The Cytoskeletal Elements of Human Retinal Pigment Epithelium: in Vitro and in Vivo
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The cytoskeletal elements of human retinal pigment epithelium: in vitro and in vivo NICOL M. McKECHNlE1*, MIKE BOULTON2, HELEN L. ROBEY1, FELICITY J. SAVAGE1 and IAN GRIERSON1 ^Departments of Pathology and ^Clinical Ophthalmology, Institute of Ophthalmology, 17-25 Cayton St, London ECIV9AT and Judd St, London WCI, UK * Author for correspondence Summary The cytoskeletal elements of normal (in situ) and identifiable. Cytokeratin 18 positive cells were cultured human retinal pigment epithelium (RPE) identified in all RPE cultures (other than early were studied by a variety of immunocytochemical primaries), regardless of passage number, age or techniques. Primary antibodies to vimentin and sex of the donor. In post-confluent cultures cyto- cytokeratins were used. Positive immunoreactivity keratin 18 cells were identified growing over cyto- for vimentin was obtained with in situ and cultured keratin 18 negative cells, suggesting an association material. The pattern of reactivity obtained with of cytokeratin 18 immunoreactivity with cell pro- antisera and monoclonals to cytokeratins was more liferation. Immunofluorescence studies of retinal complex. Cytokeratin immunoreactivity could be scar tissue from two individuals revealed the pres- demonstrated in situ and in cultured cells. The ence of numerous cytokeratin 18 positive cells. pattern of cytokeratin expression was similar to These findings indicate that RPE cells can be ident- that of simple or glandular epithelia. A monoclonal ified by their cytokeratin immunoreactivity and antibody that specifically recognizes cytokeratin 18 that the overt expression of cytokeratin 18 may be identified a population of cultured RPE cells that associated with proliferation of human RPE both in had particularly well-defined filamentous networks vitro and in vivo. within their cytoplasm. Freshly isolated RPE was cytokeratin 18 negative by immunofluorescence, Key words: RPE, cytoskeleton, keratin, cytokeratin, but upon culture cytokeratin 18 positive cells were proliferation. Introduction cells of myogenic origin, neurofilament proteins in neurones, glial fibrillary acid protein (GFAP) in glia of Proliferation of the RPE has been suspected in a variety the central nervous system and the cytokeratins in of ocular diseases including proliferative diabetic retino- epithelial cells. The cytokeratins, a related family of pathy and is known to complicate ocular trauma and about 19 polypeptides identified by two-dimensional gel retinal detachment (Grierson et al. 1987). Therefore electrophoresis, occur in varying combination in different immunocytochemical markers of the RPE would be epithelia (Moll et al. 1982). Therefore epithelia can be powerful tools in tissue culture studies and in the characterized by the cytokeratins they express. Because identification of RPE cells in pathological material. In an of their usefulness in tumour identification (Osborn & attempt to identify such markers, we have studied the Weber, 1983; Ramaekers et al. 1983a), antisera and cytoskeletal proteins expressed by cultured and patho- monoclonal antibodies to intermediate filaments are logical RPE, using antibodies to vimentin and the cyto- readily available from commercial sources. keratins. As an epithelium, the RPE would be expected to Microfilaments and microtubules occur in most cell express cytokeratins, or at least to show co-expression of types and are therefore of little potential use as cell cytokeratins with vimentin in vitro as has been demon- markers, but they can be informative with regard to strated for some other epithelial cell types (Lane et al. cellular activities such as locomotion and phagocytosis. 1983; LaRocca & Rheinwald, 1984). However, following Intermediate filaments, five types of which have been studies of chick RPE, Docherty et al. (1984) stated identified, are tissue specific (Lazarides, 1980). In gen- "although RPE is an epithelium according to all other eral vimentin occurs in mesenchymal cells, desmin in criteria, it belongs to a group of tissues (including Journal of Cell Science 91, 303-312 (1988) Printed in Great Britain © The Company of Biologists Limited 1988 303 vascular endothelium, iris and lens-forming epithelium) of this antibody. It failed to react with the epidermis but showed that have intermediate filaments composed of vimentin strong staining of all cells in the dermis. The specificity of the and possess neither cytokeratins nor desmosomes". Other monoclonal to vimentin was also confirmed by immunoblotting. Cytokeratins. An antiserum to human epidermal keratins of authors have demonstrated cytokeratin-positive immuno- 3 3 reactivity of normal and pathological RPE in human, 56X10 and 64X10 molecular weight (Schlegel et al. 1980), monkey, bovine and rabbit material (HiscotteJ al. 1984). and an antiserum of wide cross-reactivity raised to bovine muzzle epidermal keratins (Franke et al. 1978; Steinert, 1975), The present study was undertaken to determine the were obtained from Dakopattis, Denmark. These two antisera distribution and the nature of the cytoskeletal proteins are commonly used for immunodiagnostic purposes. There expressed by the RPE in vitro and in vivo and to establish reactivity has been published (Franke et al. 1978; Pinkus et al. if cytokeratin immunoreactivity could be used as a 1985; Taxy et al. 1985; Battifora & Kopinski, 1985; Makai et al. specific marker of RPE cells. The controversy concerning 1985). the expression of cytokeratins by the RPE, and our The reactivity, according to the suppliers, of the monoclonal interest in the culture and pathology of human RPE, antibodies to the cytokeratins is most easily described using the prompted us to investigate the cytokeratin immunoreac- classification system of Moll et al. (1982). Monoclonals, RSK tivity of cultured human RPE in some detail. 102 (cytokeratins 5 and 8) described by Broers et al. (1986), RSK 105 (cytokeratin 7), NCL 5D3 (cytokeratins 8, 18 and 19) described by Angus et al. (1987) and RGE 53 (cytokeratin 18) Materials and methods described by Raemaekers et al. (19836), were obtained from Eurodiagnostics. A further five monoclonal antibodies were obtained from ICN: immunobiologicals, AE1 (cytokeratins 10, Cell culture 14, 15, 16 and 18) and AE3 (cytokeratins 1, 2, 3, 4, 5, 6, 7 and Post-mortem eyes from 11 individuals with no known ophthal- 8) described by Woodcock-Mitchell et al. (1982), KG8.13 mic disease were obtained from various hospitals in London. (cytokeratins 1, 5, 6, 7, 8, 10, 11 and 18) described by Gigie? al. Age of donors ranged from 7 years to 95 years of age. Post- (1982), KG8.12 (cytokeratins 13 and 16) and KG4.62 (cyto- mortem times ranged between 24 and 48 h. keratin 19). Immunoblotting experiments using RPE cell Human RPE cells were isolated as described (Boulton et al. cytoskeletal extracts and the antibodies to the cytokeratins were 1983). In some instances cells were retained for immunocyto- conducted to confirm the specificity of the antibodies. AE1, chemical investigation (see below). Cells were grown in Ham's AE3 and RSK 105 were used for immunoblotting but not in the F10 medium supplemented with 20% foetal calf serum, 0-4% remainder of this study. glucose and antibiotics. Cultures were maintained at 37°C in a CO2 incubator (95 % air and 5 % CO2). Cells were either grown in 25 cm2 Falcon flasks or in 8-chamber tissue culture slides Gel electrophoresis and immunoblotting Cultured RPE cells were harvested from 75 cm2 flasks using (Miles Scientific). Subcultures were obtained by detachment of - EDTA (20mM in calcium/magnesium-free PBS). Cell pellets cells from their substratum with 0 25 % trypsin and then plated were extracted with 1 % Triton X-100 in PBS for 2min. The out in growth medium at an appropriate split ratio. insoluble residue was dissolved by boiling for 5 min in sodium Prior to immunostaining cultures were washed twice with dodecyl sulphate (SDS) sample buffer (Laemmli, 1970). Gel phosphate-buffered saline (PBS) and fixed with cold (-20°C) electrophoresis was performed in 12-5% polyacrylamide slab methanol and acetone for 4 and 2min, respectively. For gels, as described by Laemmli (1970). cultures grown in flasks the acetone fixation was omitted. Electrophoretically separated polypeptides were transferred Histopathological material to a nitrocellulose sheet by overnight blotting at 100 mA following the method of Towbin et al. (1979). Immunostaining Pathological material (retinal scar tissue from two individuals) of the transferred proteins was conducted as described by was obtained from Moorfields Eye Hospital following vitrec- Broers et al. (1986). Anti-mouse and anti-rabbit peroxidase tomy surgery. One specimen was removed in the treatment of conjugates were obtained from Sigma. macular pucker, the other from an eye with massive periretinal proliferation following retinal detachment. Normal human eyes were obtained following their use for corneal grafting. Material Secondary! antibodies for histological examination was frozen in liquid nitrogen, FITC- and TRITC-labelled goat anti-mouse IgG and goat anti- embedded in OCT Compound (Miles Scientific). Cryostat rabbit IgG and peroxidase-labelled goat anti-mouse IgG were sections mounted on glass slides were fixed in cold ( — 20 °C) obtained from SIGMA. Gold-labelled goat anti-mouse IgG and methanol (4min) and acetone (2min) prior to immunostaining. chemicals for the silver enhancement of immuno-gold staining In some cases when RPE cells were being isolated for culture were obtained from Janssen Pharmaceuticals.