John W. Chandler

Langerhans Cells of the Ocular Surface

Recent evidence has been collected by several investigators defining a distinct population of dendritic cells (Langerhans cells) of mesenchymal origin residing in the epidermal surfaces of many mammalian species. These cells play a dominant role in the processing of antigens presented through cutaneous surfaces and carry a Class II histocompatability antigen felt to be of central importance in the afferent arm of allograft rejection. They also possess many of the characteristics of macrophages active in the efferent arm of immunologic responses. An equivalent subset of dendritic cells (Langerhans cells) in ocular surface epithelium of the human, mouse rat, and guinea pig has been identified by enzyme histochemistry, immunofluorescence, and electron microscopy. Ocular surface Langerhans cells proliferate in the setting of corneal inflammation (remote and recent) and are depleted by topical and systemic corticosteroids. Ocular surface Langerhans cells may play a central role in ocular contact hypersensitivity, corneal allograft rejection, and ocular surface immune surveillance.

Immunologic Defense Mechanisms of the Ocular Surface

The ocular surface represents an immunologic microcosm in which certain local immunologic and paraimmunologic defense systems analogous or identical to those of other mucosal surfaces are operative. These defenses include a resident normal flora; intrinsic anatomic barriers; secretion of mucous and certain chemical bacteriostatics and bacteriolytics; local humoral (slgA) antibody secretion; and local T-lymphocyte cellular responses. A series of sophisticated mechanisms has evolved to defend the ocular surface against diverse environmental pathogens. Often, the activation of one system leads to the subsequent activation of another, providing a highly integrated, series of mechanisms for host defense. And, it is clear that close integration of these various mechanisms provides a highly efficient amplification system for host defense, so that when one mechanism fails to deal with the invading pathogen the next mechanism is then efficiently initiated. Only in those situations where the pathogen overwhelms these defense systems, or is of such a persistent nature that it becomes resistant to removal by these various mechanisms, or alternatively is able to subvert its identity and, therefore, evade these mechanisms do organisms infect and cause disease. It is undoubtedly the complex interaction of all of these defense systems, both paraimmunologic and immunologic, which finally determines the integrity of the ocular surface.

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

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 trans-planted into s.c. abdominal pouches of C57BL/6 recipi-ents demostrated rejection In contrast, corneas pre-treated with UV-A, -B, or -C at 150 mJ/cm2showed 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/cm.2 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.

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

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.

Monoclonal antibodies to rabbit corneal keratan sulfate proteoglycan

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/ml unlabelled rabbit corneal proteoglycan. Human corneal proteoglycan, 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-beta-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. The antibodies appear to recognise a carbohydrate structure found only on corneal keratan sulfate proteoglycan. This structure can serve as a basis for separation. This structure can serve as a basis for separation of corneal proteoglycan types using antibody affinity chromatography.

Rejection of murine heterotopic corneal transplants

A murine heterotopic corneal transplant model has been developed using s.c. abdominal pouches as recipient sites. Donor-recipient genetic disparities involving H-2 antigens alone or H-2 plus non-H-2 antigens result in high rates of rejection. In addition, donor-recipient disparities involving non-H-2 antigens alone or H-Y antigen also result in significant, although lower, rates of rejection. In comparison, pretreatment of the donor grafts by hyperbaric oxygen, removal of the epithelial layer, or soaking in anti-la antibody plus complement results in statistically significant reductions in the rejection rates as compared with fresh, untreated donor tissue. These observations suggest that cells bearing Ia antigens (i.e., Langerhans cells) in the epithelial layer of donor corneas play a major role in host sensitization and subsequent rejection following corneal transplantation in this model. However, other antigens may play a role when Ia antigens are depleted from donor corneas.

Experimental corneal allograft rejection: Description of murine model and a new hypothesis of immunopathogenesis

A murine model of heterotopic corneal transplantation has been developed. Whole corneas were removed from either inbred Balb/cJ or C57B1/6J mice and inserted into an abdominal subcutaneous pouch in C57B1/6J recipient mice. Fresh donor tissue, donor corneas cultured at 37°C in 5 per cent CO2-95 per cent room air, or donor corneas cultured at 37°C in two atmospheres of 98 per cent 02 - 2 per cent CO2 (hyperbaric oxygen) were utilized. Twenty-one days later, the recipients were sacrificed and the heterotopic grafts were examined in a masked fashion. Rejected grafts were edematous, opaque, and vascularized. In contast, unrejected grafts were clear and nonvascularized. These results were further confirmed by histological examination of the donor grafts in a masked fashion. Control syngeneic grafts were not rejected. The fresh allografts were usually rejected (91%) as were the allografts cultured in CO2 - room air (90%). Thus, this murine model of heterotopic corneal transplantation appears to be useful for the...

Filamentary Keratitis Following Penetrating Keratoplasty

Filamentary keratitis is a problem associated with a wide variety of ocular conditions. Of 114 patients receiving penetrating keratoplasty, 31 (27.2%) were noted to have filaments. Those patients receiving transplants for keratoconus were noted to have the highest incidence (12 of 31, 39%), while transplants following herpetic scars were not associated with filaments (0 of 11) in this series. Suturing technique or relation of donor button-to-graft site were not apparently important factors in the development of postoperative filamentary keratitis.

Dynamic Shifts in Corneal Topography after Radial and Transverse Keratotomy

PURPOSE The authors aimed to quantitate the dynamic patterns of change in corneal topography after multistaged radial and transverse keratotomy using digitized video-keratography. METHODS Single and paired radial and transverse keratotomies, with videokeratoscopy between each stage and at the end of the procedure, were performed on fresh animal cadaver eyes using an artificial orbit system. RESULTS All incisions led to central flattening. A single radial keratotomy caused flattening adjacent to the incision, and steepening 180 degrees away. A paired radial keratotomy caused increased flattening in the meridian of the incisions, and less flattening 90 degrees away. A single transverse incision caused steepening adjacent to the incision and diffuse flattening elsewhere. A paired transverse incision caused flattening near the optical center along the meridian bisecting the incisions and steepening 90 degrees away. CONCLUSION The authors have demonstrated that computerized videokeratography can be used successfully to systematically quantitate dioptric shifts in multiple hemimeridians and measurement zone diameters after refractive surgery.

Localization and characterization of binding sites for endothelin in Harder's gland in rabbits.

The characterization and localization of binding sites for endothelin-1 (ET-1) labeled with iodine 125I were investigated in homogenized tissues and sections of Harders glands of normal rabbits. The membrane of Harders glands was harvested and incubated with 125I-ET-1 (0.25-1 nmol/L) in 20 +/- 4 mg of protein per 0.25 mL at 37 degrees C for 90 min in the presence of protease inhibitors. Specific labeling was assessed by coincubating unlabeled ET-1, ET-2, ET-3 and other unrelated cytokines. The tissue labeled with 125I-ET-1 was collected by filtration and counted in a gamma counter. For an in vitro autoradiography study, 15 microns cryostat sections were incubated with 125I-ET-1 (0.1 nmol/L). They were fixed, dipped in liquid emulsion and kept for 6 days before development. Membrane counting showed that the binding of 125I-ET-1 to Harders gland was saturable. Scatchard data analysis revealed one class of binding with a dissociation constant (Kd) of 0.33 nmol/L and a maximal density of binding (Bmax) of 794 attomole/mg of protein. The binding was inhibited most by ET-1, followed by ET-2 and then ET-3 but not by unrelated peptides. Emulsion-dipped slides with sections showed specific high-density labeling mainly over structures identified from serial sections stained by hematoxylin-eosin as the walls of capillaries, arterioles, arteries, and veins of the glands. Less dense binding was found in both white and pink lobes of the gland. No binding was found in fat and connective tissues. The distribution of endothelin action sites in the glandular blood vessels and Harders gland suggests that the peptide may have a role in the regulation of blood circulation and glandular secretion in the normal rabbit.