Advertisement

Detection of Cell Membrane-Bound CD46 Using Flow Cytometry

  • Martin Kolev
  • Claudia Kemper
Part of the Methods in Molecular Biology book series (MIMB, volume 1100)

Abstract

CD46 is an important regulator of the complement system by preventing unwanted deposition of the complement activation products and opsonins C3b/C4b onto self-tissue. Recently, intracellular signals mediated by CD46 activation on several distinct human cell types have demonstrated that CD46 also plays decisive roles in immuneregulation. The growing recognition of CD46 as key regulator in several vital biological processes, led to increased demand in sensitive methods for monitoring CD46 expression and changes thereof on cells and in tissues. Here we describe a method, which allows for studying CD46 expression on the surface of cells using specific antibodies in combination with fluorescence-activated cell sorting (FACS) analysis.

Key words

CD46 MCP Complement Flow cytometry Antibodies 

Notes

Acknowledgements

This work is supported by an MRC Research Grant (Grant no. G1002165 awarded to CK), the Medical Research Council Centre for Transplantation, Guy’s Hospital, King’s College and the Department of Health, National Institute for Health Research comprehensive Biomedical Research Centre award to Guy’s & St. Thomas’ NHS Foundation Trust in partnership with King’s College London and King’s College Hospital NHS Foundation Trust.

References

  1. 1.
    Purcell D, Deacon N, Andrew S, McKenzie I (1990) Human non-lineage antigen, CD46 (HuLy-m5): purification and partial sequencing demonstrates structural homology with complement-regulating glycoproteins. Immunogenetics 31:21–28PubMedCrossRefGoogle Scholar
  2. 2.
    Liszewski M, Post T, Atkinson J (1991) Membrane cofactor protein (MCP or CD46): newest member of the regulators of complement activation gene cluster. Annu Rev Immunol 9:431–455PubMedCrossRefGoogle Scholar
  3. 3.
    Liszewski MK, Atkinson JP (1996) Membrane cofactor protein (MCP; CD46). Isoforms differ in protection against the classical pathway of complement. J Immunol 156:4415–4421PubMedGoogle Scholar
  4. 4.
    Hakulinen J, Junnikkala S, Sorsa T, Meri S (2004) Complement inhibitor membrane cofactor protein (MCP; CD46) is constitutively shed from cancer cell membranes in vesicles and converted by a metalloproteinase to a functionally active soluble form. Eur J Immunol 34:2620–2629PubMedCrossRefGoogle Scholar
  5. 5.
    Seya T, Hara T, Iwata K, Kuriyama S, Hasegawa T, Nagase Y et al (1995) Purification and functional properties of soluble forms of membrane cofactor protein (CD46) of complement: identification of forms increased in cancer patients’ sera. Int Immunol 7:727–736PubMedCrossRefGoogle Scholar
  6. 6.
    Kawano M (2000) Complement regulatory proteins and autoimmunity. Arch Immunol Ther Exp (Warsz) 48:367–372Google Scholar
  7. 7.
    Fett AL, Hermann MM, Muether PS, Kirchhof B, Fauser S (2012) Immunohistochemical localization of complement regulatory proteins in the human retina. Histol Histopathol 27:357–364PubMedGoogle Scholar
  8. 8.
    Saunders R, Abarrategui-Garrido C, Frémeaux-Bacchi V, Goicoechea de Jorge E, Goodship T, López Trascasa M et al (2007) The interactive Factor H-atypical hemolytic uremic syndrome mutation database and website: update and integration of membrane cofactor protein and Factor I mutations with structural models. Hum Mutat 28(3):222–234PubMedCrossRefGoogle Scholar
  9. 9.
    Westra D, Volokhina E, van der Heijden E, Vos A, Huigen M, Jansen J et al (2010) Genetic disorders in complement (regulating) genes in patients with atypical haemolytic uraemic syndrome (aHUS). Nephrol Dial Transplant 25:2195–2202PubMedCrossRefGoogle Scholar
  10. 10.
    Riley RC, Kemper C, Leung M, Atkinson JP (2002) Characterization of human membrane cofactor protein (MCP; CD46) on spermatozoa. Mol Reprod Dev 62:534–546PubMedCrossRefGoogle Scholar
  11. 11.
    Kawamoto A, Ohashi K, Kishikawa H, Zhu LQ, Azuma C, Murata Y (1999) Two-color fluorescence staining of lectin and anti-CD46 antibody to assess acrosomal status. Fertil Steril 71:497–501PubMedCrossRefGoogle Scholar
  12. 12.
    Ruseva MM, Hughes TR, Donev RM, Sivasankar B, Pickering MC, Wu X et al (2009) Crry deficiency in complement sufficient mice: C3 consumption occurs without associated renal injury. Mol Immunol 46:803–811PubMedCrossRefGoogle Scholar
  13. 13.
    Segerman A, Atkinson J, Marttila M, Dennerquist V, Wadell G, Arnberg N (2003) Adenovirus type 11 uses CD46 as a cellular receptor. J Virol 77:9183–9191PubMedCrossRefGoogle Scholar
  14. 14.
    Santoro F, Greenstone H, Insinga A, Liszewski M, Atkinson J, Lusso P et al (2003) Interaction of glycoprotein H of human herpesvirus 6 with the cellular receptor CD46. J Biol Chem 278:25964–25969PubMedCrossRefGoogle Scholar
  15. 15.
    Cattaneo R (2004) Four viruses, two bacteria, and one receptor: membrane cofactor protein (CD46) as pathogens’ magnet. J Virol 78:4385–4388PubMedCrossRefGoogle Scholar
  16. 16.
    Astier A, Trescol-Biémont M, Azocar O, Lamouille B, Rabourdin-Combe C (2000) Cutting edge: CD46, a new costimulatory molecule for T cells, that induces p120CBL and LAT phosphorylation. J Immunol 164:6091–6095PubMedGoogle Scholar
  17. 17.
    Kemper C, Chan A, Green J, Brett K, Murphy K, Atkinson J (2003) Activation of human CD4+ cells with CD3 and CD46 induces a T-regulatory cell 1 phenotype. Nature 421:388–392PubMedCrossRefGoogle Scholar
  18. 18.
    Cardone J, Le Friec G, Vantourout P, Roberts A, Fuchs A, Jackson I et al (2010) Complement regulator CD46 temporally regulates cytokine production by conventional and unconventional T cells. Nat Immunol 11:862–871PubMedCrossRefGoogle Scholar
  19. 19.
    Le Friec G, Sheppard D, Whiteman P, Karsten CM, Al-Tilib Shamoun S et al (2012) The novel Jagged1 and CD46 interaction on T lymphocytes mediates a critical defence mechanism of human adaptive Th1 immunity. Nat Immunol 13:1213–1221PubMedCrossRefGoogle Scholar
  20. 20.
    Astier AL, Meiffren G, Freeman S, Hafler DA (2006) Alterations in CD46-mediated Tr1 regulatory T cells in patients with multiple sclerosis. J Clin Invest 116:3252–3257PubMedCrossRefGoogle Scholar
  21. 21.
    Cardone J, Al-Shouli S, Kemper C (2011) A novel role for CD46 in wound repair. Front Immunol 2:28PubMedCrossRefGoogle Scholar
  22. 22.
    Hofman P, Hsi B, Manie S, Fenichel P, Thyss A, Rossi B (1994) High expression of the antigen recognized by the monoclonal antibody GB24 on human breast carcinomas: a preventive mechanism of malignant tumor cells against complement attack? Breast Cancer Res Treat 32:213–219PubMedCrossRefGoogle Scholar
  23. 23.
    Cho SW, Oglesby TJ, Hsi BL, Adams EM, Atkinson JP (1991) Characterization of three monoclonal antibodies to membrane co-factor protein (MCP) of the complement system and quantification of MCP by radioassay. Clin Exp Immunol 83:257–261PubMedCrossRefGoogle Scholar
  24. 24.
    Manchester M, Valsamakis A, Kaufman R, Liszewski MK, Alvarez J, Atkinson JP et al (1995) Measles virus and C3 binding sites are distinct on membrane cofactor protein (CD46). Proc Natl Acad Sci U S A 92:2303–2307PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Martin Kolev
    • 1
  • Claudia Kemper
    • 1
  1. 1.Division of Transplantation Immunology and Mucosal Biology, MRC Centre for TransplantationKing’s College London, Guy’s HospitalLondonUK

Personalised recommendations