Morula cells as the major immunomodulatory hemocytes in ascidians: evidences from the colonial species Botryllus schlosseri.

Immunocytochemical methods were used to study the presence and distribution of IL-1-alpha- and TNF-alpha-like molecules in the hemocytes of the colonial ascidian Botryllus schlosseri. Only a few unstimulated hemocytes were positive to both the antibodies used. When the hemocytes were stimulated with either mannan or phorbol 12-mono-myristate, the phagocytes were not significantly changed in their number, staining intensity, or cell morphology. In contrast, stimulated morula cells were intensely labeled, indicating that these cells play an important immunomodulatory role.

Purification and characterization of a humoral opsonin, with specificity for D-galactose, in the colonial ascidian Botryllus schlosseri.

A humoral agglutinin from the hemolysate of the colonial ascidian Botryllus schlosseri was purified by affinity chromatography. This agglutinin does not require metal cations for its activity and is specific for derivatives of D-galactose. On SDS-PAGE analysis, it was resolved in two bands, of 17 and 19 kDa in reducing conditions and 15 and 16 kDa in non-reducing conditions. This behavior is due to the establishment of disulfide bridge between the thiols of cysteine, well represented in the molecule as revealed by amino acid analysis. The latter also indicated high percentages of hydrophilic residues, probably involved in sugar recognition. The lectin is an opsonin, as it increases both the phagocytic index and the number of phagocytized yeast cells. The hypothesis that this Botryllus agglutinin belongs to the galectin family of lectins is discussed.

Phenoloxidase and cytotoxicity in the compound ascidian Botryllus schlosseri.

The vacuoles of morula cells (MC) of the colonial ascidian Botryllus schlosseri contain phenoloxidase (PO). As the release of their vacuolar content at the border of incompatible contacting colonies is associated with the formation of necrotic masses which characterize the rejection reaction, the role of PO in Botryllus cytotoxicity was investigated. When hemocytes are incubated with blood plasma from incompatible (heterologous) colonies, MC degranulate and, after 60 min, the cytotoxicity index becomes significantly greater than that observed in controls incubated with autologous plasma. The rise in cell mortality is completely inhibited by the addition of PO inhibitors sodium benzoate, tropolone and phenylthiourea, and serine protease inhibitors phenylmethylsulfonyl fluoride, benzamidine, N-tosyl-L-phenylalanine chloromethyl ketone and N-tosyl-L-lysine chloromethyl ketone. The addition of either reducing agents L-cysteine and ascorbic acid or reactive oxygen species scavenger enzymes superoxide dismutase and catalase has a similar effect. Significant inhibition of cytotoxicity is also observed with the quinone scavenger, 3-methyl-2-benzothiazolinone hydrazone. In the presence of sodium benzoate and phenylthiourea, there is a significant reduction in the number, size and color intensity of necrotic masses along the contact border of incompatible colonies. A significant increase in superoxide anion production, completely inhibited by sodium benzoate, is observed when hemocytes are incubated with heterologous blood plasma. These results indicate that: (i) PO is the enzyme responsible for the cytotoxicity observed in both hemocyte cultures and rejection reactions; (ii) PO is present inside MC vacuoles as a proenzyme which is activated, upon release, by humoral proteases; (iii) cytotoxicity appears to be mainly due to oxidative stress generated by PO during oxidation of polyphenols to quinones without the involvement of other oxidases such as NADPH oxidase and peroxidase.

Calcium homeostasis and yeast phagocytosis in hemocytes of the colonial ascidian Botryllus schlosseri.

This paper reports the effects of drugs affecting the homeostasis of cytosolic-free calcium on in vitro yeast phagocytosis by hemocytes of the colonial ascidian Botryllus schlosseri. Significant inhibition of phagocytosis is observed after exposure of hemocytes to 10 microM or higher concentrations of thimerosal, which is known to deplete intracellular calcium stores in mammalian cells. The two calcium channel blockers nifedipine and verapamil significantly decrease the phagocytic index, the minimum effective concentrations being 10 and 50 microM, respectively. As these substances have no effects at lower concentrations, they probably act through the inhibition of Ca(2+)-ATPase activity, required to restock intracellular calcium stores, due to their interaction with calmodulin. Analogously, pimozide, which suppresses ATPase activity by interacting with calmodulin, and thapsigargin, which inhibits Ca(2+)-ATPase activity, significantly reduce the phagocytic index. Moreover, nifedipine, by altering cytosolic calcium homeostasis, also lowers the production of superoxide anion associated with phagocytosis. Results indicate that in ascidians, as in mammals, a rise in intracellular calcium is required for phagocyte activation and induction of the respiratory burst.

Phagocytosis in the colonial ascidian Botryllus schlosseri.

Phagocytosis by Botryllus schlosseri hemocytes is influenced by temperature, pH, concentration, and physicochemical properties of the test particles and requires Ca2+ or Mg2+ ions to occur. Phagocytes recognize glucosyl or mannosyl residues on the surface of yeast cells, and a respiratory burst is associated with phagocytosis, as indicated by increased superoxide production. Factors that enhance phagocytosis of yeast, sheep red blood cells, and latex beads and reduce the uptake of yeast and sheep erythrocytes are present in the plasma.

Chimeras and histocompatibility in the colonial ascidian Botryllus schlosseri.

Chimeras of B. schlosseri were prepared by pairwise combination of colonies sharing one allele at the fusibility gene locus (AD = AC chimeras). A frequent resorption of one of the partners was observed and the resorption time was shown to be significantly correlated with the relative size, the resorbing partner being usually the larger. Both in the whole chimeras, AD = AC, and in the separated partners, (AD)AC, the fusibility of AC was frequently altered. In AD = AC the fusion did not prevent entirely AC fusion with BC. The fusion frequency with BC was significantly higher than with BD and significantly higher in (AD)AC than in AD = AC. Repeated rejections with BC or both BC and BD, repeated fusions with BD, simultaneous or successive fusions and rejections especially with BD, over a period of several months, indicated a long lasting competitive interaction of AD and AC in (AD)AC chimeras. The persistence in these chimeras of the AD cell population was also confirmed by the chimeric electrophoretic pattern of PGI in most of them.