Expression of interleukin-1 receptor antagonist in human cornea

The purpose of this study was to confirm the expression of interleukin-1 receptor antagonist (IL-1 Ra) in the human cornea. Four samples of human ex vivo corneal epithelium were obtained from patients undergoing photorefractive keratectomy. RT-PCR was performed using mRNA isolated from the corneal epithelium and oligo-dT primers. PCR was performed on the cDNA products using primers specific for human IL-1 Ra. The PCR products were subcloned and sequenced. Human cornea sections were prepared from eyes enucleated for choroidal melanoma. Immunocytochemistry was performed using goat anti-mouse polyclonal IL-1 Ra IgG and NL-577 conjugated donkey anti-goat IgG. IL-1 Ra mRNA was expressed in all ex vivo corneal epithelium samples as confirmed by sequencing of the PCR products. Immunofluorescence studies revealed strongest expression of IL-1 Ra in the superficial apical layer of corneal epithelium. Expression of IL-1 Ra may represent an endogenous mechanism of down-regulating the effects of epithelial- and tear-derived IL-1α and IL-1β on the intact epithelium in the unwounded cornea and stromal cells after injury.     

白介素-1受体拮抗剂滴眼液治疗大鼠高危角膜移植免疫排斥反应

目的　观察白介素 1受体拮抗剂 (IL 1ra)滴眼液对高危角膜移植排斥反应的治疗作用。方法　SD大鼠为供体 ,Wistar大鼠为受体。缝线法诱导角膜新生血管后行角膜移植。对照组用生理盐水点眼 ,实验组分别用IL 1ra 1、3、5mg/mL点眼。同时设IL 1ra 1mg/mL结膜下注射组。观察植片的存活情况并检测植片中CD1阳性细胞。结果　与对照组相比 ,IL 1ra 3、5mg/mL组和结膜下注射组均可明显延长植片的存活时间 (P <0 0 5 )并减少植片中CD1阳性细胞的浸润。结论　IL 1ra滴眼液可以用于治疗高危角膜移植免疫排斥反应     

Apoptosis in the initiation, modulation and termination of the corneal wound healing response

Stromal keratocyte apoptosis has been well-characterized as an early initiating event of the corneal wound healing response, triggering subsequent cellular processes that include bone marrow-derived cell infiltration, proliferation and migration of residual keratocyte cells, and, in some circumstances, generation of myofibroblast cells. Recent studies, however, have suggested a more general role for apoptosis in the overall stromal wound healing response that includes modulation and termination functions. This review article highlights, and ties together, recent studies that have demonstrated the important role apoptosis likely plays in weeks to months following an initial insult to the cornea-depending on the type and extent of corneal injury.     

Topical application of culture supernatant from human amniotic epithelial cells suppresses inflammatory reactions in cornea

Human amniotic epithelial cells (HAEC) may be a source of soluble anti-inflammatory factors. The purpose of this study is to determine the effect of topically applied HAEC culture supernatant on corneal inflammatory reactions. HAEC were obtained from a placenta and cultured for 48 hr, and the supernatant was collected. The conditioned medium from HAEC contained small amounts of human interleukin-1 receptor antagonist (IL-1ra). Intrastromal sutures were placed in the cornea of BALB/c mice to induce corneal neovascularisation. Superficial cauterisation was applied to induce recruitment or activation of antigen presenting cells (APCs) in the cornea without neovascularisation. HAEC conditioned medium, placebo, or recombinant human IL-1ra was topically applied three times daily for 2 weeks. Suture-induced corneal neovascularisation was evaluated microscopically for 8 weeks. The cauterised corneas were harvested at 2 weeks, and the MHC class II(+) APCs were quantified by immunofluorescent staining and confocal microscopy. Inflammatory cytokine gene expression in the cauterised corneas was analyzed by a multiprobe ribonuclease protection assay. Conditioned medium from HAEC led to a profound suppression of corneal neovascularisation and fewer MHC class II(+) APCs in the epithelium. In contrast, human IL-1ra was only slightly effective in suppressing corneal inflammatory reactions. mRNA expression of murine IL-1ra and IL-1beta in the cauterised corneas was markedly suppressed after application of the conditioned medium. These results suggest that HAEC are a source of soluble anti-inflammatory factors and that conditioned medium from HAEC contains factors other than IL-1ra that suppress corneal inflammation.     

Expression of the interleukin 1 receptor antagonist in the normal human cornea

The human cornea has been shown to express a number of inflammatory cytokines including IL-1, IL-6 and IL-8. In view of the potent proinflammatory activities of interleukin-1 (IL-1), regulatory mechanisms should be present in the human cornea to control IL-1 mediated inflammatory and immune responses. This is important for the maintenance of the integrity and transparency of the cornea. To test this hypothesis, the authors determined the presence of IL-1 receptor antagonist (IL-tra) in the normal human cornea using an enzyme-linked immunosorbent assay (ELISA). IL-tra is a natural antagonist of IL-1 and competes with IL-1 for the binding to its receptors thereby blocking the inflammatory response. Corneas were either tested immediately or after a 24-hour culture period. Furthermore, the authors separately analyzed the three layers of the cornea. Their results present evidence for the constitutive expression of the IL-tra protein in the normal human cornea and show that both epithelial and stromal cells produce IL-1ra. The epithelial cells are the major source of corneal IL-1ra immunoreactivity, and secrete IL-1ra during culture. Stromal cells contain detectable, albeit low amounts of cell associated IL-1ra. No IL-1ra was detected in the endothelial cell layer. A more accurate understanding of the balance between IL-1 and IL-1ra in ocular tissues and the role of the IL-1ra under physiologie and pathophysiologic conditions will be necessary for an eventual use of IL-1 receptor antagonist as a therapeutical tool     

IL-1受体拮抗剂防治大鼠角膜移植排斥反应的实验研究

目的　从免疫病理学角度研究白细胞介素 1受体拮抗剂 (IL 1ra)防治角膜移植排斥反应。方法　将受体大鼠随机分为 4组 ,I、II、III组自手术之日起每日结膜下分别注射IL 1ra 5 0、10 0、2 0 0 μg。IV组 (对照组 )结膜下注射等体积生理盐水。观察各实验组IL 1ra和对照组的植片存活时间 ,用免疫组织化学染色方法检测IL 1ra及对照组角膜移植片中CD4 细胞、CD8 细胞的表达。结果　I、II、III组植片存活时间分别为 (11.0 0±1 4 6 )、(12 2 3± 1.13)、(13.5 8± 1.0 7)d ,IV组植片存活时间为 (7.85± 1.5 8)d。各用药组与对照组比较存活时间显著延长 (P <0 .0 1)。排斥的角膜植片大量表达CD4 、CD8 细胞 ,IL 1ra明显抑制这 2种细胞的表达。结论　结膜下注射IL 1ra可抑制角膜移植术后免疫排斥反应 ,减轻免疫性炎症反应 ,延长植片存活时间。IL 1ra剂量越大 ,效果越好。     

白细胞介素-1受体拮抗剂防治角膜移植免疫排斥反应的实验研究

目的观察白细胞介素-1受体拮抗剂(IL-1ra)抑制大鼠正位穿透性角膜移植术后免疫排斥反应、促进植片存活的作用.方法将主要组织相容性系统完全不同的近交系F344大鼠的角膜植片移植到另一近交系LOU大鼠的角膜上,将手术成功大鼠分为实验Ⅰ、Ⅱ、Ⅲ组及对照Ⅳ组,均于术后第1d起结膜下分别注射50、100、200μg的IL-1ra,对照组(Ⅳ组)结膜下注射等量生理盐水,连续2周.对角膜植片存活情况进行评分,观察其临床效果.结果各实验组角膜植片平均存活天数(meansurvivaltime,MST)分别为(11.13±1.46)d、(12.00±1.60)d及(13.44±1.13)d,明显高于对照组(8.00±1.25)d,差异有显著性(t=0.00,P＜0.01).IL-1ra200μg组与50μg组间比较,差异有显著性(t=0.00,P＜0.01).结论结膜下注射IL-1ra可抑制角膜移植术后免疫排斥反应,减少新生血管的生成,减轻免疫性炎性反应,延长角膜植片的存活时间.IL-1ra的剂量越大,效果越好.     

Aquaporin-1-facilitated keratocyte migration in cell culture and in vivo corneal wound healing models

Aquaporin-1 (AQP1) water channels are expressed in corneal keratocytes, which become activated and migrate following corneal wounding. The purpose of this study was to investigate the role of AQP1 in keratocyte migration. Keratocyte primary cell cultures from wildtype and AQP1-null mice were compared, as well as keratocyte cultures from pig cornea in which AQP1 expression was modulated by RNAi knockdown and adenovirus-mediated overexpression. AQP1 expression was found in a plasma membrane pattern in corneal stromal and cultured keratocytes. Osmotic water permeability, as measured by calcein fluorescence quenching, was AQP1-dependent in cultured keratocytes, as was keratocyte migration following a scratch wound. Keratocyte migration in vivo was compared in wildtype and AQP1 knockout mice by histology and immunofluorescence of corneal sections at different times after partial-thickness corneal stromal debridement. AQP1 expression in keratocytes was increased by 24 h after corneal debridement. Wound healing and keratocyte appearance near the wound margin were significantly reduced in AQP1 knockout mice, and the number of neutrophils was increased. These results implicate AQP1 water permeability as a new determinant of keratocyte migration in cornea.     

IL-1ra基因修饰角膜内皮细胞及其表达

目的构建真核细胞内表达的重组质粒PEGFPhIL1ra,对角膜内皮细胞进行基因修饰,为构建白细胞介素1受体拮抗剂(interleukin1receptorantagonist,IL1ra)基因化角膜内皮细胞移植膜奠定基础。方法以人cDNA文库为模板进行PCR扩增,获得人IL1racDNA片断,插入PEGFPC2载体,构建真核表达质粒PEGFPhIL1ra。以阳离子聚合物为介导,对饲细胞培养下的角膜内皮细胞(cornealendothelialcells,CECs)进行体外转染。通过绿色荧光蛋白(greenfluorescentprotein,GFP)示踪检测转染效率,蛋白免疫印迹检测外源性IL1ra基因在角膜内皮细胞内表达强度。结果以cDNA文库为模板扩增出hIL1racDNA,通过酶切、DNA测序证实PEGFPhIL1ra构建正确。20%～25%的转染角膜内皮细胞中有绿色荧光。Westernblotting检测可见转染角膜内皮细胞内有相对分子量为44000的hIL1raGFP融合蛋白表达,3d时蛋白表达水平达到峰值,并且持续至5d,7d时表达水平开始回落,9d时表达处于较低水平,对照组在观察期内均未见蛋白表达。结论阳离子聚合物介导下重组质粒PEGFPhIL1ra可对角膜内皮细胞实现有效转染并且持续表达IL1ra蛋白。     

Local suppression of IL-1 by receptor antagonist in the rat model of corneal alkali injury

Proinflammatory cytokines, including interleukin-1 (IL-1) have been implicated in the inflammation that follows corneal alkali injury. The purpose of this series of experiments was to test whether topically applied interleukin-1 receptor antagonist (IL-1ra) could suppress corneal inflammation and promote transparency after alkali injury. Alkali injury was induced on day 0 by application of 1N NaOH to both eyes of Wistar rats (n = 28). Immediately thereafter, eyes received either topical IL-1ra (20 mg ml(-1)) in 0.2% sodium hyaluronate or vehicle alone three times daily during days 0-14. Biomicroscopic features including corneal opacity and neovascularization were assessed using a standard grading scheme. Inflammation was quantified histologically. Corneas excised at day 3 and 7 (randomly selected six eyes in each group per time point studied) were homogenized, and levels of IL-1alpha, IL-1beta, IL-6, IL-8, IL-10, and RANTES were quantified by enzyme-linked immunosorbent assay. Epithelial wound healing was examined by computed analysis of fluorescein stained corneal photographs taken daily until day 14. According to these evaluations, eyes treated with IL-1ra maintained corneal transparency with minimal neovascular invasion. Additionally, corneal damage and cell infiltration were reduced on day 7 (infiltration cells were almost 40% decreased). All cytokine/chemokine levels in IL-1ra treated eyes were significantly lower at day 3, and IL-6 and IL-10 remained significantly lower at day 7 compared to vehicle-treated eyes. IL-1ra treatment retarded epithelial wound healing in the early stage (day 1-4); however, subsequently IL-1ra treated eyes had enhanced healing with full epithelial closure at nearly the same time point as vehicle-treated eyes (day 10). We conclude that local antagonism of IL-1 after alkali injury can significantly decrease corneal inflammation and lead to enhanced corneal transparency.     

Stromal interleukin-1 expression in the cornea after haze-associated injury

The purpose of this study was to determine whether myofibroblasts or other cells in the stroma in the cornea produce interleukin (IL)-1α or IL-1β that could modulate myofibroblast viability in corneas with haze after photorefractive keratectomy (PRK). Twenty-four female rabbits had haze-generating PRK for 9 diopters of myopia and were sacrificed at 1 week, 2 weeks, 3 weeks or 4 weeks after surgery. Corneal rims were removed, frozen in OCT at −80 °C, and analyzed by immunocytochemistry using primary antibodies to IL-1α, IL-1β and alpha smooth muscle actin (SMA). Double immunostaining was performed for the co-localization of SMA with IL-1α or IL-1β. Central dense haze and peripheral slight haze regions of each cornea were analyzed. SMA+ cells that expressed IL-1α protein were detected in both regions of the corneas at most time points following PRK. However, in the haze region at the 1, 3 and 4 week time points, significantly more (p < 0.01) SMA+ cells did not express IL-1α. Also, in the haze region at all three time points, significantly more (p < 0.01) SMA- cells than SMA+ cells expressed interleukin-1α protein. IL-1β expression patterns in SMA+ and SMA- stromal cells was similar to that of IL-1α after PRK. Previous studies have demonstrated that IL-1α or IL-1β triggers myofibroblast apoptosis in vitro, depending on the available concentration of apoptosis-suppressive TGFβ. This study demonstrates that SMA- cells such as corneal fibroblasts, keratocytes, or inflammatory cells may produce IL-1α and/or IL-1β that could act in paracrine fashion to regulate myofibroblast apoptosis—especially in the region where there is haze in the cornea after PRK was performed and SMA+ myofibroblasts are present at higher density. However, some SMA+ myofibroblasts themselves produce IL-1α and/or IL-1β, suggesting that myofibroblast viability could also be regulated via autocrine mechanisms.     

高糖对人角膜上皮细胞创伤修复中Cyclin D1 蛋白表达的影响

目的： 研究高糖环境对人角膜上皮细胞损伤修复的影响,初步探讨Cyclin D1蛋白在高糖培养的角膜上皮细胞创伤愈合中表达的意义。

方法： 模拟糖尿病角膜病变的高糖微环境,人角膜上皮细胞经复苏、培养传代后,分别设置等体积蒸馏水的DMEM完全培养基的正常对照组和含25mmol/L葡萄糖的DMEM完全培养基高糖处理组的两组细胞,细胞长满后进行划伤刺激,倒置相差显微镜下观察比较各组细胞生长状况及其变化,于不同时间点(0、12、24、48和72h)利用Western blot检测分析高糖培养的角膜上皮细胞中Cyclin D1蛋白的表达,qRT-PCR检测Cyclin D1 mRNA水平相对表达情况。

结果： 体外高糖处理条件下,人角膜上皮细胞划伤后修复速度减慢,漂浮细胞增多,细胞重新贴壁少,细胞间距增加,随着高糖处理时间的延长,细胞状态变差,生长速度慢; 正常组细胞损伤修复较快, 细胞密度增大,且形态规则,细胞膜光滑。Western blot 检测划伤刺激上调Cyclin D1的表达,但随着时间延长上调作用呈逐渐减弱,两组Cyclin D1最高表达量均出现在12h。高糖处理组Cyclin D1表达量低于正常对照组。qRT-PCR结果显示：高糖处理后,Cyclin D1 mRNA表达呈上调趋势,但随着高糖处理时间延长,上调作用减弱,且在48h处mRNA水平恢复至与对照组同一水平。

结论： 在角膜上皮细胞创伤愈合过程中,高糖呈负性调节,抑制Cyclin D1蛋白的表达,且与角膜上皮细胞增殖能力下降及凋亡相关。     

Keratinocyte growth factor-2 and autologous serum potentiate the regenerative effect of mesenchymal stem cells in cornea damage in rats

AIM:To investigate the healing process after severe corneal epithelial damage in rats treated with mesenchymal stem cells (MSCs) cultured with or without keratinocyte growth factor (KGF-2) and autologous serum (AS) on amniotic membrane (AM). Many patients are blind and devastated by severe ocular surface diseases due to limbal stem cell deficiency. Bone marrow-derived MSCs are potential sources for cell-based tissue engineering to repair or replace the corneal tissue, having the potential to differentiate to epithelial cells.METHODS:The study included 5 groups each including 10 female “Sprague Dawley” rats in addition to 20 male rats used as bone marrow donors. Group I rats received AM+MSCs, Group II rats AM+MSCs cultured with KGF-2, Group III rats AM+MSCs cultured with KGF-2+AS, Group IV rats only AM and Group V rats, none. AS was derived from blood drawn from male rats and bone marrow was obtained from the femur and tibia bones of the same animals. Therapeutic effect was evaluated with clinical, histopathological and immunohistochemical assessment. MSC engraftment was demonstrated via detection of donor genotype (Y+) in the recipient tissue (X) with polymerase chain reaction.RESULTS:Corneal healing was significantly better in Groups I-III rats treated with MSC transplantation compared to Group IV and Group V rats with supportive treatment only. The best results were obtained in Group III rats with 90% transparency, 70% lack of neovascularization, and 100% epithelium damage limited to less than 1/4 of cornea.CONCLUSION: We suggest that culture of MSCs with KGF-2 and AS on AM is effective in corneal repair in case of irreversible damage to limbal stem cells.     

IL-1ra对大鼠高危角膜植片IL-1β影响的实验研究

目的 探讨炎性细胞因子白细胞介素-1(interleukin-1,IL-1)β在高危角膜移植免疫排斥反应中的作用及IL-1受体拮抗剂(IL-1 receptor antagonist,IL-1ra)抑制大鼠高危角膜移植免疫排斥反应的作用机制.方法 诱导新生血管后的大鼠行穿透性角膜移植术,受体鼠随机分为3组:生理盐水对照组、10 g·L-1 环孢霉素A(cyclosporin A,CsA)滴眼液治疗组、50 mg·L-1的IL-1ra滴眼液治疗组,每组15只.分别采用原位杂交和免疫组织化学方法 检测术后不同时间点各组大鼠角膜移植物IL-1β mRNA及蛋白表达情况.结果 正常大鼠角膜IL-1β mRNA在上皮细胞基底层微量表达,IL-1β蛋白无表达;术后不同时间点各移植组角膜植片上皮层、基质层均可检测到IL-1β mRNA及蛋白的阳性表达.术后同一时间点不同移植组相比: IL-1β mRNA及蛋白的表达依次减低顺序为生理盐水对照组、10 g·L-1 CsA组、50 mg·L-1 IL-1ra 组,差异有显著统计学意义(P＜0.01);在急性排斥期,与10 g·L-1 CsA滴眼液组相比,IL-1ra组降低更明显,两治疗组间比较差异有显著统计学意义(P＜0.01).结论 IL-1ra可通过影响IL-1β的表达抑制高危角膜移植免疫排斥反应,减少新生血管生成,减轻免疫性炎性反应,延长角膜植片的存活时间.     

白细胞介素—1拮抗剂治疗重度角膜烧伤初步疗效观察

目的：观察白细胞介素－１受体拮抗剂治疗重度角膜烧伤的效果，方法；采用ＩＬ－Ｉｒａ局部点眼法，或／和结膜下注射法治疗５例（５只眼），结果：观察２￣６个月，所有治疗眼视力均有增进，４只眼治愈遗留瘢痕，１只眼角膜混浊减轻，结论：临床资料显示ＩＬ－ｌｒａ对眼部炎症可能提供一种新的治疗途径。     

Sigma-1受体对视网膜神经节细胞保护机制的研究进展

视神经病变是一类以视网膜神经节细胞(retinal ganglion cells, RGC)及轴突受损为主要特征的致盲性疾病,发病机制复杂且治疗手段有限。Sigma-1受体(Sigma-1 receptor, S1R)是一种内质网上的分子伴侣蛋白,视网膜中含量丰富,其中高表达于神经节细胞层。近年来,S1R作为神经退行性疾病的治疗靶点备受关注。越来越多研究表明S1R参与调节多种细胞功能,包括Ca²⁺稳态、内质网应激反应、氧化应激反应、神经营养因子分泌和胶质细胞活化等,在神经退行性疾病中发挥神经保护作用。视觉系统中,研究发现激动S1R同样具有保护作用,可明显改善RGC丢失及功能减低,部分逆转损伤,维持结构完整;相反S1R缺陷则会恶化疾病进展或提高退行性疾病易感性。本文综述了S1R对视网膜中RGC的保护作用及其机制的研究进展,旨在深入了解其功能及机制,为视神经病变治疗提供新靶点。     

Effect of GLC756, a novel mixed dopamine D1 receptor antagonist and dopamine D2 receptor agonist, on TNF-alpha release in vitro from activated rat mast cells

Tumor necrosis factor-alpha (TNF-alpha) is released from activated mast cells via an IgE-dependent mechanisms, and plays a crucial role in ocular allergic inflammation. This study examined the influence of three antiglaucoma drugs differing in their chemical structure and pharmacological profile (i.e. latanoprost, timolol, GLC756) on TNF-alpha release from activated rat mast cells. A rat basophilic leukemia mast cell line (RBL-2H3) was activated via IgE/anti-IgE. Rat mast cells were incubated with latanoprost, timolol, GLC756 or betamethasone (positive control) at concentrations of 0.1, 1, 10 and 30 microM. TNF-alpha concentration in supernatant was measured by ELISA 5 h post-activation. Compared to controls, the prostaglandin derivative latanoprost and the beta-blocker timolol in the concentration range 0.1-30 microM, had no significant effect on TNF-alpha release from rat mast cells measured 5h after activation. By contrast, the dopaminergic drug GLC756 compared to controls in the concentration range 1-30 microM significantly inhibited TNF-alpha release from activated rat mast cells in a concentration-dependent manner. The positive control betamethasone inhibited TNF-alpha release almost completely at all concentrations tested. In conclusion, the results of this study suggest that latanoprost and timolol do not reduce inflammation triggered by activated mast cells. By contrast, the dopaminergic drug GLC756 inhibited TNF-alpha release from activated mast cells, suggesting an palliative potential of dopaminergic compounds on allergic conjunctivitis in topical glaucoma medication.     

Effect of CS-088, an angiotensin AT1 receptor antagonist, on intraocular pressure in glaucomatous monkey eyes

To evaluate the effect of CS-088, an angiotensin AT1 receptor antagonist, on intraocular pressure (IOP) in monkey eyes with unilateral laser-induced glaucoma. A multiple-dose study was performed in 8 glaucomatous monkey eyes. One 50 microl drop of CS-088, 2% or 4%, was topically applied to the glaucomatous eye at 9:30 a.m. and 3:30 p.m. for 5 consecutive days. IOP was measured hourly for 6 hours beginning at 9:30 a.m. for one baseline day, one vehicle-treated day, and daily for 5 days of treatment with CS-088. The washout period between the two drug concentrations was at least 2 weeks. Twice daily administration of 2 % CS-088 for 5 days did not reduce the IOP until the third dose on day 2 of the treatment regimen. A significant (p<0.02) reduction in IOP began 1 hour after the third dose, and lasted for 3 hours. The maximum reduction in IOP was 5.3+/- 0.8 (mean+/-SEM) mmHg (15%) (p<0.001), with the longest duration of IOP reduction of at least 6 hours after dosing on day 5. The 4% dose of CS-088 reduced (p<0.05) IOP from 1 to 5 hours after the first dose. The maximum reduction in IOP was 6.9+/-1.0 mmHg (20%), with the longest duration of IOP reduction of at least 18 hours after administration on day 5. Both 2% and 4% CS-088 showed enhancement of the ocular hypotensive effect with repeated dosing. 4% CS-088 produced greater (p<0.05) IOP reduction with longer duration of action than 2%. Topically applied CS-088, a new antagonist drug at the angiotensin AT1 receptor, reduced IOP in glaucomatous monkey eyes in a dose-dependent manner.