Th17/IL-17及其单克隆抗体在大鼠小肠移植免疫调控中的作用研究
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摘要
器官移植是脏器功能衰竭终末期的有效、常规性治疗手段。随着器官移植技术的不断发展,许多器官的移植已经成为现实。常用于移植的器官包括角膜、心脏、肺、胰腺、肝脏、小肠、肾脏、骨髓等。而在我国,异基因器官移植在所有移植病例中占了绝大多数。其在给家庭和个人带来希望的同时也面临着重大的问题,即移植后严重的急性和慢性排斥反应,它是导致受体死亡的主要原因。
小肠移植是肠衰竭患者最为理想的临床标准治疗方法。虽然小肠移植的临床尝试与开展已有半个世纪,但是其效果与肝脏、肾脏和心脏移植相比仍有很大差距。究其原因,主要是因为小肠富含淋巴组织和大量的组织相容性抗原,患者在小肠移植术后会产生强烈的宿主抗移植物反应、移植物抗宿主反应和严重的全身感染。因此,对小肠移植术后产生免疫排斥反应机制的深入研究及相应对策的实施将是改善小肠移植预后的关键。
目前小肠移植排斥反应的机制尚不完全清楚。在小肠移植排斥反应的发生发展过程中,多种免疫细胞和细胞因子参与了其中。而T细胞的分化增殖所介导的CTL和B细胞以及调节性T细胞在小肠移植术后免疫排斥的发生中发挥了重要作用。此外,参与免疫应答的细胞因子在该过程中也发挥了极其重要的作用。IL-2和IFN-γ是Th1细胞分泌的主要细胞因子,具有广泛的免疫调控和免疫增强作用,可促进T细胞、B细胞、NK细胞和巨噬细胞的活化,以加重移植排斥反应。TNF、IL-6、IL-8在加重排斥反应过程中也具有类似的作用。而IL-4通过抑制IL-1、IL-6和TNF的分泌,抑制T、B细胞的活化而抑制移植排斥反应。TGF-β、IL-10和内皮素ET则可以下调移植术后的免疫排斥反应。
Th17是近年来新发现的一个CD4+ T淋巴细胞的亚群,其不同于Th1、Th2细胞亚群,主要表达IL-17。IL-17是一种作用很强的促炎因子,诱导促炎细胞因子如IL-6、IL-1β、TNF-α、IL-8的表达,引起细胞浸润和组织破坏。其主要的靶细胞为上皮细胞,内皮细胞及成纤维细胞等。研究表明,其在自身免疫性疾病、炎症、肿瘤和移植免疫等疾病中发挥了重要作用。IL-17通过与IL-17R结合发挥生物学效应,而这些靶细胞在小肠组织中含量尤为丰富。由IL-17介导的炎症反应在小肠组织中更为严重。因此,我们设想其生物学功能可能与小肠移植排斥反应的发生、发展有密切的关系。
FK506是一种新型高效的免疫抑制剂,较CsA有更强的免疫抑制作用,使小肠作为单一移植物已成为可能,在移植患者中得到了广泛应用。FK506通过抑制移植排斥相关细胞因子(IL-2等)的产生进而抑制T细胞活化,以抑制移植后免疫排斥反应。此外,FK506还可以抑制排斥反应早期淋巴细胞在移植部位的聚集以及其它炎症细胞的趋化。FK506对免疫排斥的抑制作用是否也有Th17/IL-17的参与目前仍不清楚。FK506虽然在临床小肠移植术后广泛应用,但因其使用剂量大,严重的肾毒性和神经毒性等毒副作用仍不容忽视。因此,深入研究小肠移植免疫排斥的分子机理并开发新的抗排斥药物势在必行。
鉴于Th17/IL-17在免疫反应中的重要作用,本课题采用大鼠同种异体小肠移植模型和人小肠移植活检标本研究Th17/IL-17是否参与了小肠移植术后的免疫排斥反应,研究Th17/IL-17在FK506抗排斥反应中可能的作用,探索性地观察IL-17单克隆抗体在小肠移植术后免疫排斥中的保护作用,为小肠移植术后排斥反应的发生机制的研究提供了依据,为IL-17单克隆抗体的临床应用奠定了实验基础。主要结论如下:
1、证明了人活体小肠移植术后移植肠Th17/IL-17的表达与急性排斥反应正相关。
为了探索在人小肠移植术后移植肠Th17/IL-17与排斥反应之间的关系,我们收集、整理了我科1999年-2003年间的4例活体血缘性小肠移植患者围手术期移植肠活检标本及相关资料,用激光共聚焦显微镜检测了移植肠浸润CD4+IL-17+ Th17细胞的表达情况,证明了小肠移植患者术后移植肠Th17/IL-17的表达与急性排斥反应的相关性。提示,Th17/IL-17表达水平的升高可能在患者小肠移植术后强烈的免疫排斥反应过程中发挥了重要作用。IL-17可能成为小肠移植急性排斥反应早期重要的预警指标和特异、敏感的诊断指标之一。
2、构建了稳定可靠的同种异体小肠移植动物模型。
为了通过动物实验进一步验证上述假设,本课题采用一种自制、可调控的旋转持续静脉输液装置,与节段性异位小肠移植术相结合,构建了近交系大鼠F344→LEW的同种异体小肠移植模型。结果显示,该输液装置能成功地为实验大鼠连续输液1月以上,改善受体的血供和营养,显著提高移植成功率(95.0%),为小肠移植免疫的研究提供了良好的实验基础。
3、小肠移植大鼠的移植肠内Th17/IL-17表达也与急性排斥反应正相关。
为了验证在大鼠小肠移植术后移植肠内Th17/IL-17表达是否也与急性排斥反应正相关。我们利用实时定量PCR、Western blot、激光共聚焦显微镜和ELISA等方法检测了移植肠及血清Th17/IL-17的表达,同时检测了移植物中相关细胞因子和转录因子的表达。结果显示,IL-17mRNA在正常小肠组织中低表达。在异基因移植未治疗组大鼠急性排斥反应期IL-17和RORγt mRNA表达持续升高。细胞因子IL-1β、IL-6、TNF-α、IL-8和IFN-γmRNA在移植后第3,5,7天的表达也呈上升趋势。相反,IL-4、TGF-β和Foxp3 mRNA在异基因移植未治疗组术后低表达,3,5,7天呈逐渐下降趋势。
4、Th17/IL-17可能参与了FK506对移植排斥的抑制作用。
为了探讨FK506对移植排斥的抑制作用是否有可能通过Th17/IL-17发挥作用,在大鼠小肠移植术后给予FK506抗排斥治疗,然后用上述方法检测了相关细胞和因子的表达。结果发现,FK506治疗能明显降低IL-17的表达,减少移植肠内Th17细胞的表达,抑制移植肠急性排斥反应的发生,移植物中TGF-βmRNA和Foxp3 mRNA的表达升高。说明Th17/IL-17参与了FK506对移植排斥的抑制过程。
5、制备了IL-17单克隆抗体,应用IL-17单克隆抗体治疗可以在一定程度上抑制小肠移植术后急性排斥反应,延长移植物的存活时间。
为了探索IL-17单克隆抗体在抑制移植排斥中的作用,本研究利用杂交瘤克隆,制备抗大鼠IL-17mAb,并用于大鼠小肠移植术后的早期治疗。我们发现,IL-17mAb治疗组大鼠术后移植肠IL-17、IL-1β、IL-6、TNF-α、IL-8 mRNA表达降低,而IL-4和TGF-βmRNA表达缓慢升高。结果显示,与异基因移植未治疗组相比,IL-17mAb治疗能明显延长大鼠存活时间,术后移植肠组织病理学检查无排斥反应依据。说明,IL-17mAb治疗可以有效抑制大鼠小肠移植术后的急性排斥反应,明显改善移植物的生存。
6、结论
通过本课题的研究,发现人小肠移植术后移植肠内有Th17/IL-17的表达,且其表达程度与术后急性排斥反应正相关。为了探索Th17/IL-17是否参与了小肠移植术后的免疫排斥反应,本研究通过构建同种异体大鼠小肠移植模型,进一步研究发现,大鼠小肠移植术后移植肠内也存在Th17/IL-17的表达,其表达与移植物免疫排斥程度呈正相关。同时发现移植肠RORγt、IL-1β、IL-6、TNF-α、IL-8和IFN-γmRNA在移植后第3,5,7天的表达升高,而IL-4、TGF-β和Foxp3 mRNA在异基因移植未治疗组术后低表达,3,5,7天呈逐渐下降趋势。
FK506治疗能明显降低IL-17的表达,抑制移植肠急性排斥反应的发生,升高移植物中TGF-βmRNA和Foxp3 mRNA的表达。说明Th17/IL-17参与了FK506对移植排斥反应的抑制过程;移植术后早期应用IL-17mAb可以抑制移植肠急性排斥反应的发生。
以上结果说明,Th17/IL-17参与了小肠移植术后急性排斥反应的发生,且许多细胞因子也参与其中,FK506可能通过下调小肠移植后移植肠内和血清Th17/IL-17的表达而抑制急性排斥反应的发生。IL-17mAb的早期应用证实了其在抑制移植排斥反应中的有效性,为临床小肠移植急性排斥反应的防治和诱导免疫耐受提供了新的思路和方向。
Organ transplantation is an effective and conventional treatment of the terminal stage of organ failure. The advance of transplantation technology makes many kinds of organ transplantation possible namely transplantation of cornea, heart, lung, pancreas, liver, small bowel, kidney, marrow and so on. In China, allogene organ transplantation cases make up the majority, which brings hope as well as causes trouble to patients and their family. Severe acute and chronic rejections are the main factors that lead to the death of acceptors.
Small bowel transplantation is the most ideal clinical treatment of bowel failure. Clinical trial and practice of small bowel transplantation has been done for half of the contrary, but compared with the therapeutic effect of liver, kidney and heart transplantation, the small bowel transplantation is less effective. It is mainly because small bowel has redundant lymphoid tissue and histocompatibility antigen and after allogenic small bowel transplantation patients may suffer strong host versus graft reaction (HVGR), graft versus host reaction (GVHR) and severe infection. Therefore, it is crucial to have in-depth study of rejection mechanism and its treatment in order to improve prognosis.
Nowadays the mechanism underlying allograft rejection remains largely unclear. Many immunocytes and cytokines are involved in the small bowel transplantation process. T lymphocytes mediated CTL, B cells and regulatory T cells (Treg) play a decisive role in immune reaction of small bowel transplantation. Cytokines involved in immune response also have a key role in this process. IL-2 and IFN-γwhich are the main cytokines secreted by Th1, have extensive function of immunoregulation and immunoenhancement. They can improve the activation of T cells, B cells, NK cells and macrophages and intensify the rejection. TNF, IL-6 and IL-8 play similar role in intensifying rejection. In contrast, IL-4 may suppress the activation of T cells and B cells so as to suppress the rejection. GF-β, IL-10 and ET can down-regulate the rejection.
Th17 is a newly detected subset of CD4+ T, which differs from Th1 and Th2 subsets, mainly expresses IL-17. IL-17 is a proinflammatory factor with strong function, which can induce proinflammatory cytokine such as the expression of IL-6, IL-1β, TNF-αand IL-8 thereby causing cellular infiltration and tissue damage. Its major target cells include epithelial cells, endothelial cells and fibroblasts. Previous studies show that IL-17 is an important factor in autoimmune, disease inflammation, tumor and transplantation immunity. IL-17 exerts biological effects through combining with IL-17R and these target cells are particularly rich in small bowel tissue. IL-17 mediated inflammation is more severe in small bowel tissue. Therefore, we infer that biological function of IL-17 has close relation with small bowel rejection.
FK506 is a new and highly effective immunosuppressive agent, which is extensively used in organ transplantation. It may suppress the produce of rejection related cytokines thereby suppressing the activation of T cells, as a result, suppressing the rejection. Besides, FK506 can prevent aggregation of lymphocytes in the early rejection and chemotaxis of inflammatory cells. But whether Th17 or IL-17 participates in the FK506 suppression of rejection or not remains unknown. Although FK506 is used extensively after clinical small bowel transplantation, severe side effects such as renal toxicity and neurotoxicity caused by large dose of FK506 still can not be neglected. So it is imperative to do in-depth study of rejection mechanism and develop new anti-rejection medicine.
Since Th17 and IL-17 may play important roles in immune reaction, we detect the functions of Th17 and IL-17 in the process of transplantation rejection and FK506-mediated anti-rejection by using rat small bowel allograft transplantation model and biopsy specimen of human small bowel transplantation. The protective function of IL-17 monoclonal antibody in immunological rejection is further investigated after small bowel transplantation. Thus, the study may push further insight into the mechanisms underlying small bowel transplantation related rejection and lay experimental basis for clinical application of IL-17 monoclonal antibody. The major findings are as follows.
1. The expression of Th17/IL-17 is positively correlated with the intensity of acute rejection after living-related small bowel transplantation
In order to examine the relation between Th17/IL-17 expression and acute rejection after living-related small bowel transplantation, we collect biopsy specimens from 4 cases of living-related small bowel transplantation from 1999 to 2003. All the clinical information is available. The expression of CD4+, IL-17+, and Th17 is further detected by LSCM. The data show that the increased expression of Th17/IL-17 is correlated with the high intensity of acute rejection after living-related small bowel transplantation. IL-17 may be considered as one of the early and sensitive warning markers for acute rejection.
2. Establishment of a stable and reliable allograft animal model
We establish one allograft model of inbred F344→LEW rat by combining the self-made controllable swivel intravenous infusion device with the technique of segmental heterotopic small intestine transplantation. The results suggest that this device may successfully ensure continuance infusion for more than 1 month in rats, improving the blood supply and nutrition of the acceptors and evidently raising the survival rate of grafts (95.0%), thus providing one valuable tool for the experimental study of small intestine transplantation immunity.
3. The expression of Th17/IL-17 in grafts is positively correlated with the intensity of acute rejection
In order to examine the relation between the expression of Th17/IL-17 in grafts and acute rejection, we detect the expression of Th17/IL-17 and relative cytokines and transcription factor by using real time quantified PCR, Western blot, LSCM and ELISA. The results show that IL-17mRNA is decreased in normal small bowel tissues. The mRNA expression of IL-17 and RORγt is found increased gradually during acute rejection period in the control group. The mRNA expression of IL-1β, IL-6, TNF-α, IL-8 and IFN-γis found increased on postoperative day 3, 5 and 7, respectively. On the contrary, the mRNA expression of IL-4, TGF-βand Foxp3 is decreased in control group, coupled with downtrend on postoperative day 3, 5 and 7.
4. The role of Th17/IL-17 in FK506-related rejection suppression
In order to investigate the role of Th17/IL-17 in FK506-related rejection suppression, we treat the rats received small bowel transplantation with FK506 and detect the expression of relative cytokines and transcription factors using the methods as described above. The results show that FK506 treatment may decrease the expression of IL-17 as well as Th17 in grafts, thus suppressing acute rejection and increasing TGF-βmRNA and Foxp3 mRNA expression in grafts. The data confirm that Th17/IL-17 may be involved in FK506-related rejection suppression.
5. Establishment of IL-17 mAb and application of IL-17 mAb in suppressing acute rejection and prolonging the survival time of grafts
For the sake of exploring the function of IL-17 mAb in rejection suppression, the anti-rat IL-17 mAb is established by using hybridoma clone and is further used in the early treatment after small bowel transplantation. We found that the expression of IL-17, IL-1β, IL-6, TNF-α, IL-8 mRNA is decreased in experimental group treated with IL-17mAb, while the expression of IL-4 and TGF-βmRNA increase slowly. The results show that the survival time of experimental group treated with IL-17mAb is significantly longer than that of untreatment group. The results of histopathologic examination show no sign of rejection. The data implies that IL-17mAb treatment may effectively suppress rejection after rat small bowel transplantation and remarkably improve the survival of grafts.
6. Conclusion
We found that Th17/IL-17 expression existed in grafts after human small bowel transplantation and it positively correlated with acute rejection. In order to decide whether Th17/IL-17 expression was involved in rejection, we established rat allograft small bowel transplantation model and further study confirmed that Th17/IL-17 expression also existed in grafts and it positively correlated with rejection. In addition, RORγt mRNA of grafts and expression of IL-1β, IL-6, TNF-α, IL-8 and IFN-γmRNA increased on postoperative day 3, 5, and 7. On the contrary, low expression of IL-4, TGF-βand Foxp3 mRNA had been detected in untreatment group, coupled with downtrend on postoperative day 3, 5 and 7.
FK506 treatment could decrease IL-17 expression, thereby suppressing acute rejection, increase TGF-βand Foxp3 mRNA expression in grafts. It indicated that Th17/IL-17 is involved in FK506 induced rejection suppression. Likewise, early treatment with IL-17mAb after transplantation may suppress acute rejection.
From the above results we may draw the conclusion that Th17/IL-17 and many cytokines participated in the acute rejection after small bowel transplantation. FK506 may suppress acute rejection through downregulating Th17/IL-17 expression. Early administration of IL-17mAb monoclonal antibody effectively inhibited acute rejection, which might pave the way for diagnosis and treatment of acute rejection and induced immunotolerance in clinic.
小肠移植是肠衰竭患者最为理想的临床标准治疗方法。虽然小肠移植的临床尝试与开展已有半个世纪,但是其效果与肝脏、肾脏和心脏移植相比仍有很大差距。究其原因,主要是因为小肠富含淋巴组织和大量的组织相容性抗原,患者在小肠移植术后会产生强烈的宿主抗移植物反应、移植物抗宿主反应和严重的全身感染。因此,对小肠移植术后产生免疫排斥反应机制的深入研究及相应对策的实施将是改善小肠移植预后的关键。
目前小肠移植排斥反应的机制尚不完全清楚。在小肠移植排斥反应的发生发展过程中,多种免疫细胞和细胞因子参与了其中。而T细胞的分化增殖所介导的CTL和B细胞以及调节性T细胞在小肠移植术后免疫排斥的发生中发挥了重要作用。此外,参与免疫应答的细胞因子在该过程中也发挥了极其重要的作用。IL-2和IFN-γ是Th1细胞分泌的主要细胞因子,具有广泛的免疫调控和免疫增强作用,可促进T细胞、B细胞、NK细胞和巨噬细胞的活化,以加重移植排斥反应。TNF、IL-6、IL-8在加重排斥反应过程中也具有类似的作用。而IL-4通过抑制IL-1、IL-6和TNF的分泌,抑制T、B细胞的活化而抑制移植排斥反应。TGF-β、IL-10和内皮素ET则可以下调移植术后的免疫排斥反应。
Th17是近年来新发现的一个CD4+ T淋巴细胞的亚群,其不同于Th1、Th2细胞亚群,主要表达IL-17。IL-17是一种作用很强的促炎因子,诱导促炎细胞因子如IL-6、IL-1β、TNF-α、IL-8的表达,引起细胞浸润和组织破坏。其主要的靶细胞为上皮细胞,内皮细胞及成纤维细胞等。研究表明,其在自身免疫性疾病、炎症、肿瘤和移植免疫等疾病中发挥了重要作用。IL-17通过与IL-17R结合发挥生物学效应,而这些靶细胞在小肠组织中含量尤为丰富。由IL-17介导的炎症反应在小肠组织中更为严重。因此,我们设想其生物学功能可能与小肠移植排斥反应的发生、发展有密切的关系。
FK506是一种新型高效的免疫抑制剂,较CsA有更强的免疫抑制作用,使小肠作为单一移植物已成为可能,在移植患者中得到了广泛应用。FK506通过抑制移植排斥相关细胞因子(IL-2等)的产生进而抑制T细胞活化,以抑制移植后免疫排斥反应。此外,FK506还可以抑制排斥反应早期淋巴细胞在移植部位的聚集以及其它炎症细胞的趋化。FK506对免疫排斥的抑制作用是否也有Th17/IL-17的参与目前仍不清楚。FK506虽然在临床小肠移植术后广泛应用,但因其使用剂量大,严重的肾毒性和神经毒性等毒副作用仍不容忽视。因此,深入研究小肠移植免疫排斥的分子机理并开发新的抗排斥药物势在必行。
鉴于Th17/IL-17在免疫反应中的重要作用,本课题采用大鼠同种异体小肠移植模型和人小肠移植活检标本研究Th17/IL-17是否参与了小肠移植术后的免疫排斥反应,研究Th17/IL-17在FK506抗排斥反应中可能的作用,探索性地观察IL-17单克隆抗体在小肠移植术后免疫排斥中的保护作用,为小肠移植术后排斥反应的发生机制的研究提供了依据,为IL-17单克隆抗体的临床应用奠定了实验基础。主要结论如下:
1、证明了人活体小肠移植术后移植肠Th17/IL-17的表达与急性排斥反应正相关。
为了探索在人小肠移植术后移植肠Th17/IL-17与排斥反应之间的关系,我们收集、整理了我科1999年-2003年间的4例活体血缘性小肠移植患者围手术期移植肠活检标本及相关资料,用激光共聚焦显微镜检测了移植肠浸润CD4+IL-17+ Th17细胞的表达情况,证明了小肠移植患者术后移植肠Th17/IL-17的表达与急性排斥反应的相关性。提示,Th17/IL-17表达水平的升高可能在患者小肠移植术后强烈的免疫排斥反应过程中发挥了重要作用。IL-17可能成为小肠移植急性排斥反应早期重要的预警指标和特异、敏感的诊断指标之一。
2、构建了稳定可靠的同种异体小肠移植动物模型。
为了通过动物实验进一步验证上述假设,本课题采用一种自制、可调控的旋转持续静脉输液装置,与节段性异位小肠移植术相结合,构建了近交系大鼠F344→LEW的同种异体小肠移植模型。结果显示,该输液装置能成功地为实验大鼠连续输液1月以上,改善受体的血供和营养,显著提高移植成功率(95.0%),为小肠移植免疫的研究提供了良好的实验基础。
3、小肠移植大鼠的移植肠内Th17/IL-17表达也与急性排斥反应正相关。
为了验证在大鼠小肠移植术后移植肠内Th17/IL-17表达是否也与急性排斥反应正相关。我们利用实时定量PCR、Western blot、激光共聚焦显微镜和ELISA等方法检测了移植肠及血清Th17/IL-17的表达,同时检测了移植物中相关细胞因子和转录因子的表达。结果显示,IL-17mRNA在正常小肠组织中低表达。在异基因移植未治疗组大鼠急性排斥反应期IL-17和RORγt mRNA表达持续升高。细胞因子IL-1β、IL-6、TNF-α、IL-8和IFN-γmRNA在移植后第3,5,7天的表达也呈上升趋势。相反,IL-4、TGF-β和Foxp3 mRNA在异基因移植未治疗组术后低表达,3,5,7天呈逐渐下降趋势。
4、Th17/IL-17可能参与了FK506对移植排斥的抑制作用。
为了探讨FK506对移植排斥的抑制作用是否有可能通过Th17/IL-17发挥作用,在大鼠小肠移植术后给予FK506抗排斥治疗,然后用上述方法检测了相关细胞和因子的表达。结果发现,FK506治疗能明显降低IL-17的表达,减少移植肠内Th17细胞的表达,抑制移植肠急性排斥反应的发生,移植物中TGF-βmRNA和Foxp3 mRNA的表达升高。说明Th17/IL-17参与了FK506对移植排斥的抑制过程。
5、制备了IL-17单克隆抗体,应用IL-17单克隆抗体治疗可以在一定程度上抑制小肠移植术后急性排斥反应,延长移植物的存活时间。
为了探索IL-17单克隆抗体在抑制移植排斥中的作用,本研究利用杂交瘤克隆,制备抗大鼠IL-17mAb,并用于大鼠小肠移植术后的早期治疗。我们发现,IL-17mAb治疗组大鼠术后移植肠IL-17、IL-1β、IL-6、TNF-α、IL-8 mRNA表达降低,而IL-4和TGF-βmRNA表达缓慢升高。结果显示,与异基因移植未治疗组相比,IL-17mAb治疗能明显延长大鼠存活时间,术后移植肠组织病理学检查无排斥反应依据。说明,IL-17mAb治疗可以有效抑制大鼠小肠移植术后的急性排斥反应,明显改善移植物的生存。
6、结论
通过本课题的研究,发现人小肠移植术后移植肠内有Th17/IL-17的表达,且其表达程度与术后急性排斥反应正相关。为了探索Th17/IL-17是否参与了小肠移植术后的免疫排斥反应,本研究通过构建同种异体大鼠小肠移植模型,进一步研究发现,大鼠小肠移植术后移植肠内也存在Th17/IL-17的表达,其表达与移植物免疫排斥程度呈正相关。同时发现移植肠RORγt、IL-1β、IL-6、TNF-α、IL-8和IFN-γmRNA在移植后第3,5,7天的表达升高,而IL-4、TGF-β和Foxp3 mRNA在异基因移植未治疗组术后低表达,3,5,7天呈逐渐下降趋势。
FK506治疗能明显降低IL-17的表达,抑制移植肠急性排斥反应的发生,升高移植物中TGF-βmRNA和Foxp3 mRNA的表达。说明Th17/IL-17参与了FK506对移植排斥反应的抑制过程;移植术后早期应用IL-17mAb可以抑制移植肠急性排斥反应的发生。
以上结果说明,Th17/IL-17参与了小肠移植术后急性排斥反应的发生,且许多细胞因子也参与其中,FK506可能通过下调小肠移植后移植肠内和血清Th17/IL-17的表达而抑制急性排斥反应的发生。IL-17mAb的早期应用证实了其在抑制移植排斥反应中的有效性,为临床小肠移植急性排斥反应的防治和诱导免疫耐受提供了新的思路和方向。
Organ transplantation is an effective and conventional treatment of the terminal stage of organ failure. The advance of transplantation technology makes many kinds of organ transplantation possible namely transplantation of cornea, heart, lung, pancreas, liver, small bowel, kidney, marrow and so on. In China, allogene organ transplantation cases make up the majority, which brings hope as well as causes trouble to patients and their family. Severe acute and chronic rejections are the main factors that lead to the death of acceptors.
Small bowel transplantation is the most ideal clinical treatment of bowel failure. Clinical trial and practice of small bowel transplantation has been done for half of the contrary, but compared with the therapeutic effect of liver, kidney and heart transplantation, the small bowel transplantation is less effective. It is mainly because small bowel has redundant lymphoid tissue and histocompatibility antigen and after allogenic small bowel transplantation patients may suffer strong host versus graft reaction (HVGR), graft versus host reaction (GVHR) and severe infection. Therefore, it is crucial to have in-depth study of rejection mechanism and its treatment in order to improve prognosis.
Nowadays the mechanism underlying allograft rejection remains largely unclear. Many immunocytes and cytokines are involved in the small bowel transplantation process. T lymphocytes mediated CTL, B cells and regulatory T cells (Treg) play a decisive role in immune reaction of small bowel transplantation. Cytokines involved in immune response also have a key role in this process. IL-2 and IFN-γwhich are the main cytokines secreted by Th1, have extensive function of immunoregulation and immunoenhancement. They can improve the activation of T cells, B cells, NK cells and macrophages and intensify the rejection. TNF, IL-6 and IL-8 play similar role in intensifying rejection. In contrast, IL-4 may suppress the activation of T cells and B cells so as to suppress the rejection. GF-β, IL-10 and ET can down-regulate the rejection.
Th17 is a newly detected subset of CD4+ T, which differs from Th1 and Th2 subsets, mainly expresses IL-17. IL-17 is a proinflammatory factor with strong function, which can induce proinflammatory cytokine such as the expression of IL-6, IL-1β, TNF-αand IL-8 thereby causing cellular infiltration and tissue damage. Its major target cells include epithelial cells, endothelial cells and fibroblasts. Previous studies show that IL-17 is an important factor in autoimmune, disease inflammation, tumor and transplantation immunity. IL-17 exerts biological effects through combining with IL-17R and these target cells are particularly rich in small bowel tissue. IL-17 mediated inflammation is more severe in small bowel tissue. Therefore, we infer that biological function of IL-17 has close relation with small bowel rejection.
FK506 is a new and highly effective immunosuppressive agent, which is extensively used in organ transplantation. It may suppress the produce of rejection related cytokines thereby suppressing the activation of T cells, as a result, suppressing the rejection. Besides, FK506 can prevent aggregation of lymphocytes in the early rejection and chemotaxis of inflammatory cells. But whether Th17 or IL-17 participates in the FK506 suppression of rejection or not remains unknown. Although FK506 is used extensively after clinical small bowel transplantation, severe side effects such as renal toxicity and neurotoxicity caused by large dose of FK506 still can not be neglected. So it is imperative to do in-depth study of rejection mechanism and develop new anti-rejection medicine.
Since Th17 and IL-17 may play important roles in immune reaction, we detect the functions of Th17 and IL-17 in the process of transplantation rejection and FK506-mediated anti-rejection by using rat small bowel allograft transplantation model and biopsy specimen of human small bowel transplantation. The protective function of IL-17 monoclonal antibody in immunological rejection is further investigated after small bowel transplantation. Thus, the study may push further insight into the mechanisms underlying small bowel transplantation related rejection and lay experimental basis for clinical application of IL-17 monoclonal antibody. The major findings are as follows.
1. The expression of Th17/IL-17 is positively correlated with the intensity of acute rejection after living-related small bowel transplantation
In order to examine the relation between Th17/IL-17 expression and acute rejection after living-related small bowel transplantation, we collect biopsy specimens from 4 cases of living-related small bowel transplantation from 1999 to 2003. All the clinical information is available. The expression of CD4+, IL-17+, and Th17 is further detected by LSCM. The data show that the increased expression of Th17/IL-17 is correlated with the high intensity of acute rejection after living-related small bowel transplantation. IL-17 may be considered as one of the early and sensitive warning markers for acute rejection.
2. Establishment of a stable and reliable allograft animal model
We establish one allograft model of inbred F344→LEW rat by combining the self-made controllable swivel intravenous infusion device with the technique of segmental heterotopic small intestine transplantation. The results suggest that this device may successfully ensure continuance infusion for more than 1 month in rats, improving the blood supply and nutrition of the acceptors and evidently raising the survival rate of grafts (95.0%), thus providing one valuable tool for the experimental study of small intestine transplantation immunity.
3. The expression of Th17/IL-17 in grafts is positively correlated with the intensity of acute rejection
In order to examine the relation between the expression of Th17/IL-17 in grafts and acute rejection, we detect the expression of Th17/IL-17 and relative cytokines and transcription factor by using real time quantified PCR, Western blot, LSCM and ELISA. The results show that IL-17mRNA is decreased in normal small bowel tissues. The mRNA expression of IL-17 and RORγt is found increased gradually during acute rejection period in the control group. The mRNA expression of IL-1β, IL-6, TNF-α, IL-8 and IFN-γis found increased on postoperative day 3, 5 and 7, respectively. On the contrary, the mRNA expression of IL-4, TGF-βand Foxp3 is decreased in control group, coupled with downtrend on postoperative day 3, 5 and 7.
4. The role of Th17/IL-17 in FK506-related rejection suppression
In order to investigate the role of Th17/IL-17 in FK506-related rejection suppression, we treat the rats received small bowel transplantation with FK506 and detect the expression of relative cytokines and transcription factors using the methods as described above. The results show that FK506 treatment may decrease the expression of IL-17 as well as Th17 in grafts, thus suppressing acute rejection and increasing TGF-βmRNA and Foxp3 mRNA expression in grafts. The data confirm that Th17/IL-17 may be involved in FK506-related rejection suppression.
5. Establishment of IL-17 mAb and application of IL-17 mAb in suppressing acute rejection and prolonging the survival time of grafts
For the sake of exploring the function of IL-17 mAb in rejection suppression, the anti-rat IL-17 mAb is established by using hybridoma clone and is further used in the early treatment after small bowel transplantation. We found that the expression of IL-17, IL-1β, IL-6, TNF-α, IL-8 mRNA is decreased in experimental group treated with IL-17mAb, while the expression of IL-4 and TGF-βmRNA increase slowly. The results show that the survival time of experimental group treated with IL-17mAb is significantly longer than that of untreatment group. The results of histopathologic examination show no sign of rejection. The data implies that IL-17mAb treatment may effectively suppress rejection after rat small bowel transplantation and remarkably improve the survival of grafts.
6. Conclusion
We found that Th17/IL-17 expression existed in grafts after human small bowel transplantation and it positively correlated with acute rejection. In order to decide whether Th17/IL-17 expression was involved in rejection, we established rat allograft small bowel transplantation model and further study confirmed that Th17/IL-17 expression also existed in grafts and it positively correlated with rejection. In addition, RORγt mRNA of grafts and expression of IL-1β, IL-6, TNF-α, IL-8 and IFN-γmRNA increased on postoperative day 3, 5, and 7. On the contrary, low expression of IL-4, TGF-βand Foxp3 mRNA had been detected in untreatment group, coupled with downtrend on postoperative day 3, 5 and 7.
FK506 treatment could decrease IL-17 expression, thereby suppressing acute rejection, increase TGF-βand Foxp3 mRNA expression in grafts. It indicated that Th17/IL-17 is involved in FK506 induced rejection suppression. Likewise, early treatment with IL-17mAb after transplantation may suppress acute rejection.
From the above results we may draw the conclusion that Th17/IL-17 and many cytokines participated in the acute rejection after small bowel transplantation. FK506 may suppress acute rejection through downregulating Th17/IL-17 expression. Early administration of IL-17mAb monoclonal antibody effectively inhibited acute rejection, which might pave the way for diagnosis and treatment of acute rejection and induced immunotolerance in clinic.
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