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RESEARCH PRODUCT

D.P14 In vitro cytotoxic activity against erythrocytes by ascidians hemocytes: Target/effector interactions

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The cell-mediated, calcium-dependent cytotoxic activity against mammalian erythrocytes by the hemocytes of the ascidians Ciona intestinalis and Stye/a plicata was investigated by in vitro cytotoxic assay (HCA). Hemocytes/erythrocytes mixtures were performed in artificial sea water or TBS enriched with 10 mM CaC12, at 25° or 3rC, and the released haemoglobin was estimated by reading the absorbance at 541 nm (Parrinello et al., D.C.I., 17:19, 1993). With the aim to examine the cytotoxic mechanism involved, inhibition experiments with lipidic components of the erythrocyte membrane have been carried out in TBS-Ca. Among the phospholipids tested sphingomyelin is an inhibitor of the Ciona intestinalis HCA. Thin layer chromatography of the clorophorm-methanol extract from hemocyte debris (about 50% HCA) rule out the possibility that the lytic action might depend on a membrane bound sphingomyelinase. The inhibition capacity of the ceramide and phosphorylcholine suggests that the whole sphingomyelin molecule is involved in binding cytolysins. Experiments are in progress to examine the involvement of phospholipase A2 and C. In order to identify the hemocytes responsible for HCA, the hemocytes populations were separated on a Percoll density gradient. Previous results have shown that granulocytes may be the effector hemocytes.