Inhibitation Of Acute Rejection In Rat Liver Transplantation By Peptide Nucleicacid Against Chemokine Receptor CCR7 In Dendritic Cells

Organ transplant rejection is mediated primarily by T cells.Antigen-presenting cells (APCs) that play a fundamental role in the initiation of the immune response capture and transfer information from the outside world to the cells of the adaptive immune system.Dendritic cells (DCs) are the most potent APCs and play a central role in initiating the primary immune response due to their unique ability to stimulate naive T cells. Following encounter with foreign antigens, they rapidly undergo a process called maturation. Maturation involves an increase in the antigen-presenting ability and in the migration of the cells to secondary lymphoid organs, where they present antigens to naive T cells and induce specific immune responses. During maturation DCs up-regulate surface expression of chemokine receptor CCR7. Ligands for CCR7, which are constitutively expressed at high levels in lymph nodes (LNs), are powerful attractants that direct DCs to these tissues. Peptide nucleic acid (PNA) is a nucleic acid analogs with an achiral polyamide backbone consisting of N-glycine units.The purine or pyrimidine bases are joined to the each unit via a methylene carbonyl linker to target the complementary nucleic acid. PNA binds to complementary RNA or DNA in a parallel or antiparallel orientation following the Watson-Crick base-pairing rules. The uncharged nature of the PNA oligomers enhances the stability of the hybrid PNA/DNA (RNA) duplexes as compared to the natural homoduplexes. The non-natural character of the PNA makes PNA oligomers highly resistant to protease and nuclease attacks. These properties of PNA oligomers suggest that they could potentially serve as efficient antisense reagents and represent a promising tool for manipulating gene function. Objective This study was conducted (1)to observe the trafficking kinetics and role of dendritic cells (DCs) in transplantation immunity using a orthotopic rat liver transplantation model with acute rejection and without rejection, and sites for allosensitization were also analyzed and discussed.(2) to knock-down the gene expression of CCR7 in DCs in vitro by using antisense peptide nucleic acid (PNA) so that a suppression of chemotaxis activity and the proapoptotic result can be achieved.(3) to investigate the effect of down-regulating gene expression of chemokine receptor CCR7 in DCs in vivo by using antisense PNA to suppress the acute rejection in rat liver allograft transplantation. By all these experiments,we want to elucidate the exact functions and possible mechanisms of DCs in the rat liver allograft rejection and indicate that the strategy of using PNA against the CCR7 gene may be a promising approach in trapping migratory capacity of DCs and inducing allogeneic tolerance.Methods (1)Models of orthotopic rat liver transplantation were performed by cuff techniques for two vascular anastomoses with some modification. Rats were randomly divided into the acute rejection group(SD rats receiving Wistar livers as donors) and the immune tolerance group(Wistar rat receiving Wistar liver). Graft livers and host celiac lymph nodes (n=4) of each group were sampled respectively at days3, 5, and 7 after the transplantation.The acute rejection was graded according to liver histopathological changes. The dynamic state of DCs number within graft and lymph tissues was detected by means of immunohistochemical staining and image analysis. T-cell active proliferative response in lymph nodes was also studied. (2)DCs isolated from rat bone marrow cells were induced and cultured. Antisense PNA were designed for targeting at the AUG coding region of CCR7 mRNA and incubated with rat bone marrow derived DCs,and setting up random PNA groups and blank control groups for comparison. At hours24, 48, and 72 time points,the expression of CCR7 in incubated DCs samples were observed with immunocytochemical staining, western blotting and RT-PCR respectively, chemotaxis assays and flow cytometric analysis were also performed on DCs to examine the chmotaxis activities and apoptosis rate. (3)Rat liver transplantation model with acute rejection was performed as stated above. Antisense PNA was injected into graft portal vein during the cold-storage stage and tail vein after transplantation. Setting up random PNA groups and blank control groups for comparison,the recipient mean survival time was recorded. Graft livers and host celiac lymph nodes (n=4) of each group were sampled at days 7 and 10 postoperation. Liver histopathological changes were observed and DCs number and T-cell active proliferative response in lymph nodes was measured by immunohistology.Results (1)Rats in acute rejection group shew typical acute rejection with severely histopathological changes on the post-operative day 5, otherwise,rats in tolerance group shew mild morphologic changes and no acute rejection.At day 3 after transplantation, the number of DCs in allograft portal areas significantly increased and reached its peak at day 5 and then gradually decreased in number at day 7.A significant number of DCs were detected in the allogeneic host celiac lymph nodes paracortical areas from day 3 after transplantation, and displayed a continuous increasing trend for next several days. Active proliferation of T cells at a periphery of T-cell area of the celiac lymph nodes was triggered as early as day 3 after transplantation and precede to the histopathological changes of allograft. (2) Compared with random and blank control groups,at h48,DCs treated by antisense PNA shew significantly lower the protein expression of CCR7 but not the mRNA level,and at h72,the expression levels of CCR7 were all decreased significantly. The inhibited chmotaxis activity and higher apoptosis rate of DCs correlated with suppressed expression of CCR7 were observed.(3)The results shew that rats in antisense PNA group survive significantly longer compared to those in control group,and that DCs number in lymph nodes were considerably reduced and the T-cell active proliferation was significantly suppressed compared with controls.Conclusions (1) The donor liver of Wistar rats and recipient SD rats combination is a high rejection model and the combination of Wistar to Wistar shows no rejection, but immune tolerance. This study reveals that allogeneic liver transplantation induces the accelerated migrating of DCs within the allograft and host lymphoid tissues. DCs delivers a strong and sustained stimulation among T cells in the lymphoid tissues where effector cells are predominantly sensitized and rejected the graft eventually, indicating migrated DCs are crucial for allosensitization and that targeting of CCR7 may be a potential approach for manipulation of DCs trafficking to secondary lymphoid tissue after liver transplantation.(2) Antisense PNA targeting CCR7 can knock down the gene expression of CCR7 in DCs so as to suppress the chmotaxis activity of DCs and demonstrate the proapoptotic results in vitro. (3) The results of the present study demonstrates that the strategy of using PNA against the CCR7 gene in vivo may be a promising approach in trapping migratory capacity and blocking antigen presentation of DCs to suppress the alloreactive immune response and prolong allograft survival. The above results also show a significant though modest prolongation of allograft survival in rats down-regulating the chemokine receptor CCR7 ,and rats reject fully allogeneic liver allografts in a delayed fashion, but are not tolerant.