Basic Research Open Access
Copyright ©The Author(s) 2004. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Feb 15, 2004; 10(4): 573-578
Published online Feb 15, 2004. doi: 10.3748/wjg.v10.i4.573
Augmented regeneration of partial liver allograft induced by nuclear factor-κB decoy oligodeoxynucleotides-modified dendritic cells
Ming-Qing Xu, Jian-Ping Gong, Ming-Man Zhang, Lü-Nan Yan, Department of General Surgery, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China
Yu-Ping Suo, West China Second University Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China
Author contributions: All authors contributed equally to the work.
Supported by the Postdoctoral Science Foundation of China, No. 2003033531
Correspondence to: Professor Lü-Nan Yan, Department of General Surgery, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China. xumingqing0018@163.com Telephone: +86-28-85582968
Received: August 26, 2003
Revised: September 6, 2003
Accepted: September 25, 2003
Published online: February 15, 2004

Abstract

AIM: To investigate the effect of NF-κB decoy oligodeoxynuleotides (ODNs)-modified dendritic cells (DCs) on regeneration of partial liver allograft.

METHODS: Bone marrow (BM)-derived DCs from SD rats were propagated in the presence of GM-CSF or GM-CSF + IL-4 to obtain immature DCs or mature DCs, respectively. GM-CSF-propagated DCs were treated with double-strand NF-κB decoy ODNs containing two NF-κB binding sites or scrambled ODNs. Allogeneic (SD rat to LEW rat) 50% partial liver transplantation was performed. Normal saline (group A), GM-CSF-propagated DCs (group B), GM-CSF + IL-4-propagated DCs (group C), and GM-CSF + NF-κB decoy ODNs (group D) or scrambled ODNs-propagated DCs (group E) were injected intravenously into recipient LEW rats 7 days prior to liver transplantation and immediately after transplantation. DNA synthesis (BrdU labeling) and apoptosis of hepatocytes were detected with immunostaining and TUNEL staining postoperative 24 h, 48 h, 72 h and 84 h, respectively. Liver graft-resident NK cell activity, hepatic IFN-γ mRNA expression and recipient serum IFN-γ level at the time of the maximal liver allograft regeneration were measured with 51Cr release assay, semiquantitative RT-PCR and ELISA, respectively.

RESULTS: Regeneration of liver allograft was markedly promoted by NF-κB decoy ODNs-modified immature DCs but was significantly suppressed by mature DCs, the DNA synthesis of hepatocytes peaked at postoperative 72 h in group A, group B and group E rats, whereas the DNA synthesis of hepatocytes peaked at postoperative 84 h in group C rats and 48 h in group D rats, respectively. The maximal BrdU labeling index of hepatocytes in group D rats was significantly higher than that in the other groups rats. NF-κB decoy ODNs-modified immature DCs markedly suppressed but mature DCs markedly promoted apoptosis of hepatocytes, liver-resident NK cell activity, hepatic IFN-γ mRNA expression and recipient serum IFN-γ production. At the time of the maximal regeneration of liver allograft, the minimal apoptosis of hepatocytes, the minimal activity of liver-resident NK cells, the minimal hepatic IFN-γ mRNA expression and serum IFN-γ production were detected in group D rats. The apoptotic index of hepatocytes, the activity of liver-resident NK cells, the hepatic IFN-γ mRNA expression level and the serum IFN-γ level in group D rats were significantly lower than that in the other groups rats at the time of the maximal regeneration of liver allograft.

CONCLUSION: The data suggest that the augmented regeneration of partial liver allograft induced by NF-κB decoy ODNs-modified DCs may be attributable to the reduced apoptotic hepatocytes, the suppressed activity of liver-resident NK cells and the reduced IFN-γ production.




INTRODUCTION

Complete and prompt liver regeneration after living liver donation and transplantation could occur in both donor and recipient in most circumstances[1-5]. Particularly in the transplant recipients, rejection[6,7] and ischemic injury[8] could result in inadequate regeneration, leading to hepatic insufficiency or overt liver failure. Early recognition and correction of these complications maximize the chance of liver graft salvage and good outcome for the patient.

Liver regeneration may be regulated cooperatively not only by humoral factors such as hormones, growth factors and growth inhibitory factors, but also by the immune system. It has been reported that regenerating hepatocytes became sensitive to the cytotoxic activity of normal liver-resident NK cells from partially hepatectomized mice[9-15]. It has also been reported that during the acute phase of regeneration after partial hepatectomy in rats, NK cell functions were temporary suppressed, followed by their recovery at the termination of regeneration[14], suggesting that such a selective suppression of NK cell functions during the acute phase represents an important regulatory mechanism for liver regeneration in the presence of hepatic NK cells. These observations indicate that liver-resident NK cells may be involved in regulating the extent of hepatocyte regeneration.

A recent study revealed that NKT cells proliferated in the liver after administration of IL-12[16], were cytotoxic effector cells and the main antimetastatic lymphocyte population in the liver. NKT cells are thought to be responsible for the recognition and regulation of not only malignant cell proliferation but also benign cell proliferation, suggesting that NKT cells may be involved in the regulation of hepatocyte regeneration[17].

It is accepted that both donor and recipient DCs mediate the rejection of graft in organ transplantation. Several studies have shown that immature donor DCs, deficient in surface costimulatory molecules, could induce T-cell hyporesponsiveness[18-20] and prolong graft survival in unmodified hosts[21-24]. Other studies showed that DCs treated with NF-κB decoy oligodeoxynuleoides (ODNs) containing specific NF-κB binding sites, which are maintained in an immature state, could induce tolerance of cardiac allograft[25,26]. Our recent study showed that NF-κB decoy ODNs-modified DCs could induce tolerance of rat liver allograft by promoting apoptosis of liver graft-infiltrating cells (GIC) in the portal areas and suppressing IFN-γ mRNA expression in the liver graft[27].

We hypothesize that the enhanced apoptosis of liver graft-infiltrating cells by NF-κB decoy ODNs-modified DCs can reduce the total number of liver-resident NK cells and suppress the cytotoxicity of NK cells to the regenerating hepatocytes, and consequently promote liver graft regeneration. In the present study we reported for the first time that NF-κB decoy ODNs-modified DCs could promote regeneration of partial liver allograft by suppressing hepatocyte apoptosis, liver-resident NK cell activity and IFN-γ production.

MATERIALS AND METHODS
NF-κB decoy ODNs

Double-stranded NF-κB decoy ODNs or scrambled ODNs (as a control for NF-κB decoy ODNs) were generated using equimolar amounts of single-stranded sense and antisense phosphorothioate-modified oligonucleotide containing two NF-κB binding sites (sense sequence 5’-AGGGACTTTCCGCTG-GGGACTTTCC-3’, NF-κB binding sites bold lines and underlined)[25] and scrambled oligonucleotide (sense sequence 5’-TTGCCGTACCTGACTTAGCC-3’)[28]. Sense and antisense strands of each oligonucleotide were mixed in the presence of 150 mM PBS, heated to 100 °C, and allowed to cool to room temperature to obtain double-stranded DNA.

Propagation of bone marrow-derived DC populations

Bone marrow cells harvested from femurs of normal SD rats were cultured in 24-well plates (2 × 106 per well) in 2 ml of RPMI 1640 complete medium supplemented with antibiotics, 10% fetal calf serum (FCS) and 4.0 ng/ml recombinant rat GM-CSF to obtain immature DCs. In addition to GM-CSF, 10 ng/ml recombinant rat IL-4 was added to cultures to obtain mature DCs. To select plates, 10 μM NF-κB decoy or scrambled ODNs was added at the initiation of culture of DCs[25]. Cytokine-enriched medium was refreshed every 2 days, after gentle swirling of the plates, half of the old medium was aspirated and an equivalent volume of fresh, cytokine-supplemented medium was added. Thus, nonadherent granulocytes were depleted without dislodging clusters of developing DCs attached loosely to a monolayer of plastic-adherent macrophages. Nonadherent cells released spontaneously from the clusters were harvested after 7 days.

Liver transplantation

Eighty male LEW rats and eighty male SD rats weighing 250-300 g were used in all the experiments. Allogeneic liver transplantations were performed using a combination of SD rats with LEW rats. All operations were performed under ether anesthesia in sterile conditions. Orthotopic 50% partial liver transplantations were performed according to the method described in our previous study[29]. Normal saline (group A), 1 × 107 GM-CSF-propagated DCs (group B), 1 × 107 GM-CSF + IL-4-propagated DCs (group C), and 1 × 107 GM-CSF + NF-κB decoy ODNs or scrambled ODNs-propagated DCs (group D or group E) were injected intravenously through the penile vein into recipient LEW rats 7 days prior to liver transplantation and immediately after liver transplantation, respectively. Liver graft samples and blood samples (n = 8) were harvested at 24 h, 48 h, 72 h and 84 h postoperatively. Part of the liver grafts was immediately used for isolation of NK cells.

Part of the liver graft tissues was immediately frozen in liquid nitrogen and kept at -80 °C for mRNA extraction and part of the liver graft tissues was preserved in 10% formalin for liver regeneration and apoptosis detection.

Liver allograft regeneration detection (BrdU labeling index)

Regeneration extent of liver allograft was indicated by BrdU labeling index of hepatocytes. The BrdU labeling index was determined as described previously. Briefly, BrdU administered intravenously at a dose of 50 mg/kg 30 min before death to measure DNA synthesis in the regenerative liver. Deparaffinized liver sections were incubated with anti-BrdU antibody for 60 min. Immunostaining for BrdU was performed by the avidin-biotin-immunoperoxidase method using ABC kit. A total of 1000 hepatocytes were counted and the labeling index was calculated.

Apoptosis of hepatocytes

Apoptotic cells in tissue sections were detected with the in situ cell death detection kit. The liver graft tissue sections were dewaxed and rehydrated according to standard protocols. Tissue sections were incubated with proteinase K(20 μg/ml in 10 mM Tris/HCl, pH 7.4-8.0) for 15 to 30 min at 21-37 °C. Endogenous peroxidase activity was quenched with blocking solution (0.3% H2O2 in methanol) for 30 min at room temperature before exposure to TUNEL reaction mixture at 37 °C for 60 min. After washed in stop wash buffer, POD (peroxidase) was added to react for 30 min at 37 °C. DAB-substrate was used for color development, and the sections were counterstained with Harrs’ hematoxylin. TUNEL staining was mounted under glass coverslip and analysed under light microscope.

Activity of liver graft-resident NK cells

Hepatic mononuclear cells (MNCs) were isolated by the sinusoidal lavage method of Bouwens and Wisse[30]. The cells were obtained by portal vein perfusion with 1% EDTA in PBS at 37 °C and a pressure of 50 H2O, and washed. Then, 30 mL of perfused fluid was collected from the inferior vena cava, and the erythrocytes and granulocytes were separated by the Ficoll (density 1.077) at 400 g for 25 min at 25 °C. NK activity was measured by the 51Cr release method. After washed with PBS, RPMI 1640 medium containing 10% fetal bovine serum was added and the cell count was adjusted to 1 × 106/ml. Next, the cultured K562 cells were harvested by centrifugation, 50 to 100 μl of 51Cr was added, and the mixture was incubated at 37 °C for 1 h. Also after the cells were washed with PBS, RPMI 1640 medium containing 10% fetal bovine serum was added and the cell count was adjusted to 1 × 106/ml. Then the target cells were placed in each well of a microplate. 1 N HCl was added to obtain maximum release and RPMI 1640 medium containing 10% fetal bovine serum was added to obtain spontaneous release (controls). Liver MNCs were added to the other wells at an effector /target ratio of 20. Centrifugation was performed for 5 min at 800 rpm, using a plate centrifuge, followed by incubation for 3.5 h in 5% CO2. The culture supernatant was collected from each well and radioactivity was measured with a g-scintillation counter. NK cells activity was calculated as follows:

Math 1

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Semiquantitative RT-PCR assay for IFN-γ mRNA expression in liver graft

IFN-γ mRNA expression was determined by semiquantitative reverse transcription-PCR amplification in contrast with house-keeping gene β-actin. Total RNA from 10 mg liver allograft tissue was extracted using TripureTM reagent. First-strand cDNA was transcribed from 1 μg RNA using AMV and an Oligo (dT15) primer. PCR was performed in a 25 μl reaction system containing 10 μl cDNA, 2 μl 10 mM dNTP, 2.5 μl 10 × buffer, 2.5 μl 25 mmol.L-1 MgCl2, 2 μl specific primer, 5 μl water and 1 μl Taq (35 cycles: at 95 °C for 60 seconds, at 59 °C for 90 seconds, and at 72 °C for 10 seconds). The primers[31,32] used in PCR reactions were as follows: IFN-γ, 5’ primer 5’-AAGACAACCAGGCCATCAGCA-3’, 3’ primer 5’-AGCCACAGTGTGAGTTCAGTC-3’, to give a 547 bp product. β-actin, 5’ primer 5’-ATGCCATCCTGCGTCTGG ACCTGGC-3’, 3’ primer 5’-AGCATTTGCGGTGCACGAT GGAGGG-3’, to give a 607 bp product. PCR products of each sample were subjected to electrophoresis in a 15 g.L-1 agarose gel containing 0.5 mg.L-1 ethidium bromide. Densitometrical analysis using NIH image software was performed for semiquantification of PCR products, and mRNA expression was evaluated by the band-intensity ratio of IFN-γ to β-actin, and presented as percent of β-actin (%).

Statistical analysis

Statistic analysis of data was performed using t-test, P < 0.05 was considered statistically significant.

RESULTS
Augmented regeneration of partial liver allograft by NF-κB decoy ODNs-modified DCs

DNA synthesis by regenerating hepatocytes in the liver graft is shown in Figure 1. The BrdU labeling index of hepatocytes in liver graft peaked at postoperative 72 h in group A, group B and group E rats, whereas the BrdU labeling index of hepatocytes in liver graft peaked at postoperative 96 h in group C rats and 48 h in group D rats, respectively. The maximum BrdU labeling index of hepatocytes was 28.07 ± 4.53% for group A rats, 34.46 ± 6.72% for group B rats, 19.81 ± 3.65% for group C rats, 48.62 ± 9.07% for group D rats and 35.12 ± 6.37% for group E rats, respectively. The maximum BrdU labeling index of hepatocytes was significantly higher in group D rats but significantly lower in group C rats than that in the other groups rats (P < 0.01 or P < 0.001). The data suggested that regeneration of partial liver allograft was promoted by NF-κB decoy ODNs-modified DCs.

Figure 1
Figure 1 Augmented regeneration of partial liver allograft by NF-κB decoy ODNs-modified DCs (× 200). BrdU-positive hepa-tocytes in the liver allografts from group C rats (A) and group D rats (B).
Suppression of apoptosis of hepatocytes by NF-κB decoy ODNs-modified DCs

Some studies showed that liver regeneration was a process involving cell proliferation and inhibition of apoptosis[33-35]. To determine the augmented regeneration of liver allografts induced by NF-κB decoy ODNs-treated DCs was associated with the inhibited apoptotic death of hepatocytes, apoptotic activity in the liver graft at the time of maximal liver allograft regeneration was examined by TUNEL staining analysis. TUNEL staining of the liver graft sections revealed that the apoptosis index of hepatocytes was 13.07 ± 3.27% for group A rats, 9.32 ± 2.15% for group B rats, 17.76 ± 3.89% for group C rats, 5.42 ± 1.76% for group D rats and 9.75 ± 2.53% for group E rats, respectively. The apoptosis index of hepatocytes was significantly lower in group D rats but significantly higher in group C rats than that in the other groups rats (P < 0.05 or P < 0.01 or P < 0.001). These data strongly suggested that the augmented regeneration of the partial liver allograft induced by NF-κB decoy ODNs-modified DCs might be associated with the significantly suppressed apoptotic death of hepatocytes (Figure 2).

Figure 2
Figure 2 Suppression of apoptosis of hepatocytes in partial liver allografts by NF-κB decoy ODNs-modified DCs (× 400). Apoptosis of hepatocytes in the liver allografts from group C rats (A) and group D rats (B).
Suppression of activity of liver allograft-resident NK cells by NF-κB decoy ODNs-modified DCs

Activity of the liver graft-resident NK cells at the time of the maximal liver allograft regeneration was measured with 51Cr release assay. A certain degree of activity of liver graft-resident NK cells was measured in group A rats. Activity of the liver graft-resident NK cells was partially suppressed by administration of immature DCs (group B and group E) but was significantly elevated by administration of IL-4 stimulated mature DCs (Group C), whereas the activity of the liver graft-resident NK cells was significantly suppressed by administration of NF-κB decoy ODNs-modified DCs (group D). The activity value of the liver graft-resident NK cells was 36.47 ± 6.32% for group A rats, 21.58 ± 4.61% for group B rats, 52.93 ± 7.73% for group C rats, 8.43 ± 2.18% for group D rats, and 23.06 ± 5.27% for group E rats, respectively. The activity of the liver graft-resident NK cells was significantly lower in group D rats but markedly higher in group C rats than that in the other groups rats (P < 0.001). The results suggested that the augmented regeneration of the partial liver allograft induced by NF-κB decoy ODNs-modified DCs might be associated with the markedly suppressed activity of the liver allograft-resident NK cells.

Suppression of IFN-γ production by NF-κB decoy ODNs-modified DCs

To determine the relationship of specific immunoregulator cytokine production to the regeneration of partial liver allograft, IFN-γ mRNA expression in the liver graft and serum level of IFN-γ at the time of the maximal liver allograft regeneration were examined by RT-PCR assay and ELISA(as shown in Figure 3 and Figure 4), respectively. A certain level of hepatic IFN-γ mRNA expression and a certain serum level of IFN-γ were detected in group A rats. Hepatic INF-γ mRNA expression and serum INF-γ production were partially down-regulated by administration of immature DCs (group B and group E) but were significantly up-regulated by administration of IL-4 stimulated mature DCs (Group C), whereas the hepatic INF-γ mRNA expression and serum INF-γ production were significantly suppressed by administration of NF-κB decoy ODNs-modified DCs (group D). The hepatic INF-γ mRNA expression level and the serum INF-γ level were markedly lower in group D rats but markedly higher in group C rats than that in the other groups rats. The results suggested that the augmented regeneration of the partial liver allograft induced by NF-κB decoy ODNs-modified DCs might be associated with the markedly suppressed hepatic IFN-γ mRNA expression and serum INF-γ production.

Figure 3
Figure 3 Suppression of IFN-γ mRNA expression in the liver allografts by NF-κB decoy ODNs-modified DCs. aP < 0.05 vs group A, bP < 0.001 vs group A, cP < 0.001 vs group B, dP < 0.001 vs group A, eP < 0.001 vs group B, fP < 0.001 vs group C, gP > 0.05 vs group B.
Figure 4
Figure 4 Down-regulation of serum IFN-γ level in recipient rats by NF-κB decoy ODNs-modified DCs. aP < 0.001 vs group A, cP < 0.001 vs group A , eP < 0.001 vs group B, gP < 0.001 vs group A, iP < 0.001 vs group B, kP < 0.001 vs group C, nP > 0.05 vs group B.
DISCUSSION

Our and other studies have shown that NF-κB decoy ODNs-modified immature DCs, which are stably deficient in surface costimulatory molecules and allosimulatory capacity, could inhibit alloantigen-specific T cell response and prolong cardiac and liver allograft survival in unmodified recipients[25-27]. NF-κB also has been found to be an important transcriptional regulator of liver regeneration[36-39]. NF-κB could prevent hepatocytes from undergoing apoptosis during development and liver regeneration, and inhibition of NF-κB activation could induce apoptosis but not proliferation of hepatocytes. Whether the immature DCs modified with NF-κB decoy ODNs, which inhibit NF-κB activation, can interfere with the liver allograft regeneration is unknown. To study the effect of NF-κB decoy ODNs-modified DCs on hepatic regeneration after liver transplantation, we measured the BrdU labeling index of hepatocytes in the partial liver allograft. Uchiyama et al[6] demonstrated that DNA synthesis in liver was slower after an orthotopic transplantation than after a partial hepatectomy. In our partial liver transplantation model, the BrdU labeling index of hepatocytes peaked at 72 h after a allogeneic transplantation, in contrast to the maximum DNA synthesis at 24 h that has been documented after a partial hepatectomy[40]. In the present study immature DCs or mature DCs were administered to recipient rats 7 days prior to liver transplantation and immediately after liver transplantation to detect whether NF-κB decoy ODNs-modified DCs could improve the delayed liver graft regeneration. Our results showed that partial liver graft DNA synthesis peaked earlier in group D rats (pretreatment with NF-κB decoy ODNs-modified immature DCs) but slower in group C rats (pretreatment with mature DCs) than that in the other groups rats. The maximum BrdU labeling index of hepatocytes in group D rats was significantly higher than that in the other groups rats, whereas the maximum BrdU labeling index of hepatocytes in group C rats was significantly lower than that in the other groups rats. In the present study, we also found that pretreatment with NF-κB decoy ODNs-modified DCs could reduce apoptosis of hepatocytes in the liver graft, however, pretreatment with mature DCs could promote apoptosis of hepatocytes in the liver graft. In situ TUNEL staining of the liver graft sections revealed that the maximum apoptosis of hepatocytes was detected in group C rats and the minimum apoptosis of hepatocytes was detected in group D rats. Our results suggested that NF-κB decoy ODNs-modified DCs could suppress apoptosis of hepatocytes and promote partial liver allograft regeneration.

The mechanisms by which NF-κB decoy ODNs-modified DCs promote regeneration of partial liver allograft remain to be clarified. In the present study, we found that the augmented regeneration of liver graft induced by NF-κB decoy ODNs-modified DCs might be associated with the reduced activity of liver-resident NK cells and the decreased IFN-γ production. A negative correlation was observed between liver regeneration and immune reaction (NK cell activity) in other studies[11,12], for the liver-resident and spleen-resident NK cells showed specific cytotoxicity against regenerating hepatocytes. Previous studies have shown that the liver grafts contained large numbers of NK cells and NK like cells with early lymphocyte activation before transplantation, and there was an influx of recipient NK and NK-like cells into the liver graft immediately after revascularization[41] and their cytotoxic activity was significantly augmented in the rejected liver allograft[42,43]. Other experimental studies have demonstrated that FasL expression on activated NK cells was augmented[44,45], and FasL ligation to Fas expressed on hepatocytes could mediate hepatocytes apoptosis[46,47]. Moreover, NK cells produce IFN-γ, which is involved in the aggravation of fulminant hepatitis. Thus, a decrease in NK cell activity may lead to reduction of IFN-γ. Although the definite role of NK cell activity and IFN-γ in liver regeneration is to be clarified, a rapid and profound suppression of a variety of spontaneous functions of NK cells in the liver or blood at the initiation phase of liver regeneration has been shown, including the rapidly decreased NK cell numbers and NK cell activity[11,12]. Vujanovic et al[14] found that depletion of NK cells by specific antibody accelerated rat liver regeneration after 70% partial hepatectomy. Tanigawa and Tamura[11,12] also found that FK506 and augmenter of liver regeneration promoted liver regeneration by reducing NK cell activity. Liu et al[9] recently suggested that the enhanced liver regeneration by oral ursodesoxycholic acid was mediated by suppression of NK cell activity in partially hepatectomized rats, and interleukin-2, a potent inducer of allograft rejection, could completely or partially block the decrease in NK activity and the increase in hepatocyte mitotic index. IFN-γ is secreted predominantly by activated T lymphocytes and activated NK cells interact in vitro in a complex network of mediators with other immune cytokines. IFN-γ is a potent stimulus for monocytes and macrophages that increases their expression of class II MHC antigens and Fc receptors. It also enhances TNF-α secretion. IFN-γ is also known to control the expression of mitochondrial transcription factor A, a nuclear gene responsible for mitochondrial metabolism. Moreover, IFN-γ could inhibit liver regeneration by stimulating MHC class II antigens expression by Kupffer cells in the regenerating liver[48]. These MHC class II antigen-positive Kupffer cells act as antigen-presenting cells and present hepatocyte as antigen, the so-called abnormal “self ”, to helper and cytotoxic T cells. Both types of T cells, in turn, may suppress hepatocyte proliferation. In this respect, a synergistic effect was also observed with the combination of IL-2 and IFN-γ. A recent study demonstrated that administration of IFN-γ inhibited liver regeneration by decreasing the mitochondrial transcription factor A expression[10]. These data suggested that augmentation of NK cell activity and IFN-γ production could inhibit liver regeneration. In the present study, we found that liver allograft-resident NK cell activity and hepatic IFN-γ mRNA expression and serum INF-γ production at the time of the maximal liver allograft regeneration were significantly suppressed by pretreatment with NF-κB decoy ODNs-modified immature DCs, whereas liver allograft-resident NK cell activity and hepatic IFN-γ mRNA expression and serum INF-γ production at the time of the maximal liver allograft regeneration were significantly elevated by pretreatment with mature DCs. The results suggested that there was a negative relationship between liver allograft regeneration and NK cell activity and IFN-γ production in the present study.

The mechanisms by which NF-κB decoy ODNs-modified immature DCs suppress the activity of liver allograft-resident NK cells and IFN-γ production after liver transplantation are still to be clarified. In our previous study we found in vitro and in vivo evidences that the stably immature NF-κB decoy ODNs-treated DCs could suppress T cell allostimulatory ability, promote apoptotic death of live graft-infiltrating lymphocytes, and consequently suppress Th1 immunostimulatory cytokines such as IL-2 and IFN-γ mRNA expression in the liver graft[27]. We hypothesized that the enhanced apoptosis of liver graft-infiltrating lymphocytes by NF-κB decoy ODNs-modified DCs could reduce the total number of the activated liver-resident NK cells and suppress the NK cell cytotoxicity to the regenerating hepatocytes. At the same time, the enhanced apoptosis of liver graft-infiltrating lymphocytes could decrease IFN-γ mRNA expression and IFN-γ production in the liver graft.

In summary, pretreatment with NF-κB decoy ODNs-modified immature DCs can suppress liver allograft-resident NK cell activity and IFN-γ production. The suppressed liver allograft-resident NK cell activity and IFN-γ production, in turn, may suppress hepatocyte apoptosis and promote regeneration of partial liver allograft.

Footnotes

Edited by Wu XN and Wang XL

References
1.  Kido M, Ku Y, Fukumoto T, Tominaga M, Iwasaki T, Ogata S, Takenaga M, Takahashi M, Kuroda Y, Tahara S. Significant role of middle hepatic vein in remnant liver regeneration of right-lobe living donors. Transplantation. 2003;75:1598-1600.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 40]  [Cited by in F6Publishing: 39]  [Article Influence: 1.9]  [Reference Citation Analysis (0)]
2.  Eguchi S, Yanaga K, Sugiyama N, Okudaira S, Furui J, Kanematsu T. Relationship between portal venous flow and liver regeneration in patients after living donor right-lobe liver transplantation. Liver Transpl. 2003;9:547-551.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 75]  [Cited by in F6Publishing: 81]  [Article Influence: 3.9]  [Reference Citation Analysis (0)]
3.  Maetani Y, Itoh K, Egawa H, Shibata T, Ametani F, Kubo T, Kiuchi T, Tanaka K, Konishi J. Factors influencing liver regeneration following living-donor liver transplantation of the right hepatic lobe. Transplantation. 2003;75:97-102.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 69]  [Cited by in F6Publishing: 71]  [Article Influence: 3.4]  [Reference Citation Analysis (0)]
4.  Kamel IR, Erbay N, Warmbrand G, Kruskal JB, Pomfret EA, Raptopoulos V. Liver regeneration after living adult right lobe transplantation. Abdom Imaging. 2003;28:53-57.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 40]  [Cited by in F6Publishing: 40]  [Article Influence: 1.9]  [Reference Citation Analysis (0)]
5.  Marcos A, Fisher RA, Ham JM, Shiffman ML, Sanyal AJ, Luketic VA, Sterling RK, Fulcher AS, Posner MP. Liver regeneration and function in donor and recipient after right lobe adult to adult living donor liver transplantation. Transplantation. 2000;69:1375-1379.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 258]  [Cited by in F6Publishing: 236]  [Article Influence: 9.8]  [Reference Citation Analysis (0)]
6.  Uchiyama H, Yanaga K, Nishizaki T, Soejima Y, Yoshizumi T, Sugimachi K. Effects of deletion variant of hepatocyte growth factor on reduced-size liver transplantation in rats. Transplantation. 1999;68:39-44.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 19]  [Cited by in F6Publishing: 21]  [Article Influence: 0.8]  [Reference Citation Analysis (0)]
7.  Selzner N, Selzner M, Tian Y, Kadry Z, Clavien PA. Cold ischemia decreases liver regeneration after partial liver transplantation in the rat: A TNF-alpha/IL-6-dependent mechanism. Hepatology. 2002;36:812-818.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 71]  [Cited by in F6Publishing: 90]  [Article Influence: 4.1]  [Reference Citation Analysis (0)]
8.  Cheung ST, Tsui TY, Wang WL, Yang ZF, Wong SY, Ip YC, Luk J, Fan ST. Liver as an ideal target for gene therapy: expression of CTLA4Ig by retroviral gene transfer. J Gastroenterol Hepatol. 2002;17:1008-1014.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 23]  [Cited by in F6Publishing: 23]  [Article Influence: 1.0]  [Reference Citation Analysis (0)]
9.  Liu L, Sakaguchi T, Cui X, Shirai Y, Nishimaki T, Hatakeyama K. Liver regeneration enhanced by orally administered ursodesoxycholic acid is mediated by immunosuppression in partially hepatectomized rats. Am J Chin Med. 2002;30:119-126.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 20]  [Cited by in F6Publishing: 22]  [Article Influence: 1.0]  [Reference Citation Analysis (0)]
10.  Polimeno L, Margiotta M, Marangi L, Lisowsky T, Azzarone A, Ierardi E, Frassanito MA, Francavilla R, Francavilla A. Molecular mechanisms of augmenter of liver regeneration as immunoregulator: its effect on interferon-gamma expression in rat liver. Dig Liver Dis. 2000;32:217-225.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 34]  [Cited by in F6Publishing: 41]  [Article Influence: 1.7]  [Reference Citation Analysis (0)]
11.  Tanigawa K, Sakaida I, Masuhara M, Hagiya M, Okita K. Augmenter of liver regeneration (ALR) may promote liver regeneration by reducing natural killer (NK) cell activity in human liver diseases. J Gastroenterol. 2000;35:112-119.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 60]  [Cited by in F6Publishing: 65]  [Article Influence: 2.7]  [Reference Citation Analysis (0)]
12.  Tamura F, Masuhara A, Sakaida I, Fukumoto E, Nakamura T, Okita K. FK506 promotes liver regeneration by suppressing natural killer cell activity. J Gastroenterol Hepatol. 1998;13:703-708.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 39]  [Cited by in F6Publishing: 39]  [Article Influence: 1.5]  [Reference Citation Analysis (0)]
13.  Francavilla A, Vujanovic NL, Polimeno L, Azzarone A, Iacobellis A, Deleo A, Hagiya M, Whiteside TL, Starzl TE. The in vivo effect of hepatotrophic factors augmenter of liver regeneration, hepatocyte growth factor, and insulin-like growth factor-II on liver natural killer cell functions. Hepatology. 1997;25:411-415.  [PubMed]  [DOI]  [Cited in This Article: ]
14.  Vujanovic NL, Polimeno L, Azzarone A, Francavilla A, Chambers WH, Starzl TE, Herberman RB, Whiteside TL. Changes of liver-resident NK cells during liver regeneration in rats. J Immunol. 1995;154:6324-6338.  [PubMed]  [DOI]  [Cited in This Article: ]
15.  Ohnishi H, Muto Y, Maeda T, Hayashi T, Nagaki M, Yamada T, Shimazaki M, Yamada Y, Sugihara J, Moriwaki H. Natural killer cell may impair liver regeneration in fulminant hepatic failure. Gastroenterol Jpn. 1993;28 Suppl 4:40-44; discussion 53-56.  [PubMed]  [DOI]  [Cited in This Article: ]
16.  Matsushita T, Ando K, Kimura K, Ohnishi H, Imawari M, Muto Y, Moriwaki H. IL-12 induces specific cytotoxicity against regenerating hepatocytes in vivo. Int Immunol. 1999;11:657-665.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 28]  [Cited by in F6Publishing: 28]  [Article Influence: 1.1]  [Reference Citation Analysis (0)]
17.  Sato Y, Farges O, Buffello D, Bismuth H. Intra- and extrahepatic leukocytes and cytokine mRNA expression during liver regeneration after partial hepatectomy in rats. Dig Dis Sci. 1999;44:806-816.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 12]  [Cited by in F6Publishing: 12]  [Article Influence: 0.5]  [Reference Citation Analysis (0)]
18.  Lee WC, Zhong C, Qian S, Wan Y, Gauldie J, Mi Z, Robbins PD, Thomson AW, Lu L. Phenotype, function, and in vivo migration and survival of allogeneic dendritic cell progenitors genetically engineered to express TGF-beta. Transplantation. 1998;66:1810-1817.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 92]  [Cited by in F6Publishing: 95]  [Article Influence: 3.7]  [Reference Citation Analysis (0)]
19.  Hayamizu K, Huie P, Sibley RK, Strober S. Monocyte-derived dendritic cell precursors facilitate tolerance to heart allografts after total lymphoid irradiation. Transplantation. 1998;66:1285-1291.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 57]  [Cited by in F6Publishing: 59]  [Article Influence: 2.3]  [Reference Citation Analysis (0)]
20.  Khanna A, Steptoe RJ, Antonysamy MA, Li W, Thomson AW. Donor bone marrow potentiates the effect of tacrolimus on nonvascularized heart allograft survival: association with microchimerism and growth of donor dendritic cell progenitors from recipient bone marrow. Transplantation. 1998;65:479-485.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 15]  [Cited by in F6Publishing: 15]  [Article Influence: 0.6]  [Reference Citation Analysis (0)]
21.  Lu L, Li W, Zhong C, Qian S, Fung JJ, Thomson AW, Starzl TE. Increased apoptosis of immunoreactive host cells and augmented donor leukocyte chimerism, not sustained inhibition of B7 molecule expression are associated with prolonged cardiac allograft survival in mice preconditioned with immature donor dendritic cells plus anti-CD40L mAb. Transplantation. 1999;68:747-757.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 67]  [Cited by in F6Publishing: 67]  [Article Influence: 2.7]  [Reference Citation Analysis (0)]
22.  Lutz MB, Suri RM, Niimi M, Ogilvie AL, Kukutsch NA, Rössner S, Schuler G, Austyn JM. Immature dendritic cells generated with low doses of GM-CSF in the absence of IL-4 are maturation resistant and prolong allograft survival in vivo. Eur J Immunol. 2000;30:1813-1822.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in F6Publishing: 7]  [Reference Citation Analysis (0)]
23.  Chiang YJ, Lu L, Fung JJ, Qian S. Liver-derived dendritic cells induce donor-specific hyporesponsiveness: use of sponge implant as a cell transplant model. Cell Transplant. 2001;10:343-350.  [PubMed]  [DOI]  [Cited in This Article: ]
24.  Yoo-Ott KA, Schiller H, Fändrich F, Oswald H, Richter K, Xhu XF, Kampen WU, Krönke M, Zavazava N. Co-transplantation of donor-derived hepatocytes induces long-term tolerance to cardiac allografts in a rat model. Transplantation. 2000;69:2538-2546.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 11]  [Cited by in F6Publishing: 11]  [Article Influence: 0.5]  [Reference Citation Analysis (0)]
25.  Giannoukakis N, Bonham CA, Qian S, Chen Z, Peng L, Harnaha J, Li W, Thomson AW, Fung JJ, Robbins PD. Prolongation of cardiac allograft survival using dendritic cells treated with NF-kB decoy oligodeoxyribonucleotides. Mol Ther. 2000;1:430-437.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 114]  [Cited by in F6Publishing: 122]  [Article Influence: 5.1]  [Reference Citation Analysis (0)]
26.  Bonham CA, Peng L, Liang X, Chen Z, Wang L, Ma L, Hackstein H, Robbins PD, Thomson AW, Fung JJ. Marked prolongation of cardiac allograft survival by dendritic cells genetically engineered with NF-kappa B oligodeoxyribonucleotide decoys and adenoviral vectors encoding CTLA4-Ig. J Immunol. 2002;169:3382-3391.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 101]  [Cited by in F6Publishing: 106]  [Article Influence: 4.8]  [Reference Citation Analysis (0)]
27.  Xu MQ, Suo YP, Gong JP, Wang F, Yan LN; Tolerance of rat liver allograft induced by nuclear factor-B( decoy oligodeoxynucleoides-modified dendritic cells. World J Gastroenterol (In process).  .  [PubMed]  [DOI]  [Cited in This Article: ]
28.  Abeyama K, Eng W, Jester JV, Vink AA, Edelbaum D, Cockerell CJ, Bergstresser PR, Takashima A. A role for NF-kappaB-dependent gene transactivation in sunburn. J Clin Invest. 2000;105:1751-1759.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 132]  [Cited by in F6Publishing: 123]  [Article Influence: 5.1]  [Reference Citation Analysis (0)]
29.  Xu MQ, Yao ZX. Functional changes of dendritic cells derived from allogeneic partial liver graft undergoing acute rejection in rats. World J Gastroenterol. 2003;9:141-147.  [PubMed]  [DOI]  [Cited in This Article: ]
30.  Bouwens L, Wisse E. Immuno-electron microscopic characterization of large granular lymphocytes (natural killer cells) from rat liver. Eur J Immunol. 1987;17:1423-1428.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 37]  [Cited by in F6Publishing: 38]  [Article Influence: 1.0]  [Reference Citation Analysis (0)]
31.  Ikejima K, Enomoto N, Iimuro Y, Ikejima A, Fang D, Xu J, Forman DT, Brenner DA, Thurman RG. Estrogen increases sensitivity of hepatic Kupffer cells to endotoxin. Am J Physiol. 1998;274:G669-G676.  [PubMed]  [DOI]  [Cited in This Article: ]
32.  McKnight AJ, Barclay AN, Mason DW. Molecular cloning of rat interleukin 4 cDNA and analysis of the cytokine repertoire of subsets of CD4+ T cells. Eur J Immunol. 1991;21:1187-1194.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 158]  [Cited by in F6Publishing: 168]  [Article Influence: 5.1]  [Reference Citation Analysis (0)]
33.  Plümpe J, Malek NP, Bock CT, Rakemann T, Manns MP, Trautwein C. NF-kappaB determines between apoptosis and proliferation in hepatocytes during liver regeneration. Am J Physiol Gastrointest Liver Physiol. 2000;278:G173-G183.  [PubMed]  [DOI]  [Cited in This Article: ]
34.  Taira K, Hiroyasu S, Shiraishi M, Muto Y, Koji T. Role of the Fas system in liver regeneration after a partial hepatectomy in rats. Eur Surg Res. 2001;33:334-341.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 14]  [Cited by in F6Publishing: 15]  [Article Influence: 0.7]  [Reference Citation Analysis (0)]
35.  Masson S, Scotté M, Garnier S, François A, Hiron M, Ténière P, Fallu J, Salier JP, Daveau M. Differential expression of apoptosis-associated genes post-hepatectomy in cirrhotic vs. normal rats. Apoptosis. 2000;5:173-179.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 20]  [Cited by in F6Publishing: 21]  [Article Influence: 0.9]  [Reference Citation Analysis (0)]
36.  Kirillova I, Chaisson M, Fausto N. Tumor necrosis factor induces DNA replication in hepatic cells through nuclear factor kappaB activation. Cell Growth Differ. 1999;10:819-828.  [PubMed]  [DOI]  [Cited in This Article: ]
37.  Brenner DA. Signal transduction during liver regeneration. J Gastroenterol Hepatol. 1998;13 Suppl:S93-S95.  [PubMed]  [DOI]  [Cited in This Article: ]
38.  Iimuro Y, Nishiura T, Hellerbrand C, Behrns KE, Schoonhoven R, Grisham JW, Brenner DA. NFkappaB prevents apoptosis and liver dysfunction during liver regeneration. J Clin Invest. 1998;101:802-811.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 371]  [Cited by in F6Publishing: 355]  [Article Influence: 13.7]  [Reference Citation Analysis (0)]
39.  Chaisson ML, Brooling JT, Ladiges W, Tsai S, Fausto N. Hepatocyte-specific inhibition of NF-kappaB leads to apoptosis after TNF treatment, but not after partial hepatectomy. J Clin Invest. 2002;110:193-202.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 134]  [Cited by in F6Publishing: 127]  [Article Influence: 5.8]  [Reference Citation Analysis (0)]
40.  Xu M, Han B, Xue L, Gong J. [Ursodeoxycholic acid promotes liver regeneration after partial hepatectomy in bile duct obstructive rats]. Zhonghua Ganzangbing Zazhi. 2002;10:103-105.  [PubMed]  [DOI]  [Cited in This Article: ]
41.  Navarro F, Portalès P, Candon S, Pruvot FR, Pageaux G, Fabre JM, Domergue J, Clot J. Natural killer cell and alphabeta and gammadelta lymphocyte traffic into the liver graft immediately after liver transplantation. Transplantation. 2000;69:633-639.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 20]  [Cited by in F6Publishing: 20]  [Article Influence: 0.8]  [Reference Citation Analysis (0)]
42.  Li W, Lu L, Wang Z, Wang L, Fung JJ, Thomson AW, Qian S. Il-12 antagonism enhances apoptotic death of T cells within hepatic allografts from Flt3 ligand-treated donors and promotes graft acceptance. J Immunol. 2001;166:5619-5628.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 56]  [Cited by in F6Publishing: 57]  [Article Influence: 2.5]  [Reference Citation Analysis (0)]
43.  Navarro F, Portalès P, Pageaux JP, Perrigault PF, Fabre JM, Domergue J, Clot J. Activated sub-populations of lymphocytes and natural killer cells in normal liver and liver grafts before transplantation. Liver. 1998;18:259-263.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 9]  [Cited by in F6Publishing: 7]  [Article Influence: 0.3]  [Reference Citation Analysis (0)]
44.  Hsieh CL, Obara H, Ogura Y, Martinez OM, Krams SM. NK cells and transplantation. Transpl Immunol. 2002;9:111-114.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 31]  [Cited by in F6Publishing: 36]  [Article Influence: 1.6]  [Reference Citation Analysis (0)]
45.  Kojima Y, Kawasaki-Koyanagi A, Sueyoshi N, Kanai A, Yagita H, Okumura K. Localization of Fas ligand in cytoplasmic granules of CD8+ cytotoxic T lymphocytes and natural killer cells: participation of Fas ligand in granule exocytosis model of cytotoxicity. Biochem Biophys Res Commun. 2002;296:328-336.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 30]  [Cited by in F6Publishing: 31]  [Article Influence: 1.4]  [Reference Citation Analysis (0)]
46.  Ariki N, Morimoto Y, Yagi T, Oyama T, Cyouda Y, Sadamori H, Inagaki M, Urushihara N, Iwagaki H, Tanaka N. Activated T cells and soluble molecules in the portal venous blood of patients with cholestatic and hepatitis C virus-positive liver cirrhosis. Possible promotion of Fas/FasL-mediated apoptosis in the bile-duct cells and hepatocyte injury. J Int Med Res. 2003;31:170-180.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 5]  [Cited by in F6Publishing: 6]  [Article Influence: 0.3]  [Reference Citation Analysis (0)]
47.  Wang J, Li W, Min J, Ou Q, Chen J. Fas siRNA reduces apoptotic cell death of allogeneic-transplanted hepatocytes in mouse spleen. Transplant Proc. 2003;35:1594-1595.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 19]  [Cited by in F6Publishing: 21]  [Article Influence: 1.0]  [Reference Citation Analysis (0)]
48.  Sato Y, Tsukada K, Matsumoto Y, Abo T. Interferon-gamma inhibits liver regeneration by stimulating major histocompatibility complex class II antigen expression by regenerating liver. Hepatology. 1993;18:340-346.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 33]  [Cited by in F6Publishing: 32]  [Article Influence: 1.0]  [Reference Citation Analysis (0)]