Osamu Kurata

Neutrophilic granulocytes in carp, Cyprinus carpio, possess a spontaneous cytotoxic activity

This study demonstrates for the first time that carp (Cyprinus carpio) neutrophilic granulocytes from the head kidney possess potent spontaneous cytotoxic activity against several human tumor cell lines. Carp head kidney cells isolated at a density of 1.09 g/mL contained more than 90% neutrophilic granulocytes. These cells were round and approximately 10 millimicrons in diameter with reniform or polymorphic nuclei and slightly eosinophilic cytoplasm when stained with Giemsa. Electron microscopy revealed that the cytoplasm contained numerous oval granules, some of which contained a dense rod-shaped core. The neutrophilic granulocytes readily formed conjugates with the human target cells and rapidly killed them. The neutrophilic granulocytes killed human derived target cells better than murine derived target cells. Inhibition of cytotoxicity by catalase suggested that the production of H2O2 is involved as a mediator in the cytotoxic reaction. The size and granularity of the carp effector cells indicate that they are different from the small agranular nonspecific cytotoxic cells (NCC) described in the channel catfish.

Activation of carp leukocytes by a galactose-binding protein from Aphanomyces piscicida.

This study demonstrated that a galactose-binding protein (GBP) produced by a fish pathogenic water mold, Aphanomyces piscicida, activates carp leukocytes. Leukocytes were separated from the head kidney and peripheral blood using Percoll density centrifugation. A flow cytometric analysis revealed that GBP binds with many cells and a variety of cell types including lymphocytes, granulocytes and thrombocytes. Intracellular calcium flux of the peripheral blood leukocytes induced by stimulation with GBP was confirmed by counting the fluo-3 loaded cells whose fluorescence increased after the stimulation using flow cytometry. The percentage of cells in which a calcium flux was induced peaked 1 min after the stimulation. Approximately 6% of the cells specifically responded 1 min after the stimulation. The proliferation response was determined by the level of BrdU uptake by the leukocytes after the stimulation. Cell proliferation was observed 2, 4 and 6 days after stimulation with GBP. The expression of cytokines IL-1beta and TGF-beta1 in the peripheral blood leukocytes, after the stimulation was evaluated by a semi-quantitative reverse-transcriptase polymerase chain reaction. Increased expression of IL-1beta was observed 4h after stimulation with GBP. Variation of TGF-beta1 expression under the same conditions was not observed. The kinetics of intracellular calcium flux and the level of IL-1beta expression induced by GBP stimulation were different from those induced by phytohemagglutinin stimulation. These results confirmed that GBP is a pathogenic microbial component that can induce cell activation. GBP seems to induce the inflammatory response observed in the Aphanomyces infection.

Accommodation of carp natural killer-like cells to environmental temperatures

Abstract To assess cytotoxic activity of carp ( Cyprinus carpio ), head kidney leukocytes were examined with special reference to the effects of rearing water temperature and of assay temperature. Leukocytes as effector cells were separated from head kidney using Histopaque 1077 and cytotoxic activities were measured by the release of 51 Cr from target cells which were K562, human chronic myelogenous leukemia cells. Cytotoxic activity of leukocytes from carp kept at higher temperature (25 °C) was lower at lower assay temperature (10 °C) than at higher assay temperature (25 °C). However, activity from carp acclimated to low temperature (10 °C) increased on 10 °C assay and decreased on 25 °C assay. Cellular composition of effector cells changed in connection with the above phenomenon. In carp acclimated to 10 °C, small lymphocytes decreased while the others, e.g. large lymphocytes, granulocytes and macrophages, increased. Accommodation of carp natural killer-like cells to environmental temperatures may be regulated by changing the cellular composition of effector cells.

A new fluorochromasia method using a fluorescence microplate reader for assay of cytotoxic activity of carp leucocytes

A new fluorochromasia method using a fluorescence microplate reader has been established to assay spontaneous cytotoxic activity of carp leucocytes. This method is characterized by using propidium iodide (PI) for staining dead target cells and a fluorescence microplate reader for measurement of the fluorescence of PI. K562 as target cells were prepared in 96-well flat-bottom microplates, and carp leucocytes were added as effector cells. After 2.5 h incubation, PI was added to each well. After an additional 1.5 h incubation, fluorescence of each well was measured. Correlation between this method and 51Cr-release assay was obtained. The results demonstrated that this new fluorochromasia method can be used to assay cytotoxic activity of carp leucocytes.