Skip to main content
PMC home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1995 Nov 1;182(5):1545–1556. doi: 10.1084/jem.182.5.1545

Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl

PMCID: PMC2192182  PMID: 7595224

Abstract

A critical event during programmed cell death (PCD) appears to be the acquisition of plasma membrane (PM) changes that allows phagocytes to recognize and engulf these cells before they rupture. The majority of PCD seen in higher organisms exhibits strikingly similar morphological features, and this form of PCD has been termed apoptosis. The nature of the PM changes that occur on apoptotic cells remains poorly defined. In this study, we have used a phosphatidylserine (PS)-binding protein (annexin V) as a specific probe to detect redistribution of this phospholipid, which is normally confined to the inner PM leaflet, during apoptosis. Here we show that PS externalization is an early and widespread event during apoptosis of a variety of murine and human cell types, regardless of the initiating stimulus, and precedes several other events normally associated with this mode of cell death. We also report that, under conditions in which the morphological features of apoptosis were prevented (macromolecular synthesis inhibition, overexpression of Bcl-2 or Abl), the appearance of PS on the external leaflet of the PM was similarly prevented. These data are compatible with the notion that activation of an inside-outside PS translocase is an early and widespread event during apoptosis.

Full Text

The Full Text of this article is available as a PDF (1.2 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andree H. A., Reutelingsperger C. P., Hauptmann R., Hemker H. C., Hermens W. T., Willems G. M. Binding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers. J Biol Chem. 1990 Mar 25;265(9):4923–4928. [PubMed] [Google Scholar]
  2. Bevers E. M., Tilly R. H., Senden J. M., Comfurius P., Zwaal R. F. Exposure of endogenous phosphatidylserine at the outer surface of stimulated platelets is reversed by restoration of aminophospholipid translocase activity. Biochemistry. 1989 Mar 21;28(6):2382–2387. doi: 10.1021/bi00432a007. [DOI] [PubMed] [Google Scholar]
  3. Brunner T., Mogil R. J., LaFace D., Yoo N. J., Mahboubi A., Echeverri F., Martin S. J., Force W. R., Lynch D. H., Ware C. F. Cell-autonomous Fas (CD95)/Fas-ligand interaction mediates activation-induced apoptosis in T-cell hybridomas. Nature. 1995 Feb 2;373(6513):441–444. doi: 10.1038/373441a0. [DOI] [PubMed] [Google Scholar]
  4. Cohen J. J., Duke R. C., Fadok V. A., Sellins K. S. Apoptosis and programmed cell death in immunity. Annu Rev Immunol. 1992;10:267–293. doi: 10.1146/annurev.iy.10.040192.001411. [DOI] [PubMed] [Google Scholar]
  5. Dachary-Prigent J., Freyssinet J. M., Pasquet J. M., Carron J. C., Nurden A. T. Annexin V as a probe of aminophospholipid exposure and platelet membrane vesiculation: a flow cytometry study showing a role for free sulfhydryl groups. Blood. 1993 May 15;81(10):2554–2565. [PubMed] [Google Scholar]
  6. Darzynkiewicz Z., Bruno S., Del Bino G., Gorczyca W., Hotz M. A., Lassota P., Traganos F. Features of apoptotic cells measured by flow cytometry. Cytometry. 1992;13(8):795–808. doi: 10.1002/cyto.990130802. [DOI] [PubMed] [Google Scholar]
  7. Duvall E., Wyllie A. H., Morris R. G. Macrophage recognition of cells undergoing programmed cell death (apoptosis). Immunology. 1985 Oct;56(2):351–358. [PMC free article] [PubMed] [Google Scholar]
  8. Ellis R. E., Yuan J. Y., Horvitz H. R. Mechanisms and functions of cell death. Annu Rev Cell Biol. 1991;7:663–698. doi: 10.1146/annurev.cb.07.110191.003311. [DOI] [PubMed] [Google Scholar]
  9. Fadok V. A., Laszlo D. J., Noble P. W., Weinstein L., Riches D. W., Henson P. M. Particle digestibility is required for induction of the phosphatidylserine recognition mechanism used by murine macrophages to phagocytose apoptotic cells. J Immunol. 1993 Oct 15;151(8):4274–4285. [PubMed] [Google Scholar]
  10. Fadok V. A., Savill J. S., Haslett C., Bratton D. L., Doherty D. E., Campbell P. A., Henson P. M. Different populations of macrophages use either the vitronectin receptor or the phosphatidylserine receptor to recognize and remove apoptotic cells. J Immunol. 1992 Dec 15;149(12):4029–4035. [PubMed] [Google Scholar]
  11. Fadok V. A., Voelker D. R., Campbell P. A., Cohen J. J., Bratton D. L., Henson P. M. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol. 1992 Apr 1;148(7):2207–2216. [PubMed] [Google Scholar]
  12. Flora P. K., Gregory C. D. Recognition of apoptotic cells by human macrophages: inhibition by a monocyte/macrophage-specific monoclonal antibody. Eur J Immunol. 1994 Nov;24(11):2625–2632. doi: 10.1002/eji.1830241109. [DOI] [PubMed] [Google Scholar]
  13. Hall S. E., Savill J. S., Henson P. M., Haslett C. Apoptotic neutrophils are phagocytosed by fibroblasts with participation of the fibroblast vitronectin receptor and involvement of a mannose/fucose-specific lectin. J Immunol. 1994 Oct 1;153(7):3218–3227. [PubMed] [Google Scholar]
  14. Haslett C., Savill J. S., Whyte M. K., Stern M., Dransfield I., Meagher L. C. Granulocyte apoptosis and the control of inflammation. Philos Trans R Soc Lond B Biol Sci. 1994 Aug 30;345(1313):327–333. doi: 10.1098/rstb.1994.0113. [DOI] [PubMed] [Google Scholar]
  15. Higgins C. F. Flip-flop: the transmembrane translocation of lipids. Cell. 1994 Nov 4;79(3):393–395. doi: 10.1016/0092-8674(94)90248-8. [DOI] [PubMed] [Google Scholar]
  16. Kerr J. F., Wyllie A. H., Currie A. R. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 1972 Aug;26(4):239–257. doi: 10.1038/bjc.1972.33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kishimoto H., Surh C. D., Sprent J. Upregulation of surface markers on dying thymocytes. J Exp Med. 1995 Feb 1;181(2):649–655. doi: 10.1084/jem.181.2.649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Koopman G., Reutelingsperger C. P., Kuijten G. A., Keehnen R. M., Pals S. T., van Oers M. H. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood. 1994 Sep 1;84(5):1415–1420. [PubMed] [Google Scholar]
  19. Korsmeyer S. J. Bcl-2: a repressor of lymphocyte death. Immunol Today. 1992 Aug;13(8):285–288. doi: 10.1016/0167-5699(92)90037-8. [DOI] [PubMed] [Google Scholar]
  20. Lagasse E., Weissman I. L. bcl-2 inhibits apoptosis of neutrophils but not their engulfment by macrophages. J Exp Med. 1994 Mar 1;179(3):1047–1052. doi: 10.1084/jem.179.3.1047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lennon S. V., Martin S. J., Cotter T. G. Dose-dependent induction of apoptosis in human tumour cell lines by widely diverging stimuli. Cell Prolif. 1991 Mar;24(2):203–214. doi: 10.1111/j.1365-2184.1991.tb01150.x. [DOI] [PubMed] [Google Scholar]
  22. Martin S. J., Cotter T. G. Ultraviolet B irradiation of human leukaemia HL-60 cells in vitro induces apoptosis. Int J Radiat Biol. 1991 Apr;59(4):1001–1016. doi: 10.1080/09553009114550891. [DOI] [PubMed] [Google Scholar]
  23. Martin S. J., Lennon S. V., Bonham A. M., Cotter T. G. Induction of apoptosis (programmed cell death) in human leukemic HL-60 cells by inhibition of RNA or protein synthesis. J Immunol. 1990 Sep 15;145(6):1859–1867. [PubMed] [Google Scholar]
  24. Martin S. J., Matear P. M., Vyakarnam A. HIV-1 infection of human CD4+ T cells in vitro. Differential induction of apoptosis in these cells. J Immunol. 1994 Jan 1;152(1):330–342. [PubMed] [Google Scholar]
  25. Martin S. J., O'Brien G. A., Nishioka W. K., McGahon A. J., Mahboubi A., Saido T. C., Green D. R. Proteolysis of fodrin (non-erythroid spectrin) during apoptosis. J Biol Chem. 1995 Mar 24;270(12):6425–6428. doi: 10.1074/jbc.270.12.6425. [DOI] [PubMed] [Google Scholar]
  26. Maurer-Fogy I., Reutelingsperger C. P., Pieters J., Bodo G., Stratowa C., Hauptmann R. Cloning and expression of cDNA for human vascular anticoagulant, a Ca2+-dependent phospholipid-binding protein. Eur J Biochem. 1988 Jul 1;174(4):585–592. doi: 10.1111/j.1432-1033.1988.tb14139.x. [DOI] [PubMed] [Google Scholar]
  27. McGahon A. J., Cotter T. G., Green D. R. The abl oncogene family and apoptosis. Cell Death Differ. 1994;1(2):77–83. [PubMed] [Google Scholar]
  28. McGahon A. J., Martin S. J., Bissonnette R. P., Mahboubi A., Shi Y., Mogil R. J., Nishioka W. K., Green D. R. The end of the (cell) line: methods for the study of apoptosis in vitro. Methods Cell Biol. 1995;46:153–185. doi: 10.1016/s0091-679x(08)61929-9. [DOI] [PubMed] [Google Scholar]
  29. Morris R. G., Hargreaves A. D., Duvall E., Wyllie A. H. Hormone-induced cell death. 2. Surface changes in thymocytes undergoing apoptosis. Am J Pathol. 1984 Jun;115(3):426–436. [PMC free article] [PubMed] [Google Scholar]
  30. Newman S. L., Henson J. E., Henson P. M. Phagocytosis of senescent neutrophils by human monocyte-derived macrophages and rabbit inflammatory macrophages. J Exp Med. 1982 Aug 1;156(2):430–442. doi: 10.1084/jem.156.2.430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ogasawara J., Watanabe-Fukunaga R., Adachi M., Matsuzawa A., Kasugai T., Kitamura Y., Itoh N., Suda T., Nagata S. Lethal effect of the anti-Fas antibody in mice. Nature. 1993 Aug 26;364(6440):806–809. doi: 10.1038/364806a0. [DOI] [PubMed] [Google Scholar]
  32. Raynal P., Pollard H. B. Annexins: the problem of assessing the biological role for a gene family of multifunctional calcium- and phospholipid-binding proteins. Biochim Biophys Acta. 1994 Apr 5;1197(1):63–93. doi: 10.1016/0304-4157(94)90019-1. [DOI] [PubMed] [Google Scholar]
  33. Reutelingsperger C. P., Hornstra G., Hemker H. C. Isolation and partial purification of a novel anticoagulant from arteries of human umbilical cord. Eur J Biochem. 1985 Sep 16;151(3):625–629. doi: 10.1111/j.1432-1033.1985.tb09150.x. [DOI] [PubMed] [Google Scholar]
  34. Rouvier E., Luciani M. F., Golstein P. Fas involvement in Ca(2+)-independent T cell-mediated cytotoxicity. J Exp Med. 1993 Jan 1;177(1):195–200. doi: 10.1084/jem.177.1.195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ruetz S., Gros P. Phosphatidylcholine translocase: a physiological role for the mdr2 gene. Cell. 1994 Jul 1;77(7):1071–1081. doi: 10.1016/0092-8674(94)90446-4. [DOI] [PubMed] [Google Scholar]
  36. Savill J. S., Henson P. M., Haslett C. Phagocytosis of aged human neutrophils by macrophages is mediated by a novel "charge-sensitive" recognition mechanism. J Clin Invest. 1989 Nov;84(5):1518–1527. doi: 10.1172/JCI114328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Savill J. S., Wyllie A. H., Henson J. E., Walport M. J., Henson P. M., Haslett C. Macrophage phagocytosis of aging neutrophils in inflammation. Programmed cell death in the neutrophil leads to its recognition by macrophages. J Clin Invest. 1989 Mar;83(3):865–875. doi: 10.1172/JCI113970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Savill J., Dransfield I., Hogg N., Haslett C. Vitronectin receptor-mediated phagocytosis of cells undergoing apoptosis. Nature. 1990 Jan 11;343(6254):170–173. doi: 10.1038/343170a0. [DOI] [PubMed] [Google Scholar]
  39. Savill J., Fadok V., Henson P., Haslett C. Phagocyte recognition of cells undergoing apoptosis. Immunol Today. 1993 Mar;14(3):131–136. doi: 10.1016/0167-5699(93)90215-7. [DOI] [PubMed] [Google Scholar]
  40. Savill J., Hogg N., Ren Y., Haslett C. Thrombospondin cooperates with CD36 and the vitronectin receptor in macrophage recognition of neutrophils undergoing apoptosis. J Clin Invest. 1992 Oct;90(4):1513–1522. doi: 10.1172/JCI116019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Shi Y. F., Sahai B. M., Green D. R. Cyclosporin A inhibits activation-induced cell death in T-cell hybridomas and thymocytes. Nature. 1989 Jun 22;339(6226):625–626. doi: 10.1038/339625a0. [DOI] [PubMed] [Google Scholar]
  42. Suda T., Nagata S. Purification and characterization of the Fas-ligand that induces apoptosis. J Exp Med. 1994 Mar 1;179(3):873–879. doi: 10.1084/jem.179.3.873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sune A., Bette-Bobillo P., Bienvenüe A., Fellmann P., Devaux P. F. Selective outside-inside translocation of aminophospholipids in human platelets. Biochemistry. 1987 Jun 2;26(11):2972–2978. doi: 10.1021/bi00385a003. [DOI] [PubMed] [Google Scholar]
  44. Tait J. F., Gibson D., Fujikawa K. Phospholipid binding properties of human placental anticoagulant protein-I, a member of the lipocortin family. J Biol Chem. 1989 May 15;264(14):7944–7949. [PubMed] [Google Scholar]
  45. Thiagarajan P., Tait J. F. Binding of annexin V/placental anticoagulant protein I to platelets. Evidence for phosphatidylserine exposure in the procoagulant response of activated platelets. J Biol Chem. 1990 Oct 15;265(29):17420–17423. [PubMed] [Google Scholar]
  46. Williamson P., Bateman J., Kozarsky K., Mattocks K., Hermanowicz N., Choe H. R., Schlegel R. A. Involvement of spectrin in the maintenance of phase-state asymmetry in the erythrocyte membrane. Cell. 1982 Oct;30(3):725–733. doi: 10.1016/0092-8674(82)90277-x. [DOI] [PubMed] [Google Scholar]
  47. Wyllie A. H., Kerr J. F., Currie A. R. Cell death: the significance of apoptosis. Int Rev Cytol. 1980;68:251–306. doi: 10.1016/s0074-7696(08)62312-8. [DOI] [PubMed] [Google Scholar]
  48. Wyllie A. H., Morris R. G., Smith A. L., Dunlop D. Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis. J Pathol. 1984 Jan;142(1):67–77. doi: 10.1002/path.1711420112. [DOI] [PubMed] [Google Scholar]
  49. Yonehara S., Ishii A., Yonehara M. A cell-killing monoclonal antibody (anti-Fas) to a cell surface antigen co-downregulated with the receptor of tumor necrosis factor. J Exp Med. 1989 May 1;169(5):1747–1756. doi: 10.1084/jem.169.5.1747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. van Meer G. Transport and sorting of membrane lipids. Curr Opin Cell Biol. 1993 Aug;5(4):661–673. doi: 10.1016/0955-0674(93)90137-f. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES