Molecular cloning and functional characterization of guinea pig IL-12

Ikuo Shiratori12, Misako Matsumoto1, Shoutaro Tsuji1, Midori Nomura1, Kumao Toyoshima1 and Tsukasa Seya12

1 Department of Immunology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Higashinari-ku, Osaka 537-8511, Japan
2 Department of Molecular Immunology, Nara Institute of Science and Technology, Ikoma, Nara 631-0101, Japan

Correspondence to: T. Seya, Department of Immunology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Higashinari-ku, Osaka 537-8511, Japan


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
IL-12 is a heterodimeric cytokine that plays a central role in cell-mediated immunity. We cloned complete cDNAs of guinea pig homologues of IL-12 p35 and p40 subunits, and compared their functional properties with human IL-12. Both p35 and p40 mRNA were constitutively expressed in the testis and peritoneal macrophages. On immunoblotting, anti-guinea pig p40 antibody detected the constitutive expression of p40 protein in the testis, while in macrophages it was induced in response to lipopolysaccharide. An unidentified 200-kDa macromolecule was also expressed in the testis. All recombinant hybrid heterodimer p70 (guinea pig p70, human p70 and two interspecies heterodimers) exerted proliferative activity toward concanavalin A-primed guinea pig and human lymphoblasts in a dose-dependent manner. A similar tendency was observed in IFN-{gamma} production in IL-2-treated human lymphocytes. All hybrid heterodimers also induced IFN-{gamma} mRNA from IL-2-treated guinea pig splenocytes. Thus, unlike the current concept that the p35 subunit determines the species incompatibility of IL-12 in humans and mice, p35 has marginal ability to define its species-specific functional expression between humans and guinea pigs. In addition, constitutive expression of IL-12 or related molecules in the testis indicated a potential role of this molecule in regulation of physiological or pathophysiological conditions in the reproductive system.

Keywords: comparative immunology, cytokines, guinea pigs


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
IL-12 is a novel cytokine composed of two disulfide-linked subunits, p35 and p40 (1,2). The p35 subunit shares structural similarities with IL-6 and granulocyte colony stimulating factor (G-CSF), whereas the p40 subunit is structurally related to the extracellular portion of hematopoietic cytokine receptors, IL-6 receptor (R) and ciliary neurotrophic factor R.

IL-12 is an early proinflammatory cytokine produced predominantly by macrophages and other antigen-presenting cells (APC) (1,2). IL-12 promotes the differentiation of naive T cells into Th1 cells, induction of IFN-{gamma} by acting on T and NK cells, and suppression of IgE production, which are essential for eliciting optimal Th1 responses and, hence, cell-mediated immunity. IL-12 also potentiates NK-mediated and cytotoxic T lymphocyte (CTL)-dependent cytolysis. All these functions of IL-12 were confirmed by not only in vitro studies in both human and murine systems, but also in vivo mouse models including IL-12-knockout mice (3), which reinforced the importance of IL-12 in an early stage of establishment of a Th1-dominant state followed by a variety of cellular responses in infectious, allergic and tumor immunity.

Human rIL-12 failed to express the reported immune modulatory activity toward murine lymphocytes because of its species specificity governed by the p35 subunit (4,5), while it exerted these activities toward non-human primates with a wide dose range (6,7). Thus, its activity could not be evaluated in mice, with either ex vivo or in vivo challenge. Moreover, some structural and functional discrepancies between human and mouse systems have been reported. In humans, two subunits (ß1 and ß2) of the IL-12R are needed to facilitate sufficient binding of human IL-12 to the receptor. In contrast, in mice, the ß1 subunit may be sufficient to sustain mouse IL-12 binding, while the ß2 subunit is necessary for signal transduction (1,810). Likewise, binding affinity of human antagonistic p40 homodimer to the receptor is pretty low compared to that of mouse p40 homodimer that competes with the p70 with similar binding affinity to the receptor (1113). These previously reported results indicated that murine IL-12 is not a simple functional substitute for human IL-12. Therefore, it is necessary to characterize other potential small animal models for in vivo analysis of IL-12.

It has been reported that hormonally and immunologically, guinea pigs are more similar to humans than are rodents (14). Particularly, the guinea pig model of tuberculosis (TB) has certain advantages over the mouse model because of similar immune responses with humans, like generating potent skin-test reactions (delayed-type hypersensitivity) and developing substantial necrosis in the lungs after TB exposure, which eventually causes sufficient damage to the lung architecture to be fatal (1416). Protective immunity to TB is mediated by Th1 cells and triggered by the innate immune system including IL-12 (16,17).

Here, we cloned cDNAs of the two subunits of guinea pig IL-12, and compared its predicted structure and function with human, mouse and other species counterparts. Our findings demonstrated that guinea pig IL-12 showed the highest similarity to humans and, unlike mouse IL-12 p35, guinea pig p35 has no ability to define its species-specific functional expression. In addition, IL-12 (both p35 and p40) mRNA, p40 protein and a 200-kDa molecule antigenically related to the p40 subunit were constitutively expressed in the guinea pig testis.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals
All procedure were approved by Institutional Animal Care and Use Committee of Osaka Medical Center for Cancer and Cardiovascular Diseases. Guinea pigs, Hartley strain, 10- to 15-week-old males (Japan SLC, Shizuoka, Japan), were used in this study.

Preparation of cells and mitogenic stimulation
Guinea pig spleen cells were harvested from minced tissue and dispersed through mesh screens. Erythrocytes were lysed with Tris–NH4Cl and the cells were washed 3 times in RPMI 1640 medium (Life Technologies, Gaithersburg, MD) containing 10% FCS. Peritoneal macrophages elicited by thioglycollate medium (TGC) were isolated as described (18). Cells (1x107 cells/ml) were stimulated with 10 µg/ml of lipopolysaccharide (LPS; Escherichia coli 0127: B8; Difco, Detroit, MI) or 5 µg/ml of concanavalin A (Con A; Honen, Tokyo, Japan) in a humidified 5% CO2 incubator for 12–24 h at 37°C.

RNA extraction
Total RNAs from the harvested cells or frozen guinea pig tissues were isolated using Trizol (Life Technologies) according to the manufacturer's instructions. mRNA was prepared by oligo(dT) priming of total RNA isolated from LPS-stimulated peritoneal macrophages or testis using a mRNA purification kit (Amersham Pharmacia Biotech, Piscataway, NJ).

Cloning and sequencing of guinea pig IL-12 p35 and p40 subunits
First, guinea pig IL-12 p35 and p40 partial cDNAs were generated using the standard RT-PCR method. Briefly, first-strand cDNAs were reverse-transcribed from random-primed RNA templates prepared from LPS-stimulated peritoneal macrophages using Superscript II reverse transcriptase (Life Technologies). Typical nested-PCR was performed with degenerate primers synthesized according to the conserved amino acid sequences between human and mouse counterparts (Table 1Go. Second, full-length IL-12 p35, p40 cDNAs were generated using a Marathon cDNA amplification kit (Clontech, Palo Alto, CA). In this process, PCR from a macrophage cDNA template was performed using p35, p40 gene-specific internal primers (Table 1Go) paired with either the AP1- or the AP2-specific end primers. The PCR products were ligated directly into the pCR2.1 TA cloning vector (Invitrogen, Carlsbad, CA). In each experiment, eight to 10 clones were sequenced across both strands using a model 373A automated DNA sequencer (PE Applied Biosystems, Foster City, CA).


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Table 1 Primers used for cloning of guinea pig IL-12 p35, p40 subunits

 
Sequence data analysis
Nucleotide and protein sequences obtained were compared with currently available sequences in the GenBank database using the BLAST program. Multiple alignments of IL-12 p35 and p40 protein sequences were made with the GENETYX-MAC program (version 9.0), and the signal peptide cleavage sites were predicted using the signal IP program (version 2.0).

Preparation of other cytokines and ß-actin cDNA using RT-PCR
Since human IL-12 p35, p40 and guinea pig IFN-{gamma} sequences are present in the GenBank data base (accession nos M65271, M65272 and AF058395 respectively), we synthesized appropriate primers and constructed open reading frames (ORF) or partial cDNAs using the standard RT-PCR method. ß-Actin was used as a control housekeeping gene. The primer sets used for the detection of guinea pig ß-actin were those reported by Rottman et al. (19). RNA templates for cloning of human IL-12 p35 and p40 ORF cDNAs were isolated from 12 h LPS (10 µg/ml)-stimulated human monocytes as described below. Clones were characterized and sequenced as described above.

Northern blotting analysis
Total RNA (10 µg) was resolved by electrophoresis on agarose gels containing formaldehyde, transferred onto Hybond N+ membranes (Amersham Pharmacia Biotech) and hybridized with 32P-labeled cytokine cDNA probes by the standard method. The blots were rehybridized with a ß-actin probe to estimate the relative amounts of RNA loaded. The mRNA level was estimated from radioactivity of the hybridized probe by phosphoimaging or autoradiography. For IL-12 probes, the membranes were first probed with the p35 probe, stripped and reprobed with the p40 cDNA probe. Exposure times on Hyper film MP (Amersham Pharmacia Biotech) were 5 days (p35) and 7 days (p40) at –80°C with an intensifying screen. The IFN-{gamma}-probed filters were exposed for 4 days and each of the ß-actin-probed filters were exposed for 1 day at –80°C.

Purification of E. coli-expressed p40 and immunization
Predicted guinea pig mature p40 (amino acids 23–332, Fig. 1bGo) cDNA was ligated into the E. coli expression vector pET-28b (Novagen, Madison, WI), and used to transform competent E. coli BL21 (DE3) cells (Novagen). N-terminal (His)6-tagged p40 was purified from the soluble fraction by Ni-nitrilotriacetic acid–agarose (Qiagen, Valencia, CA) chromatography according to the manufacturer's instructions. For preparation of anti-guinea pig p40 antibodies, 50 µg of purified p40 polypeptide was injected with complete Freund's adjuvant (Difco) into a rabbit every week. Three days after the fourth immunization, antiserum was collected and the polyclonal antibodies were purified by precipitation with ammonium sulfate at 33% saturation.



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Fig. 1. Nucleotide and deduced amino acid sequences of p35 (a) and p40 (b) subunits. Potential N-linked glycosylation sites are boxed. The predicted signal peptide cleavage sites are indicated by arrowheads and cysteine residues are outlined. The peptide sequences used to generate nested-PCR primers are marked with arrows indicating the direction of amplification. These guinea pig nucleotide sequences in this figure will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession nos AB025723 (p35) and AB025724 (p40).

 
Expression of IL-12 cDNA clones in COS cells
Human or guinea pig IL-12 p35 and p40 ORF cDNAs were subcloned into the pME18S mammalian expression vector. COS7 cells were grown in DMEM (Life Technologies) containing 10% FCS and transfected with Lipofectin reagent (Life Technologies) according to the manufacturer's instructions. Alternatively, for IL-12 bioassay, each transfection contained 5 µg of each IL-12 subunit expression vector per 10-cm dish. After 3 h, transfected cells were washed and cultured in AIM-V medium (Life Technologies) for 24 h. After incubation, culture supernatants were harvested, concentrated 10-fold using a SpeedVac, dialyzed against D-PBS for 16 h at 4°C and sterilized by filtration. For Western blotting analysis, each transfection contained 2.5 µg of each IL-12 subunit expression vector per 3-cm dish and transfected cells were cultured in DMEM containing 1% FCS for 24 h.

Detection of recombinant hybrid IL-12 p70 by ELISA
The levels of various forms of hybrid IL-12 p70 (p35/p40) secreted into the COS cell culture supernatants were determined by ELISA using human IL-12 p70 immunoassay and human IL-12 p40 immunoassay (Genzyme/Techne, Minneapolis, MN) according to the manufacturer's instructions.

Western blotting analysis
For detection of various forms of hybrid p70 secreted into the COS cell culture supernatants or endogenous IL-12 in macrophage culture supernatant, aliquots of 250 µl of culture supernatants were concentrated using an equal volume of 10% trichloroacetic acid and solubilized in reducing or non-reducing sample buffer. Samples were resolved by SDS–PAGE and transferred onto PVDF membranes (Millipore, Bedford, MA). The blots were blocked with 5% non-fat milk and treated with rabbit anti-guinea pig p40 polyclonal antibody. After washing, the blots were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (BioRad, Hercules, CA) and developed with ECL (Amersham Pharmacia Biotech). In some experiments, the blotted membranes were reprobed by washing twice with 50 mM Tris buffer, pH 6.8, containing 2% SDS and 100 mM 2-mercaptoethanol for 15 min at 50°C, blocked and treated with goat anti-human IL-12 p70 polyclonal antibody (Genzyme/Techne). After washing, the blots were incubated with HRP-conjugated swine anti-goat IgG (Biosource, Lewisville, TX). For detection of endogenous p40 in testis, the tissue was homogenized in 20 mM Tris-buffered saline, pH 7.4, containing 1 mM PMSF, 10 mM EDTA and 1 mg/ml iodoacetic acid. The homogenates were centrifuged and 30 µg of proteins of supernatants were mixed with reducing or non-reducing sample buffer.

IL-12 proliferation assay
Human peripheral blood mononuclear cells (PBMC) were collected with methylcellulose sedimentation followed by centrifugation on a Ficoll-Hypaque cushion. Human PBMC or guinea pig splenocytes (2x106 cells/ml) were cultured in RPMI 1640 containing 10% FCS and 5 µg/ml Con A. After 3 days, the Con A-primed lymphoblasts were harvested, washed and resuspended in RPMI 1640 with 10% FCS at 1.0x106 cells/ml. Aliquots of 200 µl of the cell suspension were mixed with 10 µl aliquots of serial dilution of hybrid p70-containing culture supernatants in 96-well plates. Cells were further incubated for 48 h with [3H]thymidine (New England Nuclear, Boston, MA) added at a final concentration of 1 µCi/well for the last 24 h of incubation. In blocking experiments, the human or guinea pig Con A-primed lymphoblasts (2.0x105 cells) were cultured with 10 µl of human p35/p40-transfected COS cell supernatant in the presence of goat anti-human IL-12 p70 polyclonal antibodies (20 µg/ml) (Genzyme/Techne; cat. no. 42219) or polyclonal goat IgG (20 µg/ml) (Sigma; cat. no. I5256). The assay was performed as described above. All samples were assayed in triplicate.

IFN-{gamma} induction assay
Human peripheral blood lymphocytes (PBL) (CD14) were isolated from PBMC by negative selection using CD14 magnetic beads (Miltenyi Biotec, Bergish Gladbach, Germany). PBL were suspended in RPMI 1640 containing 10% FCS and 10 ng/ml recombinant human IL-2 (R & D Systems, Minneapolis, MN) at 1.0x107 cells/ml. Aliquots of 200 µl of the cell suspension were mixed with 10 µl aliquots of serial dilution of hybrid p70-containing culture supernatants in 96-well plates, and the cultures were incubated for 36 h at 37°C. The levels of IFN-{gamma} in culture supernatants were determined by ELISA using human IFN-{gamma} ELISA system (Amersham Pharmacia Biotech) according to the manufacturer's instructions. All samples were assayed in triplicate. Production of IFN-{gamma} by guinea pig splenocytes in response to hybrid p70 was assessed by Northern blotting analysis. Guinea pig splenocytes were suspended in 10% FCS/RPMI 1640 with or without 10 ng/ml recombinant human IL-2 at 4.0x106 cells/ml. Aliquots of 3 ml of the cell suspension were mixed with 30 µl aliquots of hybrid p70-containing culture supernatants (10-fold) in six-well plates. The cultures were incubated for 36 h at 37°C. After incubation, cells were harvested and lysed in Trizol. RNA extraction and Northern blotting analysis were conducted as described above.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cloning and sequencing of guinea pig IL-12
Partial cDNAs of guinea pig IL-12 p35 and p40 subunits were cloned from mRNA of guinea pig peritoneal macrophages that had been stimulated with LPS. Full-length cDNAs of these subunits were obtained with a combination of 5' and 3' rapid amplification of cDNA ends (RACE)-PCR methods. The nucleotide sequences and the predicted amino acid sequences of these subunits are shown in Fig. 1Go.

The predicted guinea pig IL-12 p35 was a 221-amino-acid precursor protein with a 22-amino-acid signal sequence (as determined using the signal IP program). Human IL-12 p35 has been reported to consist of two protein isoforms (p35 short and p35 long) yielded by alternative usage of the translational initiation Met codon (20,21). Guinea pig p35 was a homologue of human p35 short, since the initiation Met codon at the appropriate position for p35 long was absent in guinea pig p35 (data not shown). Recognition of a monobasic cleavage site for disparate processing of the signal peptide of IL-12 has been reported in human p35 (22). This site, His18, of human p35 short was not conserved in the guinea pig p35. The precursor form of p35 possessed nine Cys residues and four possible N-glycosylation sites. The primary sequence of guinea pig p35 was compared with those of human, mouse and other species (Fig. 2aGo). Seven of the nine Cys residues and one of the four N-glycosylation motifs were conserved among the all species examined. Incidentally, the complete HXXLAR (X = any residue) sequence in the putative monobasic cleavage site described above was only conserved among human, mouse, horse and cat p35 subunits. The homology (%) toward guinea pig p35 subunit is 54–74% among the species examined and the low score tendency was observed in the rodent (mouse and rat) counterparts. On the contrary, the highest score was observed in human p35.



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Fig. 2. Alignment of IL-12 p35 (a) and p40 (b) protein sequences. All non-guinea pig IL-12 protein sequences were obtained from GenBank and the species are indicated. Residues identical among these species are boxed. Potential N-linked glycosylation sites are in green and cysteine residues are in red. The HXXLAR monobasic cleavage recognition sequences of p35 are in orange, and hallmark residues of the Ig domain, fibronectin type III domain and hematopoietin receptor domain of p40 are shaded (blue). The homology (%) toward guinea pig counterparts (g) are shown at the end of each sequence. Accession nos of these sequences are as follows: M65271/M65272 [human (hu) p35/p40], M86672/M86671 [mouse (m) p35/p40], AAD51364/NP-072133 [rat (r) p35/p40], AAA73897/AAA75356 [pig (p) p35/p40], P54349/P46282 [bovine (b) p35/p40], AAD51976/AAB61368 [sheep (s) p35/p40], Q9XSQ6/ Q9XSQ5 [horse (ho) p35/p40] and AAB93836/AAB93835 [cat (c) p35/p40].

 
The predicted guinea pig IL-12 p40 was a 332-amino-acid precursor polypeptide with a 22-amino-acid signal sequence. A partial cDNA sequence of guinea pig p40 (position 270–777, Fig. 1bGo) was reported by Scarozza et al. (accession no. AF097507) (23,24). The precursor possessed 10 Cys residues and one possible N-glycosylation site. The primary sequence of guinea pig p40 was compared with those of human, mouse and other species (Fig. 2bGo). Nine of the 10 Cys residues and one possible N-glycosylation site were conserved across the species. Guinea pig, bovine and sheep p40 have only one N-glycosylation site at the same position, and in human p40, N-linked sugars were identified at the conserved N-glycosylation site, Asp222, suggesting the presence of N-linked sugars at this Asp in guinea pig p40 (25). The homology (%) toward guinea pig p40 subunit are 61–79% among the species examined and, again, the low score tendency was observed in the rodent (mouse and rat) counterparts, and the highest score was observed in human p40. The primary sequence of guinea pig p40 was also 20.5% identical to the extracellular portion of human IL-6R, and the Ig domain, fibronectin type III domain and hematopoietin receptor domain in human p40 were also conserved completely in the guinea pig p40 as well as other species, except for mouse and rat p40 (26).

Expression of IL-12 subunit mRNA in guinea pig tissues
Northern blotting analysis using the guinea pig p40 and p35 ORF cDNAs as probes was performed to assess the signal intensity in each organ, and in mitogen-stimulated macrophages and splenocytes of guinea pigs (Fig. 3aGo). Predominant p35 mRNA (2.0 kb) expression was found in the testis, and p40 mRNA in the testis (2.5 kb) and to lesser levels in the kidney (2.0–2.2 kb). The transcript size of the p40 message in the testis and TGC-elicited macrophages was slightly bigger than those of kidney and cDNA clones. We analyzed the nucleotide sequences of p35 and p40 expressed in the testis by the 5' and 3' RACE technique to confirm that the ORF of these subunits (p35; 168–666 bp, p40; 49–999 bp, Fig. 1Go) were identical to those obtained from macrophages. No significant expression of IL-12 message was found in the lymphoid tissues (thymus and spleen). Scarozza et al. reported that p40 mRNA levels were low or undetectable in intact splenocytes, but increased as a function of time after stimulation with Con A (23). Under our experimental conditions, however, Con A-stimulated spleen cells did not express IL-12. Likewise, no significant difference of IL-12 (both p35 and p40) message was observed between unstimulated and LPS-stimulated macrophages.



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Fig. 3. Tissue distribution of IL-12 (p35 and p40) mRNA and p40 protein. (a) Northern blotting of the indicated guinea pig tissues and cells using p35 and p40 ORF cDNA probes. Each lane contained 10 µg of total RNA as described in Methods. RNA size markers are shown on the right. (b) Detection of endogenous expression of p40 by Western blotting using anti-guinea pig p40 antibodies. For Western blotting, macrophages were cultured in RPMI 1640 containing 1% FCS. Lane 1, testis extracts (30 µg); lane 2, macrophage culture supernatant (LPS–); lane 3, macrophage culture supernatant (LPS+); lane 4, guinea pig p70-transfected COS cell culture supernatant (see Fig. 4Go); and lane 5, testis extracts (30 µg). All these bands except for the 50-kDa bands seen in lanes 1 and 5 were specific because these bands were not observed on the same membrane reprobed with pre-immune IgG (data not shown). Mobilities of p40, p70, p40/p40 and the unknown 200-kDa form are indicated on the right. Mol. wt shown on the left. NR, non-reduced; R, reduced.

 
Detection of endogenous expression of p40 by immunoblotting
We next produced an antibody against guinea pig recombinant p40 and tested protein expression of p40 by immunoblotting (Fig. 3bGo). The specific 40-kDa band was identified as a p40 protein in the extracts of the testis and the culture supernatants of peritoneal macrophages. In contrast to the result of Northern blotting of macrophages, the expression (and secretion) of endogenous p40 was up-regulated by LPS stimulation. A testis-specific 200-kDa band was detected only in the non-reducing gel, suggesting the presence of a disulfide-linked macromolecule antigenically related to the p40 subunit. It has been reported that production of human and mouse IL-12 by cell lines and APC in several activation stages resulted in the secretion of a 5- to 500-fold excess of free p40 (p40 and p40/p40) relative to the IL-12 heterodimer (1,2). In our experimental conditions, little p70 formation was also observed in guinea pig tissues and macrophages tested.

Characterization of recombinant hybrid IL-12 p70
For the following IL-12 bioassays, COS7 cells were co-transfected with vectors expressing each subunit of human or guinea pig IL-12 (hp35/hp40, gp35/gp40, hp35/gp40 and gp35/hp40), and the amounts of recombinant hybrid p35/p40 in the culture supernatants were assessed by ELISA for human p70 and p40 (Fig. 4aGo). Incidentally, essential amino acids for heterodimerization in the two subunits are all conserved across humans, guinea pigs and mice based on recent crystal structure analysis (27). Only supernatants from COS cells co-transfected with hp35 and hp40 (hp35/hp40) or gp35 and hp40 (gp35/hp40) resulted in detection of heterodimer p70 (18.5 versus 0.4 ng/ml) as well as p40 monomer or homodimer (1.5 versus 3.0 ng/ml). No other combination of transfection including mock transfection allowed us to detect the production of p70 or p40 in these human ELISA systems.



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Fig. 4. Detection of various forms of human IL-12 p70 (hp35/hp40), guinea pig p70 (gp35/gp40) or hybrid p70 proteins consisting of human p35 and guinea pig p40 (hp35/gp40) or guinea pig p35 and human p40 (gp35/hp40) subunits by ELISA and Western blotting. The levels of secreted hybrid p70 in the COS cell culture supernatant were measured by ELISA (a). Mock-transfected COS cell supernatant (no input DNA) was used as a negative control. Values are expressed as the concentration of IL-12 (p40 or p70) in 1xculture supernatant and as means ± SD of triplicate determinations. These results are representative of two separate experiments with similar results using two different sets of transfected COS cell-conditioned medium. The secreted p70 hybrids in the culture supernatants were also detected by Western blotting analysis with anti-guinea pig p40 (b) or anti-human p70 (c). Lane1, hp35/hp40; lane 2, gp35/gp40; lane 3, hp35/gp40; lane 4, gp35/hp40; and lane 5, mock. Mol. wt shown on the left. g, guinea pig; h, human; ND, not detectable.

 
In contrast, on Western blotting analyses, anti-guinea pig p40 antibodies detected gp40 and cross-reactive hp40 (40 kDa) in the four samples under reducing conditions (Fig. 4bGo, lanes 1–4). Under non-reducing conditions, heterodimer (70 kDa) and homodimer (80 kDa) were detected in lanes with gp35/gp40 and hp35/gp40. In addition, anti-human p70 antibodies recognized both 35 and 40 kDa moieties in the reducing gel (Fig. 4cGo). Strong signals were detected in the lanes with hp35 or hp40 and weak signals in the lanes with gp35 or gp40. Again, cross-reactive gp35 recognition and less effective gp40 recognition were confirmed. In all lanes (Fig. 4cGo, lanes1–4), heterodimer was observed under non-reducing conditions with different signal intensities. These bands were specific because no band was detected in mock transfectants. Molecular heterogeneity probably reflects the presence of polymorphic variants due to post-translational modification (20). Based on these results, we confirmed the expression of each hybrid proteins.

IL-12-dependent lymphoblast proliferation assay
The growth response of human PBMC and guinea pig spleen cells to various forms of IL-12 p70 hybrid was examined by cell proliferation assay (Fig. 5a and bGo). [3H]Thymidine incorporation was measured after treatment of lymphocytes with Con A and then the COS cell culture supernatants containing recombinant p70 hybrids. All hybrids induced both human and guinea pig Con A-blast proliferation in a dose-dependent manner. In guinea pig Con A blasts, the growth rates induced by hp35/hp40 and gp35/hp40 were indistinguishable, and slightly higher than those by hp35/gp40 and gp35/gp40. In addition, the neutralizing anti-human p70 antibodies completely blocked the proliferation of both human and guinea pig-hp35/hp40-treated lymphoblasts (Fig. 5cGo). Taken together, these growth responses were clearly dependent on recombinant p35/p40.



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Fig. 5. Proliferation of Con A blasts in response to hybrid IL-12 p70. Proliferation of Con A-primed human PBMC (a) or guinea pig splenocytes (b) in response to the various forms of hybrid IL-12 was measured as described in Methods. `COS CS conc 101' means COS cell culture supernatant concentrated 10-fold. (c) In blocking experiments, all COS CS were used at 100 (conc) with 20 µg/ml of blocking antibodies or isotype controls. Values are expressed as means ± SD of triplicate determinations. These results are representative of four separate experiments using two different sets of transfected COS cell-conditioned medium.

 
Schouenhaut et al. reported that the hybrid IL-12 containing the human p35 subunit did not induce proliferation of mouse splenocytes (4). However, in guinea pigs, splenocytes were induced to proliferate even by the hybrid IL-12 containing hp35. Under our experimental conditions, 20–50 µg/ml of specific anti-guinea pig p40 antibodies did not block the gp35/gp40 hybrid activity for reasons as yet unknown (data not shown), and Con A pretreatment resulted in the most pronounced synergy effect on both human and guinea pig cell proliferation compared with pretreatment with other reagents, i.e. phytohemagglutinin and/or human rIL-2 (data not shown).

IL-12-dependent IFN-{gamma} induction assay
IFN-{gamma} levels in the culture supernatants of human PBL treated with various p70 hybrids plus human rIL-2 were measured by ELISA (Fig. 6aGo). The levels of IFN-{gamma} induced by the hybrids were very high in hp35/hp40, moderate in gp35/hp40 and hp35/gp40, and low but significant in gp35/gp40. A similar tendency was observed solely with the hybrid (no addition of IL-2) although the efficiency of IFN-{gamma} production was very low (Fig. 6bGo). Taking these results together, all of the p70 hybrids induced IFN-{gamma} production by human PBL and synergized effectively with IL-2.



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Fig. 6. IFN-{gamma} production by human PBL in response to hybrid IL-12 p70. The ability of human PBL to produce IFN-{gamma} in response to the various forms of hybrid p70 was determined by ELISA as described in Methods. Human PBL were cultured with the hybrid p70- or mock-transfected COS cell supernatant in culture media containing human rIL-2 (a) or none (b). Values are expressed as the concentration of IFN-{gamma} in culture supernatant and as means ± SD of triplicate determinations. These results are representative of two separate experiments with similar results using PBL from two healthy donors.

 
Production of IFN-{gamma} in guinea pig splenocytes by the p70 hybrids was estimated by Northern blotting analysis (Fig. 7aGo). The level of guinea pig IFN-{gamma} message in intact splenocytes was below the limit of detection and addition of human rIL-2 to the cells augmented IFN-{gamma} mRNA production (28,29). Further addition of IL-12 hybrids potentiated IFN-{gamma} mRNA production. The relative amounts of the messages, which were compensated by ß-actin densities in each lane, are shown in Fig. 7(b)Go: all hybrids increased the IFN-{gamma} mRNA production by 1.7- to 2.2-fold.



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Fig. 7. IFN-{gamma} production of guinea pig splenocytes in response to hybrid IL-12. The ability of guinea pig splenocytes to produce IFN-{gamma} in response to the various forms of hybrid p70 was determined by Northern blotting analysis (a). Guinea pig splenocytes were cultured with the indicated combinations of hybrid p70- or mock control-supernatant and/or human rIL-2. Each lane contained 10 µg of total RNA. Membranes were probed with guinea pig IFN-{gamma} partial cDNA (158-bp) probe. Hybridization signals were also quantified using a phosphoimager (b). Values are expressed as relative units, i.e. the ratio of the hybridization signal of IFN-{gamma} mRNA to that of ß-actin mRNA.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Here, we identified the structural and functional properties of guinea pig IL-12. Our results can be summarized as follows. (i) The predicted primary structures of guinea pig p35 and p40 showed the highest similarities toward human counterparts among those of the species tested. (ii) The strongest signals of IL-12 (both p35 and p40) message and p40 protein were detected in the testis out of the guinea pig tissues tested. (iii) All hybrid IL-12 p70 (p35/p40) consisting of human and guinea pig IL-12 subunits more or less exhibited the IL-12 function in both human and guinea pig cells.

IL-12 is a major innate immune factor produced predominantly by macrophages and other APC, and functionally encompassing innate and acquired immunity for balancing of optimal cell-mediated immune responses (1,2). It has been reported that the p35 subunit governs the species specificity of IL-12 function (4,5). This concept was proposed based on the results with hybrid heterodimers (p70) consisting of human and mouse p35 and p40 subunits. However, this concept is not the case between human and non-human primates, and based on the present results cannot be generalized between humans and rodents (6,7). In this study, functional complementation was established with hybrid heterodimers consisting of human and guinea pig p35 and p40 subunits. Both human PBMC (or PBL) and guinea pig splenocytes responded to p70 hybrids with different sensitivity in terms of cell proliferation (Fig. 5Go) and IFN-{gamma} production (Figs 6 and 7GoGo). Accordingly, guinea pig IL-12 must be a functional substitute for human IL-12. This is in part attributable to the high degree of structural similarity between human and guinea pig IL-12 subunits. As COS7 cells expressed not only p70 but also free p40 (monomer or homodimer) (Fig. 4a–cGo), the differences in sensitivity of hybrid p70 between human PBMC (or PBL) and guinea pig splenocytes may be due to differences in binding affinity of antagonistic p40 homodimers to the receptor (1113). Therefore, the molecular structure of the IL-12 receptor and intermolecular association between IL-12 and its receptor complex must be elucidated in guinea pigs.

The tissue distribution profile of the guinea pig IL-12 based on the results of both northern and Western blotting analyses is intriguing (Fig. 3Go). There have been no previous reports that the testes constitutively express p35 and p40 messages and the p40 protein. It is notable in this study that the p40 protein was the main product and the unidentified 200-kDa macromolecule antigenically related to the p40 protein was found in the testis. Judging from the analogy of IL-12 to IL-6 family proteins, the relationship between p35 and p40 should be a ligand–receptor interaction. Thus, it is not surprising that each subunit of IL-12 can couple with different partners. Actually, p35 forms a complex with an Epstein–Barr virus-induced gene (EBI3), i.e. p35/EBI3, whose function has not yet been identified (30). p40 can choose a p19 (19-kDa) protein, closely related to p35, as a disulfide-bridged partner to exert IL-12-like cytokine activity(31). This p19/p40 complex was named IL-23 and its functional profile was found to be in part distinct from that of IL-12. Hence, the relevant 200-kDa molecule may reflect the presence of an alternative candidate of the p40-containing proteins. Analysis of the composition of the 200-kDa molecule is in progress in our laboratory.

The role of IL-12 in the testis also remains to be clarified. A variety of cytokines have been reported to have roles as paracrine mediators under physiological or pathophysiological conditions in the testis of rodents. IL-1{alpha} is constitutively expressed in rat Sertoli cells and its expression levels are dependent on the stage of the seminiferous epithelial cycle; both the IL-1 bioactivity and message were diminished in stage VII of the cycle or in the seminiferous tubules depleted of germ cells (32). Accordingly, some reports suggested that IL-1{alpha} may participate in DNA replication in the testicular germ cells (3234). Regarding IL-12, only one preliminary report suggested that rat Leydig cells cultured in vitro produce the IL-12 p40 subunit in a luteinizing hormone (LH) concentration-dependent manner in parallel with testosterone induction (35). The testosterone induced by LH is considered to act on the stage VII Sertoli cells, which in turn participate in paracrine regulation of germ cells. However, the function of this inducible IL-12 in the testis and distribution of the IL-12 receptor in the testis remain unknown, and therefore further analyses are needed to evaluate their physiological significance in the testis.

Mice have been used as an animal model for human immune diseases as their gene disruption procedure was established. However, as a human model, mice are somewhat different, particularly in the innate immune system. The complement system differs between mice and humans (36). Toll-like receptor (TLR) 2 activation leads to killing of intracellular Mycobacterium tuberculosis, but the mode of bactericidal activity again differs between murine and human macrophages (37). Species differences are also observed in APC-derived initial cytokines including IL-23 (31). At least, these innate factors are difficult to analyze using the mouse as a human analogue. In contrast, many studies of the comparative biology of the guinea pig have revealed a number of marked similarities between this species and humans, especially in the field of innate immunity (14). These fundamental similarities bear directly or indirectly on the relevance of the guinea pig as a species to model human infectious disease, especially TB (1416). It has been accepted that the guinea pig, as well as the mouse and rat, belongs to the rodent family. However, recent molecular phylogenic studies suggested that the guinea pig may not be part of the order Rodentia (38,39) and also support this concept regarding the IL-12 molecule in this study (Fig. 2Go and phylogenic analyses, data not shown). In addition, molecular cloning of guinea pig CD1 suggested that guinea pigs have eight isoforms which are homologues of human group 1 CD1b, CD1c subclasses and CD1e, while muroid rodents (mice and rats) have only homologues of human group2 CD1d subclasses (40). CD1 is a family of non-polymorphic genes that seems to have evolved to present lipid and glycolipid antigen including lipoarabinomannan and mycolic acids of TB (41). As group 1 and group 2 CD1 proteins have distinct roles in the host immune responses with respect to the types of antigens presented by each group, this issue may provide a key to the analysis of the different responses of guinea pigs and mice to TB (14,15).

A regrettable point is that compared to the mouse or human immune system, the guinea pig immune system lags far behind in its analysis. Molecular cloning of guinea pig cytokines, surface adhesion molecules and innate immune-related molecules should help establish guinea pig as a model animal. We have cloned IL-18 and IL-23 (the p19 subunit) as well as IL-12. Myd88, an adaptor molecule for TLR, was also cloned (Shiratori et al., unpublished). Although these results are not published in this communication, accumulating evidence supports our idea that the guinea pig innate system more resembles that of humans than that of mice.

In summary, IL-12 is involved in the initial steps of infection, allergy and protection against tumor progression. Most studies including gene therapy are being developed using mice. Here, we propose that the guinea pig will be an alternative small animal model for clinical application of IL-12 and will provide certain advantages over the mouse in some points, especially in vivo studies analyzing the effects of human rIL-12 in infectious disease, allergy and cancer. Further molecular analyses will be needed to confirm this proposal.


    Acknowledgments
 
We are grateful to Drs H. Akedo, M. Tatsuta and H. Koyama (Osaka Medical Center for Cancer, Osaka) for support of this work and valuable discussions. We thank Drs I. Azuma and K. Hazeki for thoughtful discussions. Thanks are also due to Drs N. A. Begum, K. Shida, M. Taniguchi and S. Kikkawa for technical assistance. This work was supported in part by Organization for Pharmaceutical Safety and Research (OPSR), Grant-in-Aids from the Ministry of Culture, Technology and Sciences, and Grant-in-Aids from the Ministry of Health and Welfare, of Japan.


    Abbreviations
 
Con A concanavalin A
CTL cytotoxic T lymphocyte
G-CSF granulocyte colony-stimulating factor
HRP horseradish peroxidase
LH luteinizing hormone
LPS lipopolysaccharide
ORF open reading frame
PBL peripheral blood lymphocyte
PBMC peripheral blood mononuclear cell
R receptor
RACE rapid amplification of cDNA ends
TB tuberculosis
TGC thioglycollate medium
TLR Toll-like receptor

    Notes
 
Transmitting editor: L. L. Lanier

Received 2 April 2001, accepted 30 May 2001.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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