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 a nd ladder-type DNA fragmentation were detected among freshly isolated peripheral lymphocytes ex vivo. Here we report that one alteration tha t can be detected among splenocytes stimulated with lymphocyte-depleti ng doses of dexamethasone (DEX) in vivo is a reduced uptake of 3,3'dih exyloxacarbocyanine iodide (DiOC(6)[3]), a fluorochrome which incorpor ates into cells dependent upon their mitochondrial transmembrane poten tial (Delta Psi(m)). In contrast, ex vivo isolated splenocytes still l acked established signs of programmed cell death (PCD): DNA degradatio n into high or low molecular weight fragments, ultrastructural changes of chromatin arrangement and endoplasmatic reticulum, loss in viabili ty, or accumulation of intracellular peroxides. Moreover, no changes i n cell membrane potential could be detected. A reduced Delta Psi(m) ha s 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 viv o, followed by cytofluorometric purification of viable Delta Psi(m)(lo w) splenic T cells ex vivo, revealed that this fraction of cells is ir reversibly committed to undergoing DNA fragmentation. Immediately afte r purification neither Delta Psi(m)(low), nor Delta Psi(m)(high) cells , exhibit detectable DNA fragmentation. However, after short-term cult ure (37 degrees C, 1 h) Delta Psi(m)(low) cells show endonucleolysis, followed by cytolysis several hours later. Incubation of Delta Psi(m)( low) cells in the presence of excess amount of the GC receptor antagon ist RU-38486 (which displaces DEX from the GC receptor), cytokines tha t inhibit DEX-induced cell death, or cycloheximide fails to prevent cy tolysis. The antioxidant, N-acetylcysteine, as well as linomide, an ag ent that effectively inhibits DEX or superantigen-induced lymphocyte d epletion in vivo, also stabilize the DiOC(6)(3) uptake. In contrast, t he endonuclease inhibitor, aurintricarboxylic acid acts at later stage s of apoptosis and only retards the transition from the viable Delta P si(m)(low) 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 deat h, preceding other alterations of cellular physiology, and thus allowi ng for the ex vivo assessment of PCD.