Functions Of TNF-?1 And TNF-?2 In Large Yellow Croaker On Monocyte/Macrophage Activation

Tumor necrosis factor-??TNF-??plays crucial roles in cell development,proliferation,apoptosis,inflammation,and immune response.There are two or even three isoforms of TNF-?in some fishes,but the functional differences between different TNF-?isoforms are still uncertain and need to be further explored.A TNF-?isoform has been reported in large yellow croaker,which is named TNF-?1 here.In this study,we identified a novel TNF-?homologue?Lc TNF-?2?from large yellow croaker?Larimichthys crocea?,which shares a low amino acid sequence identity to the previously reported large yellow croaker TNF-??Lc TNF-?1?.The open reading frame of Lc TNF-?2 is 714 nucleotides long,encoding a peptide of 237 amino acids?aa?.The deduced Lc TNF-?2 protein contains a 23-aa transmembrane region,a tumor necrosis factor-?converting enzyme?TACE?restriction site at residues T⁷¹/L⁷²,a TNF family signature(I¹⁰⁸?F¹³⁵),and two conserved cysteine residues(C³⁹and C¹⁷⁹),as found in other known TNF-?sequences.Both Lc TNF-?1 and Lc TNF-?2 were constitutively expressed in all examined tissues and significantly up-regulated in the spleen and head kidney by Vibrio alginolyticus.Their transcripts were also detected in primary head kidney monocytes/macrophages?MO/M?s?,lymphocytes?PKLs?,granulocytes?PKGs?,and large yellow croaker head kidney?LYCK?cell line and significantly increased in MO/M?s by inactivated Vibrio alginolyticus.Recombinant Lc TNF-?1 and Lc TNF-?2 proteins?r Lc TNF-?1and r Lc TNF-?2?produced in Pichia pastoris not only significantly increased the production of reactive oxygen species?ROS?,but also promoted the expression of proinflammatory cytokines?IL-1?,IL-6,IL-8,and TNF-?1?in MO/M?s from large yellow croaker.After stimulation with r Lc TNF-?1 and r Lc TNF-?2,the production of nitrogen oxide?NO?and the expression of inducible NO synthase?i NOS?gene were significantly increased.However,only r Lc TNF-?1 remarkedly enhanced the phagocytosis of MO/M?s and increased the expression of TNF-?2 in MO/M?s.All these results indicated that Lc TNF-?1 and Lc TNF-?2 have the similar but differential roles in promoting activation of MO/M?s from head kidney of large yellow croaker.Through this study,we have a further understood the relationship between TNF-?and macrophage function,and further proved that TNF-?plays an important role in innate immune resonse.