Colony specificity and chemotaxis in the compound ascidian Botryllus schlosseri.

We re-investigated the behavior of hemocytes during the non-fusion (rejection) reaction between genetically incompatible colonies of the ascidian Botryllus schlosseri. In the course of the reaction, hemocytes - mainly morula cells - crowd inside the blind ends of marginal vascular vessels (known as ampullae) of the colonial leading edge (LE) facing the foreign colony which suggests the occurrence of chemotactic attraction of circulating hemocytes towards the ampullar lumen. Then, cells migrate, through the ampullar tips, into the partially fused tunics and contribute to the formation of the necrotic spots along the contact borders which characterize the reaction. Studies on histological sections clearly indicate that, although morula cell concentration is always higher in ampullae of the LE than in those of the lateral (L) part of the colony, their frequency significantly increases in LE ampullae of rejecting colonies with respect to LE ampullae of both fusing and isolated colonies. In addition, in vitro chemotaxis experiments demonstrated that blood plasma from incompatible colonies can stimulate morula cell migration through polycarbonate filters and this passage is inhibited by antibodies raised against mammalian pro-inflammatory cytokines. The possible nature and role of molecules recognized by anti-cytokine antibodies in hemocyte migration are discussed.

Cellular aspects of allorecognition in the compound ascidian Botryllus schlosseri.

We studied changes in the morphology of morula cells, a common haemocyte type in botryllid ascidians, during both the rejection reaction (occurring between contacting, genetically incompatible colonies) and fusion (occurring between compatible colonies), and in short-term cultures of haemocytes incubated with heterologous or autologous blood plasma. In both the rejection reaction and haemocyte cultures in the presence of heterologous blood plasma, we observed alterations in morula cells, consistent with a degranulation event, and their expression of molecules recognised by anti-IL-1-alpha- and anti-TNF-alpha-antibodies. Anti-cytokine-antibodies markedly reduced the extent of the in vitro cytotoxicity, when haemocytes were exposed to heterologous blood plasma. In addition, the increase in the production of nitrite ions and the decrease of the in vitro cytotoxicity by the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester, suggest the role of nitric oxide in cell death. These results provide new clues to understand the process of rejection reaction in botryllid ascidians.

Oxidative stress induces cytotoxicity during rejection reaction in the compound ascidian Botryllus schlosseri.

When genetically incompatible colonies of the compound ascidian Botryllus schlosseri contact each other, a rejection reaction occurs, characterised by the appearance of cytotoxic foci along the touching borders. In the course of this reaction, morula cells, a common haemocyte-type in ascidians, release their vacuolar content, mainly phenoloxidase and its polyphenol substrata, upon the recognition of soluble factors diffusing from the alien colony through the partially fused tunic. In a previous paper, we demonstrated the relationship between phenoloxidase and cytotoxicity. Here, we investigated the effects of superoxide dismutase, catalase and sorbitol (scavengers of superoxide anions, peroxides and hydroxyl radicals, respectively) on the cytotoxicity observed in haemocyte cultures incubated with heterologous blood plasma. Although the above compounds have no effects on morula cell degranulation and phenoloxidase activity, they suppress cell death, suggesting that oxidative stress plays a key role in in vitro cytotoxicity. In addition, sorbitol reduces the extent of the cytotoxicity occurring in the rejection reaction between incompatible colonies, which stresses the important role of hydroxyl radicals in this process. The observation of a decrease in total and non-protein thiols in haemocytes previously incubated with heterologous blood plasma fits the hypothesis of oxidative stress as the main cause of phenoloxidase-related cytotoxicity.

Phagocyte spreading and phagocytosis in the compound ascidian Botryllus schlosseri: evidence for an integrin-like, RGD-dependent recognition mechanism.

The involvement of integrins in phagocyte spreading and phagocytosis was investigated in the compound ascidian Botryllus schlosseri. The number of spreading cells was significantly reduced when adhesion occurred in the presence of the tetrapeptide Arg--Gly--Asp--Ser (RGDS), but not of Arg--Gly--Glu--Ser (RGES) indicating the involvement of RGD-mediated adhesion mechanisms in phagocyte spreading. The significant decrease of the fraction of spreading cells in the presence of Botryllus blood plasma suggests the presence of RGD-containing molecules in the blood of our species. The increase in the same index when blood plasma-coated slides as well as fibrinogen- and fibronectin-coated coverslips were used, fits with the above hypothesis. Adhesion in the presence of RGDS leads to a consistent alteration of the actin cytoskeleton, in agreement with the known role of integrin adhesion in microfilament organization. Phagocytosis was greatly reduced by RGDS in the incubation medium, but not by RGES, and was significantly increased by coating yeast cells with fibronectin or blood plasma. Both spreading and phagocytic capability were severely inhibited by wortmannin, suggesting the importance of phosphatidylinositol-3-kinase in integrin-mediated signal transduction in ascidians.