Retinal Pigment Epithelial Cells in Epiretinal Membranes: an Immunohistochemical Study

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Br J Ophthalmol: first published as 10.1136/bjo.68.10.708 on 1 October 1984. Downloaded from

British Journal of Ophthalmology, 1984, 68, 708-715

Retinal pigment epithelial cells in epiretinal membranes: an immunohistochemical study

P. S. HISCOTT,' I. GRIERSON,' AND D. McLEOD2 From the 'Department ofPathology, Institute ofOphthalmology, London, and the 2Surgical Vitreoretinal Unit, Moorfields Eye Hospital, London

SUMMARY Immunohistochemical techniques were used to identify cells containing cytokeratins in sections or tissue-culture monolayers from ocular (reference) tissues and also from 22 epiretinal membranes obtained during closed microsurgery for macular pucker or massive preretinal retraction. Results of cytokeratin immunostaining in reference tissues indicated that this is a valuable means of determining the contribution and distribution of epithelial cells in epiretinal membranes, and that the epithelial cells in the membranes were probably derived from the retinal pigment epithelium. Epithelial cells were identified in 17 of the 22 epiretinal membranes, but they did not usually constitute the predominant cell type. We concluded that the fibroblasts or fibroblast-like cells thought to be responsible for the contraction of epiretinal membranes are seldom of retinal pigment epithelial origin. Biomicroscopic pigmentation of a membrane was shown to be a poor guide to its epithelial cell population.

Epiretinal membranes (ERMs) are cellular prolifera- cytoskeletal proteins of epithelial cells-prekeratin tions on the retinal surface which sometimes compli- and keratin-to demonstrate the contribution and cate the natural history or surgical treatment of distribution of epithelial cells in ERMs. Sections of http://bjo.bmj.com/ rhegmatogenous retinal detachment. Contraction of surgically excised ERMs, together with cells derived an ERM localised to the posterior retina produces from ERMs in tissue culture, were investigated fol- macular pucker, while more widespread ERM con- lowing closed intraocular microsurgery for macular traction causes massive preretinal retraction (MPR) pucker and MPR mainly complicating rhegmato- or proliferative vitreoretinopathy. Retinal pigment genous retinal detachment. epithelial (RPE) cells have been implicated as major contributors to such proliferations and as progenitors Material and methods on September 28, 2021 by guest. Protected copyright. of the fibroblasts or fibroblast-like cells responsible for membrane contraction. '" STANDARDS Histopathological analysis has suggested that meta- To confirm the specificity of immunostaining within plastic cells derived from a variety of sources may be the ERMs, and to provide intensity standards for present in ERMs,4-" but morphological and ultra- each immunohistochemical procedure, a variety of structural criteria are an unreliable means of demon- tissues (Table 1) and tissue culture monolayers (Table strating the precise origins ofcomponent cells. Tissue 2) were assessed for prekeratin and keratin content. culture and immunohistochemical techniques have These materials were derived from section or dissec- recently been employed to elucidate further their tion of the following globes: 4 adult albino rabbit cellular derivation,'2-'5 but so far only the glial cell eyes, 2 bovine eyes, 2 adult owl monkey eyes, an contribution to ERMs has been comprehensively albino human eye (20 weeks gestation), and 3 adult investigated.'5 In view of the possible importance of human eyebank globes. In addition 4 human eyes RPE cells in the pathophysiology of ERMs, we have containing retinal detachments were studied. used immunohistochemical markers to two of the EPIRETINAL MEMBRANES Correspondence to Dr Ian Grierson, Pathology Department, Insti- Excised ERMs were obtained from 22 eyes under- tute of Ophthalmology, 17/25 Cayton Strect, London ECIV 9AT. going closed intraocular microsurgery for macular 708 Br J Ophthalmol: first published as 10.1136/bjo.68.10.708 on 1 October 1984. Downloaded from

Retinalpigment epithelial cells in epiretinal membranes: an immunohistochemical study 709

Table I Tissue standards for immunohistochemical Table 2 Tissue culturestandardsfor immunohistochemical staining staining

Intensity of Intensity of immunostaining immunostaining FIP Prekeratin Keratin FIP Prekeratin Keratin Corneal epithelium Bovine corneal epithelium F + + + +++ Adult albino rabbit F & P + + + NP Human lens epithelium F NP + Adult bovine F +++ +++ Bovine retinal pigment Adult owl monkey P +++ +++ epithelium F + + + Fetal albino human P + + + +++ Human retinal pigment Adult human F&P + + +++ epithelium F&P ++ ++ Corneal endothelium Adult albino rabbit F & P + NP F=immunofluorescent technique. P=immunoperoxidase tech- Adult bovine F - - nique. NP=not performed. Adult owl monkey P - +/- Fetal albino human P Adult human F&P +1- +1- standards and eight of the ERMs were fixed in 10% Lens epithelium formol-saline, dehydrated in graded alcohols and Adult albino rabbit F & P +/- NP embedded in paraffin wax. In an attempt to enhance Adult owl monkey P Fetal albino human P visualisation of immunostaining, globes containing Adult human F & P heavily pigmented tissues were bleached by various Iris epithelium bleaching methods. Of these, only 0-25% potassium Adult albino rabbit F & P +/- NP permanganate followed by oxalic acid preserved Fetal albino human P + + Adult human F&P +1- +1- tissue integrity and produced effective bleaching. Ciliary epithelium Portions of bovine cornea and the remaining eight Adult albino rabbit F & P - NP ERMs were placed in OCT embedding media (Lab- Adult human F & P Neuroretinal tissue Tek Products, Illinois) and immediately frozen in Adult owl monkey P liquid nitrogen. At least four wax or frozen sections Fetal albino human P from each tissue were then prepared for immuno- Adult human F & P peroxidase or immunofluorescent staining as pre- Retinalpigment epithelium Adult albino rabbit F & P + NP viously described. 15 Sections were also taken for con- Fetal albino human P + + ventional (haematoxylin and eosin) staining. Adult human F & P + + http://bjo.bmj.com/ Metaplastic adult human P + + + + Tissue culture monolayers. Monolayers to be used as reference standards and primary monolayers grow- F=immunofluorescent technique. P= immunoperoxidase tcch- ing from ERM explants were thoroughly washed in nique. NP=not performed. phosphate-buffered saline (PBS, pH 7.6) to remove culture medium. They were then air-dried for 20 pucker (14 eyes) or MPR (8 eyes); aetiological factors minutes before immunohistochemical staining.

and clinical circumstances surrounding the surgery on September 28, 2021 by guest. Protected copyright. were recorded. A standard proforma documenting IMMUNOHISTOCHEMISTRY FOR KERATINS the biomicroscopic features of each membrane was Cytokeratins differ both biochemically and immuno- also completed by the surgeon; in this investigation logically between various epithelia,'6'8 so we used particular attention was paid to the presence or two different antibodies, each with a wide spectrum absence of visible pigmentation in the ERM prior to ofreactivity, in order to minimise false-negative stain- its removal from the retinal surface. ing: (a) antiprekeratin (Miles Scientific; Slough, Sixteen of the peeled ERMs were embedded England) raised in guinea-pigs with fetal bovine hoof directly for histopathological and immunohisto- and diluted between 1:10 and 1:50 in TRIS-buffered chemical analysis (Table 3). Monolayers of cells were saline (TBS, pH 7-6). (b) antikeratin (Dako Corp., grown in tissue culture from the remaining 6 ERMs California, USA) raised in rabbits with human by a method previously described,'4 (Table 4). Only stratum corneum from the foot and diluted between ERMs with a prominent cellular component (as dis- 1:30 and 1:200 in TBS. tinct from late collagenous scars) were included in Immunoperoxidase demonstration of prekeratin this study. was conducted by a standard indirect peroxidase technique as outlined by Mukai and Rosai.'9 For SPECIMEN PREPARATION keratin, immunoperoxidase staining was achieved Eyes and epiretinal membranes. Globes to be used as with a peroxidase-antiperoxidase method previously Br J Ophthalmol: first published as 10.1136/bjo.68.10.708 on 1 October 1984. Downloaded from

710 P. S. Hiscott, I. Grierson, and D. McLeod

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Retinalpigment epithelial cells in epiretinal membranes: an immunohistochemical study 711 http://bjo.bmj.com/ on September 28, 2021 by guest. Protected copyright. Fig. 1 Dewaxed 7Rsm sections stained by Fig. 2 Immunofluorescentstainingforprekeratin and immunoperoxidase methods to demonstrate prekeratin and keratin infrozen sections and cell monolayers. (a and b) keratin; allsections were counterstained with haematoxylin. Frozen sectionsfrom two ERMs stainedforprekeratin; in (a) (a) Normal human RPE stainedforprekeratin; the reaction epithelial cells abound, whereas in (b) epithelial cells are rare product is brown (x820). (b) Control section as in (a), but (x 130). (c and d) Frozen sectionsfrom two ERMs stained excluding theprimary antibody; no reaction product is for keratin; in (c) epithelial cells are prominent, exhibiting evident (x 820). (c) Human RPE cells grown in vitro and either moderate or intensefluorescence, whereas in (d) only a stained to demonstrate keratin; a distinctive brown reaction smallfocus ofpositively staining cells can be identified product can be seen in the cytoplasm ofthe cells (x 165). (d) (x530). (e) Frozen section from an ERMstainedfor Human corneal epithelium stained to demonstrate keratin; a prekeratin; note the elongated spindle shape ofone ofthe dark brown reaction product is present in all layers (X 245). positively staining cells (x530). (f) Monolayergrowingfrom (e and f) ERMs stainedfor keratin; there are many positive an ERM in tissue culture stainedfor keratin; the epithelial cells in (e), but epithelial cells are virtually absent in (f) cells havepositive cytoplasmic and negative nuclear (x330). (g and h) ERMs stainedforprekeratin; in (g) the fluorescence; note thefilamentous arrangement ofthe positive cells scattered within the membrane are kite-shaped cytokeratins (x 660). likefibroblasts, whereas in (h), the epithelial cells are cuboidal and virtually restricted to a surface monolayer (x165 and x330). Br J Ophthalmol: first published as 10.1136/bjo.68.10.708 on 1 October 1984. Downloaded from

712 P. S. Hiscott, 1. Grierson, and D. McLeod

Table 3 Immunohistochemistry of 16 embedded peeled epiretinal membranes

No. MPI Aetiology Biomicroscopic FIP % cells % cells Intensity Intensity Distribution MPR pigmentation prekeratin keratin prekeratin keratin ofepithelial positive positive staining staining cell componiet?t I MP Multiple surgery for large tear and RRD; Non-pigmented P 90/0 9(% + + + + + + Focus/diffuse postop. uveitis 2 MP Surgery for multiple largc brcaks and Non-pigmented P 6()% 40% + ++ +++ Focus/laycr/ RRD; diffuse 3 MP Multiple surgery for multipie breaks and Pigmented F 40% 40% + + + + + Layer/diffusc RRD; vitreous haemorrhage 4 MP Surgery for multiple tcars and RRD; post- Non-pigmented P 25% l()% + + + Diffusc op. vitrcous haemorrhage 5 MP Vitreous haemorrhage after blunt trauma; Non-pigmentcd P 25% 20% + + + + Diffusc no retinal brcaks 6 MP Silicone oil exchange for rccurrent RRD; Non-pigmented F 20% 20% + + + + Focus later peel under oil 7 MP Posterior penetrating injury; rctinal Non-pigmented F 10(% NP + NP Diffuse incarceration and vitreous haemorrhage 8 MP Multiple surgery for posterior penctrating Pigmented F 5% NP + NP Diffusc injury and RRD; retinal incarceration 9 MP Silicone oil exchange for giant tear and Non-pigmcnted P 0%/0 ()0% total RRD; later peel under oil 10 MP Central retinal vein occlusion and vitrcous Pigmcnted F (0% (0% haemorrhagc 11 MPR Surgery for aphakic RRD; postop. Pigmented F 50% 50% + + +++ Focus/diffusc vitreous hacmorrhagc 12 MPR Surgery for large break and RRD in high Pigmented P 2()% 2t)% + + + Focus/diffuse myopia 13 MPR Acute retinal necrosis syndrome; multipie Non-pigmcnted P 15% 1(% + + + + Diffuse breaks and RRD 14 MPR Surgery for multiple posterior breaks Pigmented F 1(% 1O0 + + + Focus/diffuse and RRD 15 MPR Multiple surgery for multiple breaks and Non-pigmented P 0%/0 (0% RRD 16 MPR Multiple surgery for aphakic RRD with Non-pigmented F (% (/0% vitreous haemorrhage and endophthalmitis http://bjo.bmj.com/

MP=macular pucker. RRD=rhegmatogenous retinal detachment. MPR=massivc preretinal retraction. NP= not performed. P=immuno- peoxidase technique. F=immunofluorescent tcchnique. described.'5 To highlight immunostaining in pig- fluorescent staining for both prekeratin and keratin mented tissues we employed two staining substrates on sections and cell monolayers was performed by yielding different coloured reaction products: 3,3 the indirect immunofluorescent technique previously on September 28, 2021 by guest. Protected copyright. diaminobenzidine tetrahydrochloride (DAB) or 3- employed.'4 '' Control sections or monolayers were amino-9-ethylcarbazole (AEC), which gave brown processed in each immunostaining procedure with and pink reaction products respectively. Immuno- omission or substitution of the primary antibody. Table 4 Immunofluorescence ofsix tissue culture monolayersfrom peeled epiretinal membranes No. MPI Aetiology Biomicroscopic '/, cells Initensity Intensity of MPR pigmentation positive ofprekeratini keratin stai'nintg staining 1 MP Multiple surgery for large tear and RRD Non-pigmented 25% NP ++ + 2 MP Multiple surgery for largedialysis and RRD after blunt trauma Pigmented 1(% NP + + 3 MP 'Idiopathic' epimacular membrane and peripheral retinoschisis Non-pigmented 10% ++ + ++ + without breaks 4 MP Extensive cryotherapy for small flat horseshoe tear Non-pigmented 0(%o 5 MPR Silicone oil exchangefor giant tear and RRD; later peel under oil Pigmented 5)0% + + + ± + 6 MPR Multiple surgery for dialysis and RRD after blunt trauma; retinal Non-pigmented 50% +++ + + incarceration

MP=macular pucker. RRD=rhegmatogenous retinal detachment. MPR=rmassivc preretinal rctraction. NP=not performed. Br J Ophthalmol: first published as 10.1136/bjo.68.10.708 on 1 October 1984. Downloaded from

Retinalpigment epithelial cells in epiretinal membranes: an immunohistochemical study 713

Using 'in situ' and cultured corneal epithelium as tensity was observed between cells in individual reference standards for prekeratin and keratin specimens (Fig. 2c). labelling, we evaluated staining of the various tissues The distribution of epithelial cells within the 12 and cultured monolayers taking into account the anti- positively staining ERMs was variable, but three body dilution at which the tissue or cells stained. For patterns (diffuse, foci, and layers) were recognised both immunoperoxidase staining ('P' in Tables 1, 2, occurring either singly or in combination (Table 3). and 3) and immunofluorescence ('F' in Tables 1, 2, 3, Foci were defined as circumscribed aggregates ofcells and 4), intensity of staining was assessed as follows: (Fig. 2d) and were present in six of the 12 ERMs. A layer of cells forming a single lamina on a surface of - no staining the ERM occurred in two specimens (Fig. lh). In 11 -/+ inconsistent staining + weak staining ERMs, however, isolated cells scattered throughout + + moderate staining the membrane were recognised (Figs. le, g; 2a, c). + + + intense staining. Irrespective of their pattern of distribution the epithelial cells usually had a cuboidal or oval shape If intensity of staining varied between cells within a (Figs. lh, 2c); the remainder ofthe positively staining specimen, the mean result was recorded. In the peeled cells looked like fibroblasts, that is, they were either ERMs or their monolayers the percentage of cells kite-shaped or spindle-shaped (Figs. lg, 2e). How- staining positively for prekeratin or keratin was also ever, such fibroblast-like epithelial cells were present recorded. These semiquantitative assessments were in only four ERMs, and the great majority of fibro- made by two observers (P.S.H. and I.G.) indepen- blast-like cells were negatively staining. In only one dently and subsequently averaged. membrane did fibroblast-shaped epithelial cells pre- dominate (Fig. lg; specimen 1 in Table 3). Results MONOLAYERS FROM ERMS STAN DARDS Five of the six tissue culture monolayers growing RPE cells in situ stained positively for cytokeratins from ERM explants contained cells which stained (Table 1; Figs. la, b): the staining patterns for keratin positively for cytokeratins, sometimes in substantial and prekeratin were similar but more consistent numbers (Table 4). The proportions of cells fluor- results were obtained with the prekeratin marker. escing after antiprekeratin and antikeratin prepara- Metaplastic RPE cells beneath detached human tion were judged to be equal in each monolayer (as retina and bovine and human RPE cells in vitro shown in a single column in Table 4). However, these

showed more impressive (moderate) staining for results may not represent the proportion of epithelial http://bjo.bmj.com/ keratins than did normal RPE cells (Tables 1 and 2; cells in the ERM explant because the culture medium Fig. ic). The only cells to show intense staining for may selectively enhance or repress the growth of the cytokeratin markers, either in the globes or in individual cell types. As in pure monolayers of RPE tissue culture, were corneal epithelial cells (Tables 1 cells, the melanin content of staining cells decreased and 2; Fig. ld). All the other cells examined were with progressive cell division in culture and the stain- either weakly positive, negative, or variable in staining intensity for epithelial markers was more pro- ing pattern (Tables 1 and 2). nounced than that seen in RPE cells in situ. The positively staining cells grown from ERMs adopted a on September 28, 2021 by guest. Protected copyright. EMBEDDED ERMS flattened oval shape and showed a bright filamentous Twelve of the 16 embedded ERM-specimens con- pattern in their cytoplasm which was particularly tained epithelial cells as shown by immunohisto- strong around the nucleus (Fig. 2f). The negatively chemical staining for keratin and prekeratin (Figs. staining cells in the monolayers had the morpho- le-h; 2a-e). The estimated epithelial cell contribution logical and locomotory characteristics of inflamma- in the ERMs ranged from approximately 90% to 5% tory cells, glial cells, and/or fibroblasts. (Table 3). In the majority of ERMS, however, only 25% or less of the cells were positively staining for CLINICO-IMMUNOHISTOCHEMICAL CORRELATION prekeratin and keratin, and only one specimen had a The presence of clinically recognisable pigmentation predominant epithelial cell component. in an ERM (eight specimens in Tables 3 and 4) cor- In most ERMs equal numbers of cells stained for related well with subsequent immunohistochemical prekeratin and for keratin; in four ERMs, however, demonstration of an epithelial cell component in the cells staining for prekeratin outnumbered cells stain- embedded ERM or tissue culture monolayer. The ing for keratin (Table 3). Overall the intensity of only exception (specimen 10 in Table 3) was a pig- prekeratin staining parallelled that of keratin, and mented ERM removed during microsurgery for a with both antibodies some variation in staining in- vitreous haemorrhage in which haemosiderin laden Br J Ophthalmol: first published as 10.1136/bjo.68.10.708 on 1 October 1984. Downloaded from

714 P. S. Hiscott, 1. Grierson, and D. McLeod macrophages were responsible for the ERM pig- tion in ERMs and potential confusion with fibrous mentation. Conversely, the absence of biomicro- astrocytes.8 Conversely, the cytokeratins of epithelial scopically apparent pigmentation in an ERM cells are retained in long-term tissue culture and provided no clue to the epithelial cell content; 10 of during neoplastic transformation22-26 and frequently the 14 non-pigmented membranes had a significant become more prominent-for example, keratin epithelial cell component (Tables 3 and 4), and the synthesis demonstrated during squamous metaplasia embedded ERM with the greatest proportion of of RPE cells in organ culture.27 We have shown that, positively staining cells (specimen 1 in Table 3) was both in tissue culture and during metaplasia in the clinically non-pigmented. Identification of the subretinal space, RPE cells stain more intensely for reasons for the presence or absence of biomicro- cytokeratins than normally despite their altered scopic pigmentation in an ERM was attempted. Most morphology. Thus, while the reliability of the ultra- melanin-containing cells were positive for epithelial structural criteria which can be used to identify RPE markers, but not all. Substantial numbers of cells cells decreases with cellular transformation, the without melanin in their cytoplasm were positively reliability ofimmunohistochemical identification may staining, and extracellular melanin granules were well increase. Furthermore, light microscopic abundant in some specimens. Thus such factors as immunohistochemistry on multiple sections of ERM incorporation of melanin by non-epithelial cells, pro- specimens permits more representative tissue gressive melanin-loss from RPE cells after each cell sampling for analysis compared with electron micro- division, cocooning of cells in collagen and clumping scopy. For these reasons we consider immunostaining of melanin-containing cells all tend to make bio- to be a powerful technique in the elucidation of the microscopic estimation of pigmentation a poor guide contribution and distribution of epithelial cells in to epithelial cell content in ERMs. Our histochemical ERMs. estimates of epithelial cell contribution in ERMs Apart from penetrating injuries, the clinical cir- showed that those classified as 'pigmented' had on cumstances surrounding the development of ERMs average a 21% epithelial cell content, whereas non- suggest a limited number of sources from which epi- pigmented ERMs were not dissimilar with a 22% thelial cells could gain access to the retinal surface. epithelial cell content. Because of the well established association of retinal Overall, epimacular membranes removed from break formation, RPE cell dispersion intothe vitreous eyes with traction detachment localised to the cavity, and development of ERMs, it appears likely posterior pole (MP) contained a somewhat greater that most epithelial cells in ERMs would be of RPE proportion of epithelial cells than did ERMs peeled cell origin. Furthermore, of all the intraocular epi- from bullously elevated immobile retina (MPR) thelia 'in situ' in our study, RPE cells stained for http://bjo.bmj.com/ (Table 3). Fourteen of the 17 ERMs with an epithelial keratins the most consistently (albeit weakly), and cell component in sections or monolayers were showed marked enhancement of immunofluorescent removed from eyes with a history of(orin the presence staining in tissue culture. For these reasons we believe of) a rhegmatogenous retinal detachment (Tables 3 that positively stainingcells in ourERMswere derived and4). Ofthe remainingthree ERMs which contained from the RPE (though a ciliary or iris epithelial origin or grew positively staining cells two (specimen 5 in could not be entirely excluded).

Table 3; specimen 3 in Table 4) were obtained from on September 28, 2021 by guest. Protected copyright. eyes with no clinically identified retinal break, and SIGNIFICANCE OF RPE CELLS IN ERMS the origin ofthis epithelial cell component is therefore RPE cells in ERMs may adopt the morphology of obscure. Four of the five ERMs with no positively macrophages or fibroblasts, while others resemble staining cells in sections or monolayers were removed normal RPE.2-4 In this study we identified oval cells, from eyes which contained a retinal break (Tables 3 spindle-shaped cells, and cuboidal cells which were and 4), so the presence of exposed RPE cells on positively staining for epithelial markers. Moreover, Bruch's membrane did not necessarily ensure an epi- the staining characteristics in embedded specimens thelial cell component in the associated ERM. and monolayers were broadly comparable for both prekeratin and keratin, so it is unlikely that the use of Discussion additional markers to cytokeratins would demonstrate a further complement of epithelial cells in the When RPE cells are implanted into the vitreous cavity ERMs. For these reasons, and bearing in mind our of rabbits and monkeys, their morphology changes so careful use of controls and standards, we consider they may no longer resemble RPE cells 'in situ.'2021 that cells staining negatively for prekeratin and Human RPE cells can similarly change in their keratin in the ERMs and monolayers were not of appearance if they are displaced on to the retinal RPE origin. surface,5 with consequent difficulties in cell identifica- For the first time, it has been possible specifically to Br J Ophthalmol: first published as 10.1136/bjo.68.10.708 on 1 October 1984. Downloaded from

Retinalpigment epithelial cells in epiretinal membranes: an immunohistochemical study 715 identify fibroblast-like cells of epithelial origin in 6 Clarkson JG, Green WR, Massof D. A histopathologic review of ERMs. in one embedded the 168 cases of preretinal membrane. Am J Ophthalmol 1977; 84: Indeed, specimen epi- 1-17. thelial component (including fibroblast-like cells) was 7 Green WR, Kenyon KR, Michels RG, Gilbert HD, De La Cruz so prominent as apparently to confirm the 'RPE cell Z. Ultrastructurc of epiretinal membranes causing macular hypothesis' of ERM formation and contraction.2-5 pucker after retinal reattachment surgery. Trans Ophthalmol Soc However, in a majority of our ERMs (most of which UK 1979; 99: 65-77. 8 Kampik A, Kenyon KR, Michels RG, Grecn WR, Dc La Cruz were associated with rhegmatogenous retinal detach- Z. Epirctinal and vitrcous mcmbrancs: Comparativc study of 56 ment) epithelial cells were either absent or formed cascs. Arc/h Ophthaltnol 1981; 99: 1445-54. only a minor component comprising cells scattered 9 Harada T, Chauvaud D, Pouliquen Y. An electron microscopic diffusely throughout the specimen. These positively study of the epiretinal membrane of human eyes. Albrecht von Graefes Arch Klin Ophthalmol 1981; 215: 327-39. staining but isolated cells might represent wandering 10 Hamilton CW, Chandler DB, Klintworth GK, Machemer R. A macrophages derived from RPE cells2 or RPE cells transmission and scanningelectron microscopicstudy of surgically incidentally caught up within developing ERMs excised preretinal membrane proliferations in diabetes mellitus. following dispersion from Bruch's membrane, Am J Ophthalmol 1982; 94: 473-88. 11 Trese M, Chandler DB, Machemer R. Macular pucker: II. showering throughout the retrohyaloid space, and Ultrastructure. Albrecht von GraefesA rch Klin Ophthalmol 1983; settling on to the retina. 221: 16-26. Most fibroblast-like cells in our ERMs were not of 12 Ncwsomc DA, Rodrigues MM, Machemer R. Human massivc epithelial cell origin (as judged by negative staining perirctinal prolifcration. In vitro charactcristics of ccilular components. Arch Opihthalinol 1981; 99: 873-80. for prekeratin and keratin). The origin of these non- 13 Rodrigucs MM, Ncwsomc DA, Machemcr R. Furthcr char- staining fibroblasts remains obscure. We have pre- actcrization of cpiretinal mcmbrancs in human massive viously shown that they are also unlikely to be of glial periretinal prolifcraition. Curr Ekve Res 1981; 6: 311-5. origin on the basis of anti-GFAP staining,"5 neither 14 Hiscott PS, Gricrson 1, Hitchins CA, Rahi AHS, McLeod D. are Epiretinal membranes in vitro. Trans Ophthalmol Soc UK 1983; they derived from vascular endothelial cells as 103: 89-102. judged by negative staining with anti-factor VIII 15 Hiscott PS, Grierson 1, Trombetta CJ, Rahi AHS, Marshall J, related antibody.'4 Thus, while accepting that bio- McLeod D. Retinal and epiretinal glia: an immunohistochemical microscopic pigmentation of an ERM strongly study. Br J Ophthalmol 1984, in press. an RPE cell and while conceding 16 Lec LD, Baden HP. A neutral soluble high molecular weight suggests component, keratin from epidermis. Biochem Biophys Res Comm 1978; 81: that many non-pigmented ERMs also have a RPE 366-73. cell component, we consider that the fibroblasts or 17 Sun TT, Tseng SCG, Nelson W, Jarvinen M, Woodcock-Mitchell fibroblast-like cells generally held to be responsiblefor J, Huang JW. Tissue distribution of keratin antigens: Studies ERM contraction are seldom ofRPE origin. using monoclonal antibodies. ARVO abstracts: supplement to Invest Ophthalmol'Visual Sci 1982; 22: 73. 18 Kinoshita S, Friend J, Kiorpes TC, Thoft RA. Keratin-like http://bjo.bmj.com/ proteins in corneal and conjunctival epithelium are different. We thank Mr R. J. Cooling, Mr Z. Gregor, and Mr P. K. Leaver who Invest Ophthalmol Visual Sci 1983; 24: 577-81. provided many of the surgical specimens, and Mr G. Nunn for 19 Mukai K, Rosai J. Applications of immunoperoxidase techniques ensuring their safe collection. Dr A. H. S. Rahi provided valuable in surgical pathology. In: Fenoglio CM, Wolff M, eds. Progress technical guidance, and technical assistance was provided by in surgicalpathology. New York: Masson, 1980: 15-49. R. Alexander, J. Prasad, and E. Robins. We are also indebted to 20 Meuller-Jensen K, Machemer R, Azarnia R. Autotransplanta- Miss Heather Lucas and Mrs Pat Goodwin for secretarial assistance. tion of retinal pigment epithelium in intravitreal diffusion Our research has been funded by the TFC Frost Charity Trust, the chamber. Am J Ophthalmol 1975; 80: 530-7. Wellcome Trust (grant no. 10998/1.5), the Muirhead Settlement, the 21 Mandelcorn MS, Machemer R, Fineberg E, Hersch SB. Prolifera- on September 28, 2021 by guest. Protected copyright. Moorfields Locally Organised Research Scheme and the Moorfieids tion and metaplasia of intravitreal retinal pigment epithelium cell Endowment Fund. autotransplants. Am J Ophthalmol 1975; 80: 227-37. 22 Franke WW, Schmid E, Weber K, Osborn M. HeLa cells contain intermediate-sized filaments of the prekeratin type. Exp Cell Res 1979; 118: 95-109. References 23 Sieinski W, Dorsett B, loachim HL. Identification of prekeratin by immunofluorescence staining in the differential diagnosis of I Smith TR. Pathological findings after retinal surgery. In: tumours. Hum Pathol 1981; 12: 452-7. Schepens CL, ed. Importance of vitreous body in retinal surgery 24 Gabbiani G, Kapanci Y, Barazzone P, Franke WW. Immuno- with special emphasis on reoperations. St Louis: Mosby, 1960: chemical identification of intermediate-sized filaments in human 61-73. ncoplastic cells. A diagnostic aid for the surgical pathologist. Am 2 Machemer R, Laqua H. Pigment epithelium proliferation in J Pathol 1981; 106: 206-16. retinal detachment (massive periretinal proliferation). Am J 25 Altmannsberger M, Weber K, Hdlscher A, Schaucr A, Osborn Ophthalmol 1975; 80:1-23. M. Antibodies to intermediate filaments as diagnostic tools. 3 Machemer R. Pathogenesis and classification of massive peri- Human gastrointestinal carcinomas express prekeratin. Lab retinal proliferation. BrJ Ophthalmol 1978; 62: 737-47. Invest 1982; 46: 52t)-6. 4 Machemer R, Van Horn D, Aaberg TM. Pigment epithelial 26 Said JW. Immunohistochemical localization of keratin proteins proliferation in human retinal detachment with massive peri- in tumour diagnosis. Hum Pathol 1983; 14: 1017-9. retinal proliferation. AmJ Ophthalmol 1978; 85: 181-91. 27 Barr-Nea L, Barishak YR. Behaviour of retinal pigment epi- 5 Machemer R, Aaberg TM. Vitreous. C'urrenit Ophthalmology thelium in organ culture conditions. Ophthalmic Res 1972; 4: Monographs. New York: Grune and Straitton, 1979: 19-29. 321-7.