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

We identified and characterized two isoforms of tumor necrosis factor-alpha (TNF alpha) from the goldfish, TNFo alpha-1 and TNF alpha-2. At the protein level, goldfish TNF alpha-1 and TNF alpha-2 were most homologous to carp TNF alpha-1 and TNF alpha-2, respectively. Phytogenetically, the two goldfish isoforms grouped most closely with the carp TNF alpha soforms and TNF species of other cyprinids. Real-time PCR analysis revealed constitutive expression of goldfish TNF alpha-1 and TNF alpha-2 in all tissues with TNF alpha-2 mRNA levels higher than TNF alpha-1 in all tissues examined. A modest up-regutation in expressions of goldfish TNF alpha-1 and TNF alpha-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 alpha-2 was subsequently expressed using a prokaryotic expression system and the recombinant molecule (rTNF alpha-2) was functionally characterized. The rTNF alpha-2 induce a dose-dependent chemotactic response and enhanced phagocytosis of primary goldfish macrophages. Furthermore, rTNF alpha-2 primed the respiratory burst in monocytes and induced nitric oxide production of primary goldfish macrophages. Our results indicate that goldfish TNF alpha is a central regulatory and effector cytokine of inflammatory and antimicrobial responses of the goldfish. (c) 2007 Elsevier Ltd. All rights reserved.