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

Abstract

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