Our results suggest that IL-33 expression in hepatocytes is partially dependent on perforin, but not on FasL or TNFα in acute hepatitis. Furthermore, we show that TRAIL is essential for inducing IL-33 expression in hepatocytes during T-cell-mediated hepatitis in mice or in cultured murine hepatocytes. AST, aspartate aminotransferase; ALT, alanine aminotransferase; ConA, concanavalin A; D-GalN, D-galactosamine; FasL, Fas ligand; IL-33, interleukin 33;

IL-1RAcP, interleukin-1 receptor accessory protein; TRAIL, tumor necrosis factor related apoptosis inducing ligand; TNFR1/2, tumor necrosis factor receptor 1 or 2; WT, wildtype. The C57Bl/6 WT mice (8-10-week-old, Janvier, Le Genest-sur-isle, France) AZD6244 supplier were injected intravenously with ConA (Sigma-Aldrich, St. Louis, MO) to induce acute hepatitis at a dose of 20 mg/kg body weight.

Mice were sacrificed from 6 to 10 hours postinjection. Intraperitoneal injection of anti-Fas/Jo2 antibody (Purified Hamster Anti-Mouse CD95, BD Pharmingen) agonist antibody was administered at a dose of 0.15 μg/g of body weight to induce hepatic injury and PLX-4720 nmr mice were sacrificed at 2, 4, 6, 10, and 24 hours postinjection. Recombinant murine (rm)-TNFα (PeproTech, USA) was injected intravenously (10 μg/kg body weight) alone or in combination with D-galactosamine (D-GalN, Sigma) at a dose of 15 mg/mouse (intraperitoneal) in WT mice and sacrificed at 8 hours postinjection. Mice C57Bl/6 perforin-KO, TRAIL-KO and IL-33-KO (provided by Dr.

Jean-Philippe Girard26 and bred in our local animal facility) were injected intravenously with ConA (20 mg/kg body weight) and sacrificed at the designated timepoints. The C57Bl/6 CD1d-KO mice were primed with ConA for 2 hours followed by injection of rm-TRAIL (30 μg/mouse, intravenous, PeproTech, USA). Mice were sacrificed 8 hours after injection of ConA. In each experiment the control mice were treated with phosphate-buffered saline (PBS) or vehicle only. All the mice were bred in specific pathogen-free conditions in the local animal house facilities and all treatment protocols were in accordance with the French laws and the institution’s guidelines for animal welfare (agreement of M. Samson #3596). The histopathological and serum 上海皓元 biochemical analysis was performed as reported.2 The protocol and conditions for RNA extraction, RT-PCR, and qPCR were the same as reported earlier by our laboratory2, 3 using specific primers for 18S, IL-33, FasL, Fas, TRAIL, DR5, TNFα, TNFR1, and TNFR2 (Table 1). The relative gene expression was normalized against 18S gene expression. The control mice in each treatment group served as a reference for messenger RNA (mRNA) expression (control mRNA level was arbitrarily taken as 1). Cryosections or paraformaldehyde-fixed and paraffin-embedded mouse liver sections (7 μm) followed by antigen retrieval were incubated with primary antibody (goat IgG antimouse IL-33, R&D Systems) in a Ventana automated machine (Ventana Medical Systems, USA).