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From the Divisions of 1 Cell Biology, 4 Molecular Genetics, and 6 Pathology, Institute of Ophthalmology, University College London, London, United Kingdom; 2 Neurotech SA, Evry, France; 5 Institut Cochin de Génétique Moléculaire, Centre National de la Recherche Scientifique, Unité Propre de Recherche 415, Paris, France; and the 7 Department of Psychology, University of Sheffield, Sheffield, United Kingdom. 3 Present affiliations: Department of Molecular Biology and Genetics, Bogazici University, Istanbul, Turkey; and the 8 Moran Eye Center, University of Utah, Salt Lake City, Utah.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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METHODS. Primary human RPE cells were genetically modified by transfecting with a plasmid encoding the simian virus (SV)40 large T antigen. After characterization, two cell lines, designated h1RPE-7 and h1RPE-116, were chosen for further investigation, along with the spontaneously derived RPE cell line ARPE-19. Factors reported to be important in RPE and photoreceptor cell function and survival in vivo were examined.
RESULTS. Both h1RPE-7 and h1RPE-116 cells exhibited epithelial morphology,
expressed cytokeratins, and displayed junctional distribution of ZO-1,
p100-p120 and ß-catenin. The cells expressed mRNA for RPE65 and
cellular retinaldehyde-binding protein (CRALBP) and the trophic and
growth factors brain-derived neurotropic factor (BDNF), ciliary
neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF),
pigment epithelium–derived factor (PEDF), nerve growth factor (NGF),
platelet-derived growth factor (PDGF)-
, insulin-like growth factor
(IGF)-1, and vascular endothelial growth factor (VEGF). Secreted BDNF,
bFGF, and VEGF, but not CNTF, were identified in cell supernatants. The
cell lines constitutively expressed HLA-ABC, CD54, CD58, and CD59.
After activation with IFN-
both HLA-ABC and CD54 were upregulated,
and the expression of HLA-DR was induced. Both cell lines failed to
express CD80, CD86, CD40, or CD48 in vitro and in a mixed lymphocyte
reaction were unable to induce T-cell proliferation. Fas ligand (CD95L)
was not detected in vitro by RT-PCR. Similar results were obtained with
the ARPE-19 cell line.
CONCLUSIONS. RPE lines h1RPE-7 and h1RPE-116 retain many of the morphologic and biochemical characteristics of RPE cells in vivo and may serve as a source of cells for in vitro analysis of RPE cell function, as well as for orthotopic transplantation studies.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One cell line in particular, spontaneously arising ARPE-19, has been a valuable source of human RPE cells, despite the unknown nature of the immortalizing event conferring immortality. However, the exact profile of RPE-specific characteristics of this cell line remain unknown, and there is a strong case for the development of further RPE cell lines that may express specific characteristics of interest. Furthermore, in in vivo RPE transplantation studies, particularly if these are ultimately intended for treatment of patients, a known genetic modification may be more desirable than an undefined genetic alteration.
We have previously reported that the subretinal transplantation of RPE cells into the Royal College of Surgeons (RCS) rat delays photoreceptor cell death4 5 and, along with others,6 7 8 9 have proposed that this may be an effective treatment strategy for diseases such as AMD. To overcome some of the limitations imposed by using primary cultures harvested from donor eyes, such as inadequate supply and difficulty in screening for pathogens, the use of cell lines as a source of RPE cells for orthotopic transplantation may prove advantageous. Despite some major potential problems associated with this approach, such as the cells’ safety and ability to function effectively in vivo, it remains a promising strategy. In this study we therefore set out to develop additional human RPE cell lines with extended life span by transfection with the simian virus (SV)40 large T antigen. The generation of a range of cell phenotypes after expression of SV40 large T antigen allows for the systematic selection of lines that exhibit both normal RPE characteristics and a nontransformed phenotype. Furthermore, this approach enables us to generate a pathogen-free cell line generated from an individual with a recorded lineage. The cell lines generated in this study were then tested alongside ARPE-19 cells for RPE-specific and epithelial characteristics and for factors that are believed to be important in cell survival and in the support of photoreceptor cells in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Isolation and Culture of Human RPE Cells
After local ethics committee approval and according to national
guidelines and in compliance with the tenets of the Declaration of
Helsinki, a human eye was obtained from a 50-year-old female white
donor, who underwent exenteration due to conjunctival disease, and the
anterior segment, iris, and lens were removed. The posterior segment
was placed in sterile culture medium consisting of Ham’s F-10, 20%
FCS, 2 mM L-glutamine, 100 IU/mL penicillin, 100 µg/mL
streptomycin, and 1 µg/mL amphotericin-B and was stored overnight in
the dark at 4°C to facilitate separation of the retina from the RPE.
Vitreous and neural retina were gently teased away from the optic
nerve, and the eye cup was dissected into three segments. Cloning rings
were placed on the exposed RPE cell layer, and cells were rinsed with
0.02% EDTA for 5 minutes. Trypsin-EDTA (0.25%/0.02%) was pipetted
into the cloning rings and incubated at 37°C for 45 minutes to detach
RPE from Bruch’s membrane. RPE cells, released by gentle trituration,
were plated onto FCS-coated tissue culture flasks, and after 10
minutes, culture medium was added. Medium was changed every 2 to 3
days, and all cells were cultured at 37°C in 5%
CO2. For comparative studies, primary cultures of
human RPE cells were generated from additional donor eyes, using the
same procedure, and the spontaneously arising human RPE cell line,
ARPE-19 was obtained from ATCC (CRL-2302; Rockville, MD) and grown as
previously reported.10
Genetic Modification of Human RPE Cells
The primary human RPE culture, derived from the single donor
described earlier, was transfected with RSV puro (Neurotech
S.A., Evry, France; which encodes a puromycin-selectable marker) and
either a construct encoding SV40 large T antigen (pVim Twt) or SV40
large T antigen that was deleted for the small T antigen (pVim T
t).
The constructs were generously provided by Denise Paulin,
University of Paris, Paris, France).11
This construct was
chosen, because expression of the SV40 large T antigen is known to
result in a stable cell phenotype and an associated extension of life
span, but without induction of cellular transformation. Transfection
was performed in six-well plates seeded with 2 x
104 cells/cm2. pVimTwt or
pVimTt (0.9 µg) was combined with 0.1 µg of RSV puro in 100 µL
serum-free medium containing 3 µL transfection reagent (FuGene;
Roche). After a 48-hour incubation, cells were trypsinized and replated
into Petri dishes and cultured in medium containing 1 µg/mL puromycin
to select for transfected cells. Surviving colonies were removed from
Petri dishes with cloning rings and cultured in 20% FCS, 2 mM
L-glutamine, 100 IU penicillin with 100 µg/mL
streptomycin, and 1 µg/mL puromycin. One hundred twenty-six cell
lines with large Tt and 42 cell lines with wild-type large T antigen
were generated. Each cell line was assessed by phase-contrast
microscopy for ability to form contact-inhibited monolayers of an
epithelial cobblestone phenotype and, using morphologic criteria, 18
cell lines were selected for further characterization.
Characterization of RPE Cells
Immunocytochemistry.
Primary human RPE cultures, the ARPE-19 cell line, and the 18 selected
genetically modified cell lines were cultured to confluence on
eight-well chamber slides (Gibco) for immunocytochemical
characterization. Cells were fixed in either 3.7% paraformaldehyde for
10 minutes or, for cytokeratin detection, ice-cold methanol-acetone
(1:1). Cells were permeabilized with 0.25% Triton X-100 and PBS for 10
minutes and blocked for 15 minutes with 10% goat serum and PBS.
Primary antibodies were diluted in blocking solution, added to the
cells and incubated for 1 hour at room temperature. Detection was
achieved using FITC- or TRITC-conjugated secondary antibodies (1:50)
added for 1 hour in the dark. After washing, cells were mounted, and
the cell expression and distribution recorded by epifluorescence and
confocal laser scanning microscopy. Omission of the primary or
secondary antibody in each case served as the negative control. On the
basis of morphologic appearance and the expression of cytokeratins and
SV40 large T and junctional molecules, two cell lines, generated as
described earlier, were selected for further analysis. One cell line,
designated h1RPE-7, originated from the SV40 large T antigen deleted
for the small T antigen–transfected parent line, and another cell
line, designated h1RPE-116, originated from transfection with the SV40
large T wild-type antigen. All subsequent analyses described in the
following sections were performed on these two cell lines between
passages 13 and 22.
Ultrastructure.
RPE cells were grown to confluence on 24-well tissue culture plates and
fixed with one-half strength Karnovsky’s fixative (2% formaldehyde,
2% glutaraldehyde, 0.2 M sodium cacodylate buffer, and 6.5 mM calcium
chloride). The monolayer was postfixed in 1% osmium tetroxide for 1
hour, washed, and dehydrated through ascending concentrations of
ethanol. For transmission electron microscopy (TEM), the monolayers
were embedded in resin and ultrathin sections cut and counterstained
with uranyl acetate and lead citrate. The sections were viewed in a
transmission electron microscope (model 1010; JEOL, Herts, UK). For
scanning electron microscopy (SEM), the monolayers were critical-point
dried with CO2 and sputter coated with 20 nm of gold and
then viewed on a scanning electron microscope (model 6100; JEOL).
Reverse Transcription–Polymerase Chain Reaction.
Expression of mRNAs encoding for discriminating markers, for growth and
trophic factors deemed to be important in photoreceptor cell function
and survival, and for Fas ligand (FasL; CD95L) were examined using
reverse transcription–polymerase chain reaction (RT-PCR). The three
cell lines—h1RPE-7, h1RPE-116, and ARPE-19—were investigated for the
expression of mRNA for the discriminative cellular markers RPE65 and
cellular retinaldehyde-binding protein (CRALBP). The growth and trophic
factors investigated were pigment epithelium–derived factor (PEDF),
brain-derived neurotrophic factor (BDNF), basic fibroblast growth
factor (bFGF), ciliary neurotrophic factor (CNTF), platelet-derived
growth factor (PDGF)-, insulin-like growth factor (IGF-1), nerve
growth factor (NGF), and vascular endothelial growth factor (VEGF).
Oligonucleotide primers complimentary to the 5' and 3' ends
were used in RT-PCR studies (Table 1)
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Secreted Growth and Trophic Factors.
Cell culture supernatants were collected from either RPE cells derived
from the original donor or from h1RPE-7, h1RPE-116, and ARPE-19 cell
lines 2 weeks after reaching confluence. Cell supernatants were
collected after 5 days of conditioning and centrifuged to remove
particulates. The concentrations of bFGF, BDNF, CNTF, and VEGF (R&D
Systems), human interferon (hIFN)- (Amersham PLC, Amersham, UK) and
human interferon (hIFN)-ß (Biosource International, Camarillo, CA)
were measured using commercially available enzyme-linked immunosorbent
assays (ELISA) in accordance with the manufacturer’s protocol.
Flow Cytometry.
The expression of potentially important immunologic molecules was
evaluated in h1RPE-7, h1RPE-116, and ARPE-19 cell lines and in
third-passage human RPE cells by flow cytometry. Cells were grown in
six-well tissue culture dishes and were left either untreated or
treated with 100 to 1000 U/mL IFN- (R&D Systems) for 48 or 72 hours.
After treatment, the cells were prepared as a single-cell suspension by
treating with 0.5 mL of 1 mg/mL collagenase for 10 to 15 minutes at
37°C. The cells were pelleted by centrifugation and washed by
resuspension in 1 mL sterile PBS. Each cell suspension was then
pelleted and resuspended in 100 µL PBS, with or without the
appropriate primary antibody and incubated on ice for 1 hour. Two PBS
washes were performed and the cells were incubated with a secondary
FITC-conjugated antibody for 1 hour on ice in the dark, washed with
PBS, and resuspended in 200 to 500 µL PBS for flow cytometric
analysis. The cell surface expression of major histocompatibility
complex (MHC) class I (HLA-ABC) and class II (HLA-DR) antigens; the
costimulatory molecules intercellular adhesion molecule (ICAM)-1
(CD54), CD40, B7-1 (CD80), and B7-2 (CD86); and the potential CD2
ligands CD48, CD58, and CD59 were evaluated by flow cytometry (FACScan;
BD Biosciences, Oxford, UK). The data were analyzed on computer
(CellQuest software; BD Biosciences).
Assessment of Tumorigenicity
To assess the tumorigenic capacity of h1RPE-7 and h1RPE-116
cells in vivo, 10 x 106 cells suspended in
200 µL PBS and 10 mM glucose were injected subcutaneously (SC) into
irradiated athymic Swiss nude mice (n = 10 animals for
each clone). As a positive control, U87 human glioblastoma cells were
also injected SC at the same cell density (n = 5
animals). Animals were assessed by daily gross examination and after 3
and 15 weeks by histologic analysis of the graft site. At 3 or 15 weeks
after injection the nude mice were perfusion fixed with 4%
paraformaldehyde and PBS. The tissue was then postfixed in 4%
paraformaldehyde and PBS for 2 hours at 4°C, followed by
cryoprotection in 20% sucrose and PBS overnight at 4°C and finally
embedded in optimal cutting temperature compound (TissueTek; Miles
Laboratories, Elkhart, IN). Tissue was frozen for 30 seconds at
-40°C in isopentane and liquid nitrogen and stored at -20°C until
sectioned. Sections were cut (14 µm) and processed for hematoxylin
and eosin staining. Tissue sections were finally dehydrated and mounted
in resin (Eukitt; Agar Scientific, Essex, UK). Animal care was in
accordance with the ARVO Statement for the Use of Animals in Ophthalmic
and Vision Research. Home Office (United Kingdom) regulations for the
care and use of laboratory animals and the provisions of the UK Animals
(Scientific Procedures) Act (1986) were observed at all times.
Anchorage-independent growth, which is a function of cell transformation and tumorigenicity, was assessed using a soft agar colony assay. Low melting temperature agarose (0.5% in culture medium; Seaplaque; Novara Group, Ltd, Staffs, UK) was plated onto 24-well plates and left to gel. The cell lines h1RPE-7 and h1RPE-116 and the human glioblastoma cell line U87 were then suspended in a second layer of 0.33% agarose (7 x 104 cells per well), plated into individual wells, and placed at 4°C for 5 minutes to set. Media were then added to each well and incubated at 37°C with regular changes. The wells were examined by phase-contrast microscopy every 2 to 3 days for up to 40 days for the presence of cell aggregates or foci.
T-Cell Proliferation Assay: Mixed-Lymphocyte Reaction
Human peripheral blood mononuclear cells (PBMCs) were harvested
from a healthy human donor by density-gradient centrifugation
(Ficoll-Paque; Pharmacia Biotech AB, Uppsala, Sweden). The PBMCs were
then plated onto plastic Petri dishes for 45 minutes at
37°C-5%CO2 to allow monocytes to adhere. The
nonadherent cells were then removed, and the enriched population of T
lymphocytes was cocultured (2 x 105
cells/well) with either stimulated (IFN- at 100 U/mL for 48 hours)
or unstimulated human RPE cell lines that had previously been
irradiated (240 Gy). The RPE cell lines, h1RPE-7, h1RPE-116, and
ARPE-19 were plated at various concentrations, 2 x
104, 1 x 104,
0.5 x 104, and 0.25 x
104 cells/well in triplicate in 96-well plates in
200 µL RPMI 1640 (Life Technologies) supplemented with 10% FCS,
penicillin 100 U/mL, and streptomycin 100 µg/mL. Two irradiated (120
Gy), allogeneic human B-cell lines (MOU, 9050; SA, 9001; from the Tenth
International Histocompatibility Workshop, New York, 1987) were
used as a positive control at the same concentrations as the RPE cells.
The allogeneic T-cells were harvested, and proliferation was assessed
on day 6, after an 18-hour pulse with
[3H]-thymidine (0.5 µCi/well), by measuring
incorporated [3H]-thymidine by liquid
scintillation spectroscopy. Background uptake of
[3H]-thymidine into the irradiated cells alone
was subtracted from the harvested T-cell data.
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Results |
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The barrier function and polarization of RPE cells is considered to be highly dependent on the integrity of specialized junctions. Analysis of junctional proteins revealed strong peripheral staining of ZO-1 in both primary RPE and the cell lines h1RPE-7 and h1RPE-116 (Figs. 1G 1H 1I) . The staining pattern of ZO-1 at the point of cell–cell contact was largely continuous but exhibited occasional discontinuities. The adherens junctional proteins, ß-catenin (Fig. 1J 1K 1L) and p100-p120 (data not shown) were also expressed at the cell border, but in a more diffuse pattern characteristic of these molecules. Similar results were obtained for the cell line ARPE-19 (data not shown).
Reverse Transcription–Polymerase Chain Reaction
The discriminatory molecules RPE-65, an RPE cell–specific
molecule that is thought to play an important role in the
RPE-photoreceptor vitamin A cycle, and CRALBP, which is involved in the
regeneration of visual pigment, were both detected by RT-PCR in
h1RPE-7, h1RPE-116, and ARPE-19 cell lines producing amplicons of the
predicted size (Fig. 3)
and correct sequence.
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, PEDF, IGF-1, and VEGF (Fig. 3) . Sequence analysis of all
RT-PCR products showed amplified sequences were 100% concordant with
published human sequences (data not shown). Fas and FasL are a receptor-ligand pair involved in the induction of apoptotic cell death. FasL is a type II transmembrane protein and a member of the TNF family that binds to Fas (CD95) and induces apoptosis of the Fas-expressing cells. The expression of FasL by grafted RPE cells, therefore, may be a critical determinant in their ability to attenuate the rejection process through FasL-mediated apoptosis of host T cells after transplantation. The cell lines h1RPE-7, h1RPE-116, and ARPE-19 as well as a third-passage human RPE cell culture, were all found to be negative for FasL mRNA. However, T cells assessed under identical stringent conditions were clearly positive for FasL (Fig. 4) , demonstrating that in vitro, these RPE cell lines do not express FasL.
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hIFN-ß were not detected above the limit of the assays.
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The anchorage-independent growth of both h1RPE-7 and h1RPE-116 was determined by growing cells in soft agar. These cell lines failed to form aggregates or colony foci over a 40-day culture period (data not shown), indicating that they are both nontransformed and nontumorigenic.
Expression of Molecules of Potential Immunologic Importance
The expression of surface-expressed molecules of potential
immunologic importance was determined in resting or IFN-–activated
(100–1000 U/mL for 48 or 72 hours) cells by flow cytometry. RPE cell
lines h1RPE-7, h1RPE-116, and ARPE-19 were all found to constitutively
express MHC class I (HLA-ABC). After activation with IFN-, class I
was upregulated, and the expression of MHC class II (HLA-DR) was
induced (Fig. 6)
. Similar results were obtained for third-passage human RPE cells.
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. The costimulatory molecules
B7-1 (CD80) and B7-2 (CD86) were not expressed on the three cell lines,
nor were they expressed on early-passage human RPE cells under either
resting conditions or after activation by IFN- (Fig. 6)
.
Furthermore, CD40 expression was negligible on the three cell lines but
was found on a proportion of the third-passage human RPE cells. No
differences were observed between groups activated with 100 or 1000
U/mL IFN- for either 48 or 72 hours. The CD2 ligand, CD58 (lymphocyte function-associated antigen-3), was also found to be constitutively expressed by RPE cells and remained unchanged after cytokine activation (Fig. 6) . The expression of the two potential low-affinity CD2 ligands, CD48 and CD59, was also investigated. Both h1RPE-7 and h1RPE-116 failed to express CD48 in vitro, whereas both ARPE-19 and third-passage human RPE cells expressed low levels. Conversely, CD59 was strongly expressed by all RPE cells (Fig. 6) .
The haplotype of h1RPE-7 and h1RPE-116, as determined by PCR–sequence specific priming (SSP), was found to be HLA-A 3;32, -B 44;62, -C 9;5, -DR 1;4, and -DQ 5;7.
T-Cell Proliferation Assay
To evaluate the potential of the RPE cell lines to induce T-cell
proliferation, a mixed-lymphocyte reaction assay was performed. The
three RPE cell lines—h1RPE-7, h1RPE-116, and ARPE-19—did not induce
significant T-cell proliferation (Fig. 7)
irrespective of IFN- activation or the number of RPE cells used
(data not shown). As expected, the two B-cell lines used as positive
controls were able to induce significant T-cell proliferation in the
absence of other costimulatory factors, particularly at a concentration
of 2 x 104 cells/well (Fig. 7)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transfection of a primary culture of human RPE cells with SV40 large T antigen gave rise to a large number of cell lines exhibiting a wide range of characteristics. On the basis of morphologic phenotype and preliminary screening for characteristic markers, two lines were selected. Using established assays for determining tumorigenicity, we demonstrated that the two new cell lines generated here did not proliferate in irradiated athymic nude mice and did not show anchorage-independent growth. Moreover, in subretinal grafting studies using the h1RPE-7 and ARPE-19 cell lines in the RCS rat we did not observe tumor formation up to 6 months after grafting.12
For RPE cells to exhibit barrier function and to undergo apical and
basolateral polarization, they must be capable of the successful
assembly of adherence junctions and the subsequent formation of
functional tight junctions, the latter of which are composed of a
complex of transmembrane and intracellular proteins including ZO-1. It
has previously been shown that a continuous unbroken
expression of ZO-1 correlates with functional junctions, whereas a loss
or reduction in ZO-1 expression is associated with breakdown of barrier
function. Both h1RPE-7 and h1RPE-116 cell lines expressed ZO-1 as a
near-continuous belt at the point of cell–cell contact in a pattern
identical with primary cultures of RPE and to ARPE-19 (data not shown).
The pattern of expression, however, showed occasional flaws or
discontinuities, and this was consistent with our inability to record
transmonolayer electrical resistances above 30 to 40
/cm2 under normal conditions (Kanuga et al.,
unpublished observations, 2000). These results, however, are
consistent with previously reported transmonolayer electrical
resistances of ARPE-19 cells in the absence of specialized
media.10
The adherens junction components, p100-p120 and
ß-catenin, were also found to be strongly expressed in vitro at the
point of cell–cell contact in the two cell lines in a manner identical
with that observed in primary RPE cells and ARPE-19. Although the
junctional expression of these molecules is a prerequisite for
the formation of fully functional junctions, their appearance per se
does not necessarily indicate functional junctions, because this is
dependent on the correct assembly of the junctional complex as a whole.
Although we did not investigate the associated question of cell
polarization in this study, it is interesting to note that when grafted
subretinally into RCS rats, both h1RPE-7 and ARPE-19 cell lines are
able to integrate with the host RPE, synthesize pigment granules, and
express them at their apical face, which suggests some degree of
polarization.12
Clearly, further work is required to
evaluate the capacity of these cells to exhibit functional polarity and
the factors controlling this process.
The participation of RPE cells in the visual cycle is well documented. These cells are engaged in the synthesis and storage of retinyl esters, isomerization of all-trans-retinoids to 11-cis-retinoids and the eventual conversion of retinol to retinal. The expression of genes implicated in retinal vitamin A metabolism is therefore an essential property for any RPE cell line. RPE65, a tissue-specific, highly conserved protein, is present at high levels in vivo and is thought to play an important role in the RPE-photoreceptor vitamin-A cycle.13 14 In this study, we demonstrated mRNA expression of both the full-length and nested transcript of RPE65 in h1RPE-7 and h1RPE-116 cells. We also demonstrated the presence of mRNA for CRALBP in the two cell lines. This important protein is involved in the regeneration of visual pigment and has been implicated in autosomal recessive retinitis pigmentosa.15 The finding that these genes are also expressed in ARPE-19 is consistent with a previous report.10
Recent observations have dramatically illustrated the effects of either a single growth and trophic factor or a combination of these on photoreceptor viability in dystrophic animal models. In particular bFGF, BDNF, and CNTF have been shown to be neuroprotective in the retina.16 17 18 19 20 21 The capacity of the RPE cells to produce these factors was therefore investigated and bFGF, BDNF, and CNTF were all expressed at the transcriptional level, although only bFGF and BDNF were detected in conditioned supernatant. bFGF secretion by h1RPE-7 and h1RPE-116 cells was found to be significantly higher than that produced by primary RPE or ARPE-19 cells and in concentrations that are physiologically relevant.22 23 These studies demonstrate the capacity of these cells to produce potentially important neurotrophic factors that may protect photoreceptors and enhance survival, although it remains to be shown whether these factors are produced once the cells are grafted into the retina.
The eye has been described as an immune-privileged site, with its immunity attributed in part to the expression of FasL, particularly at the site of the blood–ocular barriers.24 FasL is a type II integral membrane protein homologous with the TNF receptor and is important for protection against inflammatory processes. In the eye it has been reported to be constitutively expressed on RPE cells where it promotes apoptosis of invading Fas-positive immune cells.25 26 Thus, FasL expression by RPE cells may play an essential role in local immune regulation and in a transplantation setting in the prevention of immune rejection of transplanted cell lines.27 The in vitro detection of FasL on RPE, however, has been problematic, because of reported problems associated with certain FasL antibodies.28 29 In immunocytochemical assays we were also unable to show the presence of surface expressed FasL on RPE cells in vitro (data not shown), and this prompted us to restrict our investigation to the use of RT-PCR for the detection of FasL. The three cell lines that we studied, as well as the early-passage human RPE cells, were all found to be negative for FasL at the transcriptional level. It is likely, therefore, that, as previously reported,29 FasL is expressed only on RPE cells in vivo. It remains to be determined, however, whether these RPE cell lines are able to express this important immunomodulatory molecule when reintroduced subretinally.
The expression of MHC class II molecules on RPE has also been the
subject of much conjecture in relation to their ability to act as
antigen-presenting cells. Furthermore, in transplantation studies it
has been suggested that RPE allografts are vulnerable to rejection as a
result of the host immune response being directed against
transplantation autoantigens.30
31
Using
immunocytochemical (not shown) and flow cytometric analysis, we have
shown that the RPE cell lines did not express MHC class II
constitutively but that it was induced after cytokine activation. These
results are consistent with previous reports describing MHC expression
in human RPE cells.32
The expression of costimulatory
molecules would also determine whether these cells are potentially
capable of inducing T-cell activation. We found that, under in vitro
conditions, the three cell lines expressed the adhesion–costimulatory
molecule ICAM-1 (CD54) but did not express B7-1 (CD80) or B7-2 (CD86)
in either resting or IFN-–activated cells. These results are
generally consistent with other studies performed on primary or
early-passage cultures of RPE cells, in which CD54 has been shown to be
expressed,33
but not CD80 and CD86.33
34
35
However, unlike a previous study in human fetal RPE
cells,34
we were unable to confirm the expression of CD40
on the RPE cell lines, although we observed a significant population of
positive cells in the early-passage human RPE cells. This may indicate
that the cell lines have lost the capacity to express CD40.
The alternative CD2-mediated pathway for T-cell activation
has been suggested as a possible mechanism by which RPE cells may also
activate T cells.35
We have shown that the three cell
lines, h1RRPE-7, h1RPE-116, and ARPE-19, express similar levels of the
CD2 ligand, CD58, under both resting conditions and after
IFN- activation. Both CD4836
37
and
CD5938
39
have also been proposed as possible low-affinity
ligands for human CD2, although their functional relevance in humans
has been questioned.35
40
However, in the rat, both CD48
and CD59 may act as major ligands for CD235
and, in that
these cell lines have been grafted into rat retina,12
we
investigated the expression of these ligands on the RPE
cells. Both CD58 and CD59 were expressed constitutively,
whereas CD48 was expressed only weakly on ARPE-19 and early-passage RPE
cells. It remains unclear whether in the in vivo setting either CD58 or
CD59 is able to activate T cells through this alternative pathway.
Although the cell lines were found to express MHC class II, the absence of costimulatory molecule expression suggests that they would not, at least in vitro, be able to induce T-cell proliferation. This was confirmed in a mixed lymphocyte reaction where the cell lines were unable to induce T-cell proliferation unlike a B-cell line. Should these properties be retained after transplantation of these cells into the subretinal space, it would suggest that in an allogeneic setting there would be a limited immune response, thus improving the longevity and viability of grafted cells.
In this study, the cell lines h1RPE-7 and h1RPE-116 retained many of the phenotypic features of RPE cells in vivo. The suitability of these newly generated cell lines, along with that of ARPE-19, for in vitro research of RPE function are currently under way.41 In addition, we have recently reported that subretinal transplantation of these cells in the RCS rat is able to preserve vision as assessed using histologic, behavioral, and physiological criteria.12 42 These observations highlight the value of generating carefully characterized human RPE cell lines for both in vitro and in vivo studies.
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Acknowledgements |
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Footnotes |
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Submitted for publication July 9, 2001; revised October 11, 2001; accepted October 24, 2001.
Commercial relationships policy: E (POC, LB, AK, AZ); F (RDL, PC, PA, JG); P (PA, RDL, JG); N (NK, HLW, SH, DK).
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: John Greenwood, Endothelial and Epithelial Cell Biology Research Unit, Division of Cell Biology, Institute of Ophthalmology, University College London, Bath Street, London EC1V 9EL, UK; j.greenwood{at}ucl.ac.uk
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