CXCR2-targeted mutant mice were generated by the mating of heterozygote C.129S2(B6)-Il8rbtm1Mwm/J (Il8rbtm1Mwm/Il8rb+) mice (Jackson Laboratory, Bar Harbor, MN) in the University of Michigan animal facility. CXCR2 mutant (Il8rbtm1Mwm/Il8rbtm1Mwm) mice and CXCR2 wild-type (Il8rb+/Il8rb+) mice were used in all experiments; wild-type and mutant mice were based on the mouse 129 strain. All experiments were performed in compliance with the standards for animal use and care set by the University of Michigan's committee on the use and care of animals.
Animals were fasted overnight, and APAP or an equal volume of phosphate-buffered saline (PBS) was administered intraperitoneally.9 For mortality experiments, animals received 750 or 1000 mg/kg APAP; for all other experiments, 375 mg/kg was used. On the basis of previous experiments with this strain of mouse, 750 mg/kg APAP is approximately the median lethal dose, and 375 mg/kg is approximately the 25% lethal dose.
To confirm that apoptosis is important in the APAP-induced liver injury in this model, additional experiments were performed with the pancaspase inhibitor Q-VD-OPh. Q-VD-OPh is more effective at preventing apoptosis than other inhibitors, such as ZVAD-fmk and Boc-D-fmk, and is nontoxic to cells, even at high doses.10 This compound prevents apoptosis mediated by the three major apoptotic pathways: caspase-9/3, caspase-8/10, and caspase-12.10 Q-VD-OPh (50 mg/kg; R&D Systems, Minneapolis, MN) was administered 1 hour before APAP injection; control animals received an equivalent dose of vehicle. Animals were then sacrificed according to protocol, and apoptosis was measured by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining and DNA fragmentation assay.
Aspartate Aminotransferase (AST) and Alanine Aminotransferase (ALT) Assay.
Serum AST and ALT were measured in CXCR2 knockout and wild-type mice 24, 48, and 72 hours after APAP or PBS administration. Animals were sacrificed, their blood was collected, and the serum was separated from the clotted blood by centrifugation at 4000 rpm for 15 minutes at 4°C. ALT and AST were measured with a Diagnostics ALT and AST test kit from Sigma Chemical Co. (St. Louis, MO).
Mouse livers were perfused with a perfusion medium (Gibco, Grand Island, NY) to remove intravascular blood. Ten milligrams of liver tissue was homogenized in 1 mL of PBS containing 2 mM ethylene diamine tetraacetic acid. Fifty microliters of tissue extract was used for GSH measurement. GSH was measured according to the manufacturer's instructions with a GSH assay kit (Promega, Madison, WI). Luminescence was detected with a Synergy 2 multimode microplate reader with Gen5 data analysis software (BioTek, Winooski, VT).
DNA Fragmentation Assay.
DNA was extracted from 25 mg of mouse liver. Total DNA purification was performed with a DNeasy blood and tissue kit (Qiagen, Germantown, MA) according to the manufacturer's instructions. RNA was eliminated by incubation in 5 μg/mL RNase (Roche, Indianapolis, IN). A 1.5-μg DNA aliquot was loaded onto 1.5% agarose gel for separation to assess for DNA fragmentation.
TUNEL staining was conducted with an in situ apoptosis detection kit from R&D Systems according to the manufacturer's instructions. Six to seven animals were used per time point per treatment group. At least 1000 cells (TUNEL-positive cells and TUNEL-negative cells) were counted in each of eight separate low-power fields for each animal, and the percentage of TUNEL-positive cells was calculated.
In Vivo Measurement of Hepatocyte Proliferation by Bromodeoxyuridine (BrdU) Incorporation.
Two hours prior to sacrifice, animals received 30 μg of BrdU/g of body weight intraperitoneally. Liver specimens were obtained, fixed in 4% paraformaldehyde, processed for histological analysis, and stained with the Amersham cell proliferation kit (Amersham Pharmacia Biotech, Ltd., United Kingdom). BrdU incorporation was measured at 24, 36, 48, and 72 hours. There were four to seven mice per treatment group per time point. At least 1000 cells (BrdU-positive cells and BrdU-negative cells) were counted in each of eight separate low-power fields for each animal, and the percentage of BrdU-positive cells was calculated.
Cytoplasmic and Nuclear Protein Extraction.
Hepatic cytoplasmic and nuclear proteins were extracted with the NE-PER nuclear and cytoplasmic extraction reagent kit (Pierce Biotechnology, Rockford, IL) according to the manufacturer's instructions.
Immunoprecipitation and Western Blot Analysis.
Liver samples were homogenized in a radioimmunoprecipitation assay buffer (50 mM trishydroxymethylaminomethane–hydrochloric acid, pH 7.4, 150 mM sodium chloride, 1% Nonidet P40, 0.1% sodium dodecyl sulfate, 0.25% sodium deoxycholate, 1 mM ethylene diamine tetraacetic acid, and protease and phosphatase inhibitors). Four hundred micrograms of protein was used for immunoprecipitation. The lysate was precleared with 1 μg of isotype immunoglobulin G (IgG) and 50 μL of protein A/G agarose at 4°C for 1 hour. Five micrograms of an immunoprecipitating antibody or isotype IgG was added to the supernatant, and it was rocked overnight at 4°C. Next, 50 μL of protein agarose was added, and the mixture was rocked for 2 hours at 4°C. The protein bound to the agarose conjugate was centrifuged, and the pellet was washed three times with a radioimmunoprecipitation assay buffer. Ten microliters of a 4× sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and 5 μL of 1 M dithiothreitol were added to the pellet, and the sample was boiled for 5 minutes and centrifuged. The supernatant was saved for SDS-PAGE. Fifty micrograms of the protein lysate was subjected to SDS-PAGE under reducing conditions and transferred to polyvinylidene fluoride membranes. Blots were blocked in a 5% milk solution and exposed to anti-mouse first antibodies overnight at 4°C. First, antibodies were reacted with horseradish peroxidase–conjugated secondary antibodies. All membranes were visualized with West Pico chemiluminescent substrate (Pierce Biotechnology). Gel-Pro Analyzer software (Media Cybernetics, Bethesda, MD) was used to quantify the bands obtained via western blotting. The band optical density was normalized to the optical density of the loading control band.
Reagents for Immunoprecipitation, Western Blot Analysis, and Flow Cytometry.
Antibodies for caspase-3, caspase-9, B cell lymphoma 2 (Bcl-2), B cell lymphoma extra large (Bcl-XL), phosphorylated stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK; T183/Y185), and SAPK/JNK were purchased from Cell Signaling Technology, Inc. (Danvers, MA). Monoclonal anti-human/mouse cellular inhibitor of apoptosis protein 2 (cIAP2), XIAP, phycoerythrin-labeled anti-CXCR2, and phycoerythrin-labeled rat IgG2a were purchased from R&D Systems. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), NF-κB p65, NF-κB p52, anti-phosphoserine, horseradish peroxidase–conjugated goat anti-mouse IgG, and horseradish peroxidase–conjugated goat anti-rabbit IgG were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Primary hepatocytes were isolated by collagenase perfusion. Anesthesia was induced with isoflurane inhalation, laparotomy was performed, and the inferior vena cava was cannulated with a 26-gauge angiocatheter. A liver perfusion buffer (Gibco) was used to flush the liver of intravascular blood (3 mL/minute for 10 minutes). This was followed by the infusion of a liver digest buffer (Gibco; 3 mL/minute for 10 minutes). The liver was excised from the animal, placed in a Petri dish, minced into 1-mm pieces, and gently agitated so that the cells would be dispersed in the wash buffer (Gibco). The cell suspension was filtered and washed two times at 50g at 4°C for 5 minutes. Cells were immediately used for reverse-transcription polymerase chain reaction (RT-PCR) or flow cytometry.
RNA Extraction and RT-PCR.
Hepatocytes were isolated as described previously. Mouse neutrophils were isolated from the pooled blood of three mice by differential gradient centrifugation over Percoll (Gibco). Total RNA from hepatocytes or neutrophils was isolated with an RNeasy mini kit (Qiagen) according to the manufacturer's instructions. The polymerase chain reaction (PCR) primers were designed with Primer Premier software (Premier Biosoft International) to cross exon 1 and exon 2. The sense primer was 5′-TGCTCACAAACAGCGTCGTA-3′. The anti-sense primer was 5′-TCAGGGCAAAGAACAGGTCA-3′.
Reverse transcription was performed with the QuantiTect reverse-transcription kit (Qiagen). The extracted RNA was on-column DNase-digested (RNase-free DNase set, Qiagen), and 1 μg of total RNA was reverse-transcribed into complementary DNA with the QuantiTect reverse-transcription kit (Qiagen). Real-time PCR was performed in a SYBR Green PCR master mix (Bio-Rad, Hercules, CA) with a Bio-Rad iCycler iQ multicolor real-time PCR detection system according to the following protocol: initial activation at 95°C for 15 minutes, 40 cycles at 94°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. Amplification specificity was checked by melting curve analysis and agarose gel electrophoresis.
Hepatocytes were harvested and immediately studied with flow cytometry for CXCR2 expression. Cells were washed twice in a staining buffer (Difco, Detroit, MI), resuspended in 100 μL of the staining buffer, incubated for 15 minutes at 4°C with anti-mouse CD16/32 to block Fc receptors, and incubated for 30 minutes at 4°C with phycoerythrin-labeled anti-CXCR2 or phycoerythrin-labeled rat IgG2a. Final antibody concentrations were 1 to 2 μg/100 μL of cell solution. After incubation, cells were washed twice in the staining buffer and analyzed. Flow cytometry was performed with a FACScan cytometer (Becton Dickinson, Mountain View, CA). Data were collected and analyzed with CellQuest software. At least 10,000 cells were analyzed to determine cell-surface CXCR2 expression.
Enzyme-Linked Immunosorbent Assay (ELISA).
Fifty milligrams of mouse liver was homogenized in 1-mL of a lysis buffer containing protease inhibitors. The protein concentration was measured, and the samples were adjusted to the same protein concentrations. KC and MIP2 were measured with Quantikine murine ELISA kits (R&D Systems) according to the manufacturer's instructions. The KC and MIP2 concentrations were calculated per gram of liver protein.
All data are expressed as means and standard errors of the mean. Statistical calculations were performed with GraphPad Prism 5 (GraphPad Software, Inc., CA) on a Macintosh PowerBook G4 computer. Statistical analysis was performed with an unpaired Student t test or two-way analysis of variance. Differences were considered significant if P was less than 0.05. Survival rates are presented with Kaplan-Meier curves, and significance was calculated with the log-rank test.