All procedures were performed according to the manufacturer’s instructions. Real-time PCR was performed using SYBR green Master mix reagent (Applied Biosystems) under standard conditions (10 minutes at 95°C, 40 cycles involving denaturation at 95°C for 15 seconds, annealing/extension at 60°C for 1 minute). 36B4 was the internal control gene. Relative mRNA levels were quantitated as the fold-change relative to PBS-treated wildtype (WT) mice. All assays were performed in duplicate. KCs Cell Cycle inhibitor were depleted by injecting 200

μL clodronate-liposomes intravenously. Two days later, we injected 8 μg LPS in PBS or an equal volume of PBS intravenously. KC depletion was documented as a decrease in the number of F4/80+ cells in cryostat liver sections obtained 6 days after LPS or PBS treatment. To minimize the impact of photobleaching, digital photographs were taken (five different fields/liver section, 20× magnification) using a Zeiss Axioplan 2 fluorescence microscope and cells were counted from these images. To analyze the role of IL-10 in the development of hepatomegaly in LPS-treated Aoah−/− mice, Aoah−/− mice were given 0.5 mg/mouse of rat antimouse IL-10R

antibody or isotype control Ab (generously provided by Schering-Plough/Merck) intraperitoneally on the first day after intravenous LPS injection (0.1 μg/g body weight). Livers were harvested 7 days after LPS treatment. To inhibit circulating TNF, we gave Aoah−/− mice an intraperitoneal injection of 100 μg of PEGsTNF-R1 (Amgen) 1.5 hour before SB431542 supplier administering LPS intravenously (0.2 μg/g body weight) and then every MCE公司 other day until the end of the experiment (5 days after LPS challenge). Plasma was obtained 1 hour after LPS administration to measure TNF by ELISA (BD Biosciences).

To inhibit IL-1β, Aoah−/− mice were given Anakinra (IL-1R blocker, 25 μg/mouse) 1 hour before LPS administration and then twice every day until the end of the experiment. In some experiments, mice were given both PEGsTNF-R1 and Anakinra. PEGsTNF-R1 was given intraperitoneally every other day, and Anakinra was given intraperitoneally twice daily until the end of experiment (5 to 7 days after LPS administration). To test if LPS-induced hepatomegaly in Aoah−/− mice is influenced by the sympathetic nervous system, we delivered epinephrine (beta agonist, 2 mg/kg/day), norepinephrine (alpha agonist, 2.5 mg/kg/day), metoprolol (beta-antagonist, 20 mg/kg/day), and Prazosin (alpha-antagonist, 3 mg/kg/day) by way of implantable osmotic pumps (100 μL, Alzet, Cupertino, CA) that were placed in the peritoneal cavity 6 days before intravenous LPS administration. Dexamethasone was given intraperitoneally (1 mg/kg/day) daily from 3 days before LPS challenge until the end of the experiment 7 days after challenge.