Caspase-3-like activity is necessary for IL-2 release in activated Jurkat T-cells.
The caspase family of proteases has previously been implicated in the biochemical cascade leading to apoptotic cell death. Recently caspase-3 was reported to be cleaved into its catalytically active subunits (17 and 13 kDa) following phytohemagglutinin (PHA) activation of peripheral blood mononuclear cells (C. Miossec et al., J. Biol. Chem. 272, 13459-13462). More recently, J. M. Zapata and colleagues (J. Biol. Chem. 273, 6916-6920, 1998), however, proposed that caspase-3 activity detected during T-cell activation was due to a methodological artifact related to the composition of the cell lysis buffer. Here we show that in PHA-activated Jurkat T-cells using the recommended lysis buffer detailed by Zapata et al., a caspase-3-like protease is activated and is accompanied by cleavage of PARP and alpha-spectrin into cleavage products suggestive of caspase-3 proteolytic activation. LDH release did not increase following PHA stimulation in this paradigm. Two caspase inhibitors, carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorobenzene (Z-D-DCB) and acetyl-Asp-Glu-Val-Asp-CHO, blocked IL-2 release in a dose-dependent manner. Caspase-3-like protease-generated PARP and alpha-spectrin breakdown product formation was also reduced by Z-D-DCB. In addition, Jurkat T-cells costimulated with anti-CD3 plus anti-CD28 produced significant levels of IL-2 that were also blocked by these caspase inhibitors. Importantly, IL-2 was determined in cell culture supernatants, thus avoiding a cell lysis step that might have enabled activation of caspase-3 by granzyme B. Collectively, these data support the role of caspase-3-like protease activity in Jurkat T-cell activation and demonstrate that caspase-3 like activity is necessary for IL-2 release in PHA-activated and anti-CD3/anti-CD28 costimulated Jurkat T-cells.
Parke-Davis Pharmaceutical Research, Warner-Lambert Company, 2800 Plymouth Road, Ann Arbor, Michigan, 48105, USA.
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Gilbertsen
D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism* / Q000502:physiology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology; D006801:Humans; D006868:Hydrolysis; D007376:Interleukin-2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019169:Jurkat Cells; D008213:Lymphocyte Activation / Q000187:drug effects; D009842:Oligopeptides / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000378:metabolism; D010835:Phytohemagglutinins / Q000494:pharmacology; D013601:T-Lymphocytes / Q000201:enzymology* / Q000276:immunology
D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D007376:Interleukin-2; D009842:Oligopeptides; D010835:Phytohemagglutinins; C099158:acetyl-aspartyl-glutamyl-valyl-aspartal; C093260:benzyloxycarbonyl-Asp-CH2OC(O)-2,6-dichlorobenzene; D001224:Aspartic Acid; D010447:Peptide Hydrolases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases