Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Toll-like receptor 9–dependent activation by DNA-containing immune complexes is mediated by HMGB1 and RAGE

Nature Immunology volume 8, pages 487–496 (2007)Cite this article

A Corrigendum to this article was published on 01 July 2007

This article has been updated

Abstract

Increased concentrations of DNA-containing immune complexes in the serum are associated with systemic autoimmune diseases such as lupus. Stimulation of Toll-like receptor 9 (TLR9) by DNA is important in the activation of plasmacytoid dendritic cells and B cells. Here we show that HMGB1, a nuclear DNA-binding protein released from necrotic cells, was an essential component of DNA-containing immune complexes that stimulated cytokine production through a TLR9–MyD88 pathway involving the multivalent receptor RAGE. Moreover, binding of HMGB1 to class A CpG oligodeoxynucleotides considerably augmented cytokine production by means of TLR9 and RAGE. Our data demonstrate a mechanism by which HMGB1 and RAGE activate plasmacytoid dendritic cells and B cells in response to DNA and contribute to autoimmune pathogenesis.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: HMGB1 binds to CpG-A DNA and augments cytokine production by pDCs.
Figure 2: HMGB1 B-box augments IFN-α production induced by immunostimulatory CpG-A.
Figure 3: CpG-A but not CpG-B augments the binding of HMGB1 to RAGE.
Figure 4: RAGE-TLR9–dependent IFN-α production induced by HMGB1–CpG-A.
Figure 5: HMGB1 is present in DNA-containing immune complexes: inhibition of binding by RAGE-Fc.
Figure 6: Inhibition of AM-14 B cell proliferation induced by DNA-containing immune complexes by RAGE-Fc and A box antagonist.
Figure 7: The HMGB1-CpG-A complex recruits RAGE, MyD88 and TLR9.
Figure 8: HMGB1 and RAGE mediate the induction of genes encoding type I interferons mediated by DNA-containing immune complexes present in sera from people with lupus.

Similar content being viewed by others

Change history

  • 24 May 2007

    In the version of this article initially published, the legends for Figures 3c and 5b fail to indicate that portions of these are the same experiments as in Figures 3b and 5a, respectively. The correct legends state “Binding with or without CpG-A is from the same experiment as in b” (for Fig. 3c) and “Data for PL2-3 in the presence of supernatant alone (bold lines) as detected by anti-IgG2a (left, a,b) and by anti-HMGB1 (right, a,b) are from the same experiment” (for Fig. 5b). Also, error bars are missing from Figure 3c. The errors have been corrected in the HTML and PDF versions of the article.

References

  1. Mok, C.C. & Lau, C.S. Pathogenesis of systemic lupus erythematosus. J. Clin. Pathol. 56, 481–490 (2003).

    Article  CAS  Google Scholar 

  2. Denny, M.F. et al. Accelerated macrophage apoptosis induces autoantibody formation and organ damage in systemic lupus erythematosus. J. Immunol. 176, 2095–2104 (2006).

    Article  CAS  Google Scholar 

  3. Robak, E., Sysa-Jedrzejowska, A., Robak, T. & Smolewski, P. Peripheral blood lymphocyte apoptosis and circulating dendritic cells in patients with systemic lupus erythematosus: correlation with immunological status and disease-related symptoms. Clin. Rheumatol. 25, 225–233 (2006).

    Article  Google Scholar 

  4. ter Borg, E.J., Horst, G., Hummel, E.J., Limburg, P.C. & Kallenberg, C.G. Measurement of increases in anti-double-stranded DNA antibody levels as a predictor of disease exacerbation in systemic lupus erythematosus. A long-term, prospective study. Arthritis Rheum. 33, 634–643 (1990).

    Article  CAS  Google Scholar 

  5. Magnusson, M., Magnusson, S., Vallin, H., Ronnblom, L. & Alm, G.V. Importance of CpG dinucleotides in activation of natural IFN-α-producing cells by a lupus-related oligodeoxynucleotide. Scand. J. Immunol. 54, 543–550 (2001).

    Article  CAS  Google Scholar 

  6. Sano, H. et al. Binding properties of human anti-DNA antibodies to cloned human DNA fragments. Scand. J. Immunol. 30, 51–63 (1989).

    Article  CAS  Google Scholar 

  7. Bauer, S. et al. Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc. Natl. Acad. Sci. USA 98, 9237–9242 (2001).

    Article  CAS  Google Scholar 

  8. Latz, E. et al. TLR9 signals after translocating from the ER to CpG DNA in the lysosome. Nat. Immunol. 5, 190–198 (2004).

    Article  CAS  Google Scholar 

  9. Krieg, A.M. Therapeutic potential of Toll-like receptor 9 activation. Nat. Rev. Drug Discov. 5, 471–484 (2006).

    Article  CAS  Google Scholar 

  10. Ishii, K.J. & Akira, S. Innate immune recognition of, and regulation by, DNA. Trends Immunol. 27, 525–532 (2006).

    Article  CAS  Google Scholar 

  11. Wu, C.C., Lee, J., Raz, E., Corr, M. & Carson, D.A. Necessity of oligonucleotide aggregation for toll-like receptor 9 activation. J. Biol. Chem. 279, 33071–33078 (2004).

    Article  CAS  Google Scholar 

  12. Barrat, F.J. et al. Nucleic acids of mammalian origin can act as endogenous ligands for Toll-like receptors and may promote systemic lupus erythematosus. J. Exp. Med. 202, 1131–1139 (2005).

    Article  CAS  Google Scholar 

  13. Guiducci, C. et al. Properties regulating the nature of the plasmacytoid dendritic cell response to Toll-like receptor 9 activation. J. Exp. Med. 203, 1999–2008 (2006).

    Article  CAS  Google Scholar 

  14. Barton, G.M., Kagan, J.C. & Medzhitov, R. Intracellular localization of Toll-like receptor 9 prevents recognition of self DNA but facilitates access to viral DNA. Nat. Immunol. 7, 49–56 (2006).

    Article  CAS  Google Scholar 

  15. Ronnblom, L., Eloranta, M.L. & Alm, G.V. Role of natural interferon-α producing cells (plasmacytoid dendritic cells) in autoimmunity. Autoimmunity 36, 463–472 (2003).

    Article  Google Scholar 

  16. Asselin-Paturel, C. & Trinchieri, G. Production of type I interferons: plasmacytoid dendritic cells and beyond. J. Exp. Med. 202, 461–465 (2005).

    Article  CAS  Google Scholar 

  17. Jego, G. et al. Plasmacytoid dendritic cells induce plasma cell differentiation through type I interferon and interleukin 6. Immunity 19, 225–234 (2003).

    Article  CAS  Google Scholar 

  18. Blanco, P., Palucka, A.K., Gill, M., Pascual, V. & Banchereau, J. Induction of dendritic cell differentiation by IFN-α in systemic lupus erythematosus. Science 294, 1540–1543 (2001).

    Article  CAS  Google Scholar 

  19. Crow, M.K. Interferon pathway activation in systemic lupus erythematosus. Curr. Rheumatol. Rep. 7, 463–468 (2005).

    Article  CAS  Google Scholar 

  20. Gottenberg, J.E. et al. Activation of IFN pathways and plasmacytoid dendritic cell recruitment in target organs of primary Sjogren's syndrome. Proc. Natl. Acad. Sci. USA 103, 2770–2775 (2006).

    Article  CAS  Google Scholar 

  21. Bennett, L. et al. Interferon and granulopoiesis signatures in systemic lupus erythematosus blood. J. Exp. Med. 197, 711–723 (2003).

    Article  CAS  Google Scholar 

  22. Stott, K., Tang, G.S., Lee, K.B. & Thomas, J.O. Structure of a complex of tandem HMG boxes and DNA. J. Mol. Biol. 360, 90–104 (2006).

    Article  CAS  Google Scholar 

  23. Scaffidi, P., Misteli, T. & Bianchi, M.E. Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature 418, 191–195 (2002).

    Article  CAS  Google Scholar 

  24. Wang, H. et al. HMG-1 as a late mediator of endotoxin lethality in mice. Science 285, 248–251 (1999).

    Article  CAS  Google Scholar 

  25. Qin, S. et al. Role of HMGB1 in apoptosis-mediated sepsis lethality. J. Exp. Med. 203, 1637–1642 (2006).

    Article  CAS  Google Scholar 

  26. Andersson, U. & Erlandsson-Harris, H. HMGB1 is a potent trigger of arthritis. J. Intern. Med. 255, 344–350 (2004).

    Article  CAS  Google Scholar 

  27. Yang, H. et al. Reversing established sepsis with antagonists of endogenous high-mobility group box 1. Proc. Natl. Acad. Sci. USA 101, 296–301 (2004).

    Article  CAS  Google Scholar 

  28. Kokkola, R. et al. High mobility group box chromosomal protein 1: a novel proinflammatory mediator in synovitis. Arthritis Rheum. 46, 2598–2603 (2002).

    Article  CAS  Google Scholar 

  29. Kokkola, R. et al. RAGE is the major receptor for the proinflammatory activity of HMGB1 in rodent macrophages. Scand. J. Immunol. 61, 1–9 (2005).

    Article  CAS  Google Scholar 

  30. Park, J.S. et al. High mobility group box 1 protein interacts with multiple Toll-like receptors. Am. J. Physiol. Cell Physiol. 290, C917–C924 (2006).

    Article  CAS  Google Scholar 

  31. Hori, O. et al. The receptor for advanced glycation end products (RAGE) is a cellular binding site for amphoterin. Mediation of neurite outgrowth and co-expression of rage and amphoterin in the developing nervous system. J. Biol. Chem. 270, 25752–25761 (1995).

    Article  CAS  Google Scholar 

  32. Dumitriu, I.E., Baruah, P., Bianchi, M.E., Manfredi, A.A. & Rovere-Querini, P. Requirement of HMGB1 and RAGE for the maturation of human plasmacytoid dendritic cells. Eur. J. Immunol. 35, 2184–2190 (2005).

    Article  CAS  Google Scholar 

  33. Dumitriu, I.E. et al. Release of high mobility group box 1 by dendritic cells controls T cell activation via the receptor for advanced glycation end products. J. Immunol. 174, 7506–7515 (2005).

    Article  CAS  Google Scholar 

  34. Popovic, K. et al. Increased expression of the novel proinflammatory cytokine high mobility group box chromosomal protein 1 in skin lesions of patients with lupus erythematosus. Arthritis Rheum. 52, 3639–3645 (2005).

    Article  CAS  Google Scholar 

  35. Lakowicz, J.R. On spectral relaxation in proteins. Photochem. Photobiol. 72, 421–437 (2000).

    Article  CAS  Google Scholar 

  36. Viglianti, G.A. et al. Activation of autoreactive B cells by CpG dsDNA. Immunity 19, 837–847 (2003).

    Article  CAS  Google Scholar 

  37. Marshak-Rothstein, A. et al. Comparison of CpG s-ODNs, chromatin immune complexes, and dsDNA fragment immune complexes in the TLR9-dependent activation of rheumatoid factor B cells. J. Endotoxin Res. 10, 247–251 (2004).

    Article  CAS  Google Scholar 

  38. Hua, J., Kirou, K., Lee, C. & Crow, M.K. Functional assay of type I interferon in systemic lupus erythematosus plasma and association with anti-RNA binding protein autoantibodies. Arthritis Rheum. 54, 1906–1916 (2006).

    Article  CAS  Google Scholar 

  39. Lovgren, T., Eloranta, M.L., Bave, U., Alm, G.V. & Ronnblom, L. Induction of interferon-alpha production in plasmacytoid dendritic cells by immune complexes containing nucleic acid released by necrotic or late apoptotic cells and lupus IgG. Arthritis Rheum. 50, 1861–1872 (2004).

    Article  Google Scholar 

  40. Ehlers, M., Fukuyama, H., McGaha, T.L., Aderem, A. & Ravetch, J.V. TLR9/MyD88 signaling is required for class switching to pathogenic IgG2a and 2b autoantibodies in SLE. J. Exp. Med. 203, 553–561 (2006).

    Article  CAS  Google Scholar 

  41. Goodnow, C.C. Immunology. Discriminating microbe from self suffers a double toll. Science 312, 1606–1608 (2006).

    Article  Google Scholar 

  42. Means, T.K. & Luster, A.D. Toll-like receptor activation in the pathogenesis of systemic lupus erythematosus. Ann. NY Acad. Sci. 1062, 242–251 (2005).

    Article  Google Scholar 

  43. Muller, S. et al. New EMBO members' review: the double life of HMGB1 chromatin protein: architectural factor and extracellular signal. EMBO J. 20, 4337–4340 (2001).

    Article  CAS  Google Scholar 

  44. Stott, K., Tang, G.S., Lee, K.B. & Thomas, J.O. Structure of a complex of tandem HMG boxes and DNA. J. Mol. Biol. 360, 90–104 (2006).

    Article  CAS  Google Scholar 

  45. Napolitani, G., Rinaldi, A., Bertoni, F., Sallusto, F. & Lanzavecchia, A. Selected Toll-like receptor agonist combinations synergistically trigger a T helper type 1–polarizing program in dendritic cells. Nat. Immunol. 6, 769–776 (2005).

    Article  CAS  Google Scholar 

  46. Wang, H., Yang, H. & Tracey, K.J. Extracellular role of HMGB1 in inflammation and sepsis. J. Intern. Med. 255, 320–331 (2004).

    Article  CAS  Google Scholar 

  47. Li, J. et al. Recombinant HMGB1 with cytokine-stimulating activity. J. Immunol. Methods 289, 211–223 (2004).

    Article  CAS  Google Scholar 

  48. Andersson, U. et al. High mobility group 1 protein (HMG-1) stimulates proinflammatory cytokine synthesis in human monocytes. J. Exp. Med. 192, 565–570 (2000).

    Article  CAS  Google Scholar 

  49. Degryse, B. et al. The high mobility group (HMG) boxes of the nuclear protein HMG1 induce chemotaxis and cytoskeleton reorganization in rat smooth muscle cells. J. Cell Biol. 152, 1197–1206 (2001).

    Article  CAS  Google Scholar 

  50. Palumbo, R. et al. Extracellular HMGB1, a signal of tissue damage, induces mesoangioblast migration and proliferation. J. Cell Biol. 164, 441–449 (2004).

    Article  CAS  Google Scholar 

  51. Fiuza, C. et al. Inflammation-promoting activity of HMGB1 on human microvascular endothelial cells. Blood 101, 2652–2660 (2003).

    Article  CAS  Google Scholar 

  52. Sappington, P.L. et al. HMGB1 B box increases the permeability of Caco-2 enterocytic monolayers and impairs intestinal barrier function in mice. Gastroenterology 123, 790–802 (2002).

    Article  CAS  Google Scholar 

  53. Sorci, G., Riuzzi, F., Arcuri, C., Giambanco, I. & Donato, R. Amphoterin stimulates myogenesis and counteracts the antimyogenic factors basic fibroblast growth factor and S100B via RAGE binding. Mol. Cell. Biol. 24, 4880–4894 (2004).

    Article  CAS  Google Scholar 

  54. Rong, L.L. et al. Antagonism of RAGE suppresses peripheral nerve regeneration. FASEB J. 18, 1812–1817 (2004).

    Article  CAS  Google Scholar 

  55. Taguchi, A. et al. Blockade of RAGE-amphoterin signalling suppresses tumour growth and metastases. Nature 405, 354–360 (2000).

    Article  CAS  Google Scholar 

  56. Popovic, P.J. et al. High mobility group B1 protein suppresses the human plasmacytoid dendritic cell response to TLR9 agonists. J. Immunol. 177, 8701–8707 (2006).

    Article  CAS  Google Scholar 

  57. Bonaldi, T. et al. Monocytic cells hyperacetylate chromatin protein HMGB1 to redirect it towards secretion. EMBO J. 22, 5551–5560 (2003).

    Article  CAS  Google Scholar 

  58. Leadbetter, E.A. et al. Chromatin-IgG complexes activate B cells by dual engagement of IgM and Toll-like receptors. Nature 416, 603–607 (2002).

    Article  CAS  Google Scholar 

  59. Ahmad-Nejad, P. et al. Bacterial CpG-DNA and lipopolysaccharides activate Toll-like receptors at distinct cellular compartments. Eur. J. Immunol. 32, 1958–1968 (2002).

    Article  CAS  Google Scholar 

  60. Ishihara, K., Tsutsumi, K., Kawane, S., Nakajima, M. & Kasaoka, T. The receptor for advanced glycation end-products (RAGE) directly binds to ERK by a D-domain-like docking site. FEBS Lett. 550, 107–113 (2003).

    Article  CAS  Google Scholar 

  61. Kirou, K.A. et al. Activation of the interferon-α pathway identifies a subgroup of systemic lupus erythematosus patients with distinct serologic features and active disease. Arthritis Rheum. 52, 1491–1503 (2005).

    Article  CAS  Google Scholar 

  62. Lovgren, T., Eloranta, M.L., Bave, U., Alm, G.V. & Ronnblom, L. Induction of interferon-alpha production in plasmacytoid dendritic cells by immune complexes containing nucleic acid released by necrotic or late apoptotic cells and lupus IgG. Arthritis Rheum. 50, 1861–1872 (2004).

    Article  Google Scholar 

  63. Vallin, H., Perers, A., Alm, G.V. & Ronnblom, L. Anti-double-stranded DNA antibodies and immunostimulatory plasmid DNA in combination mimic the endogenous IFN-α inducer in systemic lupus erythematosus. J. Immunol. 163, 6306–6313 (1999).

    CAS  PubMed  Google Scholar 

  64. Walker, J.M., Goodwin, G.H., Johns, E.W., Wietzes, P. & Gaastra, W. A comparison of the amino-terminal sequences of two calf-thymus chromatin non-histone proteins. Int. J. Pept. Protein Res. 9, 220–223 (1977).

    Article  CAS  Google Scholar 

  65. Chavakis, T. et al. The pattern recognition receptor (RAGE) is a counterreceptor for leukocyte integrins: a novel pathway for inflammatory cell recruitment. J. Exp. Med. 198, 1507–1515 (2003).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank M. McCarthy and J. Suzich for comments on the manuscript; J.-C. Finet for trytophan intrinsic fluorescence measurements; and L. Xu for technical assistance. TLR9- and MyD88-deficient mice were from S. Akira (Research Institute for Microbial Diseases); HMGB1-deficient MEFs were from M. Bianchi (San Raffaele University). Supported by Deutsche Forschungsgemeinschaft (DFG/SFB 405 to P.P.N.), the Alliance for Lupus Research and the National Institutes of Health (AI0677497-01).

Author information

Authors and Affiliations

  1. Research Department, Inflammation and Autoimmune Group, MedImmune, Gaithersburg, 20878, Maryland, USA

    Jane Tian, Su-Yau Mao, Bo Chen, Kannaki Senthil, Herren Wu, Stacey Drabic, Ling Ling An, Laurent Audoly, Peter A Kiener & Anthony J Coyle

  2. Department of Microbiology, Boston University School of Medicine, Boston, 02118, Massachusetts, USA

    Ana Maria Avalos & Ann Marshak-Rothstein

  3. Department of Medicine, University of Massachusetts Medical School, Worcester, 01605, Massachusetts, USA

    Peggy Parroche, Douglas Golenbock, Cherilyn Sirois, Katherine A Fitzgerald & Eicke Latz

  4. Rheumatology Research and Autoimmunity and Inflammation Program, Hospital for Special Surgery, New York, 10021, New York, USA

    Jing Hua & Mary K Crow

  5. Critical Therapeutics, Lexington, 02421, Massachusetts, USA

    Greg La Rosa

  6. Department of Medicine, University of Heidelberg, Heidelberg, D-69115, Germany

    Angelika Bierhaus & Peter Naworth

Authors
  1. Jane Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ana Maria Avalos
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Su-Yau Mao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bo Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kannaki Senthil
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Herren Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Peggy Parroche
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Stacey Drabic
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Douglas Golenbock
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Cherilyn Sirois
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Jing Hua
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Ling Ling An
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Laurent Audoly
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Greg La Rosa
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Angelika Bierhaus
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Peter Naworth
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Ann Marshak-Rothstein
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Mary K Crow
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Katherine A Fitzgerald
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Eicke Latz
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Peter A Kiener
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. Anthony J Coyle
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.T. and S.-Y.M. carried out binding assays; A.M.A. and A.M.R., the B cell assays; B.C., immunoblots; H.W., L.L.A., L.A., K.S. and G.L.R., antibody generation and protein purification; E.L., P.P., S.D., D.G., C.S. and K.A.F., AlphaScreen assays and confocal microscopy; P.N. and A.B., generation of RAGE mice; and M.K.C. and J.H., lupus serum experiments. A.J.C. and P.A.K. conceived the experiments and wrote the paper.

Corresponding author

Correspondence to Anthony J Coyle.

Ethics declarations

Competing interests

J.T., S.-Y.M., B.C., K.S., H.W., S.D., L.A., P.A.K. and A.J.C. are employees of MedImmune, developing therapeutic monoclonal antibodies to HMGB1. G.L.R. is an employee of Critical Therapeutics.

Supplementary information

Supplementary Table 1

DNA and peptide sequence. (PDF 51 kb)

Supplementary Methods (PDF 82 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tian, J., Avalos, A., Mao, SY. et al. Toll-like receptor 9–dependent activation by DNA-containing immune complexes is mediated by HMGB1 and RAGE. Nat Immunol 8, 487–496 (2007). https://doi.org/10.1038/ni1457

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni1457

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing