Abstract
BACKGROUND: To avoid acute rejection, it is necessary to use imunosuppressive drug regimen for long term to control immune state. However, imunosuppressive drug regimen of allogenic organ transplantation increases infection incidence of recipients, and induction of allograft immunological tolerance might be an ideal method for solving these problems. The long-term immunologic tolerance has been able to be induced in the experimental rodent models. Among these protocols, donor bone marrow cell (DBMC) infusion exerts an important role in the induction of allograft immunological tolerance.
OBJECTIVE: To investigate effects of combination of cyclosporine A (CsA) with DBMC infusion on heterotopic rat cardiac allograft survival time.
DESIDN: A randomized controlled animal experiment.
SETTING: Renal Disease Center, First Affiliated Hospital of Zhejing University School of Medicine.
MATERIALS: This study was performed at the Laboratory Animal Center, Zhejiang University School of Medicine between March 2002 and December 2005. Inbred male Lewis rats (n=40, serving as donors) and male BN rats (n=60, serving as recipients) of SPF grade were used in this study. The protocol was approved by the Hospital's Ethic's Committee.
METHODS: Forty rats prepared for heterotopic rat cardiac allograft were randomly divided into 4 groups, with 10 rats in each: control group, in which, rats received no treatment, CsA group, in which, rats received CsA infusion for 7 days successively; CsA +DBMC group, in which, rats received DBMCs during and 6 days after the surgery and additional 7 successive days of CsA infusion, and a DBMC group, in which, rats received DBMCs infusion during and 6 days after the surgery. In addition, BN rats that received heterotopic rat cardiac allograft served BN controls. The survival time of heteroropic rat cardiac allograft was investigated. Serum interleukin-2 level and tumor necrosis factor-α mRNA expression level in the transplanted cardiac allograft were measured. The percentage of antigen presenting cells (APC) from donor, CD3+CD25+ cells, CD4+CD25+ cells, CD86+ cells, and the ratio for CD4+CD45RC+ and CD4+CD45RC- in the recipient peripheral blood karyocytes were measured by flow cytometry 6, 12 and 18 days after surgery.
MAIN OUTCOME MEASURES: The survival time of heteroropic rat cardiac allograft, serum interleukin-2 (IL-2)level, tumor necrosis factor-α (TNF-α) mRNA expression level, rejection grading, the percentage of DBMCs in the recipient peripheral blood karyocytes, CD3+CD25+ cells, and CD4+CD25+ cells, as well as CD86 expression, and the ratio for CD4+CD45RC+ and CD4+CD45RC-.
RESULTS: Forty Lewis male rats and sixty male BN rats were all included in the final analysis. The heterotopic rat cardiac allograft survival time was longer in the CsA +DBMC group than in the control group and DBMC group (P < 0.05). Serum IL-2 level and TNF-α mRNA expression were respectively lower in the CsA +DBMC group than in the control group and DBMC group ( P < 0.05). The rejection was milder in the CsA +DBMC group than in the remaining 3 transplantation groups. In the CsA +DBMC group, CD 86 expression in the recipient peripheral blood karyocytes was markedly inhibited, and 6 and 12 days after surgery, the ratio for CD4+CD45RC+ and CD4+CD45RC- and the percentage of CD3+CD25+ were respectively lower compared to control group and DBMC group. DBMCs in the recipient peripheral blood karyocytes were more in rats that received DBMC infusion compared to rats that received no BDMC infusion.
CONCLUSION: Short-term CsA treatment combined with DBMC infusion can lower acute rejection of heterotopic rat cardiac allograft and prolongs survival time of cardiac allograft.

INTRODUCTION

Cyclosporine A (CsA)-contained immunosuppressive regimens have effectively prevented acute rejection following organ transplantation, and improved the results of short-term success rate of transplantation. Recent clinical data have demonstrated that under the condition of use of routine immunodepressive regimens, donor bone marrow cell (DBMC) infusion can reduce acute rejections and prolong the allograft survival time [1]. In the present study, we used heterotopic rat cardiac allograft to perform short-term CsA therapy and DBMC infusion to observe effects of the therapeutic regimen on heterotopoc rat cardiac allograft survival.

MATERIALS AND METHODS

Materials
This study was performed at the Laboratory Animal Center (SPF grade), Zhejiang University School of Medicine between March 2002 and December 2005. Inbred male Lewis rats (RT11, n=40) and male BN rats (RT1n, n=60) of SPF grade, aged 12 weeks, weighing 200-220 g, were provided by Laboratory Animal Institute, Chinese Academy of Medical Sciences (certificate No. 1999-017) and, employed in this study. The protocol was approved by the Hospital's Ethic's Committee.

Methods
Male BN (RT1n) and Lewis (RT11) rats were used as
heart transplant recipients and donors, respectively. The involved rats were randomized into 4 groups, with 10 rats in each group: control group, in which, rats received 1mL saline infusion through portal vein during the surgery; CsA group, in which, rats received CsA 5 mg/（kg?d）for 7 days successively; CsA +DBMC group, in which, rats received DBMC (1×108) during and 6 days after surgery and CsA 5 mg/（kg?d）for 7 days successively after surgery; DBMC group, in which, rats received DBMC infusion (1×108) during and 6 days after surgery. In addition, BN rats that received heterotopic rat cardiac allograft served as BN controls, i.e., BN control group (n=10).

Heterotopic heart transplantation
Heterotopic heart transplantations were performed using the technique described by Ono. Briefly, recipients and donors were intraperitoneally anesthetized with 50 g/L chloral hydrate (300 mg/kg, 1mL/100 g body weight). The donor heart was removed and perfused with 10 mL of cold heparinized saline (0 ℃ to 4 ℃). The aorta was anastomosed to abdominal aorta of the recipient rat. The pulmonary artery of the donor heart was anastomosed to the inferior vena cava of the recipient. The allograft took on normal sinus rhythm (200-250 times/min) after opening the recipient's aorta clamps. The heat ischemic time was < 5 minutes and cold ischemic time ranged from 30 to 45 minutes. The allografts were monitored daily by palpation. The recipients were weighted regularly after surgery.

Preparation of cell suspension
The DBMCs in the femoral bones were flushed out using sterile saline. Cell suspensions were made and lysed of red blood cells using ammonium chloride solution and washed three times with phosphate buffer solution. The bone marrow cells were re-suspended at 1×108/mL in sterile saline and perfused into the recipients through portal vein during the surgery. The number of viable DBMCs was more than percent 95 using trypan blue exclusion.

Related indices detection
Measurement of TNF-α mRNA expression in the heterotopic rat cardiac allograft: Six days after surgery, transplanted heart specimen was harvested for determining TNF-α mRNA expression according to previous report[2].
Measurement of serum IL-2 level: Blood samples were collected from rat-tail vein 6 days after surgery. Serum was tested for IL-2 level by an enzyme-linked immunosorbent assay (ELISA) procedure.
Pathological examination: Five Lewis-to-BN heart transplant recipients were randomly chosen from each group 6 days after surgery. Hearts, liver, kidney and lung of recipients were harvested and fixed in 10% phosphate-buffered formalin for subsequent hematoxylin and eosin staining and examination by light microscopy. According to the standard of the International Society for Heart Transplantation (ISHLT) [3], acute rejection was diagnosed and graded.
Flow cytometric analysis: The recipients were analyzed on days 6, 12 and 18 after surgery. Specific T-cell subsets were identified using fluorochrome-cojugated monoclonal antibodies against CD86, CD4+CD45RC+/CD4+CD45RC-, CD3+CD25+, CD4+CD25+ and anti-rat MHC II/RT1B according to the technical data sheet described.

Statistical analysis
Statistical analysis was performed by authors with SPSS 10.0 software. In serum IL-2 concentrations, data were expressed as Mean±SD, and statistical differences were calculated by the ANOVA. The allograft survival time difference was analyzed by Kaplan-Meier technique. Scores for pathological findings were examined by the Kruskal-Wallis test. Differences between groups were considered significant at P < 0.05.

RESULTS

Quantitative analysis of experimental animals
Forty Lewis male rats and sixty male BN rats were all included in the final analysis, without deletion.

Cardiac allograft survival time
In the BN control group, the cardiac allograft survival time was over 100 days. The cardiac allograft survival time was (7.2±0.4), (15.2±2.3), (21.6±3.2), and (7.8±0.8) days for control group, CsA group, CsA+DBMC group and DBMC group, respectively. The cardiac allograft survival time was longer in the CsA+DBMC group than in the DBMC group (P < 0.05).

Measurements of serum IL-2 level and myocardial TNF-α mRNA level in recipient rats (Table 1)

Table 1 shows that serum IL-2 level and myocardial TNF-α mRNA level in recipient rats were significantly higher in the control group and DBMC group than in the CsA group and CsA +DBMC group ( P < 0.05).

Rejection grading for recipient rats
In the control group, 5 rats presented with grade 5 of rejection; In the CsA group, 3 rats presented with grade 2 of rejection and 2 rats with grade 3 of rejection; In the CsA +DBMC group, 4 rats presented with grade 1 of rejection and 1 with grade 2 of rejection; and in the DBMC group, 1 rat presented with grade 1 of rejection, 2 rats with grade 2 of rejection, and 2 rats with grade 3 of rejection.

Flow cytometry detection results (Table 2)
Table 2 shows that after surgery, CD 86 expression on the karyocytes in the peripheral blood was up-regulated with time going in each group. However, on day 6 after surgery CD 86 expression was markedly inhibited in the CsA group and CsA +DBMC group. After surgery, CD3+CD25+ cells in the peripheral blood presented an ascending tendency, however, these cells did not increase obviously on days 6 and 12, but they increased markedly on day 18 after surgery. There were no obvious changes in CD4+CD25+ cells in rat peripheral blood among groups. DBMCs in the recipient peripheral blood karyocytes were more in rats that received DBMC infusion compared to rats that received no BDMC infusion. On days 6 and 12 after surgery, the ratio for CD4+CD45RC+ and CD4+CD45RC- was gradually increased in the CsA +DMBC group, and on day 18, the ratio was remarkably higher compared to BN control group.

DISCUSSION

How to prolong graft survival remains the fundamental objective up to date. The induction of allograft immunological tolerance might provide a good solution to resolve these problems. The long-term immunologic tolerance has been able to be induced in the experimental rodent models. Among these protocols, DBMC infusion plays a critical role. In our renal transplant center, our own clinical experience with DBMC infusion procedure began in September of 1999. Since then, more than 101 renal transplants have received DBMC infusion after surgery. We observed that combined with immunosuppressive drugs, DBMC infusion had significantly decreased the incidence of acute rejection [4-5]. This supports other transplant centers' observations.
In the process of T cell activation, the central step is interaction of T cell receptor/ MHC expressed on antigen-presenting cells (APC), but TCR-MHC antigenic peptides interaction alone does not induce T cell proliferation, and the end result may be the production of an anergic T cell. DBMCs are down-regulators of recipient anti-donor immunity [6]. There are a number of subpopulations of DBMC, which can inhibit the proliferative and cytotoxic responses. These included mesenchymal stem cells, immature dendritic cells, and CD34+ stem cells [6]. Immature dendritic cells are known to be able to direct immune responses toward tolerance, and donor-derived dendritic cells make up the major part of microchimerism. After engraftment of donor's bone marrow-derived cells in the recipient's thymus, intrathymic clonal deletion of donor-reactive cells is conducted by a negative selection. DBMC infusion may have their effects by providing long-term APCs that persist in the recipient and can reduce T-cell anergy. By the way, it might induce the donor-specific tolerance.
Although the mechanism of a combined therapy of CsA which deletes donor- recipient reactive lymphocytes and DBMC infusion that low-expresses costimulation are not completely understood, it appears that powerful peripherial regulatory mechanisms and /or deletion of donor- recipient reactive T cells are involved [7]. Szabo et al [8] demonstrated that CsA inhibits T cell activation by blocking calcium-dependent NFAT activation. In the present study, 6 days after DBMC infusion, the number of chimeric cells was negatively correlated to whether rejection occurred and CsA was used. But the level of costimulatory molecules in chimeric cells treated by DBMC plus CsA was significantly lower than that treated by DBMC alone. This suggests that CsA effectively blocks the proliferation of donor-reactive T cells, indirectly blocks a costimulatory pathway induced by activated T cell/DBMC contact. Study has suggested that more "space" in the thymus be created by immune ablation of recipient with CsA prior to transplantation. CsA might play a role in down-regulating the GVHD responses that might have been elicited by DBMCs [6]. Thus, routine immunosuppressive drugs plus DBMC infusion might induce more stable and safe donor-specific immunoregulation.
In the rat, CD4+ T cell subsets are distinguished by membrane expression of the high (CD45RC+) and low (CD45RC-) molecular weight isoforms of CD45. CD4+CD45RC+ and CD4+CD45RC- T cells represent functionally distinct subpopulations identified as Th1 and Th2 helper T cells, respectively [9-10]. Shirwan et al [11] reported that acute rejection was associated with the expression of Th1 cytokines, whereas long-term survival was associated with predominant expression of Th2 cytokines. The inhibition to production of T cell-derived cytokines, particularly IL-2, plays a key role in the inhibition to T cell proliferation [12]. TNF-α exerts immunologic effects and can activate various gene transcription of target cells and was shown to play a key role in the vascular endothelial cell injury in the early of acute rejection[12]. After activated by antigen recognitions, immunologic cells release TNF-αwhose biological activation or level determination can help to early diagnose the rejection and to evaluate the curative effects [13]. The present study demonstrated that 6 days after surgery serum IL-2 level and TNF-α mRNA level in the transplanted myocardium were obviously lowered in the CsA+DBMC group. These results indicated that CsA+DBMC could help to inhibit acute rejection.
Tolerance to the allografts is associated with down-regulation of TH1 cytokines and up-regulation of TH2 cytokines. In the rat renal allograft model, the induced donor-specific acceptance of vascularized organ allografts is associated with a state of immune deviation to predominance of TH2 cell function [14]. The present study demonstrated that short-term combined therapy of CsA and DBMC infusion could inhibit deviation of TH1/TH2 to TH1, but we failed to find that TH1/TH2 deviated to TH2 cells.
These results indicated that DBMC infusion might lightly prolong heterotopic rat cardiac allograft survival time. A combined therapy of DBMC infusion and short-term CsA can significantly prolong rat allograft survival time compared to intervention of DBMC infusion (P < 0.05) or intervention of CsA. The rejection degree was very mild in the CsA +DBMC group. The mechanisms may include the deviation of TH1/TH2 subsets, clonal deletion, T-cell anergy and suppressive cytokines such as IL-2. It is also possibly associated with the effect of regulatory T cells [15]. The decreased level of TNF-α mRNA expression can help to inhibit acute rejection.
Despite our failure to tolerance induction, there are encouraging results that DBMC infusion has better effects under the condition of application of routine immunodepressant. Therefore, we will further study the effects of regulatory T cells and veto cells in the DBMC infusion-induced donor-specific hyporesponsiveness, and the effects of DBMC on mediation of chronic rejection in the experimental models to provide experimental evidence for establishing clinical donor-specific hyporesponsive state.

REFERENCES

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3 The International Society for Heart Transplantation. A working formulation for the standardization of in the diagnosis of nomenclature heart and lung rejection: heart rejection study group. J Heart Transplant 1990;9(6):587-593
4 He Q, Fang LL, Li MW, et al. Dynamic variety of chimerism in peripheral mononuclear cells of renal allograft recipients received donor bone marrow cells infusions and relationship between chimerism and acute rejection. Zhonghua Shenzangbing Zazhi 2003;19(6):385-388
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环孢素A联合供者骨髓细胞输注延长同种大鼠移植心脏存活时间*☆

姜 睿1，陈江华2，何 强2，吴建永2，金 娟2( 1泸州医学院附属医院泌尿外科，四川省泸州市 646000；2浙江大学医学院附属第一医院肾脏病中心，浙江省杭州市 310003)
姜 睿☆，男，1970年生，四川省泸州市人，汉族，2005年浙江大学毕业，博士，主任医师，主要从事器官移植和泌尿外科学研究。
浙江省卫生厅重点基金（2004C23004）*
摘要
背景：同种异体器官移植后需长期使用免疫抑制剂控制机体免疫状态以免发生排斥反应，但免疫抑制治疗使受者的感染发生率增高，诱导移植免疫耐受是解决这一问题的理想方法。在啮齿类动物，已能诱导出长期免疫耐受，其中供者骨髓细胞输注在诱导免疫耐受中具有重要作用。
目的：观察环孢素A联合供者骨髓细胞输注对同种大鼠移植心脏存活时间的影响。
设计：随机对照动物实验。
单位：浙江大学医学院附属第一医院肾脏病中心。
材料：实验于2002-03/2004-12在浙江大学医学院实验动物中心完成。选用SPF级近交系雄性Lewis大鼠40只和雄性BN大鼠60只，实验方法符合动物伦理学要求。
方法：①制作Lewis→BN大鼠的异位（腹部）心脏移植模型（n=40），将40组动物模型按随机数字表法分为4组（n=10）：对照组不进行特别处理；环孢素A组术后连续7 d给予环孢素A；联合处理组分别于术中及术后第6天输注供者骨髓细胞，术后连续7 d给予环孢素A；供者骨髓细胞组分别于术中及术后第6天输注供者骨髓细胞。另以BN大鼠间的异位（腹部）心脏移植作为BN对照组（n=10）。②观察各组受体大鼠移植心脏的存活时间，检测术后第6天血清白细胞介素2含量以及移植心脏组织中肿瘤坏死因子mRNA表达水平，应用流式细胞仪检测术后第6，12，18天时受体外周血有核细胞中的供体来源细胞、CD3+CD25+细胞、CD4+CD25+细胞的百分比以及共刺激分子CD86表达水平、CD4+CD45RC+/CD4+CD45RC-比率等。
主要观察指标：各组受体大鼠移植心脏存活时间、血清白细胞介素2含量及心肌组织中肿瘤坏死因子α mRNA水平、排斥反应分级、外周血有核细胞中的供者来源细胞、CD3+CD25+细胞、CD4+CD25+细胞的百分比及共刺激分子CD86表达、CD4+CD45RC+/CD4+CD45RC-比率等。
结果：Lewis大鼠40只及雄性BN大鼠60只全部进入结果分析。①联合处理组大鼠移植心脏存活时间长于对照组和供者骨髓细胞组(P < 0.05)。②联合处理组大鼠血清白细胞介素2含量及心肌组织中肿瘤坏死因子α mRNA表达水平均低于对照组和供者骨髓细胞组(P < 0.05)。③联合处理组大鼠排斥反应程度轻于其他3个移植组。④联合处理组大鼠外周血有核细胞上CD86表达受到明显抑制；术后6，12 d联合处理组的CD4+CD45RC+/CD4+CD45RC-比率及D3+CD25+细胞百分比均低于对照组和供者骨髓细胞组；接受供者骨髓细胞输注大鼠外周血中供者来源的有核细胞多于未输注大鼠。
结论：短疗程环孢素A治疗联合供者骨髓细胞输注能减轻大鼠心脏移植急性排斥反应程度，延长移植心脏存活时间。
关键词: 心脏移植；移植物存活；环孢菌素；骨髓移植
中图分类号: R617 文献标识码: A 文章编号: 1673-8225(2008)18-03583-04
姜睿，陈江华，何强，吴建永，金娟. 环孢素A联合供者骨髓细胞输注延长同种大鼠移植心脏存活时间[J].中国组织工程研究与临床康复,2008,12(18):3583-3586
[www.zglckf.com/zglckf/ejournal/upfiles/08-18/18k-3583(ps).pdf]
(Edited by Li LY/Song LP/Wang L)