Mitochondrial perturbations define lymphocytes undergoing apoptotic depletion in vivo.
We have recently shown that lymphocyte apoptosis induced by dexamethasone or superantigens is accompanied by reduction of mitochondrial transmembrane potential (delta psi m) which precedes nuclear DNA fragmentation. Here, we demonstrate that fluorochromes such as 3,3' dihexyloxacarbocyanine iodide [DiOC6(3)] which measure delta psi m, or fluorochromes such as hydroethidine (HE) which measure mitochondrial superoxide anion production allow the identification of thymocytes or peripheral T lymphocytes which are eliminated by apoptosis in vivo. In mice bearing transgenic alpha/beta T cell receptor (TCR) specific for a class I-restricted male-specific peptide, the superoxide-mediated oxidation of HE into ethidium (Eth) is enhanced among thymocytes which are being 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 enhanced HE oxidation are also found when apoptosis is induced by a series of pathogenic agents. Thus, lethal irradiation provokes mitochondrial and nuclear signs of apoptosis, and both these alterations are absent in mice bearing a p53 null mutation, underlying the correlation between mitochondrial perturbation and nuclear apoptosis. Similarly, superantigen-triggered deletion of peripheral T cells in vivo is accompanied by enhanced HE-->Eth conversion and reduced DiOC6(3) uptake. More importantly, as compared to normal controls, CD4+ or CD8+ cells from clinically asymptomatic human immunodeficiency virus-1 (HIV-1) carriers also contain a significantly elevated percentage of cells endowed with reduced DiOC6(3) uptake. In superantigen- and HIV-induced apoptosis, the percentage of T lymphocytes with a subnormal DiOC6(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.
Castedo
M
M
CNRS-UPR420, Villejuif, France.
Macho
A
A
Zamzami
N
N
Hirsch
T
T
Marchetti
P
P
Uriel
J
J
Kroemer
G
G
D000818:Animals; D017209:Apoptosis / Q000276:immunology* / Q000528:radiation effects; D017637:Clonal Deletion; D015658:HIV Infections / Q000276:immunology / Q000378:metabolism; D006801:Humans; D008212:Lymphocyte Depletion; D051379:Mice; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000276:immunology*; D017382:Reactive Oxygen Species / Q000032:analysis; D013194:Staining and Labeling; D018089:Superantigens / Q000494:pharmacology; D013601:T-Lymphocytes / Q000276:immunology*; D016159:Tumor Suppressor Protein p53 / Q000502:physiology
D017382:Reactive Oxygen Species; D018089:Superantigens; D016159:Tumor Suppressor Protein p53