The Investigation Of Regulatory T Cells And Cytokines In Rabbit Autoimmune Dry Eye

ObjectiveDry eye is one of the most common disease in ophthalmology. Autoimmune dry eye,such as Sjogren’s syndrome (Sjogren’s syndrome, SS), is a common cause of aqueous tear deficiency dry eye. Studies suggest that immunomodulatory genes、 hormones、viruses、nerves、changes in the environment and many other factors are involved in the pathogenesis of SS. Recent studies have pointed out that the regulatory T cell dysfunction promotes the development of Sjogren’s syndrome. The purpose of this study is to establish a stable rabbit autoimmune dry eye model. Investigate regulatory T cells and some other cytokines in this model,and analyze their contribution to the pathogenesis of autoimmune dry eye.Methods1. Developed a method to obtain a preparation of rabbit lacrimal gland epithelial cells, essentially free of immune-system cells. The method relies on controlled digestion to disperse lacrimal acini, and recovers acini by filtration through various sizes of nylon mesh. Purity and integrity of the preparation were established qualitatively using light microscopy.2. lacrimal gland epithelial cells(pLGEC) and autologous peripheral blood lymphocytes(PBL) were co-cultured for4days. BrdU incorporation was tested to monitor peripheral blood lymphocytes proliferation rate.3. Induced rabbits autoimmune dry eye model:Activated PBL were injected into the ear veins of the rabbits from which they had been obtained. With day0as basline, clinical assessment were performed every2weeks after injection.Lacrimal gland and conjunctiva were collected for histopholoty HE staining.4. Lacrimal gland was harvested. Total RNA was isolated and reverse transcribed. Real-time PCR was used to analyze the mRNA expressions of IL-6、IL-10、 IL-17、TNF-a、TGF-β、IFN-γ.5. Cells were counted and examined by flow cytometry to evaluate the expression of CD4and Foxp3. 6. CD4+CD25+Treg from the spleen were sorted by Immunomagnetic beads, Peripheral blood T lymphocytes were stimulated with lacrimal gland epithelial cells(pLGEC) and were co-cultured with CD4+CD25+Treg.BrdU incorporation assay test the supression of effector T cells by CD4+CD25+Treg.Results1. Successfully obtain highly purified rabbit lacrimal gland epithelial cells.2. Cultured purified rabbit lacrimal gland epithelial cells(plGEC) and autologous peripheral blood lymphocytes(PBL) and successfully establish the system of co-cultivation.3. Successfully established rabbit autoimmune dry eye model:Tear production was reduced and BUT was less than normal. Four weeks after intravenous injection of activated lymphictes, Histopathology showed infiltrates of lymphocytes in lacrimal gland and conjunctiva.4. Relative gene expression of cytokines:inflammatory cytokines(IL-6、IL-17、 TNF-a、IFN-γ)mRNA were upregulated and anti-inflammation cytokines(IL-10、TGF-β) mRNA were downregulated in rabbit dry model.5. The proportion of CD4+CD25+regulatory T cells in Spleen and lacrimal gland decreased in rabbit dry model.6. Spleen-derived CD4+CD25+Treg inhibit the proliferation of the lacrimal gland-specific effector T cells.ConclusionsInjecting intravenously peripheral blood lymphocytes activated against lacrimal antigens induced autoimmune dacryoadenitis. In this pathological process,the suppressive capacity of CD4+CD25+Treg decline. Th17cells are overactivate. Cytokines TNF-a、IFN-γ、IL-10、GF-β、IL-6participate in the inflammatory response.