Molecular biological studies on dolphin neutrophils

基于陆生哺乳动物同源物保守序列，利用RT-PCR技术克隆了宽鼻海豚NADPH氧化酶组分（gp91^<phox>、p22^<phox>、p40^<phox>和p67^<phox>）和细胞因子（IL-1α、IL-1β、IL-1ra、IL-4、IL-8、IFNγ和TNFα）的cDNA。海豚NADPH氧化酶组分和细胞因子的核酸序列与牛同源物具有较高的相似性（77.0 ~ 95.4%），推导出的氨基酸序列也与哺乳动物同源物相似。海豚中性粒细胞通过佛波酯刺激产生超氧化物，NADPH氧化酶的特异性抑制剂抑制其超氧化物形成活性。这些结果表明，海豚中性粒细胞具有产生氧自由基的NADPH氧化酶活性。几种刺激物均可诱导氧自由基生成的呼吸爆发（RB），但不包括n -甲酰基- met -亮氨酸和牛中性粒细胞。LPS和金黄色葡萄球菌刺激的海豚中性粒细胞RB低于牛和人。结果表明，海豚中性粒细胞的杀菌活性弱于陆生哺乳动物。多种受体介导的激动剂诱导的海豚中性粒细胞RB被重组海豚TNFα增强，且呈浓度依赖性。这种与TNFα相关的启动效应是不稳定的，并且不会被多粘菌素B消除，这表明重组海豚TNFα制剂的启动效应不是由于大肠杆菌来源的LPS污染所致。这些数据表明，细胞因子在海洋哺乳动物中也作为先天免疫的调节剂。

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.