Dynamic Expression Of HLA-G During Acute Graft Rejection: Experimental And Clinical Research

BACKGROUNDOrgan and tissue transplantation is the most magnificent feat during 20th century.Up to date,it has been well believed that organ transplantation is the most effective therapeutic measure for organ failure with the development of clinical technologies,such as tissue matching,organ preservation,skills of transplantation operation and measure to anti-infection of post-operation,more and more intensive study in transplantation immunity,and novel immunosuppressants applications.Although the survival rates of both transplanted organs and recipients have been gradually increased,there are still many problems unsolved,such as acute graft rejection(AGR) and increased occurrence of infection or malignant diseases because of the long term,even over dosage,administration of immunosuppressants. It remains very difficult for physicians to keep balance between increasing dosage of immunosuppressants to prevent the occurrence of AGR and decreasing the drug level to prevent the occurrence of malignant diseases.The ideal state is to design individual therapeutic regimen for each transplant patients according to his immunological state.Unfortunately,except histopathological examination of graft biopsy,there is no reliable,non-invasive early marker to monitor the immuno-state of recipients and to predict the AGR.With the view of immunology,organ transplantation is a complex physical process,which is much similar to the pregnancy.In its nature,embryo implantation is the process of the recognition and tolerance of mother to the fetus with paternal alloantigen.Although both organ transplantation and embryo implantation are the similar allogeneic immune response,the results are completely contrary.Many kinds of proteins and molecules are involved in these process,in which the expression of HLA-G,a non-classical major histocompatibility complex(MHC)-I molecules,has attached the attention of more and more experts and scholars of embryology and pathological pregnancy.HLA-G is a novel,non-classical MHC-I antigen,with similar gene and protein structure with classical MHC-I antigen.On the contrary,however,HLA-G is a minimally polymophic HLA-I molecule.And moreover,HLA-G protein expression has very limited distribution,only restricted to extra-embryonic tissues and other immune privilege site in the physiological conditions.However,in pathological conditions,HLA-G can be expressed by some tumors,virally infected cells and cytokine-activated antigen present cells(APCs).As reported,HLA-G molecules might mediate inhibitory signals when they bind to the inhibitory receptors expressed on the NK cells,T lymphocytes and APCs to inhibit the activity of these cells and to induce the tolerance of fetus.The decrease of HLA-G expression on extra-embryo tissues might result in the pathological pregnancy,such as loss of fetus and eclampsia.The achievement of embryology cast new light on the study of immunology of organ transplantation.Do the well-tolerant patients have high level of HLA-G expression as in maternal-fetal interface? Can immunosuppressants induce HLA-G expressed effectively?.Does the variation of HLA-G expression during the process of AGR have regularity and be helpful to the diagnosis of AGR? Some study examined the level of soluble HLA-G in plasma of patients after transplantation. Different studies,however,gave different,even opposite,results.The reasons are manifold.It is noteworthy that almost every transplanted patient received immunosuppressive therapy before or at the time of operation.It is difficult to distinguish the different role of transplantation and immunosuppressants on the HLA-G,respectively.It is necessary to investigate the variation of HLA-G changes after transplantation or during AGR systematically in the absence or presence of immunosuppressants with the animal models.It has been proved that the mouse Qa-2 protein is the homolog of HLA-G. Qa-2 has similar structure and function in immunoregulation and embryonic development as HLA-G And mouse skin transplantation model is a broadly accepted model to study the immunological mechanisms of allograft rejection because of its easy surgical procedure,easy inspection,and repeatability.Therefore, inbred mouse skin transplantation model is the ideal animal model of present study.In order to investigate the value of HLA-G as a marker for diagnosis of AGR and for monitoring the effects of as well as the effects of immunosuppressive treatment,we quantitatively and dynamically monitored the Qa-2 molecules expression on different time of post-transplant in grafts,infiltrating lymphocytes, peripheral blood lymphocytes(PBLs) and soluble Qa-2 in plasma during the whole process of AGR with inbred mouse skin transplantation model and studied its relationship with the pathological changes of the grafts and immunosuppressive therapy firstly.On the basis of animal study,we further serially monitored the HLA-G expression in peripheral blood and soluble HLA-G in plasma of kidney transplant recipients with stable renal function and with acute rejection on the different time point after transplantation,respectively,compare and analysis the difference between stable patients and those suffering from AGR,and analysis the level of HLA-G with the state of immunotolerance.OBJECTIVE1,To monitor the variation of the Qa-2,the murine homolog of HLA-G, expression in grafts,infiltrating lymphocytes,PBLs and soluble Qa-2 in plasma during the whole process of AGR with mouse skin transplantation models in the absence and presence of immunosuppressants and to study its relationship with the pathological changes of the grafts and immunosuppressive therapy.2,To study the effects of different kinds and dosages immunosuppressants on the Qa-2 expression with single and combined administration and to analysis the relationship between the Qa-2 expression and the immune state of recipients.3,To serially monitor the HLA-G mRNA and protein expression on PBLs AND soluble HLA-G in plasma of kidney transplant recipients with stable renal function and with acute rejection on the different time point after transplantation, respectively.And to compare the difference between stable patients and those suffering from AGR,and analysis the level of HLA-G with the state of immunotolerance.METHODS 1,Establishment of mouse skin transplantation models:In order to investigate the differences between different inbred lines of mouse,we treated with both SPF C57BL/J(C57) and BALB/C(BA) mouse as our transplant recipients.Each kind of mice was randomly divided into three main groups:autogeneic group, groups without immunosuppressants and groups with immunosuppressants. Among them,groups with and without immunosuppressants were divided into subgroups:Normal groups(Group N) and transplant groups,which were divided into autogenous groups,syngeneic groups(Group S and SI) and allogeneic groups(Group A and Group AI).In Groups with immunosuppressants,all the mice were administrated moderate dosage of three drugs including cyclosporine A(CsA),prednisolone(Horm) and mycophenolate mofetil(MMF) by simulating clinical therapeutic regimen.Full thickness dorsal skin derived from donor mice was transplanted onto the dorsal thoraxes of the recipients.More than 80%necrosis of the skin graft was considered as macroscopic rejection.The histopathological rejection was evaluated and classified into four grades according to the scoring systems for acute mouse skin allograft rejection HE staining.2,Monitoring the post-transplant Qa-2 expression:5 mice were sacrificed every day after transplantation in the transplant groups to collect samples.The Qa-2 expression in skin grafts,PBLs and plasma was detected serially by immunohistochemistry,Sybr Green I fluorescent quantitative real-time PCR, flow cytometry(FCM) and Enzyme-linked Immunosorbent Assay(ELISA)3,The effects of immunosuppressants on the Qa-2 expression:SPF C57BL/6 mice were randomly divided into normal control group,single medication groups including CsA group,Horm group,MMF group and combined medication group.Each single medication group was divided again into five dosage subgroups.Qa-2 expression was detected on the PBLs and plasma from each group.Serum concentration of CsA of CsA groups was determined by fluorescence polarization immunoassay(FPIA).4,Serial monitoring the level of HLA-G expression:57 recipients(32 cases with stable renal function as Group stable and 25 with acute rejection proved by kidney biopsy according to the Banff 2003 system as Group AR) were enrolled in present study.PBLs,plasma and serum were collected both before and the D3,D7,W2,W3,M1,M2,M3+).For the patients with AR symptoms,samples were obtained immediately before administration of anti-inflammatory agents and at D3,D7,W2,more than W3.The level of HLA-G mRNA on the PBLs, HLA-G protein on the CD4~+ and CD8~+ T lymphocytes,and soluble HLA-G in plasma were measured by TaqMan probe real-time PCR,FCM and ELISA. whole blood concentration of FK506 was determined by microparticle enzyme immunoassay(MEIA).RESULTS1,Graft survival,macroscopic and pathological changes of skin grafts after transplantationIn this study,all surviving mice were healthy and gained weight.No rejection was found in the autogenic and syngeneic groups until the end of observation.In these groups,the grafts grew well and hair began to grow on some mice.Grafts of the allogeneic groups became hard,black,and shrank gradually until there was necrosis of more than 80%of the graft,which was regarded macroscopically as complete rejection.Mean survival time(MST) of Group A was 9 days(9±0.38) in C57 mice and 10±0.24 days in BA mice.The MST of the skin grafts of group AI was 17±0.54 days in C57 mice and 18±0.36 days in BA,much longer than that of group A(P＜0.01 vs.group SI and P＜0.01 vs.group A).Syngeneic grafts showed almost no or very slight inflammatory infiltration from 2 to 30 days after transplantation.In contrast,allografts displayed different degrees of inflammatory infiltration and tissue destruction with the development of rejection.According to the degree of lymphocyte infiltration and necrosis of skin tissue cells,pathological changes in the grafts were classified into GradeⅠto GradeⅣ.In the Group A of C57 and BA mice,GradeⅠto GradeⅣrejection appeared on D2-D4,D5-D7,D8-D10 and after D11,respectively.And in Group AI of both inbred mice,GradeⅠto GradeⅣrejection appeared on D2-D8,D9-D12,D13-D18 and after D19,respectively.There are no significant differences between two inbred lines mice.2,Variation of Qa-2 expression during AGR in the absence of immunosuppressantsNegative or weak positive Qa-2 expression was observed in the skin graft and infiltrating lymphocytes during the whole process after transplantation.In Group S,no significant changes were observed in infiltrating lymphocyte and PBLs over the post-transplant period compared with the level of pre-operation, which is normal level.While in Group A,Qa-2 expression decreased significantly when pathological rejection was Grade 2-3.The trends of sQa-2 expression in plasma were much different.It increased rapidly after operation and gradually decreased to the pre-operation level after D7.Qa-2 expression of BA mice was much lower than that of C57 ones,but the trends of variation during AGR in both inbred mice were similar.3,Variation of Qa-2 expression during AGR in the presence of immunosuppressantsStrong positive Qa-2 expression was observed on the skin grafts and infiltrating lymphocytes and Qa-2 mRNA in PBLs and Qa-2 protein on CD4~+ and Cd8~+ T lymphocytes were much higher in Group SI and early stage of Group AI compared with normal level.With the development of AGR,Qa-2 expression of lymphocyte infiltrating into the grafts reduced gradually.While sQa-2 level in plasma was much lower in groups with immunosuppressants than in those without drugs.Trends of variation in groups with immunosuppressants were similar with that in groups without drugs.4,Effects of immunosuppressants on the Qa-2 expressionDifferent kinds and dosages of immunosuppressants have different mode of effects on the Qa-2 expression.Median and high dosage of CsA,Horm and MMF and combined medication increased the Qa-2 mRNA in PBLs and Qa-2 protein level in CD4~+ and CD8~+ lymphocytes,while decreased sQa-2 level in plasma.The effects of single medication are in a dose-dependent manner,while the effect of combined medication is the most efficiently.5,Variation of HLA-G expression in patients after kidney transplantationThe level of HLA-G mRNA in PBLs,HLA-G protein on peripheral blood CD4~+ T lymphocyte and sHLA-G in plasma decreased in the first week after operation and consistently elevated thereafter to the much higher level than pre-transplant within the first month.It declined gradually afterward and kept at a higher level than normal in the recipients with stable function.At the episodes of AGR,the level of HLA-G decreased significantly in all the patients of Group AR.CONCLUSIONS1,The expression of Qa-2 reduced significantly much earlier than macroscopic graft rejection,especially in the condition of presence of immunosuppressive agents.It might be useful to diagnose ARG earlier than pathological examination.2,Immunosuppressive treatment prolonged the survival time of grafts and elevated Qa-2 expression significantly in a dose-dependent manner.Combined medication seemed to be the most safe and effective measure.3,HLA-G may be a potential marker for predicting the AGR as well as a tool to monitor the effects of immunosuppressive treatment.