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A murine model of NKT cell-mediated liver injury induced by alpha-galactosylceramide/d-galactosamine

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An Erratum to this article was published on 04 August 2006

Abstract

Natural killer-T (NKT) cells are rich in the liver. However, their involvement in liver injury is not fully understood. We developed here a new murine model of NKT-cell-activation-associated liver injury, and investigated a role of tumor necrosis factor alpha (TNF-α) and Fas in pathogenesis. We injected intraperitoneally alpha-galactosylceramide (α-GalCer), an NKT-cell stimulant, into d-galactosamine (GalN)-sensitized mice. Survival rate, pathological changes of the liver, and plasma concentrations of cytokines were studied. Alpha-GalCer/GalN administration gave a lethal effect within 7 h, making pathological changes such as massive parenchymal hemorrhage, hepatocyte apoptosis, sinusoidal endothelial cell injury, and close apposition of lymphocytes to apoptotic hepatocytes. Anti-NK1.1 mAb-pretreated mice and Vα14NKT knock out (KO) mice did not develop liver injury. Tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) were elevated at 4 h in the plasma. These cytokines were produced by hepatic lymphocytes as demonstrated by in vitro stimulation with α-GalCer. The lethal effect was suppressed in TNF-α KO mice, TNF receptor-1 KO mice, and lpr/lpr (Fas deficient) mice, whereas it was not in IFN-γ KO mice. These results indicate that the present liver injury is characterized by parenchymal hemorrhage and hepatocyte apoptosis, and mediated by TNF-α secretion and direct cytotoxicity of α-GalCer-activated NKT cells.

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Abbreviations

NK:

natural killer

NKT:

natural killer-T

α-GalCer:

alpha-galactosylceramide

GalN:

d-galactosamine

SEB:

staphylococcal enterotoxin B

TNF-α:

tumor necrosis factor alpha

IFN-γ:

interferon gamma

TNFR1:

TNF receptor-1

FasL:

Fas ligand

LPS:

lipopolysaccharide

iNKT:

invariant NKT

KO:

knock out

mAb:

monoclonal antibody

ALT:

alanine transaminase

IL:

interleukin

TUNEL:

terminal deoxynucleotidyl transferase-mediated nick end labeling

WT:

wild type

SECs:

sinusoidal endothelial cells

RT-PCR:

reverse transcription- polemerase chain reaction

References

  1. Angermuller S, Kunstle G, Tiegs G (1998) Pre-apoptotic alterations in hepatocytes of TNFα-treated galactosamine-sensitized mice. J Histochem Cytochem 46:1175–1183

    CAS  PubMed  Google Scholar 

  2. Bendelac A, Rivera MN, Park SH, Roark JH (1997) Mouse CD1-specific NK1 T cells: development, specificity, and function. Annu Rev Immunol 15:535–562

    Article  CAS  PubMed  Google Scholar 

  3. Bursch W, Taper HS, Lauer B, Schulte-Hermann R (1985) Quantitative histological and histochemical studies on the occurrence and stages of controlled cell death (apoptosis) during regression of rat liver hyperplasia. Virchows Arch B Cell Pathol Incl Mol Pathol 50:153–166

    CAS  PubMed  Google Scholar 

  4. Cardier JE, Schulte T, Kammer H, Kwak J, Cardier M (1999) Fas (CD95, APO-1) antigen expression and function in murine liver endothelial cells: implications for the regulation of apoptosis in liver endothelial cells. FASEB J 13:1950–1960

    CAS  PubMed  Google Scholar 

  5. Cauwels A, Janssen B, Waeytens A, Cuvelier C, Brouckaert P (2003) Caspase inhibition causes hyperacute tumor necrosis factor-induced shock via oxidative stress and phospholipase A2. Nat Immunol 4:387–393

    Article  CAS  PubMed  Google Scholar 

  6. Crispe IN (2003) Hepatic T cells and liver tolerance. Nat Rev Immunol 3:51–62

    Article  CAS  PubMed  Google Scholar 

  7. Cui J, Shin T, Kawano T, Sato H, Kondo E, Toura I, Kaneko Y, Koseki H, Kannno M, Taniguchi M (1997) Requirement for Vα14 NKT cells in IL-12-mediated rejection of tumors. Science 278:1623–1626

    Article  CAS  PubMed  Google Scholar 

  8. Deaciuc IV, Bagby GJ, Lang CH, Spitzer JJ (1993) Hyaluronic acid uptake by the isolated, perfused rat liver: an index of hepatic sinusoidal endothelial cell function. Hepatology 17:266–272

    Article  CAS  PubMed  Google Scholar 

  9. Diehl KH, Hull R, Morton D, Pfister R, Rabemampianina Y, Smith D, Vidal JM, van de Vorstenbosch C; European Federation of Pharmaceutical Industries Association and European Centre for the Validation of Alternative Methods (2001) A good practice guide to the administration of substances and removal of blood, including routes and volumes. J Appl Toxicol 21:15–23

    Article  CAS  PubMed  Google Scholar 

  10. Dienes HP (1989) Viral and autoimmune hepatitis. Morphologic and pathogenetic aspects of cell damage in hepatitis with potential chronicity. Verroff Pathol 132:1–107

    CAS  Google Scholar 

  11. Dini L, Pagliara P, Carla EC (2002) Phagocytosis of apoptotic cells by liver: a morphological study. Microsc Res Tech 57:530–540

    Article  PubMed  Google Scholar 

  12. Emoto M, Kaufmann SH (2003) Liver NKT cells: an account of heterogeneity. Trends Immunol 24:364–369

    Article  CAS  PubMed  Google Scholar 

  13. Galanos C, Freundenberg MA, Reutter W (1979) Galactosamine-induced sensitization to the lethal effects of endotoxin. Proc Natl Acad Sci USA 76:5939–5943

    CAS  PubMed  Google Scholar 

  14. Gantner F, Leist M, Jilg S, Germann PG, Freudenberg MA, Tiegs G (1995) Tumor necrosis factor-induced hepatic DNA fragmentation as an early marker of T cell-dependent liver injury in mice. Gastroenterology 109:166–176

    CAS  PubMed  Google Scholar 

  15. Godfrey DI, Hammond KJ, Poulton LD, Smyth MJ, Baxter AG (2000) NKT cells: facts, functions and fallacies. Immunol Today 21:573–583

    Article  CAS  PubMed  Google Scholar 

  16. Hashimoto W, Takeda K, Anzai R, Ogasawara K, Sakihara H, Sugiura K, Seki S, Kumagai K (1995) Cytotoxic NK1.1 Ag+ αβ T cells with intermediate TCR induced in the liver of mice by IL-12. J Immunol 154:4333–4340

    CAS  PubMed  Google Scholar 

  17. Hengartner MO (2000) The biochemistry of apoptosis. Nature 407:770–776

    Article  CAS  PubMed  Google Scholar 

  18. Illes Z, Kondo T, Newcombe J, Oka N, Tabira T, Yamamura T (2000) Differential expression of NK T cell V alpha24Jalpha Q invariant TCR chain in the lesionsof multiple sclerosis and chronic inflammatory demyelinating polyneuropathy. J Immunol 164:4375–4381

    CAS  PubMed  Google Scholar 

  19. Jaeschke H, Fisher MA, Lawson JA, Simmons CA, Farhood A, Jones DA (1998) Activation of caspase 3 (CPP32)-like proteases is essential for TNF-α-induced hepatic parenchymal cell apoptosis and neutrophil-mediated necrosis in a murine endotoxin shock model. J Immunol 160:3480–3486

    CAS  PubMed  Google Scholar 

  20. Kasahara I, Saitoh K, Nakamura K (2000) Apoptosis in acute hepatic failure: histopathological study of human liver tissue using the tunel method and immunohistochemistry. J Med Dent Sci 47:167–175

    CAS  PubMed  Google Scholar 

  21. Kawano T, Cui J, Koezuka Y, Toura I, Kaneko Y, Sato H, Kondo E, Harada M, Koseki H, Nakayama T et al (1998) Natural killer- like nonspecific tumor cell lysis mediated by specific ligand-activated Vα14 NKT cells. Proc Natl Acad Sci USA 95:5690–5693

    Article  CAS  PubMed  Google Scholar 

  22. Kawano T, Cui J, Koezuka Y, Toura I, Kaneko Y, Motoki K, Ueno H, Nakagawa R, Sato H, Kondo E et al (1997) CD1d-restricted and TCR-mediated activation of Vα14 NKT cells by glycosylceramides. Science 278:1626–1629

    Article  CAS  PubMed  Google Scholar 

  23. Kita H, Naidenko OV, Kronenberg M, Ansari AA, Rogers P, He XS, Koning F, Mikayama T et al. (2002) Quantitation and phenotypic analysis of natural killer T cells in primary biliary cirrhosis using a human CD1d tetramer. Gastroenterology 123:1031–1043

    Article  CAS  PubMed  Google Scholar 

  24. Künsters S, Tiegs L, Alexopoulou M, Pasparakis E, Douni G, Künstle H, Bluchmann A, Wendel K, Pfitzenmaier G, Kollias G, Grell M (1997) In vivo evidence for a functional role of both tumor necrosis factor (TNF) receptors and transmembrane TNF in experimental hepatitis. Eur J Immunol 27:2870–2875

    PubMed  Google Scholar 

  25. Künstle G, Leist M, Uhlig S, Revesz L, Feifel R, MacKenzie A, Wendel A (1997) ICE- protease inhibitors block murine liver injury and apoptosis caused by CD95 or by TNF-α. Immunol Lett 55:5–10

    Article  PubMed  Google Scholar 

  26. Künstle G, Hentze H, Germann P-G, Tiegs G, Meergans T, Wendel A (1999) Concanavalin A hepatotoxicity in mice: tumor necrosis factor-mediated organ failure independent of caspase-3-like protease activation. Hepatology 30:1241–1251

    Article  PubMed  Google Scholar 

  27. Leist M, Jäattelä M (2001) Four deaths and a funeral: from caspases to alternative mechanisms. Nat Rev Mol Cell Biol 2:589–598

    Article  CAS  PubMed  Google Scholar 

  28. Leist M, Gantner F, Bohlinger I, Germann PG, Tiegs G, Wendel A (1994) Murine hepatocyte apoptosis induced in vitro and in vivo by TNF-α requires transcriptional arrest. J Immunol 153:1778–1788

    CAS  PubMed  Google Scholar 

  29. Leist M, Gantner F, Bohlinger I, Tiegs G, Germann PG, Wendel A (1995) Tumor necrosis factor-induced hepatocyte apoptosis precedes liver failure in experimental murine shock models. Am J Pathol 146:1220–1234

    CAS  PubMed  Google Scholar 

  30. Matsuda JL, Naidenko OV, Gapin L, Nakayama T, Taniguchi M, Wang CR, Koezuka Y, Kronenberg M (2000) Tracking the response of natural killer T cells to a glycolipid antigen using CD1d tetramers. J Exp Med 192:741–754

    Article  CAS  PubMed  Google Scholar 

  31. Minagawa M, Deng Q, Liu ZX, Tsukamoto H, Dennert G (2004) Activated natural killer T cells induce liver injury by Fas and tumor necrosis factor-alpha during alcoholic consumption. Gastroenterology 126:1387–1399

    Article  CAS  PubMed  Google Scholar 

  32. Miyazawa Y, Tsutsui H, Mizuhara H, Fujiwara H, Kaneda K (1998) Involvement of intrasinusoidal hemostasis in the development of concanavalin A-induced hepatic injury in mice. Hepatology 27:497–506

    Article  CAS  PubMed  Google Scholar 

  33. Mohammed FF, Smookler DS, Taylot SE, Fingleton B, Kassiri Z, Sanchez OH, English JL, Matrisian LM, Au B, Yeh WC et al. (2004) Abnormal TNF activity in Timp3-/- mice leads to chronic hepatic inflammation and failure of liver generation. Nat Genet 36:969–977

    Article  CAS  PubMed  Google Scholar 

  34. Morikawa A, Sugiyama T, Kato Y, Koide N, Jiang GZ, Takahashi K, Tamada Y, Yokochi T (1996) Apoptotic cell death in the response of D-galactosamine-sensitized mice to lipopolysaccharide as an experimental endotoxic shock model. Infect Immun 64:734–738

    CAS  PubMed  Google Scholar 

  35. Muschen M, Warskulat U, Douillard P, Gilbert E, Haussinger D (1998) Regulation of CD95(APO-1/Fas) receptor and ligand expression by lipopolysaccharide and dexamethasone in parenchymal and nonparenchymal rat liver cells. Hepatology 27:200–208

    Article  CAS  PubMed  Google Scholar 

  36. Nagaki M, Naiki T, Brenner DA, Osawa Y, Imose M, Hayashi H, Banno Y, Nakashima S, Moriwaki H (2000) Tumor necrosis factor α prevents tumor necrosis factor receptor-mediated mouse hepatocyte apoptosis, but not Fas-mediated apoptosis: role of nuclear factor-κB. Hepatology 32:1272–1279

    Article  CAS  PubMed  Google Scholar 

  37. Nakagawa R, Nagafune I, Tazunoki Y, Ehara H, Tomura H, Iijima R, Motoki K, Kamishohara M, Seki S (2001) Mechanism of the antimetastatic effect in the liver and of the hepatocyte injury induced by α-galactosylceramide in mice. J Immunol 166:6578–6584

    CAS  PubMed  Google Scholar 

  38. Nakatani K, Kaneda K, Seki S, Nakajima Y (2004) Pit cells as liver-associated natural killer cells: morphology and function. Med Electron Microsc 37:29–36

    Article  PubMed  Google Scholar 

  39. Nowak M, Gaines GC, Rosenberg J, Minter R, Bahjat FR, Rectenwald J, Mackay SL, Edwards CK III, Moldawer LL (2000) LPS-induced liver injury in D-galactosamine- sensitized mice requires secreted TNF-α and the TNF-p55 receptor. Am J Physiol Regul Integr Comp Physiol 278:R1202–R1209

    CAS  PubMed  Google Scholar 

  40. Osman Y, Kawamura T, Naito T, Takeda K, Van Kaer L, Okumura K, Abo T (2000) Activation of hepatic NKT cells and subsequent liver injury following administration of α-galactosylceramide. Eur J Immunol 30:1919–1928

    Article  CAS  PubMed  Google Scholar 

  41. Pretet JL, Pelletier L, Bernard B, Coumes-Marquet S, Kantelip B, Mougin C (2000) Apoptosis participates to liver damage in HSV-induced fulminant hepatitis. Apoptosis 8:655–663

    Article  Google Scholar 

  42. Sass G, Heinlein S, Agli A, Bang R, Schümann J, Tiegs G (2002) Cytokine expression in three mouse models of experimental hepatitis. Cytokine 19:115–120

    Article  CAS  PubMed  Google Scholar 

  43. Seki S, Hashimoto W, Ogasawara K, Satoh M, Watanabe H, Habu Y, Hiraide H, Takeda K (1997) Antimetastatic effect of NK1+ T cells on experimental haematogenous tumor metastases in the liver and lungs of mice. Immunology 92:561–566

    Article  CAS  PubMed  Google Scholar 

  44. Shimizu Y, Margenthaler JA, Landeros K, Otomo N, Doherty G, Flye MW (2002) The resistance of P. acnes-primed interferon γ-deficient mice to low-dose lipopolysaccharide- induced acute liver injury. Hepatology 35:805–814

    Article  CAS  PubMed  Google Scholar 

  45. Sumida T, Sakamoto A, Murata H, Makino Y, Takahashi H, Yoshida S, Nishioka K, Iwamoto I, Taniguchi M (1995) Selective reduction of T cells bearing invariant Vα24 JαQ antigen receptor in patients with systemic sclerosis. J Exp Med 182:1163–1168

    Article  CAS  PubMed  Google Scholar 

  46. Sumida T, Maeda T, Taniguchi M, Nishioka K, Stohl W (1998) TCRαV24 gene expression in double negative T cells in systemic lupus erythematosus. Lupus 7:565–568

    Article  CAS  PubMed  Google Scholar 

  47. Tagawa Y, Sekikawa K, Iwakura Y (1997) Suppression of concanavalin A-induced hepatitis in IFN-γ-/- mice, but not in TNF-α-/- mice: role for IFN-γ in activating apoptosis of hepatocytes. J Immunol 159:1418–1428

    CAS  PubMed  Google Scholar 

  48. Tokushige K, Yamaguchi N, Ikeda I, Hashimoto E, Yamauchi K, Hayashi N (2000) Significance of soluble TNF receptor-I in acute-type fulminant hepatitis. Am J Gastroenterol 95:2040–2046

    Article  CAS  PubMed  Google Scholar 

  49. Trobonjaca Z, Leithäuser F, Möller P, Schirmbeck, Reimann J (2001) Activating immunity in the liver. I. Liver dendritic cells (but not hepatocytes) are potent activators of IFN-γ release by liver NKT cells. J Immunol 167:1413–1422

    CAS  PubMed  Google Scholar 

  50. Tsuji H, Mukaida N, Harada A, Kaneko S, Matsushita E, Nakanuma Y, Tsutsui H, Okamura H, Nakanishi K, Tagawa Y et al. (1999) Alleviation of lipopolysaccharide-induced acute liver injury in Propionibacterium acnes-primed IFN-γ-deficient mice by a concomitant reduction of TNF-α, IL-12, and IL-18 production. J Immunol 162:1049–1055

    CAS  PubMed  Google Scholar 

  51. Wanner GA, Mica L, Wanner-Schmid E, Kolb SA, Hentze H, Trentz O, Ertel W (1999) Inhibition of caspase activity prevents CD95-mediated hepatic microvascular perfusion failure and restores Kupffer cell clearance capacity. FASEB J 13:1239–1248

    CAS  PubMed  Google Scholar 

  52. Watanabe H, Miyaji C, Kawachi Y, Iiai T, Ohtsuka K, Iwanaga T, Takahashi-Iwanaga H, Abo T (1995) Relationships between intermediate TCR cells and NK1.1+T cells in various immune organs. NK1.1+T cells are present within a populations of intermediate TCR cells. J Immunol 155:2972–2983

    CAS  PubMed  Google Scholar 

  53. Wilson SB, Kent SC, Patton KT, Orban T, Jackson RA, Exley M, Porcelli S, Schatz DA, Atkinson MA, Balk SP (1998) Extreme Th1 bias of invaiant Valpha24 JalphaQ T cells in type 1 diabetes. Nature 391:177–181

    Article  CAS  PubMed  Google Scholar 

  54. Wolf D, Hallmann R, Sass G, Sixt M, Kusters S, Fregien B, Trauwein C, Tiegs G (2001) TNF- alpha-induced expression of adhesion molecules in the liver is under the control of TNFR1— relevance for concanavalin A-induced hepatitis. J Immunol 166:1300–1307

    CAS  PubMed  Google Scholar 

  55. Yachida S, Kokudo Y, Wakabayashi H, Maeba T, Kaneda K, Maeta H (1998) Morphological and functional alterations to sinusoidal endothelial cells in the early phase of endotoxin-induced liver failure after partial hepatectomy in rats. Virchows Arch 433:173–181

    Article  CAS  PubMed  Google Scholar 

  56. Yasuda S, Nagaki M, Moriwaki H (2002) Staphylococcal enterotoxin B induces hepatic injury and lethal shock in endotoxin-resistant C3H/HeJ mice despite a deficient macrophage response. J Endotoxin Res 8:253–261

    Article  CAS  PubMed  Google Scholar 

  57. Zafrani ES, Cazier A, Baudelot AM, Feldmann G (1984) Ultrastructural lesions of the liver in human peliosis. A report of 12 cases. Am J Pathol 114:349–359

    CAS  PubMed  Google Scholar 

  58. Zhou BR, Gumenscheimer M, Freudenberg M, Galanos C (2003) A striking correlation between lethal activity and apoptotic DNA fragmentation of liver in response of D-galactosamine-sensitized mice to a non-lethal amount of lipopolysaccharide. Acta Pharmacol Sin 24:193–198

    CAS  PubMed  Google Scholar 

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Acknowledgements

The authors thank Dr. Yoichiro Iwakura for providing us with IFN-γ-deficient mice. We are grateful to Ms. Miki Yamauchi and Mr. Masahide Sakabe, Department of Anatomy, Osaka City University, Graduate School of Medicine, for their technical supports.

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Authors and Affiliations

  1. Department of Hepatology, Osaka City University Graduate School of Medicine, 1-4-3 Asahimachi, Abeno-ku, Osaka, 545-8585, Japan

    Hideki Fujii, Shuichi Seki, Sawako Kobayashi, Takuya Kitada & Nobuyoshi Kawakita

  2. Department of Anatomy, Osaka City University Graduate School of Medicine, Osaka, Japan

    Hideki Fujii, Masaru Ikemoto, Yuji Nakajima & Kenji Kaneda

  3. Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Kawaguchi, Japan

    Keishi Adachi, Hiroko Tsutsui & Kenji Nakanishi

  4. Department of Immunology and Medical Zoology, Hyogo College of Medicine, Nishinomiya, Japan

    Hiroko Tsutsui & Kenji Nakanishi

  5. Biomedical Research Center, Osaka University Graduate School of Medicine, Suita, Japan

    Hiromi Fujiwara

  6. Pharmacology Division, National Cancer Center Research Institute, Tokyo, Japan

    Yoshinori Ikarashi

  7. Institute of Physical and Chemical Research, Research Center for Allergy and Immunology, Tokyo, Japan

    Masaru Taniguchi

  8. Chiba University Graduate School of Medicine, Chiba, Japan

    Masaru Taniguchi

  9. La Jolla Institute for Allergy and Immunology, San Diego, CA, USA

    Kronenberg Mitchell

  10. Department of Gastoroenterology, Osaka City University Graduate School of Medicine, Osaka, Japan

    Tetsuo Arakawa

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  1. Hideki Fujii
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Correspondence to Hideki Fujii.

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An erratum to this article is available at http://dx.doi.org/10.1007/s00428-006-0270-x.

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Fujii, H., Seki, S., Kobayashi, S. et al. A murine model of NKT cell-mediated liver injury induced by alpha-galactosylceramide/d-galactosamine. Virchows Arch 446, 663–673 (2005). https://doi.org/10.1007/s00428-005-1265-8

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