Structure and biology of proteoglycans.

PURPOSE. Dendritic cells (DCs) comprise a system of highly efficient antigen-presenting cells (APCs) that initiate immune responses. The purpose of this study was to examine the normal stroma for the presence of DCs and other bone marrow (BM)-derived cells. METHODS. Normal uninflamed corneas of BALB/c and other murine strains were excised, and immunofluorescence single-and double-staining for multiple markers was performed for examination by confocal microscopy. Corneal buttons were placed in culture and immunocytochemistry and flow cytometry performed. RESULTS. MHC class II + CD80 + CD86 + cells were found in the periphery of the anterior normal stroma. These cells were CD45 + , CD11c + CD11b + suggesting a BM-derived and mono-cytic DC lineage. In a surprising finding, significant numbers of MHC class II - CD80 - CD86 - cells were found in the center of the anterior stroma. These cells were also CD45 + CD11c + CD11b + but CD3 - , GR-1 - , keratan sulfate - , and CD8α - , reflecting an immature precursor phenotype of myeloid DC. In addition to DC subsets in the anterior stroma, a CD11c - CD11b + population of BM-derived cells was found primarily in the posterior stroma, representing monocytes/macrophages. These cells were rarely present in the anterior third of the normal stroma. Further, CD14 + precursor-type DCs were found throughout the stroma. These in vivo findings were not strain specific and were confirmed by immunocytochemistry and flow cytometry analyses of cells derived from corneal explants and by transmission electron microscopy. CONCLUSIONS. This study demonstrates that, in addition to the known Langerhans cells in the corneal epithelium, at least three BM-derived cell subsets reside in the normal corneal stroma.

The corneal stroma is endowed with a significant number of resident dendritic cells.

Corneal antigen presenting cells (APC), including dendritic cells (DC), were thought to reside exclusively in the peripheral cornea. Here, we present recent data from our group dem- onstrating that the central cornea is indeed en- dowed with a heterogeneous population of epithe- lial and stromal DC, which function as APC. Al- though the corneal periphery contains mature and immature resident bone marrow-derived CD11c DC, the central cornea is endowed exclusively with immature and precursor DC, both in the epithe- lium and the stroma, wherein Langerhans cells and monocytic DC reside, respectively. During inflam- mation, a majority of resident DC undergo matu- ration by overexpressing major histocompatibility complex class II and B7 (CD80/CD86) costimula- tory molecules. In addition to the DC, macro- phages are present in the posterior corneal stroma. In transplantation, donor-derived DC are able to migrate to host cervical lymph nodes and activate host T cells via the direct pathway when allografts are placed in inflamed host beds. These data revise the tenet that the cornea is immune-privileged as a result of lack of resident lymphoreticular cells and suggest that the cornea is capable of diverse cellu- lar mechanisms for antigen presentation. J. Leu- koc. Biol. 74: 000-000; 2003.

https://jlb.onlinelibrary.wiley.com/doi/pdf/10.1189/jlb.1102544

Corneal immunity is mediated by heterogeneous population of antigen-presenting cells

PURPOSE. The presence of antigen-presenting cells (APCs) such as Langerhans cells (LCs), an epithelial form of dendritic cells (DCs), in corneal tissue is critical for generation of immune responses, including graft rejection and herpetic keratitis. The purpose of this study was to characterize the distribution and major histocompatibility complex (MHC) antigen expression of corneal LCs. METHODS. Normal and inflamed corneas were excised from BALB/c mice, and immunofluorescence staining for CD11c, CD11b, CD45, CD80 (B7.1), CD86 (B7.2), CD3, and MHC class II (Ia) was performed by confocal microscopy on wholemount corneal epithelium. RESULTS. CD11c MHC class II‐positive LCs were found in the limbus and corneal periphery, but not in the center of the normal cornea. These cells were CD45 positive, exhibiting bone marrow derivation, and CD3 and CD11b negative, confirming a DC lineage. Additionally, these cells were CD80 and CD86 negative, reflecting an immature phenotype. In the central and paracentral areas, however, significant numbers of CD11c LCs were detected that expressed no MHC class II. It is important to note that although the density of the LCs declined from the limbus toward the center of the cornea, they were always present. In the inflamed cornea, the expression of MHC class II and costimulatory molecules CD80 and CD86 was significantly enhanced, and present in all parts of the cornea, in contrast to the normal cornea. CONCLUSIONS. The present study demonstrates for the first time the phenotype and distribution of MHC class II‐negative LCs in the murine corneal epithelium. In the inflamed cornea, the LCs become activated as reflected by expression of B7 costimulatory markers. These changes in activation markers may provide additional information for devising novel immunomodulatory strategies. (Invest Ophthalmol Vis Sci. 2002;43:639 ‐ 646)

Novel characterization of MHC class II-negative population of resident corneal Langerhans cell-type dendritic cells.

Publisher Summary The eye offers interesting opportunities for analyzing a panorama of immunological phenomena, including organ transplantation, immunological privilege, tolerance, and autoimmunity. This chapter addresses some of the unique immunological characteristics of the eye. The goal is not to provide an exhaustive review of ocular immunology, but to focus on the curious immunological “ground rules” peculiar to this organ. The weight of evidence suggests that the immune system is carefully regulated within the eye and, to a lesser degree, at the ocular surface. The apparent ease with which corneal grafts avoid immunological recognition suggests that the cornea is endowed with unique immunological characteristics and thus possesses an immunological privilege not shared by other organ grafts. Historically, three basic hypotheses have been offered to account for the privileged existence of corneal allografts. The simplest explanation suggests that the cornea, like certain neuronal tissues, is devoid of conventional major histocompatibility complex (MHC) antigens. Studies in rats, rabbits, and mice indicate that heterotopic transplantation of corneas to subdermal graft beds leads to rapid sensitization and swift rejection of the allografts. As for the anterior chamber, the known absence of lymphatic drainage to regional lymph nodes led early investigators to propose that the privilege of the anterior chamber could be traced to the sequestration of alloantigens within this compartment. The high incidence of spontaneous neoplasms that occurs in patients suffering from immunological disorders and in transplant recipients subjected to prolonged immunosuppression is cited as evidence in support of the immune surveillance theory. It is reasonable to suspect that the same situation occurs with immunologically privileged sites, such as the anterior chamber of the eye, where immunological censorship creates an environment that is expected to thwart the induction and expression of immunological surveillance.

Immune privilege and immune regulation in the eye.

Langerhans cells of the ocular surface.

A reduction in the incidence of rejection of mouse heterotopic corneal allografts was achieved by in vitro pretreatment of the graft with ultraviolet light. By day 21, 68% of untreated BALB/c corneas transplanted into s.c. abdominal pouches of C57BL/6 recipients demonstrated rejection. In contrast, corneas pretreated with UV-A, -B, or -C at 150 mJ/cm2 showed an incidence of rejection at day 21 of 30%, 33%, and 18%, respectively. Experiments indicate a significant reduction in rejection with doses of UV-B radiation above 75 mJ/cm2. The mechanism of this reduction is unknown, but may be due to a depletion or alteration of ocular surface Langerhans cells residing in the corneal epithelium.

Reduction in the incidence of rejection of heterotopic murine corneal transplants by pretreatment with ultraviolet radiation.

We describe a simple and reliable technique for the preparation of corneal epithelial flat mounts. The technique is based on incubation of corneas or whole globes in an EDTA solution prior to fixation. Corneal epithelial flat mounts prepared by this technique are suitable for study by immunofluorescence or enzyme histochemistry.

Immunofluorescence and histochemistry of corneal epithelial flat mounts: Use of EDTA

Six mouse hybrid cell lines were isolated which secrete antibodies to rabbit corneal proteoglycan. All six antibodies interacted with the same fraction of the proteoglycan, precipitating approximately 50% of proteoglycan labelled in the protein moiety. A radioimmunoassay using these antibodies measured concentrations as low as 1 g/m1 unlabelled rabbit corneal proteoglycan. Human corneal proteoglycanj corneal keratan sulfate, and an oligosaccharide fraction from corneal digests all interacted with the antibodies at concentrations similar to whole rabbit proteoglycan. Proteoglycans from cultured rabbit stromal fibroblasts and from sclera were 20 to 50-fold less effective in competition for antibody. Endo-β-galactosidase treatment of proteoglycan reduced antibody binding, but protease or chondroitinase treatments did not. Labelled proteoglycan separated by antibody affinity chromatography contained only keratan sulfate, whereas proteoglycan not bound to affinity columns contained only chondroitin sulfate. Th...

Monoclonal antibodies to rabbit corneal keratan sulfate proteoglycan

types. Physical or enzymatic damage to the cultured corneas resulted in marked changes in the ratio of labeled GAG types secreted. Physical damage to the epithelial or endothelial surfaces and/or mild trypsin treatment of the corneas before culture resulted in a stimulation of galactosaminoglycan secretion and a consequent decrease in the relative abundance of keratan sulfate. The presence of soy trypsin inhibitor prevented these changes. Treatment of the corneas with collagenase resulted in a decrease in all GAG secretion, particularly keratan sulfate. When corneas were removed from chilled, enucleated eyes and rinsed with solutions of soy trypsin inhibitor before incubation, the GAG secreted in culture had a similar ratio of keratan sulfate to galactosaminglycan as GAG labeled in vivo. This ratio was maintained 1 week in culture. These results suggest that secretion of the characteristic connective tissue of the corneal stroma by keratocytes is dependent on interaction between the cells and components of the stromal extracellular matrix. Invest Ophthalmol Vis Sci 24:208-213, 1983 In adult animals, corneal connective tissue is secreted by the stromal keratocytes, flattened mesenchymal cells located between the stromal lamellae. For short periods of time in organ culture, keratocytes continue to secrete characteristic stromal connective tissue components. 1 After 48 hours in organ or cell culture, marked changes take place in the nature of these secretions. A well-characterized aspect of this change is the alteration in corneal glycosaminglycan synthesis. There are two major corneal types of glycosaminoglycan (GAG), keratan sulfate and galactosaminglycan (a chondroitin sulfate-dermatan sulfate hybrid). In vivo, more than half of the total GAG secreted by keratocytes is keratan sulfate, but after 48 hours in organ or cell culture, this level decreases to 10% or less of the total. 23 Keratocytes grown in

John W. Chandler

Papers

Langerhans cells of the ocular surface.

Reduction in the incidence of rejection of heterotopic murine corneal transplants by pretreatment with ultraviolet radiation.

Immunofluorescence and histochemistry of corneal epithelial flat mounts: Use of EDTA

Monoclonal antibodies to rabbit corneal keratan sulfate proteoglycan

Corneal glycosaminoglycan synthesis in long-term organ culture.