Skip to main content

Transfection of Indoleamine 2,3 Dioxygenase in Primary Endothelial Cells

  • Protocol
  • First Online:
T-Cell Trafficking

Part of the book series: Methods in Molecular Biology ((MIMB,volume 616))

  • 1148 Accesses

Abstract

The endothelial cell plays a central role in the control of inflammatory processes. The recruitment of inflammatory leucocytes into the blood vessels is controlled by the expression of adhesion molecules on the endothelium as well as the secretion and presentation of chemokines. Indoleamine 2,3 dioxygenase (IDO) is an enzyme that metabolises tryptophan and is known to be central in the regulation of immune responses. IDO can be expressed on endothelial cells and can alter T-cell responses to the endothelium. We show that IDO can be readily transfected in primary endothelial cells and detail a method to determine the activity of the transfected protein.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Nourshargh S, Marelli-Berg FM. (2005) Transmigration through venular walls: a key regulator of leukocyte phenotype and function. Trends Immunol 26(3), 157–65.

    Article  PubMed  CAS  Google Scholar 

  2. Choi J, Enis DR, Koh KP, Shiao SL, Pober JS. (2004) T lymphocyte-endothelial cell interactions. Annu Rev Immunol 22, 683–709.

    Article  PubMed  CAS  Google Scholar 

  3. Savage CO, Hughes CC, McIntyre BW, Picard JK, Pober JS. (1993) Human CD4+ T cells proliferate to HLA-DR+ allogeneic vascular endothelium. Identification of accessory interactions. Transplantation 56(1), 128–34.

    Article  PubMed  CAS  Google Scholar 

  4. Page CS, Holloway N, Smith H, Yacoub M, Rose ML. (1994) Alloproliferative responses of purified CD4+ and CD8+ T cells to endothelial cells in the absence of contaminating accessory cells. Transplantation 57(11), 1628–37.

    PubMed  CAS  Google Scholar 

  5. Ma W, Pober JS. (1998) Human endothelial cells effectively costimulate cytokine production by, but not differentiation of, naive CD4+ T cells. J Immunol 161(5), 2158–67.

    PubMed  CAS  Google Scholar 

  6. Berg LP, James MJ, Alvarez-Iglesias M, Glennie S, Lechler RI, Marelli-Berg FM. (2002) Functional consequences of noncognate interactions between CD4+ memory T lymphocytes and the endothelium. J Immunol 168(7), 3227–34.

    PubMed  CAS  Google Scholar 

  7. Marelli-Berg FM, Frasca L, Weng L, Lombardi G, Lechler RI. (1999) Antigen recognition influences transendothelial migration of CD4+ T cells. J Immunol 162(2), 696–703.

    PubMed  CAS  Google Scholar 

  8. Mellor AL, Munn DH. (2004) IDO expression by dendritic cells: tolerance and tryptophan catabolism. Nat Rev Immunol 4(10), 762–74.

    Article  PubMed  CAS  Google Scholar 

  9. Grohmann U, Fallarino F, Puccetti P. (2003) Tolerance, DCs and tryptophan: much ado about IDO. Trends Immunol 24(5), 242–8.

    Article  PubMed  CAS  Google Scholar 

  10. Lee GK, Park HJ, Macleod M, Chandler P, Munn DH, Mellor AL. (2002) Tryptophan deprivation sensitizes activated T cells to apoptosis prior to cell division. Immunology 107(4), 452–60.

    Article  PubMed  CAS  Google Scholar 

  11. Terness P, Bauer TM, Röse L, Dufter C, Watzlik A, Simon H, Opelz G. (2002) Inhibition of allogeneic T cell proliferation by indoleamine 2,3-dioxygenase-expressing dendritic cells: mediation of suppression by tryptophan metabolites. J Exp Med 196(4), 447–57.

    Article  PubMed  CAS  Google Scholar 

  12. Däubener W, Spors B, Hucke C, Adam R, Stins M, Kim KS, Schroten H. (2001) Restriction of Toxoplasma gondii growth in human brain microvascular endothelial cells by activation of indoleamine 2,3-dioxygenase. Infect Immun 69(10), 6527–31.

    Article  PubMed  Google Scholar 

  13. Hansen AM, Ball HJ, Mitchell AJ, Miu J, Takikawa O, Hunt NH. (2004) Increased expression of indoleamine 2,3-dioxygenase in murine malaria infection is predominantly localised to the vascular endothelium. Int J Parasitol 34(12), 1309–19.

    Article  PubMed  CAS  Google Scholar 

  14. Schroten H, Spors B, Hucke C, Stins M, Kim KS, Adam R, Däubener W. (2001) Potential role of human brain microvascular endothelial cells in the pathogenesis of brain abscess: inhibition of Staphylococcus aureus by activation of indoleamine 2,3-dioxygenase. Neuropediatrics 32(4), 206–10.

    Article  PubMed  CAS  Google Scholar 

  15. Adam R, Rüssing D, Adams O, Ailyati A, Sik Kim K, Schroten H, Däubener W. (2005) Role of human brain microvascular endothelial cells during central nervous system infection. Significance of indoleamine 2,3-dioxygenase in antimicrobial defence and immunoregulation. Thromb Haemost 94(2), 341–6.

    PubMed  CAS  Google Scholar 

  16. Frumento G, Rotondo R, Tonetti M, Damonte G, Benatti U, Ferrara GB. (2002) Tryptophan-derived catabolites are responsible for inhibition of T and natural killer cell proliferation induced by indoleamine 2,3-dioxygenase. J Exp Med 196(4), 459–68.

    Article  PubMed  CAS  Google Scholar 

  17. Beutelspacher SC, Tan PH, McClure MO, Larkin DF, Lechler RI, George AJ. (2006) Expression of indoleamine 2,3-dioxygenase (IDO) by endothelial cells: implications for the control of alloresponses. Am J Transplant 6(6), 1320–30.

    Article  PubMed  CAS  Google Scholar 

  18. Fallarino F, Grohmann U, Hwang KW, Orabona C, Vacca C, Bianchi R, Belladonna ML, Fioretti MC, Alegre ML, Puccetti P. (2003) Modulation of tryptophan catabolism by regulatory T cells. Nat Immunol 4(12), 1206–12.

    Article  PubMed  CAS  Google Scholar 

  19. Puccetti P, Grohmann U. (2007) IDO and regulatory T cells: a role for reverse signalling and non-canonical NF-kappaB activation. Nat Rev Immunol 7(10), 817–23.

    Article  PubMed  CAS  Google Scholar 

  20. Beutelspacher SC, Pillai R, Watson MP, Tan PH, Tsang J, McClure MO, George AJ, Larkin DF. (2006) Function of indoleamine 2,3-dioxygenase in corneal allograft rejection and prolongation of allograft survival by over-expression. Eur J Immunol 36(3), 690–700.

    Article  PubMed  CAS  Google Scholar 

  21. Witkiewicz A, Williams TK, Cozzitorto J, Durkan B, Showalter SL, Yeo CJ, Brody JR. (2008) Expression of indoleamine 2,3-dioxygenase in metastatic pancreatic ductal adenocarcinoma recruits regulatory T cells to avoid immune detection. J Am Coll Surg 206(5), 849–54.

    Article  PubMed  Google Scholar 

  22. Munn DH, Mellor AL. (2007) Indoleamine 2,3-dioxygenase and tumor-induced tolerance. J Clin Invest 117(5), 1147–54.

    Article  PubMed  CAS  Google Scholar 

  23. Fujigaki S, Saito K, Takemura M, Maekawa N, Yamada Y, Wada H, Seishima M. (2002) L-tryptophan-L-kynurenine pathway metabolism accelerated by Toxoplasma gondii infection is abolished in gamma interferon-gene-deficient mice: cross-regulation between inducible nitric oxide synthase and indoleamine-2,3-dioxygenase. Infect Immun 70(2), 779–86.

    Article  PubMed  CAS  Google Scholar 

  24. Oh GS, Pae HO, Choi BM, Chae SC, Lee HS, Ryu DG, Chung HT. (2004) 3-Hydroxyanthranilic acid, one of metabolites of tryptophan via indoleamine 2,3-dioxygenase pathway, suppresses inducible nitric oxide synthase expression by enhancing heme oxygenase-1 expression. Biochem Biophys Res Commun 320(4), 1156–62.

    Article  PubMed  CAS  Google Scholar 

  25. Tan PH, Chan C, Xue SA, Dong R, Ananthesayanan B, Manunta M, Kerouedan C, Cheshire NJW, Wolfe JH, Haskard DO, Taylor KM, George AJT. (2004) Phenotypic and functional differences between human saphenous vein (HSVEC) and umbilical vein (HUVEC) endothelial cells. Atherosclerosis 173, 171–83.

    Article  PubMed  CAS  Google Scholar 

  26. Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, Warren JT, Bokesch H, Kenney S, Boyd MR. (1990) New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst 82(13), 1107–12.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2010 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Mouratidis, P.X.E., George, A.J.T. (2010). Transfection of Indoleamine 2,3 Dioxygenase in Primary Endothelial Cells. In: Marelli-Berg, F., Nourshargh, S. (eds) T-Cell Trafficking. Methods in Molecular Biology, vol 616. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-461-6_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-60761-461-6_10

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60761-460-9

  • Online ISBN: 978-1-60761-461-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics