Molecular biological studies on dolphin neutrophils

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The bottlenose dolphin NADPH oxidase components (gp91^<phox>, p22^<phox>, p40^<phox> and p67^<phox>) and cytokines (IL-1α, IL-1β, IL-1ra, IL-4, IL-8, IFNγ and TNFα) cDNA were cloned utilizing the RT-PCR with specific primers based on the conserved sequences of terrestrial mammalian homologs. The nucleic acid sequences of dolphin NADPH oxidase components and cytokines indicate high similarity (77.0-95.4%) with those of bovine homologs, and these deduced amino acid sequences were also similar to those of mammalian homologs.Dolphin neutrophils generated superoxide by the stimulation of phorbol ester and a specific inhibitor of NADPH oxidase suppresses its superoxide-forming activity. These results indicate that dolphin neutrophils possess NADPH oxidase activity to generate oxygen radicals. The respiratory burst (RB) accompanying oxygen radicals generation was induced by several stimulants, but not N-formyl-Met-Leu-Phe as well as bovine neutrophils. The RB of dolphin neutrophils stimulated with LPS and Staphylococcus aureus was inferior to those of bovine and human. This result suggests that the bactericidal activity of dolphin neutrophils was weaker than those of terrestrial mammals.The RB of dolphin neutrophils induced by various receptor-mediated agonists was enhanced by recombinant dolphin TNFα in a concentration-dependent manner. This TNFα-associated priming effect was hea-instable and not abolished by polymyxin B, indicating that the priming effect of recombinant dolphin TNFα preparations are not due to contamination with LPS derived from Escherichia coli. These data demonstrate that cytokines act as a modulator for innate immunity also in marine mammals.