Inhibition of CPP32-like proteases prevents granzyme B- and Fas-, but not granzyme A-based cytotoxicity exerted by CTL clones.
The perforin-facilitated entry of granzymes in target cells is a major mechanism used by CTL to induce cell death. It has been reported that granzyme B can cleave and activate the apoptotic cysteine protease p32 (CPP32)/Yama and its homologues in vitro. However, the mechanism for granzyme-based cytolysis exerted by intact CTL remains unclear. In the present work, we have used anti-CD3 mAb-redirected lysis of Fas-negative L1210 cells by CTL clones as a model to study perforin/granzyme-based cytotoxicity separately from the contribution of the Fas/Fas ligand system. N-acetyl-Asp-Glu-Val-Asp aldehyde (Ac-DEVD-CHO), a specific inhibitor of CPP32-like proteases, completely prevented the former type of lysis in 3-h assays, but not in long-term (16-h) assays. A combination of Ac-DEVD-CHO and the granzyme A inhibitor IGA (7-(phenyl-ureido)-4-chloro-3-(2-isothioureidoethoxy)-isocoumarin) inhibited long-term cytolysis. 3,4-Dichloroisocoumarin, a serine-protease inhibitor that efficiently inhibits granzyme B and poorly inhibits granzyme A, had similar effects as Ac-DEVD-CHO on anti-CD3 mAb-redirected lysis of L1210 cells. On the other hand, Fas-based cytolysis exerted by the same CTL clones on Fas-transfected L1210 cells (L1210Fas) was inhibited completely by Ac-DEVD-CHO, irrespective of the incubation time. These results suggest that granzyme B- and Fas-based cytotoxicity exerted by CTL clones converge at the level of CPP32-like protease activation, while granzyme A acts via a different, still undefined, pathway. We also demonstrate that perforin/granzyme-based cytolysis occurs without increase in the cellular ceramide content, ruling out the contribution of the sphingomyelinase pathway to this mechanism of cell death.
Anel
A
A
Department of Biochemistry and Molecular and Cellular Biology, Faculty of Sciences, University of Zaragoza, Spain.
Gamen
S
S
Alava
M A
MA
Schmitt-Verhulst
A M
AM
Piñeiro
A
A
Naval
J
J
D000818:Animals; D053148:Caspase 3; D020169:Caspases; D002518:Ceramides / Q000378:metabolism; D002999:Clone Cells; D003546:Cysteine Endopeptidases / Q000187:drug effects*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D003601:Cytotoxicity Tests, Immunologic; D003602:Cytotoxicity, Immunologic / Q000187:drug effects*; D004338:Drug Combinations; D053804:Granzymes; D006801:Humans; D007070:Immunoglobulin A / Q000494:pharmacology; D007519:Isoantigens / Q000276:immunology; D008562:Membrane Glycoproteins / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D009842:Oligopeptides / Q000494:pharmacology; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D012697:Serine Endopeptidases / Q000633:toxicity*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D019014:fas Receptor / Q000633:toxicity*
D002518:Ceramides; D015853:Cysteine Proteinase Inhibitors; D004338:Drug Combinations; D007070:Immunoglobulin A; D007519:Isoantigens; D008562:Membrane Glycoproteins; D009842:Oligopeptides; D052899:Pore Forming Cytotoxic Proteins; C099158:acetyl-aspartyl-glutamyl-valyl-aspartal; D019014:fas Receptor; D054353:Perforin; C509213:GZMB protein, human; D053804:Granzymes; C509214:Gzmb protein, mouse; D012697:Serine Endopeptidases; C509212:GZMA protein, human; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases