Haemocytes and innate immunity in the compound ascidian Botryllus schlosseri

Two types of circulating immunocytes are present in the colonial ascidian Botryllus schlosseri: cytotoxic morula cells and phagocytes. Morula cells (MC) are large, vacuolated cells which represent the most abundant blood cells, their frequency ranging from 20 to 60% of circulating haemocytes. They are the mediators of the inflammatory (rejection) reaction which occurs, in the form of a series of necrotic spots, along the contacting borders, when genetically incompatible colonies are juxtaposed. MC induce cytotoxicity in the contacting tissues through the release of the enzyme phenoloxidase (PO), probably stored as a pro-enzyme inside their granules. MC can synthesise molecules recognized by antibodies raised against the mammalian inflammatory cytokines IL-1-alpha and TNF-alpha upon a recognition of soluble foreign molecules, such as mannan and allogeneic humoral factors diffusing from one colony into the blood of the alien colony in the course of the rejection reaction. These molecules exert a chemotactic action, as demonstrated by in vitro chemotaxis experiments in which the addition of anti-IL-1-alpha and anti-TNF-alpha antibodies significantly decreases MC migration through 8 µm-filters. Phagocytes include hyaline amoebocytes and macrophage-like cells. The former are amoeboid cells, 5-10 µm in size, capable of active movements and phagocytosis, whereas the latter are large (10-15 µm in diameter) roundish cells with at least one vacuole containing ingested material, deriving form hyaline amoebocytes having engulfed foreign cells or particles. A respiratory burst is associated with phagocytosis, leading to the production of superoxide anions and other ROS, In addition, phagocytes are capable of constitutive macropinocytosis (MP) at sites of membrane ruffling along the cell leading edge. Phagocyte spreading and MP are enhanced by the recognition of molecules containing the sequence Arg-Gly-Asp (RGD), suggesting that integrin activation is involved in both the processes. The occurrence of MP is associated with increased oxygen consumption and a rise in the production of superoxide anion and ATP. Phagocytes synthesise and secrete a galectin, with opsonic activity, able to bridge phagocytes and foreign cells such as mammalian erythrocytes or yeast cells. Spreading ability and phagocytosis are significantly enhanced by exogenous IL-8 in the culture medium and the effects are coupled to modifications of the actin cytoskeleton. The supernatant form haemocyte cultures matched with yeast cells contains factor(s) able to enhance yeast ingestion by Botryllus phagocytes. When haemocytes were fractionated by density gradient centrifugation and each band was incubated with yeast, the ability to stimulate phagocytosis was found in the supernatants of fractions rich in MC. The enhancing effect is completely absent in the presence of anti-IL-1-alpha and anti-TNF-alpha antibodies. This indicates that MC actively recognize non-self molecular patterns and, as a consequence, release humoral factor(s) able to modulate phagocytosis.