Cytokine Property of Tongue Sole ISG15 Figure 5. Expression and localization of CsISG15 in lympho- cytes. (A) Tongue sole head kidney (HK) lymphocytes were infected with or without (control) megalocytivirus, and CsISG15 expression was determined by quantitative real time RT-PCR at various time points. At each point, the expression level of the control cells was set as 1. Values are shown as means6SE (N = 3). N, the number of repeated experiments. **P,0.01, *P,0.05. (B, C, and D) Tongue sole HK lymphocytes were infected with (lane 3) or without (lane 2) megalocytivirus for 4 h, and extracellular proteins were subjected to immunoblot with antibodies against CsISG15 (B) or b-actin (C). To demonstrate that the b-actin antibody was able to detect b-actin when it was present, cytoplasmic proteins were prepared and blotted with b- actin antibody (D). Lane 1, protein marker. doi:10.1371/journal.pone.0044884.g005 extracellular milieu as it was observed with human ISG15, HK lymphocytes were infected with or without megalocytivirus for 4 h, which, as shown above, was the point when drastic CsISG15 induction was observed. Extracellular proteins were prepared from the culture supernatant and subjected to immunoblot analysis with antiserum raised against recombinant CsISG15 (Figure S1 and Figure S2) or with antibody against b-actin. The results showed that CsISG15 was detected in the culture supernatant of megalocyti- virus-stimulated cells but not in that of unstimulated cells (Fig. 5). Since no b-actin was detected in the protein preparations, it is not likely that the detected CsISG15 was due to cell lysis. Figure 6. Effect of rCsISG15 and rCsISG15M on macrophage activation. (A) Tongue sole head kidney macrophages were treated with or without (control) rCsISG15, rCsISG15M, or lipopolysaccharide (LPS) for 2 h and examined for respiratory burst. (B) Tongue sole head kidney macrophages were treated with or without (control) rCsISG15 or rCsISG15M for different hours and examined for nitric oxide production. The assays were each performed four times and the results are shown as means6SE. **P,0.01, *P,0.05. doi:10.1371/journal.pone.0044884.g006 Biological effect of rCsISG15 and its dependence on the conserved LRGG motif (i) Effect on macrophage activation. Since, as observed above, CsISG15 was a secreted protein, we examined its potential biological function by determining whether it had any effect on macrophage and lymphocyte immune response. In addition, to examine the functional importance of the LRGG motif, we also prepared and analyzed the effect of a mutant CsISG15, CsISG15M, which bears alanine substitutions at the RG residues of the LRGG sequence (i.e., LRGG was mutated to LAAG). HK macrophages were treated with or without rCsISG15, rCsISG15M, or LPS for various hours and then analyzed for respiratory burst and NO production. The results showed that significantly increased respiratory burst activity was observed in cells treated with rCsISG15 and LPS but not in cells similarly treated with rCsISG15M (Fig. 6A). NO production analysis showed that macrophages treated with rCsISG15M produced significantly higher amounts of NO after 0.5 h, 1 h, 2 h, 4 h, and 12 h treatment, while macrophages treated with rCsISG15M exhibited significant NO induction after 1 h, 2 h, 4 h, and 12 h treatment, however, the induction levels induced by rCsISG15M were significantly lower than those induced by rCsISG15 (Fig. 6B). PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e44884