Intermediate filament expression by normal and diseased human corneal epithelium

Cicatricial conjunctivitis may be a sequel to systemic disorders (eg, Stevens-Johnson syndrome, cicatricial pemphigoid) or local disorders such as chemical burns. The cicatrisation is often associated with corneal epithelial changes that cause visual loss. These have been attributed to encroachment of the conjunctival epithelium over the cornea. However, the epithelial anomalies are poorly understood. We investigated the corneal epithelial changes in cicatricial conjunctivitis with an immunohistochemical study of intermediate filaments in normal and pathological specimens. Our results show that the normal corneal epithelium is immunoreactive for cytokeratin 3 (CK 3) but not cytokeratin 19 (CK 19), whereas normal conjunctival epithelium is CK 3 negative and CK 19 positive. Conjunctiva artificially transposed over the cornea (after therapeutic conjunctival flap reconstruction) retained the normal pattern of conjunctival cytokeratin expression (CK 3 negative, CK 19 positive). Conversely, the entire corneal epithelium exhibited the normal cytokeratin pattern (CK 3 positive, CK 19 negative) in 82% of Stevens-Johnson, 80% of cicatricial pemphigoid, and 69% of chemical burns specimens. The findings suggest that conjunctival encroachment is not responsible for the changes at the corneal surface in cicatricial conjunctivitis and that the abnormal corneal epithelium is derived from native corneal cells in these diseases.     

Patterns of cytokeratins and vimentin in guinea pig and mouse eye tissue: evidence for regional variations in intermediate filament expression in limbal epithelium.

The anatomical distribution of different individual cytokeratin polypeptides and of vimentin was investigated by means of immunofluorescence with 41 monoclonal antibodies in guinea pig and mouse eyes. Simple epithelial type cytokeratins 7, 8, 18, and 19 selectively decorated conjunctival goblet cell clusters in mouse specimens and a continuous superficial cell layer of the corresponding part of guinea pig conjunctiva. A changed pattern of squamous epithelial type cytokeratins was found in the limbal region of the guinea pig eye as compared to the corneal epithelium. Cytokertains 3 and 17, which stained the entire corneal epithelium, were not detected, whereas cytokeratin 4, 5 and 13 were expressed. A focal vimentin and cytokeratin coexpression in the limbus of guinea pig is interpreted as indicating corneal stem cells. Similar patterns of expressions were found in the mouse ocular surface. In both species, a cytokeratin 4 staining of basal conjunctival epithelial cells could be detected. The neuroectodermally derived epithelia of the eye such as the retinal pigment epithelium and the ciliary body epithelia expressed solely the cytokeratin pair 8/18.     

Interleukin-1 receptor mediates the interplay between CD4+ T cells and ocular resident cells to promote keratinizing squamous metaplasia in Sjögren's syndrome

Keratinizing squamous metaplasia (SQM) of the ocular mucosal epithelium is a blinding corneal disease characterized by the loss of conjunctival goblet cells (GCs), pathological ocular surface keratinization and tissue recruitment of immune cells. Using the autoimmune regulator (Aire)-deficient mouse as a model for Sj?gren's syndrome (SS)-associated SQM, we identified CD4(+) T lymphocytes as the main immune effectors driving SQM and uncovered a pathogenic role for interleukin-1 (IL-1). IL-1, a pleiotropic cytokine family enriched in ocular epithelia, governs tissue homeostasis and mucosal immunity. Here, we used adoptive transfer of autoreactive CD4(+) T cells to dissect the mechanism whereby IL-1 promotes SQM. CD4(+) T cells adoptively transferred from both Aire knockout (KO) and Aire/IL-1 receptor type 1 (IL-1R1) double KO donors conferred SQM to severe-combined immunodeficiency (scid) recipients with functional IL-1R1, but not scid recipients lacking IL-1R1. In the lacrimal gland, IL-1R1 was primarily immunolocalized to ductal epithelium surrounded by CD4(+) T cells. In the eye, IL-1R1 was expressed on local mucosal epithelial and stromal cells, but not on resident antigen-presenting cells or infiltrating immune cells. In both tissues, autoreactive CD4(+) T-cell infiltration was only observed in the presence of IL-1R1-postive resident cells. Moreover, persistent activation of IL-1R1 signaling led to chronic immune-mediated inflammation by retaining CD4(+) T cells in the local microenvironment. Following IL-1R1-dependent infiltration of CD4(+) T cells, we observed SQM hallmarks in local tissues-corneal keratinization, conjunctival GC mucin acidification and epithelial cell hyperplasia throughout the ocular surface mucosa. Proinflammatory IL-1 expression in ocular epithelial cells significantly correlated with reduced tear secretion, while CD4(+) T-cell infiltration of the lacrimal gland predicted the development of ocular SQM. Collectively, data in this study indicated a central role for IL-1 in orchestrating a functional interplay between immune cells and resident cells of SS-targeted tissues in the pathogenesis of SQM.     

Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells

BACKGROUND: Stevens-Johnson syndrome, ocular pemphigoid, and thermal or chemical burns can cause scarring and opacification of the cornea and loss of vision. Transplantation of epithelial cells from the limbus of the contralateral cornea can restore useful vision. However, this procedure requires a large limbal graft from the healthy eye and is not possible in patients who have bilateral lesions. METHODS: We took specimens of limbal epithelial cells from the healthy contralateral eyes of six patients with severe unilateral corneal disease. The epithelial cells were cultured and expanded on amniotic membrane. The amniotic membrane, together with the sheet of limbal epithelial cells, was transplanted to the denuded corneal surface of the damaged eye after superficial keratectomy to remove fibrovascular ingrowth. The mean (+/-SD) follow-up period was 15+/-2 months. RESULTS: Complete reepithelialization of the corneal surface occurred within two to four days of transplantation in all six eyes receiving transplants. By one month, the ocular surface was covered with corneal epithelium, and the clarity of the cornea was improved. In five of the six eyes receiving transplants (83 percent), the mean visual acuity improved from 20/112 to 20/45. In one patient with a chemical burn who had total opacification of the cornea, the acuity improved from the ability to count fingers at 40 cm to 20/200. No patient had recurrent neovascularization or inflammation in the transplanted area during the follow-up period. CONCLUSIONS: Transplantation of autologous limbal epithelial cells cultured on amniotic membrane is a simple and effective method of reconstructing the corneal surface and restoring useful vision in patients with unilateral deficiency of limbal epithelial cells.     

氢化可的松对小鼠角膜、结膜上皮中郎格罕细胞的影响

郎格罕细胞（LC）在角膜排斥反应中起着重要的作用，若能在角膜移植前后选择性地抑制LC的功能活性，有可能延长移植角膜的存活期。本实验选用免疫抑制剂－氢化可的松处理小鼠眼球，利用酶组化－ATP酶法和图像分析仪，观察其对角膜，结膜上皮内LC密度以及酶活性的影响，结果显示，随着氢化可的松浓度的增大，LC内ATP酶反应程序，LC密度显示下降趋势，当氢化可的松浓度增加至1．7mg/ml时，除角巩缘外，周边角膜和结膜已无ATPase阳性的LC，当浓度达到2．0mg/ml时，角巩缘也很少见ATPase阳性的LC。     

Ultrastructural observations on experimentally produced melanin pigmentation of the corneal epithelium.

Melanin pigmentation of the corneal epithelium was induced in pigmented guinea pigs by the topical application of colchicine to their eyes or by corneal cauterization with silver nitrate. With colchicine the pigmentation was preceded by the development of an abnormal corneal epithelium in which numerous cells became arrested in cell division. The corneal melanosis resulted largely from the migration of melanocytes into the corneal epithelium from the normally pigmented contiguous conjunctiva and to a lesser extent from the presence of melanin granules within corneal epithelial cells. In both models a leukocytic and vascular invasion of the cornea proceded and accompanied the migration of melanocytes into the corneal epithelium. Electron microscopy disclosed cells with the same morphology as conjunctival melanocytes between the epithelial cells of the cornea. Mature melanin granules were also present within some squamous epithelial cells as individual granules or as clusters. The ultrastructural findings are viewed in relation to how melanin granules are transferred from melanocytes to epithelial cells. Evidence is presented which suggests that malanin granule transfer may follow the fusion of the membranes of the melanocytes and epithelial cells.     

Morphological and Autoradiographic Studies on the Corneal and Limbal Epithelium of Rabbits

The investigation was centered on the morphological features of the conjunctiva–cornea transition (limbus) of the rabbit eye and the proliferative behavior of its epithelium. The eyes were processed for examination with light and electron microscopy, as well as for autoradiography after intravitreal injection of [³H]thymidine ([³H]TdR). At the sites of extraocular muscle insertion, the vascularization of the stroma extended to the peripheral cornea, and the limbal epithelium was thin with its basal stratum made up by clear cuboidal cells. In between the muscle insertions, the cuboidal clear cells, as well as the stroma blood vessels, were scarce. At the light microscope level, the basement membrane was distinct in the cornea but not in the limbus or the conjunctiva. Autoradiographs demonstrated that, at the limbus, the basal cells migrated very quickly to the suprabasal region and remained there up to the 28‐day interval. Labeled cells were identified in all epithelial layers of the cornea, including the basal one, at 21 and 28 days but not in the limbal basal clear cells. The rate of renewal of conjunctival epithelium was similar to that observed for the transition with scarce clear cells. The high‐resolution autoradiographs demonstrated that the basal cuboidal clear limbal cells exhibit a quick renewal and that they are not label‐retaining cells. These latter ones were detected all over the corneal epithelium and in the suprabasal layers of the limbus up to 28 days, in physiological conditions, without the need of stimulation by damage to the corneal epithelium. Anat Rec, 291:191–203, 2008. © 2008 Wiley‐Liss, Inc.     

活体及体外条件下对角膜上皮干细胞的定位

背景：眼表存在两种形式的上皮干细胞即角膜上皮干细胞和结膜上皮干细胞,角膜上皮干细胞在角膜上皮细胞更新和角膜透明的维持方面起着重要作用。 目的：采用活体激光扫描角膜共焦显微镜和免疫荧光染色技术相结合的方法,从活体和体外层面上对角膜上皮干细胞进行定位研究。 方法：收集2009年9月至2012年9月来河南省眼科研究所就诊的单侧角膜缘干细胞缺乏患者,使用活体激光扫描角膜共焦显微镜检查患者双眼,健侧眼为对照。扫描方位依次为中央角膜及上、下、左、右方的角膜缘,记录扫描图像并分析。眼球材料来自于河南省眼库,切取角膜中央和角膜缘组织,组织包埋剂包被、冰冻切片,切片厚度5-7 μm;免疫荧光染色技术检测p63、ABCG2、K3和Connexin 43在角膜中央及角膜缘上皮层的表达。 结果与结论：共有24例患者确诊为单侧角膜缘干细胞缺乏,活体激光扫描角膜共焦显微镜下患侧眼角膜病变区可见结膜细胞及杯状细胞;角膜缘区域Vogt栅栏状结构消失,色素细胞消失,取而代之的是大量纤维瘢痕化组织。免疫荧光染色示表达ABCG2和p63的细胞主要在角膜缘上皮基底层,尤其在近结膜侧的角膜缘及角膜缘中间部表达相对较高,而中央角膜上皮层细胞不表达;K3及Connexin43在角膜缘上皮基底细胞层不表达,中央角膜上皮全层表达。通过活体激光扫描角膜共焦显微镜观察及干细胞标记物检测显示角膜上皮干细胞主要存在于角膜缘外2/3区域的Vogt栅栏基底部及钉突结构中。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

Localization and expression of chondromodulin-I in the rat cornea

The localization and expression in the rat cornea of chondromodulin-I (ChM-I), an inhibitory angiogenesis factor, were examined by immunohistochemistry, Western blot analysis, ribonuclease protection assay, and real-time PCR assay. We found immunoreactivity for ChM-I in the epithelial layer but not the stromal layer or endothelial layer in the cornea, in addition to the positive ChM-I immunoreactivity in other sites in the eye such as the sclera, retina, and ciliary body. The ChM-I immunoreactivity was most intense at the outside of the basal cells and in their cytoplasm while the intensity of the immunoreactivity decreased gradually from the wing cells to the superficial cells in the corneal epithelial layer. No reactivity however, was detected in the Bowman's membrane or conjunctival epithelial cells which had continuity with the corneal epithelial cells. The expression of ChM-I mRNA was demonstrated in the cornea at one-third less intensity than that in the sclera with choroids and retinal pigment epithelium by ribonuclease protection assay and real-time PCR. ChM-I in the corneal epithelial layer may prevent neovascularization and maintain avascularity in the cornea.     

Porous matrix and primary-cell culture: a shared concept for skin and cornea tissue engineering

Skin and cornea both feature an epithelium firmly anchored to its underlying connective compartment: dermis for skin and stroma for cornea. A breakthrough in tissue engineering occurred in 1975 when skin stem cells were successfully amplified in culture by Rheinwald and Green. Since 1981, they are used in the clinical arena as cultured epidermal autografts for the treatment of patients with extensive burns. A similar technique has been later adapted to the amplification of limbal-epithelial cells. The basal layer of the limbal epithelium is located in a transitional zone between the cornea and the conjunctiva and contains the stem cell population of the corneal epithelium called limbal-stem cells (LSC). These cells maintain the proper renewal of the corneal epithelium by generating transit-amplifying cells that migrate from the basal layer of the limbus towards the basal layer of the cornea. Tissue-engineering protocols enable the reconstruction of three-dimensional (3D) complex tissues comprising both an epithelium and its underlying connective tissue. Our in vitro reconstruction model is based on the combined use of cells and of a natural collagen-based biodegradable polymer to produce the connective-tissue compartment. This porous substrate acts as a scaffold for fibroblasts, thereby, producing a living dermal/stromal equivalent, which once epithelialized results into a reconstructed skin/hemicornea. This paper presents the reconstruction of surface epithelia for the treatment of pathological conditions of skin and cornea and the development of 3D tissue-engineered substitutes based on a collagen-GAG-chitosan matrix for the regeneration of skin and cornea.     

Restoration of the rabbit corneal surface after total epithelial debridement and complete limbal excision

How is the corneal epithelium restored when all of it plus the limbus have been eliminated? This investigation explored the possibility that this may be achieved through the conjunctival epithelium. The corneal epithelium of the right eye of 12 rabbits (Oryctolagus cuniculus) was totally scraped followed by surgical excision of the limbus plus 1.0-1.5 mm of the adjacent conjunctiva. Antibiotics and corticosteroids were applied for 1 week after surgery. Histological and immunohistochemical techniques were used to monitor the events taking place on the eye surface 2 weeks and 1, 3 and 6 months thereafter. Initially, the corneal surface was covered by conjunctival-like epithelium. After 1 month and more prominently at 3 and 6 months an epithelium displaying the morphological features of the cornea and reacting with the AE5 antibody was covering the central region. It is likely that the corneal epithelium originated from undifferentiated cells of the conjunctiva interacting with the corneal stroma.     

Interleukin-17 in various ocular surface inflammatory diseases

Recently, the association of Th-17 cells or IL-17 with ocular inflammatory diseases such as uveitis, scleritis and dry eye syndrome was discovered. We assessed whether interleukin (IL)-17 was present in the tears of various ocular surface inflammatory diseases and the tear IL-17 concentrations were clinically correlated with various ocular surface inflammatory diseases. We measured concentrations of IL-17 in tears of normal subjects (n = 28) and patients (n = 141) with meibomian gland dysfunction (MGD), dry eye syndrome (DES), Sjögren syndrome (SS), Stevens-Johnson syndrome (SJS), graft-versus-host disease (GVHD), filamentary keratitis, and autoimmune keratitis associated with rheumatoid arthritis or systemic lupus erythematosus. Clinical epitheliopathy scores were based on the surface area of corneal and conjunctival fluorescein staining. The mean concentrations of IL-17 in tears of patients with filamentary keratitis, GVHD, autoimmune keratitis, SS, DES, MGD, SJS were significantly higher in order than that in normal subjects. Tear IL-17 concentration was significantly correlated with clinical epitheilopathy scores in the patients with systemic inflammatory disease, while tear IL-17 was not correlated with clinical severity of the cornea and conjunctiva in the dry eye patients without any systemic inflammatory disease. Tear IL-17 is likely to correlate clinically with corneal disease severity only in the patients with systemic inflammatory disease.     

Comparative anatomy of mammalian conjunctival lymphoid tissue: a putative mucosal immune site

Organized mucosa-associated lymphoid tissue (O-MALT) is defined by mucosal lymphoid follicles with unique overlying lymphoepithelia, and classically appears in tissues with a simple columnar epithelium. Within follicle-associated epithelium, goblet cells are characteristically absent, replaced by ultrastructurally distinct antigen-absorptive cells, termed M cells (or microfold cells) for the appearance of their apical cell membranes. To determine if mammalian conjunctiva, with its stratified squamous epithelium, can be considered as a site of O-MALT, we compared the light and electron microscopic anatomy of conjunctiva from fourteen species of non-human adult mammals, and the conjunctiva of human adults harvested at autopsy. Lymphoid follicles in the conjunctiva were demonstrated in all mammals studied except for mice and rats. In those mammals with conjunctival lymphoid follicles, the follicle-associated conjunctival epithelium was notable for an absence of goblet cells. Transmission electron microscopy demonstrated an intimate association of lymphocytes with surface epithelial cells, but epithelial cell morphology was uniform overlying the follicle, and other ultrastructural features of M cells were absent. Therefore, conjunctival lymphoid follicle-associated stratified squamous epithelium demonstrates some but not all features of O-MALT lymphoepithelia. Further studies are necessary to determine what role conjunctival lymphoid tissue may play in mucosal immunity.     

Conjunctivochalasis is the precursor to pterygium

Pterygium is a fibrovascular proliferative condition of the ocular surface with no known pathological mechanism. This condition affects vision due to dry eyes, astigmatism or physical occlusion of the visual axis for severe cases. The only definitive treatment for this condition is surgical excision. Interestingly, it is a lesion that may be related to UV radiation and elaboration of proteases. Conjunctivochalasis is a dry eye related condition that is exemplified by excessive conjunctiva or the mucous membrane of the front of the eye around the cornea. Both pterygium and conjunctivochalasis are associated with elaboration of matrix metalloproteinases as well as inflammatory cytokines. We propose that under specific conditions, conjunctivochalasis in the nasal part of the conjunctiva can progress to pterygium. The progression of conjunctivochalasis to pterygium may be related to special kinds of oxidative or inflammatory damage that affects only the part of the loose conjunctival tissue adjacent to the cornea. Protease expressed may then breakdown the conjunctival and corneal epithelium causing the head of pterygium to be very adherent to the cornea. This explains the fact that surgically excised pterygium tissue has stromal tissue enclosed by epithelia on both surfaces. In addition, it explains the existence of a surgical plane when an instrument is passed under the neck of the pterygium tissue but not at the apex. The implications of this hypothesis are first, treatment should be directed to the protection of conjunctivochalasis before it transforms to pterygium. This may be achieved by anti-inflammatory measures, anti-protease treatment, or preventing the triggering of the changes at the head of pterygium, such as avoidance of sunlight. Second, during resection of pterygium, it may not be necessary to resect the pterygium too extensively away from the cornea, since this effectively removes relatively normal conjunctiva.     

Using acellular porcine limbal stroma for rabbit limbal stem cell microenvironment reconstruction

To investigate the feasibility of using acellular porcine limbal stroma for limbal stem cell microenvironment reconstruction. Limbal reconstruction was performed in rabbit partial limbal defect models. Rabbits were randomly divided into four groups: acellular porcine limbal stroma, de-epithelized rabbit limbal autograft stroma, de-epithelized porcine limbal stroma and acellular porcine corneal stroma transplantation groups. In both the acellular porcine limbal stroma and de-epithelized rabbit limbal autograft stroma groups, cornea transparency and epithelium integrity were sustained and graft rejection was not observed. The basal epithelial cells of the grafts showed the K3+/P63+/Ki67+ phenotype at postoperative month 1, but it returned to K3-/P63+/Ki67+(phenotype characteristic of limbal epithelium) by postoperative months 3 and 6. In the de-epithelized porcine limbal stroma group, acute and serious immune rejection occurred by postoperative days 8-10. The basal epithelial cells of the grafts showed the K3+/P63+/Ki67+ phenotype at postoperative month 1. In the acellular porcine corneal stroma group, there were some new vessel invasion into the peripheral cornea and mild corneal opacity. The basal epithelial cells of the grafts showed the K3+/P63+/Ki67+ phenotype at postoperative months 1, 3, and 6. In conclusion, acellular porcine limbal stroma possessed very low immunogenicity, retained a good original limbal ECM microenvironment, and thus the reconstructed rabbit limbal microenvironment maintained limbal epithelial stem cell stemness and proliferation.     

Dendritic cell-epithelium interplay is a determinant factor for corneal epithelial wound repair

The functions of intraepithelial dendritic cells (DCs) are critical for mucosal innate and adaptive immunity, but little is known about the role of tissue-specific DCs in epithelial homeostasis and tissue repair. By using the epithelial debridement wound model and CD11c-diphtheria toxin receptor mice that express a CD11c promoter-driven diphtheria toxin receptor, we showed that DCs migrate along with the epithelial sheet to cover the wound and that local depletion of DCs resulted in a significant delay in epithelial wound closure. In response to wounding, migratory epithelia produce CXCL10, thymic stromal lymphopoietin, and IL-1β and its antagonist soluble IL-1 receptor antagonist (sIL-1Ra); depletion of corneal DCs reversed their elevated expressions to a different extent, suggesting a DC-mediated positive feedback loop in epithelial gene expression. Furthermore, both CXCL10 and thymic stromal lymphopoietin were localized in migratory epithelia, suggesting that epithelial cells play a key role in DC infiltration and activation in injured corneas. On the other hand, DC depletion resulted in suppressed epithelial AKT activation, increased cell apoptosis, and decreased polymorphonuclear leukocyte infiltration in the healing cornea. These results indicate that DCs and epithelium form a functional entity at mucosal surfaces for maintaining corneal homeostasis and for tissue repair.     

Detection of measles antigen in conjunctival epithelial lesions staining by lissamine green during measles virus infection

In a study in Kenya of corneal lesions occurring in children with measles virus infection, conjunctival lesions of a characteristic appearance were seen concomitant with the rash. The lesions became visible after staining by Lissamine green or Rose Bengal. In order to investigate whether these lesions were virus-induced, paired biopsies were performed on the conjunctiva of five measles patients. One biopsy was taken of a staining lesion; the other one, taken of nonstaining conjunctiva of the same eye, served as control. Measles-specific immunofluorescence on cryostate sections of the biopsies showed that in all patients the antigen occurred in the conjunctival epithelial cell layer in the staining lesions only. The coincidence of epithelial staining and presence of measles antigen suggests a viral origin of the conjunctival lesion. Fluorescent antibody technique on cryostate sections proved to be a suitable method to demonstrate the presence of virus where isolation fails and serology is not applicable.