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

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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 A2 (sPLA2) 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°C in the presence of 1 mM Ca2+ enhanced the ability of CTL extracts to induce apoptosis. This potentiation of cell death was Ca2+-dependent, thermo-resistant, and inhibited by 4-bromophenacyl bromide and scalaradial (two inhibitors of sPLA2). The involvement of an sPLA2 was confirmed by blocking the pro-apoptotic activity of the Ca2+-treated cell extract with an anti-sPLA2 polyclonal antibody. By cell fractionation assays, we showed that the pro-apoptotic sPLA2 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 sPLA2 is expressed in the cytosol of a CTL cell line and suggest that it plays an effector role in CTL-mediated cytotoxicity.

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Acknowledgements

The authors wish to thank Drs. Russolina Zingale, Paulo Melo, Bruno L. Diaz, Bartira R. Bergmann, and Leda Q. Vieira for donating reagents, animals, and inhibitors, and Drs. João Viola, George dos Reis, Rodrigo C. Bisaggio, Robson Coutinho-Silva, André Fuly, Vera Koatz, Luciana Nogaroli, Cristiane M. Cruz, Miriam Werneck, and Camila Chaves Santos for helpful discussions.

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Authors and Affiliations

  1. Laboratório de Imunobiofísica, Instituto de Biofísica Carlos Chagas Filho –UFRJ, Universidade Federal do Rio de Janeiro, Bloco G do CCS – Ilha do Fundão, 21941–590, Rio de Janeiro, Brazil

    Helio M. Costa-Junior, Flávia C. Hamaty, Rafael da Silva Farias & Pedro M. Persechini

  2. Laboratório de Físico–Química Biológica, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

    Marcelo Einicker-Lamas

  3. Laboratório de Imunoquímica, Instituto de Microbiologia Professor Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

    Maria H. da Silva

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  1. Helio M. Costa-Junior
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  2. Flávia C. Hamaty
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  3. Rafael da Silva Farias
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  4. Marcelo Einicker-Lamas
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  5. Maria H. da Silva
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  6. Pedro M. Persechini
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Correspondence to Pedro M. Persechini.

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This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), and Programa de Núcleos de Excelência (PRONEX–CNPq).

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Costa-Junior, H.M., Hamaty, F.C., da Silva Farias, R. et al. Apoptosis-inducing factor of a cytotoxic T cell line: involvement of a secretory phospholipase A2 . Cell Tissue Res 324, 255–266 (2006). https://doi.org/10.1007/s00441-005-0095-y

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