Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1997 Sep 1;100(5):972–985. doi: 10.1172/JCI119648

Role of NFkappaB in the mortality of sepsis.

H Böhrer 1, F Qiu 1, T Zimmermann 1, Y Zhang 1, T Jllmer 1, D Männel 1, B W Böttiger 1, D M Stern 1, R Waldherr 1, H D Saeger 1, R Ziegler 1, A Bierhaus 1, E Martin 1, P P Nawroth 1
PMCID: PMC508272  PMID: 9276714

Abstract

Binding activity for nuclear factor kappa B (NFkappaB) consensus probes was studied in nuclear extracts from peripheral blood mononuclear cells of 15 septic patients (10 surviving and 5 not surviving). Nonsurvivors could be distinguished from survivors by an increase in NFkappaB binding activity during the observation period (P < 0.001). The increase in NFkappaB binding activity was comparable to the APACHE-II score as a predictor of outcome. Intravenous somatic gene transfer with an expression plasmid coding for IkappaBalpha was used to investigate the role of members of the NFkappaB family in a mouse model of endotoxemia. In this model, increased NFkappaB binding activity was present after injection of LPS. Intravenous somatic gene transfer with IkappaBalpha given before LPS attenuated renal NFkappaB binding activity and increased survival. Endothelial cells and monocytes/macrophages were the major target cells for somatic gene transfer, transfected with an average transfection efficiency of 20-35%. Tissue factor, a gene under regulatory control of NFkappaB, was induced by LPS. Somatic gene transfer with a reporter plasmid containing the functional tissue factor promoter demonstrated NFkappaB-dependent stimulation by LPS. Intravenous somatic gene transfer with IkappaBalpha reduced LPS-induced renal tissue factor expression, activation of the plasmatic coagulation system (decrease of thrombin-antithrombin III complexes) and renal fibrin/fibrinogen deposition. Somatic gene transfer with an expression plasmid with tissue factor cDNA in the antisense direction (in contrast to sense or vector alone) also increased survival. Furthermore, antisense tissue factor decreased renal tissue factor expression and the activation of the plasmatic coagulation system.

Full Text

The Full Text of this article is available as a PDF (823.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alam J., Cook J. L. Reporter genes: application to the study of mammalian gene transcription. Anal Biochem. 1990 Aug 1;188(2):245–254. doi: 10.1016/0003-2697(90)90601-5. [DOI] [PubMed] [Google Scholar]
  2. Andrews N. C., Faller D. V. A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res. 1991 May 11;19(9):2499–2499. doi: 10.1093/nar/19.9.2499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ashkenazi A., Marsters S. A., Capon D. J., Chamow S. M., Figari I. S., Pennica D., Goeddel D. V., Palladino M. A., Smith D. H. Protection against endotoxic shock by a tumor necrosis factor receptor immunoadhesin. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10535–10539. doi: 10.1073/pnas.88.23.10535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bevilacqua M. P., Pober J. S., Majeau G. R., Fiers W., Cotran R. S., Gimbrone M. A., Jr Recombinant tumor necrosis factor induces procoagulant activity in cultured human vascular endothelium: characterization and comparison with the actions of interleukin 1. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4533–4537. doi: 10.1073/pnas.83.12.4533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Biemond B. J., Levi M., ten Cate H., Soule H. R., Morris L. D., Foster D. L., Bogowitz C. A., van der Poll T., Büller H. R., ten Cate J. W. Complete inhibition of endotoxin-induced coagulation activation in chimpanzees with a monoclonal Fab fragment against factor VII/VIIa. Thromb Haemost. 1995 Feb;73(2):223–230. [PubMed] [Google Scholar]
  6. Bierhaus A., Hemmer C. J., Mackman N., Kutob R., Ziegler R., Dietrich M., Nawroth P. P. Antiparasitic treatment of patients with P. falciparum malaria reduces the ability of patient serum to induce tissue factor by decreasing NF-kappa B activation. Thromb Haemost. 1995 Jan;73(1):39–48. [PubMed] [Google Scholar]
  7. Bierhaus A., Zhang Y., Deng Y., Mackman N., Quehenberger P., Haase M., Luther T., Müller M., Böhrer H., Greten J. Mechanism of the tumor necrosis factor alpha-mediated induction of endothelial tissue factor. J Biol Chem. 1995 Nov 3;270(44):26419–26432. doi: 10.1074/jbc.270.44.26419. [DOI] [PubMed] [Google Scholar]
  8. Bohmann D., Tjian R. Biochemical analysis of transcriptional activation by Jun: differential activity of c- and v-Jun. Cell. 1989 Nov 17;59(4):709–717. doi: 10.1016/0092-8674(89)90017-2. [DOI] [PubMed] [Google Scholar]
  9. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  10. Carson S. D., Johnson D. R. Consecutive enzyme cascades: complement activation at the cell surface triggers increased tissue factor activity. Blood. 1990 Jul 15;76(2):361–367. [PubMed] [Google Scholar]
  11. Colucci M., Balconi G., Lorenzet R., Pietra A., Locati D., Donati M. B., Semeraro N. Cultured human endothelial cells generate tissue factor in response to endotoxin. J Clin Invest. 1983 Jun;71(6):1893–1896. doi: 10.1172/JCI110945. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Conway E. M., Bach R., Rosenberg R. D., Konigsberg W. H. Tumor necrosis factor enhances expression of tissue factor mRNA in endothelial cells. Thromb Res. 1989 Feb 1;53(3):231–241. doi: 10.1016/0049-3848(89)90098-4. [DOI] [PubMed] [Google Scholar]
  13. Creasey A. A., Chang A. C., Feigen L., Wün T. C., Taylor F. B., Jr, Hinshaw L. B. Tissue factor pathway inhibitor reduces mortality from Escherichia coli septic shock. J Clin Invest. 1993 Jun;91(6):2850–2860. doi: 10.1172/JCI116529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Deryckere F., Gannon F. A one-hour minipreparation technique for extraction of DNA-binding proteins from animal tissues. Biotechniques. 1994 Mar;16(3):405–405. [PubMed] [Google Scholar]
  15. Ding A., Hwang S., Lander H. M., Xie Q. W. Macrophages derived from C3H/HeJ (Lpsd) mice respond to bacterial lipopolysaccharide by activating NF-kappa B. J Leukoc Biol. 1995 Jan;57(1):174–179. doi: 10.1002/jlb.57.1.174. [DOI] [PubMed] [Google Scholar]
  16. Donovan-Peluso M., George L. D., Hassett A. C. Lipopolysaccharide induction of tissue factor expression in THP-1 monocytic cells. Protein-DNA interactions with the promoter. J Biol Chem. 1994 Jan 14;269(2):1361–1369. [PubMed] [Google Scholar]
  17. Edgington T. S., Mackman N., Fan S. T., Ruf W. Cellular immune and cytokine pathways resulting in tissue factor expression and relevance to septic shock. Nouv Rev Fr Hematol. 1992;34 (Suppl):S15–S27. [PubMed] [Google Scholar]
  18. Edwards R. L., Rickles F. R. The role of human T cells (and T cell products) for monocyte tissue factor generation. J Immunol. 1980 Aug;125(2):606–609. [PubMed] [Google Scholar]
  19. Fiedler V. B., Loof I., Sander E., Voehringer V., Galanos C., Fournel M. A. Monoclonal antibody to tumor necrosis factor--alpha prevents lethal endotoxin sepsis in adult rhesus monkeys. J Lab Clin Med. 1992 Oct;120(4):574–588. [PubMed] [Google Scholar]
  20. Galanos C., Freudenberg M. A., Reutter W. Galactosamine-induced sensitization to the lethal effects of endotoxin. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5939–5943. doi: 10.1073/pnas.76.11.5939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Geng Y., Gulbins E., Altman A., Lotz M. Monocyte deactivation by interleukin 10 via inhibition of tyrosine kinase activity and the Ras signaling pathway. Proc Natl Acad Sci U S A. 1994 Aug 30;91(18):8602–8606. doi: 10.1073/pnas.91.18.8602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hartzell S., Ryder K., Lanahan A., Lau L. F., Nathan D. A growth factor-responsive gene of murine BALB/c 3T3 cells encodes a protein homologous to human tissue factor. Mol Cell Biol. 1989 Jun;9(6):2567–2573. doi: 10.1128/mcb.9.6.2567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lasch H. G., Heene D. L., Huth K., Sandritter W. Pathophysiology, clinical manifestations and therapy of consumption-coagulopathy ("Verbrauchskoagulopathie"). Am J Cardiol. 1967 Sep;20(3):381–391. doi: 10.1016/0002-9149(67)90062-8. [DOI] [PubMed] [Google Scholar]
  24. Lehmann V., Freudenberg M. A., Galanos C. Lethal toxicity of lipopolysaccharide and tumor necrosis factor in normal and D-galactosamine-treated mice. J Exp Med. 1987 Mar 1;165(3):657–663. doi: 10.1084/jem.165.3.657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Levi M., ten Cate H., Bauer K. A., van der Poll T., Edgington T. S., Büller H. R., van Deventer S. J., Hack C. E., ten Cate J. W., Rosenberg R. D. Inhibition of endotoxin-induced activation of coagulation and fibrinolysis by pentoxifylline or by a monoclonal anti-tissue factor antibody in chimpanzees. J Clin Invest. 1994 Jan;93(1):114–120. doi: 10.1172/JCI116934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lyberg T., Galdal K. S., Evensen S. A., Prydz H. Cellular cooperation in endothelial cell thromboplastin synthesis. Br J Haematol. 1983 Jan;53(1):85–95. doi: 10.1111/j.1365-2141.1983.tb01989.x. [DOI] [PubMed] [Google Scholar]
  27. Mackman N., Fowler B. J., Edgington T. S., Morrissey J. H. Functional analysis of the human tissue factor promoter and induction by serum. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2254–2258. doi: 10.1073/pnas.87.6.2254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mackman N. Protease inhibitors block lipopolysaccharide induction of tissue factor gene expression in human monocytic cells by preventing activation of c-Rel/p65 heterodimers. J Biol Chem. 1994 Oct 21;269(42):26363–26367. [PubMed] [Google Scholar]
  29. Mackman N. Regulation of the tissue factor gene. FASEB J. 1995 Jul;9(10):883–889. doi: 10.1096/fasebj.9.10.7615158. [DOI] [PubMed] [Google Scholar]
  30. Mackman N., Sawdey M. S., Keeton M. R., Loskutoff D. J. Murine tissue factor gene expression in vivo. Tissue and cell specificity and regulation by lipopolysaccharide. Am J Pathol. 1993 Jul;143(1):76–84. [PMC free article] [PubMed] [Google Scholar]
  31. Mai U. E., Perez-Perez G. I., Wahl L. M., Wahl S. M., Blaser M. J., Smith P. D. Soluble surface proteins from Helicobacter pylori activate monocytes/macrophages by lipopolysaccharide-independent mechanism. J Clin Invest. 1991 Mar;87(3):894–900. doi: 10.1172/JCI115095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Miethke T., Wahl C., Heeg K., Echtenacher B., Krammer P. H., Wagner H. T cell-mediated lethal shock triggered in mice by the superantigen staphylococcal enterotoxin B: critical role of tumor necrosis factor. J Exp Med. 1992 Jan 1;175(1):91–98. doi: 10.1084/jem.175.1.91. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mohler K. M., Sleath P. R., Fitzner J. N., Cerretti D. P., Alderson M., Kerwar S. S., Torrance D. S., Otten-Evans C., Greenstreet T., Weerawarna K. Protection against a lethal dose of endotoxin by an inhibitor of tumour necrosis factor processing. Nature. 1994 Jul 21;370(6486):218–220. doi: 10.1038/370218a0. [DOI] [PubMed] [Google Scholar]
  34. Mohri M., Spriggs D. R., Kufe D. Effects of lipopolysaccharide on phospholipase A2 activity and tumor necrosis factor expression in HL-60 cells. J Immunol. 1990 Apr 1;144(7):2678–2682. [PubMed] [Google Scholar]
  35. Moll T., Czyz M., Holzmüller H., Hofer-Warbinek R., Wagner E., Winkler H., Bach F. H., Hofer E. Regulation of the tissue factor promoter in endothelial cells. Binding of NF kappa B-, AP-1-, and Sp1-like transcription factors. J Biol Chem. 1995 Feb 24;270(8):3849–3857. doi: 10.1074/jbc.270.8.3849. [DOI] [PubMed] [Google Scholar]
  36. Nawroth P. P., Handley D. A., Esmon C. T., Stern D. M. Interleukin 1 induces endothelial cell procoagulant while suppressing cell-surface anticoagulant activity. Proc Natl Acad Sci U S A. 1986 May;83(10):3460–3464. doi: 10.1073/pnas.83.10.3460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Nawroth P. P., Stern D. M. Modulation of endothelial cell hemostatic properties by tumor necrosis factor. J Exp Med. 1986 Mar 1;163(3):740–745. doi: 10.1084/jem.163.3.740. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Nawroth P., Handley D., Matsueda G., De Waal R., Gerlach H., Blohm D., Stern D. Tumor necrosis factor/cachectin-induced intravascular fibrin formation in meth A fibrosarcomas. J Exp Med. 1988 Aug 1;168(2):637–647. doi: 10.1084/jem.168.2.637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Nolan G. P., Ghosh S., Liou H. C., Tempst P., Baltimore D. DNA binding and I kappa B inhibition of the cloned p65 subunit of NF-kappa B, a rel-related polypeptide. Cell. 1991 Mar 8;64(5):961–969. doi: 10.1016/0092-8674(91)90320-x. [DOI] [PubMed] [Google Scholar]
  40. O'Riordain M. G., O'Riordain D. S., Molloy R. G., Mannick J. A., Rodrick M. L. Dosage and timing of anti-TNF-alpha antibody treatment determine its effect of resistance to sepsis after injury. J Surg Res. 1996 Jul 15;64(1):95–101. doi: 10.1006/jsre.1996.0312. [DOI] [PubMed] [Google Scholar]
  41. Oeth P. A., Parry G. C., Kunsch C., Nantermet P., Rosen C. A., Mackman N. Lipopolysaccharide induction of tissue factor gene expression in monocytic cells is mediated by binding of c-Rel/p65 heterodimers to a kappa B-like site. Mol Cell Biol. 1994 Jun;14(6):3772–3781. doi: 10.1128/mcb.14.6.3772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Pahl H. L., Baeuerle P. A. Expression of influenza virus hemagglutinin activates transcription factor NF-kappa B. J Virol. 1995 Mar;69(3):1480–1484. doi: 10.1128/jvi.69.3.1480-1484.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Parry G. C., Mackman N. A set of inducible genes expressed by activated human monocytic and endothelial cells contain kappa B-like sites that specifically bind c-Rel-p65 heterodimers. J Biol Chem. 1994 Aug 19;269(33):20823–20825. [PubMed] [Google Scholar]
  44. Parry G. C., Mackman N. Transcriptional regulation of tissue factor expression in human endothelial cells. Arterioscler Thromb Vasc Biol. 1995 May;15(5):612–621. doi: 10.1161/01.atv.15.5.612. [DOI] [PubMed] [Google Scholar]
  45. Pfeffer K., Matsuyama T., Kündig T. M., Wakeham A., Kishihara K., Shahinian A., Wiegmann K., Ohashi P. S., Krönke M., Mak T. W. Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to endotoxic shock, yet succumb to L. monocytogenes infection. Cell. 1993 May 7;73(3):457–467. doi: 10.1016/0092-8674(93)90134-c. [DOI] [PubMed] [Google Scholar]
  46. Ray A., Hannink M., Ray B. K. Concerted participation of NF-kappa B and C/EBP heteromer in lipopolysaccharide induction of serum amyloid A gene expression in liver. J Biol Chem. 1995 Mar 31;270(13):7365–7374. doi: 10.1074/jbc.270.13.7365. [DOI] [PubMed] [Google Scholar]
  47. Read M. A., Cordle S. R., Veach R. A., Carlisle C. D., Hawiger J. Cell-free pool of CD14 mediates activation of transcription factor NF-kappa B by lipopolysaccharide in human endothelial cells. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):9887–9891. doi: 10.1073/pnas.90.21.9887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Reinhart K., Wiegand-Löhnert C., Grimminger F., Kaul M., Withington S., Treacher D., Eckart J., Willatts S., Bouza C., Krausch D. Assessment of the safety and efficacy of the monoclonal anti-tumor necrosis factor antibody-fragment, MAK 195F, in patients with sepsis and septic shock: a multicenter, randomized, placebo-controlled, dose-ranging study. Crit Care Med. 1996 May;24(5):733–742. doi: 10.1097/00003246-199605000-00003. [DOI] [PubMed] [Google Scholar]
  49. Ryan J., Brett J., Tijburg P., Bach R. R., Kisiel W., Stern D. Tumor necrosis factor-induced endothelial tissue factor is associated with subendothelial matrix vesicles but is not expressed on the apical surface. Blood. 1992 Aug 15;80(4):966–974. [PubMed] [Google Scholar]
  50. Tannenbaum S. H., Finko R., Cines D. B. Antibody and immune complexes induce tissue factor production by human endothelial cells. J Immunol. 1986 Sep 1;137(5):1532–1537. [PubMed] [Google Scholar]
  51. Taylor F. B., Jr, Chang A., Ruf W., Morrissey J. H., Hinshaw L., Catlett R., Blick K., Edgington T. S. Lethal E. coli septic shock is prevented by blocking tissue factor with monoclonal antibody. Circ Shock. 1991 Mar;33(3):127–134. [PubMed] [Google Scholar]
  52. Tebo J. M., Chaoqun W., Ohmori Y., Hamilton T. A. Murine inhibitory protein-kappa B alpha negatively regulates kappa B-dependent transcription in lipopolysaccharide-stimulated RAW 264.7 macrophages. J Immunol. 1994 Nov 15;153(10):4713–4720. [PubMed] [Google Scholar]
  53. Torti F. M., Dieckmann B., Beutler B., Cerami A., Ringold G. M. A macrophage factor inhibits adipocyte gene expression: an in vitro model of cachexia. Science. 1985 Aug 30;229(4716):867–869. doi: 10.1126/science.3839597. [DOI] [PubMed] [Google Scholar]
  54. Tracey K. J., Beutler B., Lowry S. F., Merryweather J., Wolpe S., Milsark I. W., Hariri R. J., Fahey T. J., 3rd, Zentella A., Albert J. D. Shock and tissue injury induced by recombinant human cachectin. Science. 1986 Oct 24;234(4775):470–474. doi: 10.1126/science.3764421. [DOI] [PubMed] [Google Scholar]
  55. Tracey K. J., Fong Y., Hesse D. G., Manogue K. R., Lee A. T., Kuo G. C., Lowry S. F., Cerami A. Anti-cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteraemia. Nature. 1987 Dec 17;330(6149):662–664. doi: 10.1038/330662a0. [DOI] [PubMed] [Google Scholar]
  56. Tran-Thi T. A., Decker K., Baeuerle P. A. Differential activation of transcription factors NF-kappa B and AP-1 in rat liver macrophages. Hepatology. 1995 Aug;22(2):613–619. doi: 10.1002/hep.1840220235. [DOI] [PubMed] [Google Scholar]
  57. Warr T. A., Rao L. V., Rapaport S. I. Disseminated intravascular coagulation in rabbits induced by administration of endotoxin or tissue factor: effect of anti-tissue factor antibodies and measurement of plasma extrinsic pathway inhibitor activity. Blood. 1990 Apr 1;75(7):1481–1489. [PubMed] [Google Scholar]
  58. Wheeler A. P., Bernard G. R. Applications of molecular biology and biotechnology: antibody therapy of sepsis. J Crit Care. 1996 Jun;11(2):77–94. doi: 10.1016/s0883-9441(96)90023-4. [DOI] [PubMed] [Google Scholar]
  59. Zhang Y. M., Bachmann S., Hemmer C., van Lunzen J., von Stemm A., Kern P., Dietrich M., Ziegler R., Waldherr R., Nawroth P. P. Vascular origin of Kaposi's sarcoma. Expression of leukocyte adhesion molecule-1, thrombomodulin, and tissue factor. Am J Pathol. 1994 Jan;144(1):51–59. [PMC free article] [PubMed] [Google Scholar]
  60. Zhang Y., Deng Y., Wendt T., Liliensiek B., Bierhaus A., Greten J., He W., Chen B., Hach-Wunderle V., Waldherr R. Intravenous somatic gene transfer with antisense tissue factor restores blood flow by reducing tumor necrosis factor-induced tissue factor expression and fibrin deposition in mouse meth-A sarcoma. J Clin Invest. 1996 May 15;97(10):2213–2224. doi: 10.1172/JCI118662. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Zuckerman S. H., Evans G. F. Endotoxin tolerance: in vivo regulation of tumor necrosis factor and interleukin-1 synthesis is at the transcriptional level. Cell Immunol. 1992 Apr;140(2):513–519. doi: 10.1016/0008-8749(92)90216-c. [DOI] [PubMed] [Google Scholar]
  62. Zuckerman S. H., Evans G. F., Guthrie L. Transcriptional and post-transcriptional mechanisms involved in the differential expression of LPS-induced IL-1 and TNF mRNA. Immunology. 1991 Aug;73(4):460–465. [PMC free article] [PubMed] [Google Scholar]
  63. de Wet J. R., Wood K. V., DeLuca M., Helinski D. R., Subramani S. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol. 1987 Feb;7(2):725–737. doi: 10.1128/mcb.7.2.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. van der Poll T., Levi M., van Deventer S. J., ten Cate H., Haagmans B. L., Biemond B. J., Büller H. R., Hack C. E., ten Cate J. W. Differential effects of anti-tumor necrosis factor monoclonal antibodies on systemic inflammatory responses in experimental endotoxemia in chimpanzees. Blood. 1994 Jan 15;83(2):446–451. [PubMed] [Google Scholar]
  65. von der Möhlen M. A., van der Poll T., Jansen J., Levi M., van Deventer S. J. Release of bactericidal/permeability-increasing protein in experimental endotoxemia and clinical sepsis. Role of tumor necrosis factor. J Immunol. 1996 Jun 15;156(12):4969–4973. [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES