Characterization of haemolytic activity of coelomocytes of Holothuria tubulosa

Phylogenetic analysis recognizes echinoderms as a key group of deuterostomes, therefore the species in this group are useful for the study of the evolution of innate immunity responses. In addition, this marine invertebrate lives in coastal and estuarine waters that are directly exposed to potentially pathogenic microorganisms and stressful anthropogenic factors. Coelomocyte populations seem to be essential to immune-defence functions such as phagocytosis, ROI production, cytotoxicity, synthesis and release of antimicrobial substances including lectin, cytokine, C3-like expression, prophenoloxidase activity and capsule formation. Holothurians’ coelomocyte populations contain several coelomocyte types, including phagocytes, and can form brown bodies in response to multicellular parasites. Holothuria tubulosa coelomic fluid contains three main coelomocyte categories: amoebocytes, spherulocytes and progenitor cells. The amoebocytes represent about 30 % of the total population, and in living cell preparations exhibit two distinct forms: petaloid and philopodial; spherule cells represent the numerically dominant cell type (about 60 %). Progenitor cells are present in a lesser amount (about 20 %); they are similar to lymphocytes and show a nucleus that is typically prominent with a thin rim of cytoplasm. In the present report we show that coelomocytes of Holothuria tubulosa are able to exert cytotoxic activity against different cellular targets such as rabbit or sheep erythrocytes and the human erythromyeloid leukaemia-derived cell line K562. Moreover, the unseparated coelomocyte supernatant lysate (CLS) exerts a lytic activity, even in the absence of calcium, against the same targets. Analysis of the coelomocyte lysate by overlay assays with sheep and rabbit erythrocytes on PAGE without SDS showed a protein pattern composed by two main hemolytic bands with different electrophoretic mobility. The one with low mobility (I) showed calcium independent haemolytic activity while high mobility band (II) showed calcium dependent activity. The two bands were eluted from the gel and analyzed by SDS-PAGE and stained with silver stain; band I could be separated in three components of different sizes (52, 42 and 41 kDa), and band II had a size of 43 kDa. Spherule cells seemed to be the effector cells, as shown by a plaque lysis assay. Further studies are in progress to identify the lytic proteins and the lytic mechanism at work on both a molecular and cellular level.