Apoptotic pathways are selectively activated by granzyme A and/or granzyme B in CTL-mediated target cell lysis

Abstract

Purified cytolytic T lymphocyte (CTL) proteases granzyme (gzm)A and gzmB with sublytic dose of perforin (perf) initiate distinct proapoptotic pathways. Their physiological relevance in CTL-mediated target cell apoptosis is elusive. Using ex vivo virus-immune CD8+ T cells from mice deficient in perf, gzmA and/or gzmB, and the Fas-resistant EL4.F15 tumor target cell, we show that (a) CTL from gzmA−/− or gzmB−/− mice similarly induced early proapoptotic features, such as phosphatidyl serine (PS) exposure on plasma membrane, ΔΨm loss, and reactive oxygen radical generation, though with distinct kinetics; (b) CTL from gzmA−/− but not from gzmB−/− mice activate caspase 3 and 9; (c) PS exposure induced by CTL from gzmA−/− or gzmB−/− mice is prevented, respectively, by caspase inhibitors or by reactive oxygen scavengers without interfering with target cell death; and (d) all gzm-induced apoptotic features analyzed depend critically on perf. Thus, perf is the principal regulator in CTL-mediated and gzm-facilitated intracellular processes. The ability of gzmA and gzmB to induce multiple independent cell death pathways may be the hosts response to circumvent evasion strategies of pathogens and tumors.

Mouse granzyme A induces a novel death with writhing morphology that is mechanistically distinct from granzyme B-induced apoptosis

“…Using this distinct morphology to specifically identify cells undergoing GzmA-mediated death, we analysed hundreds of individual cellular deaths to clarify the molecular mechanisms that were consistently and authentically associated with it. Our finding that GzmA induces a caspase-independent form of death without cytochrome c release agrees with previous findings, 4,5,12 but our finding that it did not involve membrane blebbing, early DC m loss or early ROS production contrasts with others. 4,5 The absence of mitochondrial damage during the early stages of GzmB À / À NK-mediated cell death was unexpected, given its prominent role in initiating the GzmA cell death pathway.…”

“…5,25 Given the lack of ROS generation as a trigger, it is unclear whether mGzmA would induce SET cleavage in vivo; however, we found that target cell treatment with recombinant mGzmA or GzmB with Pfp did induce cleavage of the SET complex component Ape-1 in vitro (data not shown). This correlates with reports that WT, GzmA À / À , GzmB À / À and GzmA À / À B À / À CTL were equally able to induce Ape-1 cleavage in target cells, 12 suggesting that SET complex cleavage may not always be a GzmA-specific event.…”

“…Indeed, it has been previously hypothesised that GzmA may induce actin cytoskeletal disruption, resulting in downstream ROS generation similar to apoptosis in yeast. 12,26 Here, we have reported the critical and novel finding that an intact actin cytoskeleton is crucial for GzmAmediated athetosis, but is not required for GzmB-mediated apoptosis. Inhibition of myosin II also caused a mild, although statistically significant reduction in GzmA-mediated death.…”

“…Early mitochondrial damage that induces increased ROS and loss of mitochondrial transmembrane potential (DC m ) is considered a critical event for GzmA-induced cell death, 4,5,12 and the presence of superoxide scavengers such as tiron can significantly inhibit GzmA-induced cell death. 5,6 The fluorescent dye tetramethylrhodamine ethyl ester (TMRE) was used with timelapse microscopy to assess whether loss of DC m is an early feature of GzmB À / À NK-mediated death.…”

“…8 In contrast, studies using GzmB À / À CTL suggest that GzmA does have a role in target cell death. 11,12 To address this controversy in a physiologically relevant context, we used intact primary mouse NKs to deliver Gzms in order to analyse the features of GzmA-mediated cell death. As shown previously, NKs from WT mice induced classic target cell apoptosis.…”