Migratory, respiratory burst and mitogenic responses of leucocytes isolated from the gills of rainbow trout (Oncorhynchus mykiss)

Leucocytes were isolated from the perfused gill of rainbow trout (Oncorhync hus mykiss) by DTT/collagenase digestion followed by nylon wool filtration. Isolated gill cells were fractioned on a 40-70% Percoll gradient into two subpopulations, top and bottom cell fractions. Electron microscopy revealed the top fraction to comprise a mixture of cell types including lymphocytes , neutrophils, macrophages, eosinophilic granule cells (EGCs), goblet cells , chloride cells, epithelial cells and cell debris. The bottom fraction con tained mainly lymphocytes. Flow-cytometric analysis indicated the pre-Perco ll population to be mainly composed of a single peak which closely matched the size/granularity profile occupied by peripheral blood lymphocytes. Only a small difference in size/granularity properties was observed between the two post-Percoll populations. Fractionated and unfractionated isolated gil l cells were studied in respect to several 'cell-mediated' immune functions . On stimulation with calcium ionophore, the isolated gill cells, following nylon wool filtration, were shown to be capable of producing chemoattracta nts for head kidney leucocytes at a dilution of 1:8. Only the bottom cell f raction exhibited migration toward a 2% dilution of trout serum while dilut ions of 0.25% and 0.5% rainbow trout serum were not chemo-attractive for ei ther head kidney or gill leucocyte populations. Respiratory burst activity was undetectable with isolated gill cells (pre-nylon wool or post-Percoll f ractions). Mitogenic responses of isolated and fractionated gill cells to L PS and PHA suggested the presence of few B-cells and a preponderance of T-c ells. (C) 1999 Academic Press.