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 headkidney leucocytes. Anat Rec Part A 272A: 415-423, 2003.

A rapid, direct assay to measure degranulation of primary granules in neutrophils from kidney of fathead minnow (Pimephales promelas Rafinesque, 1820)

“…When media with Ca 2C was used, neutrophils readily responded to stimulants and the MPO exocytosis was detectable within 1e5 min. This finding is in agreement with studies conducted on human, bovine and seabream neutrophils [12,13,15,16,30]. Cytochalasin B has been reported to be necessary for primary granule exocytosis in bovine, human, and gilthead seabream neutrophils in response to stimulants [16,30,31].…”

“…This finding is in agreement with studies conducted on human, bovine and seabream neutrophils [12,13,15,16,30]. Cytochalasin B has been reported to be necessary for primary granule exocytosis in bovine, human, and gilthead seabream neutrophils in response to stimulants [16,30,31]. Neutrophils often have been pre-treated with cyto B before being exposed to stimulants, but no significant difference was reported when bovine and seabream neutrophils have been exposed to cyto B and stimulants at the same time [16,30].…”

“…In cattle, human, and seabream neutrophils, degranulation activity in vitro has been shown to be dependent on duration of incubation time [15,16,30]. The kinetic study of MPO release in fathead minnow kidney neutrophils demonstrated that the maximum activity of up to 80% MPO release is reached after 40e60 min when CaI is used as a stimulant with or without cyto B, and stimulation with PMA and cyto B showed significantly less maximum release after 60 min (Fig.…”

“…Also, modifications of the assay have been reported for gilthead seabream (Sparus aurata L.) [30]. Granulocyte characterisation in cyprinid species (common carp, Cyprinis carpio L. and goldfish, Carassius auratus L.) describes the presence of myeloperoxidase in fish neutrophils [6,7].…”

“…Cytochalasin B has been reported to be necessary for primary granule exocytosis in bovine, human, and gilthead seabream neutrophils in response to stimulants [16,30,31]. Neutrophils often have been pre-treated with cyto B before being exposed to stimulants, but no significant difference was reported when bovine and seabream neutrophils have been exposed to cyto B and stimulants at the same time [16,30]. The addition of cyto B alone stimulated 11% MPO release in bovine neutrophils [16], but in fathead minnow kidney neutrophils exposed only to cyto B alone, the mean percentage of MPO release was 1.1% (range 0e5%).…”