Abstract
Interleukin 1 receptor antagonist (IL-1Ra) levels are elevated in the blood of patients with a variety of infectious, immune, or traumatic conditions. To examine whether IL1Ra is produced by liver cells with characteristics resembling an acute-phase protein, human primary hepatocytes isolated from liver biopsies and HepG2 hepatoma cells were stimulated with IL-1beta, IL-6, and TNFalpha. IL-1Ra was present in the supernatants of both cells, with production significantly enhanced by IL-1beta, and by the combination of IL-1beta and IL-6. The term IL-1Ra refers to two different proteins encoded by the same gene, but generated by alternative splicing of two different first exons. One isoform is secreted (17-kD sIL-1Ra), and the other isoform remains in the cytoplasm (18-kD icIL-1Ra). By Western blot analysis, the supernatants of human hepatoma (HepG2) cells contained only sIL-1Ra, whereas the lysates contained a novel smaller molecular mass isoform of 16 kD. RT-PCR and ribonuclease protection assay with RNA from HepG2 cells showed that only sIL-1Ra mRNA was expressed, and confirmed the inducing effect of IL-1beta and IL-6. Transfection studies were performed using constructs containing the promoters of either sIL-1Ra or icIL-1Ra coupled to the luciferase reporter gene. The sIL-1Ra promoter was active in HepG2 cells stimulated by IL-1beta and/or IL-6, whereas the icIL-1Ra promoter was inactive. Mutation of binding sites for transcription factors NF-kappaB and/or C/EBP within the proximal sIL-1Ra promoter led to significant decreases in response to IL-1beta and IL-6 in comparison to the wild-type promoter. Electromobility gel shift assays confirmed the presence of NF-kappaB and C/EBP binding sites within the sIL-1Ra promoter, and indicated a significant increase in the binding activities of nuclear proteins from HepG2 cells treated with IL-1beta and IL-6. In summary, sIL-1Ra, but not icIL-1Ra, is produced by hepatocytes, and is regulated by proinflammatory cytokines as an acute-phase protein. In addition, NF-kappaB and C/EBP family members are likely to play important roles in the full expression of IL-1Ra by hepatocytes during inflammatory conditions.
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- Akira S., Isshiki H., Sugita T., Tanabe O., Kinoshita S., Nishio Y., Nakajima T., Hirano T., Kishimoto T. A nuclear factor for IL-6 expression (NF-IL6) is a member of a C/EBP family. EMBO J. 1990 Jun;9(6):1897–1906. doi: 10.1002/j.1460-2075.1990.tb08316.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Alam T., An M. R., Papaconstantinou J. Differential expression of three C/EBP isoforms in multiple tissues during the acute phase response. J Biol Chem. 1992 Mar 15;267(8):5021–5024. [PubMed] [Google Scholar]
- Arend W. P., Malyak M., Smith M. F., Jr, Whisenand T. D., Slack J. L., Sims J. E., Giri J. G., Dower S. K. Binding of IL-1 alpha, IL-1 beta, and IL-1 receptor antagonist by soluble IL-1 receptors and levels of soluble IL-1 receptors in synovial fluids. J Immunol. 1994 Nov 15;153(10):4766–4774. [PubMed] [Google Scholar]
- Bargetzi M. J., Lantz M., Smith C. G., Torti F. M., Olsson I., Eisenberg S. P., Starnes H. F., Jr Interleukin-1 beta induces interleukin-1 receptor antagonist and tumor necrosis factor binding protein in humans. Cancer Res. 1993 Sep 1;53(17):4010–4013. [PubMed] [Google Scholar]
- Baumann H., Gauldie J. Regulation of hepatic acute phase plasma protein genes by hepatocyte stimulating factors and other mediators of inflammation. Mol Biol Med. 1990 Apr;7(2):147–159. [PubMed] [Google Scholar]
- Baumann H., Prowse K. R., Marinković S., Won K. A., Jahreis G. P. Stimulation of hepatic acute phase response by cytokines and glucocorticoids. Ann N Y Acad Sci. 1989;557:280-95, discussion 295-6. doi: 10.1111/j.1749-6632.1989.tb24021.x. [DOI] [PubMed] [Google Scholar]
- Baumann H., Schendel P. Interleukin-11 regulates the hepatic expression of the same plasma protein genes as interleukin-6. J Biol Chem. 1991 Oct 25;266(30):20424–20427. [PubMed] [Google Scholar]
- Baumann H., Wong G. G. Hepatocyte-stimulating factor III shares structural and functional identity with leukemia-inhibitory factor. J Immunol. 1989 Aug 15;143(4):1163–1167. [PubMed] [Google Scholar]
- Betts J. C., Cheshire J. K., Akira S., Kishimoto T., Woo P. The role of NF-kappa B and NF-IL6 transactivating factors in the synergistic activation of human serum amyloid A gene expression by interleukin-1 and interleukin-6. J Biol Chem. 1993 Dec 5;268(34):25624–25631. [PubMed] [Google Scholar]
- Brown E. A., Dare H. A., Marsh C. B., Wewers M. D. The combination of endotoxin and dexamethasone induces type II interleukin 1 receptor (IL-1r II) in monocytes: a comparison to interleukin 1 beta (IL-1 beta) and interleukin 1 receptor antagonist (IL-1ra). Cytokine. 1996 Nov;8(11):828–836. doi: 10.1006/cyto.1996.0111. [DOI] [PubMed] [Google Scholar]
- Castell J. V., Gómez-Lechón M. J., David M., Andus T., Geiger T., Trullenque R., Fabra R., Heinrich P. C. Interleukin-6 is the major regulator of acute phase protein synthesis in adult human hepatocytes. FEBS Lett. 1989 Jan 2;242(2):237–239. doi: 10.1016/0014-5793(89)80476-4. [DOI] [PubMed] [Google Scholar]
- Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
- Cominelli F., Nast C. C., Clark B. D., Schindler R., Lierena R., Eysselein V. E., Thompson R. C., Dinarello C. A. Interleukin 1 (IL-1) gene expression, synthesis, and effect of specific IL-1 receptor blockade in rabbit immune complex colitis. J Clin Invest. 1990 Sep;86(3):972–980. doi: 10.1172/JCI114799. [DOI] [PMC free article] [PubMed] [Google Scholar]
- De Benedetti F., Pignatti P., Massa M., Sartirana P., Ravelli A., Martini A. Circulating levels of interleukin 1 beta and of interleukin 1 receptor antagonist in systemic juvenile chronic arthritis. Clin Exp Rheumatol. 1995 Nov-Dec;13(6):779–784. [PubMed] [Google Scholar]
- Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dinarello C. A. Biologic basis for interleukin-1 in disease. Blood. 1996 Mar 15;87(6):2095–2147. [PubMed] [Google Scholar]
- Dripps D. J., Brandhuber B. J., Thompson R. C., Eisenberg S. P. Interleukin-1 (IL-1) receptor antagonist binds to the 80-kDa IL-1 receptor but does not initiate IL-1 signal transduction. J Biol Chem. 1991 Jun 5;266(16):10331–10336. [PubMed] [Google Scholar]
- Fujioka N., Mukaida N., Harada A., Akiyama M., Kasahara T., Kuno K., Ooi A., Mai M., Matsushima K. Preparation of specific antibodies against murine IL-1ra and the establishment of IL-1ra as an endogenous regulator of bacteria-induced fulminant hepatitis in mice. J Leukoc Biol. 1995 Jul;58(1):90–98. doi: 10.1002/jlb.58.1.90. [DOI] [PubMed] [Google Scholar]
- Gabay C., Gay-Croisier F., Roux-Lombard P., Meyer O., Maineti C., Guerne P. A., Vischer T., Dayer J. M. Elevated serum levels of interleukin-1 receptor antagonist in polymyositis/dermatomyositis. A biologic marker of disease activity with a possible role in the lack of acute-phase protein response. Arthritis Rheum. 1994 Dec;37(12):1744–1751. doi: 10.1002/art.1780371206. [DOI] [PubMed] [Google Scholar]
- Gabay C., Genin B., Mentha G., Iynedjian P. B., Roux-Lombard P., Guerne P. A. IL-1 receptor antagonist (IL-1Ra) does not inhibit the production of C-reactive protein or serum amyloid A protein by human primary hepatocytes. Differential regulation in normal and tumour cells. Clin Exp Immunol. 1995 May;100(2):306–313. doi: 10.1111/j.1365-2249.1995.tb03669.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gabay C., Silacci P., Genin B., Mentha G., Le Coultre C., Guerne P. A. Soluble interleukin-6 receptor strongly increases the production of acute-phase protein by hepatoma cells but exerts minimal changes on human primary hepatocytes. Eur J Immunol. 1995 Aug;25(8):2378–2383. doi: 10.1002/eji.1830250838. [DOI] [PubMed] [Google Scholar]
- Ganapathi M. K., Rzewnicki D., Samols D., Jiang S. L., Kushner I. Effect of combinations of cytokines and hormones on synthesis of serum amyloid A and C-reactive protein in Hep 3B cells. J Immunol. 1991 Aug 15;147(4):1261–1265. [PubMed] [Google Scholar]
- Granowitz E. V., Clark B. D., Mancilla J., Dinarello C. A. Interleukin-1 receptor antagonist competitively inhibits the binding of interleukin-1 to the type II interleukin-1 receptor. J Biol Chem. 1991 Aug 5;266(22):14147–14150. [PubMed] [Google Scholar]
- Granowitz E. V., Santos A. A., Poutsiaka D. D., Cannon J. G., Wilmore D. W., Wolff S. M., Dinarello C. A. Production of interleukin-1-receptor antagonist during experimental endotoxaemia. Lancet. 1991 Dec 7;338(8780):1423–1424. doi: 10.1016/0140-6736(91)92725-h. [DOI] [PubMed] [Google Scholar]
- Grzelak I., Olszewski W. L., Zaleska M., Durlik M., Lagiewska B., Muszynski M., Rowinski W. Blood cytokine levels rise even after minor surgical trauma. J Clin Immunol. 1996 May;16(3):159–164. doi: 10.1007/BF01540914. [DOI] [PubMed] [Google Scholar]
- Hannum C. H., Wilcox C. J., Arend W. P., Joslin F. G., Dripps D. J., Heimdal P. L., Armes L. G., Sommer A., Eisenberg S. P., Thompson R. C. Interleukin-1 receptor antagonist activity of a human interleukin-1 inhibitor. Nature. 1990 Jan 25;343(6256):336–340. doi: 10.1038/343336a0. [DOI] [PubMed] [Google Scholar]
- Haskill S., Martin G., Van Le L., Morris J., Peace A., Bigler C. F., Jaffe G. J., Hammerberg C., Sporn S. A., Fong S. cDNA cloning of an intracellular form of the human interleukin 1 receptor antagonist associated with epithelium. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3681–3685. doi: 10.1073/pnas.88.9.3681. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Heinrich P. C., Castell J. V., Andus T. Interleukin-6 and the acute phase response. Biochem J. 1990 Feb 1;265(3):621–636. doi: 10.1042/bj2650621. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Janson R. W., King T. E., Jr, Hance K. R., Arend W. P. Enhanced production of IL-1 receptor antagonist by alveolar macrophages from patients with interstitial lung disease. Am Rev Respir Dis. 1993 Aug;148(2):495–503. doi: 10.1164/ajrccm/148.2.495. [DOI] [PubMed] [Google Scholar]
- Jenkins J. K., Arend W. P. Interleukin 1 receptor antagonist production in human monocytes is induced by IL-1 alpha, IL-3, IL-4 and GM-CSF. Cytokine. 1993 Sep;5(5):407–415. doi: 10.1016/1043-4666(93)90030-9. [DOI] [PubMed] [Google Scholar]
- Jenkins J. K., Drong R. F., Shuck M. E., Bienkowski M. J., Slightom J. L., Arend W. P., Smith M. F., Jr Intracellular IL-1 receptor antagonist promoter: cell type-specific and inducible regulatory regions. J Immunol. 1997 Jan 15;158(2):748–755. [PubMed] [Google Scholar]
- Kilpatrick L., McCawley L., Nachiappan V., Greer W., Majumdar S., Korchak H. M., Douglas S. D. Alpha-1-antichymotrypsin inhibits the NADPH oxidase-enzyme complex in phorbol ester-stimulated neutrophil membranes. J Immunol. 1992 Nov 1;149(9):3059–3065. [PubMed] [Google Scholar]
- Kinoshita S., Akira S., Kishimoto T. A member of the C/EBP family, NF-IL6 beta, forms a heterodimer and transcriptionally synergizes with NF-IL6. Proc Natl Acad Sci U S A. 1992 Feb 15;89(4):1473–1476. doi: 10.1073/pnas.89.4.1473. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kushner I. The phenomenon of the acute phase response. Ann N Y Acad Sci. 1982;389:39–48. doi: 10.1111/j.1749-6632.1982.tb22124.x. [DOI] [PubMed] [Google Scholar]
- Lai C. F., Baumann H. Interleukin-1 beta induces production of granulocyte colony-stimulating factor in human hepatoma cells. Blood. 1996 May 15;87(10):4143–4148. [PubMed] [Google Scholar]
- LeClair K. P., Blanar M. A., Sharp P. A. The p50 subunit of NF-kappa B associates with the NF-IL6 transcription factor. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):8145–8149. doi: 10.1073/pnas.89.17.8145. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Malyak M., Smith M. F., Jr, Abel A. A., Arend W. P. Peripheral blood neutrophil production of interleukin-1 receptor antagonist and interleukin-1 beta. J Clin Immunol. 1994 Jan;14(1):20–30. doi: 10.1007/BF01541172. [DOI] [PubMed] [Google Scholar]
- Matsusaka T., Fujikawa K., Nishio Y., Mukaida N., Matsushima K., Kishimoto T., Akira S. Transcription factors NF-IL6 and NF-kappa B synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):10193–10197. doi: 10.1073/pnas.90.21.10193. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Natsuka S., Akira S., Nishio Y., Hashimoto S., Sugita T., Isshiki H., Kishimoto T. Macrophage differentiation-specific expression of NF-IL6, a transcription factor for interleukin-6. Blood. 1992 Jan 15;79(2):460–466. [PubMed] [Google Scholar]
- Osborn L., Kunkel S., Nabel G. J. Tumor necrosis factor alpha and interleukin 1 stimulate the human immunodeficiency virus enhancer by activation of the nuclear factor kappa B. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2336–2340. doi: 10.1073/pnas.86.7.2336. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Phillips W. G., Feldmann M., Breathnach S. M., Brennan F. M. Modulation of the IL-1 cytokine network in keratinocytes by intracellular IL-1 alpha and IL-1 receptor antagonist. Clin Exp Immunol. 1995 Jul;101(1):177–182. doi: 10.1111/j.1365-2249.1995.tb02295.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Prieur A. M., Kaufmann M. T., Griscelli C., Dayer J. M. Specific interleukin-1 inhibitor in serum and urine of children with systemic juvenile chronic arthritis. Lancet. 1987 Nov 28;2(8570):1240–1242. doi: 10.1016/s0140-6736(87)91854-x. [DOI] [PubMed] [Google Scholar]
- Ramji D. P., Vitelli A., Tronche F., Cortese R., Ciliberto G. The two C/EBP isoforms, IL-6DBP/NF-IL6 and C/EBP delta/NF-IL6 beta, are induced by IL-6 to promote acute phase gene transcription via different mechanisms. Nucleic Acids Res. 1993 Jan 25;21(2):289–294. doi: 10.1093/nar/21.2.289. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Richards C. D., Brown T. J., Shoyab M., Baumann H., Gauldie J. Recombinant oncostatin M stimulates the production of acute phase proteins in HepG2 cells and rat primary hepatocytes in vitro. J Immunol. 1992 Mar 15;148(6):1731–1736. [PubMed] [Google Scholar]
- Schooltink H., Stoyan T., Roeb E., Heinrich P. C., Rose-John S. Ciliary neurotrophic factor induces acute-phase protein expression in hepatocytes. FEBS Lett. 1992 Dec 21;314(3):280–284. doi: 10.1016/0014-5793(92)81489-9. [DOI] [PubMed] [Google Scholar]
- Seglen P. O. Preparation of isolated rat liver cells. Methods Cell Biol. 1976;13:29–83. doi: 10.1016/s0091-679x(08)61797-5. [DOI] [PubMed] [Google Scholar]
- Shirakawa F., Saito K., Bonagura C. A., Galson D. L., Fenton M. J., Webb A. C., Auron P. E. The human prointerleukin 1 beta gene requires DNA sequences both proximal and distal to the transcription start site for tissue-specific induction. Mol Cell Biol. 1993 Mar;13(3):1332–1344. doi: 10.1128/mcb.13.3.1332. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Smith M. F., Jr, Eidlen D., Arend W. P., Gutierrez-Hartmann A. LPS-induced expression of the human IL-1 receptor antagonist gene is controlled by multiple interacting promoter elements. J Immunol. 1994 Oct 15;153(8):3584–3593. [PubMed] [Google Scholar]
- Stein B., Cogswell P. C., Baldwin A. S., Jr Functional and physical associations between NF-kappa B and C/EBP family members: a Rel domain-bZIP interaction. Mol Cell Biol. 1993 Jul;13(7):3964–3974. doi: 10.1128/mcb.13.7.3964. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Steinkasserer A., Estaller C., Weiss E. H., Sim R. B. Human interleukin-1 receptor antagonist is expressed in liver. FEBS Lett. 1992 Sep 21;310(1):60–62. doi: 10.1016/0014-5793(92)81146-d. [DOI] [PubMed] [Google Scholar]
- Suzuki H., Takemura H., Kashiwagi H. Interleukin-1 receptor antagonist in patients with active systemic lupus erythematosus. Enhanced production by monocytes and correlation with disease activity. Arthritis Rheum. 1995 Aug;38(8):1055–1059. doi: 10.1002/art.1780380806. [DOI] [PubMed] [Google Scholar]
- Szalai A. J., Briles D. E., Volanakis J. E. Human C-reactive protein is protective against fatal Streptococcus pneumoniae infection in transgenic mice. J Immunol. 1995 Sep 1;155(5):2557–2563. [PubMed] [Google Scholar]
- Tilg H., Trehu E., Atkins M. B., Dinarello C. A., Mier J. W. Interleukin-6 (IL-6) as an anti-inflammatory cytokine: induction of circulating IL-1 receptor antagonist and soluble tumor necrosis factor receptor p55. Blood. 1994 Jan 1;83(1):113–118. [PubMed] [Google Scholar]
- Vannier E., Miller L. C., Dinarello C. A. Coordinated antiinflammatory effects of interleukin 4: interleukin 4 suppresses interleukin 1 production but up-regulates gene expression and synthesis of interleukin 1 receptor antagonist. Proc Natl Acad Sci U S A. 1992 May 1;89(9):4076–4080. doi: 10.1073/pnas.89.9.4076. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wooley P. H., Whalen J. D., Chapman D. L., Berger A. E., Richard K. A., Aspar D. G., Staite N. D. The effect of an interleukin-1 receptor antagonist protein on type II collagen-induced arthritis and antigen-induced arthritis in mice. Arthritis Rheum. 1993 Sep;36(9):1305–1314. doi: 10.1002/art.1780360915. [DOI] [PubMed] [Google Scholar]