LPS from Escherichia coli O14 was prepared by phenol-chloroform-petroleum ether extraction. [3H/14C]LPS was prepared using Salmonella typhimurium PR122 as described.17 Clodronate-liposomes and phosphate-buffered saline (PBS)-liposomes were prepared by J. Niederkorn (University of Texas Southwestern Mitomycin C Medical Center) using clodronate provided by Roche. Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) and Nω-Nitro-D-arginine methyl ester hydrochloride (D-NAME) were from Sigma-Aldrich (St. Louis, MO). 5-Bromo-2′-deoxy-uridine (BrdU) was from Roche Diagnostics. PEGsTNF-R1, a pegylated form of the TNF neutralizing domain of Etanercept, was provided by Amgen. Anakinra and Actemra were

purchased from Amgen and Genentech, respectively. Aoah−/− C57Bl/6 mice and μMT, Aoah−/− (double knockout) mice were produced as described.6, 18 The mice were maintained in specific pathogen-free conditions in the UT Southwestern Animal Resources Center and used for experiments when they were 5-12 weeks of age. All protocols

were approved Ku-0059436 manufacturer by the UT Southwestern Institutional Animal Care and Use Committee. PE- or Alexa Fluor 647-labeled rat antimouse F4/80 (BM8), Alexa Fluor 555 conjugated goat antirat immunoglobulin G (IgG) and Qdot 565 conjugated goat anti-fluorescein isothiocyanate (FITC) antibody were from Invitrogen. Biotin-conjugated rat antimouse CD11b (M1/70), rat antimouse CD144 (11D4.1), and FITC-labeled anti-BrdU antibody were from BD Biosciences. An agonistic monoclonal antibody (UT12) to the Toll-like receptor 4 (TLR4)/MD-2 complex was produced by S. Ohta19 and prepared as described.20 Rat antimouse interleukin (IL)-10R antibody (Ab) (YL03.1b1.3a-34ABS) and isotype control Ab (MB819.7D7.180) were generously provided by Schering-Plough MCE公司 Biopharma (Palo Alta, CA). Antibodies for flow cytometry were from BD Biosciences. Groups of three

Aoah−/− and Aoah+/+ mice were injected intravenously with UT12 IgG20 (0.0125, 0.05, 0.1, or 0.25 μg/g body weight). Livers were harvested 7 days postinjection. In another experiment, Aoah−/− and Aoah+/+ mice were injected intravenously with 0.1 μg/g body weight and studied on days 7, 14, or 21 postinjection. Aoah−/− and Aoah+/+ mice were injected intravenously with 0.5 μg E. coli O14 LPS/g body weight or an equal volume of PBS. Seven days later, animals were deeply anesthetized with isoflurane and perfused with 15 mL of PBS followed by 20 mL of fixative (4% paraformaldehyde and 1% glutaraldehyde in 0.1 M cacodylate buffer) through the left ventricle. The liver was then removed, cut into small pieces, and immersed in fixative for 1 hour at room temperature. SEM and TEM liver samples were prepared by Tom Januszewski (Molecular and Cellular Imaging Facility, UT Southwestern). The samples were examined with an XL30 ESEM SEM and a JEOL 1200 EX TEM at voltages of 30 and 120.