| BackgroundAs all we know, corneal disease has been one of the causes leading to blindness in China. Currently, keratoplasty is still the most efficient or even the only way to treat corneal blindness. Although’immune privilege’makes it the most successful solid organ transplantation, the immune rejection response of corneal transplantation is the main reason for the failure of corneal transplantation. Nonspecific immune inhibitors such as glucocorticoids, CoA etc. are used to prevent immune rejection after corneal transplantation in clinical treatment. But systemic side effects such as cancer, infection, liver, kidney, cardiovascular system disease, restrict it even though it effective in treatment. More importantly, nonspecific immune inhibitors frequently-used in clinical can prevent or treat acute rejection effectively but useless for chronic rejection. A lot of people require a second corneal transplantation because of chronic rejection. To understand the role and mechanism of a variety of immune cells and cytokines may contribute to the clinical diagnosis, prevention and treatment of corneal allograft rejection.Fibrinogen-like protein 2(FGL-2)/fibroleukin is a member of the fibrinogen-related protein superfamily, homologous to the carboxyl terminus of the P-and y-chains of the fibrinogen and it has two forms, the membrane protein form and the soluble form. When FGL-2 is expressed as a membrane-associated protein in activated macrophages and endothelial cells, it exhibits a coagulation activity capable of directly cleaving prothrombin to thrombin. The membrane-associated FGL-2 prothrombinase with the ability to directly generate thrombin plays an important role in innate immunity. Soluble FGL-2 servers as an effector of regulatory T cells (Tregs) and demonstrates immunosuppressive function to protect against tissue injuries. Fibrinogen-like protein 2(FGL-2),also known as fibroleukin, has been demonstrated to be involved in the pathogenesis of diseases including viral-induced fulminant hepatitis and Thl cytokine-in-induced fetal loss syndrome, in which fibrin deposition is a prominent feature.IL-23, which was recently identified as a heterodimeric, proinflammatory cytokine and a member of the IL-12 family in 2000 by Oppmann etc. is secreted by antigen-presenting cells. IL-23 is composed of p19 and p40 subunits and shares a common p40 subunit with IL-12. IL-23 signals through the IL-23 receptor complex, which is composed of the IL-12 receptor Pchain and the IL-23 receptor.IL-23 mediates its proinflammatory effects through the activation of effector memory T cells that secrete IL-17A. IL-23 plays an important role in autoimmune disease such as experimental autoimmune encephalomyelitis (EAE), inflammatory bowel diseases(IBD) and rheumatoid arthritis (RA) and infection. It is significantly higher in the tumor microenvironment rather than that of normal tissue by increasing matrix metalloproteinases 9 (MMP) expressing, which could promotes the formation of tumor blood vessels and inhibits CD8+T cells infiltration. Several reports have establish a critical function for the IL23/IL-17 pathway in autoimmune disease. Moreover, it also has important role in allograft rejection.Therefore, this study is trying to observe the expression of FGL-2 mRNA and IL-23/IL-17 mRNA in corneas and clarify their potential role and mechanism through establishing penetrating keratoplasty models, which may provide new insights into clinical prevention and treatment of corneal rejection in the future.Part 1 Role of FGL-2 in corneal graft rejection after allograft transplantation in ratsObjectiveThis study was designed to detect the expression of FGL-2 in corneal and investigate the effect of steroid on FGL-2 expression in rats corneal following allograft penetrating keratoplasty.Methods1.Modeling and grouping:Sixty clean female Wistar rats were randomized into normal control group, autologous cornea transplantation group and allogeneic cornea transplantation group, glucocorticoid transplantation group. Penetrating corneal transplantation was performed with the Wistar rat donors and Wistar rat receipts in the autologous cornea transplantation group, while with the SD rat donors and Wistar rat receipts in the allogeneic corneal transplantation group and glucocorticoid group. Allograft in glucocorticoid group were treated with TobraDex after the operation,2 times a day, each 1 drop, until drawing materials.2.Clinical observation and scoring criteria:The inflammatory response of the grafts was examined under the slit lamp microscope every day after operation and scored based on the criteria of Larkin. Rejection index(RI), mean survival time and survival rate were calculated within 14 days after transplantation and once every other day after 14 days, the rejection indexes (RIs) is according to opacity, edema, and neovascularization. Corneal opacity(0-4),edema(0-2), and neovascularization(0-4) were graded according to Larkin standard:Opacity (0:completely transparent; 1: mild loss of transparency; 2:moderative loss of transparency, but iris vessels is visible on retroillumination; 3:iris vessels not visible, but pupil outline visible; 4: pupil outline not visible); Edema(0:no edema; 1:moderate edema; 2:marked edema with obvious graft thickening); Neovascularization (0:without vascularization of graft; 1:vessel growth to 25% of graft radius in any quadrant; 2:vessel growth to 50% of graft radius; 3:vessel growth to 75% of graft radius; 4:vessel growth to center of graft radius).When the RI grade was 5 or opacity grade reached 3, rejection was acknowledged.3.Histopathological examination:Eyeballs in each group were randomly selected at 9 and 14 days after keratoplasty for histopathological examination, the specimens were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into serial 4μm-thick slices with HE staining, then observed and photographed them under optical microscope. The expressions of FGL-2 were assayed by immunochemistry.4.RT-PCR:Corneal grafts in each group were randomly selected at 9 and 14 days after keratoplasty stored at-80℃ for RT-PCR to detect the expression of FGL-2mRNA.ResultsThe rejection occurred in 9.8 days after operation in allograft group, and only mild edema, opacity and neovascularization of corneas were found at different degrees in 9 days after operation in autograft group and glucocorticoid group. Severe cornea edema, a lot of inflammatory cells infiltration and new vessels in stroma were seen in allograft group, and mild inflammatory response was found in autograft group and glucocorticoid group under the light microscope. Immunochemistry showed greater positive cells for FGL-2 in allogeneic group compared with autograft group and glucocorticoid group. The fold differences of FGL-2 mRNA expression in cornea after amplification was significantly different among three groups and different time points and the evident enhance of FGL-2 mRNA expression was revealed in allograft group compared with autograft group and glucocorticoid group(P0.01).ConclusionGlucocorticoid may restrain the acute allograft rejection by down—regulating the expression of FGL-2 in corneas and its signals transaction. This result suggests that glucocorticoid offer a protection from rejection of cornea after penetrating keratoplasty.Part2 Role of IL-23/IL-17 in corneal graft rejection after allograft transplantation in ratsObjectiveThis study was designed to detect the expression of IL-23/IL-17 in cornea and investigate the effect of steroid on IL-23/IL-17 expression in rats cornea following allograft penetrating keratoplasty.Methods1. Modeling and grouping:Sixty clean female Wistar rats were randomized into normal control group, autologous cornea transplantation group and allogeneic cornea transplantation group, glucocorticoid transplantation group. Penetrating corneal transplantation was performed with the Wistar rat donors and Wistar rat receipts in the autologous cornea transplantation group, while with the SD rat donors and Wistar rat receipts in the allogeneic corneal transplantation group and glucocorticoid group. Allograft in glucocorticoid group were treated with TobraDex after the operation,2 times a day, each 1 drop, until drawing materials.2.Clinical observation and scoring criteria:The inflammatory response of the grafts was examined under the slit lamp microscope every day after operation and scored based on the criteria of Larkin. Rejection index(RI), mean survival time and survival rate were calculated within 14 days after transplantation and once every other day after 14 days. the rejection indexes (RIs) is according to opacity, edema, and neovascularization. Corneal opacity(0-4),edema(0-2), and neovascularization(0-4) were graded according to Larkin standard:Opacity (0:completely transparent; 1: mild loss of transparency; 2:moderative loss of transparency, but iris vessels is visible on retroillumination; 3:iris vessels not visible, but pupil outline visible; 4: pupil outline not visible); Edema(0:no edema; 1:moderate edema; 2:marked edema with obvious graft thickening); Neovascularization (0:without vascularization of graft; 1:vessel growth to 25% of graft radius in any quadrant; 2:vessel growth to 50% of graft radius; 3:vessel growth to 75% of graft radius; 4:vessel growth to center of graft radius).When the RI grade was 5 or opacity grade reached 3, rejection was acknowledged.3. Histopathological examination:Eyeballs in each group were randomly selected at 9 and 14 days after keratoplasty for histopathological examination, the specimens were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into serial 4μm-thick slices with HE staining, then observed and photographed them under optical microscope. The expressions of FGL-2 were assayed by immunochemistry.4.RT-PCR:Corneal grafts in each group were randomly selected at 9 and 14 days after keratoplasty stored at-80 ℃ for RT-PCR to detect the expression of IL-23/IL-17mRNA.ResultsThe rejection occurred in 9.8 days after operation in allograft group, and only mild edema, opacity and neovascularization of corneas were found at different degrees in 9 days after operation in autograft group and glucocorticoid group. Severe cornea edema, a lot of inflammatory cells infiltration and new vessels in stroma were seen in allograft group, and mild inflammatory response was found in autograft group and glucocorticoid group under the light microscope. Immunochemistry showed greater positive cells for IL-23/IL-17 in allogeneic group compared with autograft group and glucocorticoid group. The fold differences of IL-23/IL-17 mRNA expression in cornea after amplification was significantly different among three groups and different time points and the evident enhance of IL-23/IL-17 mRNA expression was revealed in allograft group compared with autograft group and glucocorticoid group(P<0.01).ConclusionGlucocorticoid may restrain the acute allograft rejection by down—regulating the expression of IL-23/IL-17 in corneas and its signals transaction. This result suggests that glucocorticoid offer a protection from rejection of cornea after penetrating keratoplasty. |
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