Immunological aspects of herpetic stromal keratitis

Herpetic stromal keratitis (HSK) is an immune reaction related to herpes simplex virus (HSV) corneal infection, and has many important immunological aspects. CD4(+) T lymphocytes, especially Th1 cells, are the principal mediators for HSK. In addition, neutrophils and antigen-presenting cells play vital roles in HSK. CD8(+) T lymphocytes, B cells, and natural killer cells all participate in the pathogenesis of HSK under certain circumstances. Many molecules are involved in the pathogenesis of HSK. Th1 cytokines such as interleukin 2 (IL-2), IL-12 and interferon gamma, and inflammatory cytokines such as IL-1alpha and IL-6 are especially important ones. Among various chemokines that take part in HSK, MIP-1alpha is one of the most important aggravating factors. Vaccination therapy against HSK has been developed; glycoprotein D is a particularly promising candidate. However, the possibility of HSK exacerbation due to vaccination is the final problem to be solved before vaccination can be clinically applied to HSK. Molecular mimicry theory and bystander activation theory are the two new autoimmune theories that have been advocated. Since genuine autoimmune HSK without HSV growth can hardly be the case in clinical practice, some part of these new theories remains controversial. In the future, better understanding of the pathogenesis of HSK is essential to resolve the paradox between suppressing the immune reaction to avoid corneal scarring and preventing viral proliferation.     

Topical antisense-oligonucleotides targeting IFN-gamma mRNA improve incidence and severity of herpetic stromal keratitis by cytokine specific and sequence unspecific effects

Background Corneal infection with herpes simplex virus-1 (HSV) can cause an inflammatory eye disease termed herpetic stromal keratitis (HSK). Interferon-gamma (IFN-γ) is known to be involved in the development of this disease. In this study, antisense oligonucleotides targeting IFN-γ mRNA (IFN-γ-ASON) were investigated for their effects in experimental HSK. Methods Splenic cells were used to examine the efficacy of IFN-γ-ASON to decrease IFN-γ- release into the cell culture supernatants as measured by ELISA. Mice were corneally infected with 10⁵ PFU HSV, and IFN-γ-ASON were given subepithelially. Alternatively, mice were infected without any further treatment, received only buffer, or received control oligonucleotides (CON) to observe substance specific effects. The animals were followed up clinically for the signs of herpetic keratitis. On days 14 and 28 post infection (p.i.), animals were sacrificed, and eyes were collected for histological analysis. On day 7 p.i., infectious virus particles in the eyes were determined by a plaque assay. Results While IFN-γ-ASON diminished the content of IFN-γ in a concentration-dependent manner in vitro, CON showed no significant effects. Whereas buffer-treated and only infected mice showed severe necrotizing keratitis on day 14 p.i., this was abolished after treatment with IFN-γ-ASON, even after 28 and 52 days. CON-treated mice also showed an improved HSK on day 14, but not on day 28. The incidence of the disease was also clearly diminished after treatment with IFN-γ-ASON at all time points examined. The number of inflammatory cells in both the central and the peripheral cornea were strongly reduced after the application of IFN-γ-ASON as compared to the controls. In contrast, the infectious viral particles in eyes at day 7 p.i. did not differ between the four groups. Conclusions Topical treatment with IFN-γ-ASON induced a long-term improvement of the course and the incidence of HSK in the murine model. IFN-γ seems to be involved in a proinflammatory manner during the pathogenesis of HSK, while the antiviral defense against HSV was not affected by this topical cytokine inhibition. Unspecific CON induced a transient and cytokine independent improvement of HSK. Some of the data presented in this study were the topic of a short oral presentation held at the 102nd DOG Congress 2004 in Berlin. Financial support: DFG He1877/12-2 and Ernst und Berta Grimmke Stiftung.     

Application of FGF-2 to modulate herpetic stromal keratitis

PURPOSE: Herpetic stromal keratitis (SK) is a tissue destructive eye lesion caused by infection of herpes simplex virus-1 (HSV-1). One step by which HSV-1 enters the cell is through binding to surface heparan sulfate proteoglycans (HSPG), a process that can be inhibited by fibroblast growth factor 2 (FGF-2). The current study examined the effect of FGF-2 application on the outcome of ocular HSV infection. METHODS: Vero cells were infected with HSV-1 after preincubation with FGF-2 protein, and viral infectivity was determined by plaque reduction assay. In an in vivo study, mice were ocularly treated with FGF-2 before (plasmid DNA) or after (recombinant protein) HSV-1 infection, and SK lesion severity was observed. Results: Whereas FGF-2 had excellent antiviral effects in vitro, it was without significant inhibitory effects when given as plasmid DNA encoding FGF-2 (100 microg/application) onto the cornea of the susceptible mouse (BALB/c) before virus infection. Only minor antiviral effects of FGF-2 in vivo were initially observed. Interestingly, topical treatment of recombinant FGF-2 protein (50 ng, two times daily until day 10 postinfection) into HSV-1-infected corneas significantly reduced SK lesion severity and incidence, presumably by promoting epithelial ulcer healing. CONCLUSIONS: These results suggest that treatment of FGF-2 has therapeutic effects on herpetic SK progression via its role in wound healing.     

The granulomatous reaction in herpetic stromal keratitis: immunohistological and ultrastructural findings

A granulomatous reaction was identified in six corneal buttons obtained from patients with herpetic stromal keratitis. The inflammation, characterized by lymphocytes, plasma cells, macrophages and multinucleate giant cells, was associated with Descemet's membrane in five cases and with a break in Bowman's membrane in one case. The ultrastructural changes were documented by scanning electron microscopy in all cases. Transmission electron microscopy, performed in four cases, failed to disclose the presence of viral inclusions. Immunoperoxidase stains utilizing antibodies to the herpes simplex virus were done on all cases. Positivity (the presence of herpes simplex virus antigen or herpes simplex-like antigen) was detected within epithelium (three cases), within stroma (three cases), and within some inflammatory cells of the granulomatous reaction at Bowman's membrane. Positive staining was not detected in cells of the granulomatous reaction at Descemet's membrane. These results suggest that the reaction at Descemet's membrane, which has been found in a variety of corneal conditions, may be due to a poorly understood alteration in the antigenicity of Descemet's membrane.     

Impact of herpetic stromal immune keratitis in corneal biomechanics and innervation

Graefe's Archive for Clinical and Experimental Ophthalmology - To study corneal innervation in eyes with history of herpetic keratitis and its correlation with corneal sensitivity and...     

白细胞介素10对小鼠单纯疱疹性角膜基质炎作用的实验研究

研究白细胞介素10(IL-10)在小鼠单纯疱疹性角膜基质炎(herpetic stromal keratitis,HSK)中的作用。方法雌性BALB／c小鼠80只分为2组,每组40只;将单纯疱疹病毒1型接种于小鼠角膜上建立HSK动物模型;分别于鼠角膜接种病毒前的6h、当日及接种后2、4d,向实验组鼠的角膜内注射重组IL-10 20ng,腹腔内注射鼠重组IL-10 500 ng;对照组同步注射生理盐水;观察IL-10对小鼠HSK的发病率、临床特征、角膜病毒滴度、角膜细胞因子含量、角膜病理改变及迟发型超敏反应(DTH)的影响。结果IL-10降低了HSK发病率,减轻了HSK角膜混浊程度、角膜新生血管化程度及角膜内炎性细胞浸润,降低了角膜内IL-2和IL-6的含量,抑制了鼠的DTH反应。IL-10不影响病毒在角膜内的复制和清除。结论IL-10可抑制鼠的DTH反应和HSK的发病进展。     

Latanoprost increases the severity and recurrence of herpetic keratitis in the rabbit.

PURPOSE: To determine whether topically applied latanoprost increases the severity of acute herpes simplex keratitis, the rate of recurrence of herpes keratitis, or both, in the rabbit. METHODS: To determine the effect on severity of acute herpetic keratitis, the corneas of New Zealand white rabbits were infected with either the less-corticosteroid-sensitive McKrae strain or the corticosteroid-sensitive F(MP)E strain of herpes simplex virus type 1. Rabbits were randomly assigned to twice-a-day treatment with latanoprost 0.005%, dexamethasone sodium phosphate 0.1%, or balanced saline solution within 3 days of infection and evaluated daily for up to 13 days after infection. The severity of keratitis was graded in a masked manner. To determine the effect on recurrences of herpetic keratitis, animals infected with McKrae strain herpes simplex virus type 1 that survived to day 32 after infection were randomized to treatment with latanoprost 0.005% or balanced saline solution and evaluated for the presence of corneal lesions from postinfection day 32 to day 47. RESULTS: In the severity studies, treatment of F(MP)E-infected corneas with latanoprost or dexamethasone significantly worsened herpetic keratitis; by postinfection day 5, F(MP)E-infected eyes treated with dexamethasone or latanoprost demonstrated significantly higher severity scores than the eyes treated with balanced saline solution (P = .0001 and .008, respectively). Scores of McKrae-infected corneas treated with latanoprost or dexamethasone were not significantly different from scores of balanced saline solution-treated corneas. In the recurrence study, treatment with latanoprost significantly increased the appearance of clinical recurrences in McKrae-infected eyes, compared with balanced saline solution treatment (P = .0064). CONCLUSION: Latanoprost may worsen acute herpetic keratitis in the rabbit eye and increase the risk of recurrences in latently infected animals.     

单纯疱疹性角膜基质炎诊疗中的失误和对策

目的 研究单纯疱疹性角膜基质炎(HSK)诊疗中的失误和对策。方法 以含微量地塞米松的复方抗单疱药疱疹灵滴眼剂,≤8.0mm治疗性穿透角膜移植和≥8.5mm深板层角膜移植等三种方法,对87例92眼诊疗失误的HSK作回顾性临床分析。结果 3种治疗方法的治愈率分别为100.0％(39/39),100.0％(3/3)和98.0％(49/50),而复发率(平均随访3年7个月)分别为53.8％(21/39),0.0％(0/3)和2.1％(1/49)。结论 ①HSK临床体征变化的多样性,是导致诊疗失误的主要原因。②单独应用抗单疱药,不符合HSK的发病机理;全身大剂量和/或局部高浓度皮质类固醇,超过了HSK抗炎作用的实际需要,凸显出许多皮质类固醇的不良作用,同样会收到事与愿违的恶果。③含微量地塞米松的复方抗单疱滴眼剂,具有抑毒抗炎双重治疗作用,又避免了大剂量皮质类固醇的不良作用,对HSK疗效卓著,是迄今中外同类药物中的姣姣者。④≥8.5mm的深板层角膜移植术,能够最大限度地清除角膜基质中的病毒抗原,净化潜伏感染基地,是一种对重症HSK安全高效的治疗措施。     

细胞毒性T淋巴细胞相关抗原-4融合蛋白对鼠单纯疱疹性角膜基质炎抑制作用的研究

研究细胞毒性T淋巴细胞相关抗原-4融合蛋白(cytotoxic Tlymphocyte-associated antigen-4 immunoglobulin,CTIA-4Ig)对鼠单纯疱疹性角膜基质炎(herpetic stromal kerafifis,HSK)的抑制作用。方法用单纯疱疹病毒1型(herpes simplex virus type 1,HSV-1)接种于BALB／c鼠角膜上建立HSK动物模型,采用CTIA-4Ig阻断B7：CD28／CTIA-4协同刺激途径,抑制T淋巴细胞增殖、分化为效应细胞;观察HSK的发病率、临床特征、角膜组织学改变、角膜病毒滴度、迟发型超敏反应及抗原刺激脾细胞分泌细胞因子的情况。结果CTIA-4Ig可减少小鼠外周血中CD4^ T淋巴细胞(81．6％)及CD8^ T淋巴细胞(67．9％),阻止鼠发生HSK、减轻角膜混浊程度及角膜内炎性细胞的浸润、损伤鼠的迟发型超敏反应能力,抑制鼠脾细胞分泌辅助性T淋巴细胞1型(T-helper 1,Th1)细胞因子;但不影响角膜病毒滴度及小鼠死亡率。结论用CTIA-4Ig阻断B7：CD28／CTIA-4协同刺激途径,能够抑制T淋巴细胞增殖并抑制CIM^ Th1细胞的功能,阻止HSK发病,减轻HSK的严重程度。     

IL-17在单纯疱疹性角膜基质炎小鼠中表达的变化

目的:研究IL-17在小鼠单纯疱疹性角膜基质炎模型中的表达。方法:将1.0×106空斑单位的单纯疱疹病毒1型KOS毒株接种于BALB/c鼠的角膜上,建立HSK动物模型。免疫组织化学染色观察IL-17在角膜中的表达。分别取正常小鼠及接种病毒后的第1～3,7,10,14,21,28d,用毛细管取小鼠的左眼眼眶静脉窦血1mL,分离淋巴细胞,行荧光抗体染色,用流式细胞仪检测IL-17阳性的CD4+T细胞的表达。在裂隙灯显微镜下观察角膜的变化,检查角膜的组织学病理改变。结果:实验组中,角膜和外周血均有IL-17表达。实验组角膜基质内炎性细胞、角膜混浊程度非常严重。IL-17的表达与HSK小鼠炎症表现的严重程度成正相关(r=0.609,P<0.01)。结论:IL-17在HSK小鼠模型中呈高表达,且IL-17在HSK的发病过程中起一定作用。     

IL-10对复发性单纯疱疹性角膜基质炎抑制作用的实验研究

目的 研究白细胞介素10（IL-10）在复发性单纯疱疹性角膜基质炎（SK）中的作用。方法 采用复发性单纯疱疹性SK的BALB/c鼠模型，用紫外线B光照射鼠的角膜诱导SK复发。自紫外线照射角膜之前的6h开始，向鼠的角膜内注射重组IL-1010ng，共7次，观察IL-10对复发性S听影响。结果 IL-10减轻了复发性S听角膜混浊程度，减少了角膜内炎性细胞浸润，降低了角膜内IL-1α和IL-6的含量。IL-10没有影响SK的复发率，角膜病毒滴度及迟发型超敏反应。结论 IL-10能抑制角膜细胞产生某些细胞因子：IL-10能抑制复发性单纯疱疹性SK的发展。     

Cytokine production in a murine model of recurrent herpetic stromal keratitis

PURPOSE: To determine the pattern of cytokine production in the cornea and its relationship with viral antigens, in our murine model of recurrent ocular herpes simplex virus (HSV)-1 infection. METHODS: Six weeks after corneal inoculation with HSV-1, the eyes of latently infected and control mice were UV irradiated and examined for signs of disease and viral reactivation. The eyes of five mice with recurrent stromal disease and two controls were processed for immunohistochemistry on days 4, 7, 10, and 14 after irradiation. Sections were double stained for viral antigens and one of the following cytokines: interleukin (IL)-1ss, IL-2, IL-4, IL-6, IL-10, IL-12, and interferon (IFN)-gamma. RESULTS: Fifty percent of mice showed signs of recurrent stromal disease, the severity of which peaked on day 10 after UV irradiation. There was a large cellular infiltrate in the stroma of all the corneas with recurrent disease and the predominant cytokines were IL-1ss, IL-6, IL-10, IL-12, and IFN-gamma, all present in large numbers of cells on the days studied. There were very few cells producing IL-2 and IL-4. Control eyes had no significant cytokine-producing cells in the stroma. CONCLUSIONS: These observations suggest that recurrent herpetic stromal keratitis (HSK) may not be characterized by a classic T-helper (Th)1 or Th2 response. However, the large number of IFN-gamma(+) and IL-12(+) cells and the relative absence of IL-4 favors a Th1 response, and despite the numerous IL-10(+) cells, the overall balance of cytokine production appears to be proinflammatory.