Characterization and functional analysis of goldfish (Carassius auratus L.) tumor necrosis factor-alpha.
We identified and characterized two isoforms of tumor necrosis factor-alpha (TNFalpha) from the goldfish, TNFalpha-1 and TNFalpha-2. At the protein level, goldfish TNFalpha-1 and TNFalpha-2 were most homologous to carp TNFalpha-1 and TNFalpha-2, respectively. Phylogenetically, the two goldfish isoforms grouped most closely with the carp TNFalpha isoforms and TNF species of other cyprinids. Real-time PCR analysis revealed constitutive expression of goldfish TNFalpha-1 and TNFalpha-2 in all tissues with TNFalpha-2 mRNA levels higher than TNFalpha-1 in all tissues examined. A modest up-regulation in expressions of goldfish TNFalpha-1 and TNFalpha-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. TNFalpha-2 was subsequently expressed using a prokaryotic expression system and the recombinant molecule (rTNFalpha-2) was functionally characterized. The rTNFalpha-2 induced a dose-dependent chemotactic response and enhanced phagocytosis of primary goldfish macrophages. Furthermore, rTNFalpha-2 primed the respiratory burst in monocytes and induced nitric oxide production of primary goldfish macrophages. Our results indicate that goldfish TNFalpha is a central regulatory and effector cytokine of inflammatory and antimicrobial responses of the goldfish.
Grayfer
Leon
L
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9.
Walsh
John G
JG
Belosevic
Miodrag
M
D000595:Amino Acid Sequence; D000818:Animals; D002633:Chemotaxis; D006054:Goldfish / Q000276:immunology*; D008264:Macrophages / Q000276:immunology; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000096:biosynthesis; D016366:Open Reading Frames; D010587:Phagocytosis; D016133:Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide