Fas Mediates Apoptosis and Oxidant-Induced Cell Death
in Cultured hRPE Cells
Shunai Jiang,1 Mei-Whey H. Wu,2 Paul Sternberg,2 and Dean P. Jones1,2
PURPOSE. To investigate whether Fas ligand (FasL) and the Fas receptor system mediates apoptosis
in cultured human retinal pigment epithelial (hRPE) cells and contributes to oxidant-induced death
of hRPE cells.
METHODS. Expression of FasL and Fas in cultured hRPE cells was examined by Western blot analysis
and flow cytometry. The susceptibility of hRPE cells to Fas-mediated apoptosis was determined by
incubating cells with recombinant soluble Fas ligand (sFasL). Characteristics of apoptosis assessed
included chromatin condensation, DNA cleavage, and phosphatidylserine exposure. To investigate
the possible involvement of Fas-mediated apoptosis in oxidative killing of hRPE cells, the effects of
the oxidant tert-butylhydroperoxide (tBH) on the expression of FasL and Fas were studied. The
specificity of effects of oxidant was examined using the antioxidants glutathione and N-acetyl-Lcysteine (NAC). The requirement for the Fas pathway in tBH-induced apoptosis was investigated
using an antagonistic anti-Fas antibody ZB4 that blocks the interaction between FasL and Fas.
RESULTS. Cultured hRPE cells constitutively expressed FasL and Fas. Ligation of Fas receptor with
recombinant sFasL triggered apoptosis in hRPE cells. tBH treatment of hRPE cells resulted in
increased expression of FasL and Fas. Glutathione and NAC completely abrogated tBH-induced
increase in FasL and Fas expression and apoptosis. Blocking FasL and Fas interaction by ZB4
inhibited tBH-induced apoptosis, but only partially.
CONCLUSIONS. A functional Fas-mediated apoptotic pathway is present in cultured hRPE cells and can
be activated with sFasL or by upregulation of FasL and Fas expression with an oxidant. The
incomplete inhibition by blocking antibody indicates that the Fas pathway is involved in oxidantinduced apoptosis, but other triggering mechanisms are also important. (Invest Ophthalmol Vis Sci.
2000;41:645– 655)
T
he retinal pigment epithelium (RPE) is a single layer of
cuboidal cells situated external to the photoreceptor
layer of the retina and internal to Bruch’s membrane.1
The function of the RPE is to maintain and support the photoreceptors by phagocytosis and degradation of debris shed from
retinal outer segments. Therefore, injury to the RPE results in
degeneration of the sensory retina and may contribute to the
pathogenesis of age-related macular degeneration (ARMD), the
leading cause of blindness in the developed world.2
The molecular mechanism for the injury to RPE cells is not
understood clearly. We have recently shown that the prooxidant tert-butylhydroperoxide (tBH) induces apoptosis in
cultured hRPE cells.3 Apoptosis is a form of cell death characterized by chromatin condensation, DNA fragmentation, and
phosphatidylserine (PS) externalization.4 Besides pro-oxidants,
other stimuli such as experimental ischemia, light exposure,
and protein kinase inhibitors are also reported to trigger apo-
From the 1Department of Biochemistry and 2Department of Ophthalmology, Emory University School of Medicine, Atlanta, Georgia.
Supported by Grants EY07892, EY06360, ES09047 from the National Institutes of Health; and a grant and the Wasserman Award from
Research to Prevent Blindness.
Submitted for publication June 23, 1999; revised October 13,
1999; accepted October 26, 1999.
Commercial relationships policy: N.
Corresponding author: Paul Sternberg, Jr., Emory University
School of Medicine, Department of Ophthalmology, 1365B Clifton
Road, NE, Atlanta, GA 30322. [email protected]
ptosis in RPE in vitro.5–7 In the highly vascularized choroidal
neovascular membrane surgically excised from eyes of patients
with ARMD, extensive apoptosis in RPE cells has been observed, suggesting that apoptosis of RPE cells is involved in the
pathogenesis of ARMD.8
In many systems, apoptosis is mediated by the interaction
between Fas ligand (FasL) and Fas.9 –10 FasL is a member of the
tumor necrosis factor family of cytokines. Its receptor Fas
belongs to the tumor necrosis factor receptor superfamily.
Binding of FasL or an agonistic anti-Fas antibody to Fas initiates
a signal transduction pathway for apoptosis in susceptible
cells. FasL and Fas are constitutively expressed in a wide range
of tissues. The Fas system provides an important mechanism
for regulation of immune response and killing of tumor cells. A
malfunction of Fas system has been found to contribute to the
development of many diseases including cancer, acquired
immune deficiency syndrome, hepatitis, and autoimmune diseases.9 –10
A substantial expression of FasL has been found in the
corneal epithelium and endothelium, iris, ciliary cells, and
retina of eyes and has led to the proposal that FasL participates
in maintenance of the immune privilege of the eye by inducing
apoptosis in infiltrating, Fas-bearing, activated lymphocytes.11,12 In the normal eye, FasL is weakly expressed in the
RPE monolayer.8 However, an increased FasL expression has
been found in the RPE monolayer of the choroidal neovascular
membranes from patients with ARMD.8 The FasL expressed on
RPE cells may be necessary for immune privilege of the poste-
Investigative Ophthalmology & Visual Science, March 2000, Vol. 41, No. 3
Copyright © Association for Research in Vision and Ophthalmology
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FIGURE 1. Expression of FasL and Fas on cultured hRPE cells. (A) Western blot analysis of total FasL and
Fas protein. Lysates (50 ␮g protein) from hRPE cells were probed with mouse IgG to human FasL (250
ng/ml) or Fas (100 ng/ml). Lysates (10 ␮g protein) from human endothelial cells and human T leukemia
Jurkat cells were used as positive controls for FasL and Fas, respectively. Note that the appearance of more
FasL detection was due to longer exposure of x-ray film. (B) Flow cytometry analysis of surface FasL and
Fas. To detect FasL, hRPE cells were incubated with a rabbit anti-human FasL antibody or with rabbit IgG,
respectively, followed by an FITC-conjugated antibody against rabbit IgG. To detect Fas, hRPE cells were
incubated with a mouse anti-human Fas antibody or with mouse IgG, respectively, followed by an
FITC-conjugated antibody against mouse IgG. Cells were analyzed by flow cytometry. Data are presented
as the number of cells plotted as a function of cell fluorescent intensity that is dependent on the amount
of FasL or Fas expression in each cell. Representative data of five separate experiments are shown.
rior of the eye. However, if its expression is upregulated or if
the expression of Fas in RPE cells is increased, death signals
could be activated. Affected cells may undergo apoptosis (suicide) or cause paracrine-induced death of neighboring cells
(fratricide). Indeed, in the highly vascularized choroidal neovascular membrane from the eye of patients with ARMD, a
correlation between Fas expression and the extent of apoptosis exists, although the association between FasL expression
and apoptosis is not found.8 A study by Esser et al.13 has
demonstrated that cultured human (h)RPE cells constitutively
express Fas but are rather resistant to apoptosis by an agonistic
Fas antibody. However, upregulation of Fas expression in hRPE
by cytokines can overcome the resistance to the agonistic Fas
antibody.
Cytokine treatment is normally accompanied by oxidative
stress, and reactive oxygen intermediates (ROI) are involved in
the induction of FasL expression by cytostatic drugs and T-cell
activation.14,15 Pro-oxidants are found to upregulate FasL expression and enhance Fas-mediated apoptosis.14,15 In Fas-mediated apoptosis, generation of ROIs is an early signaling event,
and inhibiting its production with antioxidants can prevent
apoptosis.16,17 All this evidence suggests that ROI is a key
regulator of the Fas system. However, the contribution of the
Fas pathway to oxidant-induced apoptosis in the RPE cells has
not been established. Thus, in the present study, we investigated whether the Fas system mediates apoptosis in cultured
hRPE cells and whether it could be modulated by an oxidant
and contribute to oxidative injury of RPE.
MATERIALS
AND
METHODS
hRPE Cell Cultures and Treatments
hRPE cell cultures were established as previously described.18
Cells were routinely grown in Dulbecco’s modified Eagle’s
medium containing 10% fetal bovine serum, 2 mM glutamine,
100 IU/ml penicillin, and 100 ␮g/ml streptomycin in a 37°C
humidified incubator under an atmosphere of 5% CO2 in air.
Cells were passaged every 10 days.18 For experiments, cells
FIGURE 2. Chromatin condensation in sFasL-treated hRPE cells. hRPE
cells were untreated or treated with 500 ng/ml sFasL for 48 hours. Cells
were fixed, stained with PI, and visualized by confocal microscopy.
Untreated cells had nuclei with diffuse chromatin, whereas sFasLtreated cells showed small nuclei with condensed chromatin (arrows).
Data presented are representative of three independent experiments.
Bars, 3 ␮m.
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FIGURE 3. DNA cleavage in sFasLtreated hRPE cells. (A) hRPE cells
were untreated or treated with 500
ng/ml sFasL for 48 hours. Cells were
fixed, stained with TUNEL and visualized by confocal microscopy.
Untreated cells displayed only a
background level of fluorescence,
whereas sFasL-treated cells showed
intensely stained nuclei, indicating
DNA cleavage (arrows). Bars, 2 ␮m.
(B) hRPE cells were untreated or
treated with 250 ng/ml and 500
ng/ml sFasL for 48 hours and then
fixed, stained with TUNEL, and analyzed by flow cytometry. To determine the effect of ZB4, cells were
pretreated with 125 ng/ml ZB4 for 1
hour and then incubated with 250
ng/ml sFasL for 48 hours. Single-parameter histogram shows fluorescein
intensity, a measure of dUTP incorporation, plotted against cell number. R1, nonapoptotic cells. R2, apoptotic cells with dUTP incorporation
into the DNA cleavage sites. sFasL
treatment caused an increase in the
number of cells undergoing apoptosis, and this was inhibited by pretreatment of cells with an antagonistic anti-Fas antibody ZB4. Representative data of three separate
experiments are shown.
between passages 6 and 10 were taken and plated at the
following densities: 2 ⫻ 104 cells/well in 300 ␮l medium for
chambered coverglass (Lab-Tek 136439; Miles, Napierville, IL),
2 ⫻ 105 cells/well in 1.5 ml medium for 6-well plates, and 1 ⫻
106 cells in 10 ml medium for a 100-mm culture dish. Experiments were begun 2 days after plating. To trigger apoptosis,
stock solutions of tBH and recombinant sFasL (Oncogene,
Cambridge, MA) were added to the RPE cell culture without
changing the growth medium. To examine the effects of antioxidants and antagonistic anti-Fas antibody ZB4 (MBL, Watertown, MA), cells were pretreated with these agents for 1 hour
and then incubated with tBH or sFasL for the different times
indicated.
Western Blot Analysis of FasL and Fas Protein
Cells were washed once with phosphate-buffered saline (PBS)
and then lysed in a boiling solution containing 10% glycerol,
250 mM Tris (pH 6.8), 4% sodium dodecyl sulfate, and 2%
␤-mercaptoethanol. The cell lysates were immediately boiled
for 5 minutes and centrifuged at 10,000g for 5 minutes at 4°C.
The supernatant was collected, and the amount of protein was
measured by the Bradford method. Protein extracts (50 ␮g/
lane) were loaded onto a 10% sodium dodecyl sulfate–polyacrylamide gel, and the separated proteins were blotted to 0.45
␮m polyvinylidene difluoride membranes (Hybond; Amersham, Arlington Heights, IL). Nonspecific binding was blocked
by incubating the membranes overnight at 4°C in a blocking
buffer containing 5% nonfat dry milk, 0.1% Tween-20, 10 mM
Tris (pH 7.5), and 100 mM NaCl. The membranes were then
stained with 1:2500 diluted anti-Fas antibody or 1:1000 diluted
anti-FasL antibody (F37720 and F22120; Transduction Laboratories, Lexington, KY) in blocking buffer for 1 hour at room
temperature with agitation. After they were washed three
times with washing buffer containing 10 mM Tris (pH 7.5), 100
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mM NaCl, and 0.1% Tween-20, the membranes were incubated
with 1:3000 diluted horseradish peroxidase-coupled antimouse antibody in blocking buffer for 1 hour at room temperature. The specific proteins were then visualized by incubating
the membranes with reagent (Renaissance Western Blot
Chemiluminescence Reagent; NEN, Boston, MA) and exposing
the membranes to film (X-OMAT; Eastman Kodak, Rochester,
NY). Equal protein loading was verified by ponceau red treatment of membranes.
Flow Cytometric Detection of FasL and Fas on
Cell Surface
Cells (2 ⫻ 105) were washed twice in washing buffer containing 0.2% bovine serum albumin and 0.02% sodium azide in PBS
and incubated with 2.5 ␮g/ml a rabbit anti-FasL antibody C20
(Santa Cruz Biotechnology, Santa Cruz, CA) or a mouse anti-Fas
antibody DX2 (Oncogene) for 30 minutes at 4°C. Cells were
then washed, resuspended in 50 ␮l washing buffer containing
2.5 ␮g/ml fluorescein isothiocyanate (FITC)– conjugated antirabbit IgG or goat anti-mouse IgG, respectively, and incubated
for 30 minutes at 4°C. Nonstaining cells, cells stained with the
FITC-conjugated secondary antibodies alone, and cells stained
with isotypically matched control immunoglobulin were run in
parallel as negative controls. After staining, cells were washed
twice in washing buffer and resuspended in PBS. Data acquisition and analysis were performed in a flow cytometer (FACScan using the CellQuest software; Becton Dickinson, Mountain View, CA).
Visualization of Apoptotic Nuclei by Confocal
Microscopy
hRPE cells grown in chambered coverglass were incubated
with sFasL for 48 hours. Cells remaining on the coverglass after
treatment were washed twice with PBS and fixed with ice-cold
80% methanol for 30 minutes. Cells were then gently washed
three times and stained with 50 ␮g/ml propidium iodide in PBS
for 5 minutes at room temperature. The coverglass were analyzed by laser scanning confocal microscope (model 1024;
BioRad, Richmond, CA).
Assessment of Apoptosis by TdT-Mediated dUTP
Nick-End Labeling
TdT-mediated dUTP nick-end labeling (TUNEL) was performed
using a kit (In Situ Cell Death Detection kit with fluorescein;
Boehringer–Mannheim, Mannheim, Germany) according to the
standard protocol provided by the manufacturer. Briefly, after
treatment, both floating and adherent (released by trypsin)
cells were collected and washed twice with PBS. Cells were
fixed with freshly prepared 4% paraformaldehyde in PBS (pH
7.4) for 30 minutes at room temperature and then permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 2
minutes on ice. To label DNA strand breaks, cells were incubated with 50 ␮l TUNEL reaction mixture containing terminal
deoxynucleotidyl transferase and fluorescein-dUTP in the binding buffer and incubated for 1 hour at 37°C in a humidified
atmosphere. Cells were then washed twice with PBS and analyzed by flow cytometry. Alternatively, cells grown on chambered coverglasses were incubated with sFasL for 48 hours.
Cells remaining on the coverglass were washed twice with PBS
and stained with the TUNEL kit as described. The coverglasses
were analyzed by laser scanning confocal microscopy.
FIGURE 4. Externalization of PS in cells treated with sFasL. hRPE cells
were untreated or treated with various concentrations of sFasL for 48
hours. Cells were then stained with annexin V-FITC together with PI
and analyzed by flow cytometry. Data show two-parameter analysis of
fluorescein intensity of annexin V (green fluorescence, PS exposure)
and PI (cell viability). The normal (living) cells (lower left quadrant)
had low annexin V and PI staining. The early apoptotic cells (lower
right quadrant) had high annexin V staining but low PI staining.
Percentages of cells in each quadrant are indicated. sFasL-induced PS
exposure was demonstrated as increases in the number of cells stained
with annexin V but excluding PI. Results are from one experiment that
is representative of three independent experiments.
Assessment of Apoptosis by Annexin V-FITC
Staining of PS
Cells were stained with annexin V–FITC (TACS; Trevigen,
Gaithersburg, MD) according to the manufacturer’s instructions. Briefly, after treatment, both floating and adherent cells
were collected and washed twice with PBS. Cells were then
resuspended in 100 ␮l binding buffer containing 10 mM
HEPES-KOH (pH 7.4), 150 mM NaCl, 5 mM KCl, 1 mM MgCl2,
1.8 mM CaCl2, and 1 mg/ml annexin-V–FITC. After 15 minutes
of incubation at room temperature in the dark, 400 ␮l binding
buffer was added, and cells were analyzed by flow cytometry.
Total RNA Preparation and Reverse
Transcription–Polymerase Chain Reaction for Fas
mRNA and FasL mRNA
Total RNA was prepared with a kit (RNeasy, Qiagen, Chatsworth,
CA) according to the manufacturer’s instructions. Single-stranded
DNA was synthesized from RNA in a 15-␮l reaction mixture
containing 100 ng random hexamer and 200 units of murine MLV
reverse transcriptase (Gibco, Grand Island, NY). The reaction
mixture was diluted with 20 ␮l water and 2 ␮l of which was used
for polymerase chain reaction (PCR). The PCR reaction mixture
contained 10 picomoles each of forward and reverse primers and
2 units DNA polymerase (Taq; Perkin-Elmer, Branchburg, NJ).
Amplification was performed for 30 cycles in a thermal cycler.
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FIGURE 5. Upregulation of FasL expression by tBH. hRPE cells were untreated or treated with 300 ␮M tBH
for times indicated. (A) Expression of
total FasL was determined by Western blot. Data show that tBH resulted
in an increase in total FasL expression after 1 hour. (B) Expression of
surface FasL was determined by flow
cytometry. Single-parameter histogram shows plot of fluorescein intensity, a measure of FasL expression,
against cell number. Note that
stacked plots are not offset. Data
therefore show an increase in the
number of cells with high FasL expression already apparent at 1 hour.
Dot plots show two-parameter analysis of fluorescein intensity (FasL expression) and FSC (indicating cell
size). Data demonstrate that the cells
undergoing apoptosis after tBH treatment (measured as cell shrinkage, indicated by a decrease in cell size)
expressed increased FasL. (C) Expression of FasL mRNA was analyzed
by RT-PCR. The PCR products were
analyzed by agarose gel electrophoresis (top and middle). FasL and
␤-actin migrated at the predicted size
of 827 and 243 base pairs, respectively. The relative quantities of the
FasL PCR products were determined
by densitometric analysis and normalized with the density of the ␤-actin profile (bottom). The ratios of the
FasL versus the ␤-actin products
were calculated. Representative results of three to five separate experiments are shown, except the quantitative data in (C), which are the
means ⫾ SEM from five independent
experiments.
Each cycle consisted of 1 minute of denaturation at 94°C, 1
minute of annealing at 57°C, and 1 minute of extension at 72°C.
The sequences of primers used for analysis were as follows: Fas
(forward: 5⬘-CGGAGGATTGCTCAACAAC-3⬘, reverse: 5⬘-TTGGTATTCTGGGTCCG-3⬘),19 FasL (forward: 5⬘-GTTTGCTGGGGCTGGCCTGACT-3⬘, reverse: 5⬘-GGAAAGAATCCCAAAGTGCTTC3⬘),20 and ␤-actin (forward: 5⬘-CGTGG GCCGCCCTAGGCACCA3⬘, reverse: 5⬘-TTGGCCTTAGGGTTCAGGGGGG-3⬘).21 The PCR
products were separated on 1% agarose gel and visualized by
ethidium bromide staining. The quantities of the PCR products
were determined by densitometric scanning using Lynx software
(Applied Imaging, Santa Clara, CA).
RESULTS
Expression of FasL and Fas in hRPE Cells
Western blot analysis of total FasL and Fas showed that both
FasL and Fas protein were detected in cultured hRPE cells (Fig.
1A). FasL and Fas can be present within cells and on the cell
surface, but the latter form is important in the transduction of
the death signal. To determine whether FasL and Fas are expressed on the cell surface of RPE, we treated intact cells with
antibodies that react with FasL and Fas, respectively, and analyzed the cells by flow cytometry (Fig. 1B). With anti-FasL
antibody C20, we were able to detect cell surface FasL in
untreated hRPE cells, although the expression level was low.
Using anti-Fas antibody (DX2), a relatively high expression of
Fas was detected on the surface of hRPE cells.
Apoptosis in hRPE Cells by Ligation of Fas
Receptor
To investigate whether the Fas expressed in cultured hRPE
cells functionally transduces an apoptotic signal, cells were
treated with recombinant soluble Fas ligand (sFasL) that binds
to Fas and induces apoptosis in sensitive cells. Apoptosis was
assessed by three criteria, chromatin condensation, DNA cleav-
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FIGURE 6. Upregulation of Fas expression by tBH. hRPE cells were
treated, and expression of Fas was
examined as described in Figure 5.
(A) tBH resulted in an increase in
total Fas expression as detected by
Western blot. (B) tBH caused an increase in cell surface Fas as detected
by flow cytometry. Single-parameter
histogram shows an increase in the
number of cells with high Fas expression. Two-parameter dot plots show
that the cells undergoing apoptosis
after tBH treatment (shrinkage, indicated by a decrease in cell size) expressed increased Fas. (C) tBH-induced expression of Fas mRNA. Fas
and ␤-actin migrated at the predicated size of 420 and 243 base pairs,
respectively (top and middle). The
relative quantities of the Fas PCR
products were determined by densitometric analysis, and the ratios of
the Fas versus the ␤-actin products
were calculated (bottom). Representative results of three to five separate
experiments are shown, except the
quantitative data in (C), which are
the means ⫾ SEM from five independent experiments.
age, and PS exposure. The confocal microscopy images of cells
stained with the DNA-intercalating dye propidium iodide (PI)
illustrate that the nuclei of untreated cells had dispersed chromatin structure and visible nucleoli, whereas the nuclei of cells
treated with sFasL became smaller in size and condensed—that
is, sFasL-treated RPE cells underwent typical nuclear morphologic changes of apoptosis (Fig. 2).
Extensive DNA cleavage is another characteristic event
that occurs in the cells undergoing apoptosis. The cleavage of
the DNA yields double-strand breaks that can be detected after
labeling the free 3⬘-OH termini with dUTP-FITC by TUNEL
assay. The cells with DNA cleavage can then be analyzed in
individual cells by confocal microscopy and in cell populations
by flow cytometry. Confocal microscopy revealed only a background level of fluorescence in untreated cells, whereas, cells
treated with sFasL had intensely labeled nuclei (Fig. 3A). When
these cells were measured by flow cytometry, untreated cells
had relatively little fluorescence compared with apoptotic
cells. sFasL-induced apoptosis was shown by an increase in the
number of cells staining with dUTP-FITC (region R2 in Fig. 3B)
after treatment with 250 ng/ml and 500 ng/ml sFasL. Pretreatment of cells with 125 ng/ml ZB4, an antibody that blocks
interaction between FasL and Fas, blocked the sFasL-induced
increase in dUTP-FITC fluorescence. Thus, the results indicate
that sFasL-induced apoptosis is mediated through the Fas receptor.
In cells undergoing apoptosis, the membrane phospholipid PS is translocated from the inner to the outer leaflet of the
plasma membrane, thereby exposing PS to the external cellular
environment.4 Annexin V is a 35-kDa, Ca2⫹-dependent, phospholipid-binding protein that has a high affinity for PS and
binds to cells with exposed PS.22 Therefore, staining cells by
annexin V in conjugation with vital dye PI allows identification
of early apoptotic cells (annexin V–positive and PI-negative). In
the present study, untreated hRPE cells showed primarily low
fluorescence for both annexin V-FITC and PI, indicating that
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they were viable and not undergoing apoptosis (Fig. 4, lower
left quadrant). After treatment with sFasL, the portion of cells
staining with annexin V but not PI (early apoptotic cells, lower
right quadrant) was increased as the concentration of sFasL
increased, indicating sFasL treatment induced PS externalization in hRPE cells.
Taken together, these data demonstrate that hRPE cells
possess a functional Fas-mediated apoptosis pathway and
can be induced to undergo apoptosis by ligation of the Fas
receptor.
Induction of FasL and Fas Expression by tBH and
Inhibition by Antioxidants
Although RPE cells express both FasL and Fas, they may not
undergo suicide or fratricide if their expression levels are
lower than the threshold for initiating Fas ligation or if the
Fas-mediated signaling cascade is inhibited by regulatory components.9,10 However, if stimuli can induce FasL or Fas expression in RPE and result in activation of the Fas-mediated signaling pathway, apoptosis may be triggered. Our previous study
has shown that the oxidant tBH can induce apoptosis in cultured hRPE cells.3 Because oxidants upregulate FasL expression in other cell types,14,15,23 we investigated whether the Fas
pathway could mediate tBH-induced apoptosis in cultured
hRPE cells. We first examined whether tBH affects expression
of FasL protein in cultured hRPE. By Western blot, an increase
in the expression of total FasL was detected in tBH-treated
hRPE cells after 1 hour, and it decreased after 4 hours (Fig. 5A).
When cell surface FasL was measured by flow cytometry, the
histogram, plotted as FasL expression against cell number,
demonstrated that untreated hRPE cells showed a basal expression of cell surface FasL. After tBH treatment, hRPE revealed a
marked upregulation in FasL expression (Fig. 5B). The increase
started 1 hour after treatment and continued to increase during
incubation up to 6 hours. Dot plot analysis of FasL expression
versus forward side scatter (FSC, indicating cell size) showed
that the cells undergoing apoptosis after tBH treatment (shown
here as cell shrinkage with decreased FSC) had higher FasL
expression.
We next examined whether the induction of FasL by
tBH was due to enhanced transcriptional activation. hRPE
cells were treated with tBH for various times, and mRNA was
isolated, reverse transcribed, and amplified using FasL-specific primers. As shown in Fig, 5C, the FasL-specific PCR
product was detected at a very low level in untreated cells,
whereas tBH treatment resulted in a substantial increase
(3.1-fold) in FasL expression after 1 hour. In contrast, expression of ␤-actin mRNA, which was measured as a control
for equal loading and integrity of the RNA, was not affected
by tBH treatment.
To obtain further insight into the mechanism of tBHinduced apoptosis, we also analyzed Fas expression on tBH
treatment. The Western blot data show that tBH treatment
of RPE cells caused an increase in total Fas expression at 3
hours (Fig. 6A). tBH also induced cell surface Fas expression
(Fig. 6B). The increase in cell surface Fas began 1 hour after
treatment and occurred earlier than the increase in total Fas.
RT-PCR data demonstrated that tBH-induced upregulation of
Fas was also associated with transcriptional activation of
gene expression (1.4-fold increase in Fas mRNA, Fig. 6C).
Together, these results suggest that hRPE cells may first
translocate the preexisting intracellular Fas to the cell sur-
Fas-Mediated Apoptosis in RPE Cells
651
face and then transcribe and translate more Fas. Compared
with tBH-induced FasL expression, the effect of tBH on Fas
was less prominent.
The specific effect of tBH as an oxidant dependent activation of the expression of FasL and Fas was then determined
using the antioxidants glutathione (GSH) and N-acetylcysteine
(NAC). Cultured hRPE cells were first treated with GSH or NAC
for 1 hour and then incubated with tBH. As shown in Figure
7A, pretreatment of hRPE with GSH or NAC blocked induction
of FasL and Fas by tBH. When apoptosis was determined in
these cells by the TUNEL assay, it was found that the antioxidants also blocked tBH-induced apoptosis (Fig. 7B). This protection could also be seen by light microscopy, in which tBH
alone caused an increase in cells detaching from the plates,
whereas pretreatment with antioxidants protected this change
(Fig. 7C).
Inhibition of tBH-Induced Apoptosis by Blocking
FasL and Fas Interaction
In a previous study, we found that tBH induced a loss of
cytochrome c from mitochondria beginning at 2 hours and
increasing at 6 and 8 hours.3 There was a close temporal
association with a substantial activation of caspase 3–like activity by 4 hours.3 A decrease in mitochondria membrane
potential was detected as early as 2 hours, indicating that
oxidant-induced activation of the permeability transition could
directly contribute to activation of the apoptosis pathway.
However, because the permeability transition is also inhibited
by thiol antioxidants, these data do not allow any distinction
between a mitochondria-mediated activation of apoptosis and
a Fas-mediated activation of apoptosis.
To further elucidate the involvement of the Fas-mediated
death pathway in tBH-induced apoptosis, hRPE cells were
preincubated with an antagonistic anti-Fas antibody ZB4 before
treatment with tBH. This antibody completely blocked Fasmediated apoptosis in hRPE cells (Fig. 3B) and human T leukemia Jurkat cells (data not shown). However, pretreatment of
hRPE cells with ZB4 only partially inhibited tBH-induced apoptosis in hRPE cells (Fig. 8). This suggests that tBH-induced
apoptosis is mediated, at least in part, through a death signal
activated by the increased FasL that binds to the increased Fas
in RPE cells. However, the data are also consistent with another mechanism’s having a role in activation of apoptosis in
hRPE cells, such as the previously documented mitochondrialactivated pathway.3
DISCUSSION
Histopathologic studies indicate that loss of RPE is a pivotal
event in the development of ARMD.2 Circumstantial evidence
suggests that oxidative mechanisms contribute to this process
and that supplementation with antioxidants protect against
onset or progression of ARMD.24 –27 We have previously studied the cellular mechanism of oxidant-induced death in human
retinal pigment epithelium using human cell lines established
from cadaveric donors.3,18,24 The results have shown that cellular and extracellular GSH protect against oxidative stress,
apparently by different mechanisms.18,24 Our recent research
shows that after exposure to the model oxidant tBH, cells
undergo apoptosis with a sequence of process involving loss of
mitochondrial membrane potential, release of cytochrome c
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Jiang et al.
IOVS, March 2000, Vol. 41, No. 3
FIGURE 7. Antioxidants inhibit tBHinduced FasL and Fas expression and
apoptosis. hRPE cells were pretreated with GSH and NAC (mM) for
1 hour and then incubated with 300
␮M tBH for 4 hours. (A) Expressions
of cell surface FasL and Fas were determined by flow cytometry. The
percentages of FasL- or Fas-positive
cells were shown. Data are the
mean ⫾ SEM from three to four separate experiments. (B) Apoptosis
was assessed by examining DNA
cleavage with TUNEL. Dot plots
show two-parameter analysis of fluorescein intensity (dUTP incorporation) and FSC (indicating cell size).
Data show an increase in the cells
undergoing apoptosis after tBH treatment (cell shrinkage, indicated by a
decrease in cell size, and DNA cleavage, indicated by an increase in fluorescence intensity of dUTP-FITC) and
inhibition by GSH and NAC.
and activation of caspase 3-like activity.3 These results are
associated with an activation sequence in which an oxidantinduced mitochondrial permeability transition provides a triggering event, but they do not exclude the involvement of other
activating mechanisms.
In the present study, we have examined an alternative
mechanism—namely, activation by the plasma membrane Fas
receptor. Rapid progress has been achieved during recent
years in the elucidation of the signaling pathway of Fas-mediated apoptosis.9,10 Fas ligation by FasL or by agonistic anti-Fas
antibody can result in oligomerization of receptors in the cell
membrane and formation of a death-inducing signal complex
that includes the adapter protein Fas-associated death domain
(FADD) and leads to the activation of caspase 8. The activated
caspase 8 then propagates the apoptotic signal by activating
downstream proteins through proteolytic cleavage. Among the
proteins activated by this cascade are caspase 3 and BID. BID
is a proapoptotic protein that triggers mitochondrial release of
cytochrome c, which in turn activates caspases 9 and 3.28,29
Thus, in principle, activation of the Fas pathway could contribute to the mitochondrial changes in tBH-treated RPE cells that
we have previously reported.
In this study, we show that cultured hRPE cells constitutively express low levels of FasL but relatively high levels of
Fas. Under normal culture conditions, these expression levels
are insufficient to activate apoptosis because apoptosis occurred at only a low level in untreated cells. However, when
oligomerization of Fas was facilitated with a high concentration
of recombinant sFasL or the agonistic anti-Fas antibody CH-11
(data not shown), apoptosis was induced. Compared with
other Fas-expressing cells, such as human T leukemia Jurkat
cells, hRPE cells appeared less sensitive to Fas-mediated apoptosis. Only 20% to 40% of RPE cells underwent apoptosis after
48 hours, whereas more than 80% of Jurkat cells undergo
apoptosis under these condition.30 This difference may be due
to blocking of the Fas pathway in RPE cells by intracellular
inhibitors. hRPE cells are found to have a very high level of
intracellular Zn2⫹, which is a potent inhibitor of caspase 3 and
endonucleases.31–33
Our data show that, besides sFasL, Fas-mediated apoptosis
could also be induced in RPE cells by oxidative stress. Treatment of RPE cells with the oxidant tBH resulted in increased
FasL and Fas expression; blocking FasL and Fas interaction with
an antagonistic antibody inhibited tBH-induced apoptosis.
However, unlike the antioxidants that completely inhibited
tBH-induced apoptosis, the antagonist antibody only partially
inhibited apoptosis. This suggests that the Fas-mediated pathway is involved in tBH-induced apoptosis but serves as only
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IOVS, March 2000, Vol. 41, No. 3
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653
FIGURE 7. (Continued ) (C) Conventional microscopy image shows that tBH
treatment results in cell shrinkage and
detachment of RPE cells from the dish,
which can be inhibited by 3 mM GSH
and 1 mM NAC. Bars, 10 ␮m. Results in
(B) and (C) are representative of three
independent experiments.
one of multiple death-signaling mechanisms triggered by the
oxidant. Therefore, mitochondrial damage previously observed
in tBH-treated cells may occur in part through a Fas pathway
that involves sequential events including activation of caspase
8, cleavage of BID, and damage of mitochondria and also by an
alteration of mitochondria components directly by ROIs or by
a shift in intracellular or intramitochondrial redox status.34
The present findings could have significant clinical importance. Although sensitivity to oxidant-induced apoptosis by a
FIGURE 8. Effects of antagonistic anti-Fas antibody ZB4 on tBH-induced apoptosis. hRPE cells were pretreated with 125 ng/ml ZB4 for 1
hour and then treated with 300 ␮M tBH for 4 hours. Apoptosis was
assessed by examining PS externalization, as described in Figure 4.
Single-parameter histograms show fluorescein intensity of annexin
V-FITC, a measure of externalization of PS, plotted against cell number.
tBH treatment caused an increase in the number of cells with PS
exposure, and this was partially inhibited by pretreatment of cells with
an antagonistic anti-Fas antibody ZB4. Data presented are representative of five independent experiments.
mitochondrial mechanism could increase because of life-long
accumulation of genome damage in the mitochondrial DNA,
this sensitivity may not be readily reversible by antioxidant
treatments.34 However, the Fas-mediated pathway appears to
be potentiated by increased FasL and Fas expression, and this
could be suppressed by antioxidants. In vitro studies have
demonstrated that many factors can induce FasL expression
and stimulate release of sFasL. These include T-cell activation,
phagocytosis, viral infection, metalloproteinase activation, and
oxidative stress.14,15,35–38 A number of studies suggest that
oxidative damage in the RPE cells may contribute to the pathology of ARMD.18,24 –27 Therefore, a mechanism for oxidative injury of RPE cells in ARMD may involve upregulation of
FasL in T cells or other cell types. When cells with increased
FasL infiltrate to the subretinal space, they may facilitate oligomerization of Fas receptors on RPE cells and kill them. In
healthy, noninflamed eyes, Bruch’s membrane may protect
against cells infiltrating from the choriocapillaris.1 However,
Bruch’s membrane function may be impaired in inflamed eyes.
Moreover, T cells and monocyte/macrophages can also enter
the subretinal space adjacent to the RPE cells through retinal
capillaries. Indeed, the presence of T cells in the subretinal
space has been found in the rat with experimental autoimmune uveitis.39 In exudative ARMD, a choroidal neovascular
membrane grows under or through the retinal pigment epithelium through breaks in Brush’s membrane.2 The endothelial
cells of this neovascular net lack tight junctions, and therefore
fluid and blood leak into the subpigment epithelial layer of the
retina.40 This could attract more cells that express high FasL,
such as activated T cells and activated macrophages, allow
access to FasL-bearing endothelial cells, or exposure to circulating sFasL and thereby cause death of RPE cells. Thus, if
Fas-mediated apoptosis contributes to pathogenesis of ARMD,
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Jiang et al.
it may be involved both in the initiation and progression of the
disease.
Although there are no data showing an increase of sFasL
in the sera of ARMD patients, an elevation of sFasL has been
observed in several diseases including large granular lymphocytic leukemia, natural killer cell lymphoma, hemophagocytic syndrome, Diamond–Blackfan anemia, and hepatitis.41– 43 Therefore, besides the infiltrating cells that express
high FasL, a high level of serum sFasL could also facilitate Fas
ligation in Fas-expressing RPE cells and cause death of the
RPE cells.
Our present study shows that oxidative stress also caused
increases in the expression levels of FasL and Fas in RPE cells
themselves, and pretreatment of RPE cells with antioxidants
GSH and NAC can inhibit oxidant-induced FasL and Fas expression and prevent apoptosis. Therefore, oxidative stress, in
addition to its possible role in elevating FasL expression in T
cells or macrophages or serum sFasL level, also directly upregulates FasL and Fas expression in RPE cells and triggers
autocrine and paracrine death of RPE cells. Because we did not
perform the measurement of sFasL in the culture medium of
tBH-treated cells, we do not know whether RPE-derived sFasL
is involved in oxidant-induced apoptosis in our system. However, the inhibition of sFasL-induced apoptosis in RPE cells by
antioxidants (S. Jiang, M-W. H. Wu, P. Sternberg, and D. P.
Jones, unpublished data, October 1998) indicates that oxidants
have an additional role—that is, they act as an activator of the
Fas-mediated signaling pathway.
The roles of FasL and Fas in RPE cells may not be restricted
to causing death of RPE cells. A recent study by Jorgensen et
al.44 has shown that hRPE cells can induce apoptosis in several
T cell lines and human peripheral T cells through FasL and Fas
interaction. It suggests the FasL expressed in RPE cells may
have an important role in maintaining immune privilege of the
posterior of the eye. In healthy eyes, when the resting T cells
that do not express FasL or activated T cells that express low
FasL infiltrate the subretinal space, the overall interaction of
FasL and Fas between T cells and RPE cells may activate a signal
that travels from the FasL of RPE cells to the Fas of T cells and
cause the death of the T cells. This is quite possible, because T
cells are much more sensitive than RPE cells to Fas-mediated
apoptosis. Therefore, although the FasL expressed in RPE is
insufficient to cause suicide or fratricide, it is sufficient to
trigger apoptosis in infiltrating T cells and thus to ensure
immune privilege of the eye. However, in inflamed eyes, the T
cells that infiltrate to the subretinal space are activated and
have very high FasL expression. Under this situation, the communication between T cells and RPE may then trigger a death
signal that travels from the FasL of T cells to the Fas of RPE
cells, before a counterattack signal from RPE cells to T cells can
be activated.
In conclusion, the present study demonstrates that Fas
mediates apoptosis in RPE cells and that this mechanism may
contribute to oxidant-induced apoptosis. Oxidants can induce
expression of FasL and Fas in RPE cells, and antioxidant thiols
can block this increased expression and associated apoptosis in
RPE. Although incomplete inhibition by a blocking antibody
indicates that other triggering mechanism(s) also occur, the
data suggest that Fas-mediated apoptosis may be relevant to the
death of RPE cells such as occurs in the pathogenesis of ARMD.
IOVS, March 2000, Vol. 41, No. 3
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