Th1/Th2细胞因子在小鼠真菌性角膜炎中的表达

目的 通过检测Th1/Th2细胞因子在小鼠真菌性角膜炎中的表达水平,探讨机体免疫在该病发展中的作用. 方法应用角膜表层镜法建立Balb/c小鼠茄病镰刀菌性角膜炎模型,在感染后第1、3、5、7天,裂隙灯显微镜观察角膜炎特点;HE染色观察角膜病理变化;半定量RT-PCR和ELISA检测角膜中Thl细胞因子(IFN-γL-12)及Th2细胞因子(IL-4、IL-10)基因mRNA和蛋白的表达,半定量RT-PCR检测T-bet基因mRNA的表达;直线相关分析检测T-bet mRNA与IFN-比值的相关性. 结果角膜接种菌液后,早期角膜浸润混浊进行性加重,第5天后新生血管大量生长;HE染色可见第1、3天在角膜缘、角膜基质及前房中有大量的炎症细胞浸润,第5天后炎症细胞减少,角膜基质中纤维细胞逐渐增多;RT-PCR与ELISA检测结果表明,Thl细胞因子(IFN-γL-12)和Th2细胞因子(IL-4、IL-10)基因mRNA及蛋白在角膜接种菌液后均出现表达,且IFN-γL-12的表达显著强于IL-4、IL-10;IFN-γ/IL-4比值在第3天达最高值,而后逐渐降低;T-bet mRNA在第3天达最高值;T-bet mRNA的表达与IFN-γ/IL-4比值呈正相关(P＜0.05). 结论在真菌性角膜炎中,Thl/Th2型免疫应答共同参与调节机体的抗真菌免疫,但以Th1型应答为主;角膜感染真菌后第3天机体的免疫力达最强.     

甲状腺相关眼病患者血清中Th1 Th2型细胞因子平衡偏离的研究

目的 探讨血清中Th1、Th2型细胞因子平衡偏离与甲状腺相关眼病(thyroid-associated ophthalmopathy,TAO)发病、活动性及大剂量糖皮质激素治疗反应的关系.方法 选取TAO患者52例,按CAS评分标准将其分为活动期29例(CAS≥4)和非活动期23例(CAS＜4).其中活动期患者中有22例给予大剂量糖皮质激素治疗;另外选取初诊Graves病患者15例及正常对照15例,收集上述人静脉血,行激素冲击治疗的患者于冲击前后各抽血1次.采用放射免疫法检测血清中IL-4浓度,ELISA法检测IFN γ浓度.比较活动期与非活动期TAO组、GD组以及正常对照组血清上述指标的变化;评价糖皮质激素治疗对上述指标的影响.结果 ①TAO及GD患者血清中IL-4、IFNγ浓度均较对照组明显增高.②活动期TAO与非活动期TAO相比血清中IL-4、IFN7浓度无明显统计学差异,但活动期IL-4/IFN γ比值较非活动期降低,有统计学差异.③22例激素冲击治疗患者中15例有效(CAS减少≥2),7例无效(CAS减少＜2),有效组治疗后血清中IFN γ浓度明显较治疗前降低,IL-4/IFNγ比值增高,而无效组未见明显变化.结论 Th1/Th2在一定程度可以作为TAO活动性判断指标之一,活动期TAO Th2/Th1比值下降,非活动期无明显变化.在一定程度还可以作为大剂量糖皮质激素治疗疗效的判断指标之一,有效者Th2/Th1比值上升,向Th2偏移,无效者治疗前后无明显改变.     

Th1、Th2和Th17型细胞因子水平与糖尿病视网膜病变的相关性研究进展

糖尿病视网膜病变(diabetic retinopathy,DR)是目前主要的致盲眼病,其发病机制复杂且尚未明确,近年发现免疫炎症反应和细胞因子在其发病中起重要作用。Th1、Th2、Th17型细胞因子在DR中分泌异常,作用并影响了疾病的发生发展。本文就Th1、Th2、Th17型细胞因子与DR的相关性研究进展作一综述。     

TRPV1 Antagonist Suppresses Allergic Conjunctivitis in a Murine Model

Purpose: To determine the immunologic functions of TRPA1 or TRPV1 in allergic conjunctivitis (AC).

Methods: Mice were sensitized with ovalbumin (OVA), after which TRPA1 antagonist or TRPV1 antagonist was administered before topical OVA challenge. Expression of TRPV1 or TRPA1 in AC was examined by western blotting and multicolor immunofluorescence. Clinical signs, OVA-specific IgE, infiltration of inflammatory cells into conjunctivae (CJs), and Th2 cytokine in draining lymph nodes (LNs) were evaluated by microscopy, flow cytometry, and ELISA.

Results: TRPV1 expression was increased in CJs and LNs from AC mice, but TRPA1 expression was only increased in LNs. TRPV1 antagonist but not TRPA1 antagonist attenuated the clinical signs of AC and OVA-specific IgE in sera. TRPV1 antagonist furthermore inhibited the infiltration of inflammatory cells into CJ and the production of Th2 cytokines in LNs.

Conclusion: TRPV1 antagonist but not TRPA1 antagonist may ameliorate AC by suppressing the Th2 response in LNs.     

糖皮质激素对葡萄膜炎患者外周血Th1和Th2细胞因子的影响

目的探讨糖皮质激素治疗前后葡萄膜炎患者外周血单核细胞（PBMC）培养上清液Th1/Th2细胞因子的变化。方法应用酶联免疫吸附试验检测治疗前后葡萄膜炎患者PBMC上清液干扰素（IFN-γ）、白介素（IL）-2、IL-4和IL-10水平的变化,30例健康人作为正常对照。结果急性葡萄膜炎患者PBMC上清液IFN-γ、IL-2、IL-4和IL-10水平与IFN-γ/IL-4、IFN-γ/IL-10比值明显高于恢复期患者和正常对照者（P=0.000）。糖皮质激素治疗后,IFN-γ和IL-2水平、IFN-γ/IL-4和IFN-γ/IL-10比值降低（P=0.000）,IL-10水平升高（P=0.000）。治疗前不同类型葡萄膜炎组仅IFN-γ和IL-10水平差异有统计学意义（P〈0.01）,治疗后IL-2、IL-4、IL-10和IFN-γ/IL-10比值差异有统计学意义（P〈0.05）。治疗前后前葡萄膜炎和Vogt-小柳原田病患者除IL-4外,其余细胞因子及比值差异均有统计学意义（P〈0.05）;Behcet病患者除IL-4和IL-10外,其余细胞因子及比值差异均有统计学意义（P〈0.01）。治疗前后葡萄膜炎患者IFN-γ与IL-2水平间及IL-4与IL-10水平间呈正相关（P=0.000）。结论急性葡萄膜炎患者PBMC中Th1细胞因子显著升高并占优势,表明Th1/Th2细胞因子不平衡性与葡萄膜炎的发病有关。     

Demographic aspects of allergic ocular diseases and evaluation of new criteria for clinical assessment of ocular allergy

Background The clinical features of allergic ocular diseases (AOD) are characterized by their wide variety. Clinical evaluation criteria are essential not only for objective assessment in clinical trials, but also for clinical studies on etiological aspects of AOD. However, there have been no internationally established criteria for clinical evaluation and classification of the severity of AOD. We established new criteria and applied them to many cases of AOD, and evaluated their usefulness for clinical purposes. We also studied whether each clinical entity of AOD [allergic conjunctivitis (AC), atopic keratoconjunctivitis (AKC), and vernal keratoconjunctivitis (VKC)] can be distinguished by these criteria. Methods A prospective study was carried out to assess the differential diagnosis of 1079 patients with AOD (439 male and 640 female patients). Differential diagnosis of AC, AKC or VKC was made. These diseases were diagnosed and classified based on local and systemic clinical findings. Ten objective ocular clinical findings of conjunctival, limbal and corneal lesions were graded on a 4-point scale, and the total score, with a highest value of 30, was used as the clinical score. Results Among a total of 1079 cases, seasonal allergic conjunctivitis (SAC), perennial allergic conjunctivitis (PAC), AKC and VKC accounted for 876 (81.2%), 115 (10.6%), 47 (4.4%) and 41 (3.8%) respectively. The mean age in each disease was 52.9, 56.1, 25.7 and 16.6 years respectively. Total clinical score in SAC, PAC, AKC and VKC was 1.54, 2.13, 3.72 and 12.68 respectively. Both mean age and total clinical score in all combinations of two diseases showed significantly different results. Conclusions These results suggest that AOD can be classified by our new clinical grading system, and this system is sensitive enough for clinical evaluation of AOD. Re-evaluation of AOD is essential for constructing future strategies for the treatment of AOD, which are consists of various categories of ocular disorders. The authors have no financial relationship with the organization that sponsored the research. The authors have full control of all primary data, and agree to allow Graefes Archive for Clinical and Experimental Ophthalmology to review their data upon request.     

甲状腺相关眼病与Th1/Th2免疫平衡

背景 甲状腺相关眼病(GO)是自身免疫性疾病,目前其发病机制尚不清楚,近年来的研究发现Th1/Th2型免疫平衡反应的失调是导致GO的主要原因. 目的 采用脂质体包裹的人促甲状腺激素受体(hTSHR)胞外段基因重组质粒免疫同系雌性BALB/c小鼠以建立GO动物模型,研究关于在GO小鼠模型血清Th 1/Th2型免疫反应平衡状态,探讨GO的病理机制.方法 采用计算机数字随机分配法将同系6～8周龄雌性BALB/c小鼠32只分为重组质粒注射组、空白对照组、空质粒注射组和脂质体注射组.重组质粒注射组分别于实验的0、3、6周采用脂质体包裹的重组质粒pcDNA3.1 +/hTSHR289各30 μg行双胫前肌内注射,再于腹腔内注射40 μg以免疫小鼠,空质粒注射组和脂质体注射组分别采用pcDNA3.1+和脂质体以同样的方法进行免疫,空白对照组不做任何处理.分别于注射前及各次注射后第4天及第17周末处死小鼠行心脏采血,采用ELISA法检查各组小鼠血清中白细胞介素(IL)-2及IL-4质量浓度,计算IL-2/IL-4值.于初次免疫后17周末处死重组质粒注射组小鼠,取甲状腺及眼眶组织制作病理切片,采用苏木精-伊红染色法检测标本组织中炎性细胞浸润情况,对有炎性细胞浸润的标本进行免疫组织化学检测,计算B细胞特异标志物CD20阳性细胞(B)与T细胞特异标志物CD3ε阳性细胞(T)的比值,B∶T＞1.0者为Th2反应,相反者为Th1反应.结果 在第2次和第3次注射后,重组质粒注射组小鼠血清中IL-2质量浓度明显低于空白对照组、脂质体注射组、空质粒注射组,差异均有统计学意义(均P＜0.05),但组间小鼠血清IL-4质量浓度差异无统计学意义(P＞0.05);重组质粒注射组小鼠在第2次、第3次免疫后血清IL-2质量浓度明显低于免疫前及第1次免疫后,差异均有统计学意义(注射前:P=0.009、0.019;第1次注射后:P=0.002、0.004),而处死时小鼠血清IL-2质量浓度有所回升,明显高于第3次免疫后IL-2质量浓度[(44.31±1.77) pg/ml与(42.43±2.29) pg/ml],差异有统计学意义(P=0.031).空白对照组、空质粒注射组、脂质体注射组、重组质粒注射组第2次、第3次注射后血清IL-4质量浓度及IL4/IL2值均明显高于注射前和第1次注射后,其处死时重组质粒注射组小鼠血清IL-4质量浓度明显高于第3次注射后,差异均有统计学意义(均P＜0.05).处死时重组质粒注射组苏木精-伊红染色有炎性细胞浸润的7只小鼠12个眼眶标本中4个眼眶B∶T值＞1.0. 结论 采用脂质体包裹的TSH受体胞外段基因重组质粒免疫同系雌性BALB/c小鼠可成功建立GO动物模型,重组质粒注射组小鼠在初次免疫后17周内以Th1型反应为主,小鼠第2次免疫后免疫平衡开始向Th2型转变.     

Th1、Th17细胞相关因子在实验性自身免疫性葡萄膜炎大鼠中的表达及作用

目的　探讨辅助性Ｔ细胞（Ｔｈｅｌｐｅｒｃｅｌｌｓ,Ｔｈ）１、Ｔｈ１７细胞相关因子在实验性自身免疫性葡萄膜炎（ｅｘｐｅｒｉｍｅｎｔａｌａｕｔｏｉｍｍｕｎｅｕｖｅｉｔｉｓ,ＥＡＵ）中的表达及作用。方法　取清洁级纯系健康雌性Ｌｅｗｉｓ大鼠４０只随机分为ＥＡＵ组（３２只）和对照组（８只）,ＥＡＵ组用光感受器间维生素Ａ类结合蛋白诱导大鼠ＥＡＵ模型,进行临床症状评分,免疫组织化学方法检测造模后视网膜内干扰素（ｉｎｆｅｒｆｅｒｏｎ,ＩＦＮ）-γ、诱导型一氧化氮合酶（ｉｎｄｕｃｉｂｌｅｎｉｔｒｉｃｏｘｉｄｅｓｙｎｔｈａｓｅ,ｉＮＯＳ）、白细胞介素（ｉｎｔｅｒｌｅｕｋｉｎｅ,ＩＬ）-１７、ＩＬ-６的表达。ＥＬＩＳＡ法对比分析房水中各细胞因子的变化情况。结果　ＥＡＵ模型建立成功;造模后第１４天,视网膜损害以外层为主,视网膜内有大量炎细胞浸润,从而导致视网膜内结构紊乱,同时在视网膜的视锥、视杆细胞层和神经节细胞层ｉＮＯＳ、ＩＬ-１７、ＩＬ-６、ＩＦＮ-γ表达,细胞阳性率分别为２９％、４８％、５２％、７３％。在ＥＡＵ发病过程中,ＩＬ-６于造模后第７天迅速升高,第１０天达到高峰;ＩＬ-１７于造模后第１４天达到最高值,变化趋势与炎症进程一致;ＩＦＮ-γ在炎症后期仍有升高,于造模后第１６天达到最高值;ＩＬ-６、ＩＬ-１７、ＩＦＮ-γ与对照组相比,各时间点表达差异均有统计学意义（均为Ｐ＜０．０５）。ｉＮＯＳ在炎症进程中表达有所增加,但与对照组相比,各时间点的表达差异均无统计学意义（均为Ｐ＞０．０５）。结论　Ｔｈ１、Ｔｈ１７细胞相关因子调节网络共同参与实验性自身免疫性葡萄膜炎的发生发展。     

The role of Th17 immunity in chronic ocular surface disorders

Th17 cells have been implicated in the pathogenesis of numerous inflammatory and autoimmune conditions. At the ocular surface, Th17 cells have been identified as key effector cells in chronic ocular surface disease. Evidence from murine studies indicates that following differentiation and expansion, Th17 cells migrate from the lymphoid tissues to the eye, where they release inflammatory cytokines including, but not limited to, their hallmark cytokine IL-17A. As the acute phase subsides, a population of long-lived memory Th17 cells persist, which predispose hosts both to chronic inflammation and severe exacerbations of disease; of great interest is the small subset of Th17/1 cells that secrete both IL-17A and IFN-γ in acute-on-chronic disease exacerbation. Over the past decade, substantial progress has been made in deciphering how Th17 cells interact with the immune and neuroimmune pathways that mediate chronic ocular surface disease. Here, we review (i) the evidence for Th17 immunity in chronic ocular surface disease, (ii) regulatory mechanisms that constrain the Th17 immune response, and (iii) novel therapeutic strategies targeting Th17 cells.     

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.     

M2型巨噬细胞极化在胸腺基质淋巴细胞生成素及其下游分子诱导过敏性结膜炎发病机制中作用的研究进展

过敏性结膜炎(AC)是人类眼表一种常见的过敏性疾病,发病原因复杂,主要是由免疫球蛋白E或T淋巴细胞介导的一种免疫反应。目前,过敏效应阶段细胞因子之间相互作用的分子机制已明确,但对过敏上游因子的了解却知之甚少。有研究结果表明,作为过敏上游因子的胸腺基质淋巴细胞生成素(TSLP)等具有启动和促过敏作用;此外,在短豚草花粉诱导实验性AC小鼠模型中已证实M2型巨噬细胞极化在辅助性T淋巴细胞2(Th2)显性过敏性炎症中有重要作用。目前,TSLP和M2型巨噬细胞在AC发病过程中的作用机制尚未完全阐明。因此,本文中笔者就M2型巨噬细胞极化在TSLP及其下游分子诱导AC发病机制中的作用进行综述。     

Toll样受体2调节Th细胞极化对小鼠角膜移植排斥反应发生的影响

目的 探究Toll样受体2(Toll like receptor 2,TLR2)是否通过调节辅助性T细胞(T helper cells,Th)极化影响角膜移植排斥反应的发生.方法 取30只C57BL/6小鼠和30只TLR2基因敲除小鼠为受体,BALB/c小鼠为供体,在受体小鼠右眼行同种异体角膜移植术,分别为野生组和基因敲除组;取19只C57BL/6小鼠右眼行自体角膜移植术,为自体组.术后每周两次裂隙灯下观察植片水肿程度和透明度,参照Sonoda评分标准对排斥指数(reject index,RI)进行评分;术后14 d收集角膜.分别取9只C57BL/6小鼠和TLR2基因敲除小鼠作为野生对照组和基因敲除对照组,行常规HE染色和实时荧光定量PCR检测.结果 术后随时间变化各手术组植片水肿和混浊程度不一,以野生组最为严重.角膜RI评分显示术后7d、14 d、21 d,基因敲除组(0.80 ±0.83、0.90±0.55、1.35±0.99)低于野生组(1.55 ±0.94、2.25 ±0.97、2.55±1.19),差异均有统计学意义(P=0.008、0.000、0.000).HE染色显示,野生组角膜基质层出现水肿和大量炎性细胞;而基因敲除组和自体组炎症反应较轻.PCR检测显示,基因敲除组角膜Th1和Th17细胞因子(IFN-γ和IL-17)mRNA表达(1.94±0.34、2.62±0.30)比野生组(4.27±0.37、3.99±0.40)低,差异均有统计学意义(P=0.000、0.001);Th2细胞因子(IL-4)三组差异无统计学意义(P＞0.05).结论 敲除TLR2可能通过抑制Th向Th1和Th17极化,降低角膜移植排斥率.     

龙胆泻肝汤抑制Notch信号通路活化对实验性自身免疫性葡萄膜炎大鼠Th1、Th2细胞分化的影响

目的　探讨龙胆泻肝汤（LXD）对Notch信号通路活化的抑制作用及其对实验性自身免疫性葡萄膜炎（EAU）大鼠Th1、Th2细胞分化的影响。方法　将30只Lewis大鼠随机分为正常对照组、EAU模型组和LXD干预组,其中EAU模型组、LXD干预组大鼠均诱导EAU,LXD干预组大鼠造模后使用LXD每天灌胃处理,EAU模型组和正常对照组大鼠给予等量生理盐水灌胃。干预后12 d分离三组大鼠的脾脏、淋巴结和眼组织,Q-PCR检测Rbpj基因的表达,ELISA检测Rbpj、γ干扰素（IFN-γ）和白细胞介素-4（IL-4）蛋白的表达,流式细胞仪检测各组织中Th1、Th2细胞的表达水平,分析Th1/Th2细胞比例的变化。结果　干预后12 d,Q-PCR检测发现,与正常对照组大鼠相比, Rbpj mRNA在EAU模型组大鼠脾脏、淋巴结和眼组织中均呈显著上调表达(均为P<0.001);与EAU模型组相比,LXD干预组大鼠脾脏、淋巴结和眼组织中Rbpj mRNA相对表达水平均显著降低(均为P<0.01)。ELISA检测结果发现,EAU模型组大鼠脾脏、淋巴结和眼组织中Rbpj和IFN-γ蛋白表达水平均明显高于正常对照组,IL-4蛋白表达水平均明显低于正常对照组（均为P＜0.05);相比于EAU模型组,LXD干预组大鼠脾脏、淋巴结以及眼组织中Rbpj IFN-γ蛋白表达水平均显著降低,IL-4蛋白表达水平均显著升高（均为P＜0.05)。流式细胞仪检测发现,与正常对照组相比,EAU模型组大鼠脾脏、淋巴结和眼组织中Th1细胞水平均明显升高,Th2细胞水平均明显降低,Th1/Th2细胞比例失衡（均为P＜0.05）;与EAU模型组相比,LXD干预组大鼠各组织中Th1细胞水平均明显下降,Th2细胞水平均明显升高,两者细胞比例逐渐恢复均衡（均为P<0.01)。结论　LXD可通过下调EAU大鼠Notch信号通路转录因子Rbpj的表达水平抑制Notch信号通路的活化,显著促进EAU大鼠中Th1/Th2细胞比例恢复平衡并改善免疫微环境,从而达到治疗葡萄膜炎的目的。     

青光眼与Th1、Th2相关细胞因子研究进展

青光眼是导致不可逆盲的首要原因,包括视野缺损和视神经的慢性退行性病变,如视网膜神经节细胞(RGCs)的凋亡和视神经轴突的逐步缺失.目前普遍认为高眼压是青光艰的主要危险因素,降低眼压是减缓青光眼发生和发展的首选治疗方法.近年来发现免疫因素是青光眼视神经损害的非压力依赖性危险因素之一.大部分免疫,甚至非免疫性生物效应都通过细胞因子来调控,而CD4+辅助性T细胞是细胞因子产生和调节的主要来源,其中Th1和Th2相关细胞因子在青光眼的发病机制中起着不可或缺的作用,并关系着RGCs的存活和凋亡.本文就近年Th1和Th2主要的相关细胞因子及Th1/Th2平衡与青光眼潜在关系的研究进展进行综述.     

树鼩茄病镰刀菌性角膜炎房水中Th1/Th2炎性因子的变化

目的 观察树鼩茄病镰刀菌性角膜炎房水中Th1/Th2炎性因子的变化,了解Th1/Th2炎性因子与本病炎症反应的关系.方法 茄病镰刀菌培养7d后收集真菌混悬液,调整孢子密度为10×109 CFU·mL-1.清洁级树鼩40只随机分为实验组30只、对照组10只,右眼为实验眼.实验组将真菌孢子混悬液50 μL注入角膜基质中央,对照组注入生理盐水50 μL.造模后第3天、第7天、第14天,流式细胞仪分析房水中白细胞介素(interleukin,IL)-1β、IL-6、IL-4、IL-10的水平;病理学检查观察浸润细胞类型.结果 Th1型细胞因子IL-1β和IL-6浓度均在造模后第7天达到高峰,造模后各时间点与对照组相比差异均具有统计学意义(均为P＜0.05);Th2细胞因子IL-10浓度在造模后第14天达到高峰,造模后第7天、第14天与对照组相比差异均具有统计学意义(均为P ＜0.05);IL-4浓度仅在造模后第7天与对照组相比差异有统计学意义(P＜0.05).病理学检查结果示:炎症细胞浸润数量造模后第7天达到高峰,以中性粒细胞为主;实验组各时间点均可见菌丝平行于基质纤维生长.结论 促炎因子IL-β和IL-6及抑炎因子IL-10在树鼩茄病镰刀菌性角膜炎炎症反应机制中发挥重要作用.     

Regulated secretion of complement factor H by RPE and its role in RPE migration

Background  Variants in the gene for complement factor H (CFH) have been implicated as a major risk factor for the development of age-related macular degeneration (AMD). Little is known, however, about the factors regulating local expression and secretion of CFH by retinal pigment epithelial cells (RPE). Methods  Cultured human early passage RPE cells, highly differentiated, polarized human RPE cultures, and bovine RPE explants were incubated in the presence or absence of recombinant human or bovine interferon-γ (IFN-γ; 25 ng/ml). CFH expression in cell lysates, and secretion into culture supernatants were examined by Western blot. CHF expression and localization was analyzed by confocal microscopy. Migration assay was performed in a modified Boyden chamber with early passage human RPE cells after stimulation with recombinant CFH protein (1–100 ng/ml). Results  CFH was expressed in the cell lysates of RPE cells, and this expression was significantly upregulated by IFN-γ. Immunoreactivity for CFH was detected in RPE cells of bovine explants and highly differentiated human RPE monolayers, and the level of immunoreactivity increased after IFN-γ stimulation. Confocal microscopy revealed that CFH was predominantly localized in the apical cytoplasm of polarized human RPE. Western blot confirmed that IFN-γ increased CFH secretion into RPE supernatants. Dose-dependent RPE cell chemotactic migration was induced by CFH. Conclusion  IFN-γ promotes CFH expression in the apical compartment of RPE cells and increases secretion of CFH into RPE culture supernatants. Furthermore, CFH promotes chemotactic migration of RPE. This study suggests that interactions between CFH and IFN-γ have the potential to play a role in the pathogenesis of AMD. Shikun He contributed equally to the work, and therefore should be considered equivalent first author     

Th1 and Th2 responses on the ocular surface in uveitis identified by CCR4 and CCR5 conjunctival expression

PURPOSE: To investigate CC chemokine receptor 4 (CCR4) and CC chemokine receptor 5 (CCR5) expression, known to be related to the Th2 and Th1 inflammatory pathways, respectively, and human leukocyte antigen-D related (HLA-DR) antigens as hallmarks for ocular surface inflammation in patients with uveitis using conjunctival impression cytologic specimens. DESIGN: Case-controlled study. METHODS: Conjunctival impression cytologic specimens were obtained from patients with anterior uveitis (n = 26), and their inflammatory profile was compared with those of patients with vernal keratoconjunctivitis (VKC; n = 24), keratoconjunctivitis sicca (KCS; n = 17), and normal subjects (n = 17). Expressions of CCR4, CCR5, and HLA-DR were analyzed using flow cytometry and were expressed by determining the percentage of cells expressing the markers in the conjunctival epithelium. RESULTS: CCR4 was overexpressed in the uveitis group (mean, 19.8% +/- 19.7% of positive cells) and in the VKC group (24.7% +/- 20.1%). CCR5 was expressed only weakly in uveitis patients (6.4% +/- 13.1%) and in the normal subjects (2.4% +/- 2.4%). HLA-DR expression by conjunctival cells was increased in the uveitis patients (57.4% +/- 21.1%) and in the KCS group (52.4% +/- 12.1%) compared with the VKC group (23.9% +/- 26.8%; P < .001) and normal subjects (22.1% +/- 19.1%; P < .001). CONCLUSIONS: CCR4, classically related to the Th2 system, and HLA-DR both were overexpressed by the conjunctival epithelium in uveitis patients, whereas CCR5, related to the Th1 system, was expressed weakly in uveitis patients. These preliminary results seem to suggest an involvement of the Th2 system on the ocular surface in uveitis. Exploration of the ocular surface in uveitis may represent a new way to understand better the immune pathways involved in this complex disease.     

Expression of TNF-α, IL-1β, and IFN-γ in Staphylococcus epidermidis slime-positive experimental endophthalmitis is closely related to clinical inflammatory scores

PURPOSE: The purpose of this study was to investigate the expression of inflammatory cytokines TNF-alpha, IL-1beta, and IFN-gamma in the vitreous after experimentally induced endophthalmitis by a Staphylococcus epidermidis slime-producing strain. METHODS: Seventy-two experimental Lewis rats received an intravitreal injection of 7000 viable organisms of Staphylococcus epidermidis slime-producing ATCC strain 35983, while 72 control rats received an intravitreal injection of sterile normal saline. Eyes were graded daily for signs of clinical inflammation and were removed 6, 12, 24, 48, 72 h, and 7 days after injection. Vitreous was obtained and titers of TNF-alpha, IL-1beta, and IFN-gamma were measured with established enzyme-linked immunosorbent assays. RESULTS: In the experimental group, the clinical inflammatory score reached maximum (4+) within 24 h, while inflammation was almost abolished by day 7 (score 0-0.5+). Statistically increased levels of TNF-alpha and IL-1beta were detected in the experimental vitreous with maximum levels observed at 12 h. IFN-gamma was also detected in the experimental vitreous and reached maximum levels at 48 h. None of the cytokines examined was detected in sera at any time point in experimental or control rats. CONCLUSIONS: The results of this study suggest that Staphylococcus epidermidis experimental endophthalmitis induces the expression of cytokines TNF-alpha, IL-1beta, and IFN-gamma in the vitreous. The time course of those cytokine expression levels is closely associated to the clinical presentation of this endophthalmitis model.