Systemic CD4(+) T cell phenotype and activation status in intermediate uveitis

AIM: To investigate peripheral blood lymphocyte phenotype in patients with intermediate uveitis using CD69, chemokine receptor, and cytokine expression. METHODS: Peripheral blood lymphocytes of 18 patients with idiopathic intermediate uveitis and 6 patients with presumed sarcoid intermediate uveitis were evaluated for CD4(+) T cell expression of CD69, CCR4, CCR5, CXCR3 and the intracellular cytokines IFNgamma, TNFalpha, and interleukin (IL)-10 by flow cytometry, and for IL-2, IL-4, IL-5, IL-10, IFNgamma, and TNFalpha production following unstimulated and activated culture using cytokine bead array and compared with healthy control subjects. RESULTS: The expression of CD69 and TNFalpha by peripheral blood CD4(+) lymphocytes of patients with idiopathic intermediate uveitis and presumed sarcoid intermediate uveitis was significantly higher than control subjects (p = 0.002 and p<0.05, respectively). The ratios of the concentrations of IL-2:IL-5 and IFNgamma:IL-5 in supernatants of activated peripheral blood lymphocyte cultures were significantly higher in patients with presumed sarcoid intermediate uveitis than control subjects. CONCLUSIONS: This study implicates TNFalpha in the pathogenesis of intermediate uveitis, highlighting the potential role of anti-TNF treatments for this disease. Studies of Th1:Th2 cytokine ratios suggested polarisation of the immune response towards Th1 in presumed sarcoid intermediate uveitis despite clinically quiescent systemic disease.     

Abnormal cellular reactivity to microbial antigens in patients with uveitis

PURPOSE: The purpose of the present study was to evaluate the cellular response to microbial antigens in patients with idiopathic uveitis. METHODS: Blood lymphocytes from 31 patients with uveitis and 24 healthy controls were cultivated with microbial antigens and analyzed by flow cytometry after staining with monoclonal antibodies against CD3, CD4, and activation markers CD69 and CD25. RESULTS: Although no difference was noted in circulating lymphocytes, the activation of T cells, detected with CD69, was higher in 24-hour blood culture from uveitis patients with Candida albicans antigen (Ca-Ag) than from controls, especially in posterior uveitis and panuveitis. Moreover, late response, detected with CD25, to different microbial antigens was higher in patient with uveitis. CONCLUSIONS: Such results suggest the role of Ca-Ag and microbial antigens in the pathogenic mechanisms of idiopathic uveitis.     

Disturbed expression of Fas/FasL on CD4+ and CD8+T cells in Behcet's disease,Vogt-Koyanagi-Harada syndrome,and idiopathic anterior uveitis

Aims: To evaluate the expression of Fas/FasL antigen on peripheral blood T lymphocytes in patients with Behcet's disease, Vogt-Koyanagi-Harada (VKH) syndrome, and idiopathic anterior uveitis. Methods: The expression of Fas and FasL on peripheral blood T lymphocytes was determined using flow cytometry in 26 patients with Behcet's disease (BD), 17 patients with VKH syndrome, 25 patients with idiopathic anterior uveitis, and 43 healthy individuals (controls). Results: A higher proportion of CD4 + T cells expressing Fas was noted in patients with Behcet's disease (25.70 ± 7.32%), VKH syndrome (19.60 ± 11.02%), and idiopathic anterior uveitis (20.81 ± 7.40%) compared with controls (14.02 ± 6.30%). The expression of Fas on CD8 + cells from patients with Behcet's disease (9.47 ± 6.97%) and VKH syndrome (6.84 ± 5.5%) was also higher than that seen in controls (3.47 ± 2.75%). There was no difference in FasL expression on T cells between patients and controls except that a lower expression of FasL on CD8+ T cells was noted in patients with idiopathic anterior uveitis. Conclusion: A disturbed expression of Fas and FasL on T cells is present in patients with Behcet's disease, VKH syndrome, and idiopathic anterior uveitis, which may be involved in the perpetuation and recurrence of uveitis.     

葡萄膜炎患者外周血CD4+CD25high调节性T细胞的表达

目的:通过检测葡萄膜炎患者外周血中CD4+CD25high调节性T细胞的表达水平,初步探讨其在葡萄膜炎发病中的作用。方法:取16例正常健康对照及30例不同类型活动期葡萄膜炎患者,采取抗凝外周血,进行三色免疫荧光染色、流式分析。结果:葡萄膜炎患者外周血中CD4+CD25highT细胞的百分率与正常健康对照(8.56%±4.22%vs5.72%±3.11%)相比,显著升高(P=0.022),而且与患者病情严重程度明显相关(P=0.007)。另外CD4+CD25highCTLA4+T细胞在葡萄膜炎患者外周血中比例较正常对照明显升高(P=0.012)。结论:葡萄膜炎患者外周血中CD4+CD25highTreg细胞的异常表达可能参与疾病的发生发展,并与疾病的活动密切相关。     

Selected markers of T cell activation in children with idiopathic uveitis

The aim of our investigation was to assess T cell activation markers (CD69, HLA-DR, CD25, CD49a) in idiopathic uveitis. T cells from children aged 9-17 with idiopathic uveitis were studied. Monoclonal antibodies against chosen markers of T cell activation were used. Expression of the CD69, HLA-DR, CD25, CD49a were determined by flow-cytometry. HLA-DR and CD49a--marker of the late activation--were significantly increased.     

葡萄膜炎患者外周血CD4^＋CD25^high调节性T细胞的表达

目的：通过检测葡萄膜炎患者外周血中CD4^＋CD25^high调节性T细胞的表达水平,初步探讨其在葡萄膜炎发病中的作用。 方法：取16例正常健康对照及30例不同类型活动期葡萄膜炎患者,采取抗凝外周血,进行三色免疫荧光染色、流式分析。 结果：葡萄膜炎患者外周血中CD4^＋CD25^highT细胞的百分率与正常健康对照（8.56%±4.22%vs5.72%±3.11%）相比,显著升高（P=0.022）,而且与患者病情严重程度明显相关（P=0.007）。另外CD4^＋CD25^highCTLA4＋T细胞在葡萄膜炎患者外周血中比例较正常对照明显升高（P=0.012）。 结论：葡萄膜炎患者外周血中CD4^＋CD25^highTreg细胞的异常表达可能参与疾病的发生发展,并与疾病的活动密切相关。     

Selected parameters of cellular immunity in uveitis in children

Evaluation of lymphocyte in children with uveitis basing on the study of T lymphocyte subpopulation in the peripheral blood using the flow cytometry method is presented. Cytometric analysis of peripheral blood lymphocytes using fluorochrome-marked monoclonal antibodies was carried out in 29 children, aged 6-18, with parasitic uveitis (group A) and with uveitis of another aetiology (group B). Control group consisted of 28 healthy children aged 6-15. The absolute values of T lymphocytes were higher in groups A (x = 1.60 +/- 0.53) and B (x = 1.81 +/- 0.40) compared to controls (x = 1.50 +/- 0.38). The lowest percentage of CD4+ lymphocytes (x = 34.25 +/- 6.06) and the highest percentage of CD8+ lymphocytes (x = 31.14 +/- 6.50) were revealed in children with parasitic uveitis. The lowest index CD4+/CD8+ (x = 1.29 +/- 0.53) in the group of children with parasitic uveitis reflects severe disorders in response regulation. However, the percentage values of NK cells were lower in the two groups of patients compared to controls. The results allow for the assumption that quantitative changes in the subpopulation of peripheral blood lymphocytes are more pronounced in parasitic uveitis.     

Chemokine receptors and early activation markers in acute anterior uveitis

PURPOSE: To study the peripheral expression of chemokine receptors and early activation markers in acute anterior uveitis (AU). METHODS: The expression of chemokine receptors CCR1, CCR2, CCR3, CCR5 and CXCR3, and activation markers CD25 (IL2R-alpha-chain), CD122 (IL2R-beta-chain) and CD69 were studied on peripheral blood mononuclear cells using flow cytometry in 10 patients with acute anterior uveitis and in 10 healthy controls. RESULTS: We found a highly significant up-regulation of CCR5 (p < 0.001) on CD4+ T-cells in the blood of patients with AU compared to in healthy controls. CD69 was significantly higher on both CD4+ and CD8+ T-cells (p < 0.05 and p < 0.001, respectively). We also found a significantly higher expression of CD25 expressing CD4+ T-cells in patients with anterior uveitis (p < 0.05). This expression was directly correlated to the severity of disease (rho = 0.720, p < 0.05). CONCLUSION: Our data show that patients with acute anterior uveitis have a specific systemic immune activation.     

Increased CD4+ expression and decreased IL-10 in the anterior chamber in idiopathic uveitis.

PURPOSE: To compare cell types and cytokines in aqueous humor from patients with uveitis either occurring in association with a systemic disease or apparently isolated and not associated with a systemic disease. METHODS: Cells were collected by centrifugation of fresh aqueous humor from uveitis and controls, and immunofluorescence techniques were performed with markers for T cells, B cells, and monocytes. Cytokines were measured in the aqueous supernatants, and serum samples were assayed for soluble interleukin-2 receptors. RESULTS: When aqueous samples from idiopathic uveitis were compared with those from uveitis associated with a systemic disease, there were increases in CD3+, CD4+ (p = 0.001), and activated CD4+ T cells (p = 0.02) and a decrease in B cells (p = 0.0013). This was not reflected in the peripheral blood where there were no differences in the cell types or in soluble interleukin-2 receptor levels. No cells were obtainable from control aqueous. Interleukins-10 and -12, interferon-gamma, and transforming growth factor-beta2 were detected in aqueous supernatants. Interleukin-10 was reduced (p = 0.024) in uveitis in comparison with controls. CONCLUSIONS: The results suggest a selective recruitment of CD4+ T cells within aqueous humor but only in idiopathic uveitis. In both disease groups there was a decrease in the immunoregulatory cytokine interleukin-10, which might enable an immune response to occur in an otherwise highly immunosuppressive microenvironment. Increases in activated CD4+ T cells combined with depressed interleukin-10 levels could partially explain why, for example, in acute anterior uveitis, the inflammatory disease is often more severe.     

CD69 expression on peripheral CD4+ T cells parallels disease activity and is reduced by mycophenolate mofetil therapy in uveitis

PURPOSE. To assess the effects of mycophenolate mofetil (MMF) therapy on T helper cell activation status, using CD69 expression and cytokine profile with flow cytometry in relation to clinical activity in uveitis. METHODS. Patients with posterior or intermediate uveitis treated with MMF (n = 10), patients with active uveitis not treated with MMF and receiving no or minimal therapy (n = 10), and healthy volunteers (n = 21) had peripheral blood lymphocyte immunofluorescence analysis for T helper cell (CD4, CD3) markers, activation status (CD69), and intracellular cytokine (interleukin [IL]-2, interferon [IFN]-gamma, and IL-4) levels. Patients were compared before and during MMF therapy in relation to T helper cell activation and clinical activity. RESULTS. Patients with active uveitis not treated with MMF and receiving no or minimal therapy had increased frequency of CD69-positive CD4 T cells (10.5% +/- 4.6%, P = 0.0007) compared with healthy volunteers (3.3% +/- 2.7%). Of all patients receiving MMF therapy, only patients with moderate to severe uveitis activity in the pre-MMF treatment group (n = 5; 15.5% +/- 5.0%, P = 0.004) had increased frequency of CD69-positive CD4 T cells compared with healthy volunteers. During MMF therapy, a significant reduction in frequency of CD69-positive CD4 T cells occurred in patients with prior moderate to severe uveitis activity (to 8.9% +/- 3.8%, P = 0.04). Levels of CD69-positive CD4 T cells in patients who had had inactive or mildly active disease (n = 5) before and during MMF therapy were comparable with levels in healthy volunteers. No significant changes in cytokine levels were found between the patient and control groups. A significant association between changes in frequency of CD69-positive CD4 T cells and changes in visual acuity (P = 0.008) and changes in vitreal haze (binocular indirect ophthalmoscopy score; P = 0.01) was observed in MMF-treated patients with prior moderate to severe uveitis activity. CONCLUSIONS. Reduction in uveitis activity during MMF therapy correlates with reduction in frequency of peripheral blood CD69-positive CD4 cells. The frequency of CD69-positive CD4 T cells is a measure of activity in posterior uveitis and may guide adequate immunosuppression.     

Activated T lymphocytes in uveitis.

Two colour flow cytometry techniques were used to assess the activation stages of peripheral and intraocular T lymphocytes in uveitis. Increased numbers of T lymphocytes bearing the interleukin-2 (IL-2) receptors were found in intraocular fluids or peripheral blood or both of 35/51 patients with uveitis. This increased expression of IL-2 receptors on lymphocytes correlated with increased expression of other early T lymphocyte activation markers, HLA-DR and L-35. Both T helper cells (Leu-3A+) and suppressor cells (Leu 2A+) were activated in vivo. A positive correlation was seen between lymphocyte activation and clinical uveitis activity. In idiopathic uveitis activation of Leu-3A lymphocytes (helper/inducer) was significantly increased, and intraocular activation of the Leu-2A lymphocytes (cytotoxic/suppressor) was significantly decreased. These data show that some patients with idiopathic uveitis have a perturbation of T helper cells. Twenty-two of 31 patients with idiopathic uveitis, not associated with systemic disease, had increased peripheral T lymphocyte activation. This finding indicates that in some inflammations believed to be restricted to the eye an abnormal systemic immune activation exists.     

人类永生化角膜内皮细胞的生物学特性

目的探讨人类永生化角膜内皮细胞在免疫反应中的分子表达及其刺激T淋巴细胞活化的能力。方法人类永生化角膜内皮细胞分别培养在含干扰素-γ（IFN-γ）（1000U／mL）和不包含IFN-γ（1000U／mL）的F99和M199培养液中,按照1：1等比例混合并含有5％小牛血清。健康志愿者采集外周血并分离外周血单核细胞（PBMCs）,应用锥虫蓝染色鉴定细胞的完整性和细胞数量。免疫组织化学染色法测定人类永生化角膜内皮细胞分别在有无IFN-γ诱导条件下的HLA—DP、DQ、DR抗原及CD40的表达。同时,体外共同培养人类永生化角膜内皮细胞和PBMCs,荧光活化细胞分类（FACS）法测定活化的T淋巴细胞中CD69的表达。结果IFN-γ能够诱导HLA—DP、DQ、DR抗原和CD40在人类永生化角膜内皮细胞上的阳性表达,表现为细胞膜和细胞质中的红色染色。CD3和CD28抗体预处理的外周血单核细胞中,20％的CD69阳性T淋巴细胞活化,无IFN-γ诱导时有10％阳性T淋巴细胞,而IFN-γ诱导组中,有12％的阳性T淋巴细胞,说明共同培养人类永生化角膜内皮细胞和PBMCs体系中,CD69阳性淋巴细胞数量增加。结论外周血中的T淋巴细胞可以被人类永生化角膜内皮细胞活化,由此人类永生化角膜内皮细胞是具有免疫学特性潜能的抗原递呈细胞。     

ICAM-1 expression on the surface of T lymphocytes in patients with uveitis: a comparative study between the eye and peripheral blood

PURPOSE: We studied the surface expression of intracellular adhesion molecule 1 (ICAM-1) on peripheral and intra-ocular T lymphocytes in patients with active uveitis. METHODS: Two-colour flow-cytometric analysis was performed on cells isolated from aqueous humour and peripheral blood of 23 patients with active uveitis and 16 control patients who were to undergo cataract extraction, in order to determine the percentage of cells expressing CD4, CD8 and ICAM-1 (CD54) molecules. RESULTS: In the aqueous humour of patients with uveitis, we found an increase in the percentage of CD4+ and CD8+ lymphocytes, co-expressing the ICAM-1 molecule as compared to control patients (p < 0.0001). In peripheral blood, these uveitis patients exhibited a significant decrease in the percentage of CD4+ICAM-1+ (p = 0.0106) and CD8+ICAM-1+ (p = 0.0014) as compared to control subjects. The comparative study of cells from the aqueous humour and peripheral blood showed that the percentage of CD4+ICAM-1+ cells was higher in the aqueous humour (p < 0.0001). Comparison of the aqueous humour and peripheral blood for CD8+ICAM-1+ cells revealed no significant differences. In addition, we found a high negative correlation for the CD8+ICAM-1+ subset between the aqueous humour and peripheral blood. CONCLUSIONS: Our results suggest a greater local participation for CD4+ICAM-1+ cells but not for CD8+ICAM-1+ cells in the pathogenesis of uveitis.     

异体角膜对人外周血T细胞及其亚群CD69分子表达的体外调节

目的 观察异体角膜体外对人外周血T细胞及其亚群CD69分子表达的调节。方法 应用异体角膜与外周血淋巴细胞体外共同培养和流式细胞分析技术(FACS)。结果 对照组T细胞CD69表达为10．3％；受角膜或佛波醇脂(PDB)激活后，T细胞CD69表达分别为31.6％和53.8％；受角膜激活后，CD4和CD8T淋巴细胞CD69表达分别为47.3％和30．3％。结论 角膜组织体外能够刺激人外周血T细胞CD69的表达和T细胞活化，早期以CD4T细胞活化更明显。     

Increased Circulating Proinflammatory T Lymphocytes in Children with Different Forms of Anterior Uveitis: Results from a Pilot Study

Purpose: To characterize peripheral blood T cells in juvenile idiopathic arthritis-associated uveitis (JIAU).

Methods: Blood samples were taken from children with JIAU (n = 18), JIA without ocular involvement (n = 11), idiopathic anterior uveitis (IAU, n = 12), and healthy controls (n = 11). Cells were stained for T cell surface markers, and intracellular cytokine staining was performed after cell stimulation and analyzed by flow cytometry.

Results: The Th1/Th2 ratio was increased in JIAU patients. Numbers of IL-13-expressing cells an level of IL-13 and IL-10 expression per cell were increased in all patient groups; whereas, percentages of IL-5-expressing T cells were decreased. Numbers of proinflammatory Th17 cells and T cells expressing CTLA-4 were increased in all patient groups; whereas, γ/δ T cell numbers were decreased. Results from JIA and IAU were similar.

Conclusion: T cell subtypes and potential T cell function are altered in pediatric patients with uveitis and arthritis as compared to healthy children.     

Behcet病患者外周血淋巴细胞共刺激分子的表达

目的观察Behcet病患者外周血淋巴细胞CD80(B7-1)、CD86(B7-2)、CD28、CTLA-4分子的表达。方法采用流式细胞三色直接免疫荧光标记法，对24例Behcet病患者(Behcet病组)和20个正常人(对照组)外周血T、B淋巴细胞亚群的共刺激分子CD80、CD86、CD28和CTLA-4的表达进行检测。结果Behcet病患者外周血CD ⁴⁺ T细胞表面 CTLA-4 分子的表达[(3.18±1.18)%]高于对照组[(1.73±0.66) %]，两者差异有显著性的意义(t=-3.722,P<0.01)；CD19⁺ B细胞表面CD86分子的表达[(4.49±1.73)%]高于对照组[(2.40±1.49) %]，两组比较差异有显著性的意义(t=－2.071,P<0.05)。比较两组间其它分子的表达，差异均无显著性的意义。结论Behcet病患者 B7、 CD28的相互作用促进了葡萄膜炎的发生，阻断其相互作用可能为今后葡萄膜炎的治疗提供一种新思路。(中华眼底病杂志,2003,19:357-359)     

CD56+ T cells in the peripheral blood of uveitis patients

AIMS—Natural killer T (NKT) cells, T lymphocytes expressing both T cell and NK cell markers, are suggested to be involved in autoimmune diseases. To examine the relation between the pathogenesis of uveitis and CD56+ T cells, which are thought to be a type of human NKT cells, we investigated peripheral CD56+ T cells in uveitis patients.METHODS—41 uveitis patients (Behçet''s disease (BD), 14; sarcoidosis (SAR), eight; Vogt-Koyanagi-Harada disease (VKH), five; idiopathic uveitis (IU), nine; and others, five) and 19 healthy controls participated in this study. Cell surface antigens of lymphocytes were analysed by use of monoclonal antibodies and flow cytometry.RESULTS—The proportion of CD56+ T cells in patients with BD was higher than in controls and in patients with SAR, VKH, IU, and others.CONCLUSION—Increased peripheral CD56+ T cells might be relevant to the pathogenesis of uveitis in BD, and increase of peripheral CD56+ T cells may be one of the laboratory findings to suggest that uveitis originates from BD.     

S antigen specific effector T cell activation detected by cytokine flow cytometry

BACKGROUND/AIMS: Effector T cell activation is particularly important in the initiation of autoimmune uveitis. This pilot study seeks to demonstrate activation of human peripheral effector T cells in response to the uveitis candidate autoantigen, retinal S antigen (SAg), using cytokine flow cytometry (CFC). METHODS: Peripheral blood mononuclear cell (PBMC) suspensions from uveitis patients and controls were stimulated with bovine SAg. Activation responses were detected by CFC. RESULTS: Electronic gating enabled analysis of CD69+, IFN-gamma+ CD4+ lymphocytes. An SAg specific response was detectable in four of 13 patients and four of eight controls. CONCLUSION: SAg specific, peripheral, effector T cell activation can be detected by CFC. Similar levels of responsiveness were seen in patient and control groups. More detailed cytokine profiling may demonstrate functional differences between the groups.     

Interferon-alpha: a key factor in autoimmune disease?

PURPOSE: Interferon (IFN)-alpha is an effective drug for treatment of uveitis in Beh?et's disease. This study was undertaken to investigate the mechanism of action of IFN-alpha in the treatment of various types of noninfectious sight-threatening uveitis. METHODS: Eleven patients with refractory uveitis, and 13 healthy individuals were enrolled. The number of circulating plasmacytoid dendritic cells (pDCs) and their capacity to produce IFN-alpha in culture on stimulation with synthetic oligodinucleotides containing the CpG-motif were studied. Peripheral blood CD4+ T-cell phenotype and activation status were evaluated by flow cytometry at 0, 2, and 8 weeks after treatment for expression of CD69, CD62L, chemokine receptors (CCR4, CXCR3, and CCR5), and intracellular cytokines (TNF-alpha, IFN-gamma, and IL-10). RESULTS: All patients experienced a positive clinical response to IFN-alpha treatment. There was no significant difference between patients and control subjects in the number of circulating pDCs, but there was a significant decrease in the capability of patients' pDCs to produce IFN-alpha in response to CpG (P < 0.001). Peripheral blood CD4+ T cells expressed reduced levels of surface CD62L (P < 0.005) as a measure of activation and higher levels of chemokine receptors CXCR3, CCR4, and CCR5 (P < 0.005, P < 0.05, and P < 0.05, respectively); in addition, intracellular T-cell IL-10 levels were increased once the treatment was initiated (P < 0.01). CONCLUSIONS: The data suggest that IFN-alpha may control uveitis by promoting induction of IL-10-producing T-cells, possibly T-regulatory cells. Dysregulation of the T-cell population in patients with uveitis may be associated with a defect in the pDCs' ability to produce IFN-alpha, which can be circumvented with administration of exogenous IFN-alpha.     

Th1 polarization of the immune response in uveitis in Behçet's disease

BACKGROUND: It has been reported that abnormalities in the balance of T-helper cells type 1/2 (Th1/Th2) may account for the pathophysiology of human autoimmune diseases. The purpose of this study was to define the role of the Th1/Th2 balance in the pathogenesis of uveitis in Beh?et's disease (BD). METHODS: From February 2003 to August 2005, we studied 31 patients with active BD. Of these patients, 21 (12 female, 9 male; mean age 35.5 [SD 10] years) presented with acute uveitis, and 10 (7 female, 3 male; mean age 34 [SD 11] years) presented with inflammatory arthritis but no prior uveitis attack. The control group consisted of 10 (7 female, 3 male; mean age 34.7 [SD 8] years) age-matched, healthy individuals. CD4+ CD26+ and CD4+ CD30+ cell surface expression of the peripheral blood CD4+ T lymphocytes was evaluated by analytic flow cytometry in order to determine percentages of Th1 and Th2 lymphocyte subsets. RESULTS: The mean percentage of CD4+ CD26+ and CD4+ CD30+ cells was 26.27 (SD 6.18) % and 2.56 (SD 0.82) %, 17.42 (SD 5.90) % and 2.86 (SD 0.72) %, and 14.99 (SD 3.96) % and 3.11 (SD 1.25) % in BD with active uveitis, BD with inflammatory arthritis but no prior uveitis attack, and control groups, respectively. T-helper 1 (Th1) cell percentage was significantly higher in the BD with active uveitis group than the BD with arthritis and no prior uveitis attack group (p = 0.001). With respect to the percentage of CD30+ Th2 cells, there was no statistical difference between the 2 BD groups (p = 0.529) or among the 3 groups (p = 0.375). INTERPRETATION: Th1 lymphocyte dominance in peripheral circulating blood may play a role in the pathogenesis of BD uveitis.