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BIOLOGICAL FUNCTIONS OF RECOMBINANT BOVINE INTERLEUKIN 6 EXPRESSED IN A BACULOVIRUS SYSTEM
Article No. cyto.1998.0499, available online at http://www.idealibrary.com on
BIOLOGICAL FUNCTIONS OF RECOMBINANT BOVINE INTERLEUKIN 6 EXPRESSED IN A BACULOVIRUS SYSTEM M. Yoshioka, Y. Mori, S. Miyazaki, T. Miyamoto, Y. Yokomizo, Y. Nakajima The cDNA encoding bovine interleukin 6 (IL-6) was obtained from messenger RNA extracted from lipopolysaccharide-stimulated bovine Kupffer cells by the reverse transcription polymerase chain reaction (RT/PCR), and cloned into the baculovirus vector pVL 1392. Insect cells (Sf21AE derived from Spodoptera frugiperda) infected with the recombinant baculovirus secreted a large amount of 23.7 kD protein into the culture medium. This protein was capable of causing increased haptoglobin production and decreased albumin production in primary cultured bovine hepatocytes. The swine and human IL-6s were also able to decrease albumin production in bovine hepatocytes. This recombinant IL-6 did not stimulate the proliferation of 7TD1 cells (a murine IL-6-dependent cell line), whereas the recombinant swine IL-6 which was expressed in the same baculovirus system, and recombinant human IL-6 derived from Escherichia coli were each capable of stimulating proliferation of 7TD1 cells, respectively. This suggests a species restriction between bovine IL-6 and murine IL-6 dependent cell lines. 1999 Academic Press
Interleukin 6 (IL-6) is a multifunctional cytokine with roles in the regulation of the immune response and the host defence reaction, and it is produced by a variety of cell types.1 The biological activities of IL-6 include differentiation of B cells, activation of T cells, growth promotion of hybridomas/plasmacytomas, proliferation of haematopoietic stem cells and stimulation of the acute phase response by hepatocytes.2–4 Over-expression of IL-6 is known to be an important feature of the pathogenesis of a number of inflammatory diseases, and may also be a good marker for the diagnosis of systemic inflammatory response syndrome (SIRS) and hypercytokinaemia. Human IL-6 consists of a single polypeptide chain of 184 amino acids. It is known that the IL-6 produced by mammalian cells is N- and O-glycosylated.5 The structural sequences of the IL-6 gene are less well conserved among mammalian species. At the protein level, bovine IL-6 shows 65, 53 and 42% homology to the porcine, human and murine IL-6s, respectively.6 From the National Institute of Animal Health, Tsukuba, Japan Correspondence to: M. Yoshioka, National Institute of Animal Health, 3-1-1 Kannondai, Tsukuba, Ibaraki 305-0856, Japan; E-mail: [email protected]ffrc.go.jp Received 11 August 1998; accepted for publication 23 December 1998 1999 Academic Press 1043–4666/99/110863+06 $30.00/0 KEY WORDS: albumin/bovine interleukin 6/haptoglobin/ recombinant protein CYTOKINE, Vol. 11, No. 11 (November), 1999: pp 863–868
Human IL-6 is capable of stimulating the proliferation of murine IL-6-dependent hybridoma cell lines,7 although IL-6 genes are also less conserved between human and mouse (42% at the amino acid level). However, a bioassay using a murine IL-6-dependent cell line is not adequate for measurement of the bovine IL-6 level.8 A sensitive and accurate method for detecting the level of IL-6 is needed for the diagnosis of many inflammatory diseases in cattle, as well as basic studies of IL-6 functions. We have cloned and expressed the amino acids-coding region of bovine IL-6 in a baculovirus system, as an initial step to establish a sensitive assay for IL-6 in cattle.
RESULTS Expression of bovine IL-6 in the baculovirus system The single-stranded cDNA encoding bovine IL-6 was amplified using PCR. The PCR products were cloned into pVL 1392 and sequenced. The derived sequence contained an open reading frame of 624 bp, encoding 208 amino acids with a predicted molecular weight of 23 758 Da. The nucleotide sequence was entirely identical to the bovine IL-6 reported by Droogmans et al.6 The resultant transfer vector pVLBoIL-6 was co-transfected with a baculovirus 863
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human IL-6 rabbit serum (data not shown). The molecular size of the secreted protein was about 23.7 kDa.
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In order to examine the activity of recombinant bovine IL-6 (rBoIL-6) on bovine cells, the induction of acute phase proteins by IL-6 was measured in primary cultured bovine hepatocytes. As shown in Figure 2, the supernatant of the cell culture that had been infected with AcBoIL-6 showed reduced albumin production (measured in an ELISA assay) in a dose-dependent manner. The same bovine hepatocytes were much less responsive to control supernatant which was derived from AcNPV wild-type virus-infected cells. Recombinant human IL-6 (rHuIL-6) expressed in E. coli, dose-dependently reduced albumin production by bovine hepatocytes, but the degree of reduction at rHuIL-6 concentrations of 1000 ng/ml was less than that obtained with supernatant containing rBoIL-6. Recombinant swine IL-6 (rSwIL-6) expressed in the baculovirus system, produced in the same baculovirus system, also reduced albumin production by bovine hepatocytes. In contrast to the decrease in albumin production, rBoIL-6 induced haptoglobin production by bovine hepatocytes (Fig. 3). Although primary cultured bovine hepatocytes secreted a small amount of haptoglobin in the non-stimulated state, rBoIL-6 increased
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Figure 1.
Expression of rBoIL-6 protein by recombinant baculovirus.
Proteins were analysed by electrophoresis on SDS-12.5% PAGE, followed by staining with Coomassie brilliant blue R-250. Lane M: marker proteins. Lane 1: supernatant of pVLBoIL-6-infected cells 3 days after infection. Lane 2: supernatant of pVLBoIL-6-infected cells 4 days after infection. Lane 3: 10-fold concentrated supernatant of non-infected cells 4 days after infection. Lane 4: supernatant of wild-type AcNPV-infected cells 4 days after infection. Lane 5: Western blot of the same samples as lane 1 with a rabbit anti ovine IL-6 antiserum.
(AcNPV) on Sf21AE cells. After cloning, the recombinant baculovirus containing bovine IL-6 (AcBoIL-6) was obtained. The secreted proteins reactive with antiovine IL-6 antiserum in the concentrated supernatant were detected by a Western blot analysis (Fig. 1). In addition, the secreted protein cross-reacted with anti-
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Albumin production by bovine hepatocytes in response to IL-6.
Albumin concentration of the culture supernatant of hepatocytes stimulated with IL-6 samples was determined with an ELISA. Samples were (i) serially diluted culture supernatant of AcBoIL-6 (rBoIL-6), (ii) AcSwIL-6 (rSwIL-6), (iii) wild type AcNPV infected cells (wild type), and (iv) rHuIL-6 expressed in E. coli (1000, 100, 10, 1, 0 ng/ml). Means and standard deviations are shown.
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Figure 3. Immunoblot analysis of haptoglobin secretion by bovine hepatocytes in response to rBoIL-6. The haptoglobin in the culture supernatant from hepatocytes stimulated with IL-6 was detected by immunoblotting. 10 l culture medium concentrated to a quarter of the original volume was separated by SDS-PAGE, blotted and reacted with anti-bovine haptoglobin. Lane M: marker proteins. Lane 1: rBoIL-6. Lane 2: 10-fold dilution of rBoIL-6. Lane 3: 100-dilution of rBoIL-6. Lane 4: Supernatant of wild-type AcNPV-infected cells. Lane 5: Supernatant of non-infected cells. Lane 6: medium control. Lane 7: positive control (500-fold dilution acute-phase calf serum).
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Proliferative response of 7TD1 cells to recombinant IL-6s.
The culture supernatant of Sf21AE cells infected with AcBoIL-6 was used to stimulate the proliferation of 7TD1 cells for 72 h. The colorimetric assay was done by the MTT method. Recombinant HuIL-6 expressed in E. coli, and rSwIL-6 expressed in baculovirus system, were simultaneously assayed as positive controls. ( ), rBoIL-6; ( ), rSwIL-6; (), rHuIL-6 (10 ng/ml); shaded area indicates negative control range.
haptoglobin secretion dose dependently. The supernatants derived from either non-infected cells or AcNPV wild type virus-infected cells, as a negative control, failed to increase haptoglobin production. Proliferation assays for the baculovirus-expressed rBoIL-6 were done using 7TD1 cells, which offer a highly sensitive proliferation assay for human, porcine and murine IL-6. As shown in Figure 4, the supernatant of Sf21AE cell culture that had been infected with AcBoIL-6, poorly stimulated proliferation of 7TD1 cells. Recombinant HuIL-6 and rSwIL-6 were assayed as positive controls. In the assay shown in Figure 4, activity of rHuIL-6 at 100 pg/ml was con-
verted into 1 standard unit. The supernatant containing rSwIL-6 strongly stimulated proliferation of 7TD1 cells, and its biological activity was calculated at over 1105 U/ml when compared with human IL-6 as a standard. This proliferation induced by rSwIL-6 was inhibited by anti-human IL-6 antiserum (data not shown). A combination of the supernatant containing rBoIL-6 and rHuIL-6 did not affect proliferation activity of rHuIL-6 (data not shown), suggesting that the supernatant containing rBoIL-6 was not cytotoxic to 7TD1 cells. The supernatant of Sf21AE cells infected with AcNPV wild type virus did not proliferate 7TD1 cells.
DISCUSSION In the present study, we succeeded in cloning and expressing bovine recombinant IL-6 in a baculovirus system. The sequence is identical to that reported by Droogmans et al.6 Recombinant BoIL-6 was unable to stimulate the proliferation of 7TD1 cells. However, this protein retained other functional activities of IL-6, in the induction of acute phase proteins (increase of haptoglobin, decrease of albumin) by bovine primary cultured hepatocytes. We tried to assess the bioactivity of bovine IL-6 by proliferation assay using 7TD1 cells, but failed to detect IL-6 in the serum or milk of cows. There are other reports in which this assay was not effective in measuring ruminant IL-6.8 On the other hand, the 7TD1 cell line has been used to test for bovine IL-6 activity.9,10 We have been able to detect chicken or swine IL-6 in serum using this cell line.11 Recently, it has been reported that a recombinant caprine IL-6 expressed in a baculovirus system stimulates the growth of 7TD1 cells.12 However, that report did not confirm that the antibodies to human IL-6 inhibited the proliferation of 7TD1 cells induced by the caprine IL-6. Rabbit antibodies to human IL-6 (Genzyme, Cambridge, MA, USA) inhibited the proliferation of 7TD1 cells induced by human IL-6, but had no effect on bovine serum-induced proliferation of 7TD1 cells (unpublished data). Amino acid sequences of mammalian IL-6s are not extensively conserved. This is especially true for the bovine and murine IL-6 sequences, which show only 42% homology. However, there is a high level of homology between the bovine IL-6 and the ovine IL-6 (95%).6 There may be a partial species barrier between ovine IL-6 and murine cells which is not present between human IL-6 and murine cells.8 In that report, the recombinant ovine IL-6 expressed by a yeast system did not cause extensive proliferation of B9 murine myeloma cells, and human IL-6 did not stimulate immunoglobulin synthesis by ovine peripheral blood
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mononuclear cells. In the present study, rBoIL-6, in addition to human and swine IL-6, was shown to induce acute phase protein synthesis. Human IL-6 could induce acute phase protein in cattle by continuous infusion,4 and stimulate the secretion of serum amyloid-A and haptoglobin of acute phase protein in bovine hepatocytes in vitro.13 A similar species barrier has been noted between human and murine IL-6. Human IL-6 binds to the murine IL-6 receptor to stimulate murine IL-6-dependent cell lines, while murine IL-6 is unable to induce acute phase protein in human hepatoma (Hep3B) cells.14 In this study, we also observed that recombinant murine IL-6 expressed in E. coli failed to induce acute phase protein in bovine hepatocytes (data not shown). It has been proposed that IL-6 folds into a bundle of four -helices (A, B, C and D).15 Four cysteine residues forming disulfide bridges at positions 72–78 and 101–111 are conserved in the IL-6 of cow as well as in other species.16 IL-6 requires an -helical structure and a positive charge at the C-terminus for its bioactivity.17,18 Two sets of leucine residues spaced at 7-residue intervals (Leu 168, 175, 182 and Leu 152, 159, 166 in the human sequence) play an important role in the receptor binding and bioactivity of IL-6.19 Compared with human IL-6, bovine IL-6 has some deleted amino acids in the mature sequence.20 Only Leu 168 and Leu 175 in the human sequence have equivalents in the bovine sequence, but Leu 182 is not present in most other IL-6 sequences. However, the substitution of Leu 168 with several other residues has no effect on bioactivity. A set of leucine residues in the human sequence (Leu 152, 159, 166) has equivalents in the rodent sequences. These absences of leucines may be important in the observed species barrier. The IL-6 receptor is composed of two polypeptides, the chain (IL-6r/gp80), an 80 kDa transmembrane glycoprotein that binds IL-6 with low affinity, and the chain (gp130), a 130 kDa transmembrane glycoprotein that binds to the IL-6-IL-6r heterodimer to form a high affinity signal transducing complex.21 Critical residues in IL-6 for specific binding to IL-6r or gp130 have been identified by site-directed mutagenesis.22 Human IL-6 binds to both human and rodent IL-6r, whereas rodent IL-6 interacts only with rodent cells.23 The common signal-transducing subunit gp130 does not discriminate between different IL-6 or IL-6r species.23,24 One region of human IL-6 (residues Gly77–Glu95) is important for the interaction of IL-6 with the IL-6r, and another region (residues Lys41– Ala56) is essential for the activation of gp130.25,26 The midregion of IL-6 that recognizes IL-6r may be involved in the species barrier between bovine and murine IL-6s.
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MATERIALS AND METHODS Construction of the bovine IL-6 cDNA recombinant transfer vector Bovine Kupffer cells were prepared by the centrifugal elutriation method as previously described,27 and were stimulated with 1 g/ml of lipopolysaccharide (LPS; O111:B4, Sigma, St Louis, MO, USA) for 3 h in RPMI1640) medium supplemented with 2% fetal calf serum. The messenger RNA was extracted from the stimulated Kupffer cells with an mRNA purification kit (Pharmacia, Uppsala, Sweden). A first strand cDNA was synthesized from 50 ng of the mRNA with an RNA PCR kit (Takara Shuzo, Kyoto, Japan). The entire coding region of bovine IL-6 cDNA was amplified by PCR with primers that were designed from the cDNA sequence reported by Droogmans et al.6 Then, EcoRI and BamHI restriction enzyme sites were added to 5 and 3 ends of the PCR products. The sequences of the forward and reverse primers were 5 -TCCGGAATTCGAACAGCTAT GAACTCCCGCTT-3 , and 5 -TGTACCTAGGATGCCCA GGAACTACCACCATC-3 , respectively. The PCR product was purified with a PCR Purification kit (Qiagen, Hilden, Germany), and ligated into the EcoRI- BamHI site of the baculovirus transfer vector pVL1392 (Pharmingen, San Diego, CA, USA), according to the protocol of the ligation kit (Pharmacia; Ready-To-Go T4 DNA Ligase). The recombinant transfer vector (pVLBoIL-6) containing bovine IL-6 cDNA was digested with EcoRI and BamHI enzymes and analysed by gel electrophoresis to certify the size and integrity of the inserted genes. Furthermore, the DNA sequence of the inserted portion was analysed by using an Autocyle Sequencing kit (Pharmacia).
Expression of Bovine IL-6 Purified DNA of the transfer vector pVLBoIL-6 and Baculogold Linearized Baculovirus DNA (Pharmingen) were mixed with Lipofectin (Gibco-BRL, Gaithersburg, MD, USA), then added to the Spodoptera frugiperda cell line Sf21AE.28 After 4 days incubation at 27C, the supernatant fluid containing recombinant baculoviruses was subjected to plaque purification. The recombinant baculovirus AcBoIL-6 that was obtained from pVLBoIL-6 and that contained bovine IL-6 cDNA was used for production of recombinant protein. Sf21AE cells were infected with AcBoIL-6 and cultured with serum-free medium Sf900II (Gibco-BRL) at 27C.
Biochemical characterization of recombinant bovine IL-6 The culture medium of Sf21AE cells infected with recombinant virus AcBoIL-6 was harvested after 3 days incubation, and centrifuged at 600g at 4C for 10 min. The culture supernatant was further centrifuged at 40 000g for 1 h, and then purified by a concentrator (300 kDa cut-off; Gottingen, Germany) to remove the virus and concentrated by ultrafiltration (10 kDa cut-off; Sartorius). The resultant culture supernatant was mixed with sample buffer (50 mM Tris-HCl pH 6.8, 5% 2-mercaptoethanol, 20% sodium dodecyl sulfate (SDS), 0.1% bromophenol blue and 10% glyerol), boiled for 3 min, and subjected to an SDS-PAGE
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analysis followed by staining with Coomassie brilliant blue R-250. Proteins separated by SDS-PAGE were also electrotransferred to nitrocellulose membranes by the standard method. Transferred membranes were blocked with a blocking reagent (Block Ace; Dainippon Seiyaku, Osaka, Japan) and were incubated with rabbit anti-recombinant ovine IL-6 antiserum (Serotech, Kidlington, UK). An alkaline phosphatase-conjugated goat antibody to rabbit IgG (Sigma) was used as a secondary antibody and then alkaline phosphatase activity was visualized by developing with a 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium chloride (BCIP/NBT) kit (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD, USA).
Cell proliferation assay Biological activity of recombinant bovine IL-6 was assayed by using the IL-6 dependent murine hybridoma 7TD1 cells as described by Van Snick et al.7 with modifications. In brief, 7TD1 cells were incubated at a density of 5104 cells per well in 96-well microplates with the test sample in RPMI1640 with 10% fetal calf serum, 100 U/ml penicillin, 100 g/ml streptomycin, in a total volume of 200 l for 3 days at 37C. The proliferation of 7TD1 cells was evaluated by using the MTT assay. Recombinant HuIL-6 developed in E. coli and SwIL-6 expressed by the baculovirus system were included in this assay as positive controls.
Primary culture of bovine hepatocytes Five male Holstein calves (up to 1 week old) were studied, and details of culturing have been described.29 Briefly, 2 ml of cell suspension (5105 cells/ml) in William’s E medium containing 5% calf serum, 10 M insulin, 5 IU/ml aprotinin, and antibiotics (100 U/ml penicillin, 100 g/ml streptomycin) were seeded on 35-mm plastic dishes (Sumitomo Bakelite, Tokyo, Japan), and the cells were incubated at 37C for 4 h in a humidified atmosphere of 5% CO2 in air. The medium was replaced, and monolayer cultures were further maintained for 24 h. The cells were washed 3 times with fresh medium and cultured for an additional 24 h in serum-free medium. Then the medium was exchanged with fresh serum-free medium and the test samples as stated in the figure legends were added. The supernatants were harvested after 24 h incubation, stored at 20C after centrifugation at 700 g10 min for bioassays.
ELISA assay of albumin The bovine albumin content of the culture medium was determined using an ELISA assay by the method of Yamanaka et al.30 Bovine serum albumin (Seikagaku Co., Tokyo, Japan) or diluted culture medium were used to coat a flat-bottom 96-well microplate, and were reacted with a purified rabbit anti-bovine albumin IgG antibody as the first antibody, and then alkaline phosphatase-conjugated antirabbit IgG goat serum (Sigma) as the second antibody for colour development.
Immunoblot analysis of haptoglobin The haptoglobin in the culture medium was identified by immunoblot analysis. Proteins separated by SDS-PAGE (5% stacking gel and 12.5% separation gel) were transferred to a
nitrocellulose membrane at 0.8 mA per 1 cm2 for 1 h. The transferred haptoglobin was reacted with purified anti-bovine haptoglobin rabbit IgG, for 30 min at room temperature. After washing with 10 mM sodium phosphate buffer (pH 7.4) containing 0.14 M NaCl and 0.05% tween 80, the membrane was treated with alkaline phosphatase substrate system.
Acknowledgements We thank Dr S. Shimizu and Dr N. Yamanaka of our institute for their valuable comments, and Dr Y. Ando and Mr T. Fujisawa of our institute for the preparation of the photographs. We also thank Dr A. Watanabe from the Hokkaido branch of our institute, for his generous gift of the antibody against bovine haptoglobin. This work was supported by RCP project grant no. RCP-4310 from the Ministry of Agriculture, Forestry and Fisheries of Japan.
REFERENCES 1. Van Snick J (1990) Interleukin-6: An overview. Annu Rev Immunol 8:253–278. 2. Geiger T, Andus T, Klapproth J, Hirano T, Kishimoto T, Heinrich PC (1988) Induction of rat acute-phase proteins by interleukin-6 in vivo. Eur J Immunol 18:717–721. 3. Heinrich PC, Castell JV, Andus T (1990) Interleukin-6 and acute phase response. Biochem J 265:621–636. 4. Nakajima Y, Momotani E, Murakami T, Ishikawa Y, Morimatsu M, Saito M, Suzuki H, Yasukawa K (1993) Induction of acute phase protein by recombinant human interleukin-6 (IL-6) in calves. Vet Immunol Immunopathol 35:385–391. 5. Lotz M (1993) Interleukin-6. Cancer Invest 11:732–742. 6. Droogmans L, Cludts I, Cleuter Y, Kettmann R, Burny A (1992) Nucleotide sequence of bovine interleukin-6 cDNA. DNA Sequence 2:411–414. 7. Van Snick J, Cayphas S, Vink A, Uyttenhove C, Coulie PG, Rubria MR, Simpson RJ (1986) Purification and NH2-terminal amino acid sequence of a T-cell-derived lymphokine with growth factor activity for B-cell hybridomas. Proc Natl Acad Sci USA 83: 9679–9683. 8. Ebrahimi B, Roy DJ, Bird P, Sargan DR (1995) Cloning, sequencing and expression of the ovine interleukin 6 gene. Cytokine 7:232–236. 9. Andrews AE, Barcham GY, Ashman K, Meeusen ENT, Brandon MR, Nash AD (1993) Molecular cloning and characterization of ruminant interleukin-6 cDNA. Immunol Cell Biol 71: 341–348. 10. Jian Z, Yang Z, Miller MS, Carter CD, Slauson DO, Bochsler PN (1995) Interleukin-6 secretion by bacterial lipopolysaccharide-stimulated bovine alveolar macrophages in vitro. Vet Immunol Immunopathol 49:51–60. 11. Nakamura K, Mitarai Y, Yoshioka M, Koizumi N, Shibahara T, Nakajima Y (1998) Serum levels of interleukin-6, alpha 1-acid protein, and corticosterone in the two-week-old chickens inoculated with Escherichia coli lipopolysaccharide. Poultry Science 77:908–911. 12. Takakura H, Mori Y, Tatsumi M (1997) Molecular cloning of caprine IL-6 cDNA and its expression in insect cells. Int Arch Allergy Immunol 113:409–416. 13. Alsemgeest SP, Van T Klooster GA, Van Miert AS, Hulskamp Koch CK, Gruys E (1996) Primary bovine hepatocytes in the study of cytokine induced acute-phase protein secretion in vitro. Vet Immunol Immunopathol 53:179–184.
868 / Yoshioka et al. 14. Fiorillo MT, Cabibbo A, Iacopetti P, Fattori E, Ciliberto G (1992) Analysis of human/mouse interleukin-6 hybrid proteins: both amino and carboxy termini of human interleukin-6 are required for in vitro receptor binding. Eur J Immunol 22:2609–2615. 15. Bazan JF (1990) Hemopoietic receptors and helical cytokines. Immunol Today 11:350–354. 16. Simpson RJ, Moritz RJ, Van Roost E, Van Snick J (1988) Characterization of a recombinant murine interleukin-6: Assignment of disulfide bonds. Biochem Biophys Res Commun 88:364–372. 17. Lutticken C, Kruttgen A, Moller C, Heinrich PC, RoseJohn S (1992) Evidence for the importance of a positive charge and a -helical structure of the C-terminus for biological activity of human IL-6. FEBS Lett 282:265–267. 18. Leebeek FW, Kariya K, Schwabe M, Fowlkes DM (1992) Identification of a receptor binding site in the carboxyl terminus human interleukin-6. J Biol Chem 267:14 832–14 838. 19. Nishimura C, Ekida T, Nomura K, Sakamoto K, Suzuki H, Yasukawa K, Kishimoto T, Arata Y (1992) Role of leucine residues in the C-terminal region of human interleukin-6 in the biological activity. FEBS Lett 311:271–275. 20. Savino R, Lahm M, Giorgio M, Cabibbo A, Tramontano A, Ciliberto G (1993) Saturation mutagenesis of the human interleukin 6 receptor-binding site: Implication for its three-dimensional structure. Proc Natl Acad Sci USA 90:4067–4071. 21. Taga T, Hibi M, Hirata Y, Yamasaki K, Yasukawa K, Matsuda T, Hirano T, Kishimoto T (1989) Interleukin-6 triggers the association of its receptor with a possible signal transducer, gp 130. Cell 58:573–581. 22. Xu GY, Yu HA, Hong J, Stahl M, McDonagh T, Kay LE, Cumming DA (1997) Solution structure of recombinant human interleukin-6. J Mol Biol 268:468–481.
CYTOKINE, Vol 11, No. 11 (November, 1999: 863–868) 23. Sugita T, Totsuka T, Saito M, Yamasaki K, Taga T, Hirano T, Kishimoto T (1990) Functional murine interleukin 6 receptor with the intracisternal A particle gene product at its cytoplasmic domain: Its possible role in plasmacytomagenesis. J Exp Med 171:2001–2009. 24. Hibi M, Murakami M, Saito M, Hirano T, Taga T, Kishimoto T (1990) Molecular cloning and expression of an IL-6 Signal Transducer, gp 130. Cell 63:1149–1157. 25. Van Dam M, Mullberg J, Schwabe M, Fowlkes DM (1993) Structure function analysis of interleukin-6 utilizing human/murine chimeric molecules. J Biol Chem 267:15 285–15 290. 26. Ehlers M, Grotzinger J, DeHon FD, Mullberg J, Brakenhoff JP, Liu J, Wollmer A, Rose-John S (1994) Identification of two novel regions of human IL-6 responsible for receptor binding and signal transduction. J Immunol 153:1744–1753. 27. Yoshioka M, Nakajima Y, Ito T, Mikami O, Tanaka S, Miyazaki S, Motoi Y (1997) Primary culture and expression of cytokine mRNAs by lipopolysaccharide in bovine Kupffer cells. Vet Immunol Immunopathol 58:155–163. 28. Felgner PL, Gadek TR, Holm M, Roma R, Chan HW, Wenz M, Northrop JP, Ringold GM, Danielsen M (1987 Lipofection: A highly efficient. lipid-mediated DNA-transfection procedure) Proc Natl Acad Sci USA 84:7413–7417. 29. Higuchi H, Katoh N, Miyamoto T, Yuasa A, Takahashi K (1994) Dexamethasone-induced haptoglobin release by calf liver parenchymal cells. Am J Vet Res 55:1080–1085. 30. Yamanaka N, Kitani H, Mikami O, Nakajima Y, Miura K (1997) Serum-free culture of adult chicken hepatocytes; morphological and biochemical characterization. Res Vet Sci 62:233–237.
BIOLOGICAL FUNCTIONS OF RECOMBINANT BOVINE INTERLEUKIN 6 EXPRESSED IN A BACULOVIRUS SYSTEM M. Yoshioka, Y. Mori, S. Miyazaki, T. Miyamoto, Y. Yokomizo, Y. Nakajima The cDNA encoding bovine interleukin 6 (IL-6) was obtained from messenger RNA extracted from lipopolysaccharide-stimulated bovine Kupffer cells by the reverse transcription polymerase chain reaction (RT/PCR), and cloned into the baculovirus vector pVL 1392. Insect cells (Sf21AE derived from Spodoptera frugiperda) infected with the recombinant baculovirus secreted a large amount of 23.7 kD protein into the culture medium. This protein was capable of causing increased haptoglobin production and decreased albumin production in primary cultured bovine hepatocytes. The swine and human IL-6s were also able to decrease albumin production in bovine hepatocytes. This recombinant IL-6 did not stimulate the proliferation of 7TD1 cells (a murine IL-6-dependent cell line), whereas the recombinant swine IL-6 which was expressed in the same baculovirus system, and recombinant human IL-6 derived from Escherichia coli were each capable of stimulating proliferation of 7TD1 cells, respectively. This suggests a species restriction between bovine IL-6 and murine IL-6 dependent cell lines. 1999 Academic Press
Interleukin 6 (IL-6) is a multifunctional cytokine with roles in the regulation of the immune response and the host defence reaction, and it is produced by a variety of cell types.1 The biological activities of IL-6 include differentiation of B cells, activation of T cells, growth promotion of hybridomas/plasmacytomas, proliferation of haematopoietic stem cells and stimulation of the acute phase response by hepatocytes.2–4 Over-expression of IL-6 is known to be an important feature of the pathogenesis of a number of inflammatory diseases, and may also be a good marker for the diagnosis of systemic inflammatory response syndrome (SIRS) and hypercytokinaemia. Human IL-6 consists of a single polypeptide chain of 184 amino acids. It is known that the IL-6 produced by mammalian cells is N- and O-glycosylated.5 The structural sequences of the IL-6 gene are less well conserved among mammalian species. At the protein level, bovine IL-6 shows 65, 53 and 42% homology to the porcine, human and murine IL-6s, respectively.6 From the National Institute of Animal Health, Tsukuba, Japan Correspondence to: M. Yoshioka, National Institute of Animal Health, 3-1-1 Kannondai, Tsukuba, Ibaraki 305-0856, Japan; E-mail: [email protected]ffrc.go.jp Received 11 August 1998; accepted for publication 23 December 1998 1999 Academic Press 1043–4666/99/110863+06 $30.00/0 KEY WORDS: albumin/bovine interleukin 6/haptoglobin/ recombinant protein CYTOKINE, Vol. 11, No. 11 (November), 1999: pp 863–868
Human IL-6 is capable of stimulating the proliferation of murine IL-6-dependent hybridoma cell lines,7 although IL-6 genes are also less conserved between human and mouse (42% at the amino acid level). However, a bioassay using a murine IL-6-dependent cell line is not adequate for measurement of the bovine IL-6 level.8 A sensitive and accurate method for detecting the level of IL-6 is needed for the diagnosis of many inflammatory diseases in cattle, as well as basic studies of IL-6 functions. We have cloned and expressed the amino acids-coding region of bovine IL-6 in a baculovirus system, as an initial step to establish a sensitive assay for IL-6 in cattle.
RESULTS Expression of bovine IL-6 in the baculovirus system The single-stranded cDNA encoding bovine IL-6 was amplified using PCR. The PCR products were cloned into pVL 1392 and sequenced. The derived sequence contained an open reading frame of 624 bp, encoding 208 amino acids with a predicted molecular weight of 23 758 Da. The nucleotide sequence was entirely identical to the bovine IL-6 reported by Droogmans et al.6 The resultant transfer vector pVLBoIL-6 was co-transfected with a baculovirus 863
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human IL-6 rabbit serum (data not shown). The molecular size of the secreted protein was about 23.7 kDa.
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In order to examine the activity of recombinant bovine IL-6 (rBoIL-6) on bovine cells, the induction of acute phase proteins by IL-6 was measured in primary cultured bovine hepatocytes. As shown in Figure 2, the supernatant of the cell culture that had been infected with AcBoIL-6 showed reduced albumin production (measured in an ELISA assay) in a dose-dependent manner. The same bovine hepatocytes were much less responsive to control supernatant which was derived from AcNPV wild-type virus-infected cells. Recombinant human IL-6 (rHuIL-6) expressed in E. coli, dose-dependently reduced albumin production by bovine hepatocytes, but the degree of reduction at rHuIL-6 concentrations of 1000 ng/ml was less than that obtained with supernatant containing rBoIL-6. Recombinant swine IL-6 (rSwIL-6) expressed in the baculovirus system, produced in the same baculovirus system, also reduced albumin production by bovine hepatocytes. In contrast to the decrease in albumin production, rBoIL-6 induced haptoglobin production by bovine hepatocytes (Fig. 3). Although primary cultured bovine hepatocytes secreted a small amount of haptoglobin in the non-stimulated state, rBoIL-6 increased
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Figure 1.
Expression of rBoIL-6 protein by recombinant baculovirus.
Proteins were analysed by electrophoresis on SDS-12.5% PAGE, followed by staining with Coomassie brilliant blue R-250. Lane M: marker proteins. Lane 1: supernatant of pVLBoIL-6-infected cells 3 days after infection. Lane 2: supernatant of pVLBoIL-6-infected cells 4 days after infection. Lane 3: 10-fold concentrated supernatant of non-infected cells 4 days after infection. Lane 4: supernatant of wild-type AcNPV-infected cells 4 days after infection. Lane 5: Western blot of the same samples as lane 1 with a rabbit anti ovine IL-6 antiserum.
(AcNPV) on Sf21AE cells. After cloning, the recombinant baculovirus containing bovine IL-6 (AcBoIL-6) was obtained. The secreted proteins reactive with antiovine IL-6 antiserum in the concentrated supernatant were detected by a Western blot analysis (Fig. 1). In addition, the secreted protein cross-reacted with anti-
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Figure 2.
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Albumin production by bovine hepatocytes in response to IL-6.
Albumin concentration of the culture supernatant of hepatocytes stimulated with IL-6 samples was determined with an ELISA. Samples were (i) serially diluted culture supernatant of AcBoIL-6 (rBoIL-6), (ii) AcSwIL-6 (rSwIL-6), (iii) wild type AcNPV infected cells (wild type), and (iv) rHuIL-6 expressed in E. coli (1000, 100, 10, 1, 0 ng/ml). Means and standard deviations are shown.
Biological functions of bovine IL-6 / 865 M
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Figure 3. Immunoblot analysis of haptoglobin secretion by bovine hepatocytes in response to rBoIL-6. The haptoglobin in the culture supernatant from hepatocytes stimulated with IL-6 was detected by immunoblotting. 10 l culture medium concentrated to a quarter of the original volume was separated by SDS-PAGE, blotted and reacted with anti-bovine haptoglobin. Lane M: marker proteins. Lane 1: rBoIL-6. Lane 2: 10-fold dilution of rBoIL-6. Lane 3: 100-dilution of rBoIL-6. Lane 4: Supernatant of wild-type AcNPV-infected cells. Lane 5: Supernatant of non-infected cells. Lane 6: medium control. Lane 7: positive control (500-fold dilution acute-phase calf serum).
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Figure 4.
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Proliferative response of 7TD1 cells to recombinant IL-6s.
The culture supernatant of Sf21AE cells infected with AcBoIL-6 was used to stimulate the proliferation of 7TD1 cells for 72 h. The colorimetric assay was done by the MTT method. Recombinant HuIL-6 expressed in E. coli, and rSwIL-6 expressed in baculovirus system, were simultaneously assayed as positive controls. ( ), rBoIL-6; ( ), rSwIL-6; (), rHuIL-6 (10 ng/ml); shaded area indicates negative control range.
haptoglobin secretion dose dependently. The supernatants derived from either non-infected cells or AcNPV wild type virus-infected cells, as a negative control, failed to increase haptoglobin production. Proliferation assays for the baculovirus-expressed rBoIL-6 were done using 7TD1 cells, which offer a highly sensitive proliferation assay for human, porcine and murine IL-6. As shown in Figure 4, the supernatant of Sf21AE cell culture that had been infected with AcBoIL-6, poorly stimulated proliferation of 7TD1 cells. Recombinant HuIL-6 and rSwIL-6 were assayed as positive controls. In the assay shown in Figure 4, activity of rHuIL-6 at 100 pg/ml was con-
verted into 1 standard unit. The supernatant containing rSwIL-6 strongly stimulated proliferation of 7TD1 cells, and its biological activity was calculated at over 1105 U/ml when compared with human IL-6 as a standard. This proliferation induced by rSwIL-6 was inhibited by anti-human IL-6 antiserum (data not shown). A combination of the supernatant containing rBoIL-6 and rHuIL-6 did not affect proliferation activity of rHuIL-6 (data not shown), suggesting that the supernatant containing rBoIL-6 was not cytotoxic to 7TD1 cells. The supernatant of Sf21AE cells infected with AcNPV wild type virus did not proliferate 7TD1 cells.
DISCUSSION In the present study, we succeeded in cloning and expressing bovine recombinant IL-6 in a baculovirus system. The sequence is identical to that reported by Droogmans et al.6 Recombinant BoIL-6 was unable to stimulate the proliferation of 7TD1 cells. However, this protein retained other functional activities of IL-6, in the induction of acute phase proteins (increase of haptoglobin, decrease of albumin) by bovine primary cultured hepatocytes. We tried to assess the bioactivity of bovine IL-6 by proliferation assay using 7TD1 cells, but failed to detect IL-6 in the serum or milk of cows. There are other reports in which this assay was not effective in measuring ruminant IL-6.8 On the other hand, the 7TD1 cell line has been used to test for bovine IL-6 activity.9,10 We have been able to detect chicken or swine IL-6 in serum using this cell line.11 Recently, it has been reported that a recombinant caprine IL-6 expressed in a baculovirus system stimulates the growth of 7TD1 cells.12 However, that report did not confirm that the antibodies to human IL-6 inhibited the proliferation of 7TD1 cells induced by the caprine IL-6. Rabbit antibodies to human IL-6 (Genzyme, Cambridge, MA, USA) inhibited the proliferation of 7TD1 cells induced by human IL-6, but had no effect on bovine serum-induced proliferation of 7TD1 cells (unpublished data). Amino acid sequences of mammalian IL-6s are not extensively conserved. This is especially true for the bovine and murine IL-6 sequences, which show only 42% homology. However, there is a high level of homology between the bovine IL-6 and the ovine IL-6 (95%).6 There may be a partial species barrier between ovine IL-6 and murine cells which is not present between human IL-6 and murine cells.8 In that report, the recombinant ovine IL-6 expressed by a yeast system did not cause extensive proliferation of B9 murine myeloma cells, and human IL-6 did not stimulate immunoglobulin synthesis by ovine peripheral blood
866 / Yoshioka et al.
mononuclear cells. In the present study, rBoIL-6, in addition to human and swine IL-6, was shown to induce acute phase protein synthesis. Human IL-6 could induce acute phase protein in cattle by continuous infusion,4 and stimulate the secretion of serum amyloid-A and haptoglobin of acute phase protein in bovine hepatocytes in vitro.13 A similar species barrier has been noted between human and murine IL-6. Human IL-6 binds to the murine IL-6 receptor to stimulate murine IL-6-dependent cell lines, while murine IL-6 is unable to induce acute phase protein in human hepatoma (Hep3B) cells.14 In this study, we also observed that recombinant murine IL-6 expressed in E. coli failed to induce acute phase protein in bovine hepatocytes (data not shown). It has been proposed that IL-6 folds into a bundle of four -helices (A, B, C and D).15 Four cysteine residues forming disulfide bridges at positions 72–78 and 101–111 are conserved in the IL-6 of cow as well as in other species.16 IL-6 requires an -helical structure and a positive charge at the C-terminus for its bioactivity.17,18 Two sets of leucine residues spaced at 7-residue intervals (Leu 168, 175, 182 and Leu 152, 159, 166 in the human sequence) play an important role in the receptor binding and bioactivity of IL-6.19 Compared with human IL-6, bovine IL-6 has some deleted amino acids in the mature sequence.20 Only Leu 168 and Leu 175 in the human sequence have equivalents in the bovine sequence, but Leu 182 is not present in most other IL-6 sequences. However, the substitution of Leu 168 with several other residues has no effect on bioactivity. A set of leucine residues in the human sequence (Leu 152, 159, 166) has equivalents in the rodent sequences. These absences of leucines may be important in the observed species barrier. The IL-6 receptor is composed of two polypeptides, the chain (IL-6r/gp80), an 80 kDa transmembrane glycoprotein that binds IL-6 with low affinity, and the chain (gp130), a 130 kDa transmembrane glycoprotein that binds to the IL-6-IL-6r heterodimer to form a high affinity signal transducing complex.21 Critical residues in IL-6 for specific binding to IL-6r or gp130 have been identified by site-directed mutagenesis.22 Human IL-6 binds to both human and rodent IL-6r, whereas rodent IL-6 interacts only with rodent cells.23 The common signal-transducing subunit gp130 does not discriminate between different IL-6 or IL-6r species.23,24 One region of human IL-6 (residues Gly77–Glu95) is important for the interaction of IL-6 with the IL-6r, and another region (residues Lys41– Ala56) is essential for the activation of gp130.25,26 The midregion of IL-6 that recognizes IL-6r may be involved in the species barrier between bovine and murine IL-6s.
CYTOKINE, Vol 11, No. 11 (November, 1999: 863–868)
MATERIALS AND METHODS Construction of the bovine IL-6 cDNA recombinant transfer vector Bovine Kupffer cells were prepared by the centrifugal elutriation method as previously described,27 and were stimulated with 1 g/ml of lipopolysaccharide (LPS; O111:B4, Sigma, St Louis, MO, USA) for 3 h in RPMI1640) medium supplemented with 2% fetal calf serum. The messenger RNA was extracted from the stimulated Kupffer cells with an mRNA purification kit (Pharmacia, Uppsala, Sweden). A first strand cDNA was synthesized from 50 ng of the mRNA with an RNA PCR kit (Takara Shuzo, Kyoto, Japan). The entire coding region of bovine IL-6 cDNA was amplified by PCR with primers that were designed from the cDNA sequence reported by Droogmans et al.6 Then, EcoRI and BamHI restriction enzyme sites were added to 5 and 3 ends of the PCR products. The sequences of the forward and reverse primers were 5 -TCCGGAATTCGAACAGCTAT GAACTCCCGCTT-3 , and 5 -TGTACCTAGGATGCCCA GGAACTACCACCATC-3 , respectively. The PCR product was purified with a PCR Purification kit (Qiagen, Hilden, Germany), and ligated into the EcoRI- BamHI site of the baculovirus transfer vector pVL1392 (Pharmingen, San Diego, CA, USA), according to the protocol of the ligation kit (Pharmacia; Ready-To-Go T4 DNA Ligase). The recombinant transfer vector (pVLBoIL-6) containing bovine IL-6 cDNA was digested with EcoRI and BamHI enzymes and analysed by gel electrophoresis to certify the size and integrity of the inserted genes. Furthermore, the DNA sequence of the inserted portion was analysed by using an Autocyle Sequencing kit (Pharmacia).
Expression of Bovine IL-6 Purified DNA of the transfer vector pVLBoIL-6 and Baculogold Linearized Baculovirus DNA (Pharmingen) were mixed with Lipofectin (Gibco-BRL, Gaithersburg, MD, USA), then added to the Spodoptera frugiperda cell line Sf21AE.28 After 4 days incubation at 27C, the supernatant fluid containing recombinant baculoviruses was subjected to plaque purification. The recombinant baculovirus AcBoIL-6 that was obtained from pVLBoIL-6 and that contained bovine IL-6 cDNA was used for production of recombinant protein. Sf21AE cells were infected with AcBoIL-6 and cultured with serum-free medium Sf900II (Gibco-BRL) at 27C.
Biochemical characterization of recombinant bovine IL-6 The culture medium of Sf21AE cells infected with recombinant virus AcBoIL-6 was harvested after 3 days incubation, and centrifuged at 600g at 4C for 10 min. The culture supernatant was further centrifuged at 40 000g for 1 h, and then purified by a concentrator (300 kDa cut-off; Gottingen, Germany) to remove the virus and concentrated by ultrafiltration (10 kDa cut-off; Sartorius). The resultant culture supernatant was mixed with sample buffer (50 mM Tris-HCl pH 6.8, 5% 2-mercaptoethanol, 20% sodium dodecyl sulfate (SDS), 0.1% bromophenol blue and 10% glyerol), boiled for 3 min, and subjected to an SDS-PAGE
Biological functions of bovine IL-6 / 867
analysis followed by staining with Coomassie brilliant blue R-250. Proteins separated by SDS-PAGE were also electrotransferred to nitrocellulose membranes by the standard method. Transferred membranes were blocked with a blocking reagent (Block Ace; Dainippon Seiyaku, Osaka, Japan) and were incubated with rabbit anti-recombinant ovine IL-6 antiserum (Serotech, Kidlington, UK). An alkaline phosphatase-conjugated goat antibody to rabbit IgG (Sigma) was used as a secondary antibody and then alkaline phosphatase activity was visualized by developing with a 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium chloride (BCIP/NBT) kit (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD, USA).
Cell proliferation assay Biological activity of recombinant bovine IL-6 was assayed by using the IL-6 dependent murine hybridoma 7TD1 cells as described by Van Snick et al.7 with modifications. In brief, 7TD1 cells were incubated at a density of 5104 cells per well in 96-well microplates with the test sample in RPMI1640 with 10% fetal calf serum, 100 U/ml penicillin, 100 g/ml streptomycin, in a total volume of 200 l for 3 days at 37C. The proliferation of 7TD1 cells was evaluated by using the MTT assay. Recombinant HuIL-6 developed in E. coli and SwIL-6 expressed by the baculovirus system were included in this assay as positive controls.
Primary culture of bovine hepatocytes Five male Holstein calves (up to 1 week old) were studied, and details of culturing have been described.29 Briefly, 2 ml of cell suspension (5105 cells/ml) in William’s E medium containing 5% calf serum, 10 M insulin, 5 IU/ml aprotinin, and antibiotics (100 U/ml penicillin, 100 g/ml streptomycin) were seeded on 35-mm plastic dishes (Sumitomo Bakelite, Tokyo, Japan), and the cells were incubated at 37C for 4 h in a humidified atmosphere of 5% CO2 in air. The medium was replaced, and monolayer cultures were further maintained for 24 h. The cells were washed 3 times with fresh medium and cultured for an additional 24 h in serum-free medium. Then the medium was exchanged with fresh serum-free medium and the test samples as stated in the figure legends were added. The supernatants were harvested after 24 h incubation, stored at 20C after centrifugation at 700 g10 min for bioassays.
ELISA assay of albumin The bovine albumin content of the culture medium was determined using an ELISA assay by the method of Yamanaka et al.30 Bovine serum albumin (Seikagaku Co., Tokyo, Japan) or diluted culture medium were used to coat a flat-bottom 96-well microplate, and were reacted with a purified rabbit anti-bovine albumin IgG antibody as the first antibody, and then alkaline phosphatase-conjugated antirabbit IgG goat serum (Sigma) as the second antibody for colour development.
Immunoblot analysis of haptoglobin The haptoglobin in the culture medium was identified by immunoblot analysis. Proteins separated by SDS-PAGE (5% stacking gel and 12.5% separation gel) were transferred to a
nitrocellulose membrane at 0.8 mA per 1 cm2 for 1 h. The transferred haptoglobin was reacted with purified anti-bovine haptoglobin rabbit IgG, for 30 min at room temperature. After washing with 10 mM sodium phosphate buffer (pH 7.4) containing 0.14 M NaCl and 0.05% tween 80, the membrane was treated with alkaline phosphatase substrate system.
Acknowledgements We thank Dr S. Shimizu and Dr N. Yamanaka of our institute for their valuable comments, and Dr Y. Ando and Mr T. Fujisawa of our institute for the preparation of the photographs. We also thank Dr A. Watanabe from the Hokkaido branch of our institute, for his generous gift of the antibody against bovine haptoglobin. This work was supported by RCP project grant no. RCP-4310 from the Ministry of Agriculture, Forestry and Fisheries of Japan.
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