The warm temperature acclimation protein (Wap65) has an important role in the inflammatory response of turbot (Scophthalmus maximus).

Wap65 is a molecule similar to the mammalian hemopexin that is a serum glycoprotein produced mainly by the liver with high affinity to heme. Its primary role is participating in iron metabolism scavenging heme that is released into the plasma and transporting it to the liver. It has been reported an important role of hemopexin in the inflammation as an acute-phase protein and its production is up-regulated by pro-inflammatory cytokines. There are also some evidences suggesting this immune-induction in fish Wap65 genes. Most teleost species presents two Wap65 genes but their physiological functions have not been completely elucidated; in fact, the transcriptional patterns of Wap65 genes to stimulatory treatments are variable and contradictory. In the present study two Wap65 genes, Wap65-1 and Wap65-2, have been characterized for the first time in turbot (Scophthalmus maximus). Their constitutive expression and differential modulation by thermal treatments, immune challenges (bacterial and viral), as well as iron supplementation, have been investigated. Both genes were mainly expressed in liver, but they were detected in all tested tissues. Whereas Wap65-1 and Wap65-2 were up-regulated by temperature rise and bacterial challenge, VHSV infection inhibited the expression of both genes. Moreover, iron-dextran administration induced only the overexpression of Wap65-1. Interestingly, these induction were observed in head kidney buy not in liver. The effect of Wap65 protein purified from turbot serum by hemin-agarose affinity chromatography was also studied to demonstrate a possible anti-inflammatory role, analyzing its inhibitory effect on leucocytes migration induced by zymosan injection to the peritoneal cavity.

Possible role of LTB4 in the antiviral activity of turbot (Scophthalmus maximus) leukocyte-derived supernatants against viral hemorrhagic septicemia virus (VHSV).

Turbot (Scophthalmus maximus) blood leukocyte-derived supernatants were tested for antiviral activity against viral hemorrhagic septicemia virus (VHSV). The assays were performed by quantifying the effect of the supernatants on the replication of VHSV in the rainbow trout (Oncorhynchus mykiss) cell line, RTG-2. Supernatants were obtained by incubating the leukocytes for 17 h at 18 degrees C in L-15 medium supplemented with 5% fetal calf serum (FCS). Testing of leukocyte supernatants indicated that antiviral activity against VHSV resulted in a viral titer reduction of 72.1%. After the supernatants were extracted with calcium ionophore A23187 treatment, the antiviral activity significantly increased, resulting in a viral titer reduction of 99.9%. In order to determine the nature of this antiviral activity, supernatants were produced from leukocytes treated for 17 h with inhibitors of eicosanoid biosynthesis, reactive oxygen intermediates and nitric oxide (NO) production. None of the inhibitors significantly suppressed the supernatant antiviral activity. The presence of oxygen radicals and NO was measured in the case of co-cultures of leukocytes and RTG-2 cells, but no significant differences were found in the VHSV-infected co-cultures compared to non-infected controls. Since previous work demonstrated that leukotriene B4 (LTB4) was present in turbot blood leukocyte-derived supernatants, we assessed the effect of the VHSV in vitro infection on turbot leukocyte LTB4 production by high performance liquid chromatography (HPLC). The levels of LTB4 were significantly increased in the supernatants after VHSV infection. Furthermore, exogenous LTB4 significantly inhibited VHSV replication in RTG-2 cells. These findings suggest that LTB4 may play a significant role in VHSV replication.