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.