Killing of isogeneic erythrocytes by neutrophils in ginbuna crucian carp (Carassius auratus langsdorfii)

Cytotoxic activity of fish neutrophils against isogeneic erythrocytes is described. Unsensitised neutrophil-rich leucocytes from clonal ginbuna crucian carp, separated by density gradient centrifugation, were cultured with isogeneic erythrocytes. Lysis of isogeneic erythrocytes was determined by measuring the release of haemoglobin into the supernatant using tetra-methylbenzidine (TMB). The level of cytotoxicity obtained was proportional to both the content of granulocytes in effector cell fractions and the effector to target cell ratio. Lysis of isogeneic erythrocytes occurred only after direct contact of neutrophils with target erythrocytes. No erythrolysis was observed when granulocytes and erythrocytes were cultured in the same medium separated by a polycarbonate membrane, thus allowing exchange of only supernatant but not cells. Erythrolysis was therefore not due to a soluble factor released into the supernatant by the granulocytes. When erythrocytes and neutrophils were cultured together by immobilising them in agarose, erythrolysis was only observed microscopically in cases where erythrocytes were in contact with neutrophils. Thus, neutrophils appear able to cause lysis of histocompatible cells.