Endothelial Cell Protein S Synthesis Is Upregulated by the Complex of IL-6 and Soluble IL-6 Receptor

Summary

We have recently demonstrated that the proinflammatory cytokine, interleukin-6 (IL-6), could upregulate the production of protein S in the human hepatoma cell line, HepG-2, but not in endothelial cells. In this study, we have demonstrated that the combination of exogenous IL-6 and soluble IL-6 receptor (sIL-6R) could significantly upregulate protein S production in both primary human umbilical vein endothelial cells (HUVEC) and in the immortalized human microvascular endothelial cell line, HMEC-1. The IL-6/sIL-6R complex was also able to rapidly induce tyrosine phosphorylation of the IL-6 transducer, gpl30. Neutralizing antibodies directed against either IL-6 or gpl30 blocked protein S upregulation by the IL-6/sIL-6R complex. It was also observed that exogenous sIL-6R could also upregulate protein S by forming a complex with IL-6 constitutively produced by the endothelial cell. Two other cytokines which also utilize the gpl30 receptor, oncostatin M (OSM) and leukemia inhibitory factor (LIF), were also able to upregulate endothelial cell protein S. This study demonstrates a mechanism that allows endothelial cells to respond to IL-6 and also illustrates the potential importance of circulating soluble receptors in the regulation of the anticoagulation pathway.

• References

• 1 Heinrich PC, Castell JV, Andus T. Interleukin-6 and the acute-phase response. Biochem J 1990; 265: 621-636
• 2 Gauldie J, Geisterfer M, Richards C. Baumann H: IL-6 regulation of the hepatic acute-phase response. In: IL-6: Physiopathology and Clinical Potential. Revel M. ed. New York: Raven Press; 1992. 88. pp 153-162
• 3 Waage A, Shalaby R, Espevik T. Tumor necrosis factor, interleukin-1, interleukin-6, interleukin-8 and interferon-gamma in septic shock. In: Cytokines in Health and Disease. Kunkel S, Remick D. (eds). New York: Marcel Dekker; 1992. pp 151-164
• 4 Shalaby MR, Waage A, Espevik T. Cytokine regulation of interleukin 6 production by human endothelial cells. Cell Immunol 1989; 121: 372-382
• 5 Rott O, Tontsch U, Fleischer B, Cash E. Interleukin-6 production in “normal” and HTLV-1 tax expressing brain-specific endothelial cells. Eur J Immunol 1993; 23: 1987-1991
• 6 Almeida GD, Porada CD, Jeors ST, Ascensao JL. Human cytomegalovirus alters interleukin-6 production by endothelial cells. Blood 1994; 83: 370-376
• 7 Taga T, Hibi M, Hirata Y, Yamasaki K, Yasukawa K, Matsuda T, Hirano T, Kishimoto T. Interleukin-6 triggers the association of its receptor with a possible signal transducer, gpl30. Cell 1989; 58: 573-581
• 8 Hibi M, Murakami M, Saito M, Hirano T, Taga T, Kishimoto T. Molecular cloning and expression of an IL-6 signal transducer, gpl30. Cell 1990; 63: 1149-1157
• 9 Kishimoto T, Akira S, Taga T. Interleukin-6 and its receptor: A paradigm for cytokines. Science 1992; 258: 593-597
• 10 Stahl N, Yancopoulos GD. The alphas, betas and kinases of cytokine receptor complexes. Cell 1993; 74: 587-590
• 11 Gearing DP, Comeau MR, Friend DJ, Gimbel SD, Thut CJ, McGourty J, Brasher KK, King JA, Gillis S, Mosley B, Ziegler SF, Cosman D. The IL-6 signal transducer gpl30. An oncostatin M receptor and affinity converter for the LIF receptor. Science 1992; 255: 1434-1437
• 12 Zhang X-G, Gu J-J, Lu Z-Y, Yasukawa K, Yancopoulos GD, Turner K, Shoyab M, Taga T, Kishimoto T, Bataille R, Klein B. Ciliary neurotropic factor, interleukin 11, leukemia inhibitory factor, and oncostatin M are growth factors for human myeloma cell lines using the interleukin 6 signal transducer gpl30. J Exp Med 1994; 177: 1337-1342
• 13 Hira Y, Taga T, Hibi M, Nakano N, Hirano T, Kishimoto T. Characterization of IL-6 receptor expression by monoclonal and polyclonal antibodies. J Immunol 1989; 143: 2900-2905
• 14 Taga T, Kishimoto T. Role of a two-chain IL-6 receptor system in immune and hematopoietic cell regulation. Crit Rev Immunol 1992; 11: 265-280
• 15 Mullberg J, Schooltink H, Stoyan T, Heinrich PC, Rose-John S. Protein kinase C activity is rate limiting for shedding of the interleukin-6 receptor. Biochem and Biophys Res Comm 1992; 189: 794-800
• 16 Mullberg J, Schooltink H, Stoyan T, Gunther M, Graeve L, Buse G, Mackiewicz A, Heinrich PC, Rose-John S. The soluble interleukin-6 receptor is generated by shedding. Eur J Immunol 1993; 23: 473-480
• 17 Yasukawa K, Saito T, Fukunaga T, Sekimori Y, Koishihara Y, Fukui H, Ohsugi Y, Matsuda T, Yawata H, Hirano T, Taga T, Kishimoto T. Purification and characterization of soluble human IL-6 receptor expressed in CHO cells. J. Biochem 1990; 108: 673-676
• 18 Mackiewicz A, Schooltink H, Heinrich PC, Rose-John S. Complex of soluble human IL-6-receptor/IL-6 up-regulates expression of acute-phase proteins. J Immunol 1992; 149: 2021-2027
• 19 Tamura T, Udagawa N, Takahashi N, Miyaura C, Tanaka S, Yamada Y, Koishihara Y, Ohsugi Y, Kumaki K, Taga T, Kishimoto T, Suda T. Soluble interleukin-6 receptor triggers osteoclast formation by interleukin 6. Proc Nat Acad Sci USA 1993; 90: 11924-11928
• 20 van der Poll T, Biiller HR, ten Cate H, Wortel CH, Bauer KA, van DeventerSJH, Hack CE, Sauerwein HP, Rosenberg RD, ten Cate JW. Activation of coagulation after administration of tumor necrosis factor to normal subjects. New Eng J Med 1990; 322: 1622-1627
• 21 Bevilacqua MP, Prober JS, Majeau GR, Fiers W, Cotran RS, Gimbrone MA. Recombinant tumor necrosis factor induces procoagulant activity in cultured human vascular endothelium: characterization and comparison with the action of interleukin-1. Proc Natl Acad Sci USA 1986; 83: 4533-4537
• 22 Conway EM, Rosenberg RD. Tumor necrosis factor suppresses transcription of the thrombomodulin gene in endothelial cells. Mol Cell Biol 1988; 8: 5588-5592
• 23 Hooper WC, Phillips DJ, Ribeiro MJA, Benson JM, George VG, Ades EW, Evatt BL. Tumor Necrosis Factor-a downregulates protein S secretion in human microvascular and umbilical vein endothelial cells but not in the HepG-2 hepatoma cell line. Blood 1994; 84: 483-489
• 24 Hooper WC, Phillips DJ, Evatt BL. IL-6 upregulates protein S expression in the HepG-2 hepatoma cells. Thromb Haemost 1995; 73: 819-824
• 25 Walker FJ. Regulation of activated protein C by protein S. J Biol Chem 1981; 256: 11128-11131
• 26 Taylor FB, Chang A, Esmon CT, D’Angelo A, Vigano-D’Angelo S, Blick KE. Protein C prevents the coagulopathic and lethal effect of E. coli infusion in the baboon. J Clin Invest 1987; 79: 918-925
• 27 Esmon CT, Taylor FB, Snow RT. Inflammation and coagulation: Linked processes potentially regulated through a common pathway mediated by protein C. Thromb Haemost 1991; 66: 160-165
• 28 Taylor F, Chang FerrellG, Mather T, Catlett R, Blick K, Esmon CT. C4b-binding protein exacerbates the host response to Escherichia coli. Blood 1991; 78: 357-363
• 29 Ades A, Candal FJ, Swerlick RA, George VG, Summers S, Bosse DC, Law-ley TJ. HMEC-1: Establishment of an immortalized human microvascular endothelial cell line. J Invest Dermatol 1992; 99: 683-690
• 30 Phillips DJ, Evatt BL, Hooper WC. Development of an ELISA for quantification of human protein S in cell culture fluids using commercial polyclonal antisera. J Immunoassay 1994; 15: 411-428
• 31 Lai RB, Rudolph D, Buckner C, Pardi D, Hooper WC. Infection with human T-lymphotropic viruses leads to constitutive expression of leukemia inhibitory factor in interleukin-6. Blood 1993; 81: 1827-1832
• 32 Podor TJ, Jirik FR, Loskutoff DJ, Carson DA. Lotz M. Human endothelial cells produce IL-6. Lack of responses to exogenous IL-6. Ann New York Acad Sci 1989; 557: 374-387
• 33 Hashimoto Y, Hirohata S, Kashiwado T, Itoh K, Ishii H. Cytokine regulation of hemostatic property and IL-6 production of human endothelial cells. Inflammation 1992; 16: 613-621
• 34 Rose-John S, Schooltink H, Lenz D, Hipp E, Dufhues G, Schmitz H, Schiel X, Hirano T, Kishimoto T, Heinrich PC. Studies on the structure and regulation of the human hepatic interleukin-6 receptor. Eur J Biochem 1990; 190: 79-83
• 35 Snyers L, DeWit L, Content J. Glucocorticoid up-regulation of high-affin- ity interleukin 6 receptors on human epithelial cells. Proc Nat Acad Sci USA 1990; 87: 2838-2842
• 36 Maruo N, Morita I, Ishizaki Y, Murota S-I. Inhibitory effects of interleukin- 6 on prostaglandin 12 production in cultured bovine vascular endothelial cells. Arch Biochem and Biophys 1992; 292: 600-4
• 37 Calderon TM, Sherman J, Wilkerson H, Hatcher VB, Berman JW. Interleukin-6 modulates c-sis gene expression in cultured human endothelial cells. Cell Immunol 1992; 143: 118-126
• 38 May LT, Torcia G, Cozzolino F, Ray A, Tatter SB, Santhanam U, Sehgal PB. Interleukin-6 gene expression in human endothelial cells: RNA start sites, multiple IL-6 proteins and inhibition of proliferation. Biochem Biophys Res Comm. 1989; 159: 991-998
• 39 Fair DS, Marlar RA, Levin EG. Human endothelial cells synthesize protein. S. Blood 1986; 67: 1168-1171
• 40 Stern D, Brett J, Harris K, Nawroth P. Participation of endothelial cells in the protein C-protein S anticoagulant pathway: The synthesis and release of protein S. J Cell Biol 1986; 102: 1971-1978
• 41 Gaillard J-P, Bataille R, Brailly H, Zuber C, Yasukawa K, Attal M, Maruo N, Taga T, Kishimoto T, Klein B. Increased and highly stable levels of functional soluble interleukin-6 receptor in sera of patients with monoclonal gammopathy. Eur J Immunol 1993; 23: 820-824
• 42 Suzuki H, Yasukowa K, Seito T, Narazaki M, Hasegawa A, Taga T, Kishimoto T. Serum soluble interleukin-6 receptor in MRL/lpr mice is elevated with age and mediates the interleukin-6 signal. Eur J Immunol 1993; 23: 1078-1082