Serum IL-12 levels were determined 3?h after CpG injection

Serum IL-12 levels were determined 3?h after CpG injection. liver injury and NKT activation in the HBs-Tg mice. Our data suggest that CD205-expressing Kupffer cells respond to RWJ-445167 CpG-ODNs and subsequently release IL-12 to promote NKT cell activation. Activated NKT cells induce liver damage through the Fas signaling pathway in HBs-Tg mice. for 10?min. Isolation of Kupffer cells Kupffer cells were isolated using a two-step collagenase perfusion method as described previously.22 The collected cells were allowed to attach to a plastic plate for 30?min for the removal of nonadherent cells. Then, cold phosphate-buffered saline (PBS) was added, and the cells were put on ice for 40?min. After the dish was tapped gently, the supernatant was collected and centrifuged. The cells were resuspended in RPMI 1640 and immediately used. Flow cytometric analysis The fluorochrome-coupled monoclonal Abs used for flow cytometry in this study included CD69, FasL, Fas, CD3, NK1.1, CD19, F4/80, CD11c, CD11b, IgG2a, Rat IgG2b, ArH IgG2, ArH IgG1 (BD PharMingen, San Diego, CA, USA), granzyme B, perforin, CD205 and TLR9 (eBioscience, San Diego, CA, USA). The PE-conjugated, PBS57-loaded CD1d tetramer was a gift from the National Institutes of Health Tetramer Core Facility (Atlanta, GA, USA). After being incubated with Fc-blocker (2.4 G2; BD PharMingen), cells were stained with the indicated monoclonal Abs for surface antigens. Intracellular staining was performed using the Cytofix/Cytoperm Plus kit (BD Biosciences, San Diego, CA, USA) and Abs to TLR9, granzyme B and perforin. The stained cells were analyzed using a flow cytometer (FACScalibur; Becton Dickinson, Franklin Lakes, NJ, USA). Analysis of cytokine and alanine aminotransferase amounts Serum examples from mice had been obtained in the indicated period factors after CpG injection. Cytokine amounts in the serum or the tradition supernatants had been assessed using cytokine-specific enzyme-linked immunosorbent assay kits based on the producers instructions (R&D program, Minneapolis, MN, USA). The serum alanine aminotransferase (ALT) amounts had been INSL4 antibody measured utilizing a commercially obtainable package (Rong Sheng, Shanghai, China). Cytotoxicity assay NKT cell-mediated cytotoxicity against hepatocytes was assessed with a 4-h aspartate aminotransferase (AST) launch assay.13 Hepatic NKT cells purified through the C57BL/6 mice as referred to above RWJ-445167 were put into the hepatocytes freshly isolated through the C57BL/6 or HBs-Tg mice in the indicated effector/focus on cell ratios. After 4?h, the supernatant was collected, and AST activity was measured. The precise cytotoxicity was determined the following: Treatment of mice with nanoparticle-encapsulated siRNA To particularly silence the manifestation of Compact disc205 on Kupffer cells in the HBs-Tg RWJ-445167 mice, siRNA focusing on Compact disc205 (siCD205, feeling 5-GCACUGGACACUGCUAAAUTT-3 and antisense 5-AUUUAGCAGUGUCCAGUGCTT-3) was designed and synthetized by GenePharma (Shanghai, China). The adverse control (siNeg) utilized was the following: feeling 5-UUCUCCGAACGUGUCACGUTT-3 and antisense 5-ACGUGACACGUUCGGAGAATT-3). Cationic, lipid-assisted poly(ethylene glycol)-b-poly(d,l-lactide) (PEG-PLA) nanoparticles had been utilized to encapsulate siCD205 or siNeg, and deliver them into Kupffer cells.15, 23 Nanoparticle-encapsulated siCD205 or siNeg (40?g per mouse) was administered towards the mice 48?h just before CpG-ODN treatment by intravenous injection. Statistical evaluation Students check. All data are demonstrated as the means.e.m. (purity from the purified NKT cells can be shown in Shape 4a). As demonstrated in Numbers c and 4b, unstimulated NKT cells shown small cytotoxicity against hepatocytes through the C57BL/6 and HBs-Tg mice (Shape 4b). Nevertheless, CpG-stimulated NKT cells demonstrated higher cytotoxicity against hepatocytes through the C57BL/6 and HBs-Tg mice than do the unstimulated NKT cells (Shape 4c). The hepatocytes through the HBs-Tg mice had been much more delicate to CpG-stimulated NKT cell-induced cytotoxicity than those through the C57BL/6 mice (Shape 4c). Furthermore, incubation with FasL-blocking Ab reduced the cytotoxicity of NKT cells against hepatocytes through the HBs-Tg mice (Shape 4d). These data claim that triggered NKT cells induce hepatocyte harm via the FasL/Fas pathway. Furthermore, the production of granzyme and perforin B by NKT cells.