MITOCHONDRIAL PERTURBATIONS DEFINE LYMPHOCYTES UNDERGOING APOPTOTIC DEPLETION IN-VIVO

We have recently shown that lymphocyte apoptosis induced by dexamethas one or superantigens is accompanied by, reduction of mitochondrial tra nsmembrane potential (Delta Psi(m)) which precedes nuclear DNA fragmen tation. Here, we demonstrate that fluorochromes such as 3,3'dihexyloxa carbocyanine iodide [DiOC(6)(3)] which measure Delta gamma(m), or fluo rochromes such as hydroethidine (HE) which measure mitochondrial super oxide anion production allow the identification of thymocytes or perip heral T lymphocytes which are eliminated by apoptosis in vivo. In mice bearing transgenic alpha/beta T cell receptor (TCR) specific for a cl ass I-restricted male-specific peptide, the superoxide-mediated oxidat ion of HE into ethidium (Eth) is enhanced among thymocytes which are b eing deleted due to negative selection (CD4(+) CD8(+) cells expressing the transgenic TCR in male mice) or lack of positive selection (CD4() CD8(-) thymocytes from female mice). Delta Psi(m) reduction and/or e nhanced HE oxidation are also found when apoptosis is induced by a ser ies of pathogenic agents. Thus, lethal irradiation provokes mitochondr ial and nuclear signs of apoptosis, and both these alterations are abs ent in mice bearing a p53 null mutation, underlying the correlation be tween mitochondrial perturbation and nuclear apoptosis. Similarly, sup erantigen-triggered deletion of peripheral T cells in vivo is accompan ied by enhanced HE --> Eth conversion and reduced DiOC(6)(3) uptake. M ore importantly, as compared to normal controls, CD4(+) or CD8(+) cell s from clinically asymptomatic human immunodeficiency virus-1 (HIV-1) carriers also contain a significantly elevated percentage of cells end owed with reduced DiOC(6)(3) uptake. In superantigen- and HIV-induced apoptosis, the percentage of T lymphocytes with a subnormal DiOC(6)(3) uptake is more important than that of cells marked by enhanced HE --> Eth conversion. In conclusion, mitochondrial alterations precede and/ or accompany nuclear signs of apoptosis induced by physiological and a variety of different pathogenic agents in vivo.