo-lecular cloning of feline interleukin 12 p35 reveals the conservation of leucine-zipper motifs present in human and murine IL-12 p35. Mol. Immunol. 31:1373.

17. Schijns, V. E., C. M. Wierda, T. W. Vahlenkamp, and M. C. Horzinek. 1997.Molecular cloning of cat interleukin-12. Immunogenetics 45:462.

18. Fehr, D., G. A. Dean, J. Huder, Z. Fan, S. Huettner, J. W. Higgins,N. C. Pedersen, and H. Lutz. 1997. Nucleotide and predicted peptide sequence offeline interleukin-12 (IL-12). DNA Seq. 8:77.

19. Okano, F., M. Satoh, and K. Yamada. 1997. Cloning and expression of the cDNAfor canine interleukin-12. J. Interferon Cytokine Res. 17:713.

20. Buttner, M., G. Belke-Louis, H. J. Rziha, C. McInnes, and O. R. Kaaden. 1998.Detection, cDNA cloning and sequencing of canine interleukin 12. Cytokine 10:241.

21. Nicolson, L., M. N. Penha-Goncalves, J. L. Keanie, N. A. Logan, D. J. Argyle,and D. E. Onions. 1999. Cloning and sequencing of horse interleukin-12 andinterleukin-18 cDNAs. Immunogenetics 50:94.

22. Foss, D. L., and M. P. Murtaugh. 1997. Molecular cloning and mRNA expressionof porcine interleukin-12. Vet. Immunol. Immunopathol. 57:121.

23. Swinburne, S. J., G. R. Russ, and R. Krishnan. 2000. Ovine interleukin 12 hasbiological activity on ovine and human activated peripheral blood mononuclearcells. Cytokine 12:1546.

24. De Rose, R., J. P. Scheerlinck, G. Casey, P. R. Wood, J. M. Tennent, andP. J. Chaplin. 2000. Ovine interleukin-12: analysis of biologic function and spe-cies comparison. J. Interferon Cytokine Res. 20:557.

25. Schmidt, P., C. Kuhn, S. Kang’a, O. Hanotte, J. Vanselow, I. Anton, C. Langner,and M. Schwerin. 2000. Interleukin-12 p35 encoding gene of cattle and sheepharbours a polymorphic T stretch in intron 4. Anim. Genet. 31:283.

26. Digby, M. R., and J. W. Lowenthal. 1995. Cloning and expression of the chickeninterferon-� gene. J. Interferon Cytokine Res. 15:939.

27. Schneider, K., F. Puehler, D. Baeuerle, S. Elvers, P. Staeheli, B. Kaspers, andK. C. Weining. 2000. cDNA cloning of biologically active chicken interleukin-18. J. Interferon Cytokine Res. 20:879.

28. Hilton, L. S., A. G. Bean, and J. W. Lowenthal. 2002. The emerging role of aviancytokines as immunotherapeutics and vaccine adjuvants. Vet. Immunol. Immu-nopathol. 85:119.

29. Staeheli, P., F. Puehler, K. Schneider, T. W. Gobel, and B. Kaspers. 2001. Cy-tokines of birds: conserved functions–a largely different look. J. Interferon Cy-tokine Res. 21:993.

30. Martin, A., H. S. Lillehoj, B. Kaspers, and L. D. Bacon. 1993. Antigen-specificT cell proliferation following coccidia infection. Poultry Sci. 72:2084.

31. Tirunagaru, V. G., L. Sofer, J. Cui, and J. Burnside. 2000. An expressed sequencetag database of T-cell-enriched activated chicken splenocytes: sequence analysisof 5251 clones. Genomics 66:144.

32. McMonagle, E. L., S. Taylor, H. van Zuilekom, L. Sanders, N. Scholtes,L. J. Keanie, C. A. Hopkins, N. A. Logan, D. Bain, D. J. Argyle, et al. 2001.Production of biologically active equine interleukin 12 through expression of

p35, p40 and single chain IL-12 in mammalian and baculovirus expression sys-tems. Equine Vet. J. 33:693.

33. Ding, A. H., C. F. Nathan, and D. J. Stuehr. 1988. Release of reactive nitrogenintermediates and reactive oxygen intermediates from mouse peritoneal macro-phages: comparison of activating cytokines and evidence for independent pro-duction. J. Immunol. 141:2407.

34. Stuehr, D. J., and C. F. Nathan. 1989. Nitric oxide. A macrophage product re-sponsible for cytostasis and respiratory inhibition in tumor target cells. J. Exp.Med. 169:1543.

35. Boardman, P. E., J. Sanz-Ezquerro, I. M. Overton, D. W. Burt, E. Bosch,W. T. Fong, C. Tickle, W. R. Brown, S. A. Wilson, and S. J. Hubbard. 2002. Acomprehensive collection of chicken cDNAs. Curr. Biol. 12:1965.

36. Trinchieri, G. 2003. Interleukin-12 and the regulation of innate resistance andadaptive immunity. Nat. Rev. Immunol. 3:133.

37. Weining, K. C., U. Schultz, U. Munster, B. Kaspers, and P. Staeheli. 1996.Biological properties of recombinant chicken interferon-�. Eur. J. Immunol. 26:2440.

38. Isaacs, A., and J. Lindenmann. 1957. Virus interference: I. The interferon. Proc.R. Soc. Lond. 147:258.

39. Schneider, K., R. Klaas, B. Kaspers, and P. Staeheli. 2001. Chicken interleukin-6.cDNA structure and biological properties. Eur. J. Biochem. 268:4200.

40. Shiratori, I., M. Matsumoto, S. Tsuji, M. Nomura, K. Toyoshima, and T. Seya.2001. Molecular cloning and functional characterization of guinea pig IL-12. Int.Immunol. 13:1129.

41. Diatchenko, L., Y. F. Lau, A. P. Campbell, A. Chenchik, F. Moqadam, B. Huang,S. Lukyanov, K. Lukyanov, N. Gurskaya, E. D. Sverdlov, and P. D. Siebert.1996. Suppression subtractive hybridization: a method for generating differen-tially regulated or tissue-specific cDNA probes and libraries. Proc. Natl. Acad.Sci. USA 93:6025.

42. Akira, S., K. Takeda, and T. Kaisho. 2001. Toll-like receptors: critical proteinslinking innate and acquired immunity. Nat. Immunol. 2:675.

43. Gillessen, S., D. Carvajal, P. Ling, F. J. Podlaski, D. L. Stremlo, P. C. Familletti,U. Gubler, D. H. Presky, A. S. Stern, and M. K. Gately. 1995. Mouse interleu-kin-12 (IL-12) p40 homodimer: a potent IL-12 antagonist. Eur. J. Immunol. 25:200.

44. Ling, P., M. K. Gately, U. Gubler, A. S. Stern, P. Lin, K. Hollfelder, C. Su,Y. C. Pan, and J. Hakimi. 1995. Human IL-12 p40 homodimer binds to the IL-12receptor but does not mediate biologic activity. J. Immunol. 154:116.

45. Germann, T., E. Rude, F. Mattner, and M. K. Gately. 1995. The IL-12 p40homodimer as a specific antagonist of the IL-12 heterodimer. Immunol. Today16:500.

46. Kumar, S., and S. B. Hedges. 1998. A molecular time scale for vertebrate evo-lution. Nature 392:917.

47. Vandaveer, S. S., G. F. Erf, and J. M. Durdik. 2001. Avian T helper one/twoimmune response balance can be shifted toward inflammation by antigen deliveryto scavenger receptors. Poultry Sci. 80:172.

48. Kaiser, P. The chicken Th2 cytokine gene cluster. In Workshop on MolecularPathogenesis of Marek’s Disease and Avian Immunology, October 6–11, 2002.Limassol, Cyprus, p. 76.

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