Hepatic Ischemia/Reperfusion Injury Involves Functional TLR4 Signaling in Nonparenchymal Cells

Animals

Male wild-type (WT) C57BL/6 (C3H/HeOuj) mice and TLR mutant (C3H/HeJ) mice (8–12 wk old) were purchased from The Jackson Laboratory. All animals were maintained in a laminar-flow specific pathogen-free atmosphere at the University of Pittsburgh (Pittsburgh, PA). Animal protocols were approved by the Animal Care and Use Committee of the University of Pittsburgh and the experiments were performed in adherence to the National Institutes of Health Guidelines for the use of laboratory animals.

Chimeric mice

Chimeric mice were produced by adoptive transfer of donor bone marrow cells into irradiated recipient animals using combinations of TLR4 WT (C3H/HeOuj) and TLR4 mutant (C3H/HeJ) mice in the following recipient/donor combinations: WT/WT, WT/Mutant, Mutant/Mutant, Mutant/WT. Recipient mice were exposed to an otherwise lethal 1000 cGy from a Cesium source (Nordion International) 6 h before receiving 2.5 × 10⁶ bone marrow cells by tail vein injection. The bone marrow cells were prepared in a sterile manner from the tibia and femur bones of the donor mice. All animals were monitored two to three times weekly for the first 2 wk to ensure successful bone marrow engraftment. The chimeric mice underwent hepatic I/R after another 8–10 wk to ensure stable engraftment.

Liver ischemia

A nonlethal model of segmental (70%) hepatic warm ischemia was used. The I/R protocol was initiated with the abdominal wall being clipped of hair and cleansed with betadine. Under sodium pentobarbital (40 mg/kg, i.p.) and methoxyflurane (inhalation) anesthesia, a midline laparotomy was performed. With the use of an operating microscope, the liver hilum was dissected free of surrounding tissue. All structures in the portal triad (hepatic artery, portal vein, and bile duct) to the left and median liver lobes were occluded with a microvascular clamp (Fine Science Tools) for 60 min, and reperfusion was initiated by removal of the clamp. This method of segmental hepatic ischemia prevents mesenteric venous congestion by permitting portal decompression through the right and caudate lobes. We have previously conducted a time course experiment to determine the optimal ischemia time period for the induction of hepatic injury (19). Less than 60 min of ischemia produced only minimal plasma transaminase elevations, whereas over 75 min of ischemia was poorly tolerated with gross evidence of poor reperfusion of the ischemic lobes. A reproducible level of hepatic damage was observed using 60 min of ischemia and was thus used for our study. After application of the clamp, the abdomen was covered with a sterile plastic wrap to minimize evaporative loss. Throughout the ischemic interval, evidence of ischemia was confirmed by visualizing the pale blanching of the ischemic lobes. The clamp was then removed and gross evidence of reperfusion based on immediate color change was assured before closing the abdomen with a continuous 4–0 diameter polypropylene suture. Either the absence of ischemic color changes or the lack of response to reperfusion was a criterion for immediate sacrifice and exclusion from further analysis. Temperature was monitored by rectal temperature probe and was maintained at 37°C by means of a warming pad and heat lamp. At the end of the observation period following reperfusion, the mice were anesthetized with inhaled methoxyflurane and were sacrificed by exsanguination. Sham animals underwent anesthesia, laparotomy, and exposure of the portal triad without hepatic ischemia. Animals were sacrificed at predetermined time points (1–6 h) after reperfusion to obtain serum and liver samples.

Gadolinium chloride treatment

Mice received either gadolinium chloride (Sigma-Aldrich) dissolved in normal saline (10 mg/kg) or normal saline alone by i.v. injection 24 h before ischemia.

NPC isolation

Live NPC were harvested using a modification of the perfusion technique of Seglen (20). The vena cava was cannulated and the liver was perfused for 3 min with 1× HBSS (Invitrogen Life Technologies) supplemented with 0.96 g of sodium bicarbonate/500 ml (Perfusate I) at a flow rate of 10 ml/min. Next, the liver was perfused with a 0.2% protease (Sigma-Aldrich) in Perfusate I (Perfusate II) for 3 min. The liver was dissected out and placed in a petri dish with Perfusate II and diced into 2- to 3-mm pieces. This slurry was then stirred in Perfusate II for 1 h at 37°C. After three washes, the cells were resuspended in culture media and counted. The NPC did not contain hepatocytes as detected by light microscopy.

Cell culture and treatment

NPC (2 × 10⁶) were plated onto 24-well plates. The initial culture medium was Williams medium E containing 10% calf serum, 15 mM HEPES, 2 mM l-glutamine, and 100 U/ml penicillin and streptomycin. NPC were allowed to attach to plates overnight. The cells were stimulated with LPS (0.01 μg/ml) and the medium was collected after 6 h.

Isolation of nuclear and cytoplasmic proteins

Frozen liver tissues were suspended in buffer containing 10 mM Tris (pH 7.5), 1.5 mM MgCl₂, 10 mM KCl, and 0.1% Triton X-100 and lysed by homogenization. Nuclei were recovered by microcentrifugation at 7500 rpm for 5 min. The supernatant containing cytoplasmic and membrane protein was collected and stored at −80°C for Western blot analysis. Nuclear proteins were extracted at 4°C by gently resuspending the nuclei pellet in buffer containing 20 mM Tris (pH 7.5), 20% glycerol, 1.5 mM MgCl₂, 420 mM NaCl, 0.2 mM EDTA, and 0.1% Triton X-100, followed by a 1-h incubation at 4°C with occasional vortexing. After microcentrifugation at 13,000 rpm for 15 min at 4°C, the supernatant containing nuclear protein was collected. Protein concentration was quantitated with bicinchoninic acid protein assay reagent (Pierce).

Liver damage assessment

To assess hepatic function and cellular injury following liver ischemia, serum alanine aminotransferase (ALT) levels were measured using the Opera Clinical Chemistry System (Bayer).

Histopathology

Formalin-fixed liver samples were embedded in paraffin and cut to 6-μm thick sections. Tissues were stained with H&E, and slides were assessed for inflammation and tissue damage.

Immunohistochemical staining

A segment of the left lobe of the liver was cryoprotected in 2% paraformaldehyde in PBS overnight and then 30% sucrose in PBS for another 24 h. The segment was then embedded and frozen in OCT compound using liquid nitrogen cooled isopentane. These samples were stored at −80°C until ready to be cut. To prepare the slides, the samples were cut into 6-μm sections using a cryostat machine and placed onto slides. They were left to dry at room temperature overnight and then stored at −20°C. Immunofluorescent staining for F4/80 was begun by first rehydrating the slides with PBS. Then, nonspecific proteins were blocked using 2% BSA for 45 min followed by rinses with 0.5% BSA. This process was followed by placing the primary Ab (F4/80; BD Biosciences) 1/100 in 0.5% BSA for 60 min. Again the slides were washed with 0.5% BSA and then the secondary Ab (goat anti-rabbit CY3; Jackson ImmunoResearch Laboratories) 1/1000 in 0.5% BSA was applied for another 60 min. A nuclear stain, Hoechst dye (bisbenzimide, 1 mg/100 ml), was applied for 30 s. The slides were rinsed with PBS and a coverslip with gelvatol, a water-soluble mounting medium (21 g of polyvinylalcohol, 52 ml of water, sodium azide, and 106 ml of 0.2 M Tris buffer) was placed. Slides were visualized using an Olympus BX51 epifluorescence microscope and digitized with an Olympus color video camera. In a blind manner, F4/80⁺ staining were counted in 10 high power fields (×400) per each section.

SDS-PAGE and Western blot analysis

Western blot analysis for phosphorylated and total kinase forms of p38 and JNK were performed as described (21). Primary polyclonal Ab to phosphorylated p38 and JNK (1/1000; Cell Signaling Technology) and primary polyclonal Ab to total p38 and JNK (1/1000; Santa Cruz Biotechnology) were used for Western blotting. Membranes were developed with the SuperSignal West Pico chemiluminescent kit (Pierce) and exposed to film.

Cytokine measurement

Serum concentrations of TNF, IL-6, and MCP-1 were determined using ELISA kits obtained from BioSource International.

SYBR Green real-time RT-PCR

Total RNA was extracted from the liver using the TRIzol reagent (Invitrogen Life Technologies) according to the manufacturer’s instruction. mRNA for TNF-α, IL-6, and inducible NO synthase and GAPDH was quantified in duplicate by SYBR Green two-step, real-time RT-PCR. After removal of potentially contaminating DNA with DNase I (Invitrogen Life Technologies), 1 μg of total RNA from each sample was used for reverse transcription with an oligo(dT) (Invitrogen Life Technologies) and a Superscript II (Invitrogen Life Technologies) to generate first-strand cDNA. PCR mixture was prepared using SYBR Green PCR Master mix (PE Applied Biosystems) using the primers as previously described (22, 23). Thermal cycling conditions were 10 min at 95°C followed by 40 cycles of 95°C for 15 s and 60°C for 1 min on an ABI PRISM 7000 Sequence Detection System (PE Applied Biosystems). Each gene expression was normalized with GAPDH mRNA content.

EMSA

NF-κB DNA binding activity was measured by EMSA using nuclear extracts from liver tissues. The NF-κB oligonucleotide (Promega) was based on the NF-κB sequence in the Ig L chain enhancer. DNA probes were prepared by end labeling with [γ-³²P]dATP (DuPont Merck Pharmaceuticals) and T4 polynucleotide kinase (Boehringer Mannheim) and purified in Tris-EDTA buffer containing NaCl (100 mM) using G-50 resin columns (Whatman). Typically, 5 μl (5–10 μg) of hepatic nuclear extract were incubated with 100,000 counts/min of ³²P-labeled oligonucleotides (0.5 ng) for 1–2 h at room temperature in a buffer containing 10 mM Tris (pH 7.6), 10% glycerol, 1 mM EDTA, 1 mg/ml BSA, and 0.2% Nonidet P-40. Additionally, 2–4 μg of poly(dI-dC) (Boehringer Mannheim) were added to the mixture as nonspecific competitor DNA. Protein-DNA complexes were resolved on 4% nondenaturing polyacrylamide gels in 0.4× running buffer containing 450 mM Tris borate and 1 μM EDTA (pH 8.0). Gels were dried after electrophoresis and subjected to autoradiography.

Statistical analysis

Results are expressed as the mean ± SEM. Group comparisons were performed using Student’s t test or ANOVA. Differences were considered significant at values of p < 0.05.

NPC from chimeric mice respond to LPS stimulation if their hemopoietic cells express functional TLR4

TLR4 chimeric mice were produced by adoptive transfer of donor bone marrow cells into irradiated recipient animals using combinations of TLR4 WT (C3H/HeOuj) and TLR4 mutant (C3H/HeJ) mice. To confirm successful engraftment of donor cells in chimeric animals, liver NPC from these mice were harvested and stimulated with LPS (10 ng/ml). As expected, NPC from WT/WT (recipient/donor) and Mutant/WT chimeric mice (animals given TLR4 WT bone marrow cells) responded to LPS stimulation with production of TNF (Fig. 1⇓A) and IL-6 (Fig. 1⇓B) production. In contrast, NPC from Mutant/Mutant and WT/Mutant chimeric mice (animals given TLR4 mutant bone marrow cells) had significantly diminished responses to LPS. From previous coculture studies, the cytokines produced in our experiments derived mainly from Kupffer cells and we have shown very similar responses when purified Kupffer cells have been used instead of NPC (24). To rule out the possibility that some of the chimeric animals reconstituted fewer Kupffer cells as an explanation for the differential responses, we also performed macrophage staining with F4/80 on liver sections. WT/WT chimeric mice had 4.3 ± 0.64 positive cells per high power field (×400) compared with 4.7 ± 1.15 positive cells for WT/Mutant chimeric mice (n = 6 per group).

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FIGURE 1.

NPC from chimeric mice respond to LPS stimulation if their hemopoietic cells express functional TLR4. NPC were harvested from TLR4 chimeric mice produced by adoptive transfer of donor bone marrow cells into irradiated recipient animals using the following combinations from TLR4 WT (C3H/HeOuj) and TLR4 mutant (C3H/HeJ) mice: WT/WT, WT/Mutant (WT/Mu), Mutant/Mutant (Mu/Mu), Mutant/WT (Mu/WT). TNF (A) or IL-6 (B) levels in NPC culture supernatant were determined by ELISA. Cells were treated with 10 ng/ml LPS for 6 h. Data are representative of three independent experiments with similar results. Data shown are combined results from triplicate wells in a single experiment.

Functional TLR4 on NPC, not hepatocytes, is required for liver I/R injury

To determine the cell type in the liver important in mediating TLR4-mediated inflammation and injury after I/R, TLR4 chimeric mice were subjected to 60 min of liver ischemia and 3 h of reperfusion. In agreement with previous reports (9, 10), Mutant/Mutant chimeric mice were protected from liver I/R compared with WT/WT mice as measured by serum ALT levels (Fig. 2⇓A). Interestingly, TLR4 WT mice that were adoptively transferred with TLR4 mutant bone marrow cells (WT/Mutant) were also protected from hepatic I/R. In contrast, serum ALT levels in TLR4 mutant mice transferred with TLR4 WT bone marrow cells (Mutant/WT) remained comparable to those of WT/WT controls. Similar results were seen using chimeric mice subjected to 60 min of liver ischemia and 6 h of reperfusion (Fig. 2⇓B). Liver histologic examination was consistent with the serum ALT estimation of liver damage (Fig. 3⇓). Severe sinusoidal congestion and hepatocellular necrosis were present in liver tissue from WT/WT and Mutant/WT chimeric mice, whereas minimal damage was noted in samples derived from Mutant/Mutant and WT/Mutant mice. Thus, intact TLR4 expression on bone marrow derived NPC is necessary to fully achieve the extent of liver I/R injury seen in WT mice.

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FIGURE 2.

Functional TLR4 on NPC, not hepatocytes, is required for hepatocellular damage after liver I/R. TLR4 chimeric mice underwent ischemia and 3 (A) to 6 (B) h of reperfusion. Serum ALT levels were analyzed as a measure of hepatocellular injury. Data represent mean ± SE (n = 6 mice per group). ∗, p < 0.05 vs WT/WT chimeric mice; †, p < 0.05 vs Mutant/Mutant chimeric mice.

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FIGURE 3.

Histopathology confirms that functional TLR4 on NPC is required for injury after liver I/R. H&E stained liver sections from TLR4 chimeric mice 6 h after reperfusion (original magnification, ×100). The recipient/donor combinations used for the chimeric mice are as follow: WT/WT (A), WT/Mutant (B), Mutant/Mutant (C), and Mutant/WT (D). Images are representative liver sections from six mice per group. Arrows indicate areas of hepatocellular necrosis.

JNK and NF-κB activation involved in TLR4-mediated liver I/R injury

Among the most proximal events in I/R injury is the activation of MAPK. A role for JNK activation in liver I/R injury has been demonstrated (25, 26). To determine the differences in MAPK activation in the TLR4 chimeric mice, we assessed phosphorylation of JNK and p38. Following I/R, phosphorylation of JNK was increased in liver tissue in WT/WT and Mutant/WT mice (Fig. 4⇓A) but less so in Mutant/Mutant and WT/Mutant mice. There was no difference in p38 phosphorylation among the chimeric mice.

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FIGURE 4.

JNK and NF-κB activation involved in TLR4-mediated liver I/R injury. A, MAPK activation was determined in TLR4 chimeric mice undergoing ischemia and 3 h of reperfusion. Western blot analysis for phosphorylated and total JNK and p38 was performed. Hepatic protein lysates from ischemic lobes were obtained. Blot shown is representative of three experiments with similar results. B, NF-κB activation during hepatic I/R injury was assessed. TLR4 chimeric mice underwent ischemia and 3 h of reperfusion. Nuclear extracts were prepared from the ischemic livers and subjected to EMSA. Assay shown is representative of three experiments with similar results.

NF-κB is a transcription factor also involved in signal transduction of a variety of extracellular stress stimuli. It is activated in the setting of hepatic I/R (27) and regulates both proinflammatory and protective responses in the liver (28, 29, 30). Using EMSA, we found increases in NF-κB DNA binding in the ischemic liver 3 h after reperfusion in WT/WT and Mutant/WT mice when compared with sham-treated animals (Fig. 4⇑B). Mice with mutant TLR4 marrow had less activation of NF-κB. The NF-κB bands were specific as confirmed by cold competition in the presence of excess unlabeled NF-κB consensus motif. We previously performed supershift studies to determine that the NF-κB complex was a heterodimer composed of both p65 and p50 subunits (30).

Expression of functional TLR4 on NPC is important in the production of inflammatory markers

Proinflammatory cytokines, including TNF and IL-6, and expression of the adhesion molecule (ICAM-1) play key roles and can be readily assessed as markers in the pathophysiology of hepatic I/R injury (31, 32). Using real-time RT-PCR, we measured steady-state mRNA levels for each of these markers in the liver following I/R (Fig. 5⇓). Chimeric mice lacking functional TLR4 (Mutant/Mutant) expressed lower levels of TNF, IL-6, and ICAM-1 hepatic mRNA compared with mice with functional TLR4 (WT/WT) after I/R. WT animals adoptively transferred with TLR4 mutant bone marrow cells (WT/Mutant) expressed lower hepatic levels of these inflammatory mediators when compared with WT/WT chimeric mice. In contrast, TLR4 mutant mice given WT bone marrow cells (Mutant/WT) expressed higher levels of IL-6 and ICAM-1 mRNA when compared with Mutant/Mutant mice. Because we observed decreases in intrahepatic production of inflammatory mediators among the TRL4 chimeric mice, we next examined systemic levels of TNF, IL-6, and MCP-1 in these mice (Fig. 6⇓). Chimeric mice adoptively transferred with TLR4 WT bone marrow cells (WT/WT and Mutant/WT) had higher serum levels of these mediators when compared with mice receiving TLR4 mutant cells (WT/Mutant and Mutant/Mutant). These results indicate that in our chimeric mice, the bone marrow-derived NPC with functional TLR4 are important in the production of inflammatory mediators observed following liver I/R injury.

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FIGURE 5.

Functional TLR4 on NPC are important in the production of hepatic inflammatory mediators. Hepatic TNF-α (A), IL-6 (B), and ICAM-1 (C) mRNA expression were measured following ischemia and 3 h of reperfusion in TLR4 chimeric mice. Results were obtained using real time RT-PCR and expressed as relative increase of mRNA expression compared with sham animals. Data represent mean ± SE (n = 6 mice per group). ∗, p < 0.05 vs WT/WT chimeric mice; †, p < 0.05 vs Mutant/Mutant chimeric mice.

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FIGURE 6.

Cirulating inflammatory mediator production dependent on functional TLR4 on NPC. Serum TNF-α (A), IL-6 (B), and MCP-1 (C) levels were measured following ischemia and 6 h of reperfusion in TLR4 chimeric mice. Data represent mean ± SE (n = 6 mice per group). ∗, p < 0.05 vs WT/WT chimeric mice; †, p < 0.05 vs Mutant/Mutant chimeric mice.

Functional TLR4 on phagocytic cells enhances liver I/R injury

Our findings suggest that liver NPC play a major role in TLR4-mediated inflammation and injury after I/R. We next sought to identify the cell types responsible for recognizing DAMP-TLR4 ligands as a result of I/R. We initially focused on phagocytic cells including Kupffer cells, which are the resident macrophages in the liver, as they produce multiple proinflammatory cytokines in response to liver I/R. We used gadolinium chloride to deplete liver Kupffer cells and other avidly phagocytic cells (i.e., dendritic cells) in both TLR4 WT and mutant animals immediately before inducing transient hepatic I/R. In agreement with previous findings demonstrating the role of Kupffer cell activity in hepatic I/R injury (33), gadolinium chloride-treated WT mice were protected from 60 min of ischemia and 6 h of reperfusion (Fig. 7⇓). Protection was not observed in TLR4 mutant mice undergoing hepatic I/R by gadolinium chloride pretreatment. Liver histologic findings were consistent with the serum ALT estimation of liver damage (Fig. 8⇓). Severe sinusoidal congestion and hepatocellular necrosis was present in liver tissue from TLR4 WT mice treated with normal saline, whereas minimal damage was noted in samples from WT mice treated with gadolinium chloride. There was also little evidence of hepatocellular injury in TLR4 mutant mice after I/R after treatment with either saline or gadolinium chloride after I/R. Depletion of Kupffer cells using gadolinium chloride was confirmed by staining liver sections for F4/80 Ag, a glycoprotein expressed by murine macrophages (Fig. 8⇓).

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FIGURE 7.

Functional TLR4 on phagocytic cells are important for liver I/R injury. TLR4 WT (C3H/HeOuj) and TLR4 mutant (C3H/HeJ) mice underwent ischemia and 6 h of reperfusion after Kupffer cell depletion with gadolinium chloride 24 h before ischemia. Serum ALT levels were analyzed as a measure of hepatocellular injury. Data represent mean ± SE (n = 6 mice per group). ∗, p < 0.05 vs WT mice.

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FIGURE 8.

Histopathology and immunohistochemistry of livers after I/R in phagocytic cell-depleted TLR4 mice. TLR4 WT (C3H/HeOuj) and TLR4 mutant (C3H/HeJ) mice underwent ischemia and 6 h of reperfusion after Kupffer cell depletion with gadolinium chloride 24 h before ischemia. H&E stained liver sections from TLR4 WT mice (A), TLR4 WT mice treated with gadolinium chloride (B), TLR4 mutant mice (C), and TLR4 mutant mice treated with gadolinium chloride (D) (original magnification, ×100). To confirm the depletion of Kupffer cells using gadolinium chloride, liver sections were stained for F4/80 Ag, a glycoprotein expressed by murine macrophages. E, Control saline treatment. F, Gadolinium chloride treatment. Original magnification was ×400. F4/80 (red) and nuclei (blue) are as shown (E and F). Images are representative liver sections from six mice per group.