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Am J Physiol Gastrointest Liver Physiol 293: G631-G639, 2007. First published July 19, 2007; doi:10.1152/ajpgi.00185.2007
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LIVER AND BILIARY TRACT

Selective inactivation of NF-{kappa}B in the liver using NF-{kappa}B decoy suppresses CCl4-induced liver injury and fibrosis

Gakuhei Son,1 Yuji Iimuro,1 Ekihiro Seki,1 Tadamichi Hirano,1 Yasufumi Kaneda,2 and Jiro Fujimoto1

1First Department of Surgery, Hyogo College of Medicine, Nishinomiya, Japan; and 2Division of Gene Therapy Science, Graduate School of Medicine, Osaka University, Suita, Japan

Submitted 27 April 2007 ; accepted in final form 13 July 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Sustained hepatic inflammation induced by various causes can lead to liver fibrosis. Transcription factor NF-{kappa}B is important in regulating inflammatory responses, especially in macrophages. We presently investigated whether an NF-{kappa}B decoy, a synthetic oligodeoxynucleotide (ODN) imitating the NF-{kappa}B binding site, inhibited the inflammatory response after CCl4 intoxication to prevent CCl4-induced hepatic injury and fibrosis. The NF-{kappa}B decoy was introduced into livers by injecting the spleens of mice, using a hemagglutinating virus of Japan (HVJ)-liposome method. ODN was transferred mainly to macrophages in normal or fibrotic livers. Increases in serum transaminases and production of inflammatory cytokines after a single challenge with CCl4 were inhibited by the NF-{kappa}B decoy, which suppressed nuclear translocation of NF-{kappa}B in liver macrophages. Liver fibrosis induced by CCl4 administration for 8 wk was suppressed by the NF-{kappa}B decoy, accompanied by diminished mRNA expression for transforming growth factor (TGF)-beta, procollagen type 1 {alpha}1, and {alpha}-smooth muscle actin (SMA). In vitro, isolated liver macrophages showed increased DNA binding activity of NF-{kappa}B and inflammatory cytokine production after hydrogen peroxide treatment; both increases were inhibited significantly by the NF-{kappa}B decoy. In contrast, NF-{kappa}B decoy transferred to isolated hepatic stellate cells (HSC) had no effect on their morphological activation or {alpha}-SMA expression, although the decoy accelerated tumor necrosis factor (TNF)-{alpha}-induced apoptosis in activated HSC. The effect of NF-{kappa}B decoy suppressing fibrosis probably results mainly from anti-inflammatory effects on liver macrophages, with a possible minor contribution from its direct proapoptotic effect on activated HSC.

HVJ liposome; liver macrophage; oligodeoxynucleotide; hydrogen peroxide

CHRONIC HEPATIC INJURY leading to fibrosis occurs in response to a variety of insults, including viral hepatitis, alcohol abuse, drugs, metabolic diseases involving an overload of iron or copper, autoimmune diseases, or congenital abnormalities (11, 27). Activated nonparenchymal cells such as liver macrophages, hepatic stellate cells (HSC), sinusoidal endothelial cells, platelets, and infiltrating inflammatory cells have been suggested to act importantly in these fibrogenic processes (2, 4, 9, 12, 21, 28, 29, 32).

Carbon tetrachloride (CCl4), a classic hepatotoxin, causes acute, reversible liver injury characterized by centrilobular necrosis, followed by hepatic regeneration and repair (8, 24, 40). This liver injury is attributed to inflammatory responses originating from CCl4-derived free radical formation in the liver and concomitant activation of nonparenchymal cells (3, 38, 39). For instance, activated Kupffer cells release a variety of chemical mediators and inflammatory cytokines in response to several radical species (5, 10). Sustained hepatic inflammation provoked by long-term treatment with CCl4 is believed to induce hepatic fibrosis through ongoing hepatocytic necrosis and production of fibrogenic cytokines acting on fibroblasts, including activated HSC (5, 25, 40).

Nuclear factor (NF)-{kappa}B, a transcription factor, known to regulate inflammatory responses in many cell types (1), ordinarily is retained in the cytoplasm in an inactive form through association with one of the I{kappa}B inhibitory proteins. Activation of NF-{kappa}B, associated with phosphorylation and subsequent degradation of I{kappa}B, occurs after exposure of cells to lipopolysaccharide (LPS), inflammatory cytokines such as tumor necrosis factor (TNF)-{alpha} and interleukin (IL)-1, oxidative stress, and other physiological and pathological stimuli (1, 7, 30). Released NF-{kappa}B complex translocates to the nucleus, where it regulates gene expression by binding to {kappa}B binding sites. Specifically, the complex binds to promoter regions of a large number of genes involved in the inflammatory response, such as those encoding intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM), IL-1, TNF-{alpha}, IL-6, and inducible nitric oxide synthase (18, 25). NF-{kappa}B binding activity was found to increase in liver macrophages and hepatocytes after CCl4 treatment in rats (23), and ongoing production of inflammatory cytokines regulated by NF-{kappa}B is believed to play a major role in CCl4-induced liver fibrosis (5, 40).

Some reports have indicated that hepatic injury induced by ischemia-reperfusion, alcohol, or endotoxin can be ameliorated by suppressing NF-{kappa}B activation in the liver, especially in liver macrophages (14, 33, 35, 36). However, whether selective and direct inactivation of NF-{kappa}B in liver macrophages can suppress CCl4-induced liver injury or fibrosis has been unclear so far, whereas nonspecific inactivation of hepatic NF-{kappa}B by some NF-{kappa}B inhibitors has been reported to be antifibrogenic (6, 26). In the present study we employed a fusigenic liposome system with hemagglutinating virus of Japan (HVJ), which selectively transfers oligodeoxynucleotides (ODNs) to liver macrophages in vivo (22), and investigated in mice whether in vivo transfer of NF-{kappa}B decoy (i.e., synthetic ODN representing the NF-{kappa}B binding site) inhibited expression of inflammatory cytokines and liver damage after a single CCl4 challenge and also whether the decoy could suppress hepatic fibrosis induced by long-term treatment with CCl4.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Male BALB/c mice (5–6 wk old) and Sprague-Dawley rats (weighing 200–300 g) were purchased from Japan CLEA (Tokyo, Japan). All mice and rats were housed at a controlled humidity of 60 ± 5% and a temperature 25 ± 1°C, with a 12:12-h light-dark cycle. All animal experiments were performed in accordance with the guidelines for animal experiments of Hyogo College of Medicine and also followed guidelines for human care set by the U.S. National Institutes of Health. All experimental protocols for animal research in the present study were submitted to and approved by the animal experimental review committee at Hyogo College of Medicine.

Synthesis of ODN and selection of sequence targets. Sequences of the double-stranded ODNs used as NF-{kappa}B decoy were 5'-CCTTGAAGGGATTTCCCTCC-3' and 3'-GGAACTTCCCTAAAGGGAGG-5' (consensus sequences are underlined). This NF-{kappa}B decoy ODN has been shown to bind the NF-{kappa}B transcriptional factor (19). Scrambled decoy/ODN (5'-TTGCCGTACCTGACTTAGCC-3', 3'-AACGGCATGGACTGAATCGG-5') was used as a control decoy (22).

Preparation of HVJ-liposome complexes. HVJ-liposome complex was prepared according to the method as described earlier (15). Mice were anesthetized with ether and the spleen was exposed through a minilaparotomy. The NF-{kappa}B decoy ODN or scrambled decoy ODN (50 nmol/mouse) encapsulated in HVJ-liposome was transferred into the portal vein through the splenic hilus by direct injection as described before (22). This procedure resulted in no mortality or morbidity during the experimental period.

Induction of acute and chronic liver injury by carbon tetrachloride. Acute liver injury in mice was induced by a single intragastric injection of 2 ml/kg of 50% (vol/vol) CCl4 in mineral oil (1:1), and the NF-{kappa}B decoy ODN encapsulated in HVJ liposome was transferred 24 h before CCl4 administration as described above. The mice were killed 1, 6, or 12 h after the CCl4 intoxication, and the livers and blood samples were taken. For the immunostaining against p65 (NF-{kappa}B), liver samples were harvested at 1 h, because it is well known that p65 quickly translocates into nucleus after stimuli. To assess neutrophil infiltration in the liver before massive hepatocyte necrosis would become obvious, mice were killed at 6 h and liver specimens were collected.

As a chronic liver injury model, mice were given CCl4 intragastrically once weekly for 8 wk. The NF-{kappa}B decoy ODN encapsulated in HVJ liposome was transferred biweekly for four times during the CCl4 treatment, because ODN transferred by the HVJ-liposome method had been reported to remain in vivo at least for 2 wk (17). Mice were killed 48 h after the last challenge with CCl4. Control mice in both experiments were injected with the scrambled decoy encapsulated in HVJ-liposome instead of the NF-{kappa}B decoy ODN by the same procedures.

Immunofluorescene staining. To examine the efficiency of NF-{kappa}B decoy ODN transfer by the HVJ-liposome method, we employed a fluorescein isothiocyanate (FITC)-labeled NF-{kappa}B decoy ODN described previously (22). FITC-labeled NF-{kappa}B decoy ODN was encapsulated in the HVJ-liposome, and normal mice were transfected with this ODN in the same manner as described above. The localization of the FITC-labeled NF-{kappa}B decoy ODN in the liver sections was determined by immunohistochemical staining as described before (22). The primary antibodies used included rat anti-mouse macrophage monoclonal antibody (F4/80, 1:100 dilution; Biomedicals, Augst, Switzerland), rabbit anti-mouse desmin polyclonal antibody (1:100; Immunon, Pittsburgh, PA), and anti-mouse platelet endothelial cell adhesion molecule 1 monoclonal antibody (MEC13.3, 1:50; BD Biosciences/Pharmingen, San Diego, CA), which recognize the murine liver macrophages, HSC, and endothelial cells, respectively. Texas red-conjugated goat anti-rat IgG (1:50 dilution; Southern Biotechnology Associates, Birmingham, AL) was used as a secondary antibody. Localization of the FITC-labeled NF-{kappa}B decoy ODN and liver macrophages, HSC, or endothelial cells was observed by using a confocal laser scanning microscope, LSM510 (Carl Zeiss, Jena, Germany) (22). To evaluate the efficiency of ODN transfer through the HVJ-liposome in fibrotic livers, localization of the FITC-NF-{kappa}B decoy ODN was analyzed during the long-term CCl4 treatment. Namely, after the 4-wk treatment with CCl4, mice were transfected with the FITC-NF-{kappa}B decoy ODN encapsulated in the HVJ-liposome, and immunohistochemical staining of the liver was performed as described above. Localization of FITC-labeled scrambled decoy was also analyzed in the same manner.

Histological examination and immunostaining. Paraffin-embedded tissues were sectioned and stained with hematoxylin and eosin, or Azan-Mallory staining was performed. Hepatic injury was examined in a blind manner. The extent of sinusoidal congestion, cytoplasmic vacuolization, and liver necrosis was semiquantitatively assessed respectively according to a scoring criteria previously published elsewhere (34). Fibrosis was also assessed by using image analysis techniques on Azan-Mallory staining slides, by using a planimetric method on the Automatic Image Analysis System (Carl Zeiss, Oberkochem, Germany).

Infiltration by neutrophils in the liver was also estimated by means of naphthol AS-D chloroacetate esterase staining (41). The number of esterase-positive polymorphonuclear cells was counted in 10 high-power fields (x400) in each sample, and mean values were calculated.

For immunohistochemistry, sections were pretreated through deparaffinization, antigen unmasking, and blocking with 1% H2O2 for 10 min and 1.5% goat normal serum for 60 min. The specimens were incubated with mouse anti-human {alpha}-smooth muscle actin ({alpha}-SMA) antibody (1:200 dilution; Thermo Electron, Waltham, MA) or anti-NF-{kappa}B p65 antibody (1:100 dilution; Santa Cruz Biotechnologies) for 60 min. After being washed, the sections were incubated with biotin-conjugated goat anti-mouse IgG secondary antibody (1:5,000) for 60 min, then with diaminobenzidene for 1–2 min, and counterstained with hematoxylin for 10 s.

Expression of 4-hydroxy-2'-nonenal (HNE) adducts and nitrotyrosine in the liver were analyzed by immunohistochemistry using a monoclonal anti-4-HNE antibody (JAICA, MHN-20, 1:5; Shizuoka, Japan) and polyclonal anti-nitrotyrosine antibody (AB5411, 1:100; Chemicon) as the primary antibody, respectively.

Liver function test. Blood samples were collected from each animal by puncture of vena cava at the time they were killed. Serum samples were analyzed for aspartate aminotransferase (AST) and alanine aminotrasferase (ALT) by automatic analysis (Japan Clinical Laboratories, Kyoto, Japan).

Real-time RT-PCR. Total RNA was extracted from the liver specimens by using a kit, ISOGEN (NIPPON GENE, Tokyo, Japan), according to the manufacturer's guidelines. RNA concentrations were measured spectrophotometrically at 260 nm. For each sample, 1 µg of total RNA was used. Reverse transcription was carried out using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) according to the manufacturer's guidelines. Primers and probes for real-time reverse transcription polymerase chain reaction (RT-PCR) were provided by Applied Biosystems as ready-to-use mixes. The product identification numbers of the mixes were Mm00441724_m1 for TGF-beta1, Mm00801666_g1 for procollagen type I {alpha}1, Mm01546133_m1 for {alpha}-SMA, and Hs99999901_s1 for eukaryotic 18s rRNA. RT-PCR was carried out by using the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems) as reported previously (42). A housekeeping gene, 18SrRNA, served as the endogenous control.

Cell separation procedures. Liver macrophages were isolated from mice by the pronase-collagenase method as previously described (31). Elutriated fraction was collected by using CR20B2 (Hitachi, Tokyo, Japan), and the cells were resuspended in RPMI 1640 containing 10% FCS and plated on collagen-coated plastic plates. HSC were isolated from male Sprague-Dawley rats (200–300 g) by liver perfusion with bacterial pronase and collagenase, followed by density gradient centrifugation with Nycodenz according to published protocols (13). Isolated HSC were seeded on a 60-mm uncoated plastic tissue culture dish and cultured in DMEM supplemented with 10% FCS and standard antibiotics in 95% air-5% CO2 humidfied atmosphere at 37°C for 5 days before experiments. Viability of liver macrophages and HSC was assessed by the Trypan blue exclusion method. The purity of liver macrophages was more than 90% according to fluorescence activated cell sorting using Mac1 antibody (20). Purity of HSC preparation was assessed by their typical light microscopic appearance and vitamin A-specific autofluorescence and was ~97%.

In vitro transfer of ODN and cell treatment. HVJ envelope vector (HVJ-E) kit, Genome ONE (Ishihara Sangyo, Osaka, Japan), was employed for in vitro experiments, and the NF-{kappa}B decoy ODN was encapsulated into the HVJ-E vector as described elsewhere (37). Isolated liver macrophages and HSC cultured for 1 day or 5 days, respectively, were transfected with the NF-{kappa}B decoy ODN or with the scrambled decoy ODN by using the Genome ONE. For fluorescent microphotography, the FITC-labeled NF-{kappa}B decoy ODN was transferred to the cells as described above; 24 h later they were rinsed with PBS, fixed in ice-cold paraformaldehyde, rinsed, and stained with antibodies against macrophage (F4/80, 1:100 dilution; Biomedicals) or {alpha}-SMA (Thermo Electron, Waltham, MA) at dilution of 1:100, followed by anti-mouse IgG at dilution of 1:250. A fluorescence image was obtained by the confocal laserscanning microscope LSM510 (Carl Zeiss, Jena, Germany).

Liver macrophages transfected with NF-{kappa}B decoy ODN or scrambled decoy ODN for 24 h were also rinsed with PBS, and serum-free RPMI 1640 and hydrogen peroxide (H2O2) at final concentration of 100, 500, and 1,000 µM were added. After the incubation at 37°C and 5% CO2 for 3 h, culture media were collected, and liver macrophages were scraped off the dishes and their nuclear extractions were obtained as described below.

Activated HSC transfected with the NF-{kappa}B decoy ODN or the scrambled decoy were incubated for 3 more days in DMEM supplemented with 10% FCS, and changes in their morphology and {alpha}-SMA expression were observed. To examine proapoptotic effects of the NF-{kappa}B decoy ODN against activated HSC, HSC transfected with the NF-{kappa}B decoy ODN or with the scrambled decoy ODN for 24 h were incubated for another 24 h with or without TNF-{alpha} (10 ng/ml) in serum-free DMEM, and TUNEL analysis was performed by using a commercially available in situ apoptosis detection kit (ApopTag, S7110, Chemicon, Temecula, CA) to determine apoptotic HSC. HSC treated with or without TNF-{alpha} for 24 h as described above were also scraped off the dishes and their nuclear extractions were obtained.

ELISA for cytokines. Mouse blood was collected into microtubes at death and centrifuged for 10 min at 5,400 g to obtain serum. Concentrations of TNF-{alpha}, IL-6, and IL-1beta in serum or in culture medium, which was obtained from isolated liver macrophages after H2O2 treatment, were measured by use of ELISA kits (Genzyme Techne, Boston, MA).

NF-{kappa}B transcription factor assay. DNA-binding activity of NF-{kappa}B p65 in the cultured cells was measured in duplicate by use of a nonradioactive NF-{kappa}B (p65) assay kit (StressXpress NF{kappa}B, Stressgen Bioreagents, Victoria, BC, Canada) according to the instructions of the manufacturer. This method combines the principle of the EMSA with the 96-well based ELISA. During the assay a double stranded biotinylated oligonucleotide containing the flanked DNA binding consensus sequence for NF-{kappa}B (5'-GGGACTTTCC-3') bound to the streptavidin-coated 96-well plate well is reacted with nuclear extract from cultured cells. TNF-{alpha}-activated HeLa cell nuclear extract was used as a positive control. The bound NF-{kappa}B transcription factor subunit p65 is detected with a specific primary antibody, followed by a highly sensitive horseradish peroxidase-conjugated secondary antibody, from which chemiluminescent signals were detected in a lumino image analyzer (LAS-1000 plus, FUJIFILM, Tokyo, Japan).

Western blot analysis. Three days after the NF-{kappa}B decoy or scrambled decoy transfection, the HSC were washed with cold PBS and solubilized in RIPA buffer. Total cell lysates obtained from the HSC were subjected to 10% SDS-PAGE, and the separated proteins were transferred to a nitrocellulose membrane. A protein band was detected by Western blot analysis using rat anti-{alpha}-SMA monoclonal antibody (1:5,000; Sigma) and rabbit anti-rat IgG conjugated to horseradish peroxidase secondary antibody (1:5,000; Santa Cruz Biotechnology, CA), employing a chemiluminescence assay system (ECL plus, Amersham Bioscience, Buckinghamshire, United Kingdom).

Statistical analysis. Data were expressed as means ± SD, and the statistical significance of differences among groups was assessed by Student's t-test or Mann-Whitney U-test appropriately. P values <0.05 were regarded as statistically significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Localization of FITC-labeled ODN transferred in vivo. Intrahepatic localization of FITC-labeled NF-{kappa}B decoy ODN transferred into normal mice with the HVJ-liposome method was analyzed. Immunofluorescence staining of liver sections using Texas red-labeled F4/80, an anti-mouse macrophage antibody, localized the ODN mainly to liver macrophages (>95%) rather than to hepatocytes, as our laboratory reported previously (22) (data not shown). A small portion of the FITC-labeled ODN was detected in HSC. Localization of FITC-labeled NF-{kappa}B decoy ODN in fibrotic livers also was investigated in mice treated with CCl4 for 4 wk to determine whether the ODN was similarly transferred into macrophages or activated HSC in the fibrotic livers. Immunofluorescence staining using a Texas red-labeled anti-{alpha}-SMA antibody indicated that a small fraction of FITC-labeled NF-{kappa}B decoy ODN accumulated in activated HSC, whereas most the ODN was localized to liver macrophages, as in normal liver (Fig. 1, A–F). Localization of FITC-labeled scrambled ODN, which was transferred in the same manner, was similar to that of NF-{kappa}B decoy ODN in both normal and fibrotic livers (data not shown).


Figure 1
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  		Fig. 1. Localization of FITC-labeled oligodeoxynucleotide (ODN) in the CCl4-induced fibrotic liver. After a 4-wk treatment with CCl4, mice were transfected with FITC-labeled NF-{kappa}B decoy ODN encapsulated in hemagglutinating virus of Japan (HVJ)-liposome, and immunohistochemical staining of the liver was performed. Twenty-four hours after the ODN transfer, liver samples were collected and observed with a confocal microscope. Immunofluorescence staining of the liver sections using Texas red-labeled anti-mouse macrophage antibody, F4/80 (B), or anti {alpha}-smooth muscle actin (SMA) antibody (E) revealed that FITC-labeled ODN mainly accumulated in the liver macrophages (Kupffer cells) and a small portion of it in the activated hepatic stellate cells (HSC). Green fluorescence is FITC, and red color (Texas red) represents hepatic macrophages (B) and activated HSC (E). Yellow color in merged figures represents hepatic cells transfected with FITC-labeled ODN (C and F). Each figure is representative of 4 animals in each experiment. Original magnification x200.

 
Effect of NF-{kappa}B decoy ODN on acute liver injury. According to histological analysis at 12 h, CCl4-exposed mice transfected with the scrambled decoy showed hepatocytic necrosis and infiltration by inflammatory cells, most evident surrounding central veins (Fig. 2A); these changes were clearly suppressed in the NF-{kappa}B decoy-treated group (Fig. 2B). Sinusoidal congestion, cytoplasmic vacuolization, and hepatocyte necrosis, which were semiquantitatively scored, were 2.6 ± 0.6, 2.7 ± 0.8, and 2.8 ± 0.8, in the scrambled decoy-treated group, respectively. Meanwhile those scores were 1.4 ± 0.5 (P < 0.01), 1.3 ± 0.5 (P < 0.01), and 1.9 ± 0.6 (P < 0.01) in the NF-{kappa}B decoy-treated group, respectively. To determine whether in vivo transfection with NF-{kappa}B decoy ODN effectively blocked NF-{kappa}B translocation into nuclei of liver macrophages, we carried out immunostaining against p65 (NF-{kappa}B, RelA). At 1 h after a single intragastric challenge of CCl4, nuclear localization of p65 was detected in nonparenchymal cells in control mice (Fig. 2C), whereas transfection with the NF-{kappa}B decoy ODN potently blocked this translocation (Fig. 2D). Nuclear translocation of p65 in hepatocytes was not detected in the both livers at this early time point. At 12 h after CCl4 administration, serum concentrations of AST and ALT had increased significantly in control mice, whereas the increase was much smaller in NF-{kappa}B decoy ODN-transfected mice (Fig. 2, E and F). Increases in serum concentrations of IL-6, TNF-{alpha}, and IL-1beta also were observed in exposed control mice with the scrambled decoy at 12 h; the increase was suppressed significantly in the NF-{kappa}B decoy group (Fig. 2, G–I). As a consequence, neutrophils infiltrating into the liver were significantly fewer with the NF-{kappa}B decoy at 6 h (Fig. 3, A–C). Lipid peroxidation in the liver after CCl4 treatment, which was detected as 4-HNE, was not attenuated by the NF-{kappa}B decoy at 6 h (Fig. 3, D–F) whereas nitrotyrosine, an index of nitration of tyrosine by peroxynitrite and/or other free radicals, was greatly suppressed at 6 h (Fig. 3, G–I).


Figure 2
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  		Fig. 2. NF-{kappa}B decoy ODN transfection in vivo ameliorates acute liver injury and inflammatory cytokine production induced by a single challenge of CCl4. Hematoxylin and eosin (H&E) staining of the liver tissue from scrambled decoy-treated control (A) and NF-{kappa}B decoy-treated animals (B) revealed that NF-{kappa}B decoy suppresses necrosis of hepatocytes and infiltration of inflammatory cells in the liver at 12 h. Immunohistochemical staining for p65 (NF-{kappa}B) in the liver at 1 h after the single administration of CCl4 shows that nuclear translocation of p65 is markedly blocked in nonparenchymal cells in the liver transfected with NF-{kappa}B decoy (C: scrambled decoy-treated control; D: NF-{kappa}B decoy-treated mice). The p65-positive cells are indicated with black arrowheads. After the single administration of CCl4, increase in serum aspartate aminotransferase (s-AST) and serum alanine aminotrasferase (s-ALT) concentrations observed in the scrambled decoy-treated control mice at 12 h is suppressed by NF-{kappa}B decoy (E and F). Increases in serum concentration of IL-6, TNF-{alpha}, and IL-1beta observed in the scrambled decoy-treated control animals at 12 h are also suppressed in the NF-{kappa}B decoy group (G–I). Data represent means ± SD of 6 animals in each group. *P < 0.05 compared with the scrambled decoy control group. A–D: representative figures of 6 animals in each group. Original magnification x200 in A–D and x400 in insets of C and D. C, central vein; P, portal vein.

 

Figure 3
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  		Fig. 3. NF-{kappa}B decoy ODN transfection in vivo suppresses infiltration of polymorphonuclear cells and production of nitrotyrosine induced by a single challenge of CCl4. The number of polymorphonuclear cells infiltrated in the scrambled decoy-treated control liver (A) is markedly suppressed in the NF-{kappa}B decoy group at 6 h (B and C). White arrowheads indicate esterase positive polymorphonuclear cells. Anti-4-hydroxy-2'-nonenal (HNE) staining (D–F) reveals that NF-{kappa}B decoy fails to suppress lipid peroxidation owing to radical stress induced by CCl4 at 6 h (D: untreated normal; E: scrambled decoy-treated control; F: NF-{kappa}B decoy group), whereas staining of nitrotyrosine (G–I) shows that NF-{kappa}B effectively blocks nitration of proteins along sinusoid after CCl4 intoxication at 6 h (G: untreated normal; H: scrambled decoy-treated control; I: NF-{kappa}B decoy group). Data represent means ± SD of 6 animals in each group. *P < 0.05 compared with the scrambled decoy control group. A–I: representative figures of 6 animals in each group. Original magnification x200.

 
Effect of NF-{kappa}B decoy ODN on CCl4-induced liver fibrosis. To determine the effect of NF-{kappa}B decoy ODN on liver fibrosis, mice were given repeated intragastric injections of CCl4 for 8 wk, and NF-{kappa}B decoy ODN was transferred biweekly during this period. In exposed control livers transfected with the scrambled decoy, degeneration of hepatocytes, infiltration by inflammatory cells, and massive necrosis were observed at 8 wk predominantly around central veins (Fig. 4A).


Figure 4
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  		Fig. 4. Inhibition of CCl4-induced hepatic injury and fibrosis by NF-{kappa}B decoy ODN. H&E staining of liver tissues (A and B) reveals that degeneration of hepatocyte, infiltration of inflammatory cells, and massive necrosis observed in the control liver transfected with the scrambled decoy (A) are markedly attenuated in the NF-{kappa}B decoy treated animals (B) (original magnification x200). CCl4 treatment for 8 wk established moderate fibrosis in the control animals transfected with the scrambled decoy (C), whereas NF-{kappa}B decoy suppressed formation of interlobular fibrosis (D) (Azan-Mallory staining, original magnification x40). This phenomenon was confirmed with semiquantitative analysis of liver fibrosis performed by image analysis techniques (NIH image) (G). The expression of {alpha}-SMA was efficiently suppressed in the liver transfected with NF-{kappa}B decoy (E: scrambled decoy-treated control; F: NF-{kappa}B decoy-treated, original magnification x40). E: increase in mRNA expression of TGF-beta1, Col1A1, and {alpha}-SMA evaluated by real-time RT-PCR was significantly attenuated with NF-{kappa}B decoy (H–J). Data represent means ± SD of 6 animals in each group. *P < 0.05. A–F: representative figures of 6 animals in each group.

 
In the NF-{kappa}B decoy group these histological changes were markedly attenuated, although some hepatocyte degeneration still existed (Fig. 4B). Semiquantitative scores for sinusoidal congestion, cytoplasmic vacuolization, and hepatocyte necrosis were 2.2 ± 0.5, 2.2 ± 0.8, and 2.2 ± 0.8, respectively, in the scrambled decoy-treated group, whereas they were 1.1 ± 0.4 (P < 0.01), 1.2 ± 0.4 (P < 0.01), and 1.3 ± 0.5 (P < 0.01), respectively, in the NF-{kappa}B decoy-treated group. Azan-Mallory staining indicated that 8 wk of treatment with CCl4 established moderate bridging fibrosis in exposed control mice with the scrambled decoy, whereas the NF-{kappa}B decoy markedly suppressed fibrosis (Fig. 4, C and D). Semiquantitative analysis of liver fibrosis by image analysis techniques showed more than 30% reduction of fibrosis in the NF-{kappa}B decoy group (Fig. 4G). Expression of {alpha}-SMA in the liver also was increased in CCl4-exposed control mice with the scrambled decoy; this expression was suppressed by the NF-{kappa}B decoy treatment (Fig. 4, E and F). Expression of mRNA encoding TGF-beta, Col1{alpha}1, and {alpha}-SMA, which were evaluated by real-time RT-PCR, increased significantly after the long-term CCl4 treatment with scrambled decoy, whereas these expressions were significantly attenuated by the NF-{kappa}B decoy transfer (Fig. 4, H–J).

Transfection of isolated liver macrophages and HSC with FITC-labeled ODN. For in vitro transfection we used an HVJ-Envelope vector kit (GenomONE). We examined transduction of FITC-labeled NF-{kappa}B decoy ODN in isolated liver macrophages and HSC by immunofluorescence staining using antibodies against liver macrophages (F4/80) and {alpha}-SMA. Transfectional efficiency of the FITC-labeled NF-{kappa}B decoy ODN was almost 100% in both isolated liver macrophages cultured for 24 h (Fig. 5A) and in HSC cultured for 5 days (Fig. 6A). FITC-labeled scrambled decoy ODN was also successfully transferred into the macrophages and activated HSC in vitro, and the transfectional efficiency was almost 100% in both cells (data not shown).


Figure 5
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  		Fig. 5. Effects of NF-{kappa}B inhibition on isolated Kupffer cells in vitro. Immunofluorescent microscopic findings reveals that most of the isolated Kupffer cells are transfected with FITC-labeled ODN using HVJ-Envelope vector kit (GenomONE) (A) (representative figures of 4 separate in vitro experiments). NF-{kappa}B transcription factor assay (B) shows that DNA binding activity of NF-{kappa}B increases after hydrogen peroxide (H2O2) treatment in the scrambled decoy-treated cells, whereas the activity was significantly inhibited by NF-{kappa}B decoy treatment. N (shaded bars), untreated normal macrophages. Inflammatory cytokine production into culture medium assessed by ELISA (C) increases after hydrogen peroxide (H2O2) treatment in the scrambled decoy-infected control cells. However, these increases are suppressed in the NF-{kappa}B decoy-treated group. Data represent means ± SD of 6 samples in each group. #P < 0.05 compared with untreated cells; *P < 0.05 compared with the scramble decoy-treated cells under the same concentrations of H2O2.

 

Figure 6
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  		Fig. 6. Inhibition of NF-{kappa}B has no effect on morphology of isolated HSC or on the expression of {alpha}-SMA, whereas it enhances TNF-{alpha}-induced apoptosis of activated HSC. Immunofluorescent microscopic findings of HSC on day 5 (A) show that most of activated HSC are transfected with FITC-labeled NF-{kappa}B decoy ODN, by using the HVJ-Envelope vector kit. {alpha}-SMA, red color; FITC, green color. B: NF-{kappa}B decoy transferred into activated HSC on day 5 had no effect on morphological finding or on the expression of {alpha}-SMA (B and C). Nuclear EMSA for NF-{kappa}B mobilization is determined by NF-{kappa}B transcription factor assay (D). Culture-activated HSC are pretreated with NF-{kappa}B decoy ODN or scrambled decoy ODN or media alone then incubated with TNF-{alpha} (10 ng/ml) for 24 h. E: activated HSC transfected with NF-{kappa}B decoy ODN or scrambled decoy ODN are treated with TNF-{alpha} for 24 h, and apoptosis is determined by TUNEL assay. Top and bottom: are TUNEL and DAPI staining, respectively. F: the number of apoptotic cells was counted in 10 randomly selected fields for a total of 200 cells. A–C and E are representative figures of 4 separate in vitro experiments. Data were presented as means ± SD (n = 4). #P < 0.05 compared with untreated cells group, *P < 0.05.

 
Suppression by the NF-{kappa}B decoy ODN of H2O2-induced liver macrophage activation in vitro. According to the NF-{kappa}B transcription factor assay, DNA binding activity of NF-{kappa}B increased markedly after treatment of liver macrophages transfected with the scrambled decoy with H2O2 for 3 h (Fig. 5B). Transfection of liver macrophages with the NF-{kappa}B decoy ODN significantly suppressed this DNA binding activity (Fig. 5B). To confirm that the NF-{kappa}B decoy ODN effectively suppressed production of proinflammatory cytokines by these cells, cytokine concentrations in culture media were assayed by using ELISA kits. Concentrations of proinflammatory cytokines, such as TNF-{alpha}, IL-6, and IL-1beta, increased significantly after the treatment with H2O2 for 3 h in the control cells. Transfection with the NF-{kappa}B decoy ODN effectively suppressed this production of cytokines (Fig. 5C). Viability of macrophages after the treatment with the highest concentration of H2O2 (1 mM), which was determined with the Trypan blue exclusion test, was over 98% at 3 h. These results indicated that NF-{kappa}B decoy transfer using the HVJ-Envelope vector kit functionally suppressed H2O2-induced activation of liver macrophages in vitro.

Enhanced TNF-{alpha}-induced apoptosis in activated HSC by NF-{kappa}B decoy ODN, without suppression of activation. NF-{kappa}B decoy ODN transfer to activated HSC precultured for 5 days had no effect on their morphological activation or expression of {alpha}-SMA, as determined by Western blotting (Fig. 6, B and C). To determine whether NF-{kappa}B decoy ODN affects apoptosis of activated HSC induced by TNF-{alpha}, HSC precultured for 5 days were treated with 10 ng/ml of TNF-{alpha} for 24 h with NF-{kappa}B decoy or scrambled decoy transfection. NF-{kappa}B transcription factor assay using nuclear extracts from these HSC indicated that DNA binding activity of NF-{kappa}B increased after treatment with TNF-{alpha} in untreated cells and cells transfected with the scrambled decoy, whereas the increase was greatly suppressed by the NF-{kappa}B decoy (Fig. 6D). Morphological analysis of apoptotic cells indicated more than 35% cell death after TNF-{alpha} treatment in NF-{kappa}B decoy-treated HSC; only 10% cell death was detected in cells transfected with the scrambled decoy (Fig. 6, E and F). These results indicated that NF-{kappa}B decoy ODN had no effect against activation of HSC; instead, the decoy accelerated TNF-{alpha}-induced apoptosis in these cells.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The various NF-{kappa}B transcription factor complexes act importantly in transcription of many genes, primarily those whose products are related to inflammation, such as interleukins and adhesion molecules (1). Our present study was designed to investigate how NF-{kappa}B inactivation by a transcription factor decoy affects CCl4-induced liver injury and fibrosis.

Most of a double-stranded ODN transferred in vivo using the HVJ-liposome method has been localized to liver macrophages (22). In the present study, NF-{kappa}B decoy ODN transferred in vivo ameliorated acute hepatic injury induced by CCl4 while suppressing production of inflammatory cytokines such as TNF-{alpha}, IL-1, and IL-6. Immunohistochemical studies of p65 showed that NF-{kappa}B decoy ODN effectively suppressed nuclear translocation of p65 in nonparenchymal cells (NPC) after CCl4 treatment (Fig. 2D). As a consequence, infiltration by neutrophils and tyrosine nitration in the liver were markedly suppressed by the NF-{kappa}B decoy (Fig. 3, B and I). Hepatic injury induced by CCl4 is believed to occur through CCl4-derived radical formation in the liver and concomitant activation of NPC (3, 38, 39). Lipid peroxidation resulting from oxidative stress in the liver clearly was increased after the CCl4 treatment, whereas the NF-{kappa}B decoy failed to suppress this lipid peroxidation (Fig. 3F). Instead, subsequent NF-{kappa}B activation in NPC was effectively blocked, and tyrosine nitration, an index of nitration of proteins, was suppressed. We hypothesized that the NF-{kappa}B decoy in vivo inhibited NF-{kappa}B activation that otherwise occurred in response to free radical stress in NPC, mainly in liver macrophages. We sought to confirm this hypothesis by adding the oxidant H2O2 to the culture medium of liver macrophages in vitro. Indeed, NF-{kappa}B activation and production of inflammatory cytokines induced by H2O2 in vitro was suppressed significantly by the NF-{kappa}B decoy (Fig. 5, B and C). We concluded that the protective effect of the NF-{kappa}B decoy ODN against CCl4-induced acute liver injury resulted mainly from suppression of NF-{kappa}B activation that ordinarily is induced by radical stress in NPC, mainly in liver macrophages.

We also investigated the effect of the NF-{kappa}B decoy ODN on liver fibrosis induced by long-term treatment with CCl4. The NF-{kappa}B decoy effectively suppressed this fibrosis, reducing mRNA expressions of TGF-beta, procollagen 1{alpha}1, and {alpha}-SMA in the liver. In the long-term in vivo model, the NF-{kappa}B decoy ODN was transferred biweekly four times, and CCl4 was administered once weekly for 8 wk. We speculated that repeated transfection with NF-{kappa}B decoy ODN effectively suppressed NF-{kappa}B activation induced by CCl4 in NPC, mainly liver macrophages, decreasing subsequent occurrence of inflammation to result in decreased fibrosis. How expression of TGF-beta in the liver was suppressed in the present study still is unclear, and no evidence indicates that the transcription factor NF-{kappa}B directly regulates transcription of TGF-beta. However, several factors released by activated Kupffer cells have been reported to stimulate HSC proliferation and promote their synthesis of collagen, proteoglycans, and hyaluronate (5, 10), and preventing the action of TNF-{alpha} and IL-1beta has been found to block induction of collagen (2). According to a recent report, depletion of the macrophage population decreases hepatic fibrosis (9), suggesting significant involvement of Kupffer cells in liver fibrosis. We demonstrated here that ODN transferred in vivo using the HVJ-liposome method also was preferentially localized to liver macrophages with a small portion of the ODN locating in the activated HSC, in moderately fibrotic livers (Fig. 1). This implies that the NF-{kappa}B decoy ODN may function effectively even when transferred to livers with ongoing fibrogenesis. Thus the antifibrogenic effect of NF-{kappa}B decoy ODN observed in this study appeared to result mainly from inhibitory effect against NF-{kappa}B activation in liver macrophages.

The contribution of apoptosis of activated HSC to resolution of liver fibrosis has been well documented (11, 16). Even though only a small portion of ODN was transferred in vivo into HSC in fibrotic livers in this study (Fig. 1), we tried to determine how the NF-{kappa}B decoy ODN affected activated HSC in vitro, where NF-{kappa}B decoy ODN per se did not affect morphology or {alpha}-SMA expression in activated HSC. We then examined the effect of the NF-{kappa}B decoy ODN on TNF-{alpha}-induced apoptosis in activated HSC in vitro, considering that a significant amount of TNF-{alpha} may be present during long-term CCl4 treatment in vivo. As we expected, the NF-{kappa}B decoy ODN suppressed DNA-binding activity of NF-{kappa}B after TNF-{alpha} treatment in activated HSC (Fig. 6D), whereas TNF-{alpha}-induced apoptosis in these cells was accelerated significantly by the NF-{kappa}B decoy ODN (Fig. 6, E and F). From these results, we conclude that apoptosis of activated HSC may, at least partly, account for the fibrosis-suppressing effect of the NF-{kappa}B decoy ODN in the long-term CCl4 model.

In summary, NF-{kappa}B decoy ODN transferred into the mouse liver was mainly identified in liver macrophages, effectively suppressing CCl4-induced NF-{kappa}B activation in these cells, subsequent inflammation, and ultimately liver fibrosis during long-term treatment with CCl4. TNF-{alpha}-induced apoptosis in activated HSC, which was accelerated by the NF-{kappa}B decoy ODN in vitro, may contribute to the fibrosis-suppressing effect of the decoy in vivo, even though its transfective efficiency in activated HSC was relatively low. These results directly indicate the critical role of liver macrophages in radical-induced liver injury and fibrosis. Such decoys may prove useful in prevention of liver fibrosis induced by sustained inflammation, where liver macrophages act importantly by producing proinflammatory cytokines.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported in part by Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science (JSPS), 17390375 to J. Fujimoto and 16390385 and 19591606 to Y. Iimuro, and by a Grant from the Science Research Promotion Fund to J. Fujimoto.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Fujimoto, First Dept. of Surgery, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan (e-mail: surg-1{at}hyo-med.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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