Molecular and immunological characterization of hr44, a human ocular component immunologically cross-reactive with antigen Ov39 of Onchocerca volvulus.

Structural similarities between host self-antigens and infectious organisms may be involved in the expression of autoimmune reactivity and development of autoimmune disease. The unique eye pathology associated with Onchocerca volvulus infection, particularly the development of posterior segment lesions, may be promoted by such autoreactive responses. Ov39 is a parasite-derived antigen that has been shown previously to be antigenically cross-reactive with a 44,000-M(r) host ocular component. A clone, designated hr44, was isolated from a cDNA library of human retina by immunoscreen using serum to Ov39. A monoclonal antibody raised to Ov39 also reacted with hr44 and gave evidence for a shared conformational epitope. The primary structure analysis showed that identities between the antigens are limited and confined to small peptides. The cross-reactivity between the antigens appears to involve T cells, since Ov39-specific T cells can be stimulated by hr44, a neural-specific antigen. Based on secondary structure prediction, hr44 has the typical features of a membrane-associated type I antigen with an amino-terminal extracellular domain. mAbs and antisera localized the antigen in the optic nerve, neural retina, retinal pigment epithelium, as well as the epithelial layers of ciliary body and iris.

Immunological crossreactivity between a cloned antigen of Onchocerca volvulus and a component of the retinal pigment epithelium.

Onchocerciasis (river blindness) is a major blinding disease in Africa, Central America, and South America. Loss of vision can be due to corneal change, optic atrophy, or chorioretinal disease. It has been suggested that autoimmunological reactions resulting from crossreactivity between parasite antigens and components of eye tissues contribute to development of ocular pathology. Using sera collected from onchocerciasis patients as a screening reagent, a cDNA clone (Ov39) has been isolated from a lambda gt11 expression library of Onchocerca volvulus. This antigen exhibits immunological crossreactivity with a component of retinal pigment epithelium cells (RPE). Antiserum raised against this recombinant peptide immunoprecipitates a 22,000 Mr antigen of adult O. volvulus and recognizes a 44,000 Mr component of bovine RPE by Western blotting. A 44,000 Mr antigen of cultured human RPE metabolically labeled with 35S-methionine can be immunoprecipitated with the same antiserum. An antigen of the same size is recognized by a rabbit antiserum raised against whole O. volvulus extract. Immunocytochemical studies on cryostat sections of the bovine eye using the antirecombinant sera localizes this antigen to the RPE.

The formation of autofluorescent granules in cultured human RPE.

Although the incomplete degradation of phagocytosed outer segment discs is thought to result in the formation of lipofuscin, there has to date been no proof of this concept. We report for the first time that cultured human retinal pigment epithelial (RPE) cells fed daily doses of isolated rod outer segments for periods of up to 3 months were capable of developing intracellular autofluorescent granules whose morphology and fluorescence characteristics were similar to lipofuscin. These autofluorescent granules were observed as early as 2 weeks following daily challenge with rod outer segments and the number of granules increased with the number of challenge doses until the experiment was terminated after 3 months of daily feeding. We also studied the effects of the antioxidant vitamin E and a drug, Centrophenoxine, which has been purported to slow the formation of lipofuscin formation in vivo. Neither of these additives given either with the challenge or subsequently had any effect on the formation of the intracellular granules. In conclusion, the development of these "lipofuscin-like" inclusions in cultured RPE may provide a model with which to study ageing processes and may provide an understanding of the increased lipofuscin accumulation observed in certain retinal pathologies.

The combined effects of light and acute ischemia on the structure of the rabbit retina: a light and electron microscopic study.

The ultrastructure of the rabbit retina has been investigated to determine the combined effects of light and ischemia. Both eyes of 16 adult Dutch rabbits were exposed to light of an intensity known to be near or below the threshold for ultrastructural changes in the retina. In addition, one eye of each animal was subjected to one of four periods of pressure-induced total acute ischemia. Exposure to light alone resulted in only minor disturbances of the receptor cell outer segments. The remainder of the retina was of normal morphologic appearance. Exposure to the combined insults of light exposure and ischemia produced considerably more damage to the inner and outer retina. Light exposure combined with short periods of ischemia (15 to 30 min) resulted in edema of the pigment epithelium and disturbances of the receptor cell inner and outer segments. Light exposure combined with longer ischemic periods (45 to 60 min) resulted in severe disturbances in the structure of the pigment epithelium, breakdown of the receptor cell outer segments, rupture of the inner segment mitochondria, and severe edema of the neural retina. The implication of this addictive or synergistic action of light and ischemia is discussed.

Investigations of cytoskeletal elements in cultured bovine meshwork cells.

An ultrastructural, immunohistochemical, and functional study was conducted on cultured bovine meshwork cells. Particular emphasis was placed on the organization of the cytoskeleton, and the cells were viewed either as whole cells or following detergent extraction. For ultrastructural examination, several modes of viewing were adopted, including a detector situated above the specimens collecting secondary electrons (SE), a detector situated beneath the specimen collecting transmitted electrons (STEM), and conventional transmission electron microscopy at 100 KV (TEM). In whole cell mounts, information was obtained about the organization of the cytoskeleton and its relationship to other cytoplasmic organelles. Extraction procedures removed much of the plasma membrane and most organelles. The nucleus and cytoskeleton remained and stress fibers were prominent. Immunohistochemistry showed that the actin content of the cytoskeleton could be preserved after detergent extraction. Detergent-extracted cells decreased their surface area when exposed to MgATP in a dose-dependent manner. The decrease in surface area was associated with disassembly of cytoskeletal stress fibers and was optimal with 1 mM MgATP. Whether or not the change in surface area could be considered a "contractile event" was discussed.

Immunologic cross-reactivity in the pathogenesis of ocular onchocerciasis.

PURPOSE: Onchocerca volvulus, a filarial worm, is a major cause of infectious blindness and inflammatory eye disease. An autoimmune cause for ocular onchocerciasis has been suggested since the identification of a recombinant antigen of O. volvulus that shows immunologic cross-reactivity with a host ocular component of 44,000 M(r). The aim of this study was to establish the distribution of the cross-reactive antigens in both host tissues and the parasite, and to determine if significant autoantibody responses to the host antigen could be detected in infected persons. METHODS: The tissue and organ distribution of the 44,000 M(r) antigen was determined by immunocytochemistry and Western blot analysis. Human autoantibody responses to the ocular antigen were demonstrated by Western blot analysis using sera collected from persons with onchocerciasis, with and without posterior segment pathology, Bancroftian filariasis, and Europeans with no filarial infection. RESULTS: The tissue distribution of the 44,000 M(r) antigen correlates with the sites of pathology in onchocerciasis and antibody reactivity against this antigen could be detected in all persons with onchocerciasis and posterior segment pathology. The antigen is also recognized by sera from persons with Bancroftian filariasis, but not from normal persons. CONCLUSIONS: A role is proposed for immunologic cross-reactivity in the pathogenesis of onchocerciasis and it is suggested that intraocular presentation of the cross-reactive parasite antigen by microfilariae is essential for the development of disease.

Retinal pathology in the Kearns-Sayre syndrome.

Examination of the retinal tissues obtained at necropsy from a 14-year-old boy with Kearns-Sayre syndrome showed marked photoreceptor and pigment epithelial cell loss in the retinal periphery and around the optic nerve head. Electron microscopy of surviving retinal pigment epithelial (RPE) cells indicated a loss of apical microvilli and basal infoldings. The RPE was unusually devoid of melanosomes and showed no evidence of phagocytosis of photoreceptor debris. The cytoplasm of the RPE contained numerous, often enlarged, mitochondria. These structural changes suggested that a breakdown in the energy dependent interrelationships between the RPE and the photoreceptor layer was responsible for the outer retinal degeneration. The finding of numerous macrophages in the subretinal space suggests a secondary inflammatory component in the retinal degeneration.

Fundus changes in (type II) mesangiocapillary glomerulonephritis simulating drusen: a histopathological report.

We report for the first time to our knowledge the histopathological findings in the eye of a patient with type II mesangiocapillary glomerulonephritis (dense deposit disease) in which a deposit of material morphologically very similar to that which is pathognomonic for the disease in the kidney was demonstrated in Bruch's membrane. The nature of the deposit in the renal lesion is unknown but is considered to represent a structural alteration secondary to a reaction with anticomplement antibody. Clinically the fundus appearance resembled that seen in drusen.

Post-traumatic hyperlipofuscinosis in the human retinal pigment epithelium.

Light microscopy (including fluorescence microscopy) and electron microscopy were applied to a study of the photoreceptor-retinal pigment epithelium (RPE) complex in a human eye which had been severely traumatised nine months prior to enucleation. The main feature of interest was a massive accumulation of lipofuscin in the retinal pigment epithelium at the posterior pole, and quantitative fluorescence microscopy provided values three times those obtained in appropriate control tissue. The photoreceptor layer was normal at the posterior pole but became progressively atrophic towards the periphery. The concentration of lipopofuscin was proportional to the degree of preservation of the retinal photoreceptors. By electron microscopy the cells in the RPE were seen to be packed with a mixture of lipofuscin granules and melanolysosomal complexes, but occasional photoreceptor phagosomes were found. Bruch's membrane and the choriocapillaris were normal. We attribute this hitherto unreported abnormality of the RPE after trauma to a dysfunction consequent on an overload of the monolayer by photoreceptor debris at the time of trauma.

Recovery of the rabbit retina after light damage (preliminary observations).

The retinae of anaesthetised Dutch rabbits were exposed to one of two intensities of white light for a period of 1 h. After exposure the animals were allowed to recover for various periods up to 4 weeks. The animals were then killed, and retinal and choroidal tissue was taken for investigation by both light and electron microscopy. Exposure to the lower intensity produced disruption of the visual cell outer segments and distension of the pigment epithelium. Recovery from this insult was rapid although disturbances in rod disc stacking and a loss of cone cell outer segments were evident 4 weeks after exposure. Exposure to the higher intensity resulted in necrosis of visual cells and pigment epithelial cells. Non-native phagocytic cells were active in the removal of cellular debris. Recovery from this insult was not observed. Four weeks after exposure much of the previously illuminated retina was reduced to disorganised Müller cells and occasional macrophages.

Light damage to the retina.

The retinae of anaesthetised Dutch rabbits were exposed to light of various known intensities for a period of 1 h. After this period the animals were sacrificed and tissue taken for investigation by light and electron microscopy. Changes were apparent in the visual cells and retinal pigment epithelium only at the highest intensities employed. The retinal pigment epithelium and the outer nuclear layer exhibited mild oedematous changes. The major components to be affected were the visual cell outer segments. Marked disruption and vesiculation were features of the cone outer segments, which was in distinct contrast to the focal damage observed in the rod outer segments.

Some aspects of radiant energy damage to the retina.

The retinae of anaesthetised Dutch rabbits were exposed to white light of various intensities * (*67--235 mW/cm2 measured at 2.5 cms from the end plate of the light guide) for a period of one h. After this period the animals were killed and tissue taken for investigation by light and electron microscopy. The higher intensities produced marked disruption of the visual cell outer and inner segments as well as a variety of morphological changes in the pigment epithelium. The highest intensity produced severe disruption of the visual cells and pigment epithelium in addition to a general disruption of the other retinal layers. Accompanying the retinal damage there was an inflammatory response in the choroid of the experimental eyes and surprisingly, at some intensities, a similar inflammatory response in the choroid of the control eye.

Qualitative observations on the variation of light induced damage to the rabbit retina.

The retinae of anaesthetised Dutch rabbits were exposed to light of various known intensities for one hour. Immediately after exposure the animals were killed and retinal and choroidal tissues were taken for investigation by electron microscopy from selected sited with the previously illuminated area. Initial qualitative observations suggested that a considerable variation in the degree of cellular damage had occurred even within retinal and choroidal tissues taken from the same eye. This variability of damage appeared to be related to intensity of illumination and patency of the choroidal vasculature. The mechanisms which could be underlying this variation of damage and possible methods of quantification are discussed.

Immunochemistry of the outer retina.

The immunochemistry of the outer retina is discussed with particular reference to photoreceptor cells, the retinal pigment epithelium and the interphotoreceptor space. The antigens identified and the techniques utilised are summarised.

Immunization with the cross-reactive antigens Ov39 from Onchocerca volvulus and hr44 from human retinal tissue induces ocular pathology and activates retinal microglia.

Antigen Ov39, derived from Onchocerca volvulus, cross-reacts on both the T and B cell level with a nonhomologous human retinal antigen, hr44. Lewis rats were immunized to investigate the potential of these antigens to induce eye disease. Histologic and immunohistologic examination of ocular tissues revealed pathologic changes as early as day 12, which included induction or up-regulation of class II and CD68-like antigen on perivascular cells, ramified retinal microglia, dendritiform cells of the iris epithelium, and ciliary epithelium and significant breakdown of anterior and posterior blood-ocular barriers. Extravascular immunoglobulin and staining for CD68-like antigen was detected in the optic nerve after immunization with Ov39. Unrelated structural abnormalities of retina and lens seen in 8% of eyes examined significantly predisposed eyes to the development of Ov39- or hr44-induced pathology. These findings suggest a role for cross-reactive immune responses in the development of ocular onchocerciasis.

The retinal pigment epithelium and other tissues immunocytochemical studies using hybridoma supernatants.

We describe the production of hybridomas producing antibody reactive with retinal pigment epithelium. The patterns of reactivity obtained when supernatants from these hybrids were used to immuno-stain ocular and non-ocular tissues are described. From the patterns of reactivity obtained we suggest that the RPE shares common features with simple and glandular epithelia as well as myoepithelial cells. These findings may lend support to the hypotheses of RPE cell migration and contraction in the formation of epiretinal membranes and retinal detachment.

The cytoskeletal elements of human retinal pigment epithelium: in vitro and in vivo.

The cytoskeletal elements of normal (in situ) and cultured human retinal pigment epithelium (RPE) were studied by a variety of immunocytochemical techniques. Primary antibodies to vimentin and cytokeratins were used. Positive immunoreactivity for vimentin was obtained with in situ and cultured material. The pattern of reactivity obtained with antisera and monoclonals to cytokeratins was more complex. Cytokeratin immunoreactivity could be demonstrated in situ and in cultured cells. The pattern of cytokeratin expression was similar to that of simple or glandular epithelia. A monoclonal antibody that specifically recognizes cytokeratin 18 identified a population of cultured RPE cells that had particularly well-defined filamentous networks within their cytoplasm. Freshly isolated RPE was cytokeratin 18 negative by immunofluorescence, but upon culture cytokeratin 18 positive cells were identifiable. Cytokeratin 18 positive cells were identified in all RPE cultures (other than early primaries), regardless of passage number, age or sex of the donor. In post-confluent cultures cytokeratin 18 cells were identified growing over cytokeratin 18 negative cells, suggesting an association of cytokeratin 18 immunoreactivity with cell proliferation. Immunofluorescence studies of retinal scar tissue from two individuals revealed the presence of numerous cytokeratin 18 positive cells. These findings indicate that RPE cells can be identified by their cytokeratin immunoreactivity and that the overt expression of cytokeratin 18 may be associated with proliferation of human RPE both in vitro and in vivo.

D-[3H]-galactose incorporation in the bovine retina: specific uptake and transport of the radiolabel in cones.

When bovine neural retinas are incubated in Krebs-Henseleit buffer with D-[3H]-galactose, autoradiography reveals that there is a rapid uptake of the tritium label into the inner segments of cones, but not of rods. Pulse--chase studies show that the label is first associated with the Golgi apparatus in the cones, then appears to travel around the nucleus and along the cone fibre (homologous to an axon) to the synaptic pedicle. The cone-specific label travels along the fibre at a rate of about 0.5-1.0 mm per day. Label is also found in endothelial cells and Müller cells, but does not persist in the Müller cells as long as in the cones. The striking difference between rod and cone labelling may reflect fundamental differences in the neurochemistry of these two photoreceptor cell types.

D-[3H]galactose incorporation into glycogen in retinal cone cells.

Previous studies have shown that bovine retinas incubated with [3H]galactose incorporated it, unmodified, into large molecules. Light and electron microscope autoradiography showed a significant proportion of the label to be in cone inner segments, and pulse-chase studies showed it was subsequently transported to the synaptic pedicles. In this report, evidence is presented to show that the galactose-labelled macromolecules are resistant to hydrolysis by proteolytic enzymes, testicular hyaluronidase, chondroitinase ABC, beta-glucosidase and beta-glucuronidase, but are readily degraded by alpha-amylase and beta-galactosidase, and to a lesser extent by beta-amylase. Treatment with alpha-amylase also leads to specific removal of radioactivity from cone inner segments and pedicles, as judged by light-microscopic autoradiography. These studies appear to indicate that the cone-specific galactose label is in glycogen or glycogen-like molecules.

Short-term organ culture of the retinal pigment epithelium in microtitration plates: ultrastructural studies.

Microtitration plates were used to culture simultaneously multiple, small (6 mm diameter) explants of bovine retinal pigment epithelium (RPE). Evaluation of tissue by light microscopy and by scanning and transmission electron microscopy after various incubation periods up to 6 h showed that RPE maintained in this system retains near normal morphology. Initially, the explanted RPE lacks apical microvilli, but during the first 2-3 h in culture recovery of apical microvilli occurs. The results suggest that the system is suitable for short-term maintenance of RPE for experimental purposes. Moreover, the ability to culture up to 16 explants from one bovine eye aids statistical evaluation of RPE behaviour under varying experimental conditions.