Mitochondrial perturbations define lymphocytes undergoing apoptotic depletion in vivo

Abstract

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.

Apoptosis in SIV infection

“…Cell death as a result of neglect occurs after the loss of mitochondrial homeostasis. 106 Spontaneous apoptosis during infection is associated with a loss of mitochondrial transmembrane potential (DC m ), suggesting that changes in mitochondrial permeability could be a central event in the regulation of T-cell death in HIV-infected individuals 107 (Figure 3b). Recently, we found that spontaneous T-cell death of pathogenic SIVmac251-infected macaques was not prevented by zVADfmk.…”

Evidence for a p23 caspase-cleaved form of p27[KIP1] involved in G1 growth arrest

“…Apoptosis was assayed by measuring phosphatidyl serine (PS)¯ipping and disruption of the mitochondrial transmembrane potential (Castedo et al, 1996). Results presented in Table 3 indicate that 24 h after the addition of DMSO, 0.7% of 7TD1 cells were apoptotic compared to the positive control obtained with anisomycin (10 mg/ml), which induced 60% of apoptosis in 7TD1 cells.…”

“…Cell cycle analysis was performed by quantifying the DNA content using propidium iodide according to Vindelùv et al (1983). Apoptosis was estimated by double-labelling using annexin-5 and a mitochondrial potential probe (Castedo et al 1996).…”

“…The CD4 + cells were also analysed for apoptosis by double labelling using annexin-5 and a mitochondrial potential probe (Castedo et al, 1996). Data shown in Figure 9b demonstrate that p23 or p27 failed to induce programmed cell death (0% in the lower right quadran).…”

Early work on the role of mitochondria in apoptosis, an interview with Guido Kroemer

“…Moreover, it could be truly shown in vivo, by labelling T lymphocytes with intravenously injected DC m -sensitive dies. So we had identified a molecular marker, DC m dissipation, that defined the subpopulation of T cells that would be eliminated in vivo in response to bacterial superantigens, glucocorticoids, 4 self-antigens 5 or infection with HIV-1. 6 We soon realized that the loss of the DC m marked an irreversible early step of the apoptotic process, observable in very different cell types, including epithelial cell lines and neurons, that it was controlled by Bcl-2-like proteins, 4,7 that it mostly occurred before caspases were activated, and that it involved, at least in some cases, permeability transition, that is a process inhibited by ligands of the adenine nucleotide translocase and the mitochondrial cyclophilin D. 8,9 However, we only became fully convinced that mitochondria would be part of the 'central executioner', when we constructed, back in 1995, a cell-free system in which we confronted purified mitochondria with purified nuclei.…”