Recombinant adenovirus vector Ad-hIL-10 protects grafts from cold ischemia-reperfusion injury following orthotopic liver transplantation in rats

Zhong-Zhou Si, Jie-Qun Li, Hai-Zhi Qi, Zhi-Jun He, Wei Hu and Yi-Ning Li

Changsha, China

Recombinant adenovirus vector Ad-hIL-10 protects grafts from cold ischemia-reperfusion injury following orthotopic liver transplantation in rats

Zhong-Zhou Si, Jie-Qun Li, Hai-Zhi Qi, Zhi-Jun He, Wei Hu and Yi-Ning Li

Changsha, China

BACKGROUND:Interleukin 10 (IL-10), a Th2 type cytokine, modulates inflammatory responses by inhibiting the production of proinflammatory cytokines. This study was designed to investigate the protective effects of adenovirusmediated human IL-10 (Ad-hIL-10) gene transfer on protecting grafts from cold ischemia-reperfusion injury following orthotopic liver transplantation in rats.

METHODS:Adenoviruses encoding hIL-10 orβ-galactosidase (Ad-lacZ) were injected via the superior mesenteric vein into prospective donor animals. The donor liver was harvested 48 hours after transduction, and stored for 12 hours at 4℃in lactated Ringer's solution prior to transplantation. The rats were divided into saline, Ad-lacZ, and Ad-hIL-10 groups. Liver function test, histopathological examination, reverse transcriptase-polymerase chain reaction (RT-PCR), and Western blotting were performed at 24 hours after transplantation in the three groups.

RESULTS:Liver function (ALT and AST) was significantly improved, and the Suzuki score was significantly decreased in the Ad-hIL-10 group. The levels of hepatic TNF-α, MIP-2, ICAM-1 mRNA, and NF-κB protein in the Ad-hIL-10 group were significantly decreased. The expression of hIL-10 mRNA was detected by RT-PCR in Ad-hIL-10-treated grafts but not in controls treated with saline or Ad-lacZ.

CONCLUSIONS:Donor pretreatment with Ad-hIL-10 downregulates the expression of proinflammatory cytokines TNF-α, MIP-2, and ICAM-1 mRNA. hIL-10 protects against hepatic cold ischemia-reperfusion injury, at least in part, bysuppressing NF-κB activation and subsequent expression of proinflammatory mediators.

(Hepatobiliary Pancreat Dis Int 2010; 9: 144-148)

adenovirus vector; interleukin 10; ischemia-reperfusion injury; gene transfer

Introduction

Ischemia-reperfusion injury (IRI), an antigenindependent component of harvesting insult, affects the outcome after transplantation and plays an important role in post-transplant complications, including primary nonfunction and acute and chronic rejection.[1,2]The development of new strategies to minimize hepatic IRI could have a major impact on the outcome after transplantation.

Interleukin 10 (IL-10), a Th2-type cytokine, is known to modulate inflammatory responses by inhibiting the production of proinflammatory cytokines such as TNF-α, IL-1, IL-6, and IL-8[3-5]and upregulating monocyte production of soluble TNF receptor and IL-1 receptor antagonist.[6]Administration of recombinant IL-10 (rIL-10) inhibits hepatic warm IRI by suppressing nuclear factorkappa B (NF-κB) activation and subsequent expression of proinflammatory mediators.[7]However, recombinant cytokines, including IL-10, are short-lived and have limited access to the tissue interstitiumin vivo.[8,9]This short half-life is likely to be an obstacle to the use of rIL-10 in transplantation.[10]Gene transfer techniques are attractive vehicles for prolonging the expression of shortlived proteins such as cytokines. Based on these data, we constructed a recombinant adenovirus vector, Ad-hIL-10, expressing the hIL-10 gene and evaluated its effects on preventing IRI in a rat model of liver transplantation.

Methods

Construction of recombinant adenoviral vectors

The hIL-10 cDNA was cloned by PCR from pCDNA3.1-hIL-10, as described,[11]then cloned into the Kpn Ⅰand Hind Ⅲ sites of pShuttle-CMV. The plasmid was linearized by restriction digestion and co-transformed into BJ5183 cells with pAdEasy-1 to produce recombinant adenovirus plasmid. This plasmid was subsequently transfected into AD-293 cells to produce packaging recombinant adenovirus granules. After identification, the desired recombinant adenovirus was purified by density gradient ultracentrifugation and titrated. Packaging recombinant adenovirus granules (Ad-hIL-10 and Ad-lacZ) with high purity and titer (2×1010IU/ml) were generated after density gradient ultracentrifugation.

Animals and surgical procedures

Sprague-Dawley rats (10-12 weeks of age, weighing 250-300 g; Experimental Animal Center, Medical College, Central South University, Changsha, China) were used as donors and recipients. All animal experiments were conducted in accordance with the guidelines approved by the Chinese Association of Laboratory Animal Care. A rat nonarterialized orthotopic liver transplantation mode was performed according to the techniques described by Kamada.[12]

Experimental design

The experiments were conducted in three groups of rats: an Ad-hIL-10 group, an Ad-lacZ control group, and a saline control group. One milliliter of Ad-hIL-10, Ad-lacZ (2×1010IU), or 0.9% saline was injected via the superior mesenteric vein into prospective donor animals after a simple laparotomy. Forty-eight hours later, the donor livers were harvested, and stored for 12 hours in lactated Ringer's solution at 4 ℃ prior to transplantation. Blood and liver tissue samples were obtained 24 hours after reperfusion (n=6).

Histology

Liver tissue was fixed in 10% buffered formalin and embedded in paraffin. Sections (5 μm) were stained with hematoxylin and eosin. Histological severity of IRI in liver grafts was graded using the Suzuki classification, in which sinusoidal congestion, hepatocyte necrosis, and ballooning degeneration are graded from 0 to 4.[13]No necrosis, congestion, or centrilobular ballooning is given a score of 0, while severe congestion, ballooning degeneration, or >60% lobular necrosis is given a value of 4. All histological evaluations were done in a doubleblinded fashion.

Expression of cytokine mRNA in liver tissue

To study the expression of cytokine mRNA, we used competitive template reverse transcription-polymerase chain reaction (RT-PCR) as described.[14]Briefly, total RNA was extracted from frozen liver samples with an RNeasy mini kit (Qiagen, Chatsworth, CA, USA). Five micrograms of RNA was reverse transcribed with oligo(dT) primers and SuperScript reverse transcriptase (Invitrogen GIBCO, USA). The primer sequences for TNF-α, hIL-10, intercellular adhesion molecule-1 (ICAM-1), macrophage-inflammatory protein-2 (MIP-2) and β-actin were: β-actin sense, 5'-CTC TTC CAG CCT TCC TTC CT-3' and antisense, 5'-TAG AGC CAC CAA TCC ACA CA-3'; ICAM-1 sense, 5'-CTC TGC TCC TGG TCC TGG T-3' and antisense, 5'-CGT GAA TGT GAT CTC CTT GG-3'; MIP-2 sense, 5'-CCT CAA CGG AAG AAC CAA AG-3' and antisense, 5'-CAA GAC ACG AAA AGG CAT GA-3'; hIL-10 sense, 5'-TCT TGC AAA ACC AAA CCA CA-3' and antisense, 5'-TGA GGG TCT TCA GGT TCT CC-3'; TNF-α sense, 5'-TAT GGC TCA GGG TCC AAC TC-3' and antisense, 5'-TGG TCA CCA AAT CAG CGT TA-3'. Various cycles of PCR were performed at the annealing temperature that was optimized empirically for each primer pair: 26 cycles, 72 ℃ (hIL-10); 30 cycles, 72 ℃ (ICAM-1); 28 cycles, 72 ℃ (TNF-α); 34 cycles, 60 ℃ (MIP-2); and 28 cycles, 72 ℃ (α-actin). PCR products were analyzed in ethidium bromide-stained 2% agarose gels, and scanned with Kodak 1D Image Analysis software (version 2.0; Eastman Kodak, Rochester, NY, USA). All samples were normalized against the respective α-actin template cDNA ratio.

Western blotting analysis

Protein was extracted from tissue samples with protein lysis buffer (50 mmol/L Tris, 150 mmol/L NaCl, 0.1% SDS, 1% sodium deoxycholate, and 1% Triton X-100, pH 7.2). Protein (30 μg/sample) in SDS-loading buffer (50 mmol/L Tris, pH 7.6, 10% glycerol, 1% SDS) was subjected to 12% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The gel was then stained with Coomassie blue for protein loading. The membrane was blocked with 3% dry milk and 0.1% Tween 20 (USB, Cleveland, OH, USA) in PBS. Polyclonal rabbit anti-rat NF-κB Ab (Santa Cruz, CA, USA) was used. The membranes were incubated with antibodies and developed according to the enhanced chemiluminescence protocol (GE Healthcare, USA). Relative quantities of NF-κB proteins were determined by densitometry (Kodak Digital Science 1D Analysis Software, Rochester, NY, USA).

Table 1. ALT and AST serum levels in rats after 24-hour reperfusion (mean±SD)

Table 2. Histologic criteria (after Suzuki) for grading liver IRI after 24-hour reperfusion (mean±SD)

Table 3. Intragraft expression of TNF-α mRNA, ICAM-1 mRNA, MIP-2 mRNA, hIL-10 mRNA, and NF-κB protein after 24-hour reperfusion (mean±SD)

Fig. A-D: RT-PCR analysis of cytokine mRNAs in liver allograft after 24-hour reperfusion. E: Effects of Ad-hIL-10 on the expression of NF-κB detected by Western blotting analysis after 24-hour reperfusion.

Statistical analysis

All data were expressed as mean±SD. Comparisons between the groups were performed using Student'sttest or one-way analysis of variance (ANOVA) when necessary. The histological samples were subjected to the Kruskal-Wallis test to search for significant differences among the groups. SPSS 14.0 software (SPSS Inc., Chicago, IL, USA) was used for all statistical analyses. APvalue less than 0.05 was considered statistically significant.

Results

Ad-hIL-10 pretreatment improves hepatocyte function and ameliorates histologic signs of IRIThe serum levels of ALT and AST were significantly lower in the Ad-hIL-10 group than in the saline and Ad-lacZ control groups at 24 hours after reperfusion (Table 1). In correlation with the serum levels of ALT and AST, the saline and Ad-lacZ control groups revealed moderate to severe hepatocyte necrosis with prominent sinusoidal or vascular congestion (Table 2).

Expression of cytokine mRNAs and NF-κB protein in the liver allografts

The expression of hIL-10 mRNA was detected by RTPCR in Ad-hIL-10-treated grafts but not in control grafts treated with saline or Ad-lacZ. Successful transduction of Ad-hIL-10 to the hepatic graft was thus confirmed at themRNA level. RT-PCR revealed markedly decreased levels of TNF-α, ICAM-1, and MIP-2 mRNA in Ad-hIL-10-treated rats, compared to the saline and Ad-lacZ control groups (Table 3 and Fig. A-D). These findings imply that hIL-10 gene transfer leads to a decrease in the expression of proinflammatory cytokine levels in the allografts. Western blotting was used to evaluate the expression of NF-κB in hepatic grafts, and significantly decreased NF-κB was noted in the Ad-hIL-10 group compared to the two control groups (Fig. E).

Discussion

In this study, we constructed a recombinant adenovirus vector to express hIL-10, and hIL-10 mRNA expression was detected by RT-PCR in Ad-hIL-10-treated grafts but not in control grafts treated with saline or Ad-lacZ. Successful transduction of Ad-hIL-10 to the hepatic graft was thus confirmed at the mRNA level. Adenoviral gene transfer of hIL-10 downregulated the expression of NF-κB and mRNA of proinflammatory mediators (MIP-2, ICAM-1, and TNF-α), thus improving liver function and preserving hepatocyte integrity and architecture. The current study complemented recent studies showing that exogenous rIL-10 inhibits liver injury caused by warm IRI in mice,[7]and that a selective gene therapy approach provides evidence that hIL-10 protects the liver graft from IRI in a clinically relevant cold IRI model.

In experimental animal models, two distinct phases in the development of organ injury induced by hepatic ischemia and reperfusion can be identified. During the initial phase, Kupffer cells are activated and release reactive oxygen species and proinflammatory cytokines, including TNF-α.[15,16]Although these products may directly injure liver parenchymal cells, organ dysfunction is minimal. The enhanced production of TNF-α, however, plays an important role in the initiation of a cascade of events that causes significant liver injury mediated by neutrophils. One of the main functions of TNF-α is the upregulation of adhesion molecules and neutrophil-attracting CXC chemokines.[17,18]The coordinated efforts of adhesion molecules such as ICAM-1, and CXC chemokines such as MIP-2, mediate the recruitment of neutrophils into the liver. Sequestered neutrophils release proteases and reactive oxygen intermediates, which directly damage hepatocytes and endothelial cells and also contribute to capillary plugging, causing hepatic hypoperfusion.[19,20]

A common link between cytokines (TNF-α), chemokines (MIP-2), and adhesion molecules (ICAM-1) involved in the development of hepatic IRI is their transcriptional regulation. Each of these mediators is controlled, at least in part, by the transcription factor NF-κB.[21-24]The primary form of NF-κB consists of a heterodimer of NF-κB (p50) and RelA (p65), which in most cells is retained in the cytoplasm complexed with inhibitory proteins of the IκB family.[25]In response to inflammatory stimuli, including oxidant stress and proinflammatory cytokines such as TNF-α or IL-1, IκB proteins are phosphorylated, ubiquinated, and degraded in a process requiring the 26S proteasome.[23]Degradation of IκB proteins unmasks the nuclear localization sequence of NF-κB subunits and induces gene transcription. Recent studies found that attenuation of NF-κB activation and subsequent reduction in TNF-α production after sustained ischemia play important roles in the protective mechanism of ischemic preconditioning against hepatic IRI.[26,27]These data suggest a central role of NF-κB activation in the initiation of hepatic IRI, and inhibition of NF-κB activation could protect against hepatic IRI. Therapeutic strategies against proinflammatory mediators in liver grafts with NF-κB decoy oligodeoxynucleotides[28]or by adenoviral IκB[29]gene transfer have already proven effective in reducing IRI. In the present study, the data showed that IRI-induced liver NF-κB activation can be inhibited by IL-10. This is in accordance with a previous study showing that IL-10 suppresses nuclear translocation of NF-κB by preserving the expression of the inhibitory IκB protein, IκBa, in lung cells.[30]

In conclusion, we have documented striking cytoprotection by Ad-hIL-10 against hepatic cold IRI. Downregulation of the proinflammatory cytokines TNF-α, MIP-2, and ICAM-1 mRNA may be associated with this protection. Given the central role of NF-κB activation in the initiation of hepatic IRI, hIL-10 protects against hepatic cold IRI, at least in part, by suppressing NF-κB activation and subsequent expression of proinflammatory mediators.

Acknowledgement

The pCDNA3.1-hIL-10 plasmid was a kind gift from Jie-Xiong Tan, MD (State Key Laboratory of Genetics, Changsha, China).

Funding:None.

Ethical approval:All animal experiments were conducted in accordance with the guidelines approved by the Chinese Association of Laboratory Animal Care.

Contributors:SZZ and LJQ proposed the study and wrote the first draft. LJQ analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. QHZ is the guarantor.

Competing interest:No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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August 10, 2009

Accepted after revision February 12, 2010

Author Affiliations: Department of Organ Transplantation, Second Xiangya Hospital, Central South University, Changsha 410011, China (Si ZZ, Li JQ, Qi HZ, He ZJ, Hu W and Li YN)

Hai-Zhi Qi, MD, Department of Organ Transplantation, Second Xiangya Hospital, Central South University, Changsha 410011, China (Tel: 86-731-85295808; Fax: 86-731-85295808; Email: szz5858@medmail.com.cn)

? 2010, Hepatobiliary Pancreat Dis Int. All rights reserved.