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1From the Department of Ophthalmology, Helsinki University Central Hospital, Helsinki, Finland; the 2Department of Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany.
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Abstract |
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METHODS. Retinas were obtained from seven normal human eyes at autopsy (age, 43–78 years). The specimens were embedded in medium-grade resin and studied by postembedding immunoelectron microscopy using the primary mouse mAb HNK-1 (Leu 7) to the HNK-1 epitope and secondary antibodies conjugated to 10-nm colloidal gold particles.
RESULTS. Prominent immunolabeling with mAb HNK-1 was observed on the outer surface of the entire plasma membrane of Müller radial glial cells, including their microvilli between the inner segments of rods and cones, on the plasma membranes of astrocytes in the ganglion cell layer, in bipolar cells in the inner nuclear layer, and in photoreceptor cells in the outer nuclear layer. Fewer gold particles were present on plasma membranes of other main types of retinal neurons, including ganglion cells. Only the outer segments of rods and cones and the endothelial cells of retinal capillaries were never labeled. In the ciliary epithelium, gold particles localized to the basement membrane of the nonpigmented and pigmented layers and to the cytoplasm of the pigmented epithelium.
CONCLUSIONS. Unlike in many other species, the HNK-1 epitope in the human retina is found on both glial and neuronal cells, including photoreceptors. This epitope potentially contributes to neuron-to-neuron and glia-to-neuron adhesion of human retinal cells.
3)Galß(14)GlcNAc,1 is a prominent developmentally regulated carbohydrate sequence present in the retina and retinal pigment epithelium of vertebrates.2 3 4 5 6 It is part of several plasma membrane and extracellular matrix glycoproteins, glycolipids, and proteoglycans involved in cell-to-cell and cell-to-substratum adhesion.6 7 8 9 In the human eye, the HNK-1 epitope can also be found in subepithelial matrix cells of the inner connective tissue layer and pigmented and nonpigmented epithelium of the ciliary body, in the posterior pigmented epithelium of the iris, in the lens capsule and zonular lamella, in scleral fibroblasts, and in optic and ciliary nerves.2 6 10 11 12 13 14 Immunoelectron microscopy has localized it to plasma membranes of the subepithelial matrix cells, microfibrillar bundles, and elastic fibers in the inner connective tissue layer, to the basement membranes of both layers of the ciliary epithelium, as well as to the cytoplasm of the pigmented ciliary epithelium.12 13 14 In the lens, the HNK-1 epitope is present in the outer three fourths of the central anterior capsule, as visualized by immunoelectron microscopy.12 13
Unlike in the anterior segment of the eye, the localization of the HNK-1 epitope in the vertebrate retina is not known in detail. Because of the extensive membrane-associated immunolabeling and the intimate approximation of retinal glial and neuronal cell membranes, light microscopy has allowed unquestionable localization of the HNK-1 epitope only to Müller radial glial cells in humans.2 3 4 15 To gain a better understanding of the detailed distribution and subcellular compartmentalization in the human retina of the HNK-1 epitope, we determined by immunoelectron microscopy which additional cell types bear this carbohydrate sequence and to which cell organelles the immunoreaction localizes.
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For postembedding immunogold labeling, tissue specimens approximately 2 x 2 mm in size were fixed within 1 to 5 hours after death in freshly prepared 4% (wt/vol) paraformaldehyde and 0.1% (vol/vol) glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 2 hours at 4°C. After fixation, the specimens were rinsed in 0.1 M cacodylate buffer containing 3.7% (wt/vol) saccharose and sequentially dehydrated in 30% (vol/vol) ethanol at 4°C, 50% (vol/vol) ethanol at 0°C, and 70% (vol/vol) ethanol at -20°C. The specimens were then infiltrated with a 1:1 mixture of 70% ethanol and medium grade resin (LR White; Electron Microscopy Sciences, Fort Washington, PA) at 4°C and several changes of pure resin at 4°C and room temperature and finally polymerized in gelatin capsules for 24 hours at 45°C. Ultrathin sections were cut with a microtome (Ultracut; Leica, Bensheim, Germany) and mounted on uncoated nickel grids. Part of each specimen was also fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), postfixed in 2% osmium tetroxide in phosphate buffer, and routinely processed for embedding in epoxy resin (Epon; Serva, Heidelberg, Germany).
The study was approved by the institutional review boards, and it adhered to the tenets of The Declaration of Helsinki.
Immunogold Labeling
The mouse monoclonal antibody, mAb HNK-1 (Leu 7; IgM, diluted 1:100; Lot 50808; BD Bioscience, San Jose, CA) was used to detect the HNK-1 epitope.16 This antibody has been found to react in immunoelectron microscopy.12 13 14 An unrelated mouse mAb to cytokeratin 14, CK B1 (IgM, diluted 1:100; Lot 100H4800; Sigma, St. Louis, MO), was used as a control to exclude nonspecific binding of antibodies of the same isotype. Goat anti-mouse antiserum that reacts with mouse IgG and IgM, conjugated to 10-nm colloidal gold particles (diluted 1:30; Bio Cell, Cardiff, Wales, UK), was used as the secondary antibody.
For postembedding immunogold labeling, ultrathin sections were incubated successively in drops of Tris-buffered saline (TBS, pH 7.4) for 5 minutes at room temperature; 0.05 M glycine in TBS for 30 minutes at room temperature, 0.5% (wt/vol) ovalbumin and 0.5% (vol/vol) teleost fish gelatin in TBS, both for 30 minutes at room temperature; and primary antibody diluted in TBS-ovalbumin overnight at 4°C. After 5 washing steps in TBS-ovalbumin-gelatin, the grids were incubated in 10-nm gold-conjugated secondary antibody diluted in TBS-ovalbumin-fish gelatin for 1 hour at room temperature. After being rinsed in TBS-ovalbumin-gelatin, TBS, and water (five times each), the sections were stained with 2.5% uranyl acetate for 3 minutes and examined with a transmission electron microscope (EM 906E; LEO, Oberkochen, Germany).
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In the retina, all layers except the outer portion of the photoreceptor cell layer reacted with mAb HNK-1. Gold particles localized to the inner limiting membrane (Fig. 1A , ilm), formed by plasma membrane of Müller cells (mc), but vitreous collagen was not labeled. In the inner retina, gold particles were present along the inner processes of Müller cells (Fig. 1A) , on the cell membranes of astrocytes (Fig. 1B , ac), and along ganglion cell (gc) axons (Figs. 1B 1C 1D) , even when adjacent Müller cell processes were not identified (Fig. 1C) . In the inner plexiform layer, gold particles were also found along Müller cell processes and, to a lesser extent, on neuronal processes (Fig. 1E , nc). Gold particles were three to four times more abundant on glial than neuronal plasma membranes in these layers.
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In the outer third of the retina, the membranes of perikarya and processes of rods and cones (Fig. 2A) and of the outer processes (Fig. 2A) and microvilli (Fig. 2B) of Müller cells were labeled with gold particles. The density of gold particles appeared equal on both cell types. Some gold particles were present on the inner segments of rods (Fig. 2B , ris) and cones (cis), where no Müller cell microvilli were identified. The outer segments of rods (Fig. 2C , ros) and cones (cos), as well as the mucopolysaccharide-rich interphotoreceptor matrix (ipm) remained unlabeled.
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The human retina differs from most vertebrate retinas, in which the HNK-1 epitope is found mainly or exclusively on neurons and predominantly in the plexiform layers. In amphibians, reptiles, birds, and mammals with rod-dominated retinas, this epitope is observed in populations of ganglion, amacrine, bipolar, and horizontal cells, as in the human retina, but apparently not in glial and photoreceptor cells.4 5 18 19 20 21 22 23 However, in these species immunoelectron microscopy has not been used, and low-grade expression by retinal glia consequently cannot be totally ruled out.
In fish and primates, the HNK-1 epitope is also found on Müller radial glial cells, analogous to the human retina.4 5 Similar immunoreactivity in tree squirrels has suggested a theory that expression of the HNK-1 epitope by mammalian radial glial cells may be linked with cone predominance.5 The density of gold particles along rods and cones of humans in this study was shown to be similar by immunoelectron microscopy, indicating that both types of photoreceptor cells nevertheless participate in HNK-1 epitope-mediated interactions in the cone-dominant human retina, in which the Müller cell was a major cell type bearing this epitope.
We did not detect any intracytoplasmic labeling for the HNK-1 epitope in the human retina, in contrast to the pigmented ciliary epithelium, in which intracytoplasmic gold particles were numerous.12 14 Although the specimens were from cadaveric eyes, which may have had diminished immunoreactivity, it seems that no more than minor amounts of molecules bearing this epitope could have been in transit in the cytoplasm. The HNK-1 epitope-bearing molecules in the retina must therefore have a slow turnover rate, as would be fitting of macromolecules that supposedly have a mainly constitutive, structural role. Alternatively, the epitope is added at the cell surface when the corresponding macromolecules become incorporated into the plasma membrane or adjacent extracellular matrix. So far, we know that the HNK-1 epitope is added at least to myelin-associated glycoprotein intracellularly.24
Although most gold particles were clearly associated with plasma membranes, it was difficult to exclude the possibility that some immunoreactivity may have been due to molecules secreted to the extracellular matrix. No immunoreactivity was observed in the interphotoreceptor matrix, however, secreted by the retinal pigment epithelial and Müller cells,25 which themselves are immunopositive for the HNK-1 epitope.2 26
Given our observation that the HNK-1 epitope is present in neurons of the human retina, including photoreceptor and bipolar cells, it is of note that retinoblastoma cells do not react to this epitope.2 3 Although considered to be mainly neuroblastic in nature, with differentiation toward photoreceptor cells and sometimes neurons of the inner nuclear layer,27 28 they seem to be unable to express the HNK-1 epitope. It is of note that retinoblastoma is a noncohesive rather than a solid cancer, which typically seeds satellites to the vitreous and can even give rise to a pseudohypopyon of floating cells in the anterior chamber. The stable expression of the HNK-1 epitope in normal human retinal neurons throughout adult life, as shown in the current study, and its absence from noncohesive retinoblastoma cells2 3 are observations consistent with the proposed structural function for the HNK-1 epitope in glial and neuronal adhesion.
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Footnotes |
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Submitted for publication May 22, 2002; revised September 23, 2002; accepted October 3, 2002.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Marita Uusitalo, Ophthalmic Pathology Laboratory, Department of Ophthalmology, Helsinki University Central Hospital, PO Box 220, (Haartmaninkatu 4 C), FIN-00029 HUS, Helsinki, Finland; marita.uusitalo{at}fimnet.fi.
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