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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Neutrophils are considered crucial effector cells in the pathophysiology of organ ischemia/reperfusion injury (IRI). Although neutrophil elastase (NE) accounts for a substantial portion of the neutrophil activity, the function of NE in liver IRI remains unclear. This study focuses on the role of NE in the mechanism of liver IRI. Partial warm ischemia was produced in the left and middle hepatic lobes of C57BL/6 mice for 90 minutes, and this was followed by 6 to 24 hours of reperfusion. Mice were treated with neutrophil elastase inhibitor (NEI; 2 mg/kg per os) at 60 minutes prior to the ischemia insult. NEI treatment significantly reduced serum alanine aminotransferase levels in comparison with controls. Histological examination of liver sections revealed that unlike in controls, NEI treatment ameliorated hepatocellular damage and decreased local neutrophil infiltration, as assessed by myeloperoxidase assay, naphthol AS-D chloroacetate esterase stains, and immunohistochemistry (anti–Ly-6G). The expression of pro-inflammatory cytokines (tumor necrosis factor alpha and interleukin 6) and chemokines [chemokine (C-X-C motif) ligand 1 (CXCL-1), CXCL-2, and CXCL-10] was significantly reduced in the NEI treatment group, along with diminished apoptosis, according to terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining and caspase-3 activity. In addition, toll-like receptor 4 (TLR4) expression was diminished in NEI-pretreated livers, and this implies a putative role of NE in the TLR4 signal transduction pathway. Thus, targeting NE represents a useful approach for preventing liver IRI and hence expanding the organ donor pool and improving the overall success of liver transplantation. Liver Transpl 15:939–947, 2009. © 2009 AASLD.

Ischemia/reperfusion injury (IRI), an exogenous Ag-independent inflammatory event, remains an important problem in clinical transplantation. Indeed, severe damage at the time of organ retrieval, preservation, and reperfusion often leads to primary graft nonfunction and may adversely affect the development of both acute and chronic rejection.1 The mechanisms underlying liver IRI are complex but are known to involve leukocyte accumulation and activation (neutrophils, Kupffer cells, and T cells), leading to the formation of reactive oxygen species (ROS), secretion of pro-inflammatory cytokines/chemokines, complement activation, and vascular cell adhesion molecule activation.2, 3 Recent studies have suggested that the sentinel toll-like receptor (TLR) system may trigger organ IRI.4–6 We have documented the essential role of TLR4 activation and endogenous TLR4 ligands in ischemia/reperfusion (IR)-mediated local inflammation leading to hepatocellular damage.4, 7

Neutrophil activation has long been considered the major effector mechanism in liver IRI.8–10 The rolling of leukocytes is an important prerequisite for adhesion and migration into tissues, and a 2-step paradigm for leukocyte recruitment has been established.11 In addition to adhesion molecules, leukocyte proteases have also been implicated in the process of leukocyte migration through the vessel wall because of their ability to disrupt endothelial cell junctional complexes12 and degrade key components of the basement membrane. However, the interactions between leukocyte proteases and the liver inflammation cascade remain unknown.

Serine proteinases produced by polymorphonuclear neutrophils mediate tissue injury at the inflammatory sites. Neutrophil elastase (NE) is a 29-kDa glycoprotein chymotrypsin-like serine protease stored in azurophil granules in its active form until it is released following neutrophil exposure to the inflammatory stimuli. Once released, NE is potentially fully active because it functions optimally in a neutral environment.13, 14 As a result, excessive release of NE degrades elastin, collagens, laminins, and other extracellular matrix components of the endothelium, thereby leading to subsequent organ damage through endothelial cell injury.15, 16

The present study was designed to examine the role of NE in a mouse liver warm IRI model. We found that treatment with neutrophil elastase inhibitor (NEI) not only attenuated otherwise fulminant liver damage and inflammatory cell recruitment resulting from IRI but also down-regulated the innate TLR4 signaling. These results suggest that targeting NE proteases represents a useful approach to preventing liver IRI in transplant recipients and hence should expand the organ donor pool and improve the overall success of liver transplantation.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Animals

Male C57BL/6 mice (8-10 weeks old) were purchased from the Jackson Laboratory (Bar Harbor, ME). Mice were housed in the University of California Los Angeles animal facility under specific pathogen-free conditions. All animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (National Institutes of Health publication 86-23, revised 1985).

Liver IRI Model

We used an established mouse model of partial warm hepatic IRI.4, 17 Briefly, mice were anesthetized with isoflurane and injected with heparin (100 U/kg), and an atraumatic clip was used to interrupt the artery/portal venous blood supply to the left and middle liver lobes. After 90 minutes of partial warm ischemia, the clamp was removed, and this initiated hepatic reperfusion. To investigate the role of NE, we used NEI (GW311616A; Sigma). Previous studies have shown dose-dependent effects of orally given GW311616A in dogs. At 0.22 mg/kg, >50% inhibition of elastase activity was achieved at 6 hours, with activity returning toward normal relatively quickly. A single dose of 2 mg/kg rapidly abolished circulating enzyme activity, with >90% inhibition maintained for 4 days.18 Mice were orally treated with NEI (2 mg/kg per os) at 60 minutes prior to the ischemia insult and then sacrificed at 6 or 24 hours after reperfusion for serum and liver sampling. Serum alanine aminotransferase (SALT) levels, an indicator of hepatocellular injury, were measured in peripheral blood with an autoanalyzer (Antech Diagnostics). Liver specimens were fixed in a 10% buffered formalin solution and embedded in paraffin. Liver paraffin sections (5 μm thick) were stained with hematoxylin and eosin and then analyzed blindly. Sham controls underwent the same procedure, but without vascular occlusion.

Serum NE Activity

Serum NE activity was determined with N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide, a highly specific synthetic substrate for NE according to the method of Yoshimura et al.19, 20 Briefly, a serum sample was incubated with a 0.1 M Tris-HCl buffer (pH 8.0) containing 0.5 M NaCl and 1 mM substrate at 37°C for 24 hours, and the amount of liberated p-nitroanilide was measured spectrophotometrically at 405 nm and was considered as NE activity.

Liver Neutrophil Infiltration

Myeloperoxidase (MPO) Assay

The activity of MPO, an enzyme specific for polymorphonuclear neutrophils, was used as an index of hepatic neutrophil accumulation.21 Briefly, the frozen tissue was thawed and placed in iced 0.5% hexadecyltrimethyl ammonium (Sigma-Aldrich) and a 50 mM potassium phosphate buffer solution (Sigma-Aldrich; pH = 5.0). Each sample was homogenized and centrifuged at 15,000 rpm for 15 minutes at 4°C. Supernatants were then mixed with hydrogen peroxide/sodium acetate and tetramethyl benzidine solutions (Sigma-Aldrich). The change in absorbance was measured by spectrophotometry at 655 nm in 1 minute. One unit of MPO activity was defined as the quantity of enzyme degrading 1 μM peroxide/minute/g of tissue at 25°C.

Naphthol AS-D Chloroacetate Esterase Stain

The accumulation of activated neutrophils in 5-μm cryostat liver sections was assessed by staining for chloroacetate esterase, a neutrophil-specific marker (naphthol AS-D chloroacetate esterase kit; Sigma-Aldrich). Polymorphonuclear cells, identified by staining and morphology, were counted in 10 high-power fields (HPFs) per section under light microscopy (×400), and the numbers of cells per HPF [mean ± standard error of the mean (SEM)] are shown.

Immunohistochemistry

Livers were snap-frozen, and cryostat sections (5 μm) were fixed in acetone. Endogenous peroxidase activity was inhibited with 0.3% hydrogen peroxidase. Sections were then blocked with 10% normal goat serum. Primary rat monoclonal antibody against mouse polymorphonuclear leukocytes (Ly-6G, 1A8; BD Pharmingen) was diluted (1/250 in 3% normal goat serum), and 100 μL was added to each section. The primary antibody was incubated for 60 minutes at room temperature. The secondary antibody, a biotinylated goat anti-rat immunoglobulin G (Vector; diluted 1:200), was incubated for 40 minutes at room temperature. Sections were incubated with immunoperoxidase (ABC kit; Vector), washed, and developed with a 3,3′-diaminobenzidine kit (Vector). Slides were counterstained with hematoxylin and rinsed with ammonia and tap water. A negative control was prepared by omission of the primary antibody. Polymorphonuclear cells, identified by staining and morphology, were counted in 10 HPFs per section under light microscopy (×400), and the numbers of cells per HPF (mean ± SEM) are shown.

Apoptosis Assay

Apoptosis in 5-μm cryostat liver sections was detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) method with an in situ cell death detection kit (Roche) according to the manufacturer's protocol. A negative control was prepared by omission of the terminal transferase. Positive controls were generated by treatment with DNase I (1 μg/mL for 10 minutes). The peroxidase activity was visualized with a diaminobenzidine substrate, which yielded a brown oxidation product; methyl green was used for counterstaining. TUNEL-positive cells were counted in 10 HPFs per section under light microscopy (×400), and the numbers of cells per HPF (mean ± SEM) are shown.

Caspase-3 Activity

Caspase-3 activity was determined in liver samples with the ApoAlert caspase-3 colorimetric assay kit (Clontech) according to the manufacturer's instructions. Briefly, the tissue was homogenized on ice and centrifuged at 15,000g for 10 minutes (4°C). One hundred micrograms of total protein from the supernatant was used to measure caspase-3 activity.22 Optical density measurements at 405 nm were performed with a microplate reader (BioTeK Instruments). Caspase activity was expressed in units, with 1 U being the amount of enzyme activity liberating 1 pmol of p-nitroanilide per minute.23

RNA Extraction and Reverse-Transcriptase Polymerase Chain Reaction (PCR)

Total RNA was extracted from the liver with the TRIzol reagent (Life Technologies). Reverse transcription was performed with 5 μg of total RNA in a first-strand complementary DNA (cDNA) synthesis kit (Fermentas). The cDNA product was amplified by PCR with primers specific for mouse cytokines and β-actin. Primers used in PCR were as follows: tumor necrosis factor alpha (TNF-α) sense (5′-AGC CCA CGT AGC AAA CCA CCA A-3′) and TNF-α antisense (5′-ACA CCC ATT CCC TTC ACA GAG CAA T-3′), interleukin 6 (IL-6) sense (5′-CAT CCA GTT GCC TTC TTG GGA-3′) and IL-6 antisense (5′-CAT TGG GAA ATT GGG GTA GGA AG-3′), chemokine (C-X-C motif) ligand 1 (CXCL-1) sense (5′-TGA GCT GCG CTG TCA GTG CCT-3′) and CXCL-1 antisense (5′-AGA AGC CAG CGT TCA CCA GA-3′), CXCL-2 sense (5′-GCT GGC CAC CAA CCA CCA GG-3′) and CXCL-2 antisense (5′-AGC GAG GCA CAT CAG GTA CG-3′), CXCL-10 sense (5′-ACC ATG AAC CCA AGT GCT GCC GTC-3′) and CXCL-10 antisense (5′-GCT TCA CTC CAG TTA AGG AGC CCT-3′), TLR4 sense (5′-CAG CTT CAA TGG TGC CAT CA-3′) and TLR4 antisense (5′-CTG CAA TCA AGA GTG CTG AG-3′), and β-actin sense (5′-GTG GGG CGC CCC AGG CAC CA-3′) and β-actin antisense (5′-CTC CTT AAT GTC ACG CAC GAT TTC-3′). PCR conditions for each primer couple were as follows: TNF-α, 94°C for 45 s, 55°C for 45 s, and 72°C for 60 s during 33 cycles; IL-6, 94°C for 45 s, 55°C for 45 s, and 72°C for 60 s during 33 cycles; CXCL-1 and CXCL-2, 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s during 35 cycles; CXCL-10, 95°C for 60 s, 63°C for 50 s, and 72°C for 50 s during 30 cycles; TLR4, 94°C for 60 s, 56°C for 60 s, and 72°C for 60 s during 34 cycles; and β-actin, 95°C for 30 s, 57°C for 30 s, and 72°C for 60 s during 30 cycles. PCR products were analyzed in ethidium bromide–stained 1% agarose gel and scanned with Kodak Digital Science 1D Analysis software (version 2.0). To compare relative levels of each gene, all samples were normalized against the β-actin template cDNA ratio.

Statistical Analysis

All data are expressed as means ± SEM. Differences between experimental groups were analyzed with an unpaired 2-tailed Student t test. All differences were considered statistically significant at a P value < 0.05.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

NE Activity Increases in Liver IRI

First, we measured serum NE activity in mice that underwent 90 minutes of warm ischemia followed by 24 hours of reperfusion. As shown in Fig. 1A, NE activity increased rapidly after reperfusion in the IR-induced group in comparison with the sham-operated group without vascular occlusion. It peaked at 3 hours (P < 0.01) and then declined by 24 hours to almost the baseline.

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Figure 1. (A) NE activity in mouse serum at 0 to 24 hours of liver reperfusion following 90 minutes of local warm ischemia. NE activity increased rapidly after reperfusion in the ischemia/reperfusion group (n = 6, solid line) in comparison with the sham-operated group (n = 3, dotted line). It peaked at 3 hours and then declined by 24 hours of reperfusion. (B) NE activity after treatment with NE inhibitor (GW311616A; 2 mg/kg per os) 60 minutes prior to the ischemia insult. Treatment inhibited NE activity at both 6 and 24 hours of reperfusion (*P < 0.01; n = 4-6/group). Means and standard errors are shown. Abbreviation: NE, neutrophil elastase.

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Inhibition of NE Activity Ameliorates Liver IRI

We analyzed the NE activity and hepatocellular function in our model. As shown in Fig. 1B, NEI treatment significantly inhibited NE activity at both 6 and 24 hours after reperfusion (P < 0.01). In parallel, the inhibition of NE prevented IR-induced hepatocellular damage. The SALT levels were significantly suppressed at both 6 and 24 hours after reperfusion in the treated group in comparison with controls (6 hours, 33,650 ± 1896 versus 13,510 ± 3404, and 24 hours, 14,483 ± 1985 versus 4810 ± 531, P < 0.01; Fig. 2A). These data correlated well with the histological criteria of the hepatocellular damage. Hence, untreated livers revealed severe lobular edema, congestion, ballooning, and hepatocellular necrosis. In contrast, treated livers showed good preservation of architecture and histological detail (Fig. 2B).

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Figure 2. Neutrophil elastase inhibitor treatment ameliorates liver ischemia/reperfusion injury. (A) Hepatocellular function. sALT levels after liver warm ischemia (90 minutes) followed by reperfusion (6 and 24 hours) were lower in the neutrophil elastase inhibitor treatment group in comparison with untreated controls (*P < 0.01; n = 4-6/group). Means and standard errors are shown. (B) Representative liver histology (hematoxylin-eosin staining; magnification, ×100) of ischemic (90 minutes) liver lobes harvested after 6 hours or reperfusion (n = 4/group). Abbreviation: sALT, serum alanine aminotransferase.

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NEI Treatment Suppresses Neutrophil Infiltration

We performed the local MPO assay in livers at 6 hours of reperfusion after 90 minutes of warm ischemia as an index of neutrophil infiltration (Fig. 3A). The MPO activity (U/g) was significantly suppressed in the treated group in comparison with controls (14.10 ± 3.12 versus 4.03 ± 0.49, P < 0.05). The MPO activity is correlated with the number of activated neutrophils assessed by the naphthol AS-D chloroacetate esterase stains.24 Indeed, activated neutrophil accumulation in treated livers at 6 hours was significantly decreased in comparison with controls (Fig. 3B; 15.66 ± 2.96 versus 5.66 ± 2.33, P < 0.05). These results were correlated with the number of Ly-6G–positive cells, a marker for neutrophil infiltration (Fig. 3C; 34.56 ± 3.09 versus 15.47 ± 5.07, P < 0.05).

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Figure 3. Neutrophil accumulation in ischemic liver lobes harvested at 6 hours of reperfusion after 90 minutes of warm ischemia with or without neutrophil elastase inhibitor treatment. (A) MPO activity (**P < 0.05; n = 6/group). (B) Upper panel: Representative liver sections stained by immunohistology with a naphthol AS-D chloroacetate esterase kit (magnification, ×400). Lower panel: Quantitation of hepatic activated neutrophil accumulation by immunohistology. The accumulation of polymorphonuclear cells (red spots shown by arrows) in treated livers was significantly decreased in comparison with untreated controls (**P < 0.05; n = 3/group). (C) Upper panel: Representative liver sections stained by Ly-6G (magnification, ×400). Lower panel: Quantitation of hepatic neutrophil infiltration by immunohistology. The infiltration of polymorphonuclear cells (dark brown spots) in treated livers was significantly decreased in comparison with untreated controls (**P < 0.05; n = 3/group). Means and standard errors are shown. Abbreviations: HPF, high-power field; MPO, myeloperoxidase.

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Inhibition of NE Activity Suppresses Pro-Inflammatory Cytokines and Chemokines

We used reverse-transcriptase PCR to analyze the expression of pro-inflammatory cytokines (TNF-α and IL-6) and chemokines {CXCL-1 [a mouse homolog of human chemokine gro-α (KC)] and CXCL-2 [macrophage inflammatory protein 2 (MIP-2)]} in the liver during IRI and calculated the ratio of post-IR and basal messenger RNA (mRNA) levels in each animal (n = 3/group). The untreated group showed significantly increased induction ratios of TNF-α (TNF-α/β-actin mRNA: 6 hours, 9.30 ± 0.10 versus 5.15 ± 0.61, P < 0.05, and 24 hours, 6.70 ± 0.47 versus 3.52 ± 2.39) and IL-6 (IL-6/β-actin mRNA: 6 hours, 1.00 ± 0.13 versus 0.14 ± 0.05, P < 0.01, and 24 hours, 0.78 ± 0.10 versus 0.14 ± 0.03) in comparison with the treated group (Fig. 4A). The CXC chemokines are considered to act predominantly on neutrophils.25 In mice, the 2 major CXC chemokines are cytokine-induced neutrophil chemoattractants [CXCL-1 (KC) and CXCL-2 (MIP-2)]. Although no major variations were detected in CXCL-1 expression at 6 hours (CXCL-1/β-actin mRNA: 4.10 ± 0.42 versus 3.98 ± 0.08), the expression at 24 hours (CXCL-1/β-actin mRNA: 3.84 ± 0.10 versus 1.55 ±0.17) was significantly reduced (P < 0.01) between the untreated and treated groups (Fig. 4B). In contrast, CXCL-2 expression was significantly reduced in the treated livers at both 6 hours (CXCL-2/β-actin mRNA: 39.64 ± 2.82 versus 18.62 ± 4.84, P < 0.05) and 24 hours (CXCL-2/β-actin mRNA: 34.74 ± 2.94 versus 16.41 ± 2.74, P < 0.01) after reperfusion.

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Figure 4. Reverse-transcriptase polymerase chain reaction–assisted detection of pro-inflammatory mediators in ischemia/reperfusion injury livers. (A) Cytokine gene expression. (B) Chemokine gene expression. (C) TLR4 and CXCL-10 gene expression in untreated and treated livers. Data were normalized to β-actin gene expression (*P < 0.01, **P < 0.05; n = 3/group). Means and standard errors are shown. Abbreviations: CXCL, chemokine (C-X-C motif) ligand; IL-6, interleukin 6; KC, a mouse homolog of human chemokine gro-α; MIP-2, macrophage inflammatory protein 2; TLR4, toll-like receptor 4; TNF-α, tumor necrosis factor alpha.

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Inhibition of NE Activity Suppresses CXCL-10 and TLR4 Expression

We measured CXCL-10 and TLR4 transcript levels in the ischemic liver lobes at 6 and 24 hours after reperfusion following 90 minutes of warm ischemia. The expression of mRNA coding for TLR4 and CXCL-10 increased sharply in the untreated group. Neutralization of NE was accompanied by the early inhibition of TLR4 expression at 6 hours (TLR4/β-actin mRNA: 6.70 ± 0.25 versus 4.45 ± 0.24, P < 0.01). This was followed by diminished CXCL-10 expression levels at 24 hours (CXCL-10/β-actin mRNA: 8.40 ± 0.71 versus 2.43 ± 0.72, P < 0.01; Fig. 4C).

NEI Treatment Suppresses Apoptosis

We evaluated hepatocyte apoptosis by TUNEL staining of livers subjected to 90 minutes of warm ischemia and 6 hours of reperfusion (Fig. 5A). Augmented hepatocyte apoptosis (TUNEL-positive cells/field), readily detectable in the untreated group, remained diminished following NEI treatment (24.23 ± 5.31 versus 9.00 ± 2.18, P < 0.05). Additionally, as shown in Fig. 5B, the enzymatic activity of caspase-3, a proapoptotic marker, was significantly reduced at 6 hours after reperfusion in the treated group in comparison with untreated controls (12.11 ± 1.53 versus 25.86 ± 3.21 U/g, P < 0.01).

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Figure 5. (A) Representative TUNEL-assisted detection of intrahepatic apoptosis in ischemic liver lobes harvested at 6 hours after 90 minutes of reperfusion. Upper panel: Liver sections were stained by the TUNEL method (magnification, ×400). Lower panel: Quantitation of intrahepatic apoptosis by the TUNEL method. The untreated group had a higher frequency of TUNEL-positive cells (dark brown spots) than the neutrophil elastase inhibitor–treated group (**P < 0.05; n = 3/group). (B) Caspase-3 activity was significantly depressed in the neutrophil elastase inhibitor–treated group in comparison with untreated controls (*P < 0.01; n = 5/group). Means and standard errors are shown. Abbreviations: HPF, high-power field; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Our results provide evidence for the role of NE in the pathogenesis of liver IRI. First, we showed that NE activity increased during the course of liver IRI. Second, inhibition of NE activity ameliorated IR-induced hepatocellular damage. We then showed that these beneficial effects after NEI treatment were accompanied by diminished local neutrophil infiltration and reduced expression of pro-inflammatory cytokines TNF-α and IL-6, chemokines CXCL-1, CXCL-2, and CXCL-10, and TLR4. Hence, we conclude that targeting NE represents a useful approach to preventing IRI that might improve the overall success of liver transplantation.

The protective effects of the NEI, sivelestat were first documented in acute lung injury associated with systemic inflammation response, which may occur after infection, surgical intervention, or traumatic or burn injury.26 Although some have shown the benefits of targeting NE, including sivelestat in the liver injury models, the details remain unclear.27–29 Therefore, here we used a commercially available NEI (GW311616A) with stronger inhibitory function than sivelestat18, 30 in an attempt to assess the role of NE in the mechanism of liver IRI. Indeed, treatment with GW311616A markedly inhibited SALT levels and prevented tissue damage at both 6 and 24 hours after reperfusion, and this confirmed the efficacy of that selective therapeutic approach to ameliorating warm ischemia–induced liver damage. This observation suggests several important interactions between NE and pro-inflammatory mediators at the local IRI site. As MPO activity was significantly decreased after GW311616A treatment, neutralization of NE may be sufficient to lessen the overall protease burden without the need for inhibition of all proteases.

Neutrophils can be triggered to express a number of mediators that can influence local inflammatory and immune responses. These include ROS, complement components, and proteases as well as a variety of cytokines and chemokines.31 The C-X-C chemokines CXCL-1 (KC) and CXCL-2 (MIP-2) are potent neutrophil chemoattractants that are important in liver IRI.32, 33 Murine CXCL-2 (MIP-2) is a chemokine considered to be functionally analogous to human IL-8 and rat neutrophil chemoattractant34 and is primarily induced by TNF-α.35 IL-6 is considered a marker for liver injury severity.36 Here, we have shown that the administration of NEI suppressed expression of TNF-α, IL-6, and chemokines [CXCL-1 (KC) and CXCL-2 (MIP-2)] in the liver.

Furthermore, TNF-α has a role as an initiator of the apoptotic cascade, and apoptosis has been shown to be an important event after reperfusion, the severity of which correlates with the degree of hepatic injury.37 In this study, a significantly increased frequency of TUNEL-positive apoptotic cells during IRI noted in control untreated livers, accompanied by markedly elevated caspase-3 activity, became markedly diminished following neutralization of NE activity.

Although expressed by both hepatocytes and nonparenchymal cells, TLR4 signaling on Kupffer cells is critical in the pathogenesis of liver IRI.38 The activation of TLR4 on the surface of macrophages in the liver and spleen represents the critical initiating event preceding the inflammatory reaction induced by endotoxin or exotoxin.39, 40 The TLR4 signaling pathway begins with endotoxin in the blood binding to lipopolysaccharide (LPS)-binding protein, which elicits LPS to anchor to the CD14 molecule and combine with the extracellular TLR4 portion. This compound mediates LPS signaling, transducing extracellular inflammatory signals into cells and with TNF-α mRNA initiating the inflammation response.41 During liver IR, portal vein occlusion results in congestion of the intestinal wall, leading to the release of gut-derived molecules, including endotoxin, into the blood stream. Thus, LPS is an obvious candidate for TLR4 activation, and this is consistent with its role as an initiating factor in liver IRI.42–44 However, liver IRI may also develop in animal models under aseptic conditions.42 Moreover, we have shown that cold-preserved rat livers develop hepatocellular damage after reperfusion ex vivo.45 Thus, although gut-derived LPS contributes to IRI, it may not actually trigger the intrahepatic inflammation cascade that culminates in tissue damage. Indeed, endogenous TLR4 ligands generated during liver IRI may trigger local inflammation leading to the hepatocellular damage.4 It has been reported that IL-8 up-regulation by NE occurs in part through the cell surface membrane–bound TLR4 in bronchial epithelium.46 Although NE may associate with increased expression of monocyte/macrophage TLR4 in sepsis,47 the direct effect of NE on TLR4 could not be discarded in Leishmania infection.48 As the question of how IR insult activates the TLR4 system remains,4 our results imply that a direct effect of NE on TLR4 molecules cannot be discarded. Hence, the inhibition of NE at 3 hours after reperfusion did suppress TLR4 expression at 6 hours, and this was followed at 24 hours by diminished CXCL-10, the downstream TLR4 effector mediator.

Figure 6 depicts our working hypothesis on cross-talk between NE and the cascade of inflammatory mediators triggered in liver IR. Activated Kupffer cells and hepatocytes both produce pro-inflammatory cytokines, including TNF-α. The latter affects surrounding hepatocytes, causing their apoptosis, and triggers neutrophil-attracting CXC chemokines to express adhesion molecules on vascular endothelial cells. Neutrophil adhesion to endothelial cells leads to transmigration from the vascular lumen into the liver parenchyma. The infiltrating neutrophil-derived NE induces inflammatory chemokine (CXCL-1 and CXCL-2) expression by neutrophils. NEI-mediated depression of CXCL-2 and other chemokines prevents recruitment of neutrophils and macrophages, and this in turn suppresses the local inflammation response. It is plausible that NE may not only accelerate IR-mediated damage due to the feedback with recruited neutrophils, resulting in direct injury to membrane components, but also have some effect on the TLR4 system by serving as a putative endogenous TLR4 ligand and causing TLR4 up-regulation on Kupffer cells and hepatocytes.

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Figure 6. Cross-talk interactions between NE and local inflammation responses in liver IRI. Activated Kupffer cells and hepatocytes produce TNF-α. The latter affects surrounding hepatocytes, causing their apoptosis, and triggers neutrophil-attracting CXC chemokines to express adhesion molecules on vascular endothelial cells. Neutrophil adhesion to endothelial cells leads to transmigration from the vascular lumen into the liver parenchyma and releases NE. NE accelerates ischemia/reperfusion-mediated damage because of the feedback with recruited neutrophils, resulting in direct injury to membrane components. Moreover, NE may also serve as a putative endogenous TLR4 ligand, causing TLR4 up-regulation on Kupffer cells and hepatocytes. Abbreviations: CXCL, chemokine (C-X-C motif) ligand; IRI, ischemia/reperfusion injury; NE, neutrophil elastase; ROS, reactive oxygen species; TLR4, toll-like receptor 4; TNF-α, tumor necrosis factor alpha.

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In conclusion, our data indicate an essential role of NE in the pathophysiology of liver IRI. NE contributes to the accumulation of neutrophils at the inflamed site and the secretion of pro-inflammatory mediators. Thus, NEI treatment should be considered a potential therapy for liver IRI. As inhibition of NE down-regulated innate TLR4 and its downstream CXCL-10 expression, this study suggests previously unrecognized NE-TLR4 cross-talk and implies a role of NE in the signal transduction pathway instrumental for liver IRI.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
  • 1
    Farmer DG, Amersi F, Kupiec-Weglinski JW, Busuttil RW. Current status of ischemia and reperfusion injury in the liver. Transplant Rev 2000; 14: 106.
  • 2
    Fondevila C, Busuttil RW, Kupiec-Weglinski JW. Hepatic ischemia/reperfusion injury—a fresh look. Exp Mol Pathol 2003; 74: 8693.
  • 3
    Teoh NC, Farrell GC. Hepatic ischemia reperfusion injury: pathogenic mechanisms and basis for hepatoprotection. J Gastroenterol Hepatol 2003; 18: 891902.
  • 4
    Zhai Y, Qiao B, Shen XD, Gao F, Busuttil RW, Cheng G, et al. Evidence for the pivotal role of endogenous toll-like receptor 4 ligands in liver ischemia and reperfusion injury. Transplantation 2008; 85: 10161022.
  • 5
    Zhai Y, Qiao B, Gao F, Shen X, Vardanian A, Busuttil RW, Kupiec-Weglinski JW. Type I, but not type II, interferon is critical in liver injury induced after ischemia and reperfusion. Hepatology 2008; 47: 199206.
  • 6
    Wu HS, Zhang JX, Wang L, Tian Y, Wang H, Rotstein O. Toll-like receptor 4 involvement in hepatic ischemia/reperfusion injury in mice. Hepatobiliary Pancreat Dis Int 2004; 3: 250253.
  • 7
    Zhai Y, Shen XD, O'Connell R, Gao F, Lassman C, Busuttil RW, et al. Cutting edge: TLR4 activation mediates liver ischemia/reperfusion inflammatory response via IFN regulatory factor 3-dependent MyD88-independent pathway. J Immunol 2004; 173: 71157119.
  • 8
    Jaeschke H. Chemokines and liver inflammation: the battle between pro- and anti-inflammatory mediators. Hepatology 1997; 25: 252253.
  • 9
    Jaeschke H, Smith CW. Mechanisms of neutrophil-induced parenchymal cell injury. J Leukoc Biol 1997; 61: 647653.
  • 10
    Jaeschke H. Mechanisms of liver injury. II. Mechanisms of neutrophil-induced liver cell injury during hepatic ischemia-reperfusion and other acute inflammatory conditions. Am J Physiol Gastrointest Liver Physiol 2006; 290: G1083G1088.
  • 11
    Jaeschke H, Hasegawa T. Role of neutrophils in acute inflammatory liver injury. Liver Int 2006; 26: 912919.
  • 12
    Moll T, Dejana E, Vestweber D. In vitro degradation of endothelial catenins by a neutrophil protease. J Cell Biol 1998; 140: 403407.
  • 13
    Janoff A, Scherer J. Mediators of inflammation in leukocyte lysosomes. IX. Elastinolytic activity in granules of human polymorphonuclear leukocytes. J Exp Med 1968; 128: 11371155.
  • 14
    Janoff A. Elastase in tissue injury. Annu Rev Med 1985; 36: 207216.
  • 15
    Mainardi CL, Dixit SN, Kang AH. Degradation of type IV (basement membrane) collagen by a proteinase isolated from human polymorphonuclear leukocyte granules. J Biol Chem 1980; 255: 54355441.
  • 16
    Mainardi CL, Hasty DL, Seyer JM, Kang AH. Specific cleavage of human type III collagen by human polymorphonuclear leukocyte elastase. J Biol Chem 1980; 255: 1200612010.
  • 17
    Tsuchihashi S, Zhai Y, Bo Q, Busuttil RW, Kupiec-Weglinski JW. Heme oxygenase-1 mediated cytoprotection against liver ischemia and reperfusion injury: inhibition of type-1 interferon signaling. Transplantation 2007; 83: 16281634.
  • 18
    Macdonald SJ, Dowle MD, Harrison LA, Shah P, Johnson MR, Inglis GG, et al. The discovery of a potent, intracellular, orally bioavailable, long duration inhibitor of human neutrophil elastase—GW311616A a development candidate. Bioorg Med Chem Lett 2001; 11: 895898.
  • 19
    Yoshimura K, Nakagawa S, Koyama S, Kobayashi T, Homma T. Roles of neutrophil elastase and superoxide anion in leukotriene B4-induced lung injury in rabbit. J Appl Physiol 1994; 76: 9196.
  • 20
    Hagio T, Nakao S, Matsuoka H, Matsumoto S, Kawabata K, Ohno H. Inhibition of neutrophil elastase activity attenuates complement-mediated lung injury in the hamster. Eur J Pharmacol 2001; 426: 131138.
  • 21
    Tsuchihashi S, Livhits M, Zhai Y, Busuttil RW, Araujo JA, Kupiec-Weglinski JW. Basal rather than induced heme oxygenase-1 levels are crucial in the antioxidant cytoprotection. J Immunol 2006; 177: 47494757.
  • 22
    Contreras JL, Vilatoba M, Eckstein C, Bilbao G, Anthony Thompson J, Eckhoff DE. Caspase-8 and caspase-3 small interfering RNA decreases ischemia/reperfusion injury to the liver in mice. Surgery 2004; 136: 390400.
  • 23
    Hamada T, Tsuchihashi S, Avanesyan A, Duarte S, Moore C, Busuttil RW, Coito AJ. Cyclooxygenase-2 deficiency enhances Th2 immune responses and impairs neutrophil recruitment in hepatic ischemia/reperfusion injury. J Immunol 2008; 180: 18431853.
  • 24
    Harada N, Okajima K, Murakami K, Usune S, Sato C, Ohshima K, Katsuragi T. Adenosine and selective A(2A) receptor agonists reduce ischemia/reperfusion injury of rat liver mainly by inhibiting leukocyte activation. J Pharmacol Exp Ther 2000; 294: 10341042.
  • 25
    Baggiolini M. Chemokines and leukocyte traffic. Nature 1998; 392: 565568.
  • 26
    Tamakuma S, Ogawa M, Aikawa N, Kubota T, Hirasawa H, Ishizaka A, et al. Relationship between neutrophil elastase and acute lung injury in humans. Pulm Pharmacol Ther 2004; 17: 271279.
  • 27
    Soejima Y, Yanaga K, Nishizaki T, Yoshizumi T, Uchiyama H, Sugimachi K. Effect of specific neutrophil elastase inhibitor on ischemia/reperfusion injury in rat liver transplantation. J Surg Res 1999; 86: 150154.
  • 28
    Tomizawa N, Ohwada S, Ohya T, Kawashima Y, Takeyoshi I, Morishita Y. The effect of neutrophil elastase inhibitor in hepatectomy with ischemia in dogs. J Surg Res 1999; 81: 230237.
  • 29
    Uchida Y, Kaibori M, Hijikawa T, Ishizaki M, Ozaki T, Tanaka H, et al. Protective effect of neutrophil elastase inhibitor (FR136706) in lethal acute liver failure induced by D-galactosamine and lipopolysaccharide in rats. J Surg Res 2008; 145: 5765.
  • 30
    Kawabata K, Hagio T, Matsuoka S. The role of neutrophil elastase in acute lung injury. Eur J Pharmacol 2002; 451: 110.
  • 31
    Scapini P, Lapinet-Vera JA, Gasperini S, Calzetti F, Bazzoni F, Cassatella MA. The neutrophil as a cellular source of chemokines. Immunol Rev 2000; 177: 195203.
  • 32
    Mosher B, Dean R, Harkema J, Remick D, Palma J, Crockett E. Inhibition of Kupffer cells reduced CXC chemokine production and liver injury. J Surg Res 2001; 99: 201210.
  • 33
    Lentsch AB, Yoshidome H, Cheadle WG, Miller FN, Edwards MJ. Chemokine involvement in hepatic ischemia/reperfusion injury in mice: roles for macrophage inflammatory protein-2 and KC. Hepatology 1998; 27: 11721177.
  • 34
    Oppenheim JJ, Zachariae CO, Mukaida N, Matsushima K. Properties of the novel proinflammatory supergene “intercrine” cytokine family. Annu Rev Immunol 1991; 9: 617648.
  • 35
    Tessier PA, Naccache PH, Clark-Lewis I, Gladue RP, Neote KS, McColl SR. Chemokine networks in vivo: involvement of C-X-C and C-C chemokines in neutrophil extravasation in vivo in response to TNF-alpha. J Immunol 1997; 159: 35953602.
  • 36
    Jin X, Zimmers TA, Perez EA, Pierce RH, Zhang Z, Koniaris LG. Paradoxical effects of short- and long-term interleukin-6 exposure on liver injury and repair. Hepatology 2006; 43: 474484.
  • 37
    Rüdiger HA, Graf R, Clavien PA. Liver ischemia: apoptosis as a central mechanism of injury. J Invest Surg 2003; 16: 149159.
  • 38
    Tsung A, Hoffman RA, Izuishi K, Critchlow ND, Nakao A, Chan MH, et al. Hepatic ischemia/reperfusion injury involves functional TLR4 signaling in nonparenchymal cells. J Immunol 2005; 175: 76617668.
  • 39
    Harju K, Glumoff V, Hallman M. Ontogeny of toll-like receptors TLR2 and TLR4 in mice. Pediatr Res 2001; 49: 8183.
  • 40
    Takeuchi O, Hoshino K, Kawai T, Sanjo H, Takada H, Ogawa T, et al. Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity 1999; 11: 443451.
  • 41
    Faure E, Equils O, Sieling PA, Thomas L, Zhang FX, Kirschning CJ, et al. Bacterial lipopolysaccharide activates NF-kappaB through toll-like receptor 4 (TLR4) in cultured human dermal endothelial cells. J Biol Chem 2000; 275: 1105811063.
  • 42
    Arai M, Mochida S, Ohno A, Arai S, Fujiwara K. Selective bowel decontamination of recipients for prevention against liver injury following orthotopic liver transplantation: evaluation with rat models. Hepatology 1998; 27: 123127.
  • 43
    Fiorini RN, Shafizadeh SF, Polito C, Rodwell DW, Cheng G, Evans Z, et al. Anti-endotoxin monoclonal antibodies are protective against hepatic ischemia/reperfusion injury in steatotic mice. Am J Transplant 2004; 4: 15671573.
  • 44
    Tsoulfas G, Takahashi Y, Ganster RW, Yagnik G, Guo Z, Fung JJ, et al. Activation of the lipopolysaccharide signaling pathway in hepatic transplantation preservation injury. Transplantation 2002; 74: 713.
  • 45
    Amersi F, Buelow R, Kato H, Ke B, Coito AJ, Shen XD, et al. Upregulation of heme oxygenase-1 protects genetically fat Zucker rat livers from ischemia/reperfusion injury. J Clin Invest 1999; 104: 16311639.
  • 46
    Devaney JM, Greene CM, Taggart CC, Carroll TP, O'Neill SJ, McElvaney NG. Neutrophil elastase up-regulates interleukin-8 via toll-like receptor 4. FEBS Lett 2003; 544: 129132.
  • 47
    Tsujimoto H, Ono S, Majima T, Kawarabayashi N, Takayama E, Kinoshita M, et al. Neutrophil elastase, MIP-2, and TLR-4 expression during human and experimental sepsis. Shock 2005; 23: 3944.
  • 48
    Ribeiro-Gomes FL, Moniz-de-Souza MC, Alexandre-Moreira MS, Dias WB, Lopes MF, Nunes MP, et al. Neutrophils activate macrophages for intracellular killing of Leishmania major through recruitment of TLR4 by neutrophil elastase. J Immunol 2007; 179: 39883994.