Studies on free radical molecules produced from kuruma shrimp hemocytes

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NO has been demonstrated to be in molluscan immunocytes and in echinoderm coelomocytes in invertebrates but not yet demonstrated in shrimp hemocytes. Mammalian macrophage produces NO by LPS stimulation which is enhanced by co-stimulation with interferon γ (IFNγ). NO production is affected by other inflammatory cytokines, which play important roles in innate immunity and are conserved from invertebrate to mammals through phylogeny. Therefore, the importance of demonstrating NO in shrimp hemocytes is not only to clarify the evidence of free-radicals other than ROI, but also to provide the tools for exploring the cytokine network in the shrimp defense system, in which cellular defense factors are regulated by a chemical mediator, the cytokines (Bogdan et al., 2000 ; Remick and Villarete, 1996).The present study demonstrates the potential chemiluminescent probe, CLA, specific for superoxide and singlet oxygen, and the NO production after co-stimulation with LPS and recombinant human interferon γ (rhIFNγ) in shrimp hemocytes.The Cypridina luciferin analogue (CLA), a highly sensitive chemiluminescence (CL) probe, was used to detect superoxide (O_2) in kuruma prawn hemocytes. The CLA-dependent CL of the shrimp hemocyte after phorbol 12-myristate 13-acetate (PMA) stimulation was inhibited by superoxide dismutase. Nitric oxide (NO) production from the shrimp hemocytes was detected by fluorometric measurement after co-stimulation with lipopolysaccharides (LPS) and recombinant human interferon γ in vitro. The supernatant of a hemocyte culture after stimulation showed an increase in the fluorescence intensity, and this intensity decreased with the addition of N^G-monomethyl-L-arginine (L-NMMA). These results show that shrimp hemocytes can produce O^-_2 and NO and these free radicals play an important role in the shrimp defense network.