Effect of mesenchymal stem cells on the expression of interleukin-17 in the retina of experimental autoimmune uveoretinitis / 中华实验眼科杂志

BackgroundStudies determined that Th17 cells are important inflammatory cell group in experimental autoimmune uveoretinitis ( EAU ).Interleukin-17 ( IL-17 ),as a marker of Th17,is involved in the occurrence and development of EAU.Mesenchymal stem cells (MSCs) play an immunomodulatory role,mainly by inhibiting the expression of Th17 in a variety of self-autoimmune disease.This is one of the current research focuses.ObjectiveThe present study was to investigate the therapeutic effect of MSCs in EAU and their impact on IL-17 expression in the retina.MethodsMSCs were isolated from the bone marrow of the femurs from 10 SPF Wistar rats and cultured and passaged.The third to fifth generations of cells were used in this experiment.EAU models were induced in 48 6-8 week-old SPF Lewis rats by subcutaneous injection of interphotoreceptor retinoid binding protein (IRBP) R16 peptide emulsified in adjuvant.EAU rats were randomly assigned to the model control group and the MSCs group.MSCs suspension (5×106) of 1 ml was injected via the rat tail vein once a day for 3 consecutive days after immunization,and the same amount of PBS was injected in the model control group in the same manner.Six matched normal Lewis rats were used as the normal control group.The inflammatory response was clinically examined under the slit lamp biomicroscope daily,and the histopathological changes of the retina were examined by hematoxylin and eosin staining on days 9,12,15 and 20.The clinical and histopathological scoring was performed according to the Caspi criteria.Expression of the IL-17 protein in the retina was detected by immunohistochemistry on 9,12,15 and 20days following molding.The use of the animals complied with the Regulations for the Administration of Affairs Concerning Experimental Animals by Tianjin Municipality Science and Technology Commission.Results MSCs showed the fusiform in shape and vortex-like growth.Flow cytometry verified that presented the positive expression for CD29 and CD44 but absent expression for CD45 and CD34.The scores of the anterior segment were significantly lower ( U=2.815,P =0.005 ; U =2.768,P =0.006 ; U =2.900,P =0.004 ; U =2.855,P =0.004 ),and the retinal inflammation scores were lower in the MSCs group than the model control group at various time points ( U =2.345,P =0.019 ; U =2.559,P =0.011 ; U =2.166,P =0.030 ; U =2.373,P =0.018 ).Im mnunochemistry showed that the expressions levels of the IL-17 protein (A value) in the rat retina were 26.47±5.68,77.78± 9.65,47.02±6.68 and 26.59±5.94 in the MSCs group on days 9,12,15 and 20,and those in the control group were 45.34±4.63,105.95± 10.74,64.11 ±9.76 and 37.02±6.51,showing a significant reduction ( t =6.305,P =0.000 ; t =4.799,P =0.001 ; t=3.540,P=0.005;t=2.900,P=0.016). ConclusionsMSCs can inhibit the aggravation of EAU and suppress the expression of IL-17 in ocular tissue.

Therapeutic effects of rapamycin on experimental autoimmune uveoretinitis / 中华实验眼科杂志

Background Studies determined that rapamycin has not only the antibiosis but also suppressing the auto-immunology.The treating effect of rapamycin on experimental autoimmune uveoretinitis (EAU) is still concerned.Objective This work was to investigate the therapeutic effect of rapamycin on EAU and study the effect on the expression of inflammatory cytokines which were secreted by T lymphocyte subgroup in EAU.Methods EAU was induced in 20 SPF male Lewis rats by subcutaneous injection of interphotoreceptor retinoid binding protein (IRBP) R16 peptide emulsified in adjuvant.The rats were randomized into model control group and rapamycin injection group and 10 rats for each group.The rapamycin of 0.2 mg/( kg · d) 0.4 ml was intraperitoneally injected for the consecutive 7 days immediately after modeling,and the equal volume of normal saline solution was used at the same fashion in the model control group and 5 normal matched rats( normal control group).The ocular manifestation of the rats were examined under the slit lamp regularly.The retinal sections of the rats were prepared in the 14 days after modeling for the histopathological examination with hematoxylin and eosin staining.The ocular signs of inflammation and histopathological severity were scored based on the criteria of Caspi.Expression of interferon-γ (IFN-γ) and interleukin-17 (IL-17) in rat retinas were detected by immunohistochemistry.This experiment followed the Regulation for the Administration of Affair Concerning Experimental Animals by Tianjin Municipal Government.Results The scores of ocular signs elevated gradually from 6 through 12 days after modeling and slowly declined after that in the model control group.A same tendency was seen in the rapamycin injection group.The significant differences were seen in the scores of ocular signs between the two groups from 6 to 14 days( P＜0.01 ).The disorder of retinal structure and infiltration of inflammatory cells were seen in the model control group,but the retina layers were normal in the rapamycin injection group.The pathological score was evidently declined in the rapamycin injection group ( 0.90 ± 0.45 ) in comparison with model control group( 3.30±0.48 ) ( t =16.541,P＜0.01 ).The expressions of the IFN-γ and IL-17 in retina located in the outer nuclear layer,inner nuclear layer,internal plexiform layer and retinal ganglion cell layer with the weakened levels(A values) in rapamycin injection group compared with model control group,showing a considerably difference between them ( IFN-γ:21.16±4.23 vs 62.14 ±7.32; IL-17:49.86±6.59 vs 124.85 ±6.33 )(q=33.334,q=56.923,P＜0.01 ).Conclusions Rapamycin down-regulates the expressions of IFN-γ and IL-17 in retina and further eliminates the inflammatory response in the rat with EAU by the suppression of Th1 and Th17 cells function in EAU.

Role of CD4+CD25+T cells in experimental autoimmune uveoretinitis / 中华实验眼科杂志

Background Experimental autoimmune uveoretinitis(EAU)is proved to be an organ-specific,T lymphocyte-mediated autoimmune and self-limited disease.Research showed that CD4+CD25+T cell may play important regulation on the course of events,but its mechanism is pending for further study. Objective Present study was to investigate the potential role of CD4+CD25+T cell in the pathogenesis of EAU. Methods Retinal Santigen(S-Ag)was isolated from bovine retinas according tO the procedure as Caspi's previously describe.0.1 ml Santigen(50μg)emulsified with complete Freund'S adjuvant was injected on footpad of 24 inbred adult female Lewis rats aged six to eight-week-old to induce EAU,and 4 normal Lewis rats were as normal control group.Slim-lamp examination was performed to observe the ocular manifestation.Retinal section was prepared in 7,12,15,21 days aher injection for the pathological examination.The pathological grading was on the lnoki'S method.The retinas,inguinal nodes and spleens of rats were obtained in 7,12,15,21 days after injection and the cellular suspension was prepared.Expression of CD4+CD25+T cells on cellular suspension was assayed using flow eytometry.This study complied with the Standard of Association for Research in Vision and Ophthalmology. ResuRs The obvious inflammatory response of the anterior segment was found in S-Ag injected eyes from 7 days through 21 days.The most serious inflammation was found in 12-15 days under the slim-lamp.The hemotoxylin and eosin staining showed the higher pathological grading from 12 to 15 days after injection,showing significant difference in comparison with 7 days and 14 days(P=0.000).In EAU model rats,expressions of CD4+CD25+T cells was significantly increased in retinas, inguinal nodes and spleens in 15 days after injection,showing evidently differences in comparison with control rats(P=0.000). Conclusion The expression level of CD4+CD25+T cells in inflammatory tissue is associated with the inflammation procedure in EAU model rats.This study indicates that CD4+CD25+T cells may play a role in the development of EAU.

Maturation-Resistant Dendritic Cells Ameliorate Experimental Autoimmune Uveoretinitis

BACKGROUND: Endogenous uveitis is a chronic inflammatory eye disease of human, which frequently leads to blindness. Experimental autoimmune uveoretinitis (EAU) is an animal disease model of human endogenous uveitis and can be induced in susceptible animals by immunization with retinal antigens. EAU resembles the key immunological characteristics of human disease in that both are CD4+ T-cell mediated diseases. Dendritic cells (DCs) are specialized antigen-presenting cells that are uniquely capable of activating naive T cells. Regulation of immune responses through modulation of DCs has thus been tried extensively. Recently our group reported that donor strain-derived immature DC pretreatment successfully controlled the adverse immune response during allogeneic transplantation. METHODS: EAU was induced by immunization with human interphotoreceptor retinoid-binding protein (IRBP) peptide(1-20). Dendritic cells were differentiated from bone marrow in the presence of recombinant GM-CSF. RESULTS: In this study, we used paraformaldehyde-fixed bone marrow-derived DCs to maintain them in an immature state. Pretreatment with fixed immature DCs, but not fixed mature DCs, ameliorated the disease progression of EAU by inhibiting uveitogenic CD4+ T cell activation and differentiation. CONCLUSION: Application of iBMDC prepared according to the protocol of this study would provide an important treatment modality for the autoimmune diseases and transplantation rejection.

Effects of Heme Oxygenase-1 Inducer and Inhibitor on Experimental Autoimmune Uveoretinitis

PURPOSE: Experimental autoimmune uveoretinitis (EAU) is an animal model of posterior uveitis and heme oxygenase-1 (HO-1) is a well-known anti-oxidant factor. However, there is no report a protective role of HO-1 on EAU in vivo. To verify that HO-1 is induced in EAU by interphotoreceptor retinoid-binding protein (IRBP), that an HO-1 inducers ameliorates the associated inflammation, and that an HO-1 inhibitor exacerbates this inflammation. METHODS: Forty four Lewis rats were given either 40 mol/kg hemin or 40 mol/kg SnPP (tin protoporphyrin IX) by intraperitoneal injection and twenty two uveitis control rats were injected with 0.5 mL of saline once daily 5-20 days after IRBP immunization inducing EAU. Three normal control rats were used for Western blotting and ELISA assay of HO-1. The clinical uveitis signs of inflammation were scored in the three groups from 0 to 4 on alternate three days. To confirm the clinical results, histological and immunohistochemical stain of HO-1 were performed on the day of peak inflammation and Western blotting and ELISA assay of HO-1 were performed on 6th, 12th and 18th day after IRBP immunization. RESULTS: Hemin, an inducer of HO-1, ameliorated the clinical signs of EAU. In contrast, SnPP-treated rats show that the severity of the clinical sign were exacerbated at the peak period of the disease. These results are roughly compatible with histological, immunoblotting, and immunohistochemical evaluations and an ELISA assay of HO-1. CONCLUSIONS: We suggest that HO-1 plays an important protective role in EAU.

The model of experimental autoimmune uveoretinitis in rats / 中国免疫学杂志

Objective:To induce experimental autoimmune uveoretinitis in Wistar for investigating the pathogenesis mechanism of uveitis.Methods:18 Wistar rats were immunized by different dosage of bovine soluble antigen(S Ag),and were detected the clinic changes dayly.The animals were anesthetized and killed when they had significant clinic EAU appearance or 3～4 weeks after immunized,the eyes were removed and followed by pathologic studies.Results:The incidence of EAU in the 3 different dosage S Ag immunized rats were 2/12 in 100 ?g S Ag group(6 rats,12 eyes),6/12 in 200 ?g S Ag group(6 rats,12 eyes) and 8/12 in 300 ?g S Ag group(6 rats,12 eyes) separately and the pathology scores were 0?16 76?11.02?17 56?9 96 separately in 3 group of rats.Conclusion:The middling sensitive Wistar rats can be used to induce EAU successly with middling purified S Ag.