A lytic mechanism based on soluble phospholypases A2 (sPLA2) and b-galactoside specific lectins is exerted by Ciona intestinalis (ascidian) unilocular refractile hemocytes against K562 cell line and mammalian erythrocytes

Abstract

Hemocytes from the ascidian Ciona intestinalis exert in vitro Ca2þ-dependent cytotoxic activity toward mammalian erythrocytes and K562 cells. To examine the lytic mechanism, hemocyte populations were separated (B1eB6 bands) through a Percoll discontinuous density gradient, the hemocyte cytotoxic activity (HCA) and the lytic activity of the hemocyte lysate supernatant (HLS) were assayed. In addition the separated hemocytes were cultured and the cell-free culture medium (CFM) assayed after 3 h culture. Results support that unilocular refractile hemocytes (URGs), enriched in B5, are cytotoxic. The B5-HLS contains lysins and the activity of B5-CFM shows that lysins can be released into a culture medium. The B5 activity was blocked by D-Galactose, a-Lactose, Lactulose, LacNAc, thiodigalactoside (TDG), L-Fucose, DMannose, D-Glucose, sphingomyelin (SM), and soluble phospholipase A2 (sPLA2) inhibitors (dibucain, quinacrine). Accordingly, HLS chemico-physical properties (alkaline medium, high thermostability, Ca2þ- dependence, trypsin treatment, protease inhibitors) and SEM observations of the affected targets suggested that sPLA2 could be responsible for changes and large alterations of the target cell membrane. An apoptotic activity, as recorded by a caspase 3, 7 assay, was found by treating K562 cells with very diluted HLS. A lytic mechanism involving sPLA2 and lectins promptly released by URGs and morula cells respectively is suggested, whereas target cell membrane SM could be a modulator of the enzyme activity.