Functional characterization of TNF-α in grass carp head kidney leukocytes: induction and involvement in the regulation of NF-κB signaling.

Tumor necrosis factor-alpha (TNF-α) is a potent regulatory cytokine, which serves as a key mediator of inflammation, immunity and apoptosis in mammals. Identification, expression and regulatory effects of TNF-α have been reported in various fish species, showing the structural and functional similarity or discrepancy between each other. In this study, TNF-α was identified from grass carp (Ctenopharyngodon idella) and the deduced grass carp TNF-α (gcTNF-α) protein possessed the TNF family signature motifs, a protease cleavage site, a transmembrane domain and two conserved cysteine residues. Further studies showed that gcTNF-α expression was induced with a rapid kinetics by immune challenge in vitro and in vivo. To characterize the function of gcTNF-α, recombinant gcTNF-α (rgcTNF-α) was prepared by using the Escherichia coli expression system. It was shown to enhance the mRNA expression of gcTNF-α and gcIL-1β in head kidney leukocytes (HKLs), confirming the biological activity of rgcTNF-α. In the same model, NF-κB inhibitor (PDTC) was able to attenuate rgcTNF-α-induced gcTNF-α mRNA expression, implying the involvement of NF-κB pathway in fish TNF-α action. This notion was reinforced by the finding that rgcTNF-α could induce the phosphorylation of IκBα in a time-dependent oscillation in HKLs, indicating a dynamical variation of NF-κB activity as seen in mammals. In addition, rgcTNF-α could up-regulate the expression of two TNF receptor-associated factors (TRAF), TRAF1 and TRAF2, in a time- and dose-dependent manner, suggesting that gcTNF-α may function as a regulator of fish NF-κB pathway. These results for the first time reveal the link of gcTNF-α to the NF-κB pathway and provide a better understanding of TNF-α signaling in teleost immunity.