Characterization of P2X7R and Its Function in the Macrophages of ayu, Plecoglossus altivelis

School of Marine Sciences, Ningbo University, Ningbo, China.
PLoS ONE (Impact Factor: 3.23). 02/2013; 8(2):e57505. DOI: 10.1371/journal.pone.0057505
Source: PubMed


P2X purinoceptor 7 (P2X7R), an ATP-gated ion channel, plays an important role during the innate immune response in mammals. However, relatively little is known about the role of P2X7R in the fish immune system. Here, we cloned a cDNA sequence encoding ayu (Plecoglossus altivelis) P2X7R (aP2X7R). The predicted protein was composed of 574 amino acid residues with a P2X family signature, two transmembrane domains, and a long C-terminal. aP2X7R transcripts were mainly distributed in ayu immune tissues and significantly increased in all tested tissues and in macrophages after Listonella anguillarum infection. The aP2X7R protein was upregulated significantly in macrophages upon bacterial challenge. An antibody against the ectodomain of aP2X7R (aEPAb) and an antagonist (oATP) were used to block aP2X7R. aP2X7R siRNA was also used to knockdown the receptor expression in ayu macrophages. Cell death induced by ATP was significantly inhibited in ayu macrophages after aEPAb, oATP, or siRNA treatment. Moreover, aP2X7R ablation also resulted in suppression of phagocytic activity and ATP-induced bacterial killing in ayu macrophages. Our results indicated that aP2X7R was upregulated after infection and mediated cell death, phagocytosis, and bacterial killing of ayu macrophages.

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    • "Total RNA was extracted from cells using RNAiso reagent (TaKaRa). Simultaneously, cells were also lysed in buffer containing protease inhibitors (20 mM Tris-HCl, 1 mM EDTA, 1% Triton X-100, 1 mM phenylmethanesulfonyl fluoride (PMSF), 10 mg ml -1 aprotinin, 10 mg ml -1 leupeptin, and 10 mg ml -1 pepstatin-A, pH 8.0), and total proteins were prepared as described previously[28]. "
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    • "The treated and untreated cells were harvested at 4, 8, 12, and 24 h post treatment, and preserved at À70 C before use. qPCR was performed using SYBR ® Premix Ex Taq™ (Perfect Real-Time) (TaKaRa) in a StepOne™ Real-Time PCR System (Applied Biosystems, Foster City, USA) as previously reported [16]. Primers for PaP2X7R gene and the internal control b-actin gene are shown in Table 1. "
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    • "2.2. Primary culture of ayu head kidney-derived MO/MF Ayu head kidney-derived MO/MF were isolated as previously described [15]. Briefly, fish were sacrificed by anesthetic overdose (50 mg/ml; 0.03% [v/v] ethylene glycol monophenyl ether). "
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