Regulation of HLA class II antigen expression on cultured corneal epithelium by interferon-gamma.

The effect of recombinant human interferon gamma (IFN-gamma) on the induction of HLA class II (HLA-DR, -DP, -DQ) antigen expression on human corneal epithelial (HCE) cells was examined in different stages of culture. Primary cultures were established with limbal explants without endothelium. HCE cells in Stage 1 and Stage 2, with cells negative and positive for the 64K corneal keratin (the marker for advanced corneal epithelial differentiation), respectively, were prepared. HCE cells in both stages were treated with IFN-gamma at a concentration of 0 to 1000 U/ml for two to six days and were stained by the avidin-biotin peroxidase complex method. Class II antigens were not detected on HCE cells in either stage without IFN-gamma treatment. IFN-gamma induced three class II antigens on HCE cells in both stages in a dose- and time-dependent manner but at different levels for each antigen (DR greater than DP greater than DQ). In addition, DQ expression was related to cell differentiation, with DQ extremely rare at Stage 1 and more frequent at Stage 2 (5% vs. 20%). These findings indicate that the induction of class II antigens on HCE cells may be regulated by IFN-gamma independently for each of the antigens and that DQ induction may depend upon the differentiation of HCE cells in culture.     

HLA antigenicity of normal and pathological corneas

Fresh human corneas and corneal buttons were studied for expression of HLA antigens. Using monoclonal antibodies in an indirect immunofluorescence assay, corneal layers were examined for class I (HLA-A, B, C) and class II (HLA-DR) histocompatibility antigens. Twenty-one human corneas were studied, 6 normal and 15 pathological: 4 buttons of allograft rejection, 9 buttons of pseudophakic bullous keratopathy. In fresh control corneas, HLA-A, B, C antigens were localized on corneal epithelium and on stromal keratocytes but were never found on endothelial cells. HLA-DR antigens were not detected on corneal epithelium, stroma or endothelium but were detected on Langerhans cells within epithelium and anterior stroma. At the corneal limbus, HLA class I-II antigens were expressed on vascular endothelium. HLA antigen distribution was modified in pathological corneas. Antigens HLA-A, B, C were induced on endothelial cells of rejected corneal allografts. Antigens HLA-DR were detected on epithelial cells, cells in the stroma of pseudophakic bullous keratopathy and also on endothelial cells of rejected corneal allografts. These results suggest that induction of class I and II antigen expression by inflammatory factors may occur in vivo. In rejected corneal allografts induction of HLA-DR antigen on corneal layers would intensify the process of rejection. This study and others have demonstrated the ability of modulation of HLA antigen expression on human corneal cells in vivo.     

Existence of small slow-cycling Langerhans cells in the limbal basal epithelium that express ABCG2

Despite the obvious importance of limbal stem cells in corneal homeostasis and tumorigenesis, little is known about their specific biological characteristics. The purpose of this study was to characterize limbal slow-cycling cells based on the expression of ABCG2 and major histocompatibility complex (MHC) class II and the cell size. Wistar rats were daily injected with 5-bromo-2-deoxyuridine (BrdU) at a dose of 5 mg/100 g for 2 weeks. After 4-week BrdU-free period, corneal tissues were excised, and immunofluorescence staining for ABCG2, BrdU, and MHC class II was performed by confocal microscopy. In another series, corneal tissues of normal rat were double immunostained for ABCG2, keratin 14, keratin 3, CD11c, and MHC class II. In addition, limbal, peripheral and central corneal epithelial sheets were isolated by Dispase II digestion and dissociated into single cell by trypsin digestion and cytospin preparations were double immunostained for ABCG2 and MHC class II. The cell size and nucleus-to-cytoplasm (N/C) ratio of limbal ABCG2+ cells were analyzed and compared with those of cells from other zones. BrdU label-retaining cells (LRCs) with expression of ABCG2 were found in the limbal epithelial basal layer, but not in other parts of the cornea. Approximately 20% of these cells were MHC class II positive. All MHC class II+ cells in the corneal epithelium were positive for CD11c, a marker for dendritic cells (DCs). Double labeling with ABCG2 and keratin 14 showed that nearly four-fifth of limbal ABCG2+ cells were positive for keratin 14 but negative for keratin 3, exhibiting an undifferentiated epithelial cell lineage. Cytospin sample analysis revealed the presence of a distinct population of smaller ABCG2+ cells with expression of MHC class II with a larger N/C ratio in the limbal epithelium. A new population of small slow-cycling cells with large N/C ratio has been found to express ABCG2 in the limbal epithelial basal layer. Some of these cells normally express MHC class II antigen. These findings may have important implications for our understanding of the characteristics of limbal slow-cycling cells.     

Expression of ABO blood group, hematopoietic, and other cell-specific antigens by cells in the human cornea

Because of controversy surrounding the expression of ABO blood group (ABH) antigens (which may act as transplantation antigens) in human cornea, we examined the expression of these antigens in a panel of normal corneas using a highly sensitive avidin-biotin complex immunoperoxidase technique. ABH antigens were expressed by the corneal epithelium from all donors in a pattern consistent with their red blood cell phenotype, but were absent from stroma and endothelium. We also examined the same corneas for cells expressing hematopoietic cell markers in an effort to determine which marrow-derived cells may be contributing (as "passenger leukocytes") to the immunogenicity of corneal allografts. Scattered cells positive for a pan-hematopoietic cell marker (T29/33) were present throughout all corneas, but no consistent expression was noted of markers for B lymphocytes, monocytes, or NK cells. Adult corneas showed occasional T lymphocytes, predominantly in the limbus. Finally, we looked for the expression by these corneas of a variety of cell-specific and structural antigens, to identify possible relationships between corneal cells and analogous cell populations. We found that corneal endothelial cells expressed one vascular endothelial antigen, (HuEE12), but not another (Factor VIII related antigen), suggesting a possible relationship between corneal endothelium and its immunologically important vascular counterpart. Various structural antigens were found in predicted associations with corneal endothelial, stromal, and epithelial cells. These findings cast new light on the antigens involved in corneal allograft reactions and the immunologic nature of some constituent corneal cells.     

Recombinant interferon-gamma induces HLA-DR expression on human corneal epithelial and endothelial cells in vitro: a preliminary report.

The effect of interferon-gamma on the expression in situ of major histocompatibility complex (MHC) products in human corneas was studied in vitro. Incubation for four days with 5 or 50 mg/l of Escherichia coli-derived recombinant human interferon-gamma resulted in the appearance de novo of MHC class II or HLA-DR antigens on variable numbers of corneal epithelial cells as well as on corneal endothelium, whereas it had no effect on the expression of MHC class I or HLA-ABC antigens. These results may help to explain the mechanism underlying the expression of HLA-DR antigens on corneal and limbal epithelium in various inflammatory eye diseases.     

Expression of vascular endothelial growth factor and its receptors in inflamed and vascularized human corneas

PURPOSE: To help further define the possible role of vascular endothelial growth factor (VEGF) in the pathogenesis of corneal neovascularization, the expression of VEGF and of its receptors Flt-1 and Flk-1 was investigated in various inflammatory corneal diseases. METHODS: Polyclonal antibodies to VEGF and its receptors were used for immunohistochemical staining of frozen sections of 38 human corneas with various degrees of neovascularization and inflammation. In addition, a panel of monoclonal antibodies was used to characterize the composition of the inflammatory infiltrates and to confirm the presence of neovascularization. Furthermore, VEGF concentrations were determined in vascularized corneas using a sensitive enzyme-linked immunosorbent assay. RESULTS: VEGF was expressed by epithelial cells, by corneal endothelial cells, by vascular endothelial cells of limbal vessels and of newly formed vessels in the stroma, and weakly by keratocytes. Furthermore, VEGF expression was often markedly increased in inflamed corneas on epithelial cells and on vascular endothelial cells, particularly in the vicinity of macrophage infiltrates, and on fibroblasts in scar tissue. Correspondingly, VEGF concentrations were significantly higher in vascularized corneas compared with normal control corneas (P < 0.001). Expression of both VEGF receptors, Flt-1 and Flk-1, was increased on endothelial cells of newly formed vessels in the stroma of inflamed corneas compared with limbal vessels of normal control corneas. In addition, Flt-1 was also expressed by corneal endothelial cells and by macrophages, whereas Flk-1 expression was lacking. CONCLUSIONS: These results demonstrate that VEGF, Flt-1, and Flk-1 are strongly expressed in inflamed and vascularized human corneas and, thus, may play an important role in corneal neovascularization.     

角膜上皮树突状细胞免疫学特性研究的现状

角膜上皮内存在的朗格罕细胞型树突状细胞具有吞噬、加工、递呈抗原及激发T细胞的能力.这些细胞是"职业的"抗原递呈细胞,在眼表免疫系统中起关键的免疫监视作用.近期,有研究证明在正常的角膜缘上皮基底层存在具有慢周期特性的、表达角膜缘干细胞标识物-ATP粘连转运蛋白家族G2成员(ABCG2)的树突状细胞.进一步研究发现,中央角膜炎症反应早期可以诱导角膜缘上皮基底层表达主要组织相容性复合体Ⅱ型抗原的树突状细胞密度增加并向角膜中央迁移.这些研究提示角膜缘上皮基底层内存在的树突状细胞是角膜炎症反应发生过程中的重要参与者.     

角膜缘微环境细胞的研究进展

角膜缘微环境细胞（limbal niche cell,LNC）是近年来发现的一种来自于角膜缘干细胞微环境且具有血管内皮祖细胞和间充质干细胞特性的多能干细胞。LNC具有分化为血管内皮细胞、角膜上皮细胞、角膜基质细胞等多种细胞的潜能。体外实验发现,LNC在三维立体培养环境中可以维持角膜缘干细胞的特性;动物实验发现,LNC可以预防和治疗碱烧伤所致的角膜缘干细胞缺乏。LNC还可以促进角膜基质的无瘢痕修复,减少修复过程中的新生血管化,提示其可以重建角膜基质。因此,在角膜缘干细胞缺乏、角膜基质损伤等疾病的治疗中,LNC可能是一种理想的治疗细胞。     

Comparison of cell-suspension and explant culture of rabbit limbal epithelial cells

Currently, most investigators directly use limbal explants to culture corneal epithelial cells. However, it has not been identified that limbal stem cells do readily migrate from the limbal explants onto culture plate or amniotic membrane carrier. In this study a cell-suspension culture system for rabbit limbal stem cells was developed and compared with the direct explant method in the aspect of stem cells content in the culture system. Rabbit limbal epithelial cells were dissociated from rabbit eyes by dispase and single cell suspension was made for cell-suspension culture. DeltaNp63 expression of cultured rabbit limbal epithelial cells by cell-suspension technique and explant technique was detected. In cell-suspension culture, isolated cell-suspension was evaluated by flow cytometric analysis for vimentin expression and residual limbal tissue after dispase treatment was examined by scanning electron microscopy. In limbal epithelial cells suspension less than 5% cells were vimentin positive. Examination of residual limbal tissue confirmed that all the limbal epithelial cells had been removed. Histological examination revealed that with cell-suspension culture the cultured epithelial cells could differentiate better than with explant technique. In cells cultured with cell-suspension, there were much more cells expressing DeltaNp63 than in explant cultured cells. In cells cultured with explants, most of DeltaNp63 labelling cells mustered around the explants, and peripheral cells on the slides were DeltaNp63 negative. These results suggested that with pure limbal epithelial cells suspension including basal cells, which could directly enter into culture system, cell-suspension culture technique was significantly superior to explant culture technique in terms of stem cells content.     

Modulation of HLA antigen expression on corneal epithelial and stromal cells

Human corneal epithelial cells and stromal fibroblasts in culture were incubated with gamma interferon or with medium conditioned by phytohemagglutinin (PHA)-stimulated mononuclear cells. The corneal cells were placed into suspension, assayed for class I (HLA-A,B,C) and class II (HLA-DR) antigens by indirect immunofluorescence, and analyzed with flow cytometry. Epithelial cells treated for 5 days with conditioned medium (CND-M) did not exhibit an increase in class I or an induction of class II antigen expression, although a trend toward increased class I antigen expression was present. Epithelial cells treated for 5 days with 250-500 U/ml of gamma interferon did not demonstrate an increase in class I but did show an induction of class II antigen expression; again, however, a trend toward increased class I antigen expression was present. Stromal fibroblasts treated for 3-5 days with CND-M exhibited an increase in class I antigen expression, but stromal fibroblasts treated for 1-5 days with CND-M did not show an induction of class II antigen expression. Stromal fibroblasts incubated for 1-5 days with 250-750 U/ml of gamma interferon demonstrated both an increase in class I and an induction of class II antigen expression. These data suggest that host lymphokines may intensify the process of corneal graft rejection by augmenting class I antigen expression on allogeneic cells. Moreover, the induction of class II antigen expression by host lymphokines on cells in transplanted corneal tissue may lead to host sensitization and subsequent allograft rejection.     

Epithelial cell characteristics of cultured human limbal explants

AIM: To determine the immunohistochemical characteristics of putative corneal epithelial stem cells remaining on limbal explants maintained in culture. METHODS: Human limbal explant cultures were generated from 25 residual corneoscleral donor rims following penetrating keratoplasty. Serial sections of these explants were studied using immunohistochemical techniques with a panel of antibodies, on day 0 and 1, 2, and 3 weeks. RESULTS: The number of epithelial cells expressing cytokeratin 19 and vimentin increased with duration in culture, while the number of cells expressing cytokeratin 3 decreased. Connexin 43 expression was lost by 1 week in culture. p63 was expressed by cells that had migrated around the explant and the number of p63 positive cells decreased with longer duration in culture. The explants were initially negative for Ki67, but the epithelial cells were positive at 1 week, and expression of Ki67 was progressively lost with increasing duration in culture. The initial uniform staining of the epithelium for epidermal growth factor receptor and alpha enolase remained unchanged at 3 weeks. CONCLUSIONS: There is an expansion of less differentiated (cytokeratin 3 negative and CK19/vimentin positive) epithelial cells on corneoscleral explants maintained in culture for 3 weeks. The pattern of expression of p63 noted in this study does not support the suggestion that it is a marker of limbal stem cells. The decline in p63 and Ki67 expression among the epithelial cells of the cultured explant button implies that as the epithelial sheet outgrowing from the explant button reaches confluence, the proliferative status of the cells remaining on the explant button declines. These findings are of clinical relevance as explants of limbal tissue are used in limbal stem cell transplantation. There is no information available to date on the fate of epithelial cells on such explants. This study provides some insight into this and suggests that an expansion of the stem cell pool or its progeny may occur in limbal explants.     

Lipopolysaccharide from Escherichia coli induces the expression of vascular endothelial growth factor via toll-like receptor 4 in human limbal fibroblasts

Corneal neovascularization can be induced by a severe ocular infection, injury or immunological diseases. The vascular endothelial growth factor (VEGF) is the main cytokine involved in this phenomenon, inducing angiogenesis from the vascularized ocular tissues. As the limbal tissue is located between conjunctival and corneal tissues, we suggest that the limbal cells are participating in the production of VEGF induced by bacterial components as LPS. In this work, RT-PCRs and immunoblots were used to investigate the expression of VEGF and other pro-angiogenic genes in primary cultures of human limbal fibroblasts (PCHLF) treated with lipopolysaccharide (LPS) from Escherichia coli. We found that the expression of VEGF was initiated at 6 h and reaches its highest expression at 72 h after stimulation with LPS. Up-regulation of toll-like receptor 4 (TLR4) after 3 h of treatment was also observed. LPS-induced the expression of VEGF in a dose-dependent manner, and the blocking of TLR4 with an anti-TLR4 antibody prevented VEGF expression. We also analyzed the molecules that modulate VEGF expression. LPS did not induce the up-regulation of LL-37 nor the hypoxia induced factor 1 alpha (HIF-1alpha) mRNA expression, however, an up-regulation of interleukin 13 receptor alpha 1 (IL-13Ralpha1) and interleukin 4 receptor alpha (IL-4Ralpha) were observed after 3 and 12 h of stimulation, respectively. The expression of interleukin 13 did not change throughout the treatment. These results suggest that TLR4, IL-13Ralpha1 and IL-4Ralpha induced by LPS in PCHLF could be playing an important role in the corneal neovascularization.     

HLA-A,B,C, and HLA-DR antigens and dendritic cells in fresh and organ culture preserved corneas

Fresh and organ-culture preserved human corneas were examined for HLA-A,B,C (class I) and HLA-DR (class II) histocompatibility antigens using mouse monoclonal antibodies and an indirect immunofluorescence technique. HLA-A,B,C antigens were detected on epithelial cells and on keratocytes, but not on endothelial cells in fresh corneas. The expression of HLA-A,B,C antigens was not significantly altered by organ-culture for a period up to 7 days. The epithelial cell layer bearing the larger part of the HLA-A,B,C antigens decreased, however, from 6-7 layers to 2-3 during organ culture. HLA-DR antigens were not detected on any of the corneal layers, but were present on scattered dendritic cells within the corneal epithelium and on cells in the corneal stroma just beneath the Bowman's membrane. Using immunofluorescence, no cells bearing HLA-DR antigens were seen in cornea sections obtained after 1 week in organ-culture. These results demonstrate that the presence of HLA antigens, particularly of the HLA-DR antigens, is affected by the organ-culture preservation of the human cornea.     

Induction of class II (Ia) alloantigen expression on corneal endothelium in vivo and in vitro

Class II (DR or Ia) alloantigens are potent inducers of cell-mediated immune responses. However, the expression of Class II alloantigens on corneal cells, except for Langerhans cells within the limbal epithelium, has not previously been described. We have induced Ia expression on cultured rabbit corneal endothelial cells with human immune interferon, and have demonstrated numerous Ia-positive cells in the endothelium during primary immunogenic uveitis in vivo. Cultured endothelial cells which had been treated with immune interferon in vitro functioned as stimulator cells in a mixed leukocyte reaction. Induction of Class II alloantigen expression by lymphokines may potentiate corneal allograft rejection.     

Heterogeneous induction of major histocompatibility complex class II antigens on corneal endothelium by interferon-gamma

The expression and distribution of major histocompatibility complex (MHC) class II gene products, HLA-DR, HLA-DQ, and the HLA-DR invariant chain, were studied on flat mounts of human corneal endothelial cells (HCEC) after in vitro incubation of donor corneas with interferon-gamma (IFN-gamma), interleukin-1 (IL-1), and IL-6, using a sensitive immunoperoxidase technique with monoclonal antibodies. Control HCEC and endothelium treated with IL-1 or IL-6 completely lacked MHC class II antigens. After treatment with 50 U/ml, 100 U/ml, 500 U/ml, and 5000 U/ml of human IFN-gamma, a mosaic-like, patchy staining for all MHC class II products was observed: part of the HCEC showed membranous and/or cytoplasmic positivity; other endothelial cells were negative. In addition, a dose-dependent response to IFN-gamma was observed: the proportion of cells expressing class II products rose with increasing doses of IFN-gamma. The induction of MHC class II antigen expression on HCEC by IFN-gamma was completely inhibited by the addition of a neutralizing antibody directed to IFN-gamma but not by IL-1 beta. The significance of these findings with respect to corneal transplantation immunology is discussed.     

Morphological study of experimental corneal neovascularization after anterior uveal ischemia

We morphologically investigated what kinds of inflammatory cells infiltrate the corneoscleral limbus by light and electron microscopy and what manner of keratocytes and vascular endothelial cells appear at the corneal limbus, when the corneal edema was aggravated severely and clinical corneal new vessels were found initially, i.e. until three days after anterior segmental ischemia in rabbit eyes. The majority of infiltrating inflammatory cells were polymorphonuclear leukocytes and the others were lymphocytes and histiocytes etc. Peripheral keratocytes around limbal vessels were stimulated and transformed to fibroblastic cells as a result of anterior segment ischemia. Proliferating endothelial cells of comparatively minor limbal vessels invaded between corneal lamellar layers toward the corneal center. Polymorphonuclear leukocytes were often found near activated keratocytes proliferating vascular endothelial cells, hear and there. Consequently, it is considered that the stimulated polymorphonuclear leukocytes play an important role when corneal neovascularization occurs after anterior segmental ischemia.     

Comparison of limbal and peripheral human corneal epithelium in tissue culture

Peripheral human corneal epithelium grows better in tissue culture than central epithelium, but it is not known whether ocular limbal epithelium grows even better than does the peripheral corneal epithelium. In this work we compared the growth kinetics of limbal and peripheral human corneal epithelial cells in tissue culture. Four 1-2 mm2 explants, removed from the limbus or from peripheral cornea (1-2 mm inside the limbus) of eye bank eyes, were grown to confluence in primary culture. Cells were then passaged at 2 X 10(5) cells per dish. At intervals thereafter, the cells were counted in a hemocytometer to determine plating efficiency and growth curves. Mitotic activity was determined 4 days after passaging by labeling cultures with 3H-thymidine and counting aliquots using the hemocytometer and scintillation counter. In the primary cultures, limbal epithelium grew as small, uniformly polygonal cells. Peripheral corneal cells grew to a variety sizes. The 24 hr plating efficiency and doubling time of limbal epithelial cells were 47 +/- 8% and 80 +/- 14 hr, respectively, while those of peripheral corneal cells were 41 +/- 10% (P less than 0.1) and 131 +/- 25 hr (P less than 0.001). The mitotic activity of limbal cells was significantly higher than that of peripheral (2.9 +/- 1.2 vs. 0.8 +/- 0.6) (P less than 0.01). These results indicate that human ocular limbal epithelium grows better in culture than does peripheral human corneal epithelium.     

Analysis of corneal inflammation following the injection of heterologous serum into the rat cornea.

The immunopathologic response following the injection of various antigens into the rat cornea was evaluated. This reaction, known as Wessely's phenomenon, was believed to be primarily triggered by antibodies and complement activation. The keratitis model was originally described in rabbits, using heterologous serum or purified proteins. In rats only, heterologous serum induced corneal inflammation with the characteristics of Wessely's phenomenon, (ie, a quiescent period of several days between antigen injection and onset of clinical signs and corneal opacification). Using rats allowed us to characterize the cellular infiltrate with immunohistochemical methods. Marked infiltration of the cornea by macrophages was observed, as was infiltration by polymorphonuclear cells, although to a lesser extent. Furthermore, T lymphocytes of the helper phenotype were demonstrated. Antibodies to complement activation product C3c showed faint staining, whereas B lymphocytes and plasma cells were absent. In addition, inflammatory cells and ocular tissues, particularly the limbal and peripheral corneal epithelium, were found to express major histocompatibility complex class II antigens during the inflammatory response. After the inflammation had subsided, macrophages and T lymphocytes remained in the corneal stroma (at least until day 30). These findings suggest that antigen-induced keratitis in rats might be mediated, at least partially, by T helper lymphocytes.     

Characterisation of rat corneal cells that take up soluble antigen: an in vivo and in vitro study

The aim of the present study was to determine the capacity of resident corneal and limbal dendritic cells (DC) and macrophages to capture antigen (Ag) in vivo and compare this to their capacity in vitro to take up Ag during organ culture conditions. To investigate Ag uptake in vivo 3 microl (30 microg) of fluorescently labelled Dextran, bovine serum albumin (BSA) or ovalbumin (OVA) were either placed on the intact ocular surface or injected into the anterior chamber (AC) or subconjunctival space of the Lewis rat eye. The presence of Ag+ cells in the cornea was assessed using intravital fluorescence video microscopy. Animals were sacrificed 24h after Ag injections or topical application and the distribution and phenotype of Ag+ cells were analysed ex vivo by fluorescence and confocal microscopic analysis of immunostained and unstained corneal tissue wholemounts or frozen sections. Corneal buttons and corneoscleral rims from naive Lewis rats were placed in organ culture conditions in the presence of LPS with or without FITC-Dextran for 48 h and 72 h. The explants were examined by epi-fluorescence microscopy and the phenotype of Ag+ cells in the supernatant from the organ cultures was analyzed by flow cytometry using a range of macrophage and DC markers. In vivo observations and microscopic examination of corneas 24h following Ag topical application failed to reveal evidence of Ag retention by ocular cells. Those in which Ag had been placed in the AC or subconjunctival space revealed Ag+ cells within the corneal stroma. The distribution of Ag+ cells displayed a centripetal gradient, the most marked uptake of Ag being by cells in the circumferential limbal zone. Immunophenotypic studies revealed that Ag uptake was predominantly performed by cells that were CD68+, CD172+ but rarely MHC class II+, a profile characteristic of macrophages. Occasional Ag+ keratocytes were noted. In vitro studies of corneal buttons placed in culture conditions revealed that cells from the limbal zone, but not the central cornea, were able to take up Ag from the supernatant. Significant numbers of the cells that had migrated from the corneal buttons and captured fluorescent labelled Ag in the presence of LPS were revealed by flow cytometry to consist of CD163+ and CD11b+ macrophages, but none expressed the DC markers CD11c or OX62 and they were also generally MHC class II(-). In conclusion the present study revealed that macrophages and keratocytes in the corneal stroma and limbal episcleral tissue have the capacity to internalise fluorescent mock Ag injected into the AC or subconjunctival space or in culture conditions. The failure to demonstrate significant Ag trapping ability by corneal or limbal stromal or epithelial DC may either be due to the rarity of such cells or their lack of Ag trapping ability.     

Localizations of epidermal growth factor receptor and proliferating cell nuclear antigen during corneal wound healing

The localization of epidermal growth factor receptor and proliferating cell nuclear antigen was demonstrated immunohistochemical to be similar in the corneal, limbal, and bulbar conjunctival epithelium, i.e., located adjacent to the artificially made corneal epithelial defect. In the course of regeneration of the corneal epithelium soon after the wounding, both epidermal growth factor receptor and proliferating cell nuclear antigen were expressed in the epithelial cells in the limbal area adjacent to the epithelial defect. After the defect was covered with several layers of regenerated epithelium, the main site of the expression of epidermal growth factor receptor and proliferating cell nuclear antigen moved to the basal layer of regenerated epithelium. The present study indicated that epidermal growth factor receptor, as expressed on the epithelial cell surface, can be considered to play an important role in epithelial cell proliferation, which is an indispensable process in corneal wound healing.