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J Exp Med. 1995 May 1; 181(5): 1661–1672.
PMCID: PMC2192017

Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo


In a number of experimental systems in which lymphocyte depletion was induced by apoptosis-inducing manipulations, no apoptotic morphology and ladder-type DNA fragmentation were detected among freshly isolated peripheral lymphocytes ex vivo. Here we report that one alteration that can be detected among splenocytes stimulated with lymphocyte-depleting doses of dexamethasone (DEX) in vivo is a reduced uptake of 3,3'dihexyloxacarbocyanine iodide (DiOC6[3]), a fluorochrome which incorporates into cells dependent upon their mitochondrial transmembrane potential (delta psi m). In contrast, ex vivo isolated splenocytes still lacked established signs of programmed cell death (PCD):DNA degradation into high or low molecular weight fragments, ultrastructural changes of chromatin arrangement and endoplasmatic reticulum, loss in viability, or accumulation of intracellular peroxides. Moreover, no changes in cell membrane potential could be detected. A reduced delta psi m has been observed in response to different agents inducing lymphoid cell depletion in vivo (superantigen and glucocorticoids [GC]), in mature T and B lymphocytes, as well as their precursors. DEX treatment in vivo, followed by cytofluorometric purification of viable delta psi mlow splenic T cells ex vivo, revealed that this fraction of cells is irreversibly committed to undergoing DNA fragmentation. Immediately after purification neither delta psi mlow, nor delta psi mhigh cells, exhibit detectable DNA fragmentation. However, after short-term culture (37 degrees C, 1 h) delta psi mlow cells show endonucleolysis, followed by cytolysis several hours later. Incubation of delta psi mlow cells in the presence of excess amount of the GC receptor antagonist RU38486 (which displaces DEX from the GC receptor), cytokines that inhibit DEX-induced cell death, or cycloheximide fails to prevent cytolysis. The antioxidant, N- acetylcysteine, as well as linomide, an agent that effectively inhibits DEX or superantigen-induced lymphocyte depletion in vivo, also stabilize the DiOC6(3) uptake. In contrast, the endonuclease inhibitor, aurintricarboxylic acid acts at later stages of apoptosis and only retards the transition from the viable delta psi mlow to the nonviable fraction. Altogether, these data suggest a sequence of PCD-associated events in which a reduction in delta psi m constitutes an obligate irreversible step of ongoing lymphocyte death, preceding other alterations of cellular physiology, and thus allowing for the ex vivo assessment of PCD.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Cohen JJ. Apoptosis. Immunol Today. 1993 Mar;14(3):126–130. [PubMed]
  • Kawabe Y, Ochi A. Programmed cell death and extrathymic reduction of Vbeta8+ CD4+ T cells in mice tolerant to Staphylococcus aureus enterotoxin B. Nature. 1991 Jan 17;349(6306):245–248. [PubMed]
  • Matsubara K, Kubota M, Adachi S, Kuwakado K, Hirota H, Wakazono Y, Akiyama Y, Mikawa H. Induction of apoptosis in childhood acute leukemia by chemotherapeutic agents: failure to detect evidence of apoptosis in vivo. Eur J Haematol. 1994 Jan;52(1):47–52. [PubMed]
  • Groux H, Torpier G, Monté D, Mouton Y, Capron A, Ameisen JC. Activation-induced death by apoptosis in CD4+ T cells from human immunodeficiency virus-infected asymptomatic individuals. J Exp Med. 1992 Feb 1;175(2):331–340. [PMC free article] [PubMed]
  • Meyaard L, Otto SA, Jonker RR, Mijnster MJ, Keet RP, Miedema F. Programmed death of T cells in HIV-1 infection. Science. 1992 Jul 10;257(5067):217–219. [PubMed]
  • Savill J, Fadok V, Henson P, Haslett C. Phagocyte recognition of cells undergoing apoptosis. Immunol Today. 1993 Mar;14(3):131–136. [PubMed]
  • Petit PX, O'Connor JE, Grunwald D, Brown SC. Analysis of the membrane potential of rat- and mouse-liver mitochondria by flow cytometry and possible applications. Eur J Biochem. 1990 Dec 12;194(2):389–397. [PubMed]
  • Gonzalo JA, González-García A, Kalland T, Hedlung G, Martínez C, Kroemer G. Linomide inhibits programmed cell death of peripheral T cells in vivo. Eur J Immunol. 1994 Jan;24(1):48–52. [PubMed]
  • Gatti S, Faggioni R, Echtenacher B, Ghezzi P. Role of tumour necrosis factor and reactive oxygen intermediates in lipopolysaccharide-induced pulmonary oedema and lethality. Clin Exp Immunol. 1993 Mar;91(3):456–461. [PMC free article] [PubMed]
  • Mogil RJ, Shi Y, Bissonnette RP, Bromley P, Yamaguchi I, Green DR. Role of DNA fragmentation in T cell activation-induced apoptosis in vitro and in vivo. J Immunol. 1994 Feb 15;152(4):1674–1683. [PubMed]
  • Gonzalo JA, González-García A, Martínez C, Kroemer G. Glucocorticoid-mediated control of the activation and clonal deletion of peripheral T cells in vivo. J Exp Med. 1993 May 1;177(5):1239–1246. [PMC free article] [PubMed]
  • Bräuner T, Hülser DF, Strasser RJ. Comparative measurements of membrane potentials with microelectrodes and voltage-sensitive dyes. Biochim Biophys Acta. 1984 Apr 11;771(2):208–216. [PubMed]
  • Johnson LV, Walsh ML, Chen LB. Localization of mitochondria in living cells with rhodamine 123. Proc Natl Acad Sci U S A. 1980 Feb;77(2):990–994. [PMC free article] [PubMed]
  • Hockenbery DM, Oltvai ZN, Yin XM, Milliman CL, Korsmeyer SJ. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell. 1993 Oct 22;75(2):241–251. [PubMed]
  • Hedley D, Chow S. Flow cytometric measurement of lipid peroxidation in vital cells using parinaric acid. Cytometry. 1992;13(7):686–692. [PubMed]
  • Brown DG, Sun XM, Cohen GM. Dexamethasone-induced apoptosis involves cleavage of DNA to large fragments prior to internucleosomal fragmentation. J Biol Chem. 1993 Feb 15;268(5):3037–3039. [PubMed]
  • Cohen GM, Sun XM, Snowden RT, Dinsdale D, Skilleter DN. Key morphological features of apoptosis may occur in the absence of internucleosomal DNA fragmentation. Biochem J. 1992 Sep 1;286(Pt 2):331–334. [PMC free article] [PubMed]
  • Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. [PMC free article] [PubMed]
  • Duke RC, Chervenak R, Cohen JJ. Endogenous endonuclease-induced DNA fragmentation: an early event in cell-mediated cytolysis. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6361–6365. [PMC free article] [PubMed]
  • Perandones CE, Illera VA, Peckham D, Stunz LL, Ashman RF. Regulation of apoptosis in vitro in mature murine spleen T cells. J Immunol. 1993 Oct 1;151(7):3521–3529. [PubMed]
  • Murgia M, Pizzo P, Sandoná D, Zanovello P, Rizzuto R, Di Virgilio F. Mitochondrial DNA is not fragmented during apoptosis. J Biol Chem. 1992 Jun 5;267(16):10939–10941. [PubMed]
  • Tepper CG, Studzinski GP. Teniposide induces nuclear but not mitochondrial DNA degradation. Cancer Res. 1992 Jun 15;52(12):3384–3390. [PubMed]
  • Schwartzman RA, Cidlowski JA. Internucleosomal deoxyribonucleic acid cleavage activity in apoptotic thymocytes: detection and endocrine regulation. Endocrinology. 1991 Feb;128(2):1190–1197. [PubMed]
  • Zubiaga AM, Munoz E, Huber BT. IL-4 and IL-2 selectively rescue Th cell subsets from glucocorticoid-induced apoptosis. J Immunol. 1992 Jul 1;149(1):107–112. [PubMed]
  • Kam JC, Szefler SJ, Surs W, Sher ER, Leung DY. Combination IL-2 and IL-4 reduces glucocorticoid receptor-binding affinity and T cell response to glucocorticoids. J Immunol. 1993 Oct 1;151(7):3460–3466. [PubMed]
  • Migliorati G, Nicoletti I, D'Adamio F, Spreca A, Pagliacci C, Riccardi C. Dexamethasone induces apoptosis in mouse natural killer cells and cytotoxic T lymphocytes. Immunology. 1994 Jan;81(1):21–26. [PMC free article] [PubMed]
  • Smith GK, Duch DS, Dev IK, Kaufmann SH. Metabolic effects and kill of human T-cell leukemia by 5-deazaacyclotetrahydrofolate, a specific inhibitor of glycineamide ribonucleotide transformylase. Cancer Res. 1992 Sep 15;52(18):4895–4903. [PubMed]
  • Nordeen SK, Young DA. Glucocorticoid action on rat thymic lymphocytes. Experiments utilizing adenosine to support cellular metabolism lead to a reassessment of catabolic hormone actions. J Biol Chem. 1976 Dec 10;251(23):7295–7303. [PubMed]
  • Vayssiere JL, Petit PX, Risler Y, Mignotte B. Commitment to apoptosis is associated with changes in mitochondrial biogenesis and activity in cell lines conditionally immortalized with simian virus 40. Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11752–11756. [PMC free article] [PubMed]
  • Wolvetang EJ, Johnson KL, Krauer K, Ralph SJ, Linnane AW. Mitochondrial respiratory chain inhibitors induce apoptosis. FEBS Lett. 1994 Feb 14;339(1-2):40–44. [PubMed]
  • Van den Bogert C, Dontje BH, Melis TE, van der Veen C, Kroon AM. Inhibition of mitochondrial protein synthesis influences the glucocorticoid sensitivity of lymphoid cells. Biochim Biophys Acta. 1988 Dec 9;972(3):302–310. [PubMed]
  • Smets LA, Van den Berg J, Acton D, Top B, Van Rooij H, Verwijs-Janssen M. BCL-2 expression and mitochondrial activity in leukemic cells with different sensitivity to glucocorticoid-induced apoptosis. Blood. 1994 Sep 1;84(5):1613–1619. [PubMed]
  • Jacobson MD, Burne JF, King MP, Miyashita T, Reed JC, Raff MC. Bcl-2 blocks apoptosis in cells lacking mitochondrial DNA. Nature. 1993 Jan 28;361(6410):365–369. [PubMed]
  • Loveland BE, Johns TG, Mackay IR, Vaillant F, Wang ZX, Hertzog PJ. Validation of the MTT dye assay for enumeration of cells in proliferative and antiproliferative assays. Biochem Int. 1992 Jul;27(3):501–510. [PubMed]
  • Skowronek P, Haferkamp O, Rödel G. A fluorescence-microscopic and flow-cytometric study of HeLa cells with an experimentally induced respiratory deficiency. Biochem Biophys Res Commun. 1992 Sep 16;187(2):991–998. [PubMed]
  • Sandstrom PA, Mannie MD, Buttke TM. Inhibition of activation-induced death in T cell hybridomas by thiol antioxidants: oxidative stress as a mediator of apoptosis. J Leukoc Biol. 1994 Feb;55(2):221–226. [PubMed]
  • Hennet T, Richter C, Peterhans E. Tumour necrosis factor-alpha induces superoxide anion generation in mitochondria of L929 cells. Biochem J. 1993 Jan 15;289(Pt 2):587–592. [PMC free article] [PubMed]
  • Vukmanović S, Zamoyska R. Anti-CD3-induced cell death in T cell hybridomas: mitochondrial failure and DNA fragmentation are distinct events. Eur J Immunol. 1991 Feb;21(2):419–424. [PubMed]
  • Sun DY, Jiang S, Zheng LM, Ojcius DM, Young JD. Separate metabolic pathways leading to DNA fragmentation and apoptotic chromatin condensation. J Exp Med. 1994 Feb 1;179(2):559–568. [PMC free article] [PubMed]
  • Iseki R, Kudo Y, Iwata M. Early mobilization of Ca2+ is not required for glucocorticoid-induced apoptosis in thymocytes. J Immunol. 1993 Nov 15;151(10):5198–5207. [PubMed]
  • Nicolaou KC, Stabila P, Esmaeli-Azad B, Wrasidlo W, Hiatt A. Cell-specific regulation of apoptosis by designed enediynes. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3142–3146. [PMC free article] [PubMed]
  • Ramírez R, Carracedo J, Zamzami N, Castedo M, Kroemer G. Pertussis toxin inhibits activation-induced cell death of human thymocytes, pre-B leukemia cells and monocytes. J Exp Med. 1994 Sep 1;180(3):1147–1152. [PMC free article] [PubMed]
  • Bouillaud F, Arechaga I, Petit PX, Raimbault S, Levi-Meyrueis C, Casteilla L, Laurent M, Rial E, Ricquier D. A sequence related to a DNA recognition element is essential for the inhibition by nucleotides of proton transport through the mitochondrial uncoupling protein. EMBO J. 1994 Apr 15;13(8):1990–1997. [PMC free article] [PubMed]
  • Crompton M, Ellinger H, Costi A. Inhibition by cyclosporin A of a Ca2+-dependent pore in heart mitochondria activated by inorganic phosphate and oxidative stress. Biochem J. 1988 Oct 1;255(1):357–360. [PMC free article] [PubMed]
  • Kan O, Baldwin SA, Whetton AD. Apoptosis is regulated by the rate of glucose transport in an interleukin 3 dependent cell line. J Exp Med. 1994 Sep 1;180(3):917–923. [PMC free article] [PubMed]

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