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1 From the Department of Ophthalmology and Visual Science, Department of Pathology, and 2 The Committee on Immunology, University of Chicago, Illinois.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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METHODS. Pure HFRPE cells were isolated and cultured. The supernatants of both
nonactivated and IFN-
–activated HFRPE cells were isolated. Cells
from the human T-cell line Jkt were incubated either in standard
culture medium or in the supernatant isolated from HFRPE cells. In the
first assay Jkt cell proliferation was measured by
[3H]thymidine incorporation. In the second assay Jkt cell
apoptosis was examined for annexin V staining by flow cytometry. In the
third assay Jkt cell division was evaluated with carboxyfluorescein
succinimidyl ester (CFSE) fluorescent dye. In the last assay the
mitochondrial transmembrane potential of Jkt cells was measured with
the cationic lipophilic fluorochrome 3,3'-dihexyloxacarbocyanine iodide
[DiOC(6)]. In all the assays the effect of supernatants
isolated from both nonactivated and IFN-–activated HFRPE cells were
compared with standard culture medium. The involvement of antiapoptotic
human gene bcl-xLwas determined
by using a Jkt cell line that was stably transfected with
bcl-xL.
RESULTS. The supernatant isolated from HFRPE cells significantly suppressed the
cell division in Jkt cells and induced apoptosis. These effects were
stronger when the supernatant was isolated from IFN-–activated
HFRPE cells. The apoptosis pathway induced by the secreted product of
HFRPE cells involved the early disruption of mitochondrial
transmembrane potential. Although the overexpression of
bcl-xL gene rescued the Jkt cells
from supernatant-induced apoptosis, it could not restore the
proliferation of Jkt cells.
CONCLUSIONS. These data suggest that HFRPE cells secrete a product that initiates an early cell cycle arrest in the human T-cell line Jkt, which is followed by the activation of an apoptotic pathway that involves the loss of mitochondrial membrane potential. The latter could be prevented by bcl-xL overexpression. Also these data suggest that the HFRPE-induced T-cell apoptosis may play a significant role in maintaining the immune privilege in the subretinal space.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We and others showed that HFRPE cells contributed to the immune
privilege in the subretinal space by induction of apoptosis in human T
cells.8
9
10
The mechanism(s) involved in the
HFRPE-mediated T-cell apoptosis are currently not well established.
However, we showed that the pathway involved in HFRPE-induced apoptosis
in T cells was Fas ligand independent.8
9
We also
demonstrated that PGE2, TGF-ß, IL-10, or
TNF-
were not involved in HFRPE’s inhibition of T-cell activation
and proliferation.9
There are reports suggesting the involvement of both cell–cell contact dependent and independent pathways in the induction of apoptosis by HFRPE cells.8 10 These contradictory findings suggest the possibility that HFRPE cells may induce apoptosis through multiple mechanisms. We previously described studies that focused largely on the characteristics of HFRPE cell-induced T-cell apoptosis in which cell–cell contact was possible.8 9 In light of these findings we performed a more detailed study to evaluate the effect of HFRPE supernatant on human Jurkat (Jkt) T-cell line in a HFRPE-free system.
We showed previously that HFRPE cells can induce apoptosis in Jkt T
cells.8
In many aspects Jkt T cells represent the
phenotype of an activated human primary T cell.11
12
One
of the aims of this study was to further characterize the mechanisms
involved in the HFRPE cell–mediated apoptosis of Jkt cells. We
analyzed therefore the effect of supernatant isolated from HFRPE cells
on the cell division of Jkt cells. We also examined the
supernatant-induced apoptosis to see if it was mediated by an
alteration in the mitochondrial membrane of Jkt cells. Major changes in
the function of mitochondrial membrane are critically important in the
early stages of apoptosis.13
14
A breakdown in the
mitochondrial transmembrane potential
(
m) mediated by opening of the
so-called mitochondrial permeability transition pores or megachannels
marks the point-of-no-return in the apoptotic process.14
It is known that various members of the bcl-2 gene family are actively involved in enhancing or preventing apoptosis, in part by regulating the physical properties of mitochondria. Specifically, the antiapoptotic bcl-2 family member bcl-xL has been shown to help maintain the integrity of mitochondrial membrane.15 We showed previously that bcl-xL overexpression can rescue the HFRPE-mediated apoptosis in Jkt T cells in an assay in which cell–cell contact was possible.8 In this study we investigated whether the overexpression of bcl-xL gene could rescue the supernatant-induced loss of mitochondrial membrane potential and subsequent apoptosis in Jkt cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preparation of HFRPE Supernatant
HFRPE cell supernatants were isolated from either activated or
nonactivated HFRPE cells. HFRPE cells were cultured until they reached
confluency. Activated HFRPE cells were obtained by their incubation
with 1000 U/ml of IFN- (Pharmingen, San Diego, CA). Nonactivated
HFRPE cells were incubated with culture medium without IFN-. After
72 hours the culture medium was discarded, and cultures were washed
twice with PBS without
Ca2+/Mg2+ (BioWhittaker)
and then reincubated with freshly prepared culture medium (without
IFN-) for an additional 48 hours. The supernatants from both
nonactivated and activated HFRPE cells were collected and centrifuged
for 5 minutes at 2000 rpm to separate the remaining cell debris.
Preparation of Jkt Cells
Two different Jkt cell lines were used in this study:
bcl-xL/Jkt or Neo/Jkt cells.
Both cell lines were generously provided by Craig B. Thompson and
Mathew G. Vander Heiden (Department of Medicine, University of
Chicago, Chicago, IL). The bcl-xL/Jkt cell
line was generated by the stable transfection of Jkt cells with both
the neomycin-resistant gene and the bcl-xL
antiapoptosis human gene (bcl-xL/Jkt). The
Neo/Jkt cell line, which served as control, was only
transfected with the neomycin-resistant gene (Neo/Jkt). Both
Jkt cell lines are type II cells, as defined by their ability to
prevent Fas-mediated apoptosis by overexpression of the antiapoptotic
bcl-2 family member
bcl-xL.15
16
The Jkt cells
were cultured in standard culture medium containing RPMI 1640
(BioWhittaker) supplemented with 20 mM glutamine (BioWhittaker), 10%
heat inactivated fetal calf serum (GIBCO, Life Technologies, Grand
Island, NY), 100 IU/ml penicillin, 100 µg/ml streptomycin
(BioWhittaker), and 20 mM HEPES (BioWhittaker). The cultures were
incubated at 37°C in a humidified mixture of 95% air and 5%
CO2. For selection purposes 0.5 mg/ml of G418
(GIBCO, Life Technologies) was added to the cultures.
Jkt Cell Proliferation Assay.
In this assay the rescue effect of bcl-xL
overexpression on supernatant-induced (supernatant isolated from either
activated or nonactivated HFRPE cells) apoptosis was analyzed by
measuring the proliferation of Jkt cells. Supernatants were isolated
from both nonactivated or IFN-–activated HFRPE cells (see above).
The bcl-xL/Jkt or Neo/Jkt cells
(serving as control) were suspended either in the isolated supernatants
or in standard culture medium (see above) in flat-bottomed
96-well-plate tissue culture clusters (Costar, Cambridge, MA). After an
incubation period of 48 hours, the cultures were pulsed with a
[3H]thymidine solution (5 mCi/ml, 2 mCi/mmol
specific activity; New England Nuclear, Boston, MA) for 24 hours before
harvesting on glass fiber filters. Incorporation of the radioactive
label was measured by liquid scintillation counting and was expressed
as the arithmetic mean of counts per minute (cpm) of triplicate
cultures. In Figure 1
the mean and SD counts per minute of for the two cell lines in standard
media was 415,000 ± 33,889 cpm. The 100% on the
y-axis in Figure 1 indicates the proliferation of either
bcl-xL/Jkt or Neo/Jkt cells in
standard culture medium.
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HFRPE cells (as mentioned above).
After 24, 48, 72, or 96 hours of incubation the percentage of cells
stained with annexin V- phycoerythrin (PE; R&D, Minneapolis, MN) was
determined by flow cytometry (FACScan). Annexin V binds to phosphatidyl
serine (exposed on the cell membrane), which is one of the earliest
indicators of cellular apoptosis. Staining procedures were performed
according to the manufacturer’s instructions (R&D).
Cell Division Rate Assay: Carboxyfluorescein Succinimidyl Ester
(CFSE) Staining.
The effect of HFRPE cell supernatant on the cell division of Jkt cells
was evaluated with the CFSE fluorescent dye.17
Carboxy-fluorescein diacetate succinimidyl ester (CFDASE), the
diacetate form of CFSE is a nontoxic chemical that readily diffuses
into cells. After binding to the plasma membrane, its acetyl groups are
cleaved by cellular esterases to generate CFSE, which is an active
fluorophore and absorbs and emits light at a characteristic
wavelength.17
Upon cell division CFSE segregates equally
between daughter cells resulting in the sequential dilution of
fluorescence intensity in successive generations. This dilution of
fluorescence is visualized with flow cytometry, tracking down the
number of cell divisions in proliferating cell populations.
A 5 mM stock solution of CFDASE (Molecular Probes Inc., Eugene, OR) was
prepared in DMSO (Sigma). The bcl-xL/Jkt
or Neo/Jkt cells were incubated in the supernatants isolated
from either nonactivated, IFN-–activated HFRPE cells or standard
culture medium (see above, serving as control). The cells were
subsequently washed in PBS (without
Ca2+/Mg2+) and were
resuspended in PBS at a concentration of 5 x
106 cells/ml. CFDASE stock solution was diluted
at a concentration of 1:1500 in PBS. The cells were gently mixed with
an equal volume of the diluted CFDASE solution for 10 minutes at room
temperature. The labeling process was quenched by adding an equal
volume of fetal calf serum (GIBCO, Life Technologies) to the cell
suspension solution followed by a 1-minute incubation. The cells were
subsequently washed with standard culture medium. The intensity of
fluorescence indicating the number of cell divisions, was visualized by
flow cytometric analysis on a FACScan. The number of cell divisions was
analyzed with the ModFit LT 2.0 software program (Verity Software,
Topsham, ME).
Mitochondrial Membrane Staining.
The cationic lypophilic fluorochrome 3,3'-dihexyloxacarbocyanine iodide
[DiOC(6)] was used to evaluate whether the supernatant-induced
apoptosis was associated with the disruption of transmembrane potential
in mitochondria.18
19
Neo/Jkt or
bcl-xL/Jkt cells were incubated with
supernatants isolated from nonactivated or IFN-–activated HFRPE
cells (as described above) for 12 or 36 hours. The supernatants were
then removed, and the cells were incubated with standard culture medium
containing 40 µM DiOC(6) for 30 minutes. Subsequently, cells were
washed with standard culture medium and immediately analyzed by flow
cytometric analysis with a FACScan.
Statistical Analysis
For Figure 1
the mean cpm of triplicates and for Figures 2
3
4
the absolute number of cells were used for statistical analysis using
a two-factor-with-replication analysis of variance (ANOVA) with
= 0.05. P values 
0.01 were accepted as
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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–activated HFRPE cells
and Jkt cells leads to apoptosis of Jkt cells.8
The
HFRPE-mediated apoptosis was cell–cell contact independent, because
the separation of the two cell types by a transwell insert did not
prevent the induction of apoptosis.8
In the assay
presented here the effect of supernatant isolated from both
nonactivated or IFN-–activated HFRPE cells on the proliferation of
Neo/Jkt or bcl-xL/Jkt cells was
evaluated in a HFRPE-free system. The two Jkt cell lines differed only
in their expression levels of bcl-xL. The
supernatant isolated from nonactivated HFRPE cells suppressed the
proliferation of both Neo/Jkt and
bcl-xL/Jkt cells similarly (P <<
0.01; Fig. 1
). This inhibitory effect was stronger in the supernatants
isolated from IFN-–activated HFRPE cells. It should be noted that
the supernatant isolated from IFN-–activated HFRPE cells did not
contain IFN- as measured by ELISA (data not shown). The filtration of supernatant through a Centripret concentrator 100 filter (Amicon, Grace Company, Beverly, MA), which allows the passage of molecules with a molecular weight of less than 100,000 kDa resulted in a similar inhibitory effect (results not shown). This preliminary characterization of the HFRPE-secreted soluble factor indicated that its molecular weight is likely to be less than 100,000 kDa.
bcl-xL Overexpression Rescued
Supernatant-Mediated Jkt Cell Apoptosis
The role and rate of apoptosis in supernatant-mediated inhibition
of Jkt cell proliferation was assessed with annexin V-PE staining.
bcl-xL/Jkt and Neo/Jkt cells
were incubated with the supernatant isolated from nonactivated or
IFN-–activated HFRPE cells for 24, 48, 72, and 96 hours. Standard
culture medium served as control. The supernatant isolated from HFRPE
cells induced an increased binding of annexin V to Neo/Jkt
cells when compared with the cells incubated with standard medium (Fig. 2A)
. This binding was further increased when Neo/Jkt cells were
incubated over a period of 96 hours with the supernatant isolated from
activated HFRPE cells (P << 0.01; Fig. 2A
). In contrast,
there was only a slight increase in the percentage cells binding
annexin V in the bcl-xL/Jkt cells when
they were treated with the supernatant isolated from either
nonactivated or activated HFRPE cells. The percentage of annexin V
staining in bcl-xL Jkt cells after
treatment with supernatant from either nonactivated or
IFN-–activated HFRPE cells was always significantly lower than the
percentage of annexin V staining of Neo/Jkt cells after the
same treatment (P << 0.01; Fig. 2B
).
These results indicate that the supernatant-induced apoptosis was rescued by bcl-xL overexpression. Unexpectedly, these findings did not correlate with the above-mentioned decrease of [3H]thymidine incorporation in the bcl-xL/Jkt cells that were treated with the HFRPE supernatant (Fig. 1) .
HFRPE Supernatant Induced a Cell Cycle Block in Jkt Cells
In Neo/Jkt cells the supernatant isolated from HFRPE
cells increased the rate of apoptosis and suppressed their
proliferation. These results were inconsistent with the findings in
bcl-xL/Jkt cells. Although the apoptosis
rate was not significantly increased in
bcl-xL/Jkt cells (annexin V assay) their
proliferation was significantly inhibited by the HFRPE supernatant.
This indicated that although bcl-xL
overexpression rescued apoptosis, it did not restore the Jkt cell
proliferation. In this assay we examined whether the
supernatant-mediated inhibition of
bcl-xL/Jkt cell proliferation
was due to a cell cycle arrest rather than apoptosis.
Jkt cells were treated with CFSE before their incubation with HFRPE supernatants. CFSE labeling identifies the progeny of the cells and analyzes the division history of individual cells that have undergone multiple rounds of cell division.17 Upon cell division CFSE segregates equally between the daughter cells, making the visualization of Jkt cell division possible. The viability of cells was assessed with PI staining.
The supernatants isolated from both nonactivated and activated HFRPE cells induced cell death in Neo/Jkt cells over the period of 96 hours (Fig. 3A ). In contrast, the bcl-xL/Jkt cells showed a significant reduction in cell death rate (P << 0.01) and a significantly higher viability after incubation with HFRPE-isolated supernatant (P << 0.01; Fig. 3B ). The CFSE staining, however, indicated a halt in the cell cycle progression similar to the Neo/Jkt cells (Table 1) .
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These findings may explain the supernatant-mediated inhibition of
bcl-xL/Jkt cell proliferation in the
absence of apoptosis. Additionally, it demonstrated that the
supernatant isolated from IFN-–activated HFRPE cells was more
potent in suppressing the cell cycle progression in Jkt cells.
HFRPE Supernatant Disrupted the Mitochondrial Transmembrane
Potential
The disruption of the mitochondrial transmembrane potential is one
of the early features of apoptosis. In this assay we evaluated whether
the supernatant-mediated apoptosis was associated with this disruption.
DiOC(6), a cationic lypophilic fluorochrome, is commonly used for
quantifying the mitochon-drial transmembrane potential
(m).18
19
Jkt cells
were pretreated with this dye and were analyzed by flow cytometry. A
decrease in fluorescein intensity seen as a bimodal distribution in the
flow cytometric analysis correlates with a decrease in
m. Neo/Jkt cells,
which were incubated with the supernatant isolated from
IFN-–activated HFRPE cells, showed a significant decrease in their
m at an early time point (12
hours), as indicated by the percentage of cells with DiOC(6) staining
(Fig. 4)
. This was further decreased at 36 hours of incubation. In contrast,
the bcl-xL/Jkt cells completely overcame
the supernatant-induced decrease in
m (P << 0.01; Fig. 4
). These findings indicated that the supernatant-induced apoptosis was
associated with a very early decrease in mitochondrial transmembrane
potential, which could be rescued by
bcl-xL gene overexpression. These findings
further confirm that at least one of the antiapoptotic effects of
bcl-xL is mediated through the
stabilization of transmembrane potential in mitochondria.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The recent discovery that apoptosis can be induced in anucleated cells implies that cytoplasmatic structures must control the apoptotic process.20 21 22 It has been shown that nuclear apoptosis is preceded by the disruption of the mitochondrial transmembrane potential. This disruption is mediated by the opening of permeability transition pores in the mitochondrial membrane.15 In this study, we showed that at early stages of apoptosis the HFRPE cell supernatant altered the mitochondrial membrane potential of Jkt cells. Further, we showed that the overexpression of bcl-xL gene prevented the disruption of mitochondrial transmembrane potential, leading to the rescue of Jkt cells from apoptosis. The function of bcl-xL as an ion channel in the outer mitochondrial membrane, which prevents its osmotic swelling, may explain the significant resistance of bcl-xL to cellular apoptosis.15 16 20 21 22 23 24 25
Although the apoptotic process was rescued in
bcl-xL/Jkt cells, their proliferation was
not restored in the presence of HFRPE cell supernatant. For a better
understanding of this block of proliferation we performed a cell cycle
analysis, which provided us with detailed information regarding the
different rounds of cellular division. Our results indicated that
the supernatant isolated from HFRPE cells delayed the cell cycle
progression of Jkt cells by one generation. The supernatant isolated
from IFN-–activated HFRPE cells induced an even longer delay (two
generations) in proliferation of Jkt cells. This delay in the cell
cycle progression explains the block of proliferation of Jkt cells
after 48 hours in the absence of apoptosis when the antiapoptotic gene
bcl-xL is overexpressed.
The supernatant isolated from IFN-–activated HFRPE cells possessed
a stronger immune-suppressive effect than the supernatant isolated from
nonactivated HFRPE cells. This may have significant clinical
implications. In vivo, during an inflammatory reaction, the activated T
cells would produce IFN- to further activate the surrounding immune
cells. The presence of IFN- upregulates the immune suppressive
behavior of the RPE cells, which in turn downregulates the inflammatory
process, limiting the spread of destruction and maintaining the immune
privilege state.
In summary, we showed that the supernatant isolated from HFRPE cells suppressed the proliferation of Jkt cells by induction of apoptosis. This is further evidence that HFRPE-induced apoptosis is mediated by soluble factors. The supernatant-induced apoptosis involved the disruption of the mitochondrial transmembrane potential. Although the overexpression of bcl-xL gene rescued the supernatant-mediated apoptosis, it was not capable of restoring the cellular proliferation. This suggests that the HFRPE supernatant-induced cell cycle arrest may precede the induction of apoptosis. RPE cells may therefore play an active role in maintaining the immune privilege in the subretinal space by secreting a factor that leads to cell cycle arrest and apoptosis in T cells.
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Footnotes |
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Submitted for publication April 13, 2000; revised July 6, 2000; accepted July 28, 2000.
Commercial relationships policy: N.
Corresponding author: Lili Farrokh-Siar, Department of Ophthalmology and Visual Science, University of Chicago, 939 East 57th Street, Chicago, IL 60637. lfarrokh{at}midway.uchicago.edu
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-positive, top
half) and viable cells (