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 (Oncorhynchus 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 contained mainly lymphocytes. Flow-cytometric analysis indicated the pre-Percoll 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 gill 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 chemoattractants for head kidney leucocytes at a dilution of 1:8. Only the bottom cell fraction exhibited migration toward a 2% dilution of trout serum while dilutions of 0·25% and 0·5% rainbow trout serum were not chemo-attractive for either head kidney or gill leucocyte populations. Respiratory burst activity was undetectable with isolated gill cells (pre-nylon wool or post- Percoll fractions). Mitogenic responses of isolated and fractionated gill cells to LPS and PHA suggested the presence of few B-cells and a preponderance of T-cells.