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 (ΔΨ(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 ΔΨ(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 ΔΨ(m)(low) splenic T cells ex vivo, revealed that this fraction of cells is irreversibly committed to undergoing DNA fragmentation. Immediately after purification neither ΔΨ(m)(low), nor ΔΨ(m)(high) cells, exhibit detectable DNA fragmentation. However, after short-term culture (37°C, 1 h) ΔΨ(m)(low) cells show endonucleolysis, followed by cytolysis several hours later. Incubation of ΔΨ(m)(low) cells in the presence of excess amount of the GC receptor antagonist RU-38486 (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 ΔΨ(m)(low) to the nonviable fraction. Altogether, these data suggest a sequence of PCD-associated events in which a reduction in ΔΨ(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.