The Study Of Local Application Of Sphingosine-1Phosphate Receptor Modulators Inhibiting Corneal Allograft Rejection In Mice

Objective: Compared with1%Cyclosporin A (CsA) eye drop, the ophthalmic gels ofSphingosine-1Phosphate (S1P) receptor modulator FTY720were applied in cornealallograft in mice. The results could confirm the effects of prolonging corneal allograftrejection by topical FTY720and CsA. The corneal grafts, peripheral bloods, spleen andcervical lymph nodes were measured by flow cytometry, Elisa, realtime-PCR,hematoxylin and eosin and immunohistochemical staining. Above these methods couldexplore the mechanism of suppression. In orfer to futher investigate the mechanismabout local use of S1P receptor modulators, CD4+CD25+regulatory T cells weremagnetically separated using a Treg separation kit. Then, these isolated cells wereco-cultured with or without S1P receptor modulators (FTY720or FTY720-P). Theproliferation of co-cultured Treg cells and mixed lymphocyte reaction were measuredby Cell Counting Kit-8(CCK-8). The levels of cytokines (IL-10and TGF-β1) in thesupernatants were measured by Elisa. The expressions of cytokines mRNA weremeasured by realtime-PCR. Above these results would help to explore the mechanismof changes on treg cells co-clutred with S1P receptor modulators directly.Methods:1. The preparation of three doses (0.1%,0.3%and0.5%) FTY720was carried on.2.45male BALB/c mice which were received corneal grafts from C57BL/6donorswere divided into5groups (blank control; low, medium and high dose FTY720;1%CsA) at random. All of mice were received the different managements aftertransplantation. Nylon corneal sutures were removed on day10to11afteroperations. The degree of opacity as well as the degree of neovascularization wasevaluated daily within the first postoperative two weeks and then three times weeklyup to day30. At the time of rejection or the end of the study, mice were sacrificed.3.30male BALB/c mice which received corneal grafts from C57BL/6donors weredivided into5groups at random. Nylon corneal sutures were removed on day10to 11after operations. Six mice in each group were sacrificed for laboratoryexamination on day14after transplantation. The cervical lymph nodes, peripheralbloods and spleen were moved. The above samples were used to measure CD4+andCD4+CD25+Foxp3+T cells by flow cytometry. Cytokines serum levels includingIL-2, L-10, IFN-γ and TGF-β1were meseasured by Elisa. Expressions of IL-2, L-10,IFN-γ, TGF-β1and Foxp3mRNA in each group of three corneas were detected byrealtime-PCR. Other three corneas in each group were fixed and stained withhematoxylin and eosin for histological examination. In order to detect the levels ofCD4+T cells and cytokins including IL-2, L-10, IFN-γand TGF-β1and CD4+Tcells in corneal grafts, corneal samples were deparaffinized and processed forimmunohistochemical analyses.4.CD4+CD25+T cells were magnetically separated using a Treg separation kit.Spleens samples were obtained form30male BALB/c mice. The efficiency ofisolation was identified by flow cytometry assay.5. All of isolated cells were cultured and activated for5days under the cultureconditions which included IL-2, antibody of antiCD3and antiCD28.6. The activated CD4+CD25+T cells were co-cultured with or without three doses(10ng/ml,100ng/ml and1000ng/ml) FTY720and FTY720-P for48houre underabove condition. The blank control group was only added in10ul DMSO. Theproliferation of co-cultured Treg cells was measured by CCK-8for5days (on Days0,1,2,3and4after co-culture).7. Mixed lymphocyte reaction assay examined the suppressive function on days5aftermixed culture by means of CCK-8.8. The levels of IL-10and TGF-β1in the supernatants of co-cultured CD4+CD25+Tcells were measured by Elisa on days2after co-culture. The expressions of IL-10,TGF-β1and Foxp3mRNA were detected by realtime-PCR at that time.Results:1. Three doses FTY720temperature-sensitive in situ forming ophthalmic gels weremanufactured successfully, which can form gel depots at body temperature.2. Compared with the control group (mean survival time (MST)13.44±0.48days),corneal grafts survival was prolonged by treatment with topical0.5%FTY720(MST24.11±1.58days) or1%CsA eye-drop group (MST25.00±1.91days) respectively(both P<0.01). 3. Topical0.5%FTY720treatment significantly increased the percentages of CD4+Tcells (p<0.05) and Treg cells (p<0.01) in the cervical lymph nodes compared withthe control group. TGF-β1mRNA transcription in corneal grafts in topical0.5%FTY720group increased (p<0.05), while IL-2and IFN-γ mRNA expression incorneal grafts in topical1%CsA group decreased (p<0.01and p<0.05respectively).The similar results of cytokines in corneal samples were identified byimmunohistochemical staining. Both topical0.5%FTY720and1%CsA treatmentreduced the infiltration of CD4+Tcells in corneal grafts.4. There were no significant differences in cytokines serum levels including IL-2, L-10,IFN-γ and TGF-β1among the5groups (both P>0.05).5. CD4+CD25+T cells were isolated successfully. The purity of positively selectedCD4+CD25+T cells was more than93%, and the percentage of Foxp3+in positivesorted cells was more than94%. The purity of selected CD4+CD25-T cells wasnearly96%, and the percentage of Foxp3+was only1.45%.6. All of isolated cells were activated completely under the cultured system includingIL-2, antibody of antiCD3and antiCD28.7. Neither FTY720nor FTY720-P enhanced the proliferations of Treg cells comparedwith the control group (P>0.05).8. The proliferation of effector T cells was suppressed significantly in medium andhigh dose FTY720-P group at a Treg/Teff cell ratio of1:1(P<0.05). At a ratio of1:1,the proliferation of Teff cells was also suppressed in the high dose FTY720group(P<0.05). At a ratio of1:4, the proliferation of Teff cells was suppressed only in thehigh dose FTY720-P group (P<0.05). There was no difference between the treatedgroups and the control group at a ratio of1:8(P>0.05).9. The percentages of CD25+and Foxp3+were enhanced in high dose FTY720-Pgroup. The levels of TGF-β1in the supernatants were enhanced in high doseFTY720group. Medium and high dose FTY720-P also enhanced the levels ofTGF-β1. TGF-β1and Foxp3mRNA expression were upregulated in high doseFTY720-P group (P<0.05).Conclusion:1. Topical0.5%FTY720opthalmic gel and1%CsA eye drop can effectively prolongthe allogeneic corneal graft survival in mice.2. Topical0.5%FTY720increases the percentage of CD4+T cells and the higher percentage of Treg cells in cervical lymph nodes.0.5%FTY720increases TGF-β1mRNA expression and decreases infiltration of CD4+T cells in corneal grafts.3.Topical1%CsA can decrease infiltration of CD4+T cells in corneal grafts, anddown-regulate the expression of IL-2and IFN-γ in the corneal grafts.4. There are no significant differences in cytokines serum levels among the5groups.5. The proliferations of Treg cells are not affected by three doses FTY720andFTY720-P.6. High dose FTY720-P can enhance immune function of co-cultrued Treg cells, whilemedium dose FTY720-P and high dose FTY720can enhance partly the function.The reason may be attributed to the higher ratios of CD25+and Foxp3+and theenhanced levels of TGF-β1and Foxp3in co-cultured Treg cells.