, Volume 9, Issue 4, pp 495–500 | Cite as

Phosphatidylserine exposure during early primary necrosis (oncosis) in JB6 cells as evidenced by immunogold labeling technique

  • O. Krysko
  • L. de Ridder
  • M. Cornelissen


Apoptotic cell death is characterized by the early exposure of phosphatidylserine (PS) at the outer surface of the plasma membrane. The aim of the present study was to examine whether PS exposure also occurs during oncosis (early primary necrosis) and to localize PS at the subcellular level, applying a pre-embedding immunogold labeling technique with biotin conjugated annexin V. The issue was addressed by using caspase-8 deficient, Bcl-2 overexpressing JB6 cells, which die by oncosis when stimulated with synthetic dsRNA. We observed by fluorescence microscopy that oncotic cells with preserved plasma membrane integrity showed PS exposure (annexin+/propidium iodide). The data was confirmed on the ultrastructural level and PS was localized in oncosis at the outer leaflet of the continuous plasma membrane with preserved trilamellar structure. In postoncotic necrotic cells the immunogold labels were found on the plasma membrane and on the intracellular membranes of the cells, which underwent plasma membrane disruption. In conclusion, this study reveals that PS externalization occurs not only in apoptosis but also in oncosis at least in our cell model system.

immunogold labeling oncosis phosphatidylserine externalization ultrastructure 


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  1. 1.
    Wyllie AH, Kerr JFR, Currie AR. Cell death: The significance of apoptosis. Int Rev Cytol 1980; 68: 251–306.PubMedGoogle Scholar
  2. 2.
    Majno G, Joris I. Apoptosis, oncosis and necrosis. An overview of the cell death. Am J Path 1995; 146: 3–15.PubMedGoogle Scholar
  3. 3.
    Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled Annexin V. J Immunol Methods 1995; 184: 39–51.CrossRefPubMedGoogle Scholar
  4. 4.
    Schlegel RA, Williamson P. Phosphatidylserine, a death knell. Cell Death Differ 2001; 8: 551–563.CrossRefPubMedGoogle Scholar
  5. 5.
    Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, Henson PM. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol 1992; 148: 2207–2216.PubMedGoogle Scholar
  6. 6.
    Fadok VA, Bratton DL, Rose DM, Pearson A, Ezekewitz RA, Henson PM. A receptor for phosphatidylserine-specific clearance of apoptotic cells. Nature 2000; 405: 85–90.PubMedGoogle Scholar
  7. 7.
    Geske FJ, Monks J, Lehman L, Fadok VA. The role of the macrophage in apoptosis: Hunter, gatherer, and regulator. Int J Hematol 2002; 76: 16–26.PubMedGoogle Scholar
  8. 8.
    van den Eijnde SM, Luijsterburg AJM, Boshart L, et al. In situ detection of apoptosis during embryogenesis with annexin V: From whole mount to ultrastructure. Cytometry 1997; 29: 313–320.PubMedGoogle Scholar
  9. 9.
    Pellicciari C, Bottone MG, Biggiogera M. Detection of apoptotic cells by Annexin V labeling at electron microcopy. Eur J Histochem 1997; 41: 211–216.PubMedGoogle Scholar
  10. 10.
    Schrevens A, van Nassauw L, Harrisson F. Histochemical demonstration of apoptotic cells in the chicken embryo using annexin V. Histochem J 1998; 30: 917–922.PubMedGoogle Scholar
  11. 11.
    Cornelissen M, Philippe J, De Sitter S, de Ridder L. Annexin V expression in apoptotic peripheral blood lymphocytes: An electron microscopic evaluation. Apoptosis 2002; 7: 41–47.PubMedGoogle Scholar
  12. 12.
    Kalai M, Van Loo G, Vanden Berghe T, et al. Tipping the balance between necrosis and apoptosis in human and murine cells treated with interferon and dsRNA. Cell Death Differ 2002; 9: 981–994.PubMedGoogle Scholar
  13. 13.
    Kawahara A, Ohsawa Y, Matsumura H, Uchiyama Y, Nagata S. 1998. Caspase independent cell killing by Fas-Associated protein with death domain. J Cell Biol 143: 1353–1360.PubMedGoogle Scholar
  14. 14.
    Matsumura H, Shimizu Y, Ohsawa Y, Kawahara A, Uchiyama Y, Nagata S. Necrotic death pathway in Fas receptor signaling. J Cell Biol 2000; 151: 1247–1255.PubMedGoogle Scholar
  15. 15.
    Van Engeland M, Nieland LJW, Ramaekers FCS, Schutte B, Reutelingsperger CPM. Annexin V-affinity assay: A review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry 1998; 31: 1–9.PubMedGoogle Scholar
  16. 16.
    Verhoven B, Krahling S, Schlegel RA, Williamson P. Regulation of phosphatidylserine exposure and phagocytosis of apoptotic T lymphocytes. Cell Death Differ 1999; 6: 262–270.PubMedGoogle Scholar
  17. 17.
    Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST, Oers MH. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 1994; 84: 1415–1420.PubMedGoogle Scholar
  18. 18.
    Hirt UA, Leist M. Rapid, noninflammatory and PS-dependent phagocytic clearance of necrotic cells. Cell Death Differ 2003; 10: 1156–1164.PubMedGoogle Scholar
  19. 19.
    Krysko DV, Brouckaert G, Kalai M, et al. Mechanisms of internalization of apoptotic and necrotic L929 cells by a macrophage cell line studied by electron microscopy. J Morphol 2003; 258: 336–345.PubMedGoogle Scholar
  20. 20.
    Henson PM, Bratton DL, Fadok VA. Apoptotic cell removal. Curr Biol 2001; 11: R795–R805.PubMedGoogle Scholar
  21. 21.
    Waring P, Lambert D, Sjaarda A, Hurne A, Beaver J. Increased cell surface exposure of phosphatidylserine on propidium iodide negative thymocytes undergoing death by necrosis. Cell Death Diff 1999; 6: 624–637.Google Scholar
  22. 22.
    Bonneau MJ, Poulin R. Spermine oxidation leads to necrosis with plasma membrane phosphatidylserine redistribution in mouse leukemia cells. Exp Cell Res 2000; 259: 23–34.PubMedGoogle Scholar
  23. 23.
    Lecoueur H, Prevost MC, Gougeon ML. Oncosis is associated with exposure of phosphatidylserine residues on the outside layer of the plasma membrane: A reconsideration of the specificity of the annexin V/propidium iodide assay. Cytometry 2001; 44: 65–72.PubMedGoogle Scholar
  24. 24.
    Lecoeur H, de Oliveira-Pinto LM, Gougeon ML. Multiparametric flow cytometric analysis of biochemical and functional events associated with apoptosis and oncosis using the 7-aminoactinomycin D assay. J Immunol Methods 2002; 265: 81–96.PubMedGoogle Scholar
  25. 25.
    Leist M, Single B, Castoldi AF, Kuhne S, Nicotera P. Intracellular adenosine triphosphate (ATP) concentrations: A switch in the decision between apoptosis and necrosis. J Exp Med 1997; 185: 1481–1486.PubMedGoogle Scholar
  26. 26.
    Martin JS, Reutelingsperger CP, McGahon AJ, et al. Early redistribution of plasma membrane phossphatidylserine is a general feature of apoptosis regardless of initiating stimulus: Inhibition by overexpression of Bcl-2 and Abl. J Exp Med 1995; 182: 1545–1556.PubMedGoogle Scholar
  27. 27.
    Mateo V, Lagneaux L, Bron D, et al. CD47 ligation induces caspase-independent cell death in chronic lymphocytic leukemia. Nature Medicine 1999; 5: 1277–1284.PubMedGoogle Scholar
  28. 28.
    Mateo V, Brown EJ, Biron G, et al. Mechanisms of CD47-induced caspase-independent cell death in normal and leukemic cells: Link between phosphatidylserine exposure and cytoskeleton organization. Blood 2002; 100: 2882–2890.PubMedGoogle Scholar
  29. 29.
    Kagan VE, Borisenko GG, Serinkan BF, et al. Appetizing rancidity of apoptotic cells for macrophages: Oxidation, externalisation, and recognition of phosphatidylserine. Am J Physiol Lung Cell Mol Physiol 2003; 285: L1–L17.PubMedGoogle Scholar
  30. 30.
    Hamon Y, Broccardo C, Chambenoit O, et al. ABC1 promotes engulfment of apoptotic cells and transbilayer redistribution of phosphatidylserine. Nat Cell Biol 2000; 2: 399–406.PubMedGoogle Scholar
  31. 31.
    Trump BF, Berezesky I, Chang Shand Phelps PC. The pathways of the cell death: Oncosis, apoptosis and necrosis. Toxicologic Path 1997; 25: 82–88.Google Scholar

Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  • O. Krysko
    • 1
  • L. de Ridder
    • 2
  • M. Cornelissen
    • 2
  1. 1.Laboratory of Clinical ChemistryFaculty of Pharmaceutical SciencesK.U. LeuvenBelgium
  2. 2.Department of Human Anatomy, Embryology, Histology and Medical PhysicsGhentBelgium

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