Objective To investigate the effect of peptidoglycan (PGN) on the secretion of pro-inflammatory cytokines by dendritic cells (DCs) and the regulation of T helper 17 (Th17) responses in experimental autoimmune uveitis. Methods Bone marrow cells from naive mice were cultured with granulocyte macrophage-colony-stimulating factor and interleukin (IL)-4 to induce DCs. DCs cultured for six days were randomly divided into two groups: PGNtreated group and control group. The DCs in PGNtreated group were stimulated with PGN and the same volume of phosphate buffered saline was added to the DCs as control group. The relative mRNA expression levels of IL-23, tumor necrotic factor alpha; (TNF-alpha;), IL-6,IL-1beta;were measured by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Peptide fragment of interphotoreceptor retinoidbinding protein (IRBP1-20)specific T cells, which were isolated from the spleen and draining lymph nodes of C57BL/6 mice immunized with IRBP1-20 peptide fragments 13 days earlier, were co-cultured with PGN-treated or untreated DCs, respectively. Total RNA from T cells cocultured for two days were isolated and the relative expression of retinoic acid receptor-related orphan receptor gamma;t (ROR-gamma;t), IL-17, T-box expression in T cells (T-bet), interferon gamma; (IFN-gamma;) mRNA were detected by realtime RT-PCR. On the second, the fifth and the seventh day, the cocultured T cells were analyzed by flow cytometry to detect the percentages of IFN-gamma;, IL-17 positive cells. Results The real-time RT-PCR results revealed that the level of IL-23, IL-1beta;, IL-6, TNF-alpha; mRNA from PGNstimulated DCs were significantly increased compared to the control group (t=-14.363, -5.627, -3.85, -28.151; P＜0.05). The level of RORgamma;t, IL-17 mRNA from the T cells cocultured with PGN-stimulated DCs were greatly increased compared with the control group (t=-5.601, -19.76；P＜0.05). However, the level of T-bet, IFN-gamma; mRNA from the T cells cocultured with PGNstimulated DCs were significantly decreased compared with the control group (t=4.717, 11.207; P＜0.05). Data of flow cytometry showed that at two days, five days, seven days after cocultured with PGN-treated DCs, the percentages of IL-17 positive T cells were increased compared to the control group (t=-2.944, -3.03, -4.81; P＜0.05), and the percentages of IFN-gamma; positive T cells had no remarkable change (t=-1.25, -0.18, -2.16; P＞0.05). Conclusion PGN can promote the secretion of Th17-related cytokines by DCs, which favors proliferation and differentiation of Th17 in experimental autoimmune uveitis.
Objective To investigate the features and main reasons of blindness induced by uveitis in China. Methods A retrospective analysis was performed on the data from 1214 patients with uveitis, referring to Zhongshan Ophthalmic Center, with special respect to the incidence of blindness in different uveitis entities, the characteristics of blindness, and possible causes for the blindness. Results In the affected 1892 eyes of 1214 patients with uveitis, 355 eyes (18.83%) were blind. The mean age at the onset of blindness was 34.38 years and the gender ratio of male to female was 1.52:1. The blindness led by panuveitis was found in 248 eyes (26.27%), including 128 (51.61%) and 73 (29.44%) blind eyes caused by Behcet’s disease and Vogt-Koyanagi-Harada syndrome. Complicated cataract, vitreous opacity and secondary glaucoma were responsible for the blindness of the patients with panuveitis［89(35.89%), 53 (21.37%), and 30 eyes (12.10%), respectively］. Blindness caused by anterior uveitis was noted in 79 eyes (10.73%) with the main reasons of complicated cataract ［56 eyes (70.89%)］and secondary glaucoma［16 eyes (20.25%)］, posterior uveitis in 15 eyes (15.63%) with the main reason of vitreous opacity ［9 eyes (60.00%)］, macular diseases in 3 eyes (20.00%), intermediate uveitis in 13 eyes (11.21%) with the main reasons of vitreous opacity［8 eyes (61.54%)］, and complicated cataract in 5 eyes (38.46%). Conclusions Uveitis is one of the important causes leading to blindness, especially in the young adults. Panuveitis, especially Behcet’s disease and Vogt-Koyanagi-Harada syndrome, are the most common entities responsible for blindness in patients with uveitis. Complicated cataract and secondary glaucoma are the main causes of blindness in uveitis. (Chin J O cul Fundus Dis, 2005, 21: 350-352)
Objective To detect the changes of function of blood-aqueous barrier in different Syndrome stages of patients with Vogt-Koyanagi-Harada (VKH) syndrome in order to provide the appropriate therapy. Methods According to clinical manifestation, 77 patients (144 eyes) with VKH syndrome were divided into 4 groups: 10 cases in posterior uvietis stage group (20 eyes), 27 in anterior uveal involvement stage group (50 eyes), 23 in recurrent anterior uvitis stage group (41 eyes), and 17 in convalescent stage group (33 eyes). The other 50 cases (100 eyes) were in the control group. Flare and cells of anterior chamber in patient with VKH Syndrome at different stages were graded and measured by laser flare and cell meter (LFCM) and slitlamp microscope. Results According to the results of slitlamp biomicroscopy, anterior chamber flare and cells were at the 0 grade in the patients at posterior uvietis stage (20 eyes). The results of LFCM examination revealed that the flare value and cells were (9.7±3.4) pc/ms and (0.9±0.6)/0.5 mm3 in posterior uvietis stage group, and (5.3±2.3) pc/ms and (0.8±0.6)/0.5 mm3 in the control group. The differences between the two groups were significant (Plt;0.001) and insignificant (P=0.899), respectively. In anterior uveal involvement stage group, the cells in anterior chamber was at grade 1+ in 25 eyes, 2+ in 19, and 3+ in 6, respectively, while the flare was at grade 1+in 27 eyes and 2+ in 23; the number of cells in anterior chamber was (13.7±6.5)/0.5 mm3，(40.8±17.6)/0.5 mm3, and (75.7±25.5)/ 0.5 mm3 respectively, and the value of flare was (31.4±12.8) pc/ms and (133.4±59.5) pc/ms. In recurrent anterior uvitis stage group, the cells in anterior chamber was at grade 1+ in 19 eyes, 2+ in 15, and 3+ in 7, respectively, while the flare was at grade 1+ in 24 eyes and 2+ in 17; the number of cells in anterior chamber was (11.2±5.4)/0.5 mm3，(29.6±14.4 )/0.5 mm3,and (69.3±22.2)/0.5 mm3, respectively, and the value of flare was (34.94±14.3) pc/ms and (150.9±83.3) pc/ms. The flare and cells in anterior chamber both in anterior uveal involvement stage and recurrent anterior uvitis stage group were higher than that in the control group (Plt;0.001). In convalescent stage group, the cells was at grade 0 in 33 eyes and the flare was at grade 0 in 15 eyes and 1+ in 18; while the number of cells was (1.0±0.7)/0.5 mm3 which was insignificantly differed from that in the control group (P=0.310), and the value of flare was (9.5±4.8) pc/ms and (30.0±12.3) pc/ms which were both higher than that in the control group (Plt;0.001). Conclusions The breakdown of blood-aqueous barrier with different degrees occurs at each stage in VKH syndrome, whereas inflammatory cells appearing in anterior chamber are only noted at some certain stages. This is very significant to offer directional and effective treatment to the patients with VKH syndrome. (Chin J Ocul Fundus Dis, 2005, 21: 363-366)
ObjectiveTo observe the expression of interleukin (IL)-17, IL-4 and interferon γ (IFN-γ) in experimental autoimmune uveoretinitis (EAU). MethodsC57BL/6 mice were immunized with interphotoreceptor retinoid-binding protein 1-20 to induce EAU. The inflammatory reaction before and on 7, 14, 21, 28 days after immunization were observed. The level of IL-17, IL-4 and IFN-γ in the serum were measured by enzyme-linked immune sorbent assay (ELISA). mRNA and protein expression of spleen and retina were analysed using quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot at the same time, respectively. ResultsThe most serious inflammatory reaction occurred at the 14th day after immunization. The highest level of IFN-γ in serum, highest mRNA and protein expression of IFN-γ in spleen and retina of mice occurred at day 7 after being immunized. The highest level of IL-17, IL-4 in serum, highest mRNA and protein expression of IL-17, IL-4 in spleen and retina of mice occurred at day 14 after being immunized. The increase degree of IL-17 was more than IFN-γ and IL-4. At 7, 14 and 21 days after immunization, compared with the pre-immunization, the level of IL-17, IL-4, IFN-γ in serum of mice were significantly increased (F=1 817.346, 268.600, 164.621; P < 0.05). There was no difference in the levels of IL-17, IL-4, IFN-γin serum of mice between pre-and 28 days after immunization (P > 0.05). At 7, 14 and 21 days after immunization, compared with the pre-immunization, the protein expression of IL-17, IL-4, IFN-γ in spleen (F=312.67, 114.250, 216.220) and retina (F=271.504, 85.370, 80.722) of mice were significantly increased (P < 0.05). There was no difference in protein expression of IL-17, IL-4, IFN-γ in spleen and retina of mice between pre-and 28 days after immunization (P > 0.05). ConclusionsThere were IL-17, IL-4 and IFN-γ expression in EAU. IL-17, IL-4 and IFN-γ play a key role in the occurrence and development of the EAU.
Objective To explore the consistency and significance of optical coherence tomography (OCT) and clinical and histopathological findings in adoptively transferred uveitis in mice. Methods The adoptively transferred experimental autoimmune uveitis (EAU) model was established by intraperitoneal injection of antigen-specific T cells in C57BL/6 mice. Since 9 days after transferred, inflammation of eyes was observed by indirect ophthalmoscope with +90D lens and record clinical scores every 3 days. The disease was divided into 6 phases including onset phase, early phase, pre-peak phase, peak phase, resolution phase and late phase of EAU, which respectively corresponding to clinical score 0.5, 1.0, 1.5 - 2.0, 2.5 - 3.0, 1.0 - 2.0 and less than 1.0. Since 9 days after transferred, the retina and retinal thickness (RT) was measured by spectralis OCT about 1 disc from the disc edge in 10 time points including 9, 11, 16, 21, 25, 30, 35, 40, 50 and 60 days after transferred. The OCT score was recorded as from 0.0 to 4.0. After transferred 9, 21 and 60 days, the mice were killed and eye balls were examined in histology. OCT score, clinical score and histology in the mouse were compared and analyzed. Results The disease was divided into onset phase, early phase, pre-peak phase and peak phase of EAU, which respectively corresponding to 9, 16, 21 and 26 days after transferred. In four phases, OCT score were 0.5, 1.0, 2.0 and 4.0 respectively. After transferred 30 days, which was in resolution phase of EAU, the inflammation cells in vitreous were decreased and OCT score was 3.0. After transferred 60 days, which was in late phase of EAU, inflammation cells in vitreous were disappeared and retina was atrophic topically. The histology showed the vitreous has slight inflammation cells and retinal structure was normal at onset of EAU. The vitreous has massive inflammation cells and retina structure was disorder at pre-peak of EAU. And in resolution phase of EAU, the inflammation cells in vitreous were slightly and retina was atrophic and thinned. The data in this study demonstrated that OCT score was well correlated with clinical score in EAU (r=0.957 9, P < 0.000 1). Conclusion OCT and clinical and histopathological findings in adoptively transferred uveitis in mice were consistency and OCT is contribute to evaluate the disease dynamically and quantifiably.
ObjectiveTo preliminarily investigate the mechanism of MMP-9 blocking CD73 detachment from RPE cells surface and preventing and treating experimental autoimmune pigment membranitis (EAU).MethodsRPE cells isolated from wild-type C57BL/6 and CD73 gene knockout (CD73-/-) mice were cultured in vitro, and treated with lipopolysaccharide and TNF-α to induce CD73 detachment from RPE surface. According to whether MMP-9 inhibitor CTK8G1150 was added at the same time (the final concentration was 5.0 mol/L) or not, RPE cells cultured in the two types of mice were respectively set as MMP-9 inhibitor intervention group and non-intervention control group. The cells in each group were treated with the intervention of a solvent, 1 μmol/L adenosine monophosphate (AMP), 1 μmol/L AMP, and 3 μmol/L 5' -α,β-methylene adenosine diphosphate (APCP) (AMP+APCP). The stimulating effect of RPE cells in different groups on CD4+ T cell proliferation was detected by tritiated thymidine incorporation. Adoptive immune induced EAU in wild-type B6 mice and CD73-/- mice, respectively. The receptor mice were randomly divided into the MMP-9 inhibitor intervention group and the non-intervention control group, and CTK8G1150 or the solvent were injected into the subretinal cavity 4, 7 and 10 days after adoptive immunity. CD73 mRNA and protein expression in RPE cells of recipient mice were detected by real-time quantitative PCR (RT-PCR) and Western blot. One-way ANOVA was used to analyze all experimental data.ResultsWhen the stimulation mode was AMP, the proliferation of CD4+ T cells in the C57BL/6 MMP-9 inhibitor intervention group decreased significantly compared with the non-intervention group (F=13.28, P＜0.01). When the stimulation mode was solvent and AMP+APCP, there was no statistically significant difference in the proliferation capacity of CD4+ T cells between the two groups (F=7.78, 6.58; P＞0.05). There was no statistically significant difference in the proliferation capacity of CD4+ T cells between the CD73-/- MMP-9 inhibitor intervention group and the non-intervention group (F=5.24, 6.12, 7.04; P＞0.05). RT-PCR results showed that there was no statistically significant difference in the relative expression of CD73 mRNA in RPE cells between the MMP-9 inhibitor group and the non-intervention control group (F=6.54, P>0.05). Western blot results showed that the expression of CD73 protein in RPE cells in the MMP-9 inhibitor group of B6 receptor mice was significantly increased compared with the control group (F=15.24, P＜0.01).ConclusionMMP-9 inhibitor blocks CD73 detachment from RPE cells surface and has a protective effect on EAU.