Lysosomes and Fas-mediated liver cell death

Abstract

A number of studies, mostly performed ex vivo, suggest that lysosomes are involved in apoptosis as a result of a release of their cathepsins into the cytosol. These enzymes could then contribute to the permeabilization of the outer mitochondrial membrane; they could also activate effector caspases. The present study aims at testing whether the membrane of liver lysosomes is disrupted during Fas-mediated cell death of hepatocytes in vivo, a process implicated in several liver pathologies. Apoptosis was induced by injecting mice with aFas (anti-Fas antibody). The state of lysosomes was assessed by determining the proportion of lysosomal enzymes (β-galactosidase, β-glucuronidase, cathepsin C and cathepsin B) present in homogenate supernatants, devoid of intact lysosomes, and by analysing the behaviour in differential and isopycnic centrifugation of β-galactosidase. Apoptosis was monitored by measuring caspase 3 activity (DEVDase) and the release of sulfite cytochrome c reductase, an enzyme located in the mitochondrial intermembrane space. Results show that an injection of 10 µg of aFas causes a rapid and large increase in DEVDase activity and in unsedimentable sulfite cytochrome c reductase. This modifies neither the proportion of unsedimentable lysosomal enzyme in the homogenates nor the behaviour of lysosomes in centrifugation. Experiments performed with a lower dose of aFas (5 µg) indicate that unsedimentable lysosomal hydrolase activity increases in the homogenate after injection but with a marked delay with respect to the increase in DEVDase activity and in unsedimentable sulfite cytochrome c reductase.Comparative experiments ex vivo performed with Jurkat cells show an increase in unsedimentable lysosomal hydrolases, but much later than caspase 3 activation, and a release of dipeptidyl peptidase III and DEVDase into culture medium. It is proposed that the weakening of lysosomes observed after aFas treatment in vivo and ex vivo results from a necrotic process that takes place late after initiation of apoptosis.

Cysteine cathepsins are not involved in Fas/CD95 signalling in primary skin fibroblasts

“…However, our results show that lysosome destabilization is a very late event in Fas/CD95 induced apoptosis and not an early event, as suggested by Brunk and Svensson [19], although this may depend on cells or cell line used. Despite the very late appearance of cells with destabilized lysosomes, our results do not support the idea that secondary necrosis is responsible for lysosome breakdown [39], as only 5–11% of late apoptotic/necrotic cells were observed even at the latest time point (18 h), whereas 30–35% of cells with destabilized lysosomes have been found already after 15 h (Figs. 1 and 2).…”

Lysosomes as “Suicide Bags” in Cell Death: Myth or Reality?

“…However, knocking out the cathepsins failed to completely block apoptosis but led only to its suppression (44 -47), arguing against an essential role of cathepsins in this pathway. This is also more consistent with recent data on Fas-induced apoptosis, where lysosomes were indeed observed to be broken but substantially later than mitochondria (48,49). These latter findings are more consistent with a bystander role of cathepsins, although their role in an amplification loop through mitochondria cannot be excluded.…”

Secondary necrosis in multicellular animals: an outcome of apoptosis with pathogenic implications

“…That lysosomal enzymes participate in secondary necrosis is also suggested by the observation that, in apoptosis induced in vitro in the epithelial cell line LLC-PK1 by S-(1,2-dichlorovinyl)-L-cysteine, inhibition of cathepsin B resulted in inhibition of secondary necrosis [123]. In support of this interpretation, recent results with caspase 3-associated apoptosis induced in Jurkat cells by treatment with anti-Fas showed that the terminal cell lysis due to secondary necrosis was concomitant with lysosome rupture [124].…”