Study Of Peripheral Blood Lymphocytes MHC-I Expression In Acute Graft Rejection

Background:Organ and tissue transplantation is one of the most medical achievements in 20^th century.As the most effective therapy when organ and tissue function become failure, the success of organ transplantation depends on the excellent surgery,the novel immunosuppressant application,the inflammation management and the advancement of transplants tissue match.However,there are many problems unsolved,such as acute rejection(AR)post-transplants and the gradually increased frequency of malignant disease because of the immunosuppressant treatment of high dose and long duration.Although graft histopathological examination remains the golden standard in the diagnosis of AR in the world,patients are unwilling to accept biopsy because it is invasive and costly.At present,some noninvasive monitoring markers,for instance, biochemistry examination(C-reactive protein),peripheral blood lymphocytes CD4/CD8 ratios,soluble HLA,CD30,cytokines,image measurement and urine examination aimed at kidney transplantation have been used in clinical practice. However,the sensitivity and specificity of these markers are not satisfactory when used for post-transplant monitoring of acute rejection.There is an urgent need for the development of more reliable and noninvasive early markers.It is well known that the major histocompatibility complex(MHC)molecules on donor cells is the major target antigen of host immune response to allogeneic tissues and plays the key role of host immunoreactions.MHC is the highly polymorphic molecules.The exon 2 and 3 of MHC-Ⅰgene were highly polymorphic and related to transplants match.Both exon 4 and exon 5 were non-polymorphic,which can be quantitatively compared among different individuals.MHC-Ⅰmolecules almost express on all nucleated cells of the body,especially abundant in the peripheral blood lymphocytes(PBLs).Lymphocytes are the important cells response to heterogeneity antigen and appear consistently in acutely rejecting transplants.Le Morvan et al observed a significant decrease of PBLs HLA-ⅠmRNA contents with increasing age and proposed that the decreased expression of peripheral HLA-Ⅰat the transcriptional level could contribute to the immunological unresponsiveness of CD8+ T cells and to the increase of infectious and malignant disease,thus the down-regulation of peripheral HLA-Ⅰtranscripts was associated with host cellular immune function depression.Wang et al also demonstrated the expression level of lymphocytes MHC-Ⅰwas associated with the activity and the apoptosis of lymphocytes.In this way,here interested me is:1)The changes of MHC expression in PBLs during the process of AR and the potential possibility of their expression as the early markers of AR.2) The relationship of MHC-Ⅰexpression between the PBLs and the somatic cell.3) Which is the better marker in the diagnosis of acute graft rejection between the MHC-Ⅰand MHC-Ⅱgene expression measurement? 4)The correlation of MHC-Ⅰexpression in mRNA transcripts level and protein expression level.5)The immunosuppressant dosage effect on PBLs MHC-ⅠmRNA expression.All of these deserve the comprehensive study in experiment.Currently,there is few research about the role of MHC-Ⅰexpression in PBLs during AR in few patients.But it is very limited and studied partially.The purified inbred mouse strain is selected as an animal model because the mouse MHC genotype is homologic with homo sapiens largely.This work was based on host cellular immunity.The aims of this study were to investigate the expression of MHC-Ⅰand -Ⅱgenes in peripheral blood lymphocytes following skin grafting and whether their expressions can be used as early markers for AR.Objective:1.To develop a real time PCR measurement semi-quantitatively detecting the expression of MHC mRNA in host peripheral blood lymphocytes before and after skin transplantation in mice2.To explore the changes regulation of MHC-ⅠmRNA expression in the whole process of acute rejection and to analysis the correlation of MHC-Ⅰgene expression and acute graft rejection by the serial analysis the transcripts and protein expression level of MHC3.To study the immunosuppressant dosage effect on the host PBLs MHC mRNA expression,different immunosuppressant single and,or,combination administrated. To investigate the relationship between the MHC-ⅠmRNA expression and CsA plasma concentration level and explore the possibility monitoring allograft acceptance by analysis the expression of MHC-ⅠmRNA on recipient peripheral blood lymphocytes,so as to evaluate the effectiveness of the immunosuppressive therapy in each recipient4.Compared with the other accepted noninvasive indexes of acute rejection,such as PBLs HLA-DR and FasL mRNAs expression,the ratio of peripheral blood lymphocytes CD4/CD8,evaluating the value of MHC-ⅠmRNA level in PBLs in allograft recipient as an early acute rejection markerMethods:1.The development of real time PCR measurementAccording to MHC gene sequence announced in GenBank,the non-polymorphic sequence in exon 4 and 5 of MHC gene were selected to design the primers, synthesized and screened,β-actin gene as the internal standard;using SYBR GreenⅠfluorescent quantitative real time PCR method to detection the expression variation of MHC mRNAs.2.In the study of the first part,peripheral blood lymphocytes MHC-Ⅰgene expression and acute rejection,all mice(n=225,untreated transplants),C57BL/6(MHC phenotype:H-2b)and BALB/C(MHC phenotype:H-2d),specific pathogen free (SPF),were randomly divided into two groups to observe skin graft rejection daily. Experimental grouping were as follows:control group(n=30),normal healthy individuals without any disposed as the pre-transplants controls;experiment group(n =195,untreated graft groups)including Ea allograft group(n=65,BALB/C mice as recipient,H-2b to H-2d),Eb allograft group(n=65,C57BL/6 mice as recipient, H-2d to H-2b),and Ec syngeneic graft group(n=65,BALB/C as recipient,H-2d to H-2d).Skin from the back of donor mice was transplanted onto that of recipient mice in allografi groups(H-2b to H-2d,and H-2d to H-2b)and in syngeneic graft groups (H-2d to H-2d).All the skin grafts were daily inspected and harvested to observe histopathology changes detecting skin graft rejection before and after transplantation every day with H-E staining.Peripheral blood samples were drawn daily and obtained peripheral blood lymphocytes,detected the MHC-Ⅰ(H-2K,H-2D),MHC-Ⅱ(H-2Ia,H-2Ie),and FasL mRNA expression with real time PCR.At the same time, the expression of membrane protein H-2K and H-2Ia antigen in CD8+ T-cell was examined with flow cytometry(FCM).3.In the study of the second part,we studied the immunosuppressant dosage effect on the host PBLs MHC mRNA expression in non-transplants mice firstly,all mice(n =90,normal healthy individuals without any disposed,BALB/C mice)were randomly divided into four groups to explore the immunosuppressant dosage which could down-regulated the expression of MHC mRNAs.Experimental grouping were as follows:control groups(n=20),including normal healthy individuals without any disposed as the pre-treated controls(n=10)and garages placebo as the treated controls(n=10);experiment group(n=70,administrated with different doses of immunosuppressant,such as 10mg/kg,20 mg/kg,40 mg/kg/,60 mg/kg/,80 mg/kg) including the groups of CsA single,prednisone single,and CsA combined with prednisone administrated.Peripheral blood lymphocytes samples of all mice were obtained after the stability of CsA plasma concentration,detected the MHC-Ⅰ(H-2K, H-2D)and MHC-Ⅱ(H-2Ia,H-2Ie)mRNA expression with real time PCR,and a monoclonal fluorescence polarization immunoassay(FPIA;Abbott)to assay the plasma concentration of CsA 2 hours after its oral administration.Secondly,we also studied the effect of immunosuppressant dose on the host PBLs MHC-ⅠmRNA expression in transplants mice.All BALB/C mice(n=285) were randomly divided into two groups to investigate the immunosuppressant dosage effect on host PBLs MHC-ⅠmRNA expression.Experimental grouping were as follows:control group(n=30),normal healthy individuals without any disposed as the pre-transplants controls;experiment group(n=255,immunosuppressant-treated graft groups)including Ea allograft group(n=85,low dose group administrated with CsA 30mg/kg combined with prednisone 30mg/kg to induce the allograft tolerance, H-2b to H-2d),Eb allograft group(n=85,high dose group administrated with CsA 60mg/kg combined with prednisone 60mg/kg,H-2b to H-2d),and Ec syngeneic graft group(n=85,low dose group with CsA 30mg/kg combined with prednisone 30mg/kg,H-2d to H-2d).Skin from the back of donor mice was transplanted onto that of recipient mice in allograft groups(H-2b to H-2d)and in syngeneic graft groups(H-2d to H-2d).All the skin grafts were daily inspected and harvested to observe histopathology changes detecting skin graft rejection before and after transplantation every day with H-E staining.Peripheral blood samples were drawn and obtained peripheral blood lymphocytes,detected the MHC-Ⅰ(H-2K,H-2D) mRNA expression with real time PCR.Results:1.The general state of mice,allograft rejection macroscopically and pathological analysisAll mice survived were healthy and gained weight.Each graft was harvested daily beginning from day 2 post-transplant and was considered absolutely rejected macroscopically when 60%or more of the graft tissue was destroyed.Compared with syngeneic graft controls growing well without skin rejection,the acute allograft rejection was first macroscopically presented at day 11 post-transplant in untreated group,at day 18 post-transplant in low dose group,and since day 26 post-transplant in high dose group.There was statistic significance between untreated groups and treated groups in skin grafts mean survival time(P＜0.05).Pathological evidence of acute graft rejection in allograft groups showed more lymphocytes and monocyte infiltrated in the epidermis.According to infiltrates percentage,the acute graft rejection was grade as four.No obvious infiltration was observed in syngeneic graft group.On the other hand,pathological evidence of acute grafts rejection demonstrated severe lymphocytes and monocytes infiltration in the epidermis in allograft groups.In allograft groups,grade 0-1 of AR appeared from day 2-4 post-transplant in untreated group,from day 2-8 post-transplant in low dose group, and from day 2-15 post-transplant in high dose group;grade 2 appeared from day 5-8 in untreated group,from day 9-15 in low dose group and from day 16-22 in high dose group;grade 3 appeared from day 9-11 in untreated group,from day 16-22 in low dose group,and since day 23 in high dose group;grade 4(severe rejection)appeared from day 12-14 in untreated group,and from day 23-26 in low dose group.It was not observed the skin-grafted severe rejection because of examination stage limited.2.Analysis of PBLs MHC-Ⅰand -ⅡmRNAs Expression.MHC-Ⅰand -ⅡmRNAs expression levels did not show any obvious changes in the syngeneic graft group.However,MHC-Ⅰand -ⅡmRNAs levels were up-regulated, showing bimodal distribution pattern in accordance with AR in allograft group.Based on the standard and validated assay,we investigated the relationship between the target gene expression,histopathological evidence and macroscopical presence in AR.In untreated group,MHC-ⅠmRNA expression increased significantly from day 5 post-transplant(corresponding to grade 2)to reach the first peak at day 7,then slowly decreased,and increased again to form the second peak at day 11,corresponding to the grade 3-4.Noticeably,the low valley point levels which appeared at day 9 post-transplant were still higher than pre-transplant levels.It was evident that the significantly increased expression of MHC-ⅠmRNA appeared earlier approximately 5 to 8 days than graft rejection manifested macroscopically.Compared with MHC-Ⅱlevel,MHC-ⅠmRNA expression level was higher and lasted longer.The expression levels of two loci of MHC-Ⅰgene,H-2K and H-2D,were also analyzed.H-2K mRNA expression(110.38)seemed higher than H-2D(70.06)in the first peak.61 of 65 (93.84%)allograft samples showed increased expression levels of H-2K mRNA at post-transplant than pre-transplant level during AR(P＜0.01).The MHC-Ⅰand -ⅡmRNAs levels also showed obvious up-regulated changes in low dose group the same as that in untreated group.However,the second peak was postponed at day 15 post-transplant.In high dose group,the MHC-ⅠmRNA levels also showed obvious up-regulated changes in mono-modal distribution pattern in accordance with AR.But it appeared at day 20 post-transplant.3.The membrane protein expression of H-2K and H-2Ia on CD8+ T-cell.The expression of membrane protein H-2K and H-2Ia antigen on CD8+ T-cell was examined with flow cytometry(FCM).It showed the median fluorescence intensity(MFI)of H-2K and H-2Ia antigen decreased at the effect of immunosuppressant,however,when acute rejection was presented,the MFI of H-2K and H-2Ia antigen was up-regulated again post-transplant compared with that of pre-transplant in low dose immunosuppressant group.4.The comparative study of PBLs MHC-ⅠmRNA expression level.Compared with that in syngeneic group,the PBLs H-2Ie(corresponding to HLA-DR in human,accepted AR diagnosis marker)mRNA expression increased significantly at day 11-14 post-transplants in untreated group and at day 15-18 in low dose group,respectively,corresponding to grade 3-4.(P＜0.05).Also,the FasL mRNA expression(another accepted AR diagnosis marker)was up-regulated in rejection group compared to non-rejection group(P＜0.05).It appeared at day 9-13 post-transplants in untreated group and at day 11-16 in low dose group,respectively, corresponding to grade 3-4.Nevertheless,the up-regulated expression of MHC-ⅠmRNA,especially H-2K mRNA during AR appeared at grade 1-4 of graft rejection, which was earlier than that of MHC-Ⅱand FasL mRNA.It indicated that the expression levels of MHC-ⅠmRNA in PBLs was more suitable to predicate the early acute rejection.5.The effect dosage on host PBLs MHC-ⅠmRNA in normal miceThe expression level of MHC-ⅠmRNA on host PBLs was associated with the dosage of CsA at the single administrated.The low-dose CsA,including 10 mg/kg, 20mg/kg,and 40mg/kg can up-regulated the expression level of PBLs MHC-Ⅰ(H-2K, H-2D)mRNA,whereas high-dose CsA,including 60mg/kg and 80mg/kg can down-regulated the expression level of PBLs MHC-Ⅰ(H-2K,H-2D)and MHC-Ⅱ(H-2Ia,H-2Ie)mRNAs in mice at the stable CsA blood concentration.CsA combinated with prednisone also can down-regulate the expression level of MHC-Ⅰ/-ⅡmRNAs in PBLs in normal mice.6.The effect dosage on host PBLs MHC-ⅠmRNA in skin-grafted miceThe administration of CsA combined prednisone can down-regulated the expression level of MHC-ⅠmRNA on host PBLs during acute rejection,which was associated with the dosage of immunosuppressant.From the dose-effect relations of view,CsA combined prednisone administrated can prolong significantly the median survival time(MST)of skin-allograft in high dose group(25.08±1.11),low dose group(17.05±1.17),compared with that in untreated group(8.2±1.04).There was a statistically significant difference between the two groups.From the dose-expression of gene relation of view,high immunosuppressant dose could decreased the expression level of host PBLs MHC-ⅠmRNA significantly compared with that in low dose group during acute graft rejection(P＜0.05).The higher immunosuppressant dose used,the more marked the expression level of MHC-ⅠmRNA decreased in PBLs during acute graft rejection.Conclusions:1.Serial detection of host PBLs MHC-ⅠmRNA expression was contributed to prediction acute graft rejection with real time PCR. 2.Elevated MHC-ⅠmRNA expression in host PBLs was highly associated with acute graft rejection in huge animal experiment.3.The expression of MHC-ⅠmRNA,especially H-2K mRNA,can be used as an early noninvasive monitoring marker for AR.