Advertisement

Hepatology International

, Volume 2, Issue 2, pp 222–230 | Cite as

Increased expression of Toll-like receptor 3 in intrahepatic biliary epithelial cells at sites of ductular reaction in diseased livers

  • Minoru NakamuraEmail author
  • Kenji Funami
  • Atsumasa Komori
  • Terufumi Yokoyama
  • Yoshihiro Aiba
  • Aiko Araki
  • Yasushi Takii
  • Masahiro Ito
  • Mutsumi Matsuyama
  • Makiko Koyabu
  • Kiyoshi Migita
  • Ken Taniguchi
  • Hikaru Fujioka
  • Hiroshi Yatsuhashi
  • Misako Matsumoto
  • Hiromi Ishibashi
  • Tsukasa Seya
Original Article

Abstract

Background Toll-like receptors (TLRs) may play active roles in both innate and adaptive immune responses in human intrahepatic biliary epithelial cells (HIBECs). The role of TLR3 expressed by HIBECs, however, remains unclear. Methods We determined the in vivo expression of TLRs in biopsy specimens derived from diseased livers immunohistochemically using a panel of monoclonal antibodies against human TLRs. We then examined the response of cultured HIBECs to a TLR3 ligand, polyinosinic–polycytidylic acid (polyI:C). Using siRNAs specific for Toll-IL-1R homology domain-containing adaptor molecule 1 (TICAM-1) and mitochondrial antiviral signaling protein (MAVS), we studied signaling pathways inducing IFN-β expression. Results The expression of TLR3 was markedly increased in biliary epithelial cells at sites of ductular reaction in diseased livers, including primary biliary cirrhosis (PBC), autoimmune hepatitis (AIH), and chronic viral hepatitis (CH) as compared to nondiseased livers. Although cultured HIBECs constitutively expressed TLR3 at both the protein and mRNA levels in vitro, the addition of polyI:C to culture media induced only minimal increases in IFN-β mRNA. In contrast, transfection of HIBECs with polyI:C induced a marked increase in mRNAs encoding a variety of chemokines/cytokines, including IFN-β, IL-6, and TNF-α. The induction of IFN-β mRNA was efficiently inhibited by an siRNA against MAVS but not against TICAM-1, indicating that the main signaling pathway for IFN-β induction following polyI:C transfection is via retinoic acid-inducible gene I (RIG-I)/melanoma differentiation-associated gene 5 (MDA5) in HIBECs. Conclusions TLR3 expression by biliary epithelial cells increased at sites of ductular reaction in diseased livers; further study will be necessary to characterize it’s in vivo physiological role.

Keywords

Primary biliary cirrhosis (PBC) Human intrahepatic biliary epithelial cells (HIBECs) Interferon beta (IFN-β) Toll-like receptor 3 (TLR3) Toll-IL-1R homology domain-containing adaptor molecule 1 (TICAM-1) Mitochondrial antiviral signaling protein (MAVS) Retinoic acid inducible gene I (RIG-I) Melanoma differentiation-associated gene 5 (MDA5) 

Abbreviations

BEC

Biliary epithelial cell

CK

Cytokeratin

dsRNA

Double stranded RNA

ER

Endoplasmic reticulum

ELISA

Enzyme-linked immunosorbent assay

GAPDH

Glyceraldehydes-3-phosphate dehydrogenase

HIBEC

Human intrahepatic biliary epithelial cell

HRP

Horseradish peroxidase

IFN

Interferon

IL

Interleukin

IRF

Interferon regulatory factor

MAVS

Mitochondrial anti-viral signaling protein

MDA5

Melanoma differentiation associated gene-5

MyD88

Myeloid differentiation factor 88

PBC

Primary biliary cirrhosis

PBMC

Peripheral blood mononuclear cells

PolyI:C

Polyinosinic–polycytidylic acid

PRR

Pattern-recognition receptor

RIG-I

Retinoic acid-inducible gene I

RT-PCR

Reverse transcription-polymerase chain reaction

siRNA

Small interfering RNA

TLR

Toll-like receptor

TNF

Tumor necrosis factor

TICAM-1

Toll-IL-1R homology domain containing adaptor molecule 1

Notes

Acknowledgments

This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Health, Labour and Welfare of Japan and Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science.

References

  1. 1.
    Akira S, Takeda K. Toll-like receptor signaling. Nat Rev Immunol 2004;4:499–511.PubMedCrossRefGoogle Scholar
  2. 2.
    Yoneyama M, Kikuchi M, Natsukawa T, Shinobu N, Imaizumi T, Miyagoshi M, et al. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat Immunol 2004;5:730–7.PubMedCrossRefGoogle Scholar
  3. 3.
    Lee MS, Kim Y-J. Pattern-recognition receptor signaling initiated from extracellular, membrane, and cytoplasmic space. Mol Cells 2007;23:1–10.PubMedGoogle Scholar
  4. 4.
    Matsumoto M, Kikkawa S, Kohase M, Miyake K, Seya T. Establishment of a monoclonal antibody against human Toll-like receptor 3 that blocks double-stranded RNA-mediated signaling. Biochem Biophys Res Commun 2002;293:1364–9.PubMedCrossRefGoogle Scholar
  5. 5.
    Matsumoto M, Funami K, Tanabe M, Hiroyuki O, Shingai M, Seto Y, et al. Subcellular localization of Toll-like receptor 3 in human dendritic cells. J Immunol 2003;171:3154–62.PubMedGoogle Scholar
  6. 6.
    Cario E, Podolsky DK. Differential alteration in intestinal epithelial cell expression of Toll-like receptor 3 (TLR3) and TLR4 in inflammatory bowel disease. Infect Immun 2000;68:7010–17.PubMedCrossRefGoogle Scholar
  7. 7.
    Furrie E, Macfarlane S, Thomson G, Macfarlane GT. Toll-like receptors-2, -3 and -4 expression patterns on human colon and their regulation by mucosal-associated bacteria. Immunology 2005;115:565–74.PubMedCrossRefGoogle Scholar
  8. 8.
    Schaefer TM, Desouza K, Fahey JV, Beagley KW, Wira CR. Toll-like receptor (TLR) expression and TLR-mediated cytokine/chemokine production by human uterine epithelial cells. Immunology 2004;112:428–36.PubMedCrossRefGoogle Scholar
  9. 9.
    Jorgenson RL, Young SL, Lesmeister MJ, Lyddon TD, Misfeldt ML. Human endometrial epithelial cells cyclically express Toll-like receptor 3(TLR3) and exhibit TLR3-dependent responses to dsRNA. Human Immunol 2004;66:469–82.CrossRefGoogle Scholar
  10. 10.
    Schaefer TM, Fahey JV, Wright JA, Wira CR. Innate immunity in the human female reproductive tract: antiviral response of uterine epithelial cells to the TLR3 agonist poly(I:C). J Immunol 2005;174:992–1002.PubMedGoogle Scholar
  11. 11.
    Guillot L, Goffic RL, Bloch S, Escriou N, Akira S, Chignard M, et al. Involvement of Toll-like receptor 3 in the immune response of lung epithelial cells to double-stranded RNA and influenza A virus. J Biol Chem 2005;280:5571–80.PubMedCrossRefGoogle Scholar
  12. 12.
    Ritter M, Mennerich D, Weith A, Seither P. Characterization of Toll-like receptors in primary lung epithelial cells: strong impact of the TLR3 ligand poly(I:C) on the regulation of Toll-like receptors, adaptor proteins and inflammatory response. J Inflamm 2005;2:16.CrossRefGoogle Scholar
  13. 13.
    Kumar A, Zhang J, Yu FSX. Toll-like receptor 3 agonist poly(I:C)-induced antiviral response in human corneal epithelial cells. Immunology 2005;117:11–21.CrossRefGoogle Scholar
  14. 14.
    Ueta M, Hamuro J, Kiyono H, Kinoshita S. Triggering of TLR3 by polyI:C in human corneal epithelial cells to induce inflammatory cytokines. Biochem Biophys Res Commun 2005;331:285–94.PubMedCrossRefGoogle Scholar
  15. 15.
    Bsibsi M, Ravid R, Gveric D, van Noort JM. Broad expression of Toll-like receptors in the human central nervous system. J Neuropathol Exp Neurol 2002;61:1013–21.PubMedGoogle Scholar
  16. 16.
    Kollisch G, Kalali BN, Voelcker V, Wallich R, Behrendt H, Ring J, et al. Various members of the Toll-like receptor family contribute to the innate immune response of human epidermal keratinocytes. Immunology 2005;114:531–41.PubMedCrossRefGoogle Scholar
  17. 17.
    Takii Y, Nakamura M, Ito M, Yokoyama T, Komori A, Shimizu-Yoshida Y, et al. Enhanced expression of type I interferon and Toll-like receptor-3 in primary biliary cirrhosis. Lab Invest 2005;85:908–20.PubMedCrossRefGoogle Scholar
  18. 18.
    Yokoyama T, Komori A, Nakamura M, Takii Y, Kamihira T, Shimoda S, et al. Human intrahepatic biliary epithelial cells function in innate immunity by producing IL-6 and IL-8 via the TLR4-NF-γB and -MAPK signaling pathways. Liver Int 2006;26:467–76.PubMedCrossRefGoogle Scholar
  19. 19.
    Sasai M, Shingai M, Funami K, Yoneyama M, Fujita T, Matsumoto M, et al. NAK-associated protein 1 participates in both the TLR3 and the cytoplasmic pathways in type I IFN induction. J Immunol 2006;177:8676–83.PubMedGoogle Scholar
  20. 20.
    Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C (T)) method. Methods 2001;25:402.PubMedCrossRefGoogle Scholar
  21. 21.
    Dalpke A, Frank J, Peter M, Heeg K. Activation of Toll-like receptor 9 by DNA from different bacterial species. Infect Immunol 2006;74:940–6.CrossRefGoogle Scholar
  22. 22.
    Kariko K, Ni H, Capodici J, Lamphier M, Weissman D. mRNA is an endogenous ligand for Toll-like receptor 3. J Biol Chem 2004;279:12542–50.PubMedCrossRefGoogle Scholar
  23. 23.
    Roelofs MF, Joosten LAB, Abdollahi-Roodsaz S, van Lieshout AWT, Sprong T, van den Hoogen FH, et al. The expression of Toll-like receptor 3 and 7 in rheumatoid arthritis synovium is increased and costimulation of Toll-like receptor 3, 4, and 7/8 results in synergistic cytokine production by dendritic cells. Arth Rheum 2005;52:2313–22.CrossRefGoogle Scholar
  24. 24.
    Brentano F, Schorr O, Gay RE, Gay S, Kyburz D. RNA released from necrotic synovial fluid cells activates rheumatoid arthritis synovial fibroblasts via Toll-like receptor 3. Arth Rheum 2005;52:2656–65.CrossRefGoogle Scholar
  25. 25.
    Harii N, Lewis CJ, Vasko V, McCall K, Benavides-Peralta U, Sun X, et al. Thyrocytes express a functional Toll-like receptor 3: overexpression can be induced by viral infection and reversed by phenylmethimazole and is associated with Hashimoto’s autoimmune thyroiditis. Mol Endocrinol 2005;19:1231–50.PubMedCrossRefGoogle Scholar
  26. 26.
    Lang KS, Georgiev P, Recher M, Navarini AA, Bergthaler A, Heikenwalder M, et al. Immunoprivileged status of the liver is controlled by Toll-like receptor 3 signaling. J Clin Invest 2006;116:2456–63.PubMedCrossRefGoogle Scholar
  27. 27.
    Wen L, Peng J, Li Z, Wong FS. The effect of innate immunity on autoimmune diabetes and the expression of Toll-like receptors on pancreatic islets. J Immunol 2004;172:3173–80.PubMedGoogle Scholar
  28. 28.
    Rasschaert J, Ladriere L, Urbain M, Dogusan Z, Katabua B, Sato S, et al. Toll-like receptor 3 and STAT-1 contribute to double-stranded RNA+ interferon-γ-induced apoptosis in primary pancreatic β-cells. J Biol Chem 2005;280:33984–91.PubMedCrossRefGoogle Scholar
  29. 29.
    Kaiser WJ, Kaufman JL, Offermann MK. IFN-γ sensitizes human umbilical vein endothelial cells to apoptosis induced by double-stranded RNA. J Immunol 2004;172:1699–710.PubMedGoogle Scholar
  30. 30.
    Salaun B, Coste I, Rissoan MC, Lebecque SJ, Renno T. TLR3 can directly trigger apoptosis in human cancer cells. J Immunol 2006;176:4894–901.PubMedGoogle Scholar
  31. 31.
    Wornle M, Schmid H, Banas B, Merkle M, Henger A, Roeder M, et al. Novel role of Toll-like receptor 3 in hepatitis C-associated glomerulonephritis. Am J Pathol 2006;168:370–86.PubMedCrossRefGoogle Scholar
  32. 32.
    Bsibsi M, Persoon-Deen C, Verwer RWH, Meeuwsen S, Ravid R, Van Noort JM. Toll-like receptor 3 on adult human astrocytes triggers production of neuroprotective mediators. Glia 2006;53:688–95.PubMedCrossRefGoogle Scholar
  33. 33.
    Vijay-Kumar M, Wu H, Aitken J, Kolachala VL, Neish AS, Sitaraman SV, et al. Activation of Toll-like receptor 3 protects against DSS-induced acute colitis. Inflamm Bowel Dis 2007;13:856–64.PubMedCrossRefGoogle Scholar
  34. 34.
    Harada K, Van de Water J, Leung PS, Coppel RL, Ansari A, Nakanuma Y, et al. In situ nucleic acid hybridization of cytokines in primary biliary cirrhosis: predominance of the TH1 subset. Hepatology 1997;25:791–6.PubMedCrossRefGoogle Scholar
  35. 35.
    Harada K, Nakanuma Y. Molecular mechanisms of cholangiopathy in primary biliary cirrhosis. Med Mol Morphol 2006;39:55–61.PubMedCrossRefGoogle Scholar
  36. 36.
    Gershwin ME, Nishio A, Ishibashi H, Lindor K. Primary biliary cirrhosis. In: Gershwin ME, Vierling JM, Manns MP, editors. Liver immunology, Chapter 20. Philadelphia, PA: Hanley & Belfus, Inc.; 2003. p. 311–27.Google Scholar
  37. 37.
    Kamihira T, Shimoda S, Nakamura M, Yokoyama T, Tkii Y, Kawano A, et al. Biliary epithelial cells regulate autoreactive T cells: implications for biliary-specific diseases. Hepatology 2005;41:151–9.PubMedCrossRefGoogle Scholar
  38. 38.
    Wang AP, Migita K, Ito M, Takii Y, Daikoku M, Yokoyama T, et al. Hepatic expression of Toll-like receptor 4 in primary biliary cirrhosis. J Autoimmun 2005;25:85–91.PubMedCrossRefGoogle Scholar
  39. 39.
    Heinz S, Haehnel V, Karaghiosoff M, Schwarzfischer L, Muller M, Krause SW, et al. Species-specific regulation of Toll-like receptor 3 genes in men and mice. J Biol Chem 2003;278:21502–9.PubMedCrossRefGoogle Scholar
  40. 40.
    Tanabe M, Kurita-Taniguchi M, Takeuchi K, Takeda M, Ayata M, Ogura H, et al. Mechanism of up-regulation of human Toll-like receptor 3 secondary to infection of measles virus-attenuated strains. Biochem Biophys Res Commun 2003;311:39–48.PubMedCrossRefGoogle Scholar
  41. 41.
    Tissari J, Siren J, Meri S, Julkunen I, Matikainen S. IFN-γ enhances TLR3-mediated antiviral cytokine expression in human endothelial and epithelial cells by up-regulating TLR3 expression. J Immunol 2005;174:4289–94.PubMedGoogle Scholar
  42. 42.
    Siren J, Imaizumi T, Sarkar D, Pietila T, Noah DL, Lin R, et al. Retinoic acid inducible gene-I and mda-5 are involved in influenza A virus-induced expression of anti-viral cytokines. Microbes Infect 2006;8:2013–20.PubMedCrossRefGoogle Scholar
  43. 43.
    Liu P, Jamaluddin M, Li K, Garofalo RP, Casola A, Brasier AR. Retinoic acid-inducible gene I mediates early antiviral response and Toll-like receptor 3 expression in respiratory syncytial virus-infected airway epithelial cells. J Virol 2007;81:1401–11.PubMedCrossRefGoogle Scholar
  44. 44.
    Theise ND, Saxena R, Portmann BC, Thung SN, Yee Y, Chiriboga L, et al. The canals of Hering and hepatic stem cells in humans. Hepatology 1999;30:1425–33.PubMedCrossRefGoogle Scholar
  45. 45.
    Zhou H, Rogler LE, Teperman L, Morgan G, Rogler C. Identification of hepatocytic and bile ductular cell lineages and candidate stem cells in bipolar ductular reactions in cirrhotic human liver. Hepatology 2007;45:716–24.PubMedCrossRefGoogle Scholar

Copyright information

© Asian Pacific Association for the Study of the Liver 2008

Authors and Affiliations

  • Minoru Nakamura
    • 1
    • 2
    Email author
  • Kenji Funami
    • 3
  • Atsumasa Komori
    • 1
  • Terufumi Yokoyama
    • 1
  • Yoshihiro Aiba
    • 1
  • Aiko Araki
    • 1
  • Yasushi Takii
    • 1
  • Masahiro Ito
    • 1
  • Mutsumi Matsuyama
    • 1
  • Makiko Koyabu
    • 1
  • Kiyoshi Migita
    • 1
  • Ken Taniguchi
    • 1
  • Hikaru Fujioka
    • 1
  • Hiroshi Yatsuhashi
    • 1
  • Misako Matsumoto
    • 3
  • Hiromi Ishibashi
    • 1
  • Tsukasa Seya
    • 3
  1. 1.Clinical Research Center National Hospital Organization (NHO) Nagasaki Medical CenterOmura, NagasakiJapan
  2. 2.Department of HepatologyNagasaki University Graduate School of Biomedical SciencesOmura, NagasakiJapan
  3. 3.Department of Microbiology and ImmunologyHokkaido University Graduate School of MedicineSapporoJapan

Personalised recommendations