Establishment Of An Acute Graft Versus Host Disease Model Following Liver Transplantation In Rats And The Preliminary Mechanisms

IntroductionAcute graft-versus-host disease following liver transplantation(LTx-aGVHD) occurs when immunocompetent donor lymphocytes originating from the transplanted liver graft undergo activation and clonal expansion,allowing them to mount a destructive immune response against recipient tissues.It is classified as cellular aGVHD,and humoral aGVHD.Cellular aGVHD(referred to as aGVHD)is directed against MHC(major histocompatibility complex)and/or mHC(minor histocompatibility complex)antigens,which often results in severe multi-system disease with a high mortality.Humoral aGVHD is commonly seen after an ABO-mismatched transplant when donor-derived lymphocytes produce antibodies to red-cell antigens,which usually causes only mild and self-limiting haemolytic anaemia of little clinical importance.LTx-aGVHD is an uncommon,but devastating complication.Since the first case described by Burdick et al in 1988,there have been about 80 cases reported up to now.The incidence of LTx-aGVHD in adult liver transplantation is just approximately 1-2%.But,it poses a major diagnostic and therapeutic challenge,and the mortality rate in published reports is as high as 85%.Previous reports of LTx-aGVHD have described the clinical characteristics in detail and focused on the risk factors,time-saving diagnostic methods and effective treatments.Due to the scarcity of reported cases,most of these conclusions are under-debate or even contradictory to each other.On the other hand,most animal models associated with liver transplantation have focused mostly on the rejection (host versus graft reaction)and no animal model of LTx-aGVHD has been established successfully until now.The development of a stable,and reproducible LTx-aGVHD model and study of its mechanism is a necessary and urgent prerequisite for development of effective clinical modalities in this disease. Materials and methods1.Animals and experimental groups:Lewis rats were used as donors,and Lewis or(LewisXBN)F1 rats were used as recipients.Orthotopic liver transplantation was performed using the technique described by Kamada and Calne without anastomosis of the hepatic artery.The experimental animals consisted of three main groups.(1)semiallogeneic liver transplantation groups(Semi-LT groups):liver transplantation was performed between Lewis rat(donor)to(LewisXBN)F1 rat(recipient).The donor splenocytes were adoptively transferred to the same recipient immediately after liver transplantation.The rats were divided into five subgroups according to the numbers of splenocytes(0,1×10⁸,2×10⁸,3×10⁸,4×10⁸,respectively)transferred.(2) syngeneic liver transplantation group(sLT group):Lewis rats received liver graft from the same strain without splenocyte transfusion.(3)semiallogeneic splenocyte transplantation groups(Semi-ST groups):Lewis splenocytes were adoptively transferred to(LewisXBN)F1 rats without liver transplantation.Rats were divided into four subgroups according to the numbers of splenocytes(1×10⁸,2×10⁸,3×10⁸, 4×10⁸,respectively)transferred.From the separate groups(three or six animals in each group),peripheral blood was obtained every 4 days after transplantation for the further study of its mechanism.Rats were sacrificed on day 16 for tissue sampling.These groups consisted of group 1,group 5,group 6 or group 10.2.Assessment of aGVHD:(1)Clinical course and animal survival:All animals were observed twice a day for typical aGVHD-related signs such as dermatitis,alopecia,weight loss,diarrhea, hunched posture and cachexia.The actuarial survival rate,and mean time to death (mean survival time,MST)were calculated after observation of 100 days. (2)Morphometric and histopathologic investigations:Tissue samples were taken at the time of death or sacrificed on day 16 post-operation.Skin,small intestine,colon and liver were pathologically evaluated.3.Peripheral blood cells and biochemical analysis: Peripheral blood was serially obtained from the tail vein once every 4 days after liver transplantation.The WBC,HGB and PLT were measured.Serum ALT,AST concentrations were measured using standard techniques with a serum analyzer.4.The content of TH1/TH2 cytokine in the serum:The level of INF-γ,IL-2,TGF-β₁ in peripheral blood serum were detected by enzyme-linked immunosorbent assay(ELISA).5.Analysis of CD4⁺ CD25⁺ regulatory T cell in the peripheral PBMCs:After the isolation of PBMCs from peripheral blood,the count of CD4⁺ CD25⁺ regulatory T cell was analyzed by Flow Cytometry.6.Analysis of chimera in the peripheral PBMCs:Male Lewis rats were used as donors,and female Lewis or(LewisXBN)F1 rats were used as recipients.Genomic DNA was prepared from PBMCs,and the quantification of sex-genotype(Y-chromatosome)was counted by Real-Time PCR. Results1.The morbidity of aGVHD and animal survival:In the Semi-LT groups,all recipients that received liver transplantation alone survived long-term as the sLT group without any evidence of aGVHD.The survival of rats diminished depending on the number of donor splenocytes transferred.In the 1×10⁸ splenocytes transfusion group,aGVHD occurred in only one recipient (16.7%).In comparison,administration of 2×10⁸,3×10⁸ and 4×10⁸ splenocytes led to a morbidity rate with aGVHD of 50%(3/6),83.3%(5/6)and100%(8/8), respectively.In the Semi-ST groups,no aGVHD happened after 1×10⁸ of Lewis splenocyte were adoptively transferred to(LewisXBN)F1 rat.When the amount of splenocytes was increased to 2×10⁸,3×10⁸ or 4×10⁸,the morbidity of lethal aGVHD was 16.7%(1/6),16.7%(1/6)and 50%(3/6),respectively.The clinical course of aGVHD was similar between Semi-LT groups and Semi-ST groups,including severe dermatitis,diffuse alopecia,diarrhea,weight loss and hunched posture.Histologic examination of the skin and intestine showed pathologic features of aGVHD in the Semi-LT groups and Semi-ST groups.In the Semi-ST groups,obvious mononuclear infiltrate within the portal tracts and sinusoids were observed in the liver.In comparison,the liver grafts were normal without obvious mononuclear infiltrate within portal tracts and sinusoids in the Semi-LT groups.Thus,a reproducible rat model of LTx-aGVHD was developed for the first time here by performing liver transplantation from Lewis to(LewisXBN)F1 rat in combination with donor splenocyte transfusion.2.Analysis of Th1/Th2 cytokine and CD4⁺ CD25⁺ regulatory T cell in peripheral blood:In the sLT group,the serum IL-2,IFN-γ,TGF-β₁ levels changed transiently after liver transplantation.The IL-2,IFN-γlevel were much higher than those in normal rats at 4 days after operation,and the TGF-β₁ levels was significantly lower than those in normal rats at the same time point.But they all returned to normal gradually,and there was no significantly difference as compared with normal rats at 16 days after liver transplantation.When the(LewisXBN)F1 recipients developed aGVHD in Semi-LT groups,the serum IL-2,IFN-γlevels increased continuously after operation,and were much higher than those in normal rats at 8,12 and 16 days after liver transplantation.But the serum TGF-β₁ levels and the count of CD4⁺ CD25⁺ regulatory T cell in the peripheral PBMCs decreased rapidly and were significantly lower than those in normal rats at 12 and 16 days after liver transplantation.In the recipients without aGVHD,the serum IL-2,IFN-γlevels kept stable and were similar to those in normal rats.But the serum TGF-β₁ levels and the count of CD4⁺ CD25⁺ regulatory T cell in the peripheral PBMCs increased gradually and were significantly higher than those in sLY group at 12 and 16 days after liver transplantation.3.Analysis of chimera in the peripheral PBMCs after liver transplantation:In the sLT group,donor cells were detected in peripheral PBMCs and the chimera ratio is 1.34%±0.37%at 4 days after liver transplantation.But this ratio decreased rapidly,and no donor cell can be detected 100 days after operation.In Semi-LT groups,the change of chimera ratio in the recipients without aGVHD was similar as the recipients in sLT group.When the(LewisXBN)F1 recipients developed aGVHD in Semi-LT groups,the chimera ratio in peripheral PBMCs increased continuously after operation,and were much higher than those in sLT group at 4,8,12 and 16 days after liver transplantation. Conclusions1.A reproducible rat model of LTx-aGVHD can be developed by performing liver transplantation from Lewis to(LewisXBN)F1 rat in combination with donor splenocyte transfusion.This relatively timesaving model has the main clinical and histologic features of aGVHD,but without the liver graft involvement, which is the most important histological difference between the transfusion-associated aGVHD and LTx-aGVHD.2.Surgical damage of liver transplantation could lead to the dysregulation of TH1/TH2 cytokines in the early time after liver transplantation,which would promote the development of aGVHD after liver transplantation.3.Dysregulated cytokine production occured during the manifestations of aGVHD after liver transplantation in the rat LTx-aGVHD model we established here.4.CD4⁺ CD25⁺ regulatory T cell may inhibited the development of aGVHD after liver transplantation by a mechanism dependent on down-regulation of TH1 cytokines and up-regulation of TH2 cytokines.5.Detection and continuously analysis of the chimera ratio in peripheral PBMCs after liver transplantation would be helpful for the diagnosis of LTx-aGVHD.