| Purpose: Corneal transplantation is the most successful form of organ transplantation, and the most important treatment for the irreversible corneal blindness. But immunological rejection remains the leading cause of the corneal graft failure. A better understanding of the mechanism of corneal graft rejection and new effective treatments for the graft failure, are required to improve the success of corneal transplantation and to restore the patient's vision. Recently, dendritic cells (DCs) are found to play a dual role in the immunological system. Apart from inducing immune response as antigen presenting cells (APCs), DCs can also induce central and peripheral tolerance. The therapy with immature or inhibiting cytokine expressing DCs to induce tolerance, is a new strategy for the organ transplant rejection. The purpose of this study is to understand the physiology and function of DCs in vitro andthe migration of DCs in vivo following subconjunctival (s.conj.) and subcutaneous (s.c.) injection, to investigate the mechanism of murine corneal allograft rejection and the role of different types of DCs in immunomodulation of corneal allograft response. Methods:1. DCs were derived from bone marrow (BM) of mice and cultured in 5% granulocyte -macrophage colony-stimulation factor (GM-CSF) RPMI 1640 medium with or without 0.2ng/ml TGFB2 for five days. The phenotype of DCs was determined by flow cytometry (FCM) and immunofluorescent staining. MLR was used to test capacity of DC or TGFB2-DC to stimulate the allogeneic T cells to proliferate. After coculture of syngeneic or allogeneic DC, TGFI32-DC for five days, the proliferation and phenotype of CD4" T cells were examined by MLR or FCM respectively.2. Cultured BMDC on day 5 were labeled with PKH2-GL green fluorescent dye. PBS alone (control) or lx!06PKH2-GL labeled DCs were injected into the subconjunctival space (s.conj.) of the right eye or into the right side of nape (s.c.) of syngeneic recipient mice. Cervical lymph nodes were then separately collected as submandibular lymph node (SMLN) and superficial cervical lymph node (SCLN) at 24h and 48h after injection. Labeled DCs were detected by confocal microscopy on LN sections or FCM analysis of LN cells.3. Using a mouse corneal graft model, eyes were evaluated by clinical observation and immunofluorescent staining at various times. The phenotype of leukocytes in the SMLN and SCLN was studied by three-color FCM analysis three days after transplantation.4. Animals were divided into three groups. Mice that received PBS alone9(la), syngeneic DC (Ib), or allogeneic DC (Ic) by s.c. injection comprised Group I; Mice that received PBS alone (Ha), syngeneic DC (lib), or syngeneic TGF132-DC (He) by s.c. injection comprised Group II; Mice that recieved PBS alone (Ilia), syngeneic DC (Illb), or syngeneic TGFB2-DC (IIIc) by s.conj. injection comprised Group III. Seven days after the first injection, in some animals, the proliferation of lymphocytes in SMLN and SCLN was examined by MLR, and LN cytokine production was tested by ELISA in the supernatants from MLR culture Corneal allograft transplantations were performed in the other animals seven days after the first injection. The phenotype of lymphocytes in SMLN and SCLN was examined by FCM three days after transplantation. Eyes were evaluated by clinical observation at various times. Results:1. BMDC were CDllc"T+, CD86+~, MHC class If, CD8a myeloid DC. Treatment with TGFB2 resulted in a significant reduction of DC expressing costimulatory molecules. The expression of CD86 was decreased from 24.75% to!3.3%, and MHC class II was decreased from 8.7% to 4.9%. The capacity of DC to stimulate allogeneic T cell to proliferate was also suppressed by TGFB2 (P<0.01). In the MLR with allogeneic splenic cells, the proliferation of CD4+ T cells stimulated by syngeneic TGFB2-DC were stronger than by syngeneic DC. In contrast, CD4+ T cells induced by allogeneic TGF132-DC showed lower proliferation than by allogeneic D...
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