Immunology of corneal allograft rejection. Associated human leucocyte populations

Human serum samples and peripheral blood mononuclear leucocytes (PBML) were collected from ten corneal transplant recipients over a two-year period at intervals before and after surgery, and during and after episodes of immunologic (rejection) reaction. Serum was monitored for the development of antibodies to histocompatibility antigens and PBML were stained with a panel of monoclonal reagents specific for T cell and for monocyte populations using indirect immunofluorescence. Patients who demonstrated an elevation in the Leu 2a+ (suppressor/cytotoxic) T cell population following an immunologic reaction ultimately accepted their grafts, whereas patients whose Leu 2a+ population was unaffected developed irreversible graft clouding. Additionally, a direct positive correlation between the development of elevated levels of lymphocytotoxic antibodies and helper T cells was noted in some patients during immunologic reactions. Neither DR-positive nor monocyte population changes demonstrated any consistent correlation with rejection episodes or final graft outcome.     

HLA-A,HLA-B and HLA-DR matching reduces the rate of corneal allograft rejection

Purpose The purpose of this study is to determine the effectiveness of HLA typing in preventing corneal allograft rejection.Methods This retrospective single-center study analyzed 459 consecutive HLA-typed patients who underwent perforating keratoplasty (PKP) between 1983 and 2001. Grafts were postoperatively transparent after donor-recipient selection by HLA-A, -B and -DR typing. Patients were divided into a low- and a high-risk group based on their preoperative diagnosis.Results The estimated 1-, 5- and 10-year graft survival (Kaplan-Meier) was 93, 88 and 67% in low-risk patients and 73, 43 and 38% in high-risk patients. We found a significant correlation between the number of HLA mismatches and the rate of allograft rejections: a donor-recipient match of two or more alleles in HLA-A, -B or -DR reduces the rejection rate by at least 10% in low-risk (10 years after PKP; P<0.04) and 40% in high-risk patients (3 years after PKP; P<0.0001). Especially HLA-B mismatches are important prognostic factors for both low- (P<0.008) and high-risk patients (P<0.003). Considering both HLA-B and -DR mismatches significantly reduces the rate of allograft rejection, particularly in high-risk patients (P<0.0001). Matching on a split typing level offers no significant advantage over broad level matching.Conclusion Clinical results confirm theories developed to explain the function of the HLA (MHC) receptor. The closest possible donor-recipient match of HLA antigens based on broad level typing significantly reduces the rate of allograft rejection and thus improves the prognosis for long-term transparency of corneal grafts in both high- and low-risk patients.This work was presented in part at the 100th meeting of the German Society of Ophthalmology, Berlin, 26–29 September 2002     

人体角膜移植内皮排斥反应的共焦显微镜研究

目的：应用共焦显微镜探讨活体角膜内皮排斥反应的早期形态学特征和变化。方法：19例(19只眼)角膜移植术后发生内皮排斥反应的人群根据角膜内皮排斥反应轻重的不同分为轻、中、重三组。应用共焦显微镜(Confoscan2．0)对角膜植片进行检查，并分析其相关因素。结果：活体角膜内皮排斥反应特有的共焦显微镜改变主要包括：①早期发现在KP及其附近的内皮细胞面有少量的免疫细胞附着，紧靠KP处的角膜基质细胞出现轻度水肿，细胞排列较混乱，胞浆中出现少量空泡状暗区。②中期发现在内皮细胞表面可见较多的免疫细胞聚集并被完全激活，基质细胞明显水肿，细胞核不清楚，基质中有免疫细胞进入；可见由高反光的免疫细胞和不明成份的组织碎片混合组成内皮排斥线。③在晚期排斥的患者中可见大量“激活”的免疫细胞在角膜内皮细胞面聚集，内皮细胞几乎完全破坏；基质细胞明显水肿，部分被“激活”且伴有大量免疫细胞聚集。结论：①内皮排斥反应的早期能够发现角膜内皮面有免疫细胞沉积；②角膜内皮面的免疫细胞沉积与角膜内皮排斥反应的轻重成正相关；③角膜内皮排斥反应实际上是一个混合排斥反应的过程；④共焦显微镜对角膜内皮排斥反应具有实时、无创、敏感、准确以及可莺蒽、可比较、可活体观察等临床价值。     

树突状细胞在角膜移植排斥反应中的免疫调节机制的探讨

目的 观察自体未成熟树突状细胞(Idc)对于小鼠同种异体角膜移植的免疫调节作用,探讨其作用机制.方法 PBS或自体Idc尾静脉注射7 d后,采用双色免疫荧光染色法及流式细胞术分析小鼠脾脏中CD4 CD28 及CD4 CTLA4 T细胞的表达.PBS或自体Idc尾静脉注射7 d后进行小鼠同种异体角膜移植,临床观察两组植片的存活时间.结果 Idc注射后,脾脏中CD4 CD28 T细胞的比例下降(P＜0.01),而CD4 CTLA 4 T细胞比例明显升高 (P＜0.01).Idc经尾静脉注射后明显延长了同种异体角膜移植片的存活时间(P＜0.01).结论 Idc可诱导CD80/CD86-CTLA-4抑制型共刺激信号替代CD80/CD86-CD28信号通路,从而阻断协同刺激信号的传递及T细胞的活化,对于同种异体角膜移植排斥反应具有免疫抑制作用.     

调节性T细胞在角膜移植排斥反应中的研究进展

角膜移植术是治疗终末期角膜疾病的常用方式,虽然角膜移植的成功率较其它器官移植高,但是术后排斥仍然是手术失败的主要原因。器官移植后排斥反应高度依赖于免疫细胞向淋巴组织或炎症部位的定向迁移和归巢,并受粘附分子和趋化因子的调控。调节性T细胞在免疫调节中起着关键性作用,通过诱导免疫耐受维持内环境稳定,在器官移植排斥反应、自身免疫性疾病及肿瘤相关研究中发挥重要作用。本篇综述主要介绍调节性T细胞参与眼部免疫耐受的相关研究,着重阐述调节性T细胞在角膜移植排斥过程中的作用、机制和应用。     

角膜移植排斥反应的共焦显微镜研究

目的：探讨角膜移植术后免疫排斥反应的共焦显微镜特征,在细胞水平为该症的基础和临床研究提供活体、三维与实时的科学依据。方法：应用NIDEK公司提供的共焦显微镜,对11例（11眼）发生角膜移植免疫排斥反应的患眼角膜在不同时期行三维实时扫描成像,并用计算机作定性与定量分析。结果：角膜移植免疫排斥反应的共焦显微镜呈现如下特征：①上皮排斥线是由较小的炎症细胞与受损的较大的上皮细胞混合形成的结构;②上皮下浸润表现为体积较小、折射率高的炎症细胞的聚集,上皮下神经纤维水肿呈串珠状改变,浅基层细胞水肿;③基质排斥反应可见角膜基质细胞水肿,胞体变形,数量减少,并见炎症细胞浸润,后者于缝线周围较明显;④内皮排斥线则是由较小的明亮的炎症细胞与核固缩胞体形态异常的内皮细胞混合而成,随着内皮排斥线的发展,内皮 细胞数量减少,体积变大呈伪足状;⑤以上排斥反应均见新生血管长入植片,并与血管壁可见游动的炎症细胞。结论：应用共焦显微镜可在活体上为角膜移植术后免疫排斥反应提供早期诊断与鉴别诊断的科学依据,并在细胞水平对排斥反应的触发与转归进行活体的动态观察。     

Immune mechanisms of corneal allograft rejection

Penetrating keratoplasty has been successfully performed on humans for over 100 years and remains the most common form of solid tissue transplantation. Although corneal allografts enjoy a remarkable degree of immune privilege, immune rejection remains the leading cause of keratoplasty failure. The immunologic basis for corneal allograft rejection was established in animal studies over 50 years ago, yet large gaps remain in our knowledge regarding the cellular and molecular mechanisms of corneal allograft rejection. The enormous redundancy in the mammalian immune system creates a condition that favors the development of multiple independent immune mechanisms that can produce corneal allograft rejection. Although there are few absolute principles, it is certain that the immune rejection of corneal allografts is (1) T cell-dependent, (1) heavily dependent upon CD4(+) T cells, (3) not restricted to either Th1 or Th2 T cell populations, and (4) dependent upon an intact repertoire of resident antigen presenting cells.     

同种异体角膜移植后角膜内皮细胞的特征表现及生物学意义

同种异体角膜移植目前仍是治疗角膜疾病最为有效的方法,但术后发生的免疫排斥所引起的角膜炎症反应和血管化,最终导致植片水肿,甚至功能丧失仍是造成手术失败的最主要原因.角膜内皮细胞作为免疫赦免及角膜正常生理功能维系的首要屏障,在眼部系统中占据着至关重要的位置.本文就同种异体角膜移植术后免疫的识别,应答和最终反应的各阶段角膜内皮所呈现的特征改变及生物学意义进行阐述.

Abstract：

Allograft corneal transplantation is currently the most effective and optimum method to treat corneal disease, however it is the immune rejection that the main reason for graft edema caused by vascularization and inflammation of the cornea and surgical failure after transplantation. Corneal endothelial cells occupy a crucial position as immune privilege and the primary barriers for maintaining normal physiological function of eye. In this paper, the various stages (recognition, response and reaction of immune system) of corneal endothelial changes in the morphological and biological characteristics had been presented after allograft corneal transplantation.     

Cyclosporine-containing collagen shields suppress corneal allograft rejection

Thirty-seven rabbit eyes with penetrating keratoplasty grafts placed in vascularized beds to enhance the possibility of graft rejection were treated with cyclosporine delivered in collagen shields or drops of olive oil. Treatment was begun either immediately after grafting or at the first sign of immune graft reaction. Mean survival time of the grafts in the collagen shield treated eyes was significantly longer than in the eyes treated with drops. In the eyes treated at the first sign of graft reaction, cyclosporine in collagen shields halted the rejection process; seven of these eyes survived the 120-day observation period, compared to one of the eyes treated with drops. These results indicate that the collagen shield is an effective delivery system for cyclosporine and the topically administered cyclosporine is effective in suppressing the initiation of graft rejection and in reversing a graft reaction in progress.     

角膜移植术后免疫排斥反应的防治

目的 为控制角膜移植术后发生排斥反应，探讨防治排斥反应的有效途径。方法 根据不同角膜病变排斥反应发生的规律给予不同期的药物联合治疗，降低和控制排斥反应。结果 129眼中47眼(36．43％)发生免疫排斥反应，其中高危角膜病变40眼，非高危角膜病变7眼。有角膜新生血管者占89．36％。排斥反应发生时间为术后3周—3年。经药物联合治疗，角膜排斥反应得到明显的抑制，有效率达63．83％。结论 免疫排斥反应的发生是多因素参与的复杂过程，尤其高危角膜病变，术后出现排斥反应的机率明显增加，发生的时间相对较早，时间跨度较长。因此应根据不同的角膜病变及手术情况，尽早应用免疫抑制剂可明显降低排斥反应的发生率。     

The impact of corneal allograft rejection on the long-term outcome of corneal transplantation

PURPOSE: To examine the influence of corneal allograft rejection on the survival of penetrating corneal transplantation, to review the status of conventional therapies to improve graft survival, and to consider prospects for alternative approaches to reduce the impact of rejection. DESIGN: Perspective, including prospective, observational cohort study. METHODS: An examination of the literature on human corneal graft rejection and data from the Australian Corneal Graft Registry, reviewed in the context of clinical experience. RESULTS: Corneal graft outcome is not improving with era. The sequelae of inflammation, whether occurring before corneal transplantation or subsequently, exert a profound influence by predisposing the graft to rejection. Of the developments that have been instrumental in reducing rejection in vascularized organ transplantation, living-related donation is not an option for corneal transplantation. However, HLA matching may be beneficial and requires reassessment. The evidence base to support the use of systemic immunosuppressive agents in corneal transplantation is thin, and topical glucocorticosteroids remain the drugs of choice to prevent or reverse rejection episodes. Experimental approaches to local allospecific immunosuppression, including the use of antibody-based reagents and gene therapy, are being developed but may be difficult to translate from the laboratory bench to the clinic. CONCLUSIONS: Corneal allograft rejection remains a major cause of graft failure. High-level evidence to vindicate the use of a particular approach or treatment to prevent or treat corneal graft rejection is lacking. In the absence of extensive data from randomized, controlled clinical trials, corneal graft registers and extrapolation from experimental models provide some clinically useful information.     

雷公藤多甙滴眼液防治角膜移植免疫排斥反应的实验研究

目的观察雷公藤多甙(TⅡ)滴眼液局部应用对角膜移植免疫排斥反应的影响.方法建立大鼠角膜移植模型,随机分组A,B,C组为SD大鼠-Wistar大鼠组间同种异体角膜移植,SD大鼠为受体,Wistar大鼠为供体,其中A组为空白对照组、B组为0.02%TⅡ滴眼液组、C组为0.03%TⅡ滴眼液组,另设D组为SD大鼠同种间对照组、E组为SD大鼠自体对照组.用裂隙灯显微镜记录及比较各组移植排斥指数(RI),包括角膜植片透明度、水肿度、新生血管程度,并比较角膜排斥发生时间.结果术后各组移植排斥指数及发生角膜移植排斥时间,A组与B组、A组与C组、B组与C组比较均有显著性差异,P＜0.05;D组和E组两对照组之间比较P＞0.05,无显著性差异.结论TⅡ滴眼液可有效地防治角膜移植免疫排斥反应,0.03%比0.02%TⅡ滴眼液更有效.     

Class II alloantigen induced on corneal endothelium. Role in corneal allograft rejection

The expression of Class II major histocompatibility complex (MHC) antigens on corneal cells can be increased in vitro by (gamma-interferon) and in vivo in inflammatory reactions. The expression of Class II MHC by corneal endothelium of New Zealand White (NZW) rabbits during the rejection of corneal allografts was demonstrated by immunoperoxidase staining. Class II MHC expression by corneal endothelial cells may facilitate rejection of corneal allografts.     

穿透性角膜移植术后慢性失功移植片的免疫学研究

目的检测穿透性角膜移植术后慢性失功移植片的免疫与非免疫相关细胞因子表达的变化,探讨免疫与非免疫因素在角膜植片慢性失功中的作用机制。设计临床实验研究。研究对象分为两组,角膜植片慢性失功（CCAD）组：穿透性角膜移植（PK）术后因CCAD导致移植失败的8例患者的角膜植片;正常对照组：由山东跟库提供的正常角膜供体3只。方法免疫组化法检测两组角膜片免疫相关细胞因子表达,结合患者的临床资料进行综合分析。主要指标角膜中CD4^＋、CD8^＋、F4／80、TGF-β、bFGF、α-SMA的表达。结果免疫组化检测显示正常对照组：角膜各层组织 、α-SMA、bFGF表达阴性,F4／80角膜基质偶见表达,TGF-β在角膜上皮层有表达。CCAD组：所有角膜植片全层均术见CD4^＋及CD8^＋T淋巴细胞浸润;5例失功植片基质层F4／80阳性表达,其余3例角膜植片全层表达阴性;所有角膜植片上皮层可见TGF-β阳性表达,基质层内可见TGF-β表达。所有失功角膜片基质层bFGF表达呈弱阳性;所有患者角膜基质内α-SMA表达阳性,后弹力层附近可见较强表达。结论穿透性角膜移植术后慢性失功移植片免疫组化检查未发现支持临床急性免疫排斥反应的证据,抗原递呈细胞及非免疫特异相关细胞因子的异常表达提示免疫与非免疫因素参与了CCAD的发生和发展。     

雷公藤对异位角膜移植抗排斥反应的研究

目的：采用雷公藤对角膜移植排斥反应进行实验性治疗。 方法以 Ｗｉｓｔａｒ大鼠异种角膜异位移植为模型，将 ３６只鼠随机分为治疗组及对照组，并在术后ｄ２、ｄ５、ｄ７、ｄ１０、ｄ１４分别取出植片做病理切片，检测外周血Ｔ淋巴细胞亚群及Ｔ淋巴细胞集落形成数。 结果雷公藤可推迟排斥反应的发生时间（排斥时间 ７．５±１．０７ｄ，对照组 ５．４±０． ９７ｄ）；有明显减少外周血中辅助 Ｔ淋巴细胞百分率的作用（术后 ｄ５治疗组 ＣＤ４＋／ＣＤ８＋为 １． ４２±０． ２７，对照组为１， ９６±０． ２６）；对大鼠外周血 Ｔ淋巴细胞集落形成能力无明显影响。 结论雷公藤多甙是一种有效的免疫抑制剂，能明显抑制角膜移植排斥反应发生时间及 ＣＤ４＋／ＣＤ８＋比值的上升。     

环孢霉素A滴眼治疗角膜移植排斥反应

应用0.5％环孢霉素A(cyclosporin A,CsA)滴眼治疗穿透性角膜移植术后发生免疫排斥患者16例(16只眼),治愈9只眼,好转6只眼,无效1只眼。随访5～24个月,其中2只眼因停药复发,1只眼于拆线后复发,继续用药或增加给药次数后治愈。研究表明0.5％CsA滴眼剂治疗术前移植床条件较好,角膜移植术后发生免疫排斥的病例可得到良好疗效;而对术前移植床条件较差,角膜移植术后发生免疫排斥的病例有一定的疗效。作者对眼局部应用CsA治疗角膜移植排斥的疗效和作用机理进行了讨论。     

白细胞介素-34在大鼠角膜移植排斥反应中的作用机制

目的　探讨白细胞介素-34（interleukin-34,IL-34）在大鼠角膜移植术后的表达情况以及在免疫排斥反应中的作用。方法　以SD大鼠为供体、Wistar大鼠为受体建立角膜移植实验模型。Wistar大鼠60只按照随机数字表法随机分为3组,B组为自体角膜移植组,C、D组行同种异体角膜移植术。另取10只为正常对照组（A组）。术后B、C组术眼滴泰利必妥眼液,D组术眼滴典必殊眼液。术后各组分别取10只大鼠判断四组角膜植片的存活情况并作生存分析。其余大鼠于术后14 d取术眼角膜植片,行组织病理学、免疫组织化学及RT-PCR检测。结果　生存分析结果提示,A组和B组角膜不发生排斥反应,D组角膜存活时间为（26.00±0.97）d,远高于C组（10.00±1.55）d（P<0.001）。HE染色结果显示,C组角膜组织各层有大量炎性细胞浸润以及新生血管形成,D组仅有少量炎性细胞、新生血管。免疫组织化学结果提示,IL-34蛋白在C组的表达量（0.089 4±0.005 6）明显高于A组（0.037 7±0.002 3）、B组（0.068 4±0.004 4）和D组（0.044 5±0.004 5）的表达量（F=145.21,P＜0.01）,且主要集中在上皮层和基质层。RT-PCR结果提示,IL-34、IL-1β、TNF-α、IL-17A的mRNA 在C组角膜组织中表达水平明显高于A组、B组和D组（均为P<0.05）。结论　IL-34参与了大鼠角膜移植术后的排斥反应,而典必殊可能通过抑制IL-34的表达及相关的信号通路,延缓排斥反应的进展。     

CTLA4-Ig抑制鼠高危角膜移植的免疫排斥反应

目的探讨细胞毒性T淋巴细胞相关性抗原4免疫球蛋白（CTLA4-Ig）对大鼠角膜移植排斥反应的抑制作用及局部抗免疫排斥反应机制。方法建立42只大鼠穿透角膜移植模型,治疗组26只术前将角膜供体置于10μg/mgCTLA4-Ig的Optisol液中,对照组16只术前角膜供体只浸泡于Optisol液24h,浸泡后的植片移植到SD大鼠右眼角膜上。术后裂隙灯显微镜观察两组角膜植片的存活情况。排斥反应植片分别行缺氧诱导因子（HIF-1α）、血管内皮生长因子（VEGF）、碱性成纤维生长因子（bFGF）等免疫组织化学SABC法染色,比较两组间各因子表达情况。结果治疗组的角膜植片混浊及血管化程度明显低于对照组,两者比较差异有统计学意义（P〈0.05）;治疗组角膜植片中HIF-1α、bFGF、VEGF表达明显低于对照组（P〈0.05）。结论CTLA4-Ig能够减轻角膜植片的急性排斥反应,可能与它能下调HIF-1α、bFGF、VEGF等因子的表达有关。     

TGFβ2-DC对小鼠同种异体角膜移植排斥反应的抑制作用

目的:观察自体TGFβ2-DC对于同种异体角膜移植术后免疫排斥反应的调节作用.方法:PBS或已摄取供体抗原的自体TGFβ2-DC经小鼠尾静脉注射7d后,建立小鼠同种异体角膜移植模型,临床观察两组植片混浊程度等情况,比较植片存活时间.结果:TGFβ2-DC注射后,植片发生移植排斥反应的时间延迟,植片存活时间明显延长,PBS组及TGFβ2-DC尾静脉组植片的存活时间分别为17.8±3.01d和37.6±1.65d(P＜0.01).结论:摄取供体抗原的不成熟自体DC可以诱导受体获得供体抗原的特异性免疫耐受,使小鼠同种异体角膜移植的免疫排斥反应延迟,植片存活时间明显延长.