TNF-α is a cytokine involved in systemic inflammation and regulation of immune cells. It is produced chiefly by activated macrophages as a membrane or secreted form. In rainbow trout, two TNF-α molecules were described previously. In this article, we report a third TNF-α (TNF-α3) that has only low identities to known trout molecules. Phylogenetic tree and synteny analyses of trout and other fish species suggest that two types (named I and II) of TNF-α exist in teleost fish. The fish type-II TNF-α has a short stalk that may impact on its enzymatic release or restrict it to a membrane-bound form. The constitutive expression of trout TNF-α3 was generally lower than the other two genes in tissues and cell lines, with the exception of the macrophage RTS-11 cell line, in which expression was higher. Expression of all three TNF-α isoforms could be modulated by crude LPS, peptidoglycan, polyinosinic:polycytidylic acid, and rIFN-γ in cell lines and primary macrophages, as well as by bacterial and viral infections. TNF-α3 is the most responsive gene at early time points post-LPS stimulation and can be highly induced by the T cell–stimulant PHA, suggesting it is a particularly important TNF-α isoform. rTNF-α3 produced in CHO cells was bioactive in different cell lines and primary macrophages. In the latter, it induced the expression of proinflammatory cytokines (IL-1β, IL-6, IL-8, IL-17C, and TNF-αs), negative regulators (SOCS1–3, TGF-β1b), antimicrobial peptides (cathelicidin-1 and hepcidin), and the macrophage growth factor IL-34, verifying its key role in the inflammatory cytokine network and macrophage biology of fish.

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A rainbow trout (Oncorhynchus mykiss) gene for tumor necrosis factor (TNF) has been cloned and sequenced. The cDNA contains an open reading frame of 738 nucleotides that translate into a 246 amino‐acid putative peptide, with a 5′ untranslated region (UTR) of 140 bp and a 3′ UTR of 506 bp. Two potential N‐linked glycosylation sites exist in the translation. The genomic sequence measures 2007 bp and contains three introns that intercept four coding exons. Expression studies using RT‐PCR have shown that the trout TNF gene is constitutively expressed in the gill and kidney of unstimulated fish. Trout TNF expression could be up‐regulated by stimulation of isolated head kidney leucocytes with lipopolysaccharide (LPS). Similarly, stimulation of a trout macrophage cell line (RTS11) with LPS resulted in an increased transcript level, as did incubation with recombinant trout interleukin (IL)‐1β. The optimal timing for induction of TNF expression in trout macrophages was determined using recombinant trout IL‐1β, where a clear induction was apparent by 2 h and peaked at 4 h. Evidence that this TNF gene is equivalent to mammalian TNF‐α is discussed.

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