Phagocytosis and peroxidase release by seabream (Sparus aurata L.) leucocytes in response to yeast cells.

Abstract

A flow cytometric method was adapted to evaluate phagocytosis by gilthead seabream leucocytes after incubation with yeast cells (Saccharomyces cerevisiae). Head-kidney leucocytes were incubated in vitro for different times in different proportions with heat-killed fluorescein isothiocyanate (FITC)-labeled yeast cells to study the kinetics of phagocytosis. Attached and internalized yeast cells were differentiated by quenching FITC-labeled S. cerevisiae with trypan blue dye. Only internalized cells kept their FITC fluorescence after quenching. Monocyte-macrophages and acidophilic granulocytes showed phagocytic activity, as demonstrated by transmission electron microscopy (TEM). From the ultrastructural features of the phagocytic process, it was observed that cytoplasmic granule membranes fused with the phagocyte membrane at the point where the yeast cell was attached to the phagocyte surface. This observation led us to adapt a colorimetric method to study peroxidase (myeloperoxidase and eosinophil peroxidase) release, since both are considered to be markers of the degranulation that occurs in seabream head-kidney leucocytes in response to yeast cells. Head-kidney leucocytes were incubated with calcium ionophore (CaI), phorbol myristate acetate (PMA), or yeast cells for different periods of time (0-30 min) to study the kinetics of peroxidase release. The results obtained indicate that CaI and yeast cells, but not PMA, stimulate the degranulation (by about 44.51% and 21.04%, respectively, at 30 min) of seabream head-kidney leucocytes.