Functional inhibition of retinal pigment epithelial cell-substrate adhesion with a monoclonal antibody against the beta 1 subunit of integrin

In the preceding report, experiments were described which identify the 2A10 antigen of retinal pigment epithelial (RPE) cells as a beta 1 subunit of the cell-surface extracellular matrix receptor protein integrin. In this article, experiments are presented which use the 2A10 and CSAT antibodies, both directed against the beta 1 subunit, to investigate the role of integrin in RPE and fibroblast (FB) cell-substrate adhesion. When added to cultures simultaneously with cells, either the 2A10 or CSAT antibodies inhibit both FB and RPE cell adhesion and spreading on laminin (LM). However, although the 2A10 antibody blocks adhesion and spreading of FB and RPE cells on fibronectin (FN), the CSAT antibody has no effect. The inhibition of the 2A10 antibody is specific for integrin-mediated adhesion; it does not affect FB or RPE cell adhesion and spreading on tissue-culture plastic. When RPE cells are first allowed to attach to and spread on FN and LM and then the 2A10 or CSAT antibody is added to the cultures, both cause detachment and rounding of RPE cells from LM, but neither has any effect on the cells already spread on FN. These results indicate that there are differences in the way FB and RPE cells interact with LM and FN. Furthermore, these results provide the first direct functional demonstration that RPE cell-substrate adhesion is mediated by integrin.     

Protein kinase C epsilon activates lens mitochondrial cytochrome c oxidase subunit IV during hypoxia

Protein kinase C (PKC) isoforms have been identified as major cellular signaling proteins that act directly in response to oxidation conditions. In retina and lens two isoforms of PKC respond to changes in oxidative stress, PKCgamma and PKCepsilon, while only PKCepsilon is found in heart. In heart the PKCepsilon acts on connexin 43 to protect from hypoxia. The presence of both isoforms in the lens led to this study to determine if lens PKCepsilon had unique targets. Both lens epithelial cells in culture and whole mouse lens were examined using PKC isoform-specific enzyme activity assays, co-immunoprecipitation, confocal microscopy, immunoblots, and light and electron microscopy. PKCepsilon was found in lens epithelium and cortex but not in the nucleus of mouse lens. The PKCepsilon isoform was activated in both epithelium and whole lens by 5% oxygen when compared to activity at 21% oxygen. In hypoxic conditions (5% oxygen) the PKCepsilon co-immunoprecipitated with the mitochondrial cytochrome c oxidase IV subunit (CytCOx). Concomitant with this the CytCOx enzyme activity was elevated and increased co-localization of CytCOx with PCKvarepsilon was observed using immunolabeling and confocal microscopy. In contrast, no hypoxia-induced activation of CytCOx was observed in lenses from the PKCepsilon knockout mice. Lens from 6-week-old PKCepsilon knockout mice had a disorganized bow region which was filled with vacuoles indicating a possible loss of mitochondria but the size of the lens was not altered. Electron microscopy demonstrated that the nuclei of the PCKepsilon knockout mice were abnormal in shape. Thus, PKCepsilon is found to be activated by hypoxia and this results in the activation of the mitochondrial protein CytCOx. This could protect the lens from mitochondrial damage under the naturally hypoxic conditions observed in this tissue. Lens oxygen levels must remain low. Elevation of oxygen which occurs during vitreal detachment or liquification is associated with cataracts. We hypothesize that elevated oxygen could cause inhibition of PKCepsilon resulting in a loss of mitochondrial protection.     

Activity distribution of cytochrome oxidase in the rat retina. A quantitative histochemical study

Cytochrome oxidase (CYO) is a key enzyme in the respiratory chain. Therefore, CYO has an important role in the cell metabolism. In the present study CYO activity in the rat retina was identified by histochemical staining. The density of the staining, corresponding to the activity of the CYO, was evaluated quantitatively by densitometry. A high CYO activity was found in the retinal pigment epithelium, in the inner segment of the photoreceptors, in the outer plexiform layer and in the inner plexiform layer. In the outer segment of the photoreceptors, in the outer nuclear layer, and in the inner nuclear layer the CYO activity was relatively low. An analysis of variance demonstrated that the precision in an estimation of a mean depends on the number of animals and the number of retinal sections per animal.     

Up-regulation of cytochrome oxidase in the retina following optic nerve injury

The purpose of this study is to investigate the cytochrome oxidase (COX) activity in the retina and optic nerve following an optic nerve injury. The optic nerve crush of one eye was carried out in Balb/c mice. A semi-quantitative RT-PCR method was then adopted to evaluate the mRNA expression of cytochrome oxidase subunit 1 (COX1) in the retina after surgery. Up-regulation of COX1 mRNA in the retina was detected by RT-PCR at 24 hr following the optic nerve injury. Total retinal mitochondrial mass measured by fluorescent intensity of MitoTracker green was not altered following the injury. COX histochemistry performed on cryostat sections showed an elevated enzyme activity of COX in the retina and in the optic nerve. In the retina, elevation of the COX activity was observed in the retinal ganglion cell layer and the overlying nerve fibre layer. The increase of COX activity began from 24 hr after injury, peaked around day 3, and maintained up to 1 week after the operation. In the optic nerve, increase of COX activity was observed in regions distal to the crush line and distributed either randomly or in a cone shape. In conclusion, both the expression of COX1 mRNA in retina and the activity of COX in inner plexiform layer and retinal ganglion cell layer were elevated following optic nerve injury without affecting total retinal mitochondrial mass. These findings suggested that one of early responses in the retina and in the optic nerve after the optic nerve injury is to scale up the energy production.     

Inhibition of cytochrome oxidase and blue-light damage in rat retina

The activity of cytochrome oxidase, outer nuclear layer thickness, and edema were quantitatively evaluated in the blue-light exposed rat retina. Dark-adapted or cyclic-light reared rats were exposed to blue light with a retinal dose of 380 kJ/m². Immediately, 1, 2, and 3 day(s) after exposure, the retinas of six rats from each adaptation group were examined. There was no difference between the dark-adapted and cyclic-light reared rats. Immediately after light exposure, cytochrome oxidase activity decreased. The activity in the inner segments remained low at day 1, while severe edema was observed in the inner and outer segments. The outer nuclear layer thickness decreased 1–3 days after exposure. The blue-light exposure inhibited cytochrome oxidase activity and caused retinal injury. Similarity of the injury process in the dark-adapted and cyclic-light reared retinas suggests that rhodopsin was not involved. The inhibition of cytochrome oxidase could be a cause of retinal damage.     

Specific labelling of ganglion cells in the cat retina by a monoclonal antibody

The introduction of monoclonal antibody technology has made it possible to produce specific markers for various retinal cell types. We report here on a monoclonal antibody, AB5, which specifically labels the retinal ganglion cells of the cat. The labelled cells could be categorized as either alpha, beta or gamma subtype based on morphological criteria. This antibody will be useful for studies of the morphology, localization and synaptic connectivity of ganglion cells in the cat retina.     

Species variations in distribution of S100 in retina. Demonstration with a monoclonal antibody and a polyclonal antiserum

The S100 protein has been found consistently in glial cells both in the central nervous system (CNS) and peripheral nervous system (PNS). However, in the retina we find substantial species variation in the distribution of this protein. Immunohistochemically, in the human retina we do not find any S100. In the rabbit retina it is present both in Müller cells and in astrocytes and in the chicken retina it is in neurons. This demonstrates how misleading it can be to use the distribution of a protein in one species to generalize about the distribution of the same protein in other species. It is also clear that even though immunohistochemical staining for the S100 protein could be used to study pathologic conditions that involve Müller cells in guinea pigs, hamster, rat, and rabbit retina it is going to be of limited value in investigations of the same conditions in the human eye.     

S-antigen in the developing retina and pineal gland: a monoclonal antibody study

Spleen cells of BALB/c mice, previously immunized with bovine retinal S-antigen, were fused with Sp2/0-Ag14 mouse myeloma cells. Two monoclonal antibodies (MAbs) MAbA9-C6 (IgG2a isotype) and MAbA1-G5 (IgG1 isotype) were selected on the basis of reactivity in an ELISA and immunofluorescent assay. In radioimmunoassay MAbA9-C6 and MAbA1-G5 do not compete and appear to define unrelated epitopes of S-antigen. Both MAbs reacted in the ELISA assay, whereas only MAbA9-C6 bound to S-antigen in fixed tissue sections. Because MAbA9-C6 was useful for immunocytochemistry, it was studied in more detail. MAbA9-C6 bound to all vertebrate retinas tested including human, bovine, guinea pig, and mice. The immunoreactivity of MAbA9-C6 also was studied in the developing rat retina and pineal gland. In the morphologically undifferentiated retina, assessed by conventional light microscopy, there was an incomplete separation of the outer neuroblastic cells. However, in the same retina a distinct zone, corresponding to S-antigen immunoreactivity, was present indicating antigenic differentiation with regard to S-antigen at this stage of retinal development. In the pineal gland, S-antigen immunoreactivity was first observed in the three day old rat and at all stages examined thereafter. The usefulness of these two MAbs in the study of the embryologic development of the retina and of the antigenic epitopes of S-antigen is discussed.     

Isolation and characterization of a mouse monoclonal antibody against human corneal endothelial cells.

In vitro cultivation of human corneal endothelial cells (HCEC) is associated with loss of typical cobblestone-like appearance during successive passages. Thus far morphology was the sole criterion for the cell's endothelial nature. Mouse monoclonal antibodies (mabs) to human corneal endothelial cells were raised using standard immunization and hybridoma isolation procedures. The specificity of mabs for human corneal endothelial cells was tested in comparison to other endothelial cell types, to fibroblasts, corneal keratocytes and to human retinal pigmented epithelial cells. In addition immunofluorescence or immunoperoxidase staining was performed with frozen tissue sections of human corneas and with various other human tissues. The mab 9.3.E reacts with cultured human corneal endothelial cells, but not with cultured human fibroblasts and human keratocytes. In frozen sections selective positivity of corneal endothelium in contrast to negativity of the other corneal cell types was confirmed. In investigated extraocular tissues positivity was observed in smooth muscle cells including related cells (i.e. Ito and mesangial cells) and in Schwann's cells and adipocytes, but apparently not in vascular endothelial cells. The mab is human-specific and binds to a protein with a molecular weight of 130 kDa mainly accumulating along cell membranes. A mouse monoclonal antibody against human corneal endothelial cells was established in vitro and was shown to be capable of differentiating corneal endothelial cells from other corneal cell types, especially from corneal keratocytes. It is, however, not cornea-specific, but also reacts with certain extraocular cell types.     

Establishment of anti-idiotypic monoclonal antibodies against anti-interphotoreceptor retinoid-binding protein monoclonal antibody

Two monoclonal antibodies (MAbs) against interphotoreceptor retinoid-binding protein (IRBP) were previously established. One of the antibodies designated as MAb-TRA4 reacted with the IRBP of multiple species. Using this MAb-TRA4, 6 clonal anti-idiotypic monoclonal antibodies (MAb-TRDy; y = 1-6) against MAb-TRA4 were established. All of the MAb-TRDy were characterized by ELISA, immunoblotting and immunohistochemical studies. It was shown by ELISA that MAb-TRDy inhibited the specific binding between IRBP and MAb-TRA4 and did not cause nonspecific binding to rat polyclonal immunoglobulins (IgG and IgM). Immunoblotting assay demonstrated that MAb-TRDy inhibited the binding between IRBP and MAb-TRA4. Immunohistochemical examination also confirmed the immunological characterization of MAb-TRDy. Thus the present results indicate that the MAb-TRDy which have been established are the anti-idiotypic MAbs against anti-IRBP MAb-TRA4. MAb-TRDy can be used as a tool to study the pathogenesis of experimental autoimmune uveitis induced by IRBP.     

Displaced ganglion cells in the retina of the monkey.

Horseradish peroxidase has been injected into the dorsal lateral geniculate nucleus of macaque monkeys to label ganglion cells of the retina by retrograde axoplasmic transport. Displaced ganglion cells, with somata in the inner nuclear layer and dendrites in the inner plexiform layer, were detected by virtue of their filling with peroxidase-positive granules. These cells were numerous in the peripapillary region, but relatively uncommon elsewhere in the retina.     

A monoclonal antibody specific to Müller cells and selective synaptic sites in the retina.

We have produced a monoclonal antibody which stains the Müller cells in the region of the photoreceptors, nerve terminals surrounding the horizontal cells, and nerve terminals in the inner plexiform layer in carp, goldfish, and white perch retinas. Electron microscopy showed that the staining in the outer and inner retina was confined to Müller cells and presynaptic terminals, respectively. In the teleost brain, the antibody stained only the optic tectum and efferent fibers from the stratum album centrale. Biochemical characterization by immunoblotting showed that this monoclonal antibody recognizes an approximately 70-kD molecule in both whole retina and brain homogenates, suggesting that the antibody recognizes an identical molecule. No staining was noted in the spleen or the liver. This monoclonal antibody appears to be specific to a molecule common to the Müller cells and presynaptic terminals in the teleost retina, and although it is present in other parts of the central nervous system, it is confined to the visual pathway.     

The monoclonal antibody GRC1 produced against human cornea recognizes a common determinant of collagen.

The monoclonal antibody GRC1 was obtained by immunizing BALB/c mice with human cornea. Screening was performed by indirect immunofluorescence in cryostatic sections of several tissues: cornea, skin, placenta, hyaline cartilage, blood vessels, and nerves. GRC1 was seen to recognize fibrillar structures in all of these tissues. The pattern of reaction was interstitial and membranous. On cornea, GRC1 reacts definitely with Bowman's membrane and diffusely with the stroma, while on skin it shows strongly positive reactivity with the papillary dermis and with the basement membrane. It also reacts on hyaline cartilage at the periphery of the condrocytic lacunae. These immunohistologic results suggest that GRC1 recognized human collagen. In order to investigate further the subtype of collagen defined by GRC1, an ELISA was performed with purified collagens of several types: I, II, III, IV, and V. The monoclonal antibody GRC1 defines a common determinant in types III, IV, and V.     

Organization and development of horizontal cells in the goldfish retina, II: Use of monoclonal antibody MH1.

We have produced and characterized a monoclonal antibody, MH1, which selectively labels rod horizontal cells and Müller cells in the goldfish retina. Biochemical and tissue distribution studies indicate that MH1 may recognize four out of five classes of intermediate filament proteins in goldfish: vimentin, desmin, glial fibrillary acidic protein (GFAP), and keratin, but not neurofilament. The intermediate filament which is labeled strongest in the retina is vimentin. In the goldfish retina, the only type of horizontal cells recognized by MH1 appear to be rod horizontal cells. This result suggests that the rod horizontal cell, an interneuron, and Müller (glial) cells share a common antigen: vimentin, which is usually only expressed in mesenchymal origin cells. The development of rod horizontal cells in the goldfish retina was also studied using MH1. The cells were not labeled by MH1 until 4-6 weeks posthatching, a stage in which the animals are already visually active. MH1 also did not label any horizontal cell in the region close to the ora terminalis in the goldfish retina. These results suggest that either the emergence and maturation of rod horizontal cells occur late during goldfish retinal development or the expression of vimentin itself occurs late in the development of rod horizontal cells.     

Immunolocalization of heat shock proteins in the retina of normal monkey eyes and monkey eyes with laser-induced glaucoma

PURPOSE: To examine the expression and localization of heat shock proteins (HSPs) in the retinas of normal and experimentally induced primate glaucoma eyes. These proteins are known to be produced in response to a variety of stresses. METHODS: Experimental glaucoma was induced in the right eyes of three adult monkeys by repeated applications of argon laser to the chamber angle. Immunostaining with a panel of antibodies against HSP 90, 70, 60, 47, and 27 was performed on retinal sections prepared from the normal and glaucomatous monkey eyes. RESULTS: The intensity of immunostaining for HSP 90, 60, and 27 was greatly enhanced in the retinas of glaucomatous eyes. Prominent reactivity was observed in the inner retinal layers, especially in the ganglion cell and nerve fiber layers. The staining intensity for HSP 70 was also moderately increased, while immunoreactivity against HSP 47 remained almost unchanged in glaucomatous retinas. Immunostaining against glial fibrillary acidic protein was increased and the immunolabeling pattern appeared to be identical with that of HSP 90 in glaucoma retinas. CONCLUSIONS: The level of HSP 90, 70, 60, and 27 in primate retinas was increased in experimentally induced ocular hypertension. The differences in expression pattern suggest that each HSP may have its unique role in responding to damage or injury related to intraocular pressure elevation.