Apoptosis-inducing factor of a cytotoxic T cell line: involvement of a secretory phospholipase A2

Abstract

Natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) kill target cells by the granule-exocytosis pathway and by the engagement of molecules belonging to the tumor necrosis factor family. The involvement of secretory phospholipase A(2) (sPLA(2)) in the cytotoxic process has been proposed in NK cells. However, its molecular identity and intracellular localization remain unknown, and its mechanism of action is poorly understood. Here, we have readdressed this issue by studying the cytotoxic activity of whole cell extracts of a CTL line. We observed that inactivation of the perforin-granzyme pathway at 37 degrees C in the presence of 1 mM Ca(2+) enhanced the ability of CTL extracts to induce apoptosis. This potentiation of cell death was Ca(2+)-dependent, thermo-resistant, and inhibited by 4-bromophenacyl bromide and scalaradial (two inhibitors of sPLA(2)). The involvement of an sPLA(2) was confirmed by blocking the pro-apoptotic activity of the Ca(2+)-treated cell extract with an anti-sPLA(2) polyclonal antibody. By cell fractionation assays, we showed that the pro-apoptotic sPLA(2) was localized in the cytoplasmic fraction but not in perforin-rich granules or plasma membrane fractions. Western blotting analysis revealed the presence of four distinct bands of 56, 29.5, 21, and 15 kDa. The highest molecular weight band was consistent with the expression of a group III sPLA2. Taken together, these data indicate that an apoptosis-inducing sPLA(2) is expressed in the cytosol of a CTL cell line and suggest that it plays an effector role in CTL-mediated cytotoxicity.