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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Previous studies demonstrated that targeted deletion of the Ron receptor tyrosine kinase (TK) domain in mice leads to marked hepatocyte protection in a well-characterized model of lipopolysaccharide (LPS)-induced acute liver failure in D-galactosamine (GalN)-sensitized mice. Hepatocyte protection in TK−/− mice was observed despite paradoxically elevated serum levels of tumor necrosis factor alpha (TNF-α). To understand the role of Ron in the liver, purified populations of Kupffer cells and hepatocytes from wildtype (TK+/+) and TK−/− mice were studied. Utilizing quantitative reverse-transcription polymerase chain reaction (RT-PCR), we demonstrated that Ron is expressed in these cell types. Moreover, we also recapitulated the protected hepatocyte phenotype and exaggerated cytokine production observed in the TK−/− mice in vivo through the use of purified cultured cells ex vivo. We show that isolated TK−/− Kupffer cells produce increased levels of TNF-α and select cytokines compared to TK+/+ cells following LPS stimulation. We also show that conditioned media from LPS-treated TK−/− Kupffer cells was more toxic to hepatocytes than control media, suggesting the exaggerated levels of cytokines produced from the TK−/− Kupffer cells are detrimental to wildtype hepatocytes. In addition, we observed that TK−/− hepatocytes were more resistant to cell death compared to TK+/+ hepatocytes, suggesting that Ron functions in both the epithelial and inflammatory cell compartments to regulate acute liver injury. These findings were confirmed in vivo in mice with hepatocyte and macrophage cell-type-specific conditional Ron deletions. Mice with Ron loss selectively in hepatocytes exhibited less liver damage and increased survival compared to mice with Ron loss in macrophages. Conclusion: We dissected cell-type-specific roles for Ron such that this receptor modulates cytokine production from Kupffer cells and inhibits hepatocyte survival in response to injury. (HEPATOLOGY 2011;)

Acute liver failure (ALF) is an often fatal condition resulting in hepatocellular apoptosis and hemorrhagic necrosis. The most frequent cause of ALF in adults is due to drug toxicity, with a wide spectrum of etiologies responsible for the remaining cases.1 The cascade of events that leads to ALF is complex and not well understood. An established model for studying acute hepatocellular injury in mice is by the coadministration of the hepatocyte-specific transcriptional inhibitor galactosamine (GalN) and the bacterial endotoxin lipopolysaccharide (LPS).2 This model is principally a macrophage/monocyte-mediated model of shock and liver injury with secreted tumor necrosis factor alpha (TNF-α) required for hepatic injury.3, 4 In this model, LPS stimulates the release of TNF-α, a pleiotropic cytokine that is capable of inducing proliferation or apoptosis in hepatocytes and other cell types,5 depending on the physiologic conditions, and numerous other cytokines and chemokines present in the microenvironment secreted from Kupffer cells, the resident tissue macrophage in the liver. After partial hepatectomy, TNF-α is crucial for tissue regeneration, whereas in the setting of a toxic insult, TNF-α induces cell death. The transcription factor nuclear factor kappaB (NF-κB) is reported to play an important role in determining which way the TNF-α balance will tilt.6

Ron is a cell surface receptor tyrosine kinase that participates in divergent processes, including modulation of inflammatory responses.7 Ron is expressed in a variety of cells but is most abundant in epithelial cells and macrophages.8 Ron is activated by its ligand, hepatocyte growth factor-like protein (HGFL), also known as macrophage-stimulating protein.9 HGFL is an 85-kDa circulating protein produced and secreted primarily by hepatocytes.10 Activation of Ron in peritoneal macrophages has been shown to stimulate macrophage shape changes, chemotaxis, adhesion, and phagocytosis.11 Ron has also been shown in alveolar and peritoneal macrophages to limit select cytokine responses in inflammatory cells through attenuation of NF-κB by a mechanism that has yet to be identified.12, 13

Previous studies from our laboratory showed increased inflammatory responses and shortened survival times in mice with a deleted Ron tyrosine kinase domain (TK−/−) compared to wildtype control mice during the induction of bacterial peritonitis and in a lung injury model.14, 15 Paradoxically, utilizing the well-characterized model of LPS/GalN induced ALF in mice, although serum levels of TNF-α were elevated, livers from TK−/− mice exhibited marked hepatocyte protection compared with controls.16 To investigate the function of Ron in regulating hepatocyte survival, purified populations of Kupffer cells and hepatocytes from wildtype and TK−/− mice were isolated. Utilizing purified cells, we recapitulated ex vivo the protected hepatocyte phenotype and exaggerated cytokine production observed in the TK−/− mice in vivo. Furthermore, by using mice with targeted deletions of Ron in hepatocytes and macrophages, we were able to substantiate our findings ex vivo. In total, our data suggests that Ron loss selectively in hepatocytes provides a survival benefit during ALF despite increased cytokine production by deregulated Kupffer cell activation.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Mice.

Ron tyrosine kinase-deficient mice (TK−/−) and floxed Ron mice (TKfl/fl) were generated as described and were backcrossed into a C57BL/6 background.7 Age-matched male mice between 14 and 24 weeks old were used for all experiments. C57BL/6 albumin-Cre and lysozyme-Cre mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Cre-expressing mice were crossed with floxed Ron (TKfl/fl) mice to create the targeted knockouts. Deletion of the Ron TK domain was determined by semiquantitative competitive polymerase chain reaction (PCR) as described.7, 17 All mice were maintained under specific pathogen-free conditions and were treated in accordance with protocols approved by the Institutional Animal Care and Use Committee of the University of Cincinnati.

Cellular Isolation and Immunocytochemistry.

Mouse Kupffer cells and hepatocytes were isolated using the technique described by Kuboki et al.18 Cell staining was performed with antibodies against F4/80 (ab6640, Abcam, Cambridge, MA), Albumin (Bethyl Laboratories, Montgomery, TX), Ron (AF431, R&D Systems, Minneapolis, MN), or isotype control antibodies. Mounting media contained DAPI for nuclear staining. THP-1 cells were purchased from the American Tissue Culture Collection (ATCC, Manassas, VA) and were differentiated with 100 ng/mL phorbol 12-myristate 13-acetate (PMA).

RNA and Quantitative Real-Time PCR.

RNA was isolated using TriZol (Invitrogen, Carlsbad, CA). One μg of RNA was converted to complementary DNA (cDNA) with the high capacity RNA to cDNA kit according to manufacturer's instructions (Applied Biosystems, Foster City, CA). Real-time PCR was performed using FastStart SYBR Green (F. Hoffmann-La Roche, Nutley, NJ). The following genes and corresponding sequences were chosen: Ron (5′-TCCC ATTGCAGGTCTGTGTAGA-3′; 5′-CGGAAGCTG TATCGTTGATGTC-3′), β-glucuronidase (GusB) (5′-TTGAGAACTGGTATAAGACGCATCAG-3′; 5′-TCT GGTACTCCTCACTGAACATGC-3′). TNF-α (5′-CAT CTTCTCAAAATTCGAGTGACAA-3′; 5′-TGGGAG TAGACAAGGTACAACCC-3′), keratinocyte chemoattractant (KC) (5′-TGCACCCAAACCGAAGTCAT-3′; 5′-TTGTCAGAAGCCAGCGTTCAC-3′), HGFL (5′-TGGTACAGTGTTCAAGGGCTCTT-3′; 5′-GCATGG CTGCTCATG-3′), and EGR1 (5′-TCTTGG TGCCTTTTGTGTGAC-3′; 5′-CTCTTCCTCGTTT TTGCTCTC-3′). Expression levels were normalized to GusB as internal control. Relative gene expression results are reported. Real-time analyses were repeated twice with similar results using samples from three independent isolations.

Cytokine Production.

Kupffer cells were plated in Williams E media supplemented with 5% fetal bovine serum (FBS). Conditioned media was generated by replacing the Kupffer cell media with fresh media plus 500 μg/mL LPS (E. coli serotype 0111:B4; Sigma, St. Louis, MO) and collected at the timepoints indicated. For the cytokine array, conditioned media was collected and incubated with the mouse cytokine antibody array from R&D Systems. Detection of replicate spots is by horseradish peroxidase-based chemiluminescence and film. Film was scanned and spots were quantitated using ImageJ from the National Institutes of Health. TNF-α levels were measured by enzyme-linked immunosorbent assay (ELISA) (R&D Systems). Recombinant HGFL was supplied by R&D Systems.

Luciferase Reporter Assays.

Twenty-four hours before LPS exposure, Kupffer cells or primary hepatocytes were transfected with an NF-κB reporter (pNF-κB luc) plasmid or an empty vector (pTAL luc), and a control plasmid expressing Renilla (pRL-TK) utilizing Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Kupffer cells were treated with LPS (1 μg/mL) in complete media for 2 hours. Hepatocytes were treated with 10 ng/mL of TNF-α for 6 hours. Cell lysates were collected and luciferase activity was determined using the Dual-Luciferase Assay System (Promega, Madison, WI). Samples were run in duplicate and averaged.

Conditioned Kupffer Media on Hepatocytes.

Conditioned media was generated by placing fresh media, with or without 500 μg/mL LPS, onto Kupffer cells. The media was subsequently collected after 1 or 2 hours. AML12 mouse hepatocytes (ATCC) were grown in DMEM/F12 media supplemented with 10% FBS and ITS (Invitrogen). Cells were pretreated with 200 ng/mL of actinomycin D (ActD) for 30 minutes. Media was then changed to 150 μL of conditioned Kupffer cell media plus 200 ng/mL ActD. After 18 hours, media was removed and cells were fixed. Cells were quantitated by crystal violet assays and hepatocyte number was calculated based on a standard curve.19 TNF-α antibody (AB-410-NA) was from R&D Systems.

TNF-α Hepatocyte Assay.

Primary hepatocytes were pretreated with 200 ng/mL of ActD in Williams E media + 5% FBS for 30 minutes. Media was then changed to include increasing amounts of recombinant mouse TNF-α (Peprotech, Rocky Hill, NJ) plus ActD. After 18 hours, media was removed and cells were fixed and quantitated by crystal violet assays.

Western Analyses.

Cells were collected and homogenized in 2× Laemmli Buffer. For HGFL determination, cells were cultured in serum-free media and the media was collected after 36 hours. Media was concentrated with Amicon Ultra-4 centrifugal filters (Millipore, Billerica, MA). Protein concentrations were determined using the Micro BCA Kit (Pierce Biotechnology, Rockford, IL). Primary antibodies used were anti-NF-κB p65, anti-pNF-κB p65 Ser536, anti-pIKKα/β, anti-IKKβ anti-Caspase-3 (Cell Signaling, Boston, MA), anti-HGFL (T-19, Santa Cruz Biotechnology, Santa Cruz, CA).

LPS and GalN Injection of Mice.

Mice were injected with 0.8 μg LPS and 30 mg GalN in saline and normalized to 30 g body weight (500 μL total volume) or GalN alone. This low dose of LPS does not alter mortality in the Ron TK-deficient mice, but when combined with GalN induces significant liver injury.7, 16 Blood was collected and plasma alanine aminotransferase (ALT) levels were determined at Shriners Hospital.

Terminal Deoxynucleotidyl Transferase-Mediated Deoxyuridine Triphosphate Nick-End Labeling (TUNEL) Analysis.

Paraffin-embedded sections of liver tissue were analyzed by TUNEL staining.16 For each liver tissue section per mouse, the number of TUNEL-positive cells in three random high-powered fields was counted by an investigator blinded to treatment group.

Statistical Analyses.

Statistical significance for all analyses was determined by Student's t test, Logrank, or one-way analysis of variance (ANOVA) using GraphPad Prism 3.03 software (La Jolla, CA). Error bars represent standard error of the mean (SEM).

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The Ron Receptor Is Expressed in Kupffer Cells and Hepatocytes.

Quantitative data directly comparing Ron expression in liver macrophages (Kupffer cells) and in liver parenchymal cells in the mouse are lacking. To examine Ron expression in mouse Kupffer cells and hepatocytes, populations of murine Kupffer cells and hepatocytes were collected. The isolated Kupffer cells ranged in purity from 90%-95% following centrifugal elutriation based on F4/80 immunostaining (Fig. 1A) and the ability of the cells to ingest fluorescent beads (data not shown). Hepatocyte identity was confirmed with albumin immunostaining and purity was over 99% (Fig. 1A). Ron protein expression was observed in isolated Kupffer cells and hepatocytes by immunocytochemistry (Fig. 1A). Quantitative real-time PCR was performed to compare Ron RNA expression levels in Kupffer cells, hepatocytes, AML12 cells, and peritoneal macrophages as shown in Fig. 1B. The expression of Ron in TK−/− Kupffer cells and hepatocytes was undetectable. AML12 cells and primary hepatocytes express and secrete HGFL (Fig. 1C).

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Figure 1. Ron is expressed in Kupffer cells and hepatocytes. (A) Representative pictures of isolated Kupffer cells and hepatocytes. Top: Immunocytochemical staining of Kupffer cells by immunofluorescence for the macrophage-specific surface marker F4/80 or for Ron compared to control staining with an isotype-matched primary antibody. Nuclei were stained with DAPI. Bottom: Representative pictures depicting albumin and Ron staining of hepatocytes with DAPI-stained nuclei. Control staining with an isotype-matched primary antibody on hepatocytes is shown. (B) Quantitative real-time PCR analysis of Ron messenger RNA (mRNA) expression from Kupffer cells, hepatocytes, AML12 cells, and peritoneal macrophages. Normalized relative Ron expression levels are expressed as the mean ± the standard error and are representative of two independent experiments. Ron expression levels are relative to β-glucuronidase as an internal control. TK−/− Kupffer cells and hepatocytes serve as a negative control. (C) HGFL is expressed at the mRNA level and is secreted by AML12 cells and primary mouse hepatocytes.

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Ron Regulates Kupffer Cell Responses to LPS.

To investigate the role of Ron as a potential mediator of Kupffer cell inflammation, we examined cytokine production from wildtype (TK+/+) and TK−/− Kupffer cells in response to LPS ex vivo. Figure 2A demonstrates that Ron loss leads to increases in TNF-α production from Kupffer cells in response to LPS. To examine the extent of cytokine changes regulated by Ron, an antibody array was utilized to simultaneously compare 40 cytokines in conditioned media from LPS-stimulated wildtype and Ron TK−/− Kupffer cells. Figure 2B displays relative values of select cytokines whereby macrophage chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-2 (MIP-2), interleukin-1 receptor antagonist (IL-1ra), and IL-6 levels were elevated ≈2-fold in the media from the Ron TK−/− cells compared to controls. Keratinocyte chemoattractant (KC) and tissue inhibitor of metalloproteinase (TIMP-1) showed moderate increases in the TK−/−-conditioned media, whereas no changes between groups were observed in the levels of IL-1a or IL-13. The results for all cytokines are listed in Supporting Information Table S1. No differences were observed in the basal conditioned media between the TK+/+ and TK−/− Kupffer cells in the absence of LPS treatment, with the exception of IL-1ra and TIMP-1 expression, which were higher basally in the TK−/− Kupffer cells and did not respond to LPS treatment (data not shown). To examine the role of HGFL in suppressing cytokine production, TK+/+ Kupffer cells were treated with HGFL and stimulated with LPS. As shown in Fig. 2C, HGFL treatment suppresses the release of TNF-α and reduces the expression of TNF-α, KC, and early growth response 1 (EGR1), well-documented NF-κB-responsive genes.

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Figure 2. Ron regulates cytokine production in Kupffer cells. (A) TNF-α ELISA of Kupffer cell media after stimulation with LPS for the indicated times. TNF-α production increases in TK+/+ and TK−/− Kupffer cells following LPS stimulation with significantly higher levels in the TK−/− media by 2 hours. *P < 0.05. (B) Densitometric analysis of an antibody-based cytokine array of select cytokines from TK+/+ and TK−/− Kupffer cells following LPS stimulation for 2 hours. Error bars are the range of two spots of each cytokine and is representative of a least two experiments. (C) TNF-α ELISA and quantitative real-time PCR of TNF-α, KC, and EGR1 showing reduced expression of NF-κB target genes in LPS-stimulated Kupffer cells after overnight treatment with 100 ng/mL HGFL. *P < 0.05.

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Loss of Ron Signaling Leads to Increased NF-κB Activity.

As demonstrated in alveolar and peritoneal macrophages, Ron functions to inhibit cytokine signaling by limiting potentially damaging hyper-signaling through the NF-κB pathway.12, 20, 21 To investigate whether the increased cytokine expression observed in Ron TK−/− Kupffer cells is associated with increased NF-κB signaling, NF-κB activation in TK+/+ and TK−/− Kupffer cells was examined by luciferase reporter assays. Kupffer cells from TK+/+ and TK−/− mice were transfected with a vector containing an NF-κB response element upstream of luciferase or an empty vector control. As shown in Fig. 3A, TK−/− Kupffer cells had significantly higher reporter activity compared to TK+/+ cells in response to LPS treatment (2.5-fold versus 1.5-fold over the basal level). Basal levels of reporter activity were similar between genotypes. Electrophoretic mobility shift assay (EMSA) analysis confirmed an increased NF-κB response in the TK−/− cells (data not shown). To support these data and further define the mechanism by which Ron may regulate cytokine production, the human macrophage cell line THP-1 was utilized. Differentiated THP-1 cells express abundant levels of Ron (data not shown). As shown in Fig. 3B, LPS stimulation induces the phosphorylation of NF-κB at Ser536, a verified marker of NF-κB transcriptional activity,22 in THP-1 cells. Treatment with HGFL decreases this phosphorylation. In addition, the stimulation of IκB kinase (IKK) phosphorylation, an upstream kinase that regulates NF-κB activation, is reduced by HGFL treatment, similar to recently reported findings.21 Similar results are also observed with Kupffer cells that exhibit less NF-κB and IKK phosphorylation in response to HGFL treatment than that observed with LPS alone (Fig. 3C and data not shown).

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Figure 3. TK−/− Kupffer cells exhibit exaggerated NF-κB signaling in response to LPS. (A) Kupffer cells from TK+/+ and TK−/− mice were transfected with a vector containing an NF-κB response element upstream of firefly luciferase and a control plasmid. After LPS stimulation for 4 hours, TK−/− Kupffer cells had dramatically higher reporter activity than TK+/+ cells. Bars represent the average of three independent experiments with at least two mice per genotype per experiment. *P < 0.002. (B) Differentiated THP-1 cells were stimulated with 500 ng/mL of LPS with or without overnight HGFL treatment. Western analysis of cell lysates depicts diminished NF-κB and IKKα/β phosphorylation in HGFL-treated cells. Samples from two independent experiments are shown. Total NF-κB, IKKβ, and tubulin serve as loading controls. (C) Western analysis of LPS-stimulated Kupffer cell lysates with or without HGFL pretreatment overnight (O/N) or for 30 minutes (30′). HGFL treatment before LPS stimulation leads to reduced NF-κB Ser536 phosphorylation (p-p65) compared to controls. Total NF-κB and tubulin serve as loading controls.

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Conditioned Media from LPS-Treated Ron TK−/− Kupffer Cells Is Detrimental to Hepatocytes.

Given the significant decrease in hepatocyte apoptosis observed in TK−/− mice in response to LPS/GalN treatment in vivo16 and the alteration of cytokine signaling observed in the TK−/− Kupffer cells ex vivo, we initially hypothesized that the altered cytokine milieu produced by the TK−/− Kupffer cells was capable of affording protection to hepatocytes during the induction of acute liver failure. To test this hypothesis, we isolated Kupffer cells from TK+/+ and TK−/− mice and stimulated them ex vivo with LPS or vehicle. Equal amounts of conditioned media were collected after 2 hours and were applied to the AML12 hepatocyte cell line in the presence of the transcriptional inhibitor actinomycin D (ActD) to emulate conditions in vitro of hepatocyte death observed in previous in vivo experiments. As shown in Fig. 4, whereas the viability of hepatocytes exposed to control media or conditioned media from either untreated TK+/+ or TK−/− Kupffer cells did not significantly differ from one another, the percent hepatocyte death was significantly increased in the presence of conditioned media from LPS-treated TK−/− Kupffer cells compared to similarly treated TK+/+ Kupffer cells. These results demonstrate that the LPS-induced cytokine milieu from the TK−/− Kupffer cells is capable of inducing increased hepatocyte death compared to control cells under these conditions. In Fig. 4C, neutralization of TNF-α in the TK−/− Kupffer cell conditioned media restored hepatocyte viability to near 100%, suggesting that TNF-α has a prominent role in inducing hepatocyte death in ActD-treated hepatocytes.

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Figure 4. Conditioned media from LPS-stimulated TK−/− Kupffer cells is detrimental to hepatocytes. (A) Kupffer cells from TK+/+ or TK−/− mice were treated in the presence or absence of LPS for 2 hours. Conditioned media was collected and applied to AML12 hepatocytes with ActD. After 18 hours the hepatocytes were fixed and quantified by crystal violet staining. (B) Media from the TK−/− Kupffer cells increased hepatocyte death by 35% compared to control cells. The data are expressed as the mean ± standard error and are representative of three independent experiments. *P < 0.05. (C) Treatment of TK−/− Kupffer cell conditioned media with an anti-TNF-α antibody for 30 minutes leads to a significant reduction in hepatocyte death to less than 5%. Goat IgG was used as a control. Bar graph is the average of three experiments. *P < 0.05.

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Ron TK−/− Hepatocytes Are Afforded Protection from TNF-α-Induced Apoptosis.

Given that our prior studies in TK−/− mice demonstrated lessened hepatocyte apoptosis in vivo, despite elevated serum levels of TNF-α, following the induction of acute liver failure by LPS/GalN treatment, we next sought to determine if Ron signaling regulates hepatocyte survival. Using a well-established method to induce apoptosis in hepatocytes by the coadministration of ActD and TNF-α,23 hepatocytes from TK+/+ and TK−/− mice were treated with increasing amounts of TNF-α for 18 hours and hepatocyte viability was subsequently assessed. As shown in Fig. 5, hepatocytes derived from TK−/− mice were significantly protected from TNF-α-induced apoptosis compared to TK+/+ hepatocytes at a TNF-α concentration range from 0.5 to 5.0 ng/mL. At a TNF-α concentration of 1 ng/mL, 90% of the TK−/− hepatocytes were viable compared to 75% viability observed in hepatocytes from wildtype mice. These data suggest that Ron signaling in hepatocytes may be an important mediator of hepatocyte survival following liver injury.

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Figure 5. TK−/− hepatocytes are protected from TNF-α-induced cell death. Primary hepatocytes from TK+/+ or TK−/− mice were treated for 30 minutes with ActD followed by the addition of increasing amounts of TNF-α. After 18 hours the hepatocytes were fixed and cell survival was determined by crystal violet staining with cell number quantitated based on a standard curve of hepatocytes. The data are expressed as the mean ± standard error and are representative of three independent experiments. *P < 0.01 compared to the corresponding control treated sample.

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Inhibition of NF-κB Signaling Attenuates the Survival of Ron TK−/− Hepatocytes.

Although we and others have shown that Ron regulates NF-κB in macrophages, including Kupffer cells (Fig. 3), nothing is known about Ron signaling in hepatocytes.12, 20 To test whether the differential hepatocyte survival observed in TK−/− hepatocytes may be due to differential NF-κB activation, we examined survival of TK+/+ and TK−/− hepatocytes in response to constant levels of TNF-α and ActD with the inclusion of increasing concentrations of the irreversible NF-κB activation inhibitor BAY 11-7085.24 As depicted in Fig. 6A, TK+/+ and TK−/− hepatocyte survival converged with increasing concentrations of Bay 11-7085. These data suggest that blunting NF-κB activation in TK−/− hepatocytes negates the survival advantage in these cells. Indeed, as shown in Fig. 6B, TK−/− hepatocytes have elevated phosphorylated NF-κB after TNF-α treatment compared to wildtype hepatocytes. Basal levels of pNF-κB did not differ between genotypes and are similar to that observed at the 6-hour timepoint (data not shown). To confirm exaggerated NF-κB signaling in TK−/− hepatocytes, NF-κB luciferase reporter assays were performed. TK+/+ and TK−/− hepatocytes were stimulated with TNF-α and reporter activity was determined after 6 hours. As shown in Fig. 6C, TK−/− hepatocytes exhibited 1.5× more luciferase activity than TK+/+ hepatocytes.

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Figure 6. NF-κB inhibition abrogates survival of TK−/− hepatocytes in response to TNF-α-induced death. (A) Hepatocytes from TK+/+ or TK−/− mice were treated for 30 minutes with ActD plus increasing amounts of the NF-κB activation inhibitor, Bay 11-7085 followed by 5 ng/mL TNF-α. After 18 hours the hepatocytes were fixed and cell survival was determined by crystal violet staining. The data are expressed as the mean ± standard error. *P < 0.01. (B) Hepatocytes from TK+/+ or TK−/− mice were treated for 30 minutes with ActD followed by 5 ng/mL TNF-α. Cells from three independent experiments of each cell type were examined after 10 minutes and 6 hours by western analysis. pNF-κB (phosphorylated p65 subunit at Ser536) band density was normalized to actin. *P < 0.05. (C) TK+/+ and TK−/− hepatocytes were transfected with a vector containing an NF-κB response element upstream of firefly luciferase and a control plasmid. After 10 ng/mL TNF-α stimulation for 6 hours, TK−/− hepatocytes had 1.5-fold higher reporter activity than TK+/+ cells. Values represent the average of two independent experiments relative to untreated cells. *P < 0.05.

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Targeted Deletion of Ron TK in Hepatocytes Leads to a Protected Phenotype In Vivo.

Our ex vivo data suggest that despite an elevated cytokine profile, including increased TNF-α, TK−/− hepatocytes are protected from damage compared to wildtype hepatocytes. In order to test our ex vivo findings in vivo, we employed Cre-LoxP technology to generate mice with cell type-specific deletion of Ron from hepatocytes (i.e., albumin-Cre [Alb-Cre or AC] Ron TKfl/fl mice) or myeloid lineage cells (i.e., lysozyme-Cre [Lys-Cre or LC] Ron TKfl/fl mice). By semiquantitative competitive PCR, the TK region of Ron appeared completely ablated in Alb-Cre Ron TKfl/fl hepatocytes (Supporting Information Fig. S1). Ron TK ablation was observed in the majority of Lys-Cre Ron TKfl/fl Kupffer cells (Supporting Information Fig. S1), ranging from ≈60%-80%.

Mice were injected with LPS/GalN, and then liver tissue was analyzed for histopathology and blood was analyzed for ALT levels. In Fig. 7A-C, hematoxylin-eosin staining of representative liver sections shows the greatest hemorrhagic necrosis and pyknotic nuclei in the Lys-Cre Ron TKfl/fl liver. Alb-Cre Ron TKfl/fl liver sections displayed the least damage of the three groups. ALT levels at 5 hours in Lys-Cre Ron TKfl/fl plasma displayed a more rapid rise than the other two groups (Fig. 7G). Unfortunately, by 6 hours a majority of Lys-Cre Ron TKfl/fl mice were moribund or dead and adequate blood could not be collected for ALT analysis. Importantly, however, at 6 hours ALT levels in Ron TKfl/fl control plasma were significantly higher than Alb-Cre Ron TKfl/fl plasma, suggesting protection of the TK−/− hepatocytes.

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Figure 7. Deletion of the Ron TK domain in hepatocytes is protective, whereas deletion in myeloid derived cells is detrimental to mice treated with LPS/GalN. Histopathology of LPS/GalN mice after 5 hours: (A) Ron TKfl/fl, (B) Alb-Cre Ron TKfl/fl, (C) Lys-Cre Ron TKfl/fl. (D-F) Representative TUNEL-stained slides (bar = 100 μm), positive nuclei stained red. (D) Ron TKfl/fl, (E) Alb-Cre Ron TKfl/fl, (F) Lys-Cre Ron TKfl/fl. (G) Plasma ALT levels of LPS/GalN treated mice at 4.5, 5, and 6 hours. At 6 hours, Ron TKfl/fl control mice had significantly higher ALT than Alb-Cre Ron TKfl/fl mice (*P < 0.05). ND, not determined, as Lys-Cre mice were moribund or dead at 6 hours. (H) Positive TUNEL counts per 100× field. *P < 0.05.

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TUNEL staining was performed on liver sections at 5 hours to assess the level of apoptosis (Fig. 7D-F). Alb-Cre Ron TKfl/fl liver had statistically lower levels of TUNEL-positive cells followed by control Ron TKfl/fl and Lys-Cre Ron TKfl/fl livers (Fig. 7H). This result is consistent with western analyses for active (cleaved) caspase-3 observed in liver lysates with the lowest levels of active caspase-3 detected in the Alb-Cre Ron TKfl/fl livers (Supporting Information Fig. S2).

To test for survival differences, mice from each group were allowed to proceed until death from ALF. Kaplan-Meier survival curves (Fig. 8A) clearly show that Lys-Cre Ron TKfl/fl mice have a significantly shorter survival time. The mean survival time of each genotype is depicted in Fig. 8B. Although Fig. 7 demonstrates protection from hepatocyte apoptosis and liver injury in the Alb-Cre Ron TKfl/fl mice compared to the other genotypes, overt survival of the Alb-Cre Ron TKfl/fl mice was longer than the Ron TKfl/fl mice but was not statistically different (P = 0.07).

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Figure 8. Alb-Cre Ron TKfl/fl and Ron TKfl/fl mice have a survival advantage over Lys-Cre Ron TKfl/fl mice treated with LPS/GalN. (A) Kaplan-Meier survival graph of mice injected with LPS/GalN. Lys-Cre Ron TKfl/fl n = 5, Ron TKfl/fl n = 8, Alb-Cre Ron TKfl/fl n = 6. Survival of the Lys-Cre Ron TKfl/fl mice was significantly less than the other two groups. Logrank test P = 0.005. (B) Comparison of survival times in hours. *P < 0.05 between Alb-Cre and Lys-Cre mice.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Here we dissected the role of Ron receptor TK in an endotoxin-induced model of acute liver failure to further studies demonstrating that mice with a global deletion of Ron TK had a protected liver injury phenotype.16 Examination of hepatocytes and Kupffer cells showed expression of Ron in both, and importantly, ex vivo experiments showed Ron signaling is critical for both suppressing hepatotoxic Kupffer cell cytokine production and for sensitizing hepatocytes to Kupffer cell-derived products such as TNF-α. Together with in vivo experiments using mice with either hepatocyte- or Kupffer cell-specific deletion of Ron TK, we determined that Ron TK signaling in both cell types has important effects on hepatocyte survival following exposure to endotoxin in this model of acute liver failure. However, the cell type likely responsible for Ron-dependent hepatic protection in this liver injury model is the hepatocyte, given that lack of Ron signaling in hepatocytes ex vivo and in vivo is sufficient to afford a hepatocyte survival benefit.

Following stimulation with LPS, TK−/− Kupffer cells have altered cytokine production ex vivo compared to TK+/+ Kupffer cells, with the most significantly elevated cytokines being IL-1ra (2.5-fold), IL-6 (2-fold), TIMP-1 (1.5-fold), MCP-1 (3.5-fold), and MIP-2 (1.9-fold). A potential mechanism for these perturbations is increased NF-κB signaling in the Ron-deficient state. These perturbations in cytokine production in response to LPS are consistent with earlier in vivo observations in this liver injury model,16 as well as in models of endotoxic shock or nickel-induced acute lung injury.12, 15, 20, 25 Interestingly, the effect of the altered cytokine milieu generated by LPS-treated TK−/− Kupffer cells was more toxic to mouse hepatocytes in vitro compared to TK+/+ Kupffer cells. This may be due to higher TNF-α levels, the composition of cytokines, or the presence of an untested cytokine in the conditioned media. Moreover, it is possible that the kinetics of cytokine expression is as important as the composition of cytokines produced.

TK−/− hepatocytes are protected compared to TK+/+ hepatocytes from TNF-α/ActD initiated cell death over the range of 0.5 to 5 ng/mL of TNF-α, which are similar to the serum TNF-α levels observed in the previously reported in vivo experiment.16 Whether the observed differences in hepatocyte viability are sufficient to explain the in vivo observations by Leonis et al.16 is not discernable by this assay. Hepatocytes plated ex vivo are without feedback mechanisms to Kupffer cells and other hepatic cell types and, thus, the magnitude of the effect of TNF-α ex vivo may not completely mimic what is observed in vivo. However, the results from the conditional deletion of Ron selectively in hepatocytes support the significance of our ex vivo culture conditions and prior in vivo experiments.

Previously, we demonstrated that the protected liver injury response to treatment with LPS/GalN in Ron TK−/− mice was associated with a 1- to 2-hour delay in the progression to death based on survival analyses.16 The lack of a more significant discrepancy in the time to mortality may be multifactorial and confounded by the sensitivity of the Ron TK−/− mice to LPS alone and by the severe necrosis and endothelial damage that is observed in the model. Despite the modest overall survival benefit of the TK−/− mice compared to controls, less liver injury was observed in the TK−/− mice as judged by liver histopathology, ALT levels, hepatocyte TUNEL staining, and the extent of hepatic apoptosis. Similar protected phenotypes were observed in the Alb-Cre Ron TKfl/fl mice as the Ron TK−/− mice compared to control mice. This is based on a reduction in liver histopathology and significantly decreased ALT levels and TUNEL staining in the Alb-Cre Ron TKfl/fl mice compared to controls. Interestingly, a 1- to 2-hour increase in survival time in the Alb-Cre Ron TKfl/fl mice was also observed compared to controls. Therefore, the data are consistent with a protected liver phenotype in the Alb-Cre Ron TKfl/fl mice compared to controls. Our data also show a significant decrease in survival of the LysCre mice and associated worsened liver phenotypes in these mice compared to Alb-Cre Ron TKfl/fl and wildtype mice and supports the premise that Ron functions in both cellular compartments in vivo.

Our results show increased NF-κB activation in hepatocytes that lack Ron signaling. NF-κB inhibition has been shown to tilt the balance of TNF-α signaling toward apoptosis in hepatocytes; the converse is found as well, that increased NF-κB signaling provides a survival benefit.6, 26-30 In addition, Ron has been shown to regulate NF-κB.12, 20, 21 Pretreatment of primary hepatocytes with the NF-κB inhibitor Bay-11-7085 abrogated the survival advantage of Ron-deficient cells, suggesting that the elevated NF-κB levels observed in the early timepoints in these cells may at least be partly responsible for the protective phenotype. Although the exact mechanism for how NF-κB activity protects cells from apoptosis is not clear, up-regulation of antiapoptotic proteins is one important effect of NF-κB signaling. Several antiapoptotic proteins can be up-regulated by TNF-α through NF-κB; however, we have seen no difference in two such proteins, C-IAP-2 or XIAP,31 between TK+/+ and TK−/− hepatocytes ex vivo following exposure to TNF-α when examined by western blotting (data not shown).

Thus, we have demonstrated that Ron signaling is detrimental to hepatocyte survival when challenged with TNF-α and that Ron is a regulator of hepatotoxic cytokine signaling in Kupffer cells. Although the exact mechanism has not been elucidated, our ex vivo and in vivo studies suggest that Ron signaling appears to limit NF-κB signaling in both hepatocytes and Kupffer cells, leading to an overall sensitization of hepatocytes to Kupffer cell-derived products. Further research on the cell-type specific effects of Ron, and on how Ron regulates NF-κB, is important in order to understand the mechanisms underlying this receptor's effects on hepatocyte survival and before positing strategies that may lead to therapies for ALF or other liver pathologies, such as obesity-related steatohepatitis and alcohol-induced liver disease, that may, in part, have liver injury mediated by endotoxin.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The authors thank William Niehaus for technical assistance.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
  • 1
    Ostapowicz G, Fontana RJ, Schiodt FV, Larson A, Davern TJ, Han SH, et al. Results of a prospective study of acute liver failure at 17 tertiary care centers in the United States. Ann Intern Med 2002; 137: 947-954.
  • 2
    Bohlinger I, Leist M, Gantner F, Angermuller S, Tiegs G, Wendel A. DNA fragmentation in mouse organs during endotoxic shock. Am J Pathol 1996; 149: 1381-1393.
  • 3
    Galanos C, Freudenberg MA. Mechanisms of endotoxin shock and endotoxin hypersensitivity. Immunobiology 1993; 187: 346-356.
  • 4
    Josephs MD, Bahjat FR, Fukuzuka K, Ksontini R, Solorzano CC, Edwards CK 3rd, et al. Lipopolysaccharide and D-galactosamine-induced hepatic injury is mediated by TNF-alpha and not by Fas ligand. Am J Physiol Regul Integr Comp Physiol 2000; 278: R1196-1201.
  • 5
    Wajant H, Pfizenmaier K, Scheurich P. Tumor necrosis factor signaling. Cell Death Differ 2003; 10: 45-65.
  • 6
    Xu Y, Bialik S, Jones BE, Iimuro Y, Kitsis RN, Srinivasan A, et al. NF-kappaB inactivation converts a hepatocyte cell line TNF-alpha response from proliferation to apoptosis. Am J Physiol 1998; 275: C1058-1066.
  • 7
    Waltz SE, Eaton L, Toney-Earley K, Hess KA, Peace BE, Ihlendorf JR, et al. Ron-mediated cytoplasmic signaling is dispensable for viability but is required to limit inflammatory responses. J Clin Invest 2001; 108: 567-576.
  • 8
    Brunelleschi S, Penengo L, Lavagno L, Santoro C, Colangelo D, Viano I, et al. Macrophage stimulating protein (MSP) evokes superoxide anion production by human macrophages of different origin. Br J Pharmacol 2001; 134: 1285-1295.
  • 9
    Ronsin C, Muscatelli F, Mattei MG, Breathnach R. A novel putative receptor protein tyrosine kinase of the met family. Oncogene 1993; 8: 1195-1202.
  • 10
    Skeel A, Yoshimura T, Showalter SD, Tanaka S, Appella E, Leonard EJ. Macrophage stimulating protein: purification, partial amino acid sequence, and cellular activity. J Exp Med 1991; 173: 1227-1234.
  • 11
    Leonard EJ, Skeel AH. Enhancement of spreading, phagocytosis and chemotaxis by macrophage stimulating protein (MSP). Adv Exp Med Biol 1979; 121B: 181-194.
  • 12
    Lentsch AB, Pathrose P, Kader S, Kuboki S, Collins MH, Waltz SE. The Ron receptor tyrosine kinase regulates acute lung injury and suppresses nuclear factor kappaB activation. Shock 2007; 27: 274-280.
  • 13
    Liu QP, Fruit K, Ward J, Correll PH. Negative regulation of macrophage activation in response to IFN-gamma and lipopolysaccharide by the STK/RON receptor tyrosine kinase. J Immunol 1999; 163: 6606-6613.
  • 14
    Caldwell CC, Martignoni A, Leonis MA, Ondiveeran HK, Fox-Robichaud AE, Waltz SE. Ron receptor tyrosine kinase-dependent hepatic neutrophil recruitment and survival benefit in a murine model of bacterial peritonitis. Crit Care Med 2008; 36: 1585-1593.
  • 15
    McDowell SA, Mallakin A, Bachurski CJ, Toney-Earley K, Prows DR, Bruno T, et al. The role of the receptor tyrosine kinase Ron in nickel-induced acute lung injury. Am J Respir Cell Mol Biol 2002; 26: 99-104.
  • 16
    Leonis MA, Toney-Earley K, Degen SJ, Waltz SE. Deletion of the Ron receptor tyrosine kinase domain in mice provides protection from endotoxin-induced acute liver failure. Hepatology 2002; 36: 1053-1060.
  • 17
    Nikolaidis NM, Kulkarni RM, Gray JK, Collins MH, Waltz SE. Ron receptor deficient alveolar myeloid cells exacerbate LPS-induced acute lung injury in the murine lung. Innate Immun 2010;17534259 1030:3725, first published on November 18, 2010. DOI: 10.1177/17534 259103:3725.
  • 18
    Kuboki S, Okaya T, Schuster R, Blanchard J, Denenberg A, Wong HR, et al. Hepatocyte NF-kappaB activation is hepatoprotective during ischemia-reperfusion injury and is augmented by ischemic hypothermia. Am J Physiol Gastrointest Liver Physiol 2007; 292: G201-207.
  • 19
    Gillies RJ, Didier N, Denton M. Determination of cell number in monolayer cultures. Anal Biochem 1986; 159: 109-113.
  • 20
    Nikolaidis NM, Gray JK, Gurusamy D, Fox W, Stuart WD, Huber N, et al. Ron receptor tyrosine kinase negatively regulates TNF-alpha production in alveolar macrophages by inhibiting NF-kappaB activity and Adam17 production. Shock 2010; 33: 197-204.
  • 21
    Ray M, Yu S, Sharda DR, Wilson CB, Liu Q, Kaushal N, et al. Inhibition of TLR4-induced IkappaB kinase activity by the RON receptor tyrosine kinase and its ligand, macrophage-stimulating protein. J Immunol 2010; 185: 7309-7316.
  • 22
    Luedde T, Heinrichsdorff J, de Lorenzi R, De Vos R, Roskams T, Pasparakis M. IKK1 and IKK2 cooperate to maintain bile duct integrity in the liver. Proc Natl Acad Sci U S A 2008; 105: 9733-9738.
  • 23
    Leist M, Gantner F, Kunstle G, Bohlinger I, Tiegs G, Bluethmann H, et al. The 55-kD tumor necrosis factor receptor and CD95 independently signal murine hepatocyte apoptosis and subsequent liver failure. Mol Med 1996; 2: 109-124.
  • 24
    Pierce JW, Schoenleber R, Jesmok G, Best J, Moore SA, Collins T, et al. Novel inhibitors of cytokine-induced IkappaBalpha phosphorylation and endothelial cell adhesion molecule expression show anti-inflammatory effects in vivo. J Biol Chem 1997; 272: 21096-21103.
  • 25
    Mallakin A, Kutcher LW, McDowell SA, Kong S, Schuster R, Lentsch AB, et al. Gene expression profiles of Mst1r-deficient mice during nickel-induced acute lung injury. Am J Respir Cell Mol Biol 2006; 34: 15-27.
  • 26
    Wullaert A, van Loo G, Heyninck K, Beyaert R. Hepatic tumor necrosis factor signaling and nuclear factor-kappaB: effects on liver homeostasis and beyond. Endocr Rev 2007; 28: 365-386.
  • 27
    Li ZW, Chu W, Hu Y, Delhase M, Deerinck T, Ellisman M, et al. The IKKbeta subunit of IkappaB kinase (IKK) is essential for nuclear factor kappaB activation and prevention of apoptosis. J Exp Med 1999; 189: 1839-1845.
  • 28
    Heyninck K, Wullaert A, Beyaert R. Nuclear factor-kappa B plays a central role in tumour necrosis factor-mediated liver disease. Biochem Pharmacol 2003; 66: 1409-1415.
  • 29
    Libert C, Van Bladel S, Brouckaert P, Shaw A, Fiers W. Involvement of the liver, but not of IL-6, in IL-1-induced desensitization to the lethal effects of tumor necrosis factor. J Immunol 1991; 146: 2625-2632.
  • 30
    Van Antwerp DJ, Martin SJ, Kafri T, Green DR, Verma IM. Suppression of TNF-alpha-induced apoptosis by NF-kappaB. Science 1996; 274: 787-789.
  • 31
    Baldwin AS. Control of oncogenesis and cancer therapy resistance by the transcription factor NF-kappaB. J Clin Invest 2001; 107: 241-246.

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
HEP_24239_sm_suppinfofig1.tif365KSupporting Information Figure 1
HEP_24239_sm_suppinfofig2.tif229KSupporting Information Figure 2
HEP_24239_sm_suppinfotab1.doc46KTable S1. Relative cytokine expression from TK+/+ and TK−/− Kupffer cells following 2 hours of LPS stimulation. Values were determined by densitometric analysis and have no units.

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