ly Effect of IL-15 on APAP Hepatitis diminish liver injury in concanavalin A or Fas ligand-induced hepatitis, whereas DC-derived IL-15 enhanced endotoxin shock injury through the liver. Moreover, IL-15 mediates the crosstalk between conventional and plasmacytoid DCs for immune activation. Interestingly, IL-15 promoted hepatocyte mitosis and liver proliferation in healthy mice and those with hepatectomy, respectively. In this study, we aimed to study the role of IL-15 in a sterile APAP-induced fulminant hepatitis model in IL-15-knockout and wild-type mice. By treatment of mice with a hepatotoxic dose of APAP and evaluation of animal 
survival, liver damage, APAP metabolism in livers and the inflammatory response, we were able to elucidate whether IL-15 was involved in APAP hepatitis. as the non-parenchymal cell fraction was centrifuged at 350 g for 5 min at 4uC and, used to analyze mononuclear cell population or KC enrichment. After perfusion, hepatocyte viability was determined by trypan blue exclusion. Hepatocytes were suspended in 10% fetal bovine serum containing William’s Essential Medium and cultured in 0.02% collagen-coated plate; after 4 hrs, the indicated concentration of APAP was added to analyze cytotoxicity to hepatocytes. The APAP toxicity was quantified by use of the Cell Counting Kit-8. NPCs underwent a 25%/50% two-step Percoll gradient to isolated KC-enriched cells. Hepatic glutathione measurement Hepatic total GSH was measured by 5,59-dithiobis assay. Frozen liver samples were homogenized with 4 volumes of 5% trichloroacetic acid, and supernatant was incubated with DTNB for 15 min. Hepatic GSH concentration was measured by colorimetry at optical density 412 nm. Materials and Methods Animals and materials Male C57BL/6J mice were purchased from National Laboratory Animal Center, Taipei, Taiwan. C57BL/6NTac/Il152/2 mice, obtained from Taconic Farms, were backcrossed to C57BL/6J background for 4 generations. This substrain, as C57BL/6J/Il152/2 mouse, was used in our later study. All mice were kept in a pathogen-free condition in compliance with institutional animal care and use committee guidelines. All chemicals were purchased from Sigma Chemical Co. unless otherwise stated. Recombinant murine IL-15, with specific activity of $26105 units/mg, was purchased from Perprotech. Rat anti-IL-15 neutralizing antibody was obtained from eBiscience. Recombinant IL-15 was intraperitoneally or subcutaneously injected 20 minutes before APAP challenge. IL-15 neutralizing antibody was intraperitoneally injected 30 minutes prior to APAP administration. APAP was dissolved in warm saline and intraperitoneally injected into overnight-fasted mice to induce hepatitis, and 350 mg/kg of APAP were generally used in this study unless otherwise mentioned. Animal survival was monitored for 5 days. 1.5 mg/kg of vinblastine or 30 mg/kg of gadolinium chloride was intravenously given to animals for neutrophil or KC elimination, respectively. Determination of cytokines by ELISA Murine IL-15, IL-1b, IFNc and TNFa levels in serums or livers were measured by ELISA kits. Serum samples were collected by heart puncture and liver proteins were collected from frozen liver samples homogenized in Scopoletin protein lysis buffer. Immunohistochemical staining of liver samples The levels of hepatic inducible NO synthase and nitrotyrosine were evaluated in deparaffinized and antigenretrieved liver sections by LSAB2 system-HRP kits. Liver sections were incubated with primary antibod
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