- Email: [email protected]
Immunosuppression by Combined Use of Cyclosporine and Resveratrol in a Rat Liver Transplantation Model
IMMUNOSUPPRESSIVES
Immunosuppression by Combined Use of Cyclosporine and Resveratrol in a Rat Liver Transplantation Model S.L. Wu, C.E. Pan, L. Yu, and K.W. Meng ABSTRACT Introduction. Despite continued progress in the development of immunosuppressive agents, allograft rejection remains an important cause of morbidity and mortality after liver transplantation. We examined the effect of intraperitoneal injection of cyclosporine (CsA) and resveratrol (Res) on allograft rejection after liver transplantation in rats. Methods. Male Sprague-Dawley rats were selected as donors and male Wistar rats as recipients for a rejection model. The recipients were divided into three groups after orthotopic liver transplantation (OLTx): in the combination group both Res (100 mg/kg) and CsA (20 mg/kg) were given by intraperitoneal route once a day, whereas in the CsA group or control group CsA (20 mg/kg) or vehicle buffer was given. The survival period, serum chemistry, production of some cytokines, activation of transcription factor NF- B, and histopathological findings were then compared among them. Results. The mean survival period after OLTx in the combination group was significantly longer than that in the CsA group or control group (P ⬍ .05 and P ⬍ .01). On posttransplant day 7, the serum albumin level significantly improved, the serum total bile acid and alanine aminotransferase levels were significantly lower, the serum interleukin-2 and interferon-␥ levels were significantly lower, and the activation of transcription factor NF-B in peripheral blood T lymphocytes was significantly suppressed in the combination group in comparison with those in the CsA group (all P ⬍ .05) or control group (all P ⬍ .01), and a histological examination revealed apparent difference in the severity of rejection between the combination group and CsA group (P ⬍ .05) or control group (P ⬍ .01). Conclusion. The combined use of CsA and Res has a stronger immunosuppressive effect on hepatocytes under allograft rejection in comparison with the sole use of CsA. Res might serve as a novel supplementary immunosuppressive agent for reducing the severity of hepatic allograft rejection in rats.
S
INCE THE INTRODUCTION of cyclosporine, (CA) and tacrolimus for the control of postoperative rejection, liver transplantation has become an established surgical technique and is now performed on patients with various terminal liver diseases. Most instances of mortality after liver transplantation are still attributed to infection, and such infection is apt to occur as a result of overimmunosuppression to treat severe rejection.1–3 Rejection is still a leading cause of morbidity and mortality after liver transplantation. To improve survival after liver transplantation,
novel strategies are still needed for the treatment of the rejection. Resveratrol (3,5,4=-trihydroxystilbene; Res) is a polypheFrom the No. 1 Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi Province, People’s Republic of China. Address reprint requests to Dr Wu Shengli, No. 1 Hospital of Xi’an Jiaotong University: Department of Hepatobiliary, No. 1 Jianking Road, Xi’an City, Shaanxi Province, 710061, People’s Republic of China. E-mail: [email protected]
0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2005.03.112
© 2005 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
2354
Transplantation Proceedings, 37, 2354 –2359 (2005)
COMBINED USE OF CYCLOSPORINE AND RESVERATROL
nol present in a limited number of plants,4 mainly in grapes, with levels up to tens of grams per Kilogram,5 where it is synthesized in response to stress conditions such as fungal infections and trauma.4 Many studies have demonstrated that this molecule exhibits a wide range of biological and pharmacological activities both in vitro and in vivo.6 A series of studies showed that Res has antioxidant properties,7 anti-inflammatory properties,8,9 and cancer-chemopreventive activity.10,11 Many of the biological activities of resveratrol, like the inhibition of cyclooxygenase,8 induction of CD95 signaling-dependent apoptosis,12 effects on the cell division cycle,13 and the modulation of NF-B activation,14 indicate a possible effect on immune response, and many in vitro experiments have proven that Res has immunomodulatory activity.15–17 To our knowledge, there has so far been no report on the in vivo immunomodulatory effect of Res on hepatic allograft after transplantation. We therefore investigated whether the combined use of Res and CsA may have any beneficial effect on hepatic allograft after transplantation.
MATERIALS AND METHODS Experimental Design Animals. Male Sprague-Dawley rats 9 to 10 weeks old weighing 190 to 210 g as donors and male Wistar rats 7 to 8 weeks weighing 190 to 210 g as recipients were purchased from the Animal Center of Xi’an Jiaotong University (Xi’an, China) and Animal Center of Shanxi Medical College (Shanxi, China), respectively. All rats were allowed free access to water and standard laboratory chow. Before operation the rats were fasted for 12 hours and only allowed free access to water. All animal protocols were approved by the Xi’an Jiaotong University Institutional Animal Care and Use Committee. Reagents. Res, dimethyl sulfoxide (DMSO), and interleukin -2 (IL-2) and interferon-gamma (INF-␥) kits were purchased from Sigma Chemical Co. RPMI-1640, HEPES, EDTA, EGTA, DTT, PMSF, and NP-40 were from Gibco BRL. CsA was purchased from Calbiochem Co. NF-B consensus oligonucleotide and single base pair mutant were from Promega. Fetal bovine serum was from SiJiQing Co (Hangzhou, China).32 P-ATP was from Beijing Isotope Co, China. Orthotopic liver transplantation. Orthotopic rat liver transplantation (OLTx) was performed by the cuff technique as described by Kamada and Calne;18 with some slight modifications. With the rat under ketamine anesthesia (75 mg/kg), the liver was gently skeletonzed and flushed with chilled lactated Ringer’s solution through the abdominal aorta. Special care was taken for minimal manipulation of the graft and portal vein and bile duct for reconstruction. The liver was harvested and stored at 4°C in lactated Ringer’s solution until transplantation. OLTx was performed without hepatic artery reconstruction. The suprahepatic vena cava was anastomosed with 7-0 Prolene continuous suture (Ethicon, Somerville, NJ, USA) and portal vein and inferior vena cava reconstruction was performed by the cuff technique. The bile duct connection was made using an intraluminal epidural catheter stent. Res and CsA administration. The Res was dissolved and sterilized in DMSO first and then diluted in RPMI-1640 to 20 mg/mL. The CsA was diluted in RPMI-1640 to 4 mg/mL. The recipients were randomly divided into three groups after OLTx: in the CsA group, 1 mL of CsA preparation and 1 mL of RPMI-1640 were
2355 administered by intraperitoneal route once a day (20 mg/kg); in the combination group, 1 mL of Res preparation and 1 mL of CsA preparation were administered by intraperitoneal route once a day (100 mg/kg and 20 mg/kg, respectively); and in the control group, 2 mL of RPMI-1640 was given. Graft survival. Six rats were left in each group until they died. The rats used to evaluate graft survival were given Res or CsA or vehicle buffer until they died. Liver function test and ELISA. Six animals in each group were killed on posttransplant day 7, for blood collection. A 6-mL blood sample was obtained from the vena cava. Two milliliters of blood was centrifuged immediately at 3000 revolutions/min at 4°C for 10 minutes and stored at ⫺80°C until the analysis. Albumin, total bile acid, and alanine aminotransferase (ALT) were assayed by standard enzymatic methods, while the serum IL-2 and INF-␥ levels were assayed by ELISA. EMSA. The remaining 4 mL of blood was used for the detection of NF-B activation in peripheral blood T lymphocytes. First, the rat’s peripheral blood mononuclear cells were separated by standard Ficoll-Hypague gradient centrifugation and then incubated in RPMI-1640 culture medium containing 10% calf serum. The cell suspension was separated from the adherent cells the next day and the T-lymphocyte subpopulation was obtained by magnetic separation column according to the manufacturer’s instruction (Miltenyi Biotec, Germany). The purity of the cell population was confirmed by means of FACScan analysis and cell viability was determined by trypan blue dye exclusion. Then the T-lymphocyte nuclear extracts were prepared by the modified procedure of Dignam et al.19 Following treatment, cells were washed three times with phosphate-buffered solution, resuspended, and incubated on ice for 15 minutes in hypotonic buffer A (10 mol/L HEPES, pH 7.9, 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1 mmol/L DTT, 0.5 mmol/L PMSF, and 0.6% NP-40). Cells were vortexed gently for lysis, and nuclei were separated from cytosolic components by centrifugation at 12,000g for 1 minute at 25°C. Nuclei were resuspended in buffer C (20 mmol/L HEPES, pH 7.9, 25% glycerol, 0.4 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L DTT, 0.5 mmol/L PMSF) and shaken for 30 minutes at 4 °C. Nuclear extracts were obtained by centrifugation at 12,000g for 10 minutes at 25°C. Protein concentration was measured by the Bradford assay (Bio-Rad). For binding reactions, nuclear extracts (10 g of protein) were incubated in a 25-L total reaction volume containing 20 mol/L HEPES, pH 7.9, 60 mmol/L NaCl, 0.1 mmol/L EDTA, 1 mmol/L DDT, 8% glycerol, and 2.55 g/mL of poly(dl-dC) (Pharmacia). Double-stranded radiolabeled NF-B oligonucleotide probe (5=CGCTTGATGAGTCAGCCGGAA-3=) was added to the mixture after preincubation for 15 minutes at 4°C, and the reaction mixture was then incubated for 20 minutes, at room temperature. Samples were loaded on 6% polyacrylamide gels in low-ionic strength 0.25 ⫻ TBE buffer (22.3 mmol/L Tris, 22.2 mmol/L borate, 0.5 mmol/L EDTA) and run at 150 V/cm with cooling. The gels were dried and analyzed by autoradiography. Histopathological examination. Liver specimens were collected from six animals in each group on posttransplant day 7 and were fixed in the 10% neutral buffered formalin. Then the specimens were embedded in paraffin and 3-m-thick sections were cut and stained with hematoxylin and eosin (H-E) staining. A single blinded pathologist examined the liver graft specimens.
2356
WU, PAN, YU ET AL
Statistical Analysis All values were expressed in the mean ⫾ SD, and the MannWhitney U test was applied for all the analyses. The survival period was compared according to the Kaplan-Meier method of analysis. A P value less than .05 was considered significant.
RESULTS
In rat liver transplantation the cold ischemic time and anhepatic time were 40 to 50 minutes and 14 to 16 minutes, respectively, and no significant difference was recognized among these groups. Graft Survival
The mean survival period in the CsA group was 18.5 ⫾ 3.4 days; in the combination group, 23.8 ⫾ 3.9 days; in the control group, 9.3 ⫾ 1.0 days. The differences between the combination group and CsA group or control group were statistically significant (P ⬍ .05 and P ⬍ .01, respectively). The survival rates in the three groups are shown in Fig 1. Liver Function Tests
Figure 2 shows serum chemistry data that reflect the liver functions after OLTx. On the posttransplant day 7 the albumin level in the CsA group was 2.81 ⫾ 0.19 g/dL; in the combination group, 3.15 ⫾ 0.12 g/dL; and 2.07 ⫾ 0.28 g/dL in the control group. The differences between the combination group and CsA group or control group were statistically significant (P ⬍ .05 and P ⬍ .01, respectively). The total bile acid in the CsA group was 149.1 ⫾ 19.0 mol/L; in the combination group, 116.2 ⫾ 6.3 mol/L; and 353.9 ⫾ 84.4 mol/L in the control group. The differences between the combination group and CsA group or control group were statistically significant (P ⬍ .05 and P ⬍ .01, respectively). ALT in CsA group was 428.7 ⫾ 55.4 U/L; in the combination group, 318.1 ⫾ 35.9 U/L; and 1137.5 ⫾ 235.4 U/L in the control group. It was significantly lower in the combination group than in the CsA group (P ⬍ .05) or control group (P ⬍ .01). ELISA
The IL-2 level in the CsA group was 193.2 ⫾ 19.1 ng/L; in the combination group, 147.2 ⫾ 17.7 ng/L, and 294.4 ⫾ 38.0
Fig 2. (A) Serum albumin levels (g/dL). (B) Serum total bile acid levels (mol/L). (C) Serum ALT levels (U/L).
ng/L in the control group. It was significantly lower in the combination group than in the CsA group (P ⬍ .05) or control group (P ⬍ .01). The INF-␥ level in the CsA group was 71.0 ⫾ 3.8 ng/L; in the combination group, 59.5 ⫾ 5.3 ng/L; and 101.3 ⫾ 14.1 ng/L in the control group. It was significantly lower in the combination group than in the CsA group (P ⬍ .05) or control group (P ⬍ .01). The serum cytokine levels on posttransplant day 7 in the three groups are shown in Fig 3. Activation of NF-B
Fig 1. Survival rate of rats after transplantation.
The activation of NF-B DNA binding activity in peripheral blood T lymphocytes in the CsA group was 261.2 ⫾ 57.7 units; in the combination groups 89.1 ⫾ 34.2 units; and 404.3 ⫾ 64.3 units in the control group. It was significantly lower in the combination group than in the CsA group (P ⬍
COMBINED USE OF CYCLOSPORINE AND RESVERATROL
2357
.05) or control group (P ⬍ .01). Figure 4 demonstrates measurable NF-B DNA binding activity in the three groups on posttransplant day 7. Histopathological Examination
All three groups showed the typical signs of severe graft rejection with intense portal infiltrate. There is an apparent difference in the severity of rejection between the combination group (Banff score 3.5 ⫾ 0.5) and CsA group (Banff score 4.8 ⫾ 0.3) or control group (Banff score 7.3 ⫾ 0.6) on the basis of Banff schema by a blinded pathologist (P ⬍ .05 and P ⬍ .01, respectively). Figure 5 shows the H-E staining of histological sections in the combination group and control group on posttransplant day 7. DISCUSSION
As rejection developed in the graft, the hepatocytes were destroyed by infiltrating T lymphocytes. Hepatic function deteriorates as the number of hepatocytes decreases, and rejection eventually kills the recipient unless immunosuppressants are given. Transcription factor NF-B and other members of the Rel homology family of transcription factors have been shown to play a pivotal role in the transcription of genes involved in immune and inflammatory responses20 and in cell proliferation and transformation.21,22 Our study revealed that the combined use of Res and CsA down-regulation NF-B activation of peripheral blood T lymphocytes and IL-2 and INF-␥ serum levels and decreased portal infiltrate of T lymphocytes in rats with severe rejection and therefore promoted albumin synthesis
Fig 3. (A) Serum IL-2 levels (g/L). (B) Serum INF-␥ levels (g/L).
Fig 4. (A) The levels of NF-B in peripheral blood T lymphocytes were analyzed by EMSA using sequence-specific radiolabeled oligonucleotide. (B) Quantitation of NF-B activity by PhosphorImager.
and prevented an elevation of total bile acid and ALT and prolonged the survival time of rats after liver transplantation. In addition, we found all these immunosuppressive effects are stronger in Res and CsA combination group than in CsA alone group. We considered the possibility that suppression of lymphocyte infiltration and cytokine production by CsA and Res may result from suppression of NF-B activation. In resting cells, NF-B remains sequestered in the cytoplasm in a functionally inactive form noncovalently bound to an inhibitory protein, IB. Upon stimulation of cells with mitogens, antigens, or cytokines, IB dissociates from the NF-B complex, allowing NF-B to translocate to the nucleus where it binds to B motifs in the promoter region of the response genes.23 CsA is a cyclic peptide with immunomodulatory properties. CsA acts within the cell by binding to cyclophilin, an immunophilin. This results in inactivation of calcineurin. Since calcineurin can inactivate IB, thus promoting the translocation of NF-B to nucleus and its transcription activity, CsA therefore can increase the number of IB in active form and thus suppress the translocation of NF-B to nucleus and its transcription activity, ultimately leading to
2358
Fig 5. (A) control group and (B) combination group. The control group showed typical signs of severe graft rejection with intense portal infiltrate spilling into lobules, while the combination group showed obviously less portal infiltration.
suppression of transcription of genes, including those for cytokines and cytokine receptors.24 The immunomodulatory effect of resveratrol has been reported with use of mouse splenic lymphocytes, lymphokine activated killer cells, mouse macrophage-like cell line RAW 264.7, and human peripheral blood T lymphocytes.15,25–28 Gao et al15 reported that Res inhibited splenic lymphocytes proliferation, induction of cytotoxic T lymphocytes, and cytokine production, at least in part through the inhibition of NF-B activation. Yu L et al25 reported that Res can suppress notably the proliferation and transformation of human lymphocytes and the combination of Res at a given concentration with CsA can enhance immune suppression. Recently, Jeong29 reported that Res increased NF-B-luciferase activations at lower dose but inhibited activation at higher dose, and the suppressive mechanism may lie in that it reduce lB␣ phosphorylation. However, the detailed mechanisms of the biphasic modulatory effects of Res still remained to be studied. In this hepatic allograft rejection model we used a relatively higher dosage of Res and compared its combination with CsA with the sole use of CsA. We have shown that
WU, PAN, YU ET AL
in this hepatic allograft rejection model, peripheral blood T lymphocytes express high levels of activated NF-B; however, after administration of Res and CsA, the NF-B activation that occurs upon stimulation with the heterogeneous antigen is partly blocked. These results are consistent with those of previous investigators. The possible mechanism may lie that both of Res and CsA have influence on NF-B of T lymphocytes and therefore lead to a stronger lmmunosuppressive effect. It is generally accepted that the immune response leading to graft rejection is accompanied by an increase of cytokine production by the primed T cell. The cytokines are essential for the differentiation, proliferation, and amplification of the T cell.30 The most important of these cytokines is IL-2, which is essential for activated T-cell proliferation31,32; INF-␥ is mainly secreted by activated T cells33 and induces MHC class I antigen expression in several kinds of cells such as lymphocytes34 and myocytes.35 Some studies have shown that the gene expression of IL-2 and INF-␥ by intragraft was specific to acute rejection, which was preceded histopathological maniestation in liver transplantation.36,37 Thus we also investigated the effects of CsA in combination with Res on the production of INF-␥ and IL-2. Our results showed that compared with CsA group, the combined use of CsA and Res can more notably suppress the serum IL-2 and INF-␥ levels of rats after liver transplantation. In conclusion, these results showed that in vivo administration of CsA and Res in liver transplantation model of rat down-regulated the serum IL-2 and INF-␥ levels, decreased the lymphocyte infiltration in allograft liver, prolonged mean survival period after OLTx, and demonstrated hepatocyte protective effect. Suppression of the activation of transcription factor NF-B in peripheral blood T lymphocytes appears to be part of the mechanism by which CsA and resveratrol inhibit the in vivo development of immunological responses. We conclude that Res might serve as a supplmentary immunosuppressive agent and, by decreasing the dosage of CsA, reduce the side effects of CsA in liver transplantation. ACKNOWLEDGMENTS We recognize Tzakis AG, in the Department of Surgery, Division of Transplantation, University of Miami School of Medicine, and Phillip Ruiz, in the department of Immunopathology, University of Miami School of Medicine, for their critical reading and expert comments.
REFERENCES 1. Klintmalm GB: Rejection therapies. Dig Dis Sci 36:1431, 1991 2. Kusne S, Dummer JS, Singh N, et al: Infections after liver transplantation: an analysis of 101 consecutive cases. Medicine 67:132, 1988 3. Berlakovich GA, Rockenschaub S, Taucher S, et al: Underlying disease as a predictor for rejection after liver transplantation. Arch Surg 133:167, 1998 4. Langcake P, Pryce RJ: The production of resveratrol by Vitis vinifera and other members of the Vitaceae as a response to infection or injury. Physiol Plant Pathol 9:77, 1976
COMBINED USE OF CYCLOSPORINE AND RESVERATROL 5. Soleas GJ, Diamandis EP, Goldberg DM: Resveratrol: a molecule whose time has come to gone? Clin Biochem 30:91, 1997 6. Frémont L: Biological effects of resveratrol. Life Sciences 66:663, 2000 7. Fauconneau B, Waffo-Teguo P, Huguet F, et al: Comparative study of radical scavenger and antioxidant properties of phenolic compounds from Vitis vinifera cell coltures using in vitro tests. Life Sci 61:2103, 1997 8. Jang M, Cai L, Udeani GO, et al: Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science 275:218, 1997 9. Subbaramaiah K, Chung WJ, Michaluart P, et al: Resveratrol inhibits cyclo-oxigenase-2 transcription and activity in phorbol ester-treated human mammary epithelial cells. J Biol Chem 273: 21875, 1998 10. Hsieh TC, Burfeind P, Laud K, et al: Cell cycle effects and control of gene expression by resveratrol in human breast carcinoma cells lines with different metastatic potential. Int J Oncol 15:245, 1999 11. Hsieh TC, Wu JM: Differential effects on growth, cells cycle arrest, and induction of apoptosis by resveratrol in human prostate cancer cell lines. Exp Cell Res 249:109, 1999 12. Clément MV, Hirpara JL, Chawdhury SA, et al: Chemopreventive agent resveratrol, a natural product derived from grapes, triggers CD95 signaling-dependent apoptosis in human tumor cells. Blood 92:996, 1998 13. Ragione FD, Cucciolla V, Borriella A, et al: Resveratrol arrests the cell division cycle at S/G2 phase transition. Biochem Biophys Res Commun 250:53, 1998 14. Holmes-McNary M, Baldwin AS Jr: Chemopreventive properties of trans-resveratrol are associated with inhibition of activation of the lkB kinase. Cancer Research 60:3477, 2000 15. Gao X, Xu YX, Janakiraman N, et al: Immunomodulatory activity of resveratrol: suppression of lymphocyte proliferation, development of cell-mediated cytotoxicity, and cytokine production. Biochem Pharmacol 62:1299, 2001 16. Wadsworth TL, Koop DR: Effects of the wine polyphenolics quercetin and resveratrol on pro-inflammatory cytokine expression in RAW 264.7 macrophages. Biochem Pharmacol 57:941, 1999 17. Gao X, Deeb D, Media J, et al: Immunomodulatory activity of resveratrol: discrepant in vitro and in vivo immunological effect. Biochem Pharmacol 66:2427, 2003 18. Kamada N, Calne RY: Orthotopic liver transplantation in the rat. Technique using cuff for portal vein anastomosis and biliary drainage. Transplantation 28:47, 1979 19. Dignam JD, Lebovitz RM, Roeder RG: Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11:1475, 1983 20. Lenardo MJ, Baltimore D: NF-B: a pleiotrophic mediator of inducible, and tissue-specific gene control. Cell 58:227, 1989 21. Bargou RC, Emmerich F, Krappmann D, et al: Constitutive nuclear factor-B-RelA activation is required for proliferation and
2359 survival of Hodgkin’s disease tumor cells. J Clin Invest 100:2961, 1997 22. Reuther JY, Reuther GW, Cortez D, et al: A requirement for NF-B activation in Bcr-Abl-mediated transformation. Genes Dev 12:968, 1998 23. Blackwell TS, Christman JW: The role of nuclear factorkappa B in cytokine gene regulation. Am J Respir Cell Mol Biol 17:3, 1997 24. Sabatini DM, Lai MM, Snyder SH: Neural roles of immunophilins and their ligands. Mol Neurobiol 15:223, 1997 25. Yu L, Wu SL, Zhang M, et al: Effect of resveratrol alone and its combination with cyclosporin A on the immune function of human peripheral blood T lymphocytes. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi 19:549, 2003 26. Boscolo P, del Signore A, Sabbioni E, et al: Effects of resveratrol on lymphocyte proliferation and cytokine release. Ann Clin Lab Sci 33:226, 2003 27. Falchetti R, Fuggetta MP, Lanzilli G, et al: Effects of resveratrol on human immune cell function. Life Sci 70:81, 2001 28. 23Feng YH, Zhou WL, Wu QL, et al: Low dose of resveratrol enhanced immune response of mice. Acta Pharmacol Sin 23:893, 2002 29. Jeong WS, Kim IW, Hu R, et al: Modulatory properties of various natural chemopreventive agents on the activation of NF-B signaling pathway. Pharm Res 21:661, 2004 30. Schwartz RH: Costimulation of T lymphocytes: the role of CD28, CTLA-4, and B7/BB1 in interleukin-2 production and immunotherapy. Cell 71:1065, 1992 31. Fitch FW, McKisic MD, Lancki DW, et al: Differential regulation of murine T lymphocyte subsets. Annu Rev Immunol 11:29, 1993 32. Seder RA, Paul WE: Acquisition of lymphokine-producing phenotype by CD4⫹ T cells. Annu Rev Immunol 12:635, 1994 33. Halloran PF, Wadgymar A, Autenried P: The regulation of expression of major histocompatibility complex products. Transplantation 41:413, 1986 34. Walrand F, Picard F, McCullough K, et al: Recombinant bovine interferon-gamma enhances expression of class I and II bovine lymphocyte antigens. Vet Immunol Immunopathol 22:379, 1989 35. Kalovidouris AE: The role of cytokines in polymyositis: interferon-gamma induces class II and enhances class I major histocompatibility complex antigen expression on cultured human muscle cells. J Lab Clin Med 120:244, 1992 36. Martinez OM, Villanueva JC, Lake J, et al: IL-2 and IL-5 gene expression in response to alloantigen in liver allograft recipients and in vitro. Transplantation 55:1159, 1993 37. Gaweco AS, Otto G, Otto HF, et al: Common and sequential overexpression patterns of T-helper cytokines during acute rejection and correlation of proinflammatory cytokine expression with chronic rejection of human liver allografts: a study under cyclosporine, FK506, and quadrupt BT563 immunosuppression. Transplant Proc 27:1152, 1995
Immunosuppression by Combined Use of Cyclosporine and Resveratrol in a Rat Liver Transplantation Model S.L. Wu, C.E. Pan, L. Yu, and K.W. Meng ABSTRACT Introduction. Despite continued progress in the development of immunosuppressive agents, allograft rejection remains an important cause of morbidity and mortality after liver transplantation. We examined the effect of intraperitoneal injection of cyclosporine (CsA) and resveratrol (Res) on allograft rejection after liver transplantation in rats. Methods. Male Sprague-Dawley rats were selected as donors and male Wistar rats as recipients for a rejection model. The recipients were divided into three groups after orthotopic liver transplantation (OLTx): in the combination group both Res (100 mg/kg) and CsA (20 mg/kg) were given by intraperitoneal route once a day, whereas in the CsA group or control group CsA (20 mg/kg) or vehicle buffer was given. The survival period, serum chemistry, production of some cytokines, activation of transcription factor NF- B, and histopathological findings were then compared among them. Results. The mean survival period after OLTx in the combination group was significantly longer than that in the CsA group or control group (P ⬍ .05 and P ⬍ .01). On posttransplant day 7, the serum albumin level significantly improved, the serum total bile acid and alanine aminotransferase levels were significantly lower, the serum interleukin-2 and interferon-␥ levels were significantly lower, and the activation of transcription factor NF-B in peripheral blood T lymphocytes was significantly suppressed in the combination group in comparison with those in the CsA group (all P ⬍ .05) or control group (all P ⬍ .01), and a histological examination revealed apparent difference in the severity of rejection between the combination group and CsA group (P ⬍ .05) or control group (P ⬍ .01). Conclusion. The combined use of CsA and Res has a stronger immunosuppressive effect on hepatocytes under allograft rejection in comparison with the sole use of CsA. Res might serve as a novel supplementary immunosuppressive agent for reducing the severity of hepatic allograft rejection in rats.
S
INCE THE INTRODUCTION of cyclosporine, (CA) and tacrolimus for the control of postoperative rejection, liver transplantation has become an established surgical technique and is now performed on patients with various terminal liver diseases. Most instances of mortality after liver transplantation are still attributed to infection, and such infection is apt to occur as a result of overimmunosuppression to treat severe rejection.1–3 Rejection is still a leading cause of morbidity and mortality after liver transplantation. To improve survival after liver transplantation,
novel strategies are still needed for the treatment of the rejection. Resveratrol (3,5,4=-trihydroxystilbene; Res) is a polypheFrom the No. 1 Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi Province, People’s Republic of China. Address reprint requests to Dr Wu Shengli, No. 1 Hospital of Xi’an Jiaotong University: Department of Hepatobiliary, No. 1 Jianking Road, Xi’an City, Shaanxi Province, 710061, People’s Republic of China. E-mail: [email protected]
0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2005.03.112
© 2005 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
2354
Transplantation Proceedings, 37, 2354 –2359 (2005)
COMBINED USE OF CYCLOSPORINE AND RESVERATROL
nol present in a limited number of plants,4 mainly in grapes, with levels up to tens of grams per Kilogram,5 where it is synthesized in response to stress conditions such as fungal infections and trauma.4 Many studies have demonstrated that this molecule exhibits a wide range of biological and pharmacological activities both in vitro and in vivo.6 A series of studies showed that Res has antioxidant properties,7 anti-inflammatory properties,8,9 and cancer-chemopreventive activity.10,11 Many of the biological activities of resveratrol, like the inhibition of cyclooxygenase,8 induction of CD95 signaling-dependent apoptosis,12 effects on the cell division cycle,13 and the modulation of NF-B activation,14 indicate a possible effect on immune response, and many in vitro experiments have proven that Res has immunomodulatory activity.15–17 To our knowledge, there has so far been no report on the in vivo immunomodulatory effect of Res on hepatic allograft after transplantation. We therefore investigated whether the combined use of Res and CsA may have any beneficial effect on hepatic allograft after transplantation.
MATERIALS AND METHODS Experimental Design Animals. Male Sprague-Dawley rats 9 to 10 weeks old weighing 190 to 210 g as donors and male Wistar rats 7 to 8 weeks weighing 190 to 210 g as recipients were purchased from the Animal Center of Xi’an Jiaotong University (Xi’an, China) and Animal Center of Shanxi Medical College (Shanxi, China), respectively. All rats were allowed free access to water and standard laboratory chow. Before operation the rats were fasted for 12 hours and only allowed free access to water. All animal protocols were approved by the Xi’an Jiaotong University Institutional Animal Care and Use Committee. Reagents. Res, dimethyl sulfoxide (DMSO), and interleukin -2 (IL-2) and interferon-gamma (INF-␥) kits were purchased from Sigma Chemical Co. RPMI-1640, HEPES, EDTA, EGTA, DTT, PMSF, and NP-40 were from Gibco BRL. CsA was purchased from Calbiochem Co. NF-B consensus oligonucleotide and single base pair mutant were from Promega. Fetal bovine serum was from SiJiQing Co (Hangzhou, China).32 P-ATP was from Beijing Isotope Co, China. Orthotopic liver transplantation. Orthotopic rat liver transplantation (OLTx) was performed by the cuff technique as described by Kamada and Calne;18 with some slight modifications. With the rat under ketamine anesthesia (75 mg/kg), the liver was gently skeletonzed and flushed with chilled lactated Ringer’s solution through the abdominal aorta. Special care was taken for minimal manipulation of the graft and portal vein and bile duct for reconstruction. The liver was harvested and stored at 4°C in lactated Ringer’s solution until transplantation. OLTx was performed without hepatic artery reconstruction. The suprahepatic vena cava was anastomosed with 7-0 Prolene continuous suture (Ethicon, Somerville, NJ, USA) and portal vein and inferior vena cava reconstruction was performed by the cuff technique. The bile duct connection was made using an intraluminal epidural catheter stent. Res and CsA administration. The Res was dissolved and sterilized in DMSO first and then diluted in RPMI-1640 to 20 mg/mL. The CsA was diluted in RPMI-1640 to 4 mg/mL. The recipients were randomly divided into three groups after OLTx: in the CsA group, 1 mL of CsA preparation and 1 mL of RPMI-1640 were
2355 administered by intraperitoneal route once a day (20 mg/kg); in the combination group, 1 mL of Res preparation and 1 mL of CsA preparation were administered by intraperitoneal route once a day (100 mg/kg and 20 mg/kg, respectively); and in the control group, 2 mL of RPMI-1640 was given. Graft survival. Six rats were left in each group until they died. The rats used to evaluate graft survival were given Res or CsA or vehicle buffer until they died. Liver function test and ELISA. Six animals in each group were killed on posttransplant day 7, for blood collection. A 6-mL blood sample was obtained from the vena cava. Two milliliters of blood was centrifuged immediately at 3000 revolutions/min at 4°C for 10 minutes and stored at ⫺80°C until the analysis. Albumin, total bile acid, and alanine aminotransferase (ALT) were assayed by standard enzymatic methods, while the serum IL-2 and INF-␥ levels were assayed by ELISA. EMSA. The remaining 4 mL of blood was used for the detection of NF-B activation in peripheral blood T lymphocytes. First, the rat’s peripheral blood mononuclear cells were separated by standard Ficoll-Hypague gradient centrifugation and then incubated in RPMI-1640 culture medium containing 10% calf serum. The cell suspension was separated from the adherent cells the next day and the T-lymphocyte subpopulation was obtained by magnetic separation column according to the manufacturer’s instruction (Miltenyi Biotec, Germany). The purity of the cell population was confirmed by means of FACScan analysis and cell viability was determined by trypan blue dye exclusion. Then the T-lymphocyte nuclear extracts were prepared by the modified procedure of Dignam et al.19 Following treatment, cells were washed three times with phosphate-buffered solution, resuspended, and incubated on ice for 15 minutes in hypotonic buffer A (10 mol/L HEPES, pH 7.9, 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1 mmol/L DTT, 0.5 mmol/L PMSF, and 0.6% NP-40). Cells were vortexed gently for lysis, and nuclei were separated from cytosolic components by centrifugation at 12,000g for 1 minute at 25°C. Nuclei were resuspended in buffer C (20 mmol/L HEPES, pH 7.9, 25% glycerol, 0.4 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L DTT, 0.5 mmol/L PMSF) and shaken for 30 minutes at 4 °C. Nuclear extracts were obtained by centrifugation at 12,000g for 10 minutes at 25°C. Protein concentration was measured by the Bradford assay (Bio-Rad). For binding reactions, nuclear extracts (10 g of protein) were incubated in a 25-L total reaction volume containing 20 mol/L HEPES, pH 7.9, 60 mmol/L NaCl, 0.1 mmol/L EDTA, 1 mmol/L DDT, 8% glycerol, and 2.55 g/mL of poly(dl-dC) (Pharmacia). Double-stranded radiolabeled NF-B oligonucleotide probe (5=CGCTTGATGAGTCAGCCGGAA-3=) was added to the mixture after preincubation for 15 minutes at 4°C, and the reaction mixture was then incubated for 20 minutes, at room temperature. Samples were loaded on 6% polyacrylamide gels in low-ionic strength 0.25 ⫻ TBE buffer (22.3 mmol/L Tris, 22.2 mmol/L borate, 0.5 mmol/L EDTA) and run at 150 V/cm with cooling. The gels were dried and analyzed by autoradiography. Histopathological examination. Liver specimens were collected from six animals in each group on posttransplant day 7 and were fixed in the 10% neutral buffered formalin. Then the specimens were embedded in paraffin and 3-m-thick sections were cut and stained with hematoxylin and eosin (H-E) staining. A single blinded pathologist examined the liver graft specimens.
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Statistical Analysis All values were expressed in the mean ⫾ SD, and the MannWhitney U test was applied for all the analyses. The survival period was compared according to the Kaplan-Meier method of analysis. A P value less than .05 was considered significant.
RESULTS
In rat liver transplantation the cold ischemic time and anhepatic time were 40 to 50 minutes and 14 to 16 minutes, respectively, and no significant difference was recognized among these groups. Graft Survival
The mean survival period in the CsA group was 18.5 ⫾ 3.4 days; in the combination group, 23.8 ⫾ 3.9 days; in the control group, 9.3 ⫾ 1.0 days. The differences between the combination group and CsA group or control group were statistically significant (P ⬍ .05 and P ⬍ .01, respectively). The survival rates in the three groups are shown in Fig 1. Liver Function Tests
Figure 2 shows serum chemistry data that reflect the liver functions after OLTx. On the posttransplant day 7 the albumin level in the CsA group was 2.81 ⫾ 0.19 g/dL; in the combination group, 3.15 ⫾ 0.12 g/dL; and 2.07 ⫾ 0.28 g/dL in the control group. The differences between the combination group and CsA group or control group were statistically significant (P ⬍ .05 and P ⬍ .01, respectively). The total bile acid in the CsA group was 149.1 ⫾ 19.0 mol/L; in the combination group, 116.2 ⫾ 6.3 mol/L; and 353.9 ⫾ 84.4 mol/L in the control group. The differences between the combination group and CsA group or control group were statistically significant (P ⬍ .05 and P ⬍ .01, respectively). ALT in CsA group was 428.7 ⫾ 55.4 U/L; in the combination group, 318.1 ⫾ 35.9 U/L; and 1137.5 ⫾ 235.4 U/L in the control group. It was significantly lower in the combination group than in the CsA group (P ⬍ .05) or control group (P ⬍ .01). ELISA
The IL-2 level in the CsA group was 193.2 ⫾ 19.1 ng/L; in the combination group, 147.2 ⫾ 17.7 ng/L, and 294.4 ⫾ 38.0
Fig 2. (A) Serum albumin levels (g/dL). (B) Serum total bile acid levels (mol/L). (C) Serum ALT levels (U/L).
ng/L in the control group. It was significantly lower in the combination group than in the CsA group (P ⬍ .05) or control group (P ⬍ .01). The INF-␥ level in the CsA group was 71.0 ⫾ 3.8 ng/L; in the combination group, 59.5 ⫾ 5.3 ng/L; and 101.3 ⫾ 14.1 ng/L in the control group. It was significantly lower in the combination group than in the CsA group (P ⬍ .05) or control group (P ⬍ .01). The serum cytokine levels on posttransplant day 7 in the three groups are shown in Fig 3. Activation of NF-B
Fig 1. Survival rate of rats after transplantation.
The activation of NF-B DNA binding activity in peripheral blood T lymphocytes in the CsA group was 261.2 ⫾ 57.7 units; in the combination groups 89.1 ⫾ 34.2 units; and 404.3 ⫾ 64.3 units in the control group. It was significantly lower in the combination group than in the CsA group (P ⬍
COMBINED USE OF CYCLOSPORINE AND RESVERATROL
2357
.05) or control group (P ⬍ .01). Figure 4 demonstrates measurable NF-B DNA binding activity in the three groups on posttransplant day 7. Histopathological Examination
All three groups showed the typical signs of severe graft rejection with intense portal infiltrate. There is an apparent difference in the severity of rejection between the combination group (Banff score 3.5 ⫾ 0.5) and CsA group (Banff score 4.8 ⫾ 0.3) or control group (Banff score 7.3 ⫾ 0.6) on the basis of Banff schema by a blinded pathologist (P ⬍ .05 and P ⬍ .01, respectively). Figure 5 shows the H-E staining of histological sections in the combination group and control group on posttransplant day 7. DISCUSSION
As rejection developed in the graft, the hepatocytes were destroyed by infiltrating T lymphocytes. Hepatic function deteriorates as the number of hepatocytes decreases, and rejection eventually kills the recipient unless immunosuppressants are given. Transcription factor NF-B and other members of the Rel homology family of transcription factors have been shown to play a pivotal role in the transcription of genes involved in immune and inflammatory responses20 and in cell proliferation and transformation.21,22 Our study revealed that the combined use of Res and CsA down-regulation NF-B activation of peripheral blood T lymphocytes and IL-2 and INF-␥ serum levels and decreased portal infiltrate of T lymphocytes in rats with severe rejection and therefore promoted albumin synthesis
Fig 3. (A) Serum IL-2 levels (g/L). (B) Serum INF-␥ levels (g/L).
Fig 4. (A) The levels of NF-B in peripheral blood T lymphocytes were analyzed by EMSA using sequence-specific radiolabeled oligonucleotide. (B) Quantitation of NF-B activity by PhosphorImager.
and prevented an elevation of total bile acid and ALT and prolonged the survival time of rats after liver transplantation. In addition, we found all these immunosuppressive effects are stronger in Res and CsA combination group than in CsA alone group. We considered the possibility that suppression of lymphocyte infiltration and cytokine production by CsA and Res may result from suppression of NF-B activation. In resting cells, NF-B remains sequestered in the cytoplasm in a functionally inactive form noncovalently bound to an inhibitory protein, IB. Upon stimulation of cells with mitogens, antigens, or cytokines, IB dissociates from the NF-B complex, allowing NF-B to translocate to the nucleus where it binds to B motifs in the promoter region of the response genes.23 CsA is a cyclic peptide with immunomodulatory properties. CsA acts within the cell by binding to cyclophilin, an immunophilin. This results in inactivation of calcineurin. Since calcineurin can inactivate IB, thus promoting the translocation of NF-B to nucleus and its transcription activity, CsA therefore can increase the number of IB in active form and thus suppress the translocation of NF-B to nucleus and its transcription activity, ultimately leading to
2358
Fig 5. (A) control group and (B) combination group. The control group showed typical signs of severe graft rejection with intense portal infiltrate spilling into lobules, while the combination group showed obviously less portal infiltration.
suppression of transcription of genes, including those for cytokines and cytokine receptors.24 The immunomodulatory effect of resveratrol has been reported with use of mouse splenic lymphocytes, lymphokine activated killer cells, mouse macrophage-like cell line RAW 264.7, and human peripheral blood T lymphocytes.15,25–28 Gao et al15 reported that Res inhibited splenic lymphocytes proliferation, induction of cytotoxic T lymphocytes, and cytokine production, at least in part through the inhibition of NF-B activation. Yu L et al25 reported that Res can suppress notably the proliferation and transformation of human lymphocytes and the combination of Res at a given concentration with CsA can enhance immune suppression. Recently, Jeong29 reported that Res increased NF-B-luciferase activations at lower dose but inhibited activation at higher dose, and the suppressive mechanism may lie in that it reduce lB␣ phosphorylation. However, the detailed mechanisms of the biphasic modulatory effects of Res still remained to be studied. In this hepatic allograft rejection model we used a relatively higher dosage of Res and compared its combination with CsA with the sole use of CsA. We have shown that
WU, PAN, YU ET AL
in this hepatic allograft rejection model, peripheral blood T lymphocytes express high levels of activated NF-B; however, after administration of Res and CsA, the NF-B activation that occurs upon stimulation with the heterogeneous antigen is partly blocked. These results are consistent with those of previous investigators. The possible mechanism may lie that both of Res and CsA have influence on NF-B of T lymphocytes and therefore lead to a stronger lmmunosuppressive effect. It is generally accepted that the immune response leading to graft rejection is accompanied by an increase of cytokine production by the primed T cell. The cytokines are essential for the differentiation, proliferation, and amplification of the T cell.30 The most important of these cytokines is IL-2, which is essential for activated T-cell proliferation31,32; INF-␥ is mainly secreted by activated T cells33 and induces MHC class I antigen expression in several kinds of cells such as lymphocytes34 and myocytes.35 Some studies have shown that the gene expression of IL-2 and INF-␥ by intragraft was specific to acute rejection, which was preceded histopathological maniestation in liver transplantation.36,37 Thus we also investigated the effects of CsA in combination with Res on the production of INF-␥ and IL-2. Our results showed that compared with CsA group, the combined use of CsA and Res can more notably suppress the serum IL-2 and INF-␥ levels of rats after liver transplantation. In conclusion, these results showed that in vivo administration of CsA and Res in liver transplantation model of rat down-regulated the serum IL-2 and INF-␥ levels, decreased the lymphocyte infiltration in allograft liver, prolonged mean survival period after OLTx, and demonstrated hepatocyte protective effect. Suppression of the activation of transcription factor NF-B in peripheral blood T lymphocytes appears to be part of the mechanism by which CsA and resveratrol inhibit the in vivo development of immunological responses. We conclude that Res might serve as a supplmentary immunosuppressive agent and, by decreasing the dosage of CsA, reduce the side effects of CsA in liver transplantation. ACKNOWLEDGMENTS We recognize Tzakis AG, in the Department of Surgery, Division of Transplantation, University of Miami School of Medicine, and Phillip Ruiz, in the department of Immunopathology, University of Miami School of Medicine, for their critical reading and expert comments.
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2359 survival of Hodgkin’s disease tumor cells. J Clin Invest 100:2961, 1997 22. Reuther JY, Reuther GW, Cortez D, et al: A requirement for NF-B activation in Bcr-Abl-mediated transformation. Genes Dev 12:968, 1998 23. Blackwell TS, Christman JW: The role of nuclear factorkappa B in cytokine gene regulation. Am J Respir Cell Mol Biol 17:3, 1997 24. Sabatini DM, Lai MM, Snyder SH: Neural roles of immunophilins and their ligands. Mol Neurobiol 15:223, 1997 25. Yu L, Wu SL, Zhang M, et al: Effect of resveratrol alone and its combination with cyclosporin A on the immune function of human peripheral blood T lymphocytes. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi 19:549, 2003 26. Boscolo P, del Signore A, Sabbioni E, et al: Effects of resveratrol on lymphocyte proliferation and cytokine release. Ann Clin Lab Sci 33:226, 2003 27. Falchetti R, Fuggetta MP, Lanzilli G, et al: Effects of resveratrol on human immune cell function. Life Sci 70:81, 2001 28. 23Feng YH, Zhou WL, Wu QL, et al: Low dose of resveratrol enhanced immune response of mice. Acta Pharmacol Sin 23:893, 2002 29. Jeong WS, Kim IW, Hu R, et al: Modulatory properties of various natural chemopreventive agents on the activation of NF-B signaling pathway. Pharm Res 21:661, 2004 30. Schwartz RH: Costimulation of T lymphocytes: the role of CD28, CTLA-4, and B7/BB1 in interleukin-2 production and immunotherapy. Cell 71:1065, 1992 31. Fitch FW, McKisic MD, Lancki DW, et al: Differential regulation of murine T lymphocyte subsets. Annu Rev Immunol 11:29, 1993 32. Seder RA, Paul WE: Acquisition of lymphokine-producing phenotype by CD4⫹ T cells. Annu Rev Immunol 12:635, 1994 33. Halloran PF, Wadgymar A, Autenried P: The regulation of expression of major histocompatibility complex products. Transplantation 41:413, 1986 34. Walrand F, Picard F, McCullough K, et al: Recombinant bovine interferon-gamma enhances expression of class I and II bovine lymphocyte antigens. Vet Immunol Immunopathol 22:379, 1989 35. Kalovidouris AE: The role of cytokines in polymyositis: interferon-gamma induces class II and enhances class I major histocompatibility complex antigen expression on cultured human muscle cells. J Lab Clin Med 120:244, 1992 36. Martinez OM, Villanueva JC, Lake J, et al: IL-2 and IL-5 gene expression in response to alloantigen in liver allograft recipients and in vitro. Transplantation 55:1159, 1993 37. Gaweco AS, Otto G, Otto HF, et al: Common and sequential overexpression patterns of T-helper cytokines during acute rejection and correlation of proinflammatory cytokine expression with chronic rejection of human liver allografts: a study under cyclosporine, FK506, and quadrupt BT563 immunosuppression. Transplant Proc 27:1152, 1995