Characterization and functional analysis of goldfish (Carassius auratus L.) tumor necrosis factor-alpha

We identified and characterized two isoforms of tumor necrosis factor-alpha (TNFα) from the goldfish, TNFα-1 and TNFα-2. At the protein level, goldfish TNFα-1 and TNFα-2 were most homologous to carp TNFα-1 and TNFα-2, respectively. Phylogenetically, the two goldfish isoforms grouped most closely with the carp TNFα isoforms and TNF species of other cyprinids. Real-time PCR analysis revealed constitutive expression of goldfish TNFα-1 and TNFα-2 in all tissues with TNFα-2 mRNA levels higher than TNFα-1 in all tissues examined. A modest up-regulation in expressions of goldfish TNFα-1 and TNFα-2 in kidney-derived monocytes and significant increase in expression of both isoforms in mature macrophages were observed in response to activation with macrophage-activating factors. TNFα-2 was subsequently expressed using a prokaryotic expression system and the recombinant molecule (rTNFα-2) was functionally characterized. The rTNFα-2 induced a dose-dependent chemotactic response and enhanced phagocytosis of primary goldfish macrophages. Furthermore, rTNFα-2 primed the respiratory burst in monocytes and induced nitric oxide production of primary goldfish macrophages. Our results indicate that goldfish TNFα is a central regulatory and effector cytokine of inflammatory and antimicrobial responses of the goldfish.