Briefly, 1 x 106 MNCs were incubated with antimouse CD3, antimouse check details CD4, antimouse CD25, antimouse CD8, antimouse CD19, antimouse NK1.1, antimouse CD11c, antimouse F4/80, and antimouse CD206 conjugated with fluorescein isothiocyanate (FITC; BD Biosciences, Franklin Lakes, NJ), phycoerythrin

(PE; BD Biosciences), peridinin chlorophyll protein (BD Biosciences), or allophycocyanin (APC; BD Biosciences). MNCs derived from livers were stained for different markers of cell subsets (i.e., CD4, CD8, NK1.1, CD11c, and F4/80) and concomitantly for the intracellular content of TNFα, IFNγ, and IL-17, -10, and -4. To this end, monoclonal antibodies were used as follows: antimouse TNFα and antimouse IL-10 conjugated with APC (BD Bioscience), antimouse IL-4, IFNγ, and IL-17 conjugated with PE. Intracellular staining for forkhead box protein 3 (Foxp3) was performed using the BD Biosciences fixation/permeabilization buffer kit. Stained cells were counted using a BD Biosciences FACSCalibur, and the results were analyzed with WinMDI software. For the detection Sotrastaurin supplier of apoptosis, the Annexin V– binding capacity of liver MNCs and splenocytes was examined by flow cytometry using the Annexin V FITC Detection Kit (BD Pharmingen, San Jose, CA), as previously described.16 Individual mouse serum was collected, and serum levels of IFNγ, TNFα, and IL-17, -4, and -10 were measured by enzyme-linked immunosorbent

assay (ELISA) using ELISA kits (R&D Systems, Minneapolis, MN). Individual spleens of Con A–untreated WT and Gal-3−/− mice were collected. The single-cell suspension of splenocytes was cultured in 24-well plates at 4 x 106 cells per well and was stimulated with 5 μg/mL of Con A (Sigma-Aldrich). After 24 hours, supernatants

were collected and cytokine concentrations were measured by ELISA kits (R&D Systems). All statistics were carried out using SPSS 13.0 for Windows software (SPSS, Inc., Chicago, IL). Results were analyzed using the Student t test. All data in nearly this study were expressed as the mean ± standard error of the mean (SEM). Values of P < 0.05 were considered as statistically significant. First, we investigated whether acute liver injury in humans would affect Gal-3 expression in the liver. Human liver tissue sections were obtained from patients suffering from acute liver disease induced by isoniazid or hepatitis B virus (HBV) and were compared to healthy controls. Compared to healthy controls (Supporting Fig. 1A), Gal-3 was strongly expressed in lining cells of hepatic sinuses both in patients with isoniazid-induced (Supporting Fig. 1B) and HBV-induced (Supporting Fig. 1C) fulminant hepatitis, suggesting a possible role of Gal-3 in liver inflammation. Next, to investigate the role of Gal-3 in experimental fulminant hepatitis, we injected Con A into WT and C57Bl/6 mice with the targeted disruption of Gal-3 gene (Gal-3−/− mice).