Acta Ophthalmologica 2011

Clearance of dying ARPE-19

cells by professional and
nonprofessional phagocytes
in vitro – implications for age-
related macular degeneration
(AMD)
Goran Petrovski,1,2 Erika Berényi,2 Morten C. Moe,3
Attila Vajas,1 László Fésüs,2 András Berta1 and Andrea Facskó1
1
Department of Ophthalmology, Medical and Health Science, Center, University of
Debrecen, Debrecen, Hungary
2
Department of Biochemistry and Molecular Biology, Medical and Health Science
Center, University of Debrecen, Debrecen, Hungary
3
Department of Ophthalmology, Center for Eye Research, Oslo, University
Hospital, Ullevål, University of Oslo, Oslo, Norway

ABSTRACT.
Purpose: Failure of retinal pigment epithelial (RPE) cells and macrophages to engulf
Introduction
different dying cells in the retina may result in accumulation of debris and development In the retina, various forms of cell
of age-related macular degeneration (AMD). The dynamics and influence of different death and phagocytosis occur on a
treatments on this clearance process can be studied in vitro using human ARPE-19 daily basis (Green & Enger 1993).
cells and macrophages as phagocytes modelling dry and wet type of AMD, Among others, retinal pigment epithe-
respectively. lial (RPE) cell apoptosis, necrosis,
Methods: Death through extracellular matrix detachment using polyHEMA-coated paraptosis and autophagic cell death
surfaces (anoikis) and UV irradiation (apoptosis) was induced in ARPE-19 cells. Two- have all been described extensively
coloured phagocytic assays were performed to quantify the amount of dying cells (Hafezi et al. 1997; Valamanesh et al.
phagocytes engulfed (flow cytometry) and for visualization (fluorescent and scanning 2007; Ryhänen et al. 2009). The clear-
electron microscopy). The effect of phosphatidylserine inhibition with recombinant ann- ance of dying cells by professional
exin-V and glucocorticoid (triamcinolone) treatment on the phagocytic process was and nonprofessional phagocytes in
tested. the retina has received much less
Results: The clearance of anoikic and apoptotic cells by nondying ARPE-19 cells over attention.
8 hr of co-incubation increased over time (at 8 hr, over 53% and 35% of the phago- Failure of dead cells to clear may
cytes contained engulfed dying cells, respectively). The human macrophages engulfed lead to drusen formation and age-
the anoikic and apoptotic ARPE-19 cells with seven and four times lower capacity, related macular degeneration (AMD)
respectively. Phosphatidylserine appearance on the dying cells did not affect, but triam- (Forrester 2003). AMD is the leading
cinolone treatment enhanced the phagocytosis of the dying cells by macrophages. cause of blindness in the elderly
Conclusions: ARPE-19 cells are more efficient in clearing anoikic than UV-induced (Gehrs et al. 2006) starting with an
apoptotic cells. Macrophages are less efficient in the clearance process than ARPE-19 asymptomatic stage in which drusen
cells. The present model can be used for studying both dry and wet type of AMD (waste material) accumulates in the
in vitro and for testing different pharmacological aspects affecting this disease.
space between the basement (Bruch’s)
membrane and the RPE layer. The
Key words: AMD – Anoikis – macrophages – nonprofessional phagocytes – phagocytosis – tri- drusen accumulation elevates the RPE
amcinolone
cells from this membrane inducing
death by detachment from the extra-
Acta Ophthalmol. 2011: 89: e30–e34 cellular matrix (ECM) also known as
ª 2010 The Authors anoikis (Gilmore 2005). A hallmark of
Acta Ophthalmologica ª 2010 Acta Ophthalmologica Scandinavica Foundation
dry AMD is pigment mottling of
doi: 10.1111/j.1755-3768.2010.02047.x RPE, accumulation of intracellular

e30

lysosomal lipofuscin and extracellular
drusen deposits that may elicit RPE
cell damage and secondarily lead to
central vision loss. In dry AMD, pro-
gressive cell death of the cells in the
retina epithelial layer occurs (Green &
Enger 1993) and because of having an
integral blood-retina barrier, only
clearance by neighbouring, nonprofes-
sional phagocytes is possible. Wet
AMD – the more advanced form
of the disease – involves proliferation
of blood vessels (neovascularization)
through the blood-retina barrier and
subretinal exudate formation (Green
& Enger 1993). This stage is marked
by professional phagocytes (macro-
phages and dendritic cells) involve-
ment, the price of which is the
accumulation of chemoattractant-rich
drusen with the ability of initiating
low-grade inflammation (Forrester
2003; Irschick et al. 2004).
The aim of this study was to inves-
tigate the clearance dynamics of dying
anoikic cells compared to classical
UV-induced apoptotic ones in the
ARPE-19 cell line. The clearance by
nonprofessional phagocytes (nondying
RPE cells) served as in vitro model for
dry AMD, while the clearance by pro-
fessional phagocytes (macrophages)
served as a model for wet AMD. In
addition, the involvement of the phos-
phatidylserine (PS) signalling pathway
as well as the effect of the glucocorti-
coid triamcinolone (TA) on the clear-
ance dynamics was investigated.
Materials and Methods
Cell culture and treatments
Dulbecco’s modified Eagle’s medium
(DMEM), poly-2-hydroxyethylmeth-
acrylate (poly-HEMA) and TA were
purchased from Sigma (Steinheim,
Germany); CFDA-SE, carboxyfluores-
ceindiacetate-succinimidyl ester and
CMTMR, 5-(and-6)-(((4-chlorometh-
yl)benzoyl) amino)tetramethylrhod-
amine were purchased from Molecular
Probes (Leiden, Netherlands); recom-
binant annexin-V was kindly provided
by Dr. Peter Zavodszky (Hungarian
Academy of Sciences); plastic tis-
sue culture flasks were purchased
from TPP (Trasadingen, Switzerland).
ARPE-19 human retina pigment
epithelial cells were kindly provided
by Prof. Stephen Moss, (UCL, Lon-
don, UK) and were cultured at 37C
under 5% CO2 in a 1:1 mixture of
DMEM and Nutrient Mixture F12
Medium supplemented with 10% fetal
calf serum (FCS) (Turowski et al.
2004). Cells were detached from the
substrate using trypsin ⁄ EDTA
(0.05:0.02%). For the induction of
anoikis, cells were plated on poly-
HEMA-covered dishes over a 24-hr
period. Apoptosis was induced by
UV-B irradiation (Roduit & Schorder-
et 2008) – 5, 10 and 20 min,
90 mJ ⁄ (cm2 min) – and the cells were
left to die for additional 8 or 24 hr
before harvesting.
Assay for cell death analysis
Cell death was assessed by the annex-
in-V-fluorescein isothiocyanate Apop-
tosis Detection Kit (MBL, Woburn,
MA, USA) according to manufac-
turer’s recommendations; the propor-
tion of stained annexin-V+ and
annexin-V+ PI+ cells was determined
by fluorescence-activated cell sorter
(FACS) analysis on BD Biosciences
flow cytometer (BD Biosciences, Miss-
issauga, ON, Canada) as previously
described (Petrovski et al. 2007).
Fluorescent and scanning electron micros-
copy (SEM)
For fluorescent microscopy, cells
grown on adherent glass cover slips
were fixed with 1% paraformaldehyde
and stained with phalloidin-FITC for
actin cytoskeleton, 2-(4-amidinophe-
nyl)-6-indolecarbamidine dihydrochlo-
ride (DAPI) for nuclear staining or
vital stains (CMTMR and CFDA-SE)
all purchased from Sigma (Steinheim,
Germany). Axiovert-150 Zeiss micro-
scope (Carl Zeiss MicroImaging
GmbH, Göttingen, Germany) was
used for image acquisition.
For scanning electron microscopy
(SEM), sample fixation was carried
out in 0.1 m sodium cacodylate buf-
fered, pH 7.4 and 2.5% glutaraldehyde
solution for 2 hr and then rinsed (three
times, 10 min) in 0.1 m sodium cacody-
late buffer, pH 7.4 and 7.5% saccha-
rose. After dehydration in an ethanol
gradient (70% ethanol (20 min), 96%
ethanol (20 min), 100% ethanol (two
times, 20 min)), sample surface coating
was performed with 30-nm-thick layer
of platinum in a Polaron E5100 sput-
ter coater (Polaron Instruments Inc.,
Hatfield, PA, USA) and analysed on
Acta Ophthalmologica 2011
XL30 ESEM electron microscope (Phi-
lips, Eindhoven, the Netherlands).
Phagocytosis assay
Human monocytes were isolated from
‘buffy coats’ of healthy blood donors
on Ficoll–Paque Plus (Amersham Bio-
sciences, Uppsala, Sweden) gradient
and magnetic separation using CD14
human microbeads (Miltenyi Biotec,
Auburn, CA, USA). Human macro-
phages were obtained through a 5-day
differentiation using 5 ng ⁄ ml macro-
phage colony-stimulating factor. Non-
dying ARPE-19 cells acting as
phagocytes were plated 24 hr before
phagocytosis to achieve confluence.
Dying ARPE-19 cells were fed to
engulfing cells 24 hr after the induc-
tion of anoikis. ARPE-19 cells that
were UV irradiated for 20 min and
turned apoptotic 24 hr later were also
used for engulfment. Inhibition with
10 mg recombinant annexin-V was
carried on 105 dying cells (30 min
before phagocytosis assay) and main-
tained throughout the assay. Pretreat-
ment of macrophages with 1 lm TA
was performed 48 hr prior to the
assay. The phagocytes, macrophages
and nondying ARPE-19 cells, were
stained with 7.5 mm CMTMR for
16 hr, washed twice in phosphate-
buffered saline (PBS) before starting
the assay. Accordingly, the cells to be
engulfed were stained with 12.5 mm
CFDA for 16 hr before the phagocy-
tosis and washed twice in PBS before
being added to the phagocytes. The
ratio of phagocytes and cells to be
engulfed was set at 1:5. The phago-
cytosis assay started when the cells to
be engulfed were added to the appro-
priate phagocytes and kept together
for 1, 4 or 8 hr. The assay was ended
by trypsinizing the phagocytic cell
mixture to remove bound but not
engulfed dying cells, centrifuging,
washing twice in PBS and fixing in
1% PBS-buffered paraformaldehyde
(pH 7.4). The phagocytosis rate was
determined by FACS analysis as per
cent phagocytic cells (CMTMR posi-
tive) that have engulfed dying cells
(positive for both CMTMR and
CFDA) (Petrovski et al. 2007).
Statistical analysis
All data represent mean of at least
three independent experiments. Paired
e31
Acta Ophthalmologica 2011

student t-test was used to test signifi- 45

30
8 hrs 24 hrs
cance (p < 0.05). postirradiation postirradiation
30
Results *

%
15

%
15
Induction of anoikis and apoptosis in
ARPE-19 cells
0 0
Anoikis initiated by detachment of 0 1 2 4 8 12 24 48 5 10 20 5 10 20
(A) (B)
cells from the ECM has been intro- Hours Minutes of UV irradiation
duced as a model for studying the
mechanism of cell death in the absence
10.5%
of ECM-derived signals in vivo (Gil- Annexin-V+/PI+
more 2005). Our ARPE-19 cells under-
went anoikis in 10% FCS of culture Annexin-V PI
when not allowed to attach to the + –
plate. Their annexin-V positivity, a + +
sign of early or reversible apoptosis
and annexin-V ⁄ PI double positivity, a
Annexin-V+
sign of late or irreversible apoptosis 23.1%
increased over time reaching 19% and (C)
33% at 24 hr of treatment, respec-
Fig. 1. Cell death induced by (A) anoikis and (B) UV irradiation in ARPE-19 cells. Per cent of
tively (Fig. 1A). UV-induced apoptosis
annexin-V and both annexin-V and PI-positive cells as assessed by fluorescence-activated cell
increased in a dose- and time-depen- sorter analysis. (C) Representative flow cytometry data for the given time point (*). Means of
dent manner also, reaching a total of three experiments ± SD are shown.
34% early and late apoptosis after
20 min of irradiation and 24-hr postir-
radiation time (Fig. 1B,C). 37.3%

Both anoikic and UV-irradiated apoptotic

ARPE-19 cells are engulfed by nondying
ARPE-19 cells and macrophages
Although phagocytosis of dying cells
by nonprofessional and professional
37.3%
phagocytes has been extensively stud-
ied, no data are available on how RPE (A) 20 μm (B)

cells dying by anoikis or apoptosis are

removed when the Bruch’s membrane
is or is not intact. In our study,
ARPE-19 cells (Fig. 2) and macro-
phages could engulf dying anoikic or
UV-irradiated apoptotic ARPE-19
cells with increasing capacity over 8-hr
time. As flow cytometric analysis
shows, ARPE-19 cells engulfed both
anoikic and apoptotic cells more heav-
ily than macrophages. At 8 hr of co- (C) (D)
incubation, about 53% and 35% of
the ARPE-19 cells contained anoikic Fig. 2. Detection of the phagocytosis of anoikic ARPE-19 cells by fluorescent, scanning elec-
and apoptotic dying cells, respectively tron microscopy and fluorescence-activated cell sorter (FACS) analysis. (A) Nondying ARPE-
(Figs 2B and 3A), while the macro- 19 cells stained with CMTMR (red) engulfing anoikic cells stained with CFDA (green). (B)
FACS analysis of the phagocytosis process – upper left quadrant and lower black peak repre-
phages contained about 8% and 9%
sent ARPE-19 cells stained with CMTMR that have not engulfed; upper right quadrant and
of the same dying cells (Fig. 3B). lower line marker represent ARPE-19 cells that have engulfed anoikic corpses stained with
CFDA. (C–D) Scanning electron microscopy of the phagocytosis process in (A); arrow heads
represent anoikic cells or corpses bound, but not yet engulfed, and arrow represents an engulfed
The engulfment process is dynamic
anoikic corpse. Co-incubation time: 4 hr.
involving cytoskeletal changes in the
engulfing cells actin cytoskeletal changes during the target seems to act as phagocytic
ARPE-19 cells engulfing anoikic clearance process. Formation of ex- tentacle (Fig. 2C) pulling bound
(Fig. 3C,D) or UV-irradiated dying quisitely localized actin polymeriza- objects towards the cell for phagocy-
cells (data not shown) undergo active tion right underneath the phagocytic tosis.

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 Acta Ophthalmologica 2011

ARPE-19 MФ between the two death pathways. Mac-

60 10 rophages recognized and engulfed both
50
types of dying ARPE-19 cells about
seven and four times less efficiently
40 than nondying ARPE-19 cells. In sup-
30 5 port of this observation, in vivo find-
%

%
20 ings show that many apoptotic cells are
phagocytosed by neighbouring cells
10
first (Fadok & Henson 1998).
0 0 Dying cells present on their surface
1 4 8 1 4 8
(A) (B) several ‘eat-me’ signals, facilitating
Hours Hours
their engulfment by professional and
nonprofessional phagocytes, one such
being externalized PS (Savill et al.
2003). Recombinant annexin-V, which
binds PS exposed on the surface of
dying cells, did not block the engulf-
ment of both types of dying cells by
(C) 20 μm (D) 20 μm neither nondying ARPE-19 cells (data
not shown) nor macrophages (Fig.
Fig. 3. Phagocytic capacity of (A) ARPE-19 cells and (B) macrophages (Ms) when challenged to 4A,B). This indicates that PS-indepen-
engulf anoikic (white bars) or UV-induced apoptotic ARPE-19 (black bars). Bars represent mean dent cell surface changes could make
values of three independent experiments ± SD; cytoskeletal changes during the phagocytosis of the dying cells ‘eatable’ by the phago-
anoikic ARPE-19 cells by nondying ARPE-19 cells. Fluorescent microscopy of (C) control and cytes. Apoptotic cells can compete for
(D) anoikic cells fed ARPE-19; actin cytoskeleton stained with phalloidin-FITC (green), anoikic
both macrophage and RPE binding.
dying cells with CMTMR (red) and nuclei with DAPI (blue). Co-incubation time: 8 hr.
The macrophages use aVb3 and RPE
cells aVb5 integrins for binding apopto-
Phosphatidylserine on dying cells does through anoikis and used an already tic cells or OSs (Finnemann & Rodri-
not, but triamcinolone treatment does established UV-irradiation apoptosis guez-Boulan 1999). Calreticulin on the
influence professional phagocytosis model (Roduit & Schorderet 2008) for surface of apoptotic cells may also ini-
Treatment of the dying anoikic and studying the dead cells’ clearance. The tiate clearance of dying cells acting
UV-irradiated apoptotic cells with nondying ARPE-19 cells were much together with PS for achieving optimal
recombinant annexin-V before and more efficient in taking up anoikic than phagocytic clearance (Gardai et al.
throughout the phagocytic assay, that UV-induced apoptotic ones, that is 2005). Changing carbohydrate compo-
is blocking PS on the surface of dying they could recognize the difference sition of glycoproteins, complement-
cells, did not inhibit the engulfment
by nondying ARPE-19 cells (data not 15 15
shown) or macrophages (Fig. 4A,B).
TA treatment of the macrophages
10 10
48 hr prior the phagocytic assay, on
%
%

the other side, enhanced the engulf-

ment of both anoikic and UV-irradi- 5 5
ated apoptotic cells in a dose- and
time-dependent manner reaching 16%
0 0
and 15%, respectively, at 8 hr of co- 1 4 8 1 4 8
(A) (B)
incubation (Fig. 4C,D). Hours Hours

Discussion 20 p = 0.02 20 p = 0.01

Insufficient clearance of dying cells in 15 15

the retina may lead to degeneration
and inflammation (Mullen & LaVail 10 10
%

1976; Edwards & Szamier 1977). The

mechanism and the dynamics of clear-
ance of shed photoreceptor OSs by
RPE cells in vitro and in vivo has been
described in detail and, to a lesser
extent, the same can be said about the
involvement of macrophages (Kirch-
hof et al. 1989; Forrester 2003).
We have described here a novel way
of inducing cell death in ARPE-19 cells
5 5
0 0
(C) 1 4 8 1 4 8
Hours (D) Hours
Fig. 4. Phagocytic capacity of macrophages challenged to engulf (A, C) anoikic (white bars) or
UV-induced apoptotic ARPE-19 cells (black bars) under recombinant annexin-V, 10 mg per 105
cells (A, B) or 1 lm triamcinolone (C, D) treatment. Bars represent mean values of three
independent experiments ± SD.

e33
Acta Ophthalmologica 2011
associated mannoproteins and expo-
sure to DNA on dying cells can act as
alternative mechanism to PS for recog-
nition of dead cells (Wilt et al. 1999;
Dini 2000; Palaniyar et al. 2004).
The process of engulfing dying cells
is dynamic and driven by a finely con-
trolled rearrangement of the actin
cytoskeleton (cytochalasin D blocking
of the actin polymerization, inhibited
the engulfment of both types of dying
retinal cells by the nondying ARPE-19
cells and the macrophages (data not
shown)). The internalization process
was characterized by actin-dependent
extension of the plasma membrane
around the particle (Figs 2C,D and 3D).
Intravitreal triamcinolone has been
widely used for treatment of intraocular
proliferative, exudative and neovascular
disorders (Jonas et al. 2005). The poten-
tial deleterious or cytotoxic effects of
glucocorticoids use has been thoroughly
analysed in RPE cells (Yeung et al.
2003; Narayanan et al. 2006). TA has
been demonstrated to induce nonapop-
totic (paraptotic) cell death in ARPE-19
cells (Valamanesh et al. 2007). Only one
study so far described an inhibiting
effect of corticosteroids on porcine
RPE-mediated phagocytosis, but they
used latex microspheres instead of
actual dying cells for engulfment. (Ka-
wahara et al. 2000). In our study, TA
enhanced the macrophage-mediated
clearance of dying retinal cells: an aug-
mentation of about two times in the
engulfing capacity of anoikic ARPE-19
cells, and about 1.5 times of UV-irradi-
ated apoptotic cells was found over the
8 hr of co-incubation (Fig. 4C,D).
Further studies are required to deter-
mine which ‘eat-me’ signals other than
PS are exposed on the surface of RPE
cells dying by anoikis, UV-induced
apoptosis or other death pathways.
Furthermore, the limitation of using
ARPE-19 cell line can be overcomed by
using human primary cells in the
future. Our experiments indicate that
the clearance of dying cells in the retina
is a dynamic process carried both
by professional and nonprofessional
phagocytes. This process can be
enhanced by TA in macrophages.
In vivo, the inner layers of the multi-
layer retina get exposed to maximum
concentrations of TA and the RPE
layer and the macrophages to least
amounts of TA diffusing from the vitre-
ous. Thus, the toxic effect of TA on the
different cell types may be different.
e34
Even at the low TA dose, we used (1 lm
versus the clinically used 1 mm) an
enhancement of the macrophage-medi-
ated phagocytosis of dying retinal cells
– a known effect of glucocorticoids on
macrophages (van der Goes et al. 2000)
could be observed. Along with the
immunosuppressive and anti-inflamma-
tory TA action, this would be a plausi-
ble target during treatment of exudative
or neovascular eye disorders. Very
likely, a combination of intravitreal TA
with other phagocytosis enhancing and
angiogenesis blocking drugs would be a
promising treatment for wet (exudative)
type of AMD (Jonas 2006).
Acknowledgements
The authors acknowledge the financial
support from the MECENATURA Grant
(University of Debrecen, Medical and
Health Science Center) and The Research
Council of Norway.
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Received on March 17th, 2010.
Accepted on October 2nd, 2010.
Correspondence:
Goran Petrovski MD, PhD
Department of Ophthalmology
Medical and Health Science Center
University of Debrecen
Nagyerdei krt. 98
4012 Debrecen
Hungary
Tel ⁄ Fax: + 36 52 255 626
Email: gokip@indi.dote.hu