The Research On Skin Transplantion Immune Tolerance Induction By Murine Immature Dendritic Cells Transfected By CCR7 Gene

Background and objectiveAllogeneic skin transplantation is the most effective method in the early coverage of extensive burn wounds. However, the host versus graft reaction (HVGR) after transplantation leads to irreversible rejection about 3 weeks later, which limits the effection of skin transplantation. And there isn't any effective treatment to induce immune tolerance after transplantation. Dendritic cells (DCs) are the most potent professional antigen-presenting cells (APC) in vivo, which play critical roles in immune reponse. Dendritic cells differentiate from immature dendritic cells (imDCs) to mature dendritic cells (mDCs), in the process, morphological characteristics, surface markers and biological functions in DCs have changed accordingly. The most distinguished ability of imDCs is inducing T cell anergy and promote alloantigen-specific tolerance, which has been verified in our last National Natural Science Foundation of China (NSFC) subjest (No.30271341). Based on the advantages, combined transplantation using imDCs or genetically-modified imDCs have seen satisfied results in kidney,liver,heart,pancreatic islet and small intestine transplantation.The phenotypical changes that take place during maturation include the blunting of the endocytic ability and the up-regulation of MHCs and costimulatory molecules that eventually transform these DCs into potent APC. Maturation takes place concomitantly with the migration of the DCs from their niches in the peripheral tissues to the lymph nodes where they arrive through the lymphatics. In this regard, during maturation the expression of the chemokine receptor-7 (CCR7) is up regulated. Through interaction with ligand MIP3β(CCL19) and SLC (CCL21), CCR7 guides the migratory DCs to the nodes, presents antigens to naive T cells and activates immune response. Recent studies have also showed that CCR7 plays an important role in cancer metastasis. Such function of CCR7 is kind of like oriented migration in DCs. If DCs became more matured, they express more CCR7, the immune system will present stronger response. Otherwise, immune tolerance is induction. However, imDCs lack the expression of CCR7 and direct less migratory DCs to lymph nodes. Long time for residing in non-T cell area put imDCs into high risks sensing of"danger signals"(tissue damage,inflammatory cytokines or pathogens), which starts the differentiation of maturation and change the important tolerance inducing functions of imDCs.Base on the the analysis above, the present study was designed to build expression of CCR7 in imDCs through the construction of recombinant adenovirus carrying murine CCR7 gene, which will afford imDCs high chemotactic ability possessed by mDCs, inhibit the maturation of imDCs in peripheral tissues and guarantee the induction of immune tolerance of imDCs in the early coverage of extensive burn woundsMethods1,Cultivation and identification of dendritic cells derived from murine marrow ImDCs isolated from BALB/c murine marrow was induced and proliferated by rmGM-CSF and rmIL-4, and harvested amount of imDCs after 5 days, obtained mDCs through the stimulation of rmTNF-α. The change of cell morphology was observed through the light microscope and the scanning electron microscope, the expression level of cell surface markers were detected application flow cytometry.2,CCR7 gene transfection immature dendritic cell derived from murine marrow and identification of its functionTransfected the Ad-ccr7 adenovirus into immature dendritic cells through gradient centrifugation methods, observed the change of cell morphology through the light microscope and the scanning electron microscope and detected the expression level of cell surface markers application flow cytometry after Ad-ccr7 adenovirus transfection it, it will be useful to observed the influence of mature state and morphology after transfection. The expression change of CCR7 mRNA and protein in immature dendritic cells after Ad-ccr7 adenovirus tansfection were detected by Real-time PCR,cell immumofluorescence and Western Blot methods. The migration ability and the stimulate proliferation ability of T lymphocyte by immature dendritic cells after Ad-ccr7 adenovirus transfection was observed by migration experiment and mixed lymphocyte reaction in vitro.3,Influence of imDCs after CCR7 gene transfection to murine allogene skin transplantionIn murine allogene skin transplantion experiments, we injected the donor imDCs,donor mDCs,donor imDCs+Ad,donor imDCs+Ad-ccr7 and donor imDCs+Ad-ccr7+IL-10 into receptor murine, observed the influence of each group cells to murine allogene skin transplantion graft survive state and observed the construction change of skin grafts through histology detection.Results1,The results of cultivation and identification of dendritic cells derived from murine marrowThe dendritic cells cultivated in vitro were observed by light mircroscope and scanning electron microscope, we discovered: the surface of immature dendritic cell was slick,rugosity and less sentus ecphyma, but the surface of mature dendritic cell was stretched out many branch ecphymas, which was different longer and strange form. The flow cytometry founded that the positive percentage of CD11c,CD80,CD83,MHCⅡin immature dendritic cell was 24.7%,9.4%,20.5% and 20.5%, but the positive percentage in mature dendritic cell was 86.5%,88.4%,76.9% and 92.4%. These demonstrated the dendritic cells cultivation in vitro consistented with the typical morphology characteristic and the level of surface markers.2,Influence of CCR7 gene transfection to immature dendritic cells derived from murine marrow2.1 We transfected Ad-ccr7 adenovirus to immature dendritic cells derived from murine marrow by high speed centrifugation method, the transfection efficiency up to 70% as MOI in 100. The morphology of immature dendritic cell after transfection Ad void vector and Ad-ccr7 adenovirus by scanning electron microscope was rugosity, rough hard sphere, the irregularity dendritic processes more than imDCs un-transfection, but the irregularity dendritic processes decreased in IL-10 group, this illustration IL-10 can slightly inhibited the maturation of imDCs. The flow cytometry founded that the positive percentage of CD11c,CD80,CD83,MHCⅡin imDCs+Ad was 40.0%,27.7%,28.5% and 37.8%, the positive percentage of CD11c,CD80,CD83,MHCⅡin imDCs+Ad-ccr7 was 37.8%,27.4%,21.3% and 37.0%, but the positive percentage in imDCs+Ad-ccr7+IL-10 was 20.5%,15.5%,21.6% and 32.9%. These demonstrated Ad-ccr7 adenovirus transfection can spur the surface marker of imDCs slightly increased, development to maturity direction, but IL-10 can slightly down regulated the expression of surface markers and inhibited the maturation of imDCs.2.2 Results of Real-time PCR manifested, the expression level of CCR7 mRNA in imDC was low, and the expression level of CCR7 mRNA in mDCs was 1.544 fold than imDCs. The expression level of CCR7 mRNA in imDCs after Ad void vector transfection was 1.115 fold than imDCs, the expression level of CCR7 mRNA in imDCs after Ad-ccr7 adenovirus transfection was 2.941 fold than imDCs, but the expression level of CCR7 mRNA in IL-10 group was 1.566 fold than imDCs, approach the normal level in mDCs. These illustrated the expression level of CCR7 mRNA in imDCs after Ad-ccr7 adenovirus transfection can increased obviously, meanwhile IL-10 can slightly inhibited the maturation of imDCs during transfection.2.3 Results of cell immunofluorescence displayed there has no expression of CCR7 in imDCs and imDCs+Ad group, but it had expression of CCR7 in mDCs. The expression of CCR7 in imDCs after Ad-ccr7 adenovirus transfection was increased, it means CCR7 gene had transfected imDCs and expressed the protein of CCR7. The results of Western Blot analysed by software Qulitity One manifested: the expression relative amount of CCR7 protein in imDCs,imDCs+Ad,imDCs+Ad-ccr7,imDCs+Ad-ccr7+IL-10 and mDCs were 0.09,0.15,0.96,0.46 and 0.35 respectively. There almost has no expression of CCR7 protein in imDCs and imDCs+Ad group, but the expression of CCR7 in imDCs after Ad-ccr7 adenovirus transfection upgraded obviously, the expression relative amount of CCR7 protein more than the normal expression in mDCs. IL-10 can inhibitted the imDCs differentiation toward maturation, but the expression of CCR7 protein can achieve the level of mDCs.2.4 The results of migration experiment in vitro displayed the migration percentage of imDCs less than 5%, but the migration percentage in mDCs,imDCs+ Ad-ccr7 and imDCs+Ad-ccr7+IL-10 group which affected by CCL19 were increased obviously than imDCs(aP<0.05). But in CCL19+anti-CCR7 mAb group, the migration percentage decreased by CCL19 antagonizer anti-CCR7 mAb, it higher than its control group respectively (dP <0.05).These indicated that the migration ability in vitro of imDCs after Ad-ccr7 adenovirus transfection and mDCs which expressed CCR7 protein can enhanced by its ligand CCL19, meanwhile anti-CCR7 mAb can rivalied the effection of its ligand CCL19 in vitro.2.5 The results of mixed lymphocyte reaction demonstrated, imDCs mixed the T lymphocytes at rate 1:5, the proliferation ability of imDCs stimulate to T lymphocytes was lower, SI was 1.6±0.1, but the proliferation ability of mDCs stimulate to T lymphocytes increased significantly, SI was 5.6±0.3 (aP<0.05). The proliferation ability of imDCs after adenovirus transfection was higher than imDCs and lower than mDCs, SI was 3.4±0.2 and 3.1±0.2 respectively. IL-10 can decreased the proliferation ability, SI was 1.8±0.1 (bP<0.05). Adenovirus transfection can partly enhance the proliferation ability of imDCs, but it still lower than mDCs, IL-10 can inhibited the proliferation ability.3,Influence of imDCs after CCR7 gene transfection to murine allogene skin transplantion3.1 Compare to control group, the MST of skin grafts in donor imDCs,donor imDCs+Ad-ccr7,donor imDCs+Ad-ccr7+IL-10 group were prolonged obviously (aP<0.05), but the MST of donor mDCs and imDCs+Ad group had no difference(P>0.05). It means the donor mDCs had no obviously influence on the MST of skin grafts, but the donor imDCs can prolong the MST of skin grafts. Meanwhile the MST of donor imDCs+Ad-ccr7+IL-10 group was significant prolongation than the MST of donor imDCs, but the MST of donor imDCs+Ad-ccr7 group has no obviously difference than donor imDCs, it manifestation the ability of induction immune tolerance of imDCs after Ad-ccr7 adenovirus transfection was higher than before the transfection, IL-10 can significant extended the MST of skin grafts.3.2 The clear epithelial structure and infiltration of inflammatory cells were observed in specimens.Conclusion1. The cells we cultured displayed typical morphlogical features of imDCs and mDCs, which were induced with lower rmGM-CSF and rmIL-4 from murine marrow.2. We successfully transfected Ad-ccr7 adenovirus to immature dendritic cells derived from murine marrow. The morphology and surface marker of imDCs after Ad-ccr7 adenovirus transfection can differentiated towards maturity direction, but IL-10 can slightly inhibited the maturation of imDCs.3. Results of Real-time PCR manifested the expression level of CCR7 mRNA in imDCs after Ad-ccr7 adenovirus transfection can increased obviously, meanwhile IL-10 can slightly inhibited the maturation of imDCs during transfection.4. Results of cell immunofluorescence and Western Blot displayed the expression of CCR7 in imDCs after Ad-ccr7 adenovirus transfection upgraded obviously. IL-10 can inhibitted the imDCs differentiated towards maturation, but the expression of CCR7 protein can achieved the level of mDCs.5. The results of migration experiment in vitro displayed the migration percentage of imDCs was lower, but the migration percentage in imDCs after Ad-ccr7 adenovirus transfection which affected by CCL19 were increased obviously than imDCs. Meanwhile anti-CCR7 mAb can slightly rivalied the effection of its ligand CCL19 in vitro.6. The results of mixed lymphocyte reaction demonstrated the proliferation ability of imDCs stimulate to T lymphocytes was lower, but the proliferation ability of mDCs stimulate to T lymphocytes increased significantly. The proliferation ability of imDCs after adenovirus transfection was higher than imDCs and lower than mDCs, IL-10 can decreased the proliferation ability.7. Results of murine allogene skin transplantion experiment demonstrated the MST of skin grafts in donor mDCs had no significant difference, but the donor imDCs after Ad-ccr7 adenovirus transfection can prolonged the MST of skin grafts, IL-10 can significant extended the MST of skin grafts.