An Experiment Research On The Immunomodulation Of Dendritic Cells On High-risk Corneal Allograft Rejection

Objective: To culture the rat bone marrow derived dendritic cell (DC) and to study the effects ofdifferent maturation status of DC on the in vivo immune response to rat orthotopic cornealtransplantation in high-risk eyes and investigate the mechanism of the immunologic tolerarceinduced by immature dendritic cell (imDC)．Methods: Bone marrow-derived DC of wistar rats were cultured with recombinant ratgranulocyte-macrophage colony stimulating factor (GM-CSF) and Interleukin (IL)-4. ImDC andmature DC (mDC) were generated without or with the stimulation of tumor necrosis factor-α(TNF-α), we define the developmental morphology, and their phenotype were analyzed by flowcytometry.60SD rats (recipient) and30Wistar rats (donor) were randomly divided into3groups:control, imDC and mDC group respectively. Using a model of orthotopic corneal transplantationin which allografts were placed in neovascularized high-risk eyes of recipient rat. Cornealneovascularization was induced by alkaline burn. Recipient in the imDC group or mDC groupswere injected imDCs or mDCs of1×106respectively1week before corneal transplantation via tailvein, control rat received the same volume of PBS. In each group,8recipients were kept fordetermination of survival time and the other12recipients were executed on days3,7and14aftertransplantation. Cornea was harvested for hematoxylin-eosin staining, RT-PCR was used to detectthe expression of Th1-type cytokine IL-12、IL-15、IL-18and Th2-type cytokine IL-4、IL-10,Western blot was used to detect the expression level of Foxp3.Result: A quantity of DC were successfully cultured, these DC were validated and identifiedthrough microscopy, scanning electron microscopy and flow cytometry analysis. Injection ofdonor derived imDC, unlike mDC and control group, significantly prolonged graft survival(P<0.05). In imDC group, the mRNA level expression of IL-12、IL-15、IL-18were significantlydecreased and IL-4、IL-10were significantly increased, lower Th1and higher Th2-type responsewas induced. The expression level of Foxp3on CD4+CD25+T cells of the imDC group wassignificantly higher than that in the control and mDC groups (P<0.05).Conclusion: The approaches of culturing DC with GM-CSF, IL-4and TNF-α could obtainadequate DC with high purity. The pretreatment with imDC can prolong the survival time of ratcorneal graft after transplantation. The mechanism of immune tolerance induced by imDC mightbe influence the balance of Th1/Th2cytokines and inhibition of T lymphocytes responsiveness byregulatory T cells.