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LIVER TRANSPLANTATION 16:357-363, 2010

ORIGINAL ARTICLE

Rapamycin and Tacrolimus Differentially Modulate Acute Graft-Versus-Host Disease in Rats After Liver Transplantation Guodong Xu,1* Linyan Wang,1,2* Wei Chen,1 Fei Xue,4 Xueli Bai,1 Liang Liang,1 Xuning Shen,1 Mangli Zhang,1 Dajing Xia,3 and Tingbo Liang1 1 Department of Hepatobiliary and Pancreatic Surgery, Key Laboratory of Combined Multiorgan Transplantation (Ministry of Public Health), First Affiliated Hospital, 2Department of General Surgery, Children’s Hospital, and 3Institute of Immunology, School of Medicine, Zhejiang University, Hangzhou, People’s Republic of China; and 4Department of Hepatobiliary and Pancreatic Surgery, Organ Transplantation Center, Henan Province People’s Hospital, Zhengzhou, Henan Province, People’s Republic of China

Acute graft-versus-host disease (aGVHD) is a serious complication of liver transplantation (LTx); it occurs in 1% to 2% of liver allograft recipients. The condition has a poor prognosis and poses major diagnostic and therapeutic challenges. A rat model of aGVHD after LTx has been developed, and a relative decrease in regulatory T (Treg) cells has been shown to be associated with this model. Interest has been expressed in the effects of different immunosuppressive agents on CD4þCD25þFoxp3þ Treg cell homeostasis. Rats with aGVHD after LTx were treated with tacrolimus (FK506), rapamycin (RAPA), or no immunosuppressive drug. Those that received RAPA survived longer (91.4 6 8.1 days) than those in the FK506 group (62.3 6 13.4 days) or the control group (22.9 6 1.2 days). Flow cytometry analysis showed that Treg cells, as a percentage of peripheral blood mononuclear cells (PBMCs), were more abundant in the RAPA group (6.8% 6 0.8%) than in the FK506 group (1.7% 6 0.4%) or the control group (2.0% 6 0.4%). Immunohistochemistry demonstrated more Foxp3þ staining of intestinal cells in the RAPA group than in the FK506 group or the control group. In conclusion, the reduced mortality induced by RAPA in a rat model of aGVHD after LTx was associated with higher percentages of CD4þCD25þFoxp3þ Treg cells in PBMCs in blood and tissues than those occurring after the administration of FK506. Liver Transpl 16:357–363, 2010. V 2009 AASLD. C

Received August 13, 2009; accepted November 15, 2009.

Acute graft-versus-host disease (aGVHD) is an uncommon (1%-2%) complication of liver transplantation (LTx) and is associated with a high mortality rate (85%-90%). aGVHD poses major diagnostic and therapeutic challenges.1 Most patients with aGVHD after LTx develop overwhelming sepsis, marrow aplasia, and multiorgan failure. Current therapeutic ap-

proaches for aGVHD after LTx are controversial. They include increasing the dosage of immunosuppressive drugs1–3; decreasing the dosage of, or discontinuing, immunosuppressive drugs4–6; administering antilymphocyte therapy, such as anti-thymocyte globulin, anti-lymphocyte globulin, or the monoclonal antilymphocyte agent OKT31,3,7; administering anti–

Abbreviations: aGVHD, acute graft-versus-host disease; FK506, tacrolimus; Foxp3, forkhead box P3; GVHD, graft-versus-host disease; IL, interleukin; LC, lymphocyte cell; LTx, liver transplantation; PBMC, peripheral blood mononuclear cell; RAPA, rapamycin; TGF-b1, transforming growth factor b1; Treg, regulatory T. This study was supported by grants from the National Natural Science Funds for Distinguished Young Scholars (30925033), the National Natural Science Fund of China (30772054), and the Natural Science Fund of Zhejiang Province (Y207420, Z2080283, and Y2090368). *These authors contributed equally to this study. Address reprint requests to Tingbo Liang, M.D, Ph.D., Department of Hepatobiliary and Pancreatic Surgery, Key Laboratory of Combined Multiorgan Transplantation (Ministry of Public Health), First Affiliated Hospital, School of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou 310003, People’s Republic of China. Telephone: 086-571-87236688; FAX: 086-571-87236688; E-mail: [email protected] DOI 10.1002/lt.22003 Published online in Wiley InterScience (www.interscience.wiley.com).

C 2009 American Association for the Study of Liver Diseases. V

358 XU ET AL.

interleukin 2 (IL-2) receptor antibodies, such as daclizumab or basiliximab8,9; and infusing host immune cells.5,10 The precise mechanisms responsible for the development of aGVHD after LTx are uncertain. Recent literature on this condition is limited to reports of individual patients or small groups of patients. The development of a stable, reproducible model of aGVHD after LTx would appear to be desirable to facilitate (1) the determination of whether changes in the status of the immune system occur that may contribute to its pathogenesis and (2) the development of new approaches that may be effective in its treatment. Over a period of several years, we have developed a model of aGVHD following LTx in the donor-dominant, 1-way major histocompatibility complex matching rat.11 This model has been shown to be associated with a relative decrease in regulatory T (Treg) cells.12 The aim of this study was to investigate the effects of tacrolimus (FK506) and rapamycin (RAPA) on the rat model of aGVHD after LTx and to explore associated changes in the status of the immune system that may have pathological relevance.

MATERIALS AND METHODS

The experimental animals were divided into 3 groups: 1. Control group of F1 rats that received no treatment. 2. FK506 (Astellas Ireland, Killorglin, Ireland) group in which FK506 at 1 mg/kg/day was administered by gastric perfusion from days 8 to 15 after LTx to achieve whole blood trough concentrations of approximately 10 to 12 ng/mL. FK506 was diluted in a vehicle containing phosphatebuffered saline. 3. RAPA (Huadong Medicine Group Co., Ltd., Hangzhou, China) group in which RAPA at 1 mg/kg/ day was administered by gastric perfusion from days 8 to 15 after LTx to achieve whole blood trough concentrations of 2 to 3 ng/mL.

Assessment of aGVHD Clinical Course and Survival All animals were assessed twice daily for signs typical of aGVHD, such as dermatitis, alopecia, weight loss, diarrhea, hunched posture, and cachexia. The actuarial survival rate and mean time to death (mean survival time) were calculated after an observation period of 100 days.

Animals Rats weighing 200 to 300 g were used. Donors were male Lewis (RT11) rats. Recipients were female Lewis and (Lewis$ BN#) F1 (RT11/n) rats. Animals were purchased from Beijing Vital River Co. Both donors and recipients were housed in an animal facility under specific pathogen-free conditions. All experiments were conducted according to the guidelines of the Animal Ethics Committee of Zhejiang University.

Model of aGVHD After LTx and Experimental Groups A rat model of aGVHD was generated as previously reported.11 In brief, orthotopic LTx was undertaken with the technique described by Kamada and Calne13 without anastomosis of the hepatic artery. No antibiotics were administered. Freshly prepared Lewis splenocytes were injected into (Lewis BN) F1 recipients via the dorsal penile vein immediately after LTx (within 30 minutes). Splenocytes (4 108) were infused into each rat. To determine appropriate doses of FK506 and RAPA, the effects of administering daily doses of 0.5, 1.0, and 2.0 mg/kg of each drug were assessed. Features of aGVHD developed in rats treated with either RAPA or FK506 at 0.5 mg/kg/day; continuing treatment was associated with aGVHD of increasing severity. In contrast, a large proportion of rats treated with RAPA or FK506 at 2 mg/kg/day died from either severe infection or drug toxicity. A 1 mg/ kg/day dose of either drug was found to be most effective in treating rats with aGVHD after LTx.

Morphometric and Histopathological Investigations Tissue samples were taken at the time of death or at the time of sacrifice on day 16 after LTx. Samples of skin and intestine were subjected to microscopic evaluation. Each sample was fixed in 10% buffered neutral formalin and embedded in paraffin, and sections (4 lm thick) were stained with hematoxylin and eosin. Slides were coded without reference to experimental groups and examined by a pathologist who was blinded to the code. Microscopic abnormalities typically associated with aGVHD were noted.

Immunohistochemistry Immunohistochemistry was undertaken with paraffin sections (4 lm thick) and a 1:100 dilution of monoclonal antibodies directed against forkhead box P3 (Foxp3; Biolegend, San Diego, CA). The procedures described in the Biolegend instructions were carried out. Slides were coded and evaluated by light microscopy. The number of Foxp3þ cells in 1 mm2 of tissue was independently evaluated in 10 reticules (10 0.1 mm2) that were randomly selected in a single section by 2 investigators blinded to the code. A total of 5 sections per sample were examined.

Flow Cytometry Analysis Phenotypic analysis of Treg cells was undertaken by 3-color flow cytometry. The following fluorescent monoclonal antibodies were used: antibody to CD4–

LIVER TRANSPLANTATION.DOI 10.1002/lt. Published on behalf of the American Association for the Study of Liver Diseases

RAPAMYCIN AND FK506 MODULATE ACUTE GVHD AFTER LTX 359

as triple-positive staining cells (CD4þ, CD25þ, and Foxp3þ) and were expressed as a percentage of all CD25þFoxp3þ cells.

Statistical Analysis All data were analyzed with SPSS software (version 15.0 for Windows) and were expressed as means and standard deviations. Survival analysis and intergroup comparisons were evaluated by an analysis of variance, which was followed by the determination of the least significant difference. A P value < 0.05 was considered to be statistically significant.

RESULTS Clinical Course and Pathology of aGVHD In the rat model, all recipients of liver allografts developed aGVHD. The first features of aGVHD occurred 7 to 10 days after LTx. Severe dermatitis developed predominantly on the ears, foot pads, and genitalia. Subsequently, allograft recipients in the control group began to undergo diffuse depilation. In an advanced stage of the syndrome, rats developed diarrhea and increasing cachexia and exhibited a distinctive hunched posture; weight loss was severe. Death occurred 19 to 29 days after LTx. In the FK506 and RAPA groups after 4 or 8 days of drug administration and in rats that survived more than 100 days, signs of aGVHD gradually abated; dermatitis, alopecia, diarrhea, and cachexia resolved, and the body weight stabilized. However, rats in the FK506 and RAPA groups that died exhibited signs of the disease and body weight changes similar to those of allograft recipients in the control group. Histological examination of the skin revealed pathological features of aGVHD in the control group. In the epidermis and dermis of the control group, there was intense infiltration of mononuclear cells (Fig. 1A); in the FK506 group, there was sparse infiltration of mononuclear cells in the skin (Fig. 1B); and in the RAPA group, there was no obvious infiltration of mononuclear cells in the skin (Fig. 1C). Figure 1. Representative skin biopsy samples obtained at the time of death or at the time of sacrifice on day 16 after liver transplantation (hematoxylin and eosin, original magnification 3200). (A) Control group: the epidermis and dermis were infiltrated by many mononuclear cells. (B) Tacrolimus group: some mononuclear cells infiltrated the skin. (C) Rapamycin group: there was no obvious mononuclear cell infiltration of the skin.

fluorescein isothiocyanate, antibody to CD25–phycoerythrin, and antibody to Foxp3–phycoerythrin–cyanine 5 (eBiosciences Pharmingen, United States). For the Foxp3 antibody, an isotype-matched control antibody was used to determine the intensity of background staining. After staining, cells were washed and analyzed by flow cytometry (Cytomics FC500, Beckman Coulter, United States). Treg cells were identified

Mortality and Survival Time Data on mortality due to aGVHD in the different groups after LTx are summarized in Table 1 and Fig. 2. In the control group, all recipients died 19 to 29 days after LTx. The mortality rate in the FK506 group, which was 50% (4/8), was significantly lower than that in the control group. In the RAPA group, in which only 1 allograft recipient died (on day 31 after LTx), the mortality rate was significantly lower than that in both the control and FK506 groups.

Immunohistochemical Expression of Foxp3 The expression of Foxp3 in intestinal tissue of the control, FK506, and RAPA groups was 0.9% 6 0.4% (Fig. 3A), 1.0% 6 0.5% (Fig. 3B), and 3.8% 6 1.5%


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TABLE 1. Mortality of Acute Graft-Versus-Host Disease, Survival Time, and Mean Survival Time in Different Groups Group

n

Mortality

Survival Time [days (number of animals)]

Mean Survival Time (days)

Control Tacrolimus Rapamycin

8 8 8

100% (8/8) 50% (4/8) 12.5% (1/8)

19, 20, 21 (2), 23 (2), 27, 29 21, 23, 24, 30, >100 (4) 31, >100 (7)

22.9 62.3* 91.4*,y

*P < 0.05 versus the control group. y P < 0.05 versus the tacrolimus group.

(Fig. 3C), respectively. Expression was significantly higher in the RAPA group than in either the control group or the FK506 group (Table 2).

Frequency of Treg Cells The expression of CD4, CD25, and intracellular Foxp3 by peripheral blood mononuclear cells (PBMCs) was used to identify Treg cells. Triple-positive cells (expressing CD4, CD25, and Foxp3) were designated Treg cells. Representative dot plots of the flow cytometry analysis from a liver allograft recipient are shown in Fig. 4. We previously demonstrated that after LTx, Treg cells in a group with aGVHD accounted for 4.6% 6 0.7% and 3.7% 6 0.5% of PBMCs on days 4 and 8 after LTx, respectively.12 In this study, Treg cells in the control group decreased to 3.1% 6 0.4% and 2.0% 6 0.4% of PBMCs by days 12 and 16 after LTx, respectively. Analogous data for Treg cells in the FK506 group were 3.0% 6 0.5% and 1.7% 6 0.4% on days 12 and 16 after LTx, respectively, and in the RAPA group, they were 4.8% 6 0.5% and 6.8% 6 0.8%, respectively; differences between the control and FK506 groups were not significant, but the percentages for the RAPA group were significantly higher than corresponding percentages in either the control group or the FK506 group.

DISCUSSION aGVHD after LTx was first described by Burdick and colleagues in 1988.14 So far, a total of 81 patients with aGVHD after LTx have been reported.15 According to data in the United Network for Organ Sharing registry, the incidence of graft-versus-host disease (GVHD) in allograft recipients is only 0.1%.16 A possible explanation for the low incidence of GVHD in the United Network for Organ Sharing registry is the difficulty in diagnosing this entity. At present, therapeutic approaches for aGVHD after LTx are controversial. Attempts to treat aGVHD in this setting have included increasing or withdrawing immunosuppression, but the approaches that have been tried have often been ineffective. The mortality rate from aGVHD is greater than 75%. Treg cells are crucial in maintaining self-tolerance17,18; they contribute to the induction of tolerance after solid organ transplantation19 and provide protection from the lethal effects of GVHD in models of bone marrow transplantation.20–22 Recently, it has been

Figure 2. Kaplan-Meier survival curves for the 3 experimental groups. Censored indicates allograft recipients that were still alive after the final time point. The mortality in the RAPA group was significantly lower than that in the control and FK506 groups. *P < 0.05 versus the control group. ~P < 0.05 versus the FK506 group. Abbreviations: FK506, tacrolimus; RAPA, rapamycin.

shown that adoptively transferred exogenous Treg cells can inhibit the development of GVHD.23–25 A unique transcription factor, Foxp3, has been found to be specifically expressed by Treg cells. Foxp3 encodes a forkhead/winged-helix transcriptional repressor (designated Scurfin) that may be expressed specifically by CD4þCD25þ Treg cells and is associated with their development and function.26 Previously, we developed a rat model of aGVHD after LTx11 and showed that this model is associated with a relative decrease in Treg cells.12 Modulation of the function of Treg cells is emerging as a potentially promising strategy for suppressing GVHD. Investigations of the effects of different immunosuppressive agents on Treg cell homeostasis in the model of aGVHD after LTx appear to have considerable potential significance. Immunosuppression that prevents the activation and expansion of effector T cells and allows the activation and suppressor function of Treg cells may have important implications for clinical trials.



TABLE 2. Expression of Foxp3 in the Intestine by Immunohistochemistry in Different Groups Group

n

Foxp3þ/LC (%)

Control Tacrolimus Rapamycin

8 8 8

0.9 6 0.4 1.0 6 0.5 3.8 6 1.5*,y

*P < 0.05 versus the control group. y P < 0.05 versus the tacrolimus group.

Figure 3. Expression of forkhead box P3 in intestinal tissue from (A) the control group, (B) the tacrolimus group, and (C) the rapamycin group (original magnification 3400). The expression of forkhead box P3 in intestinal tissue in the rapamycin group was greater than that in the control and tacrolimus groups.

RAPA (a mammalian target of rapamycin inhibitor) and FK506 (a calcineurin inhibitor) are effective in preventing allograft rejection in transplanted patients, and these drugs are widely applied in such patients to achieve this goal. Both immunosuppressive drugs are potent and moderately well tolerated. However, their effects on allograft-destructive effector T cells and allograft-protective Treg cells are fundamentally different. Several studies have indicated that calcineurin

Figure 4. Changes in the frequency of Treg cells as a percentage of peripheral blood mononuclear cells in allograft recipients in the control (n 5 8), FK506 (n 5 8), and RAPA (n 5 8) groups. These percentages decreased progressively in the control group, especially by days 12 and 16 after LTx. Similar decreases occurred in the FK506 group (P > 0.05). In the RAPA group, corresponding percentages were higher than those in the control and FK506 groups on days 12 and 16 after LTx (P < 0.05).

inhibitors reduce the expression of Foxp3 by human Treg cells, and they have adverse effects on the generation, survival, and function of these cells; in contrast, mammalian target of rapamycin inhibitors have little or no effect on Treg cells.27,28 RAPA not only induces expansion of CD4þCD25þFoxp3þTreg cells ex vivo but also induces dose-dependent de novo expression of Foxp3 by murine alloantigen-specific T cells.27,29 Interestingly, a recent study has suggested that RAPA may induce regulatory functions in conventional CD4þ T cells in culture.30 This inference is consistent with the results of this study. RAPA is administered as a treatment for corticosteroid-refractory aGVHD31,32 and as prophylaxis for the development of aGVHD after bone marrow


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transplantation.33,34 However, so far there is a paucity of data on the use of RAPA in the treatment of aGVHD after LTx. In this study, we found that the reduction in mortality induced by the administration of RAPA in liver allograft recipients with aGVHD was significantly greater than that in the control and FK506 groups. The administration of RAPA was also associated with Treg cells constituting an increasing percentage of PBMCs and with the expression of Foxp3 in tissues. The survival rate in the FK506 group was significantly lower than that in the RAPA group but higher than that in the control group. Although tissue infiltration with PBMCs can be inhibited by RAPA, Treg cells, as a percentage of PBMCs, and the expression of Foxp3 were reduced, and in some allograft recipients in which the development of aGVHD progressed, death occurred. Recently, transforming growth factor b1 (TGF-b1) and IL-2 signaling has been shown to be critically important for the survival and function of Treg cells, even after these cells have differentiated.35–42 The mechanism for the observed changes in Treg cells is uncertain. On day 16 after LTx, serum levels of IL-2 in liver allograft recipients in the FK506 group were significantly lower than those in the other 2 groups, and serum levels of IL-2 in the RAPA group were significantly lower than those in the control group. IL-2 is secreted mainly by activated effector T cells, which are inhibited by RAPA (data not shown). FK506 is a potent inhibitor of phosphatase calcineurin, which is essential for T cell activation. By inhibiting calcineurin, this drug suppresses the production of IL-2 and related cytokines by the prevention of downstream activation of the transcription factor, the nuclear factor of activated T cells.43 RAPA (sirolimus), a potent inhibitor of the IL-2 signaling pathway, is an immunosuppressant that is structurally similar to FK506, but it does not inhibit calcineurin. It inhibits T cell activation by interfering with signals induced by the T cell growth factors IL-2 and IL-4.44,45 Furthermore, levels of T helper 2 cytokine (TGF-b1) in the RAPA group were significantly higher than those in the other 2 groups; these levels had decreased markedly by day 16 after LTx (data not shown). Previously, it has been demonstrated that RAPA not only induces TGF-b1 expression but also acts synergistically with TGF-b1 in the induction of Treg cells.27,46 Although FK506 is known to induce TGF-b1 expression, it did not augment Foxp3 expression induced by TGF-b1.28 In conclusion, RAPA administration significantly reduces mortality in a rat model of aGVHD after LTx. This effect of RAPA is associated with a greater increase in the percentage of PBMCs in peripheral blood and tissues that are CD4þCD25þFoxp3þ Treg cells than that occurring after the administration of FK506 to the model.

ACKNOWLEDGMENT The authors thank Haiyang Xie, Lin Zhou, and Da Yu for their technical support. They thank Huiping Wang

for undertaking the morphometric and histopathological investigations.

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