Retinal and ciliary body pigment epithelium suppress activation of T lymphocytes via transforming growth factor beta

The ocular microenvironment is immunosuppressive and anti-inflammatory. Pigment epithelial (PE) cells isolated from the eye possess a new property of suppressing T cell receptor-dependent activation of T cells in vitro. This property depends on their capacity to produce cell-surface and soluble inhibitory molecules. The iris pigment epithelia (IPE) do so through direct cell-to-cell contact with na?ve T cells, and this suppressive contact is mediated by interactions between B7 and membrane-bound TGFbeta that are expressed constitutively on IPE. We have now examined whether other ocular PE cells, e.g., retinal pigment epithelia (RPE) and ciliary body pigment epithelia (CBPE), have a similar suppressive property by a similar process. We have found that RPE and CBPE significantly suppress the activation of bystander T cells via soluble inhibitory factors. RPE and CBPE secrete different soluble inhibitory factors including TGFbeta1 and TGFbeta2. Although IPE cells suppress the activation of bystander T cells by membrane-bound TGFbeta, the RPE and CBPE do so by soluble forms of active TGFbeta through mechanisms independent of cell contact. These ocular PE cells are capable modifying T cell function by enhancing production of regulatory cytokines including TGFbeta. We propose that this mechanism of suppression via TGFbeta ensures that soluble active TGFbeta is released into the ocular microenvironment in order to create the immune privilege of the posterior segment of the eye.     

Th17/Treg细胞与自身免疫性葡萄膜炎的研究进展

葡萄膜炎是一组常见的由自身免疫紊乱导致的眼部炎症性致盲性眼病,现代研究认为主要是由CD4⁺T 细胞介导产生。CD4⁺T 细胞主要分为Th1,Th2,Th17和 Treg 细胞(CD4⁺CD25⁺Foxp3⁺调节性T细胞)四个亚群。以往研究主要集中在Th1,Th2细胞亚群,近年来研究表明,Th17 细胞是诱导自身免疫性葡萄膜炎发病的主要原因,而Treg细胞是负向调控葡萄膜炎的主要因素。因此,Th17和 Treg 细胞在葡萄膜炎的发病及病程演变过程中发挥重要作用,本文就Th17 细胞、Treg 细胞与自身免疫性葡萄膜炎的相关研究进展进行综述。     

High glucose‐induced apoptosis in bovine retinal pericytes is associated with transforming growth factor β and βIG‐H3: βIG‐H3 induces apoptosis in retinal pericytes by releasing Arg‐Gly‐Asp peptides

Background: Transforming growth factor β (TGF‐β) plays an important role in diabetic retinopathy. βIG‐H3 is a downstream target molecule of TGF‐β that may participate in the pathogenesis of diabetic retinopathy and in particular in the loss of pericytes during early pathological changes. Methods: We observed bovine retinal pericytes apoptosis and the increased expression of TGF‐β and βIG‐H3 induced by high concentrations of glucose in the cell culture media. An anti‐TGF‐β antibody was used to block glucose‐induced retinal pericytes apoptosis. Retinal pericytes were also transfected with cDNA encodings either wild‐type or mutant βIG‐H3 lacking Arg‐Gly‐Asp (RGD) sequences in order to validate the effects of βIG‐H3 and RGD signalling on retinal pericytes apoptosis. Results: A cell death‐detecting enzyme‐linked immunosorbent assay revealed that 25 mM glucose significantly increased cell death compared with 5.5 mM glucose after 5 or 7 days of exposure (P < 0.01). High glucose significantly increased the TGF‐β levels as compared with 5.5 mM glucose after 5 days, and βIG‐H3 levels after 3, 5 and 7 days of exposure (P < 0.01). TGF‐β increased cell death and βIG‐H3 levels in a dose‐dependent manner, with a maximal effect observed at 1 ng/mL. An anti‐TGF‐β antibody nearly completely blocked high glucose‐induced cell death. Wild‐type βIG‐H3‐transfected cells showed a significant increase in cell death as compared with mutant βIG‐H3‐transfected (Mycb‐c) cells, untransfected or mock‐transfected cells. Conclusion: These results suggest that hyperglycaemia‐induced expression of TGF‐β and βIG‐H3 contributes to accelerated retinal pericytes apoptosis. βIG‐H3 induces pericytes apoptosis through its RGD motif, which may constitute an important pathogenic mechanism leading to pericytes loss in diabetic retinopathy.     

Inhibition of cellular transfer of experimental autoimmune uveoretinitis by Rapamycin

The crucial role of CD4⁺ T cells in mediating uveitis is well recognized. One treatment strategy of non-infectious uveitis therefore seeks to inhibit T cell function. For that purpose the authors have evaluated the efficacy of Rapamycin (RAPA), an inhibitor of lymphocyte response to growth factors. To reproduce as best as possible the immune system condition during active disease, the adoptive transfer of activated T cells was used to induce experimental autoimmune uveoretinitis (EAU). Treatment with RAPA was delivered by continuous intravenous infusion. The results showed a complete inhibition of EAU transfer at the RAPA dose of 0.1 mg/kg/day. They indicate that RAPA could be a useful immunosuppressant for uveitis therapy.     

Inducible co-stimulator (ICOS) is upregulated in experimental autoimmune uveoretinitis

Purpose To study the expression of inducible co-stimulator (ICOS) and its association with T cell effector function in experimental autoimmune uveoretinitis (EAU).Methods Eighteen Lewis rats were immunized by retinal S-antigen (50 μg) emulsified in complete Freund’s adjuvant (CFA). Twelve normal rats served as normal controls and 18 receiving injection of CFA and PBS as CFA controls for studying the influence of CFA on the expression of ICOS in CD4⁺CD25⁺ T cells. ICOS expression on cells from the spleens, inguinal nodes and retinae on day 0 (normal rats), 7, 13 and 21 was investigated using fluorescent quantitative real-time-PCR and Western blot. Expression of B7RP-1, an ICOS ligand, was also studied by Western blot. The phenotype of the cells from the aforementioned three tissues was identified with flow cytometry using antibodies to ICOS, CD4 and CD25. ICOS⁺ cells from the lymph nodes, and spleens on day 13 were magnetically sorted and cultured with S-antigen to study the cytokines production with enzyme-linked immunosorbent assay.Result An obvious uveitis was induced in all the immunized rats on day 13 after S-antigen immunization. The mRNA and protein of ICOS were scarcely detectable in normal rat spleens. In EAU rats, an up-regulation of ICOS could be observed on day 7 and was very pronounced on day 13, followed by a decrease on day 21 in the spleens, draining nodes and retinae. Similarly, B7RP-1 expression seemed to be up-regulated during EAU. Flow cytometry showed that ICOS⁺ cells were mostly CD4 positive. Kinetics of ICOS⁺CD4⁺CD25⁺ T cells was similar to that of ICOS⁺ cells. CFA alone was also able to induce increased expression of ICOS in CD4⁺CD25⁺ T cells. IFN-γ was secreted predominantly by ICOS⁺ T cells.Conclusion ICOS expression is upregulated in association with T cell effector capacity in EAU. It is presumed that the ICOS/B7RP-1 costimulatory pathway may play a role in the development of EAU.This study is supported by the Fund for Innovative Research Groups of China (30321004), Specialized Research Fund for the Doctoral Program of Higher Education in China (20030558077).     

CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript>Tregs express an increased LAG-3 and CTLA-4 in anterior chamber-associated immune deviation

Background Regulatory CD4⁺CD25⁺ T cells have been proven to be essential for maintenance of peripheral tolerance and autoimmune diseases. ACAID is a model of immune privilege in the eye. Relatively little is known about the role and phenotype of these regulatory CD4⁺CD25⁺ T cells in ACAID. Methods Injection of OVA into the anterior chamber of BALB/C mice was performed to induce ACAID. The frequencies of splenic CD4⁺CD25⁺ Tregs and the expression of CTLA-4 and LAG-3 on these cells were determined by flow cytometry. Magnetic cell sorting was used to isolate CD4⁺CD25⁺ and CD4⁺CD25⁻T cells. The function of CD4⁺CD25⁺ T cells was detected by in vitro immunosuppression assays and in vivo adoptive transfer. Results ACAID was successfully induced following an i.c. injection of OVA. Frequencies of CD4⁺CD25⁺ and Tregs were significantly increased in ACAID mice as compared to those in controls. The CD4⁺CD25⁺ T cells stimulated with OVA in ACAID mice showed a stronger suppressive ability in vitro than those seen in non-ACAID mice. CD4⁺CD25⁺ T cells from ACAID mice, but not from non-ACAID mice, were able to suppress DTH responses in an antigen-specific manner following adoptive transfer. The frequencies of CTLA-4 or LAG-3 on Tregs in ACAID mice were higher as compared with those in naive mice. Conclusion Splenic CD4⁺CD25⁺Foxp3⁺T cells expressing CTLA4 and LAG3 play an important role in the induction of ACAID. This study is supported by the Fund for the National Natural Science Foundation (30400487), Research Group Fund of Guangdong Province Natural Science (2005-04) and Innovation Research Groups of China (30321004).     

Rapamycin ameliorates experimental autoimmune uveoretinitis by inhibiting Th1/Th2/Th17 cells and upregulating CD4+CD25+ Foxp3 regulatory T cells

AIM:To determine the effects of rapamycin on experimental autoimmune uveoretinitis (EAU) and investigate of role of rapamycin on T cell subsets in the disease. METHODS:EAU was induced in rats using peptides 1169 to 1191 of the interphotoreceptor binding protein (IRBP). Rapamycin (0.2 mg/kg/d) was administrated by intraperitoneal injection for a consecutive 7d after immunization. Th1/Th2/Th17 cytokines, TGF-β1, and IL-6 produced by lymphocyteswere measured by ELISA, while Th17 cells and CD4+CD25+ regulatory T cells (Tregs) from rat spleen were detected by flow cytometry. RESULTS: Intraperitoneal treatment immediately after immunization dramatically ameliorated the clinical course of EAU. Clinical responses were associated with reduced retinal inflammatory cell infiltration and tissue destruction. Rapamycin induced suppression of Th1/Th2/Th17 cytokines, including IFN-γ, IL-2, IL-17, IL-4, and IL-10 release from T lymphocytes of EAU rats, in vitro. Rapamycin also significantly increased TGF-β1 production but had no effect on IL-6 productionof T lymphocytes from EAU rats in vitro. Furthermore, rapamycin decreased the ratio of Th17 cells/CD4+T cells and upregulated Tregs in EAU, as detected by flow cytometry. CONCLUSION: Rapamycin effectively interferes with T cell mediated autoimmune uveitis by inhibiting antigen-specific T cell functions and enhancing Tregs in EAU. Rapamycin is a promising new alternative as an adjunct corticosteroid-sparing agent for treating uveitis.     

儿童眼肌型重症肌无力免疫相关发病机制的初步探讨

目的:通过分析儿童眼肌型重症肌无力患儿外周血中免疫球蛋白、补体及T细胞亚群的含量,初步探讨体液免疫和细胞免疫在儿童眼肌型重症肌无力发病中的作用。方法:采用免疫比浊法检测37例儿童眼肌型重症肌无力患儿及健康对照儿童外周血中IgG,IgA,IgM,补体C3及C4的含量,流式细胞术检测12例儿童眼肌型重症肌无力患儿及健康对照儿童外周血中CD3+T淋巴细胞、CD4+T淋巴细胞及CD8+T淋巴细胞的含量,独立样本t检验进行数据统计分析。结果:儿童眼肌型重症肌无力患儿外周血IgA,IgM及CD3+T淋巴细胞、CD8+T淋巴细胞值与健康儿童比较,均无显著性差异(P>0.05),IgG,补体C3及C4的含量低于健康儿童(P<0.05)。CD4+T淋巴细胞值高于对照儿童(P<0.05)。结论:补体C3,C4及CD4+T淋巴细胞在儿童眼肌型重症肌无力的发病机制中起着重要作用。     

Adhesion molecules in iris biopsy specimens from patients with uveitis

BACKGROUND/AIMS—Earlier studies on intraocular tissue have demonstrated that T lymphocytes play a major role in the pathogenesis of uveitis. Adhesion molecules are immunoregulatory molecules for the interaction between T lymphocytes and vascular endothelium and they play an important role in the recruitment of specific T lymphocytes from the circulation into inflamed tissue. In uveitis an increased expression of some of these adhesion molecules may be expected.
METHODS—The presence of adhesion molecules was investigated in iris biopsy specimens from 11 patients with uveitis and eight controls (patients with primary open angle glaucoma) immunohistochemically with a panel of monoclonal antibodies: LECAM (CD 62L), ICAM-1 (CD 54), LFA-1 (CD 11a/18), VCAM-1 (CD 106), VLA-4 (CD 49d), and HECA-452, a marker for high endothelial venules.
RESULTS—Positive staining for ICAM-1, LFA-1 and VCAM-1 was found in the iris in a significantly higher number of uveitis patients than in controls. The remaining adhesion molecules were also found in a higher number of uveitis patients than in controls, but this difference did not reach statistical significance.
CONCLUSION—An increased expression of adhesion molecules was found in the iris of patients with uveitis, indicating an immunoregulatory function for adhesion molecules in the pathogenesis of uveitis.

Keywords: adhesion molecules; uveitis; iris     

Demystifying viral anterior uveitis: A review

A viral aetiology should be suspected when anterior uveitis is accompanied by ocular hypertension, diffuse stellate keratic precipitates or the presence of iris atrophy. The most common viruses associated with anterior uveitis include herpes simplex virus, varicella‐zoster virus, cytomegalovirus and rubella virus. They may present as the following: Firstly, granulomatous cluster of small and medium‐sized keratic precipitates in Arlt's triangle, with or without corneal scars, suggestive of herpes simplex or varicella‐zoster virus infection. Secondly, Posner‐Schlossman syndrome with few medium‐sized keratic precipitates, minimal anterior chamber cells and extremely high intraocular pressure; this is mainly associated with cytomegalovirus. Thirdly, Fuchs uveitis syndrome, with fine stellate keratic precipitates diffusely distributed over the corneal endothelium, with diffuse iris stromal atrophy but without posterior synechiae, is associated mainly with rubella or cytomegalovirus infection. In rubella, the onset is in the second to third decade. It presents with posterior subcapsular cataract, may have iris heterochromia and often develops vitritis without macular oedema. Cytomegalovirus affects predominantly Asian males in the fifth to seventh decade, the keratic precipitates may be pigmented or appear in coin‐like pattern or develop nodular endothelial lesions, but rarely vitritis. Eyes with cytomegalovirus tend to have lower endothelial cell counts than the fellow eye. As their ocular manifestations are variable and may overlap considerably, viral AU can pose a diagnostic dilemma. Thus, quantitative polymerase chain reaction or Goldmann‐Witmer coefficient assay from aqueous humour samples are preferred to confirm the aetiology and determine the disease severity as this impacts the treatment.     

艾滋病并发巨细胞病毒性视网膜炎32例临床分析

【摘要】 目的 分析获得性免疫缺陷综合征（艾滋病）患者发生巨细胞病毒性视网膜炎及免疫恢复 性葡萄膜炎的疾病特征。设计 回顾性病例系列。研究对象 北京佑安医院眼科诊治的艾滋病合并巨 细胞病毒性视网膜炎患者32例。方法 对上述患者进行与艾滋病相关的免疫学检测;并进行视力、 眼压、眼前节检查、彩色眼底照相及荧光素眼底血管造影等眼科检查,并观察其临床特征。主要指 标 视力、眼压、眼底、CD4+T细胞计数。结果 32例患者的视力为光感～1.0;眼压8~16 mm Hg;眼 前节检查8例患者（4例为免疫恢复性葡萄膜炎）可见眼前节反应阳性（前房闪辉及角膜后KP）;32 例患者彩色眼底像均可见典型巨细胞病毒性视网膜炎眼底改变;8例（25%）CD4+T细胞＜10个/μL ,视力为光感～0.8,其中3例发生免疫恢复性葡萄膜炎,1例发生视网膜脱离;15例（46.88%） CD4+T细胞在10～50个/μL,视力为眼前指数～1.0,其中1例发生免疫恢复性葡萄膜炎,1例发生视 网膜脱离;9例（28.12%）CD4+T细胞＞50个/μL,无患者发生免疫恢复性葡萄膜炎及视网膜脱离。 19例患者（含4例免疫恢复性葡萄膜炎患者）合并其他全身机会性感染。结论 CD4+T细胞＜50个/μ L的患者更易患巨细胞病毒性视网膜炎,其视力预后差; CD4+T细胞
＜10个/μL的患者更易发生免疫恢复性葡萄膜炎,且眼前节反应明显,更易合并其他全身机会性感 染。（眼科, 2012, 21: 405-408）     

Glucosamine inhibits epithelial‐to‐mesenchymal transition and migration of retinal pigment epithelium cells in culture and morphologic changes in a mouse model of proliferative vitreoretinopathy

Purpose: To explore the effect of glucosamine (GlcN) on transforming growth factor (TGF)‐β signalling and several processes involved in proliferative vitreoretinopathy (PVR). Methods: We evaluated the surface levels of TGF‐β receptor and its binding of TGF‐β in ARPE‐19 cells. Release of cytokines and collagen, and expression of signalling intermediates were quantified. Migration was qualitatively and quantitatively examined. The morphology of cells undergoing PVR in vitro and in a mouse PVR model was observed. Results: Glucosamine reduced the surface levels of TGF‐β receptor and the ability of ARPE‐19 cells to bind TGF‐β. In ARPE‐19 cells, TGF‐β1 plus epidermal growth factor (EGF) or TGF‐β2 increased the expression of alpha‐smooth muscle actin (α‐SMA) and decreased the expression of zona occludens protein (ZO‐1). Transforming growth factor‐(β2) also caused the release of platelet‐derived growth factor (PDGF), connective tissue growth factor (CTGF) and type 1 collagen and increased the phosphorylation of SMAD2 and SMAD3. Platelet‐derived growth factor and CTGF stimulated cell migration, and TGF‐β2 stimulated wound closure, contraction of collagen and changes in cell morphology. Conclusions: Treatment with GlcN counteracted all of these effects, and its administration in the mouse model reduced the morphologic appearance of PVR. Glucosamine could inhibit the TGF‐β signalling pathway in retinal pigment epithelium cells and several of the downstream events associated with epithelial–mesenchymal transition and PVR.     

Capacity of ocular infiltrating T helper type 1 cells of patients with non-infectious uveitis to produce chemokines

BACKGROUND/AIMS: Chemokines are key molecules that initiate leucocyte infiltration to the inflammatory site. The involvement of chemokines in uveitis is well studied, yet the source of this molecule in the inflamed eye is not clearly identified. The possible sources of chemokines are ocular resident cells or the inflammatory cells infiltrated to the eye. Here the authors examined whether ocular infiltrating T cells of uveitis patients do produce chemokines. METHODS: T cell clones (TCCs) were established from ocular infiltrating cells of patients with non-infectious uveitis. TCCs were characterised using flow cytometry. Spontaneous production of chemokines by TCCs was evaluated by ELISA. RESULTS: TCCs from ocular infiltrating cells were revealed to be memory activated Th1 type CD4 positive cells. Those TCCs produced larger amounts of chemokines than TCCs from peripheral blood mononuclear cells of uveitis or healthy donors. CONCLUSIONS: The present data indicate that ocular infiltrating T cells of patients with non-infectious uveitis produce chemokines and recruit further infiltrating lymphoid cells. Such T cells may have roles in the prolonged/chronic state of non-infectious uveitis.     

Combined Low Dose Cyclosporine and Prednisone Down-Regulate Natural Killer Cell-Like Effector Functions of CD8brightCD56+ T Cells in Patients with Active Behçet Uveitis

Purpose: To investigate the changes of effector-related phenotypic markers and the natural killer (NK)-like effector functions of CD8^brightCD56+ T cells in patients with Behçet uveitis after combined cyclosporine and prednisone (Cs/Pd) treatment. Methods: Ten patients with active Behçet panuveitis and 10 healthy controls were prospectively recruited in this study. The effector-related surface markers (CD27, CD62L, CD11b, HLA-DR, CD94, NKG2D), chemokine receptors (CXCR1, CXCR3, CCR4, CCR5), and intracellular perforin of circulating CD8^brightCD56+ T cells were determined by flow cytometric analysis before and after two months' treatment. NK-like cytotoxicity of ex vivo CD8^brightCD56+ T cells against K562 was measured by standard ⁵¹Cr release assay. Results: The expression levels of effector-related molecules on CD8^brightCD56+ T cells normalized after treatment. The expression levels of CXCR1 and CCR5 were down-regulated on CD8^brightCD56+ T cells after treatment. The amounts of preformed intracellular perforin of CD8^brightCD56+ T cells were reduced to the normal levels. Furthermore, the NK-like cytolytic capacities of CD8^brightCD56+ T cells were decreased after treatment. Conclusions: Our results suggest that the combined Cs/Pd treatments in active Behçet uveitis may downregulate the NK-like effector functions of CD8^brightCD56+ T cells.