Effects of Th2 cytokines on expression of collagen,MMP-1, and TIMP-1 in conjunctival fibroblasts

PURPOSE: To determine whether cytokines involved in chronic allergic conjunctival disorders may affect formation of giant papillae and tissue remodeling. METHODS: Conjunctival fibroblast cultures were challenged with different concentrations of human recombinant interleukin (IL)-4, IL-13, interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha. Procollagens I (PIP) and III (PIIIP), matrix metalloproteinase (MMP)-1 and -9, and tissue inhibitor of metalloproteinase (TIMP)-1 were measured in supernatants, and their respective mRNAs were evaluated by RT-PCR. RESULTS: IL-4 and -13 (10 ng/mL) significantly increased production and expression of PIP compared with nonstimulated cells, whereas IFN-gamma elicited the opposite effect, at both the protein and mRNA levels. Both IL-4 and -13 significantly decreased production of MMP-1 and increased that of TIMP-1, whereas TNF-alpha increased production of MMP-1 and -9. Expression of MMP-1 was reduced by IL-4 and increased by the other tested cytokines, whereas expression of TIMP-1 was increased by all tested cytokines. CONCLUSIONS: IL-4 and -13 increased production of collagen and modified the equilibrium between MMP-1 and its inhibitor, TIMP-1. These effects were partially opposed by IFN-gamma and TNF-alpha.     

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。     

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。     

Soluble Fas ligand expression in the ocular fluids of uveitis patients

PURPOSE. The expression of Fas ligand (FasL) in ocular tissues is thought to play a critical role in maintaining immune privilege in the eye. In this study, to clarify the involvement of the Fas-FasL system in inflammatory processes of the eye,we examined soluble FasL (sFasL) in ocular inflammation. METHODS. Using ELISA systems recently developed, sFasL concentrations in aqueous humor (AH) and/or vitreous fluid (VF) were measured. AH was obtained from 17 eyes of 17 uveitis patients and from 12 eyes of 12 non-uveitis (cataract) patients. VF was obtained from 22 eyes of 22 uveitis patients and 7 eyes of 7 non-uveitis (macular hole) patients. Serum levels of sFasL were also determined. RESULTS. sFasL in AH and VF was below the detection limit of the ELISA systems in all non-uveitis eyes. On the other hand, sFasL was detected in AH from uveitis patients where it measured 367.0 +/- 154.7 pg/ml (mean +/- SEM). sFasL was also detected in VF from uveitis patients where it measured 1132.2 +/- 281.7 pg/ml. None of the sera from patients with or without uveitis contained a detectable level of sFas L. CONCLUSIONS. sFasL levels in AH and VF are elevated in the eye during ocular inflammation. Fas-FasL mediated apoptosis may play an important role in the regulation of inflammation during uveitis.     

高渗透压对兔眼表上皮和黏蛋白5AC表达的影响

目的 探讨高渗透压对兔眼表上皮和黏蛋白(MUC)5AC表达的影响.方法 实验研究.将18只健康新西兰大白兔采用计算机随机数字法分为3组,分别为高渗盐溶液(500 mmol/L)、生理盐水(308 mmol/L)滴眼组及空白对照组,每组6只兔.实验第0、7、14天测定泪液分泌量与泪膜破裂时间(BUT),结膜印迹细胞学测定杯状细胞密度.实验第7、14天处死动物,行角结膜光镜和电镜观察,结膜组织末端脱氧核苷酸转移酶介导的脱脲苷三磷酸缺口末端标记法(TUNEL)检测,第14天的结膜组织另行MUC5AC表达的免疫组织化学染色和免疫印迹法检测.同一时间点上泪液分泌量值、BUT值和杯状细胞密度的组间比较采用单因素方差分析法,进一步两两比较采用Bonferroni法;若方差不齐则采用Kruskal-Wallis H检验.组内不同时间点比较采用配对t检验.结果 实验第7和第14天,高渗液组BUT分别为(7.6±2.5)和(7.0±2.3)s,较第0天的(10.3±2.5)s显著缩短(t=5.800,4.950;均P＜0.01),与空白对照和生理盐水组相比亦明显缩短(F=8.030,P＜0.01).高渗液组结膜杯状细胞密度明显减少,第7和第14天分别为(19.5±16.6)和(32.3±18.2)个/mm2,与第0天(75.7±43.4)个/mm2相比,差异有统计学意义(t=5.319,2.970;均P＜0.05).高渗盐溶液滴眼第14天,光镜下可见结膜上皮下炎性细胞浸润,角膜上皮部分脱落;扫描电镜下可见角膜上皮细胞肿胀、脱屑、微绒毛减少;透射电镜下可见角膜上皮细胞问连接松弛,基底层上皮细胞内自噬空泡增多,结膜上皮微绒毛减少,杯状细胞内分泌颗粒减少;TUNEL检测结膜上皮可见明显的阳性染色;结膜组织免疫组织化学染色和免疫印迹检测则发现MUC5AC表达明显低于其他两组.生理盐水组与空白对照组的结膜杯状细胞密度及角结膜上皮细胞结构在实验中则均无明显变化.结论 眼表高渗压力可破坏角结膜上皮细胞结构,造成杯状细胞密度降低和MUC5AC表达水平下降,并导致泪膜稳定性下降.

Abstract：

Objective To investigate the effects of hyperosmotic stress on rabbit ocular surface and mucin 5AC (MUC5AC) expression. Methods Experimental study. Eighteen New Zealand white rabbits were randomly divided into three groups with equal number as hyperosmolar saline solution (HOSS,500 mmol/L) group, normal saline (NS, 308 mmol/L) group and blank control group respectively. In HOSS and NS groups, the HOSS and NS eye drops were instilled on bilateral eyes six times every day for 14 days. On day 0, 7 and 14, Schirmer Ⅰ test and tear break-up time (BUT) were measured and conjunctival impression cytology specimens were collected. On day 7 and 14, cornea and conjunctiva were harvested for Hematoxylin and Eosin (HE) staining, scanning and transmission electron microscopy observation and conjunctival TUNEL examination. On day 14, the conjunctiva were also harvested for immunology histological staining and western blot to evaluate the expression of MUC5AC. Results In HOSS group, the BUT on day 7 and 14 was (7.6±2. 5) and (7.0±2. 3) s respectively which was significantly shorter than the (10.3±2. 5) s on day 0(t=5. 800,4. 950; P ＜ 0.01), and also significantly shorter than the BUT in NS and control groups (F=8.030, P ＜ 0.01). But the Schirmer Ⅰ test value did not change obviously in and between all those three groups. The mean conjunctival goblet cell (GC) density in HOSS group on day 7 and 14 was (19.5±16.6) and (32.3±18.2) cells/mm2 respectively which was also significantly lower than the (75.7±43.4) cells/mm2 on day 0 (t=5.319,2. 970; P ＜ 0.05). However the GC density did not change obviously in other two groups with time. After instillation of HOSS for 14 days,subepithelial inflammatory cell infiltration was showed on conjunctival tissue specimens and decreased epithelial layers and evident desquamation were found in the cornea specimens by the HE staining. Under the electron microscope, decreased microvilli and loosened intercellular junction in the superficial epithelium and increased autophagic vesicles in basal epithelium were observed in the cornea in HOSS group; and decreased microvilli and mucous granules were found in the conjunctiva in HOSS group. Obvious TUNEL positive staining was showed in the conjunctiva in the HOSS group. Also the MUC5AC immunology histological staining and western blot indicated decreased MUC5AC protein expression in HOSS group. Conclusion Hyperosmotic stress destroyed the structure of ocular surface epithelium, induced the decrease of conjunctival goblet cell density and MUC5AC expression, and led to the decreased tear film stability.     

Suppression of ocular inflammation in endotoxin-induced uveitis by blocking the angiotensin II type 1 receptor

PURPOSE: To examine whether the angiotensin II type 1 receptor (AT1-R) signaling plays a role in ocular inflammation in endotoxin-induced uveitis (EIU). METHODS: EIU was induced in C57BL/6 mice by a single intraperitoneal injection of 150 mug lipopolysaccharide (LPS). Tissue localization, mRNA expression, and protein levels of AT1-R in murine retinas were examined by immunohistochemistry, RT-PCR, and Western blot analyses, respectively. Telmisartan, an AT1-R antagonist widely used as an antihypertensive agent, was administered intraperitoneally at a dose of 10 mg/kg daily for 5 days until the injection of LPS. Twenty-four hours after administration, leukocyte adhesion to the retinal vasculature was evaluated with a concanavalin A lectin perfusion-labeling technique. Retinal mRNA and protein levels of intercellular adhesion molecule (ICAM)-1 were examined by RT-PCR and ELISA, respectively. Protein concentration and inflammatory cells in the aqueous humor were also measured. RESULTS: Retinal vessels were positive for AT1-R. In mice with EIU, retinal AT1-R mRNA and protein levels were significantly increased when compared to the normal control. EIU animals also showed significant increases in the number of inflammatory cells infiltrating the anterior chamber and adhering to the retinal vessels and in retinal ICAM-1 levels. Administration of telmisartan to EIU mice resulted in significant suppression of retinal ICAM-1 expression and leukocyte adhesion and infiltration compared with vehicle treatment. Protein concentration in the aqueous humor of telmisartan-treated EIU mice tended to be lower than that of vehicle-treated EIU mice, but the difference was not statistically significant. CONCLUSIONS: AT1-R signaling blockade inhibited retinal ICAM-1 upregulation and leukocyte adhesion and infiltration in the EIU model. These results suggest the potential use of an AT1-R antagonist as a therapeutic agent to reduce ocular inflammation.     

Impression cytology study of conjunctival epithelial phenotypes on the healing ocular surface after pterygium excision

PURPOSE: To compare the process of conjunctival epithelial regeneration after three types of pterygium excision procedures. METHODS: Thirty-eight patients (45 eyes) with primary pterygium were randomly assigned to a bare-sclera procedure (group 1, 15 eyes of 12 patients), bare-sclera with intraoperative mitomycin C (MMC 0.02% for 30 seconds; group 2, 15 eyes of 14 patients), or pterygium excision with conjunctival autografting (group 3, 15 eyes of 12 patients). Controls were healthy fellow eyes and seven eyes of age- and sex-matched subjects. Impression cytology was performed preoperatively, at 1 and 2 weeks, and at 1, 3, 6, and 12 months after surgery. The nucleus-to-cytoplasm (N/C) ratio of nongoblet epithelial cells and goblet cell density (GCD) in the pterygial area were calculated and compared over time across treatment groups. RESULTS: Pterygium excision wounds healed in a similar four-stage process in all groups, but at different rates and with different final results. The N/C ratio was highest at about 1 month postoperatively in groups 1 and 2 and at 2 weeks in group 3, before gradually returning to control levels. Preoperatively, the GCD in treated eyes was almost twice that in control eyes (p = 0.001) but fell to zero immediately postoperatively. Goblet cells first appeared (with the most rapidly increased density) in group 3, followed by group 1. At 12 months, the mean GCD in groups 1 and 3 were not significantly different from those in controls, whereas the mean GCD in group 2 was still less than that of control (p = 0.02). CONCLUSIONS: Healing of conjunctiva is delayed by MMC and is promoted by autografting. Even 1 year after surgery, the ocular surface remains abnormal with respect to epithelial phenotypes in eyes treated by any of the three techniques.     

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。     

Effects of IL-4 on conjunctival fibroblasts: possible role in ocular cicatricial pemphigoid

PURPOSE: Increased stromal accumulation of macrophages and submucosal fibrosis due to excessive accumulation of collagens are central histologic features in ocular cicatricial pemphigoid (OCP). Interleukin (IL)-4 plays an important role in both the inflammatory and fibrotic events in several human and experimental diseases. In the present study, the possible role of IL-4 in the pathogenesis of OCP was investigated. METHODS: Biopsy specimens from the conjunctivae of 10 patients with OCP and 5 normal subjects were studied for the expression of IL-4 by immunohistochemistry. The expression level of IL-4 was also examined in conjunctival fibroblasts of normal control subjects and patients with OCP. The effects of IL-4 in the induction of inflammatory and fibrogenic molecules was studied in IL-4-treated conjunctival fibroblasts, and the expression levels of macrophage colony-stimulating factor (m-CSF), heat shock protein (HSP)-47 and type I collagen was determined by quantitative real-time PCR. The level of IL-4 was also measured by enzyme-linked immunosorbent assay (ELISA) in serum samples obtained from patients with OCP during active stage and remission and were compared with the levels in control sera. RESULTS: Compared with the weak expression of IL-4 in the normal conjunctival sections, an increased expression of IL-4 was noted in conjunctival sections of patients with OCP. A similar increase in the expression of IL-4 was also detected in fibroblasts isolated from conjunctiva of patients with OCP, compared with control fibroblasts. Real-time PCR and ELISA detected a significantly increased level of m-CSF, at both the mRNA and protein levels in IL-4-stimulated cells. Similarly, IL-4 treatment resulted in the induction of type I collagen and collagen-binding HSP47 by conjunctival fibroblasts, as detected by real-time PCR. However, no apparent changes in the levels of IL-4 were detected by ELISA in serum samples of patients with OCP and control subjects. CONCLUSIONS: Increased conjunctival expression of IL-4 may play an important role in the regulation of local accumulation of macrophages (by inducing m-CSF), and matrix accumulation (by inducing HSP47 and collagen) during conjunctival scarring in patients with OCP. IL-4, therefore, may augment or enhance both conjunctival inflammatory and subsequent fibrotic responses in patients with OCP.     

养阴润目丸对干眼模型大鼠结膜上皮细胞CXCR3和CCR5表达的影响

目的 研究养阴润目丸对干眼模型大鼠结膜上皮细胞CXC型趋化因子受体3(CXC chemokine receptor 3,CXCR3)、CC型趋化因子受体5(CC chemokine receptor 5,CCR5)表达的影响.方法 采用随机数字表法将60只SD雄性大鼠分为5组,每组12只,分别为正常组、假手术组、养阴润目丸组、新泪然组、模型组,除正常组与假手术组外均采用去势法制造干眼模型.造模成功后用药3个月,分别进行泪液分泌试验及泪膜破裂时间的检测,取大鼠结膜上皮组织,采用免疫组织化学法检测CX-CR3、CCR5的表达.结果 用药3个月后,养阴润目丸组、新泪然组泪液分泌量分别为(9.60±1.04)mm和(9.00±1.37)mm,均明显高于模型组的(6.40±0.84) mm(均为P＜0.01);两组泪膜破裂时间分别为(9.61±0.82)s和(9.12±0.69)s,均明显长于模型组的(6.21±0.72)s(均为P＜0.01).养阴润目丸组、新泪然组结膜上皮细胞中CXCR3的表达分别为11.40±1.75和12.71±2.46,均明显少于模型组的29.39±2.48(均为P＜0.05);两组结膜上皮细胞中的CCR5表达分别为26.33±4.17和28.15±4.52,均明显少于模型组的38.78±6.60(均为P＜0.05).结论 养阴润目丸治疗能增加泪液分泌量,延长泪膜破裂时间,下调结膜上皮细胞中CXCR3、CCR5的表达.提示干眼的发病机制与炎症密切相关,养阴润目丸对干眼有一定的治疗作用.