Experimental Study On The Effects Of Anti-CD25 Monoclonal Antibody In Prevention Of Rat Corneal Allograft Rejection Reaction

Corneal transplantation is a sight restoring surgery done for corneal blindness which is due to corneal disease,such as keratoconus,corneal degeneration,corneal dystrophy,corneal scar,active keratitis.Penetrating keratoplasty(PKP) in "normal-risk" eyes shows a low incidence of allograft rejection for the normal cornea lack of blood vessels and lympgatic vessels and location a relative immune privileged site.However,PKP in "high-risk" eyes shows a high incidence of allograft rejection for immune privilege eroded by the sequelae of inflammation and neovascularization. Corneal transplantation is one of the most successful organ transplantations,but immunological rejection reaction is still the major cause of human corneal allograft failure.Corneal allograft rejection reaction is a comparative complicated immune reaction process,its exact pathogenesis still needs to be resolved.Recently,many studies have shown that the cytokine plays an important role in corneal allograft rejection reaction.Helper T lymphocytes(Th) are the cells which can secrete kinds of cytokines at most,and also are the key cells of immune regulation.Its key roles are mainly performed by complicated cytokine network,so they are closely associated with corneal allograft rejection.Based on the different kinds of cytokines Th cells produce,they are divided into two groups:Th1 and Th2 subsets.Th1 cells predominantly produce IFN-γand interleukin(IL)-2,and so on,which mediate cellular immunity involving delayed type hypersensitivity(DTH) and cytotoxic T lymphocyte reaction.Th2 cells produce IL-4,IL-10,and so on,which activate B cells to promote antibody production and mediate humoral immunity.Th1 and Th2 cells are mutually interactional and inhibitory.Recently,relative studies have shown that corneal allograft rejection is mainly mediated by the Th1 type immune response, which leads to a delayed-type hypersensitivity reaction.But some studies have also shown that Th2 type cytokines are associated with allograft immune rejection,the survival of grafts can be prolonged by using Th2 type cytokines inhibitor.Therefore, to further investigate the interaction with different kinds of cytokines in corneal allograft rejection reaction and the expressions of cytokines before and after allograft rejection is not only to offer theory basis for elucidating the mechanism of allograft immune rejection,but also to offer an important means for discovering and diagnosing the allograft rejection as early as possible to lower the incidence of allograft rejection and heighten the incidence of allograft survival.CD4+CD25+regulatory T cells(CD4+CD25+Tr) have anergy and immunosuppressive activity,and play a crucial role in prevention of autoimmunity diseases and mediating transplantation tolerance.FOXP3 is the intracellular molecule expressed in the CD4+CD25+Tr.Many experiments have shown that the transcription factor FOXP3 plays a key role in CD4+CD25+Tr development and function and represents a specific molecular marker for these cells.Recombinant adeno-associated virus vector carrying mouse FOXP3 gene was used to transduce CD4(+)CD25(-) T cells.These FOXP3-transduced T cells are similar to naturally occurring CD4+CD25+Tr cells which show immunosuppressive activity in vitro. Because FOXP3 is a critical regulator of CD4+CD25+Tr cells development and function,it has important significance to investigate the role of FOXP3 in the allograft rejection of rat penetrating keratoplasty,which may help to know the relationship between the effector T cells(Teff) and CD4+CD25+Tr cells in allograft immune rejection.CD25,also known as IL-2R alpha chain,is not only the marker of activated T cells but also the marker of CD4+CD25+Tr.Maintained CD25 high expression on CD4+CD25+Tr is helpful to promote its immunity suppression function,while significantly enhanced CD25 expression on effector T cells is most likely associated with T cell proliferation and graft rejection.The leading cause of corneal graft failure is T cell mediated immune rejection.Current immunosuppressive drugs used to prevent or treat corneal graft rejection include corticosteroids and cyclosporin-A,and so on.However,long-term use of these agents may come into being serious complications,such as infection,cataract,glaucoma and nephrotoxicity.Therefore, development of more effective and low side-effect immunomodulatory strategies for the prevention of corneal graft rejection has important significance.Using anti-CD25 monoclonal antibody(anti-CD25 mAb) therapy has been shown to effectively prevent acute rejection of renal allografts in human being.But the reports about using anti-CD25 mAb in prevention of corneal allograft immune rejection reaction are few.We know that anti-CD25 mAb can inhibit the function of CD25 by binding CD25,so using anti-CD25 mAb has an effect on the Teff and the CD4+CD25+Tr.To preferably analyze the effect of anti-CD25 mAb on the Teff and the CD4+CD25+Tr and determine its prevention and cure roles in prevention of rat corneal allograft rejection reaction,we first study the roles of Th1/Th2 cytokines(IFN-γ/IL-4) and CD4+CD25+Tr(FOXP3) in the allograft rejection reaction of rat penetrating keratoplasty(PKP).At last,we construct a eukaryotic expression vector containing rat FOXP3 gene for the research of FOXP3 gene therapy on the rat corneal allograft immune rejection reaction.Based on those researchs,this topic plans to conduct the following research:(1) To study the role of Th1/Th2 cytokines(IFN-γ/IL-4) in the allograft rejection reaction of rat PKP.(2) To study the role of CD4+CD25+Tr(FOXP3) in the allograft rejection reaction of rat PKP.(3) To investigate the effects of anti-CD25 mAb/with dexamethasone on Th1/Th2 cytokines(IFN-γ/IL-4) in corneal allograft rejection reaction.(4) To investigate the effects of anti-CD25 mAb/with dexamethasone on CD4+CD25+Tr(FOXP3) in corneal allograft rejection reaction.(5) To investigate the toxicity of anti-CD25 mAb on rat cornea and the effects of anti-CD25 mAb/with dexamethasone in prevention of corneal allograft rejection reaction.(6) To construct a eukaryotic expression vector containing rat FOXP3 gene for the research of FOXP3 gene therapy on the rat corneal allograft immune rejection reaction.This research is divided into four parts as follows:PartⅠExperimental study of IFN-γ,IL-4 and CD25 in the rat corneal allograft rejection reactionObjective To investigate the role of IFN-γ,IL-4 and CD25 in the allograft rejection reaction of rat PKP.Methods The Wistar rat was used as a donor,and the SD rat was a recipient. Routine PKP was performed.57 SD rats were randomly divided into three groups: Group A(n=9),normal group;Group B(n=24) and Group C(n=24),allograft groups, were treated with N.S subconjunctival injection and dexamethasone(100ug) subconjunctival injection respectively.N.S and dexamethasone were injected five times on postoperative day 0,2,4,6 and 8 respectively.The grafts were evaluated clinically by means of Larkin's scoring criterion and a rejection reaction was observed with a slit lamp microscope.The serum and aqueous humour levels of IFN-γand IL-4 were measured and the expressions of IFN-γand CD25 mRNA in grafts were detected respectively by using enzyme linked immunosorbent assay(ELISA) and reverse transcription polymerase chain reaction(RT-PCR) before and after corneal transplantation.The expression of CD3+CD25+T/CD3+T in peripheral lymphocytes was measured by flow cytometry(FCM) before operation at 6d,11d,24d after operation.Immunohistochemical assay about CD4,CD8 and CD25 in corneal graft was performed at day 11 in Group B and Group C.Results The rejection time was different in Group B and Group C.The average transplant survival time in group B was(10.583±1.084)d,which in group C was(16.417±1.379)d with a statistically significant difference between them(P＜0.05). There were no expressions of IFN-γand CD25 mRNA in the normal cornea.As compared with Group C,the expressions of IFN-γand CD25 mRNA in grafts were markedly increased in Group B(P＜0.05) at 11d after PKP.In Group B:The mean IFN-γlevel in serum was increased during the rejection,reaching to(13.307±2.540)pg/ml at 11d,and descending at 24d.As compared with the mean IFN-γlevel in serum at 6d and in the normal SD rats,the mean IFN-γlevel in serum at 11d was marked increased(P＜0.05).The mean IL-4 level in serum,however,was not statistically significant difference before and after the operation(P＞0.05).The mean IFN-γand IL-4 levels in aqueous humour were increased remarkably at 6d.At 11d, the mean IFN-γlevel increased and rose to the maximum,(155.947±23.807)pg/ml; the mean IL-4 level,however,was not marked increased.At 24d,the mean IFN-γand IL-4 levels in aqueous humour were decreased.The mean CD3+CD25+T/CD3+T level after operation was significantly higher at 6d and 11d than that before operation. At 11d after operation,the expressions of CD4,CD8 and CD25 in grafts were obvious by immunohistochemical assay.In Group C:The expressions of IFN-γ,IL-4 and CD3+CD25+T/CD3+T were markedly decreased during the rejection compared with Group B.Conclusion The results suggest that IFN-γ,IL-4 and CD25 may play important roles during the rat corneal allograft rejection reaction.Increasing the expression of IL-4 and decreasing the expressions of IFN-γand CD25 may help to inhibit corneal allograft rejection reaction.Monitoring the expressions of IFN-γand CD25 in peripheral blood after operation can help to indicate the degree of local immune reaction and predict the occurrence of corneal allograft rejection reaction.PartⅡExperimental study of FOXP3 in the rat corneal allograft rejection reactionObjective To investigate the role of FOXP3 in the allograft rejection reaction of rat PKP.Methods The Wistar rat was used as a donor,and the SD rat was a recipient. Routine PKP was performed.57 SD rats were randomly divided into three groups: Group A(n=9),normal group;Group B(n=24) and Group C(n=24),allograft groups, were treated with N.S subconjunctival injection and dexamethasone(100ug) subconjunctival injection respectively.N.S and dexamethasone were injected five times on postoperative day 0,2,4,6 and 8 respectively.The grafts were evaluated clinically by means of Larkin's scoring criterion and a rejection reaction was observed with a slit lamp microscope.The expression of FOXP3 mRNA in grafts was detected by using RT-PCR before and after corneal transplantation.The expressions of CD4+CD25+T and CD4+FOXP3+T in peripheral lymphocytes were measured by FCM before operation at 6d,11d,24d after operation.Immunohistochemical assay about CD4,CD8,CD25 and FOXP3 in corneal graft was performed at day 11 in Group B and Group C.Results The rejection time was different in Group B and Group C.The average transplant survival time in group B was(10.583±1.084)d,which in group C was(16.417±1.379)d with a statistically significant difference between them(P＜0.05). There was no expression of FOXP3 mRNA in the normal cornea.As compared with Group C,the expression of FOXP3 mRNA in grafts was markedly decreased in Group B(P＜0.05) at 11d after PKP.In Group B:The mean CD4+CD25+T/CD4+T level was significantly higher,and the mean CD4+FOXP3+T/CD4+T level was significantly lower at 11d after operation than that before operation.At 11d after operation,the expressions of CD4,CD8 and CD25 in grafts were stronger,and the FOXP3 was also detected by immunohistochemical assay.In Group C:The expression of CD4+CD25+T/CD4+T was markedly decreased,and the expressions of FOXP3 mRNA and CD4+FOXP3+T/CD4+T were markedly increased during the rejection compared with Group B.Conclusion The results suggest that FOXP3 may play important roles in the course of the rat corneal allograft rejection reaction.Increasing the expression of FOXP3 in the graft or increasing the content of CD4+CD25+Tr in peripheral blood can help to inhibit corneal allograft rejection reaction.PartⅢExperimental study on the effects of anti-CD25 monoclonal antibody in prevention of rat corneal allograft rejection reactionObjective To investigate the toxicity of anti-CD25 mAb on rat cornea and the effects of anti-CD25 mAb on Teff and CD4+CD25+Tr in corneal allograft rejection. To study the effects of anti-CD25 mAb in prevention of corneal allograft rejection reaction.Methods 1.Twelve SD rats were randomly divided into four groups.Each group was treated with N.S,50ug anti-CD25 mAb,100ug anti-CD25 mAb and 200ug anti-CD25 mAb subconjunctival injection respectively and was injected five times on day 0,2,4,6 and 8 respectively.Rat corneas were observed with a slit lamp microscope daily.At the 9th day,right corneas of SD rats were evaluated with histology and transmission electron microscope(TEM).2.The Wistar rat was used as a donor,and the SD rat was a recipient.Routine PKP was performed.93 SD rats were randomly divided into five groups:Group A(n=9),normal group;Group B(n=24), Group C(n=18),Group D(n=18) and Group E(n=24),allograft groups,were treated with N.S,100ug anti-CD25 mAb,100ug anti-CD25 mAb with 50ug dexamethasone and 100ug dexamethasone subconjunctival injection five times on postoperative day 0, 2,4,6 and 8 respectively.The grafts were evaluated by means of Larkin's scoring criteria and a rejection reaction was observed with a slit lamp microscope.The aqueous humour levels of IFN-γand IL-4 were measured and the expressions of IFN-γ,CD25 and FOXP3 mRNA in grafts were detected respectively by using ELISA and RT-PCR before and after corneal transplantation.The expressions of CD4+CD25+T/CD4+T and CD4+FOXP3+T/CD4+T in peripheral lymphocytes were measured by FCM before operation at 6d,11d,24d after operation.Results 1.No significant corneal toxicity was observed in 50ug anti-CD25 mAb treated group and 100ug anti-CD25 mAb treated group.Corneal stroma-cells and endothelial cells were swollen in 200ug anti-CD25 mAb treated group.2.The rejection time was different in four Groups.The average transplant survival time in Group B was(10.583±1.084)d,which in group C,Group D and Group E was (13.167±1.169)d,(17.333±2.160)d,(16.417±1.379)d respectively.Compared with Group B,the survival time of grafts was markedly prolonged in group C,Group D and Group E(P＜0.05).There were no expressions of IFN-γ,CD25 and FOXP3 mRNA in the normal cornea.At 11d after PKP,compared with Group C,Group D and Group E,the expressions of IFN-γand CD25 mRNA in grafts were markedly increased in Group B(P＜0.05);as compared with Group D and Group E,the expressions of FOXP3 mRNA in grafts were markedly decreased in Group C(P＜0.05). At 6d and 11d after PKP,compared with homeochronous Group B,the mean IFN-γlevel in aqueous humour was decreased remarkably in Group C,Group D and Group E(P＜0.05);At 11d after PKP,compared with Group C,the mean IFN-γlevel in aqueous humour was decreased remarkably in Group D and Group E(P＜0.05).At 6d and 11d after PKP,compared with homeochronous Group B,the mean IL-4 level in aqueous humour was increased remarkably in Group C(P＜0.05),while the mean IL-4 level in aqueous humour was decreased remarkably in Group D and Group E(P＜0.05); At 6d and 11d after PKP,compared with homeochronous Group C,the mean IL-4 level in aqueous humour was decreased remarkably in Group D and Group E(P＜0.05). At 11d after PKP,compared with Group B,the mean CD4+CD25+T/CD4+T level in peripheral lymphocytes was significantly lower in Group C,Group D and Group E(P＜0.05);as compared with Group D and Group E,the mean CD4+CD25+T/CD4+T level in peripheral lymphocytes was significantly lower in Group C(P＜0.05).At 11d after PKP,compared with Group B,the mean CD4+FOXP3+T/CD4+T level in peripheral lymphocytes was no statistical significance in Group C(P＞0.05),while the mean CD4+FOXP3+T/CD4+T level in peripheral lymphocytes was significantly higher in Group D and Group E(P＜0.05);as compared with Group D and Group E,the mean CD4+FOXP3+T/CD4+T level in peripheral lymphocytes was significantly lower in Group C(P＜0.05).Conclusions 1.No significant corneal toxicity is found in 50ug anti-CD25 mAb treated Group and in 100ug anti-CD25 mAb treated Group.2.The results suggest that Teff and CD4+CD25+Tr play important roles in the course of the rat corneal allograft rejection reaction.Decreasing the expression of Teff and increasing the expression of CD4+CD25+Tr have important significance in prevention of corneal allograft rejection.Using anti-CD25 mAb in prevention of corneal allograft rejection inhibits the Teff,at the same time the CD4+CD25+Tr is also inhibited. While using anti-CD25 mAb with low dose dexamethasone can inhibit the Teff and promote relatively the CD4+CD25+Tr,which has impotant roles in prevention of corneal allograft rejection reaction.PartⅣCloning of rat FOXP3 gene and construction of its eukaryotic expression vectorObjective To construct a eukaryotic expression vector containing rat FOXP3 gene for the research of FOXP3 gene therapy on the rat corneal allograft immune rejection reaction.Methods The rat FOXP3 gene was obtained from the SD rat splenic cells by RT-PCR.The PCR production digested by EcoRⅠand XhoⅠwas inserted into pcDNA3.1 to construct a recombinant plasmid,named as pcDNA3.1-FOXP3.the pcDNA3.1-FOXP3 was detected with DNA sequencing.Results FOXP3 gene with a length of 1290bp was successfully cloned from rat splenic cells by RT-PCR.DNA sequencing indicated that the pcDNA3.1-FOXP3 was correctly cloned.Conclusion The recombinant plasmid pcDNA3.1-FOXP3 is successfully constructed,which lays a foundation for further study of FOXP3 gene therapy on the rat corneal allograft immune rejection reaction. We can get following conclusions:1.IFN-γ,IL-4 and CD25 may play important roles during the rat corneal allograft rejection reaction.Monitoring the expressions of IFN-γand CD25 in peripheral blood after operation can help to indicate the degree of local immune reaction and predict the occurrence of corneal allograft rejection reaction.Increasing the expression of IL-4 and decreasing the expressions of IFN-γand CD25 may help to inhibit corneal allograft rejection reaction.2.FOXP3 may play important roles in the course of the rat corneal allograft rejection reaction.Increasing the expression of FOXP3 in the graft or increasing the content of CD4+CD25+Tr in peripheral blood can help to inhibit corneal allograft rejection reaction.3.No significant corneal toxicity was observed in 100ug anti-CD25 mAb treated group.4.Decreasing the expressions of IFN-γand increasing the expressions of IL-4 have a positive role in inhibiting corneal allograft rejection reaction.Inhibition of the expressions of IFN-γand IL-4,however,led to a statistically significant prolongation of transplant survival.5.Teff and CD4+CD25+Tr play important roles in the course of the rat corneal allograft rejection reaction.Decreasing the expression of Teff and increasing the expression of CD4+CD25+Tr have important significance in prevention of corneal allograft rejection.Using anti-CD25 mAb in prevention of corneal allograft rejection inhibits the Teff,at the same time the CD4+CD25+Tr is also inhibited.While using anti-CD25 mAb with low dose dexamethasone can inhibit the Teff and promote relatively the CD4+CD25+Tr,which has impotant roles in prevention of corneal allograft rejection reaction.6.The recombinant plasmid pcDNA3.1-FOXP3 is successfully constructed, which lays a foundation for further study of FOXP3 gene therapy on the rat corneal allograft immune rejection reaction.