Inflammation Research

, Volume 44, Issue 11, pp 475–481 | Cite as

Metallothionein induction in cultured rat hepatocytes by arthritic rat serum, activated macrophages, interleukin-6, interleukin-11 and leukaemia inhibitory factor

  • P. Coyle
  • J. C. Philcox
  • A. M. Rofe


Potential mediators of hepatic metallothionein (MT) synthesis in adjuvant-induced arthritis were investigated in cultured rat hepatocytes. Sera from arthritic rats (14 d post-adjuvant treatment) in the presence of Zn (50 µmol/L) + dexamethasone (Dex; 1 µmol/L) increased metallothionein (MT) accumulation by 34% above that obtained with control rat serum with Zn + Dex. Endogenous IL-6 activity in serum from arthritic rats was 93 ± 49 U/mL and was undetectable in control rat serum. The activities of TNF, IL-1 and corticosterone concentrations were the same in control and arthritic rats. The accumulation of MT in hepatocytes in the presence of Zn (10 µmol/L) + Dex (1 µmol/L) was enhanced 29% and 49% by media from lipopolysaccharide (LPS)-stimulated peritoneal macrophage (PMM) and Kupffer cell cultures (KCM), respectively. The response with PMM and KCM was quantitatively the same as that with interleukin-6 (IL-6). Analysis of PMM and KCM showed activities of 1,000–10,000 U/mL for IL-6, 100–1000 U/mL for TNF and <10,000 U/mL for IL-1, the latter detected only in PMM. LPS alone enhanced the accumulation of MT above Zn + Dex in a dose dependent manner. A significant LPS response was obtained at 5 mg/L with a maximal stimulation above Zn + Dex of 38% at 10 mg/L. This direct stimulation of MT by LPS was not part of the response observed with PMM and KCM where the final LPS concentration in culture was only 0.1 mg/L. Other cytokines capable of synergy with Zn + Dex on MT synthesis were investigated. Interleukin-11 (IL-11) increased the Zn + Dex induction in a dose dependent manner with maximal stimulation at 100 U/mL of 40%. A small stimulation of 12% above Zn + Dex was obtained with leukaemia inhibitory factor (LIF) at concentrations greater than 100 U/mL. No enhancement of the Zn + Dex response was obtained with interleukin-3 (1000 U/mL), interleukin-4 (10 µg/L), platelet activating factor (5 nmol/L) or granulocyte-colony stimulating factor (5 µg/L). Neither IL-11 nor LIF enhanced the response obtained with Zn + Dex + IL-6. The results demonstrate that mediators present in arthritic rat serum and in LPS-stimulated PMM and KCM cause a quantitatively similar response on MT accumulation as IL-6. IL-11 and to a lesser extent LIF, are also potential mediators of MT synthesis in inflammation.

Key words

Rats Metallothionein LPS Cytokines Hepatocytes 



















kupffer cell media


leukaemia inhibitory factor


granulocyte-colony stimulating factor






platelet activating factor


peritoneal macrophage media


tumour necrocis factor-α


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. [1]
    Cousins RJ. Absorption, transport and hepatic metabolism of copper and zinc: special reference to metallothionein and caeruloplasmin. Physiol Rev 1985;65:238–309.PubMedGoogle Scholar
  2. [2]
    Bremner I, Beattie JH. Metallothionein and the trace minerals. Ann Rev Nutr 1990;10:63–83.Google Scholar
  3. [3]
    Rofe AM, Philcox JC, Haynes DR, Whitehouse MW, Coyle P. Changes in plasma zinc, copper, iron, and hepatic metallothionein in adjuvant-induced arthritis treated with cyclosporin. Biol Trace Elem Res 1992;34:237–48.PubMedGoogle Scholar
  4. [4]
    Rofe AM, Philcox JC, Haynes DR and Coyle P. Wasting in adjuvant-induced arthritis and its relationship to plasma zinc, copper and liver metallothionein. Agents Actions 1994;40:60–2.Google Scholar
  5. [5]
    Leisten JC, Gaarde WA, Scholz W. Interleukin-6 serum levels correlate with footpad swelling in adjuvant-induced arthritic lewis rats treated with cyclosporin A or indomethacin. Clin Immunol Immunopathol 1990;56:108–15.PubMedGoogle Scholar
  6. [6]
    Thiesen-Pop P, Pape H, Müller-Peddinghaus R. Interleukin-6 (IL-6) in adjuvant arthritis of rats and its pharmacological modulations. Int J Immunopharmacol 1992;14:565–71.PubMedGoogle Scholar
  7. [7]
    Schroeder JJ, Cousins RJ. Interleukin-6 regulates metallothionein gene expression and zinc metabolism in hepatocyte monolayer cultures. Proc Natl Acad Sci USA 1990;87:3137–41.PubMedGoogle Scholar
  8. [8]
    Coyle P, Philcox JC, Rofe AM. Metallothionein induction in freshly isolated rat hepatocytes. Biol Trace Elem Res 1993;36:35–49.PubMedGoogle Scholar
  9. [9]
    Coyle P, Philcox JC, Rofe AM. Corticosterone enhances the zinc and interleukin-6-mediated induction of metallothionein in cultured rat hepatocytes. J Nutr 1993;123:1464–70.PubMedGoogle Scholar
  10. [10]
    Gauldie J, Richards C, Harnish D, Lansorp P, Baumann H. Interferonβ-2/B-cell stimulating factor type 2 shares identity with monocyte-derived hepatocyte-stimulating factor and regulates the major acute phase protein response in liver cell. Proc Natl Acad Sci USA 1987;84:7251–5.PubMedGoogle Scholar
  11. [11]
    Koj A. The role of interleukin-6 as the hepatocyte stimulating factor in the network of inflammatory cytokines. Ann NY Acad Sci 1989;557:1–8.Google Scholar
  12. [12]
    Baumann H, Schendel P. Interleukin-11 regulates the hepatic expression of the same plasma protein genes as interleukin-6. J Biol Chem 1991;266:20424–7.PubMedGoogle Scholar
  13. [13]
    Yang YC, Yin T. Interleukin-11 and its receptors. Biofactors 1992;4:15–21.PubMedGoogle Scholar
  14. [14]
    Richards CD, Brown TJ, Shoyab M, Baumann H, Gauldie J. Recombinant oncostatin M stimulates the production of acute phase proteins in HepG2 cells and rat primary hepatocytes in vitro. J Immunol 1992;148:1731–6.PubMedGoogle Scholar
  15. [15]
    Kordula T, Rokita H, Koj A, Fiers W, Gauldie J, Baumann H. Effects of interleukin-6 and leukaemia inhibitory factor on the acute phase response and DNA synthesis in cultured rat hepatocytes. Lymphokine Cytokine Res 1991;10:23–6.PubMedGoogle Scholar
  16. [16]
    Fey GH, Hattori M, Northemann W, Abraham LJ, Baumann MF, Braciak TA, et al. Regulation of rat liver acute phase genes by IL-6 and production of a hepatocyte stimulating factor by rat hepatoma cells. Am NY Acad Sci 1989;557:317–31.Google Scholar
  17. [17]
    Northemann W, Braciak TA, Hattori M, Lee F, Fey GH. Structure of the rat interleukin-6 gene and its expression in macrophage-derived cells. J Biol Chem 1989;264:16072–82.PubMedGoogle Scholar
  18. [18]
    Castell JV, Andus T, Kuns D, Heinrich PC. Interleukin-6: The major regulator of acute-phase protein synthesis in man and rat. Ann NY Acad Sci 1989;557:87–102.PubMedGoogle Scholar
  19. [19]
    West MA, Billiar TR, Mazuski JE, Curran RD, Cerra FB, Simmons RL. Endotoxin modulation of hepatocyte secretory and cellular protein synthesis is mediated by Kupffer cells. Arch Surg 1988;123:1400–5.PubMedGoogle Scholar
  20. [20]
    Wardle EN. Kupffer cells and their function. Liver 1987;7:63–75.PubMedGoogle Scholar
  21. [21]
    Rofe AM, James HM, Bais R, Edwards JB, Conyers RAJ. The production of (14C)oxalate during the metabolism of (14C) carbohydrates in isolated rat hepatocytes. Aust J Exp Biol Med Sci 1980;58:103–16.PubMedGoogle Scholar
  22. [22]
    Eaton LD, Toal BF. Evaluation of the Cd/hemoglobin assay for the rapid determination of metallothionein in biological tissues. Toxicol Appl Pharmacol 1982;66:134–42.PubMedGoogle Scholar
  23. [23]
    Smedsrod B, Pertoft H, Eggertsen G, Sundstrom C. Functional and morphological characterization of cultures of Kupffer cells and liver endothelial cells prepared by means of density separation in percoll, and selective substrate adherence. Cell Tissue Res 1985;241:639–49.PubMedGoogle Scholar
  24. [24]
    Haynes DR, Garrett IR, Vernon-Roberts B. Effect of gold salt treatment on receptor binding activity of monocytes and macrophages isolated from rats with adjuvant arthritis. Rheumatol Int 1988;8:159–64.PubMedGoogle Scholar
  25. [25]
    Haylock DN, To LB, Dowse TL, Juttner CA, Simmons PJ. Ex vivo expansion and maturation of peripheral blood CD34+cells into the myeloid lineage. Blood 1992;80:1405–12.PubMedGoogle Scholar
  26. [26]
    Thornhill MH, Haskard DO. IL-4 regulates endothelial cell activation by IL-1, tumor necrosis factor or INF-γ. J Immunol 1990;145:865–72.PubMedGoogle Scholar
  27. [27]
    Hamer DH. Metallothionein. Ann Rev Biochem 1986;55:913–51.PubMedGoogle Scholar
  28. [28]
    Cousins RJ. Absorption, transport, and hepatic metabolism of copper and zinc: special reference to metallothionein and caeruloplasmin. Physiol Rev 1985;65:238–309.PubMedGoogle Scholar
  29. [29]
    Berry MN, Edwards AM, Barritt GJ. Isolated hepatocytes preparation, properties and applications. In: Burdon RH, van Knippenberg PH editors. Amsterdam, New York and Oxford: Elsevier, 1991:460.Google Scholar
  30. [30]
    Baumann H, Gauldie J. Regulation of hepatic acute phase plasma protein genes by hepatocyte stimulating factors and other mediators of inflammation. Mol Biol Med 1990;7:147–59.PubMedGoogle Scholar
  31. [31]
    Castell JV, Gomez-Lechon MJ, David M, Fabra R, Trullenque R, Heinrich PC. Acute-phase response of human hepatocytes: Regulation of acute-phase protein synthesis by interleukin-6. Hepatol 1990;12:1179–86.Google Scholar
  32. [32]
    Fey GH, Hattori M, Hocke G, Brechner T, Baffat G, Baumann M, et al. Gene regulation by interleukin-6. Biochimie 1991;73:47–50.PubMedGoogle Scholar
  33. [33]
    De SK, McMaster MT, Andrews GK. Endotoxin induction of murine metallothionein gene expression. J Biol Chem 1990;265:15267–74.PubMedGoogle Scholar
  34. [34]
    Durnam DM, Hoffman JS, Quaife CJ, Benditt EP, Chen HY, Brinster RL, et al. Induction of mouse metallothionein-1 mRNA by bacterial endotoxin is independent of metals and glucocorticoid hormones. Proc Natl Acad Sci USA 1984;81:1053–6.PubMedGoogle Scholar
  35. [35]
    Liu J, Liu YP, Sendelbach LE, Klaassen CD. Endotoxin induction of hepatic metallothionein is mediated through cytokines. Toxicol Appl Pharmacol 1991;109:235–40.PubMedGoogle Scholar
  36. [36]
    Friedman RL, Stark GR.α-Interferon-induced transcription of HLA and metallothionein genes containing homologous upstream sequences. Nature 1985;314:637–9.PubMedGoogle Scholar
  37. [37]
    Kusari J, Tiwari RK, Kumar R, Sen GC. Expression of interferon-inducible genes in RD-114 cells. J Virol 1987;61:1524–31.PubMedGoogle Scholar
  38. [38]
    Rietschel ET, Kirikie T, Schade FU, Mamat U, Schmidt G, Loppnow H, Ulmer AJ, Zahringer U, Seydel U, Padova FD, Schreier M, Brade H. Bacterial endotoxin: molecular relationships of structure to activity and function. FASEB J 1994;8:217–25.PubMedGoogle Scholar
  39. [39]
    Portoles MT, Ainaga MJ, Pagani R. The induction of lipid peroxidation by E. coli lipolysaccharide on rat hepatocytes as an important factor in the etiology of endotoxic liver damage. Biochim Biophys Acta 1993;1158:287–92.PubMedGoogle Scholar
  40. [40]
    Baumann H, Wong GG. Hepatocyte-stimulating factor III shares structural and function identity with leukemia inhibitory factor. J Immunol 1989;143:1163–7.PubMedGoogle Scholar
  41. [41]
    Gasull T, Giralt M, Hernandez J, Martinez P, Bremner I, Hidalgo J. Regulation of metallothionein concentrations in rat brain: Effect of glucocorticoids, zinc, copper and endotoxin. Am J Physiol 1994;255:E760–7.Google Scholar

Copyright information

© Birkhäuser Verlag, Basel 1995

Authors and Affiliations

  • P. Coyle
    • 1
  • J. C. Philcox
    • 1
  • A. M. Rofe
    • 1
  1. 1.Division of Clinical BiochemistryInstitute of Medical and Veterinary ScienceAdelaideAustralia

Personalised recommendations