Abstract
Uridine 5'-diphosphate (UDP)-activated purinergic receptor P2Y₆ is a member of a G-protein-coupled purinergic receptor family that plays an important role in mammalian innate immunity. However, the role of the P2Y₆ receptor (P2Y₆R) in fish immunity has not been investigated. In this report, we characterized a P2Y₆R gene from Japanese flounder (Paralichthys olivaceus) and examined its role in fish innate immunity. Sequence analysis reveals that the Japanese flounder P2Y₆R protein is conserved and possesses four potential glycosylation sites. Quantitative real-time RT-PCR analysis shows that P2Y₆R is broadly distributed in all examined Japanese flounder tissues with dominant expression in the liver. In addition, P2Y₆R gene expression was up-regulated in head kidney macrophages (HKMs) upon lipopolysaccharides (LPS) and poly(I:C) stimulations but down-regulated by LPS challenge in peripheral blood leukocytes (PBLs). Furthermore, pharmacological inhibition of the endogenous P2Y₆ receptor activity by the potently selective P2Y₆R antagonist, MRS 2578, greatly up-regulated pro-inflammatory cytokine interleukin (IL)-1β, IL-6 and TNF-α gene expression in PBL cells treated with UDP. Moreover, LPS- and poly(I:C)-induced gene expression of IL-1β and TNF-α in Japanese flounder PBL cells was attenuated significantly by inhibition of P2Y₆R activity with antagonist MRS 2578. Collectively, we, for the first time, showed the involvement of functional purinergic P2Y₆R in fish innate immunity.