Animal Experiment Of MHC-I Expression And Its Application In Clinical During Acute Graft Rejection

BACKGROUND Organ transplantation is the most magnificent feat during 20th century. Extensive use of organ transplantation saved lives of numeric patients who had organ failures. However, there are still many problems unsolved. Among them,acute graft rejection (AGR)after transplantation is the main cause for that the graft function is decrease or even lose, and it's also the main cause for that result in chronic graft rejection. To identify and prevent AGR at the early stage of rejection can retrieve allograft.At present, graft biopsy and histopathological examination remains the "golden standard" to diagnose AGR. It is hard to be used as routine method because of its Achilles'heel, such as recurrent error of sampling, complications and especially its invasive injuries. At the same time, some non-invasive monitoring markers, such as imaging examination, biochemistry examination and antibodies examination, have not yet been used for diagnosis of AGR in clinical practice because of their poor sensitivity and specificity. So there is an urgent need to develop more reliable and non-invasive early markers to monitor the immuno-state of transplanted patients and to predict the AGR.MHC is a closely linked gene cluster located in one chromosome of mammals and codes a group of proteins, which are the key molecules participating in antigen presenting and activation of T lymphocytes, and MHC-Ⅰmolecule play important and extensive roles in immunoregulation. Classic MHC molecules have great polymorphism, and mismatched MHC gene between donor and receipt result in graft rejection after transplantation. In research area of transplantation, most researches focus on MHC of graft itself and antibody to graft in host serum. It is notable that T lymphocyte is one of the cells bearing most abundant MHC-Ⅰmolecules. Some researches argued that HLA-A and HLA-B transcription decreased with ageing in peripheral blood leucocytes and might be a monitor of failed immune function in aged person; MHC-Ⅰmolecule played a protective role for memory T cell; MHC-Ⅰmolecule prolonged the survival time of CD4 positive T cell. Our previous study showed that the expression of MHC-ⅠmRNA increased when acute rejection occurred. As a molecule that exercises the biological function, does MHC-Ⅰprotein also elevate when AGR occurs? And how is its application in clinic? Is MHC-ⅠmRNA or MHC-Ⅰprotein appreciable to predict AGR?In order to investigate the value of MHC-Ⅰas a marker for diagnosis of AGR, we made inbred mouse skin transplantation model to monitored quantitatively and dynamically the MHC-Ⅰmolecules expression in peripheral blood lymphocytes (PBLs),grafts and infiltrating lymphocytes on different times during the whole process of AGR and studied the relationship among MHC-Ⅰexpression, the pathological changes of the grafts and immunosuppressive therapy firstly. Then we further serially monitored the HLA-Ⅰexpression in peripheral blood CD4 and CD8 T lymphocyte of 53 kidney transplant recipients who showed stable renal function or suffered from acute rejection at the different time points after transplantation, compared and analyzed the difference between stable patients and AGR patients and analyzed the level of HLA-Ⅰwith the state of immunotolerance. This research explored the feasibility of MHC-Ⅰas a marker for predicting AGR through both animal experiment and clinical study. OBJECTIVE1. To establish the mouse skin transplantation model to observe the macroscopic and histopathological changes of skin graft after transplantation and classified rejection into 4 grades according to the scoring systems for acute mouse skin allograft rejection.2. To explore the changes of MHC-ⅠmRNA expression and protein during the whole process of acute rejection and which is better for predicting AR? 3. Compared with the other noninvasive index of AR, MHC-II, to evaluate the value of MHC-I in PBLs as AR marker.4. To serially measure respectively the HLA-I protein and mRNA expression in PBLs of kidney transplant recipients with stable renal function and with acute rejection at the different time points after transplantation. And to compare the difference between stable patients and those suffering from AGR, and analyze the level of HLA-I with the state of imrnunotolerance.METHOD1. Experimental groups:All 270 BALB/C recipient mice were randomly divided into three groups:untreated graft groups, immunosuppressive-treated graft groups and bacterial infection groups. Untreated graft groups and immunosuppressive-treated graft groups were further divided into non-transplant groups, syngeneic groups(group S and group SI) and allogeneic groups(group A and group AI). In groups with immunosuppresants, all the mice were administrated moderate dosage of three drugs including cyclosporine A (CsA), prednisolone (Horm) and mycophenolate mofetil (MMF) according to clinical therapeutic regimen. Enterococcus were injected into BALB/C mice enterocoelia to construct bacterical infectious group. Full-thickness skin from the backs of C57BL/6 mice (H-2b,donor) was transplanted onto that of Balb/C mice(H-2d,recipient) in allograft group(H-2b to H-2d) and in syngeneic graft group(H-2d to H-2d).2. Establishment of mouse skin transplantation models and collection of samples: Full thickness dorsal skin derived from donor mice was transplanted onto the dorsal thoraxes of the recipients. Six recipient mice at each time point were analyzed systematically every two days as following:blood samples were collected and anticogulated by EDTA-K2; Skin grafts were obtained from the sacrificed mice at the same time as blood were collected, and then fixed in 4%formalin solution, ready for hematoxylineosin stain and immunohistochemistry detection. For normal control group and bacterical infectious groups, anticogulant blood sample were collected to be ready to use.3. Detecting blood samples:To detect MHC-I, MHC-II MFI on different subtypes of lymphocytes (CD4+T, CD8+T) by FCM and MHC-I, MHC-II mRNA expression levels by FQ-PCR.4. Examination of mice graft skin samples:all the graft skin samples were cut into 5-μm sections, and stained with hematoxylineosin(HE). The skin rejection was classified into 4 grades according to the extent of lymphocytes infiltration. The dynamic histological changes of graft rejection were recorded. MHC-I expression in the skin graft was examined by immunohistochemistry.5. Serial monitoring the level of HLA-I expression:53 recipients (21-60 years old) who had undergone renal transplantations from March 2008 to September 2010 (33 cases with stable renal function as Group stable and 20 with acute rejection proved by kidney biopsy according to the Banff 2003 system as Group AR) were enrolled in present study. PBLs were collected both before transplant and the D3, D7, W2, W3, Ml, M2, M3+ after transplant. For the patients with AR symptoms, samples were obtained immediately before administration of anti-inflammatory agents and at D3, D7, more than W2. The level of HLA-I mRNA in the PBLs, HLA-I protein on the CD4+ and CD8+ T lymphocytes were measured by real-time RT-PCR and FCM respectively.RESULTS1. Skin grafts' macroscopy and pathological analysis All experimental mice gained weight during the trail process. The skin graft of mice in syngraft group grew well, no rejection was found until the end of observation. In A group and AI group, the graft gradually looked hard, black and shrank, and up to 80% graft necrosis took place at the 11th day and 22th day after transplantation respectively. In the syn grafts group (group C), there was no obvious infiltration in the epidermis, and could be considered as grade 0 or grade 1. In the whole process of trail in group A, the different degrees of infiltration of lymphocytes and monocytes were observed successively in the epidermis. During 2-4th,4-8th,8-12 th,12-16th day post-operation, pathological analysis showed grade 1,2,3,4 respectively. In group AI, pathological grade 1,2,3,4 was corresponding to 2-8th,8-14th,14-20th,20-26th day post-operation. 2. The expression of MHC-I in PBLs in bacterial infection group Compared with normal control group, at 8 hours after mice peritoneum were administrated with Enterococcus, the amount of white blood cells increased significantly, but the expression of MHC-I in PBLs, whether on mRNA level or protein level, has no changes.3. The expression of MHC-I during AR in the absence of immunosuppressantsIn 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, MHC-I expression varied significantly at different times post-operation.Whether on protein level detected by FCM or on mRNA level detected by real time RT-PCR, MHC-I expression had the similar trends of variation during AGR. In group A, it increased rapidly at sixth day after operation and gradually decreased to the pre-operation level at D14. The time period when MHC-I increased was corresponding to pathological rejection Grade 2-4.Positive MHC-I expression was observed in the skin graft and infiltrating lymphocytes during the whole process after transplantation. The MHC-I expression increased when AGR occurred.4. The expression of MHC-I during AGR in the presence of immunosuppressantsIn Group SI, no significant changes were observed in infiltrating lymphocyte and PBLs over the post-transplant period compared with the level of pre-operation. Compared with S group, Group SI showed a decreased MHC-I expression, which demonstrated that immunosuppressants could decrease MHC-I expression.Whether on protein level detected by FCM or on mRNA level detected by real time RT-PCR, MHC-I expression has the similar trends of variation during AGR. In Group AI, it increased rapidly at eighth day after operation, reached a highest level at D16, and gradually decreased to the pre-operation level at D24. The time period when MHC-I increased was corresponding to pathological rejection Grade 2-4.The intensity of MHC-I expression was lower in the skin graft and infiltrating lymphocytes during the whole process after transplantation in group SI than in group S. The MHC-Ⅰexpression increased when AR occurred.5. The expression of MHC-Ⅰand MHC-Ⅱin PBLs during AGRThe expression of MHC-ⅡmRNA had the similar trend with MHC-ⅠmRNA during AGR, but the range of variation was smaller than that of MHC-ⅠmRNA. Compared with MHC-Ⅰprotein, The expression level of MHC-Ⅱprotein was lower and lasted shorter.6. Variation of HLA-Ⅰexpression in patients after kidney transplantationThe level of HLA-Ⅰprotein on peripheral blood CD4+T lymphocyte and CD8+T lymphocyte had the similar trend. HLA-I on T lymphocyte showed a slight increase in the first week after operation and then rapidly decreased to the pre-operation level in the recipients with stable function. At the episodes of AGR, the level of HLA-Ⅰincreased significantly in all the patients of Group AR.CONCLUSIONS1. Animal experiment showed that at early stage of AGR, when is corresponding to pathological gradeⅠ-Ⅱ, the expression of both MHC-Ⅰprotein and mRNA increased, and lasted to pathological gradeⅣ. The expression of both MHC-Ⅰprotein and mRNA can predict AGR.2. Clinical application further demonstrated that when AGR occurred in kidney transplantation, the expression of MHC-Ⅰin PBLs increased, especially on protein levels. The expression of MHC-Ⅰprotein on PBLs can be a marker for predicting AGR.