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Induces Apoptosis and Expression of Inflammation-Related Proteins in Chang Conjunctival Cells
1 From the Services d’Ophthalmologie et 3 d’Immunohématologie, Hôpital Ambroise Paré, AP-HP, Université René Descartes Paris V, Boulogne, France; and 2 Laboratoire de Biologie Cellulaire, Institut National de la Santé et de la Recherche Médicale U327, Faculté de Médecine Xavier Bichat, Université Paris VII, France.
Abstract
PURPOSE. The purpose of this study was to investigate the effect of
interferon (IFN)
on cell viability, cell growth, and apoptosis and
on expression of apoptotic and inflammation-related proteins in
epithelial conjunctival cells in vitro. Some aspects of transduction
pathways of IFN-induced alterations were also investigated,
especially the role of protein kinase C (PKC) and IFN
transcriptional factor STAT1.
METHODS. A human conjunctival cell line was treated with different
concentrations (30 and 300 U/ml) of human recombinant IFN. After 24,
48, and 72 hours of treatment, cell viability and relative cell number
were studied with 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium
bromide (MTT) and crystal violet colorimetric assays. The apoptotic
process was sought by phase-contrast microscopy,
4',6'-diamidino-2-phenylindole dihydrochloride (DAPI) staining, and
transmission electron microscopy and was confirmed by DNA
electrophoresis and immunoblotting of poly(ADP-ribose) polymerase
(PARP). The cell cycle and expression of apoptotic proteins Fas, bax,
and p53; of inflammation-related proteins HLA-DR and intercellular
adhesion molecule (ICAM)-1; and of IFN signal-transducing factor
STAT1 were evaluated by flow cytometry and/or western blot analysis. To
investigate PKC-related transduction pathways, two PKC modulators,
12-O-tetradecanoyl-phorbol-13-acetate (TPA) and staurosporine,
were applied for 3 hours, followed by IFN treatment for 72 hours.
Moreover, the effects of PKC depletion were studied after a 24-hour
application of TPA, also followed by IFN treatment for 72 hours.
Then, Fas, ICAM-1, and HLA-DR expressions were studied by flow
cytometry.
RESULTS. IFN at 30 U/ml induced no change in cell cycle and in cell
viability. Cell viability significantly decreased after 48 hours of
treatment with 300 U/ml IFN, associated with cell cycle alterations
(decrease in number of cells in the S–M phase), apoptotic chromatin
condensation and fragmentation, ladder pattern on DNA electrophoresis
assay, and cleavage of PARP. Moreover, IFN-treated cells
overexpressed plasma membrane Fas, HLA-DR, and ICAM-1 in a dose- and
time-dependent manner, and STAT1 in both nuclear and cytosolic cell
fractions. Only 300 U/ml IFN-treated cells overexpressed bax,
whereas Bcl-2 and p53 proteins were not modified. HLA-DR and Fas were
upregulated after addition of staurosporine or after PKC-depleting
treatment and repressed with TPA. Staurosporine, PKC depletion, and TPA
all enhanced ICAM-1 expression.
CONCLUSIONS. In our model, IFN induced expression of inflammatory molecules
and apoptotic mediators, cell growth arrest, and apoptosis of Chang
conjunctival cells. Moreover, our results suggest that activation of
PKC is not involved in some IFN cellular effects that possibly imply
the upregulation and nuclear translocation of STAT1. IFN-induced
apoptosis could explain in part the recently reported coexistence of
inflammation and programmed cell death in ocular surface inflammatory
disorders such as Sjögren’s syndrome.
Apoptosis is one of the forms of cell death which, in opposition
to necrosis, can be induced by specific stimuli (such as an interaction
between death receptors and their ligands) and engages well-defined
signal transduction pathways and effector mechanisms.1
2
It is a genetically programmed process, marked by cytoplasm shrinkage,
membrane blebbing, chromatin condensation, and DNA fragmentation. Three
families of genes and their products play a crucial role in induction
and control of apoptosis.3
4
These are the death receptor
family (Fas receptor and its ligand and tumor necrosis factor (TNF)-
receptors rTNF and TNF), the Bcl-2 family, and the family of
caspases. The Bcl-2 family comprises two groups of proteins that either
protect (Bcl-2, bcl-Xl) or sensibilize (bax, bak, bad) the cell to
undergo apoptosis. Caspases are cysteine proteases that act in cascade
in initiating and executing programmed cell death.
IFN, a 25-kDa glycoprotein, plays a crucial role in vivo in
many different types of immune responses, such as delayed-type
hypersensitivity, inflammation, or graft rejection5
6
and
is involved in pathogenesis of inflammatory diseases, for instance of
Sjögren’s syndrome.7
8
9
10
This cytokine is secreted
exclusively by T cells (cytotoxic and Th1) and natural killer cells. It
induces antiviral6
and antiproliferative
activities,11
stimulates macrophages,11
and
controls the expression of several adhesion molecules and surface cell
receptors (e.g., intercellular adhesion molecule [ICAM]-1, Fas
antigen),12
13
14
of several cytokines and of major
histocompatibility complex (MHC) class I and II
molecules.15
16
Class II MHC antigens such as HLA-DR play
a crucial role in the initiation of immune responses. Their expression
by epithelial cells may enable them to act as antigen-presenting cells
and to interact with helper T lymphocytes in immune processes. ICAM-1
is one of the accessory molecules essential for communication between
lymphocytes and other cells (e.g., epithelial cells) and for control of
leukocyte migration and adhesion to different target tissues in
inflammatory process.
In vitro, IFN is also among the earliest polypeptides
found to inhibit growth and proliferation in cultured
cells.17
18
Recently, its proapoptotic action has been
demonstrated in some tumoral cell lines.18
19
20
Both its apoptotic and immune effects depend on IFN-induced
cellular transcriptional changes. Molecular mechanisms of IFN signal
transduction imply the activation, by phosphorylation on tyrosine, of
specific latent transcriptional factors or STATs (signal transducers
and activators of transcription), which induce activation or repression
of numerous genes.21
22
The protein tyrosine kinase–STAT
pathway has been shown to be critical for IFN-induced expression of
ICAM-1,23
for growth arrest, and for
apoptosis.17
24
25
Moreover, IFN was reported to
stimulate the PKC-related pathway in many cellular
systems,26
27
28
29
and this action was reported to be
mandatory in the cytokine-mediated upregulation of
ICAM-1.30
31
32
PKC activation was also shown to participate
in IFN-induced expression of HLA-DR and Fas antigen in some cellular
systems.29
33
34
In the eye, IFN levels are increased in some inflammatory ocular
surface disorders, such as corneal allograft
rejection35
36
or Sjögren’s
syndrome.37
38
39
Moreover, in Sjögren’s syndrome,
this cytokine is presumed to be one of the principal molecules
responsible for stimulation of salivary, conjunctival, and lacrimal
expression of inflammation-related molecules including HLA-DR and
ICAM-1, which are constantly upregulated in this disorder and are even
used as markers of the pathologic state in clinical
practice.38
40
41
42
43
44
In addition to inflammation, apoptotic
changes were shown in glandular and ocular surface tissues of patients
with Sjögren’s syndrome and in Sjögren-like animal
models,42
45
46
and their dependence on lymphocytic
cytokines such as IFN seems most likely. Apoptosis and inflammation
are both resolved with appropriate causal treatment, such as
cyclosporin A,46
a fact that could argue for the common
origin of both processes.
We studied the inflammatory and apoptotic potentials of IFN in a
human continuous conjunctival cell line. The concentrations of IFN
that we applied were 10 times higher than those detected in human serum
or in different tissues in normal or pathologic
states,47
48
49
50
but they were comparable to the
concentrations of IFN secreted by normal human peripheral blood
mononuclear cells in in vitro conditions.51
We used the
inflammatory markers HLA-DR and ICAM-1 as proof of efficiency of IFN
inflammatory stimulation. We thus showed that the proapoptotic action
of IFN accompanies expression of inflammatory molecules and thus
could help to explain the coexistence of inflammation and apoptosis in
ocular surface disorders in human tissues in Sjögren’s syndrome.
Materials and Methods
Reagents
Eagle’s minimum essential medium, fetal calf serum, and
trypsin-EDTA were purchased from Gibco (Paisley, Scotland); human
recombinant IFN from Pepro Tech (Rocky Hill, NJ); and
12-O-tetradecanoyl-phorbol-13-acetate (TPA), staurosporine,
crystal violet, 4',6'-diamidino-2-phenylindole dihydrochloride (DAPI),
and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT)
from Sigma (St. Louis, MO). Antibodies specific for the following human
antigens were used: anti-Fas (UB2, fluorescein isothiocyanate [FITC
]-conjugated; Immunotech, Marseille, France), anti-p53 (DO7, purified
and FITC-conjugated; Pharmingen, San Diego, CA), anti-Bcl-2 (N-19,
purified; Pharmingen), anti-bax (4F11, purified; Immunotech),
anti-poly(ADP-ribose) polymerase (PARP; C2-10, purified; Pharmingen),
anti-STAT1 (purified; Transduction Laboratories, Lexington, KY),
anti-HLA-DR (Immu-357, FITC-conjugated; Immunotech), and anti-ICAM-1
(6.5B5, purified; Dako, Trappes, France). Control antibodies (mouse
FITC-conjugated IgG1 and IgG2
, mouse phycoerythrin
(PE)-conjugated IgG1) were purchased from Immunotech. Staining
solutions for cell cycle (DNA Prep Stain) were from Coulter (Miami,
FL).
Conjunctival Cell Line Culture
A human conjunctival cell line (Wong–Kilbourne derivative
of Chang conjunctiva, clone 1-5c-4, ATCC CCL-20.2) was cultured under
standard conditions (5% CO2-95% humidified air,
37°C) in Eagle’s minimal essential medium supplemented with 10%
fetal calf serum, 2 mM L-glutamine, 50 mg/ml streptomycin,
and 50 IU/ml penicillin. Cells were plated at a density of 10,000
cells/well in 96-well plates (Falcon, Becton Dickinson Labware,
Plymouth, UK) for MTT and crystal violet assays. Cells were plated in
75-cm2 flasks (Falcon) for flow cytometry,
western blot analysis, DNA electrophoresis, and transmission electron
microscopy and on 20-mm2 permanox chamber slide
systems (Laboratory-Tek; Nalge Nunc, Naperville, IL), 25,000
cells/chamber, for other morphologic studies. Cells were treated with
IFN at least 24 hours after the passage (1:4 split ratio at
confluence).
IFN Treatment
IFN was dissolved in culture medium at concentrations of 30 and
300 U/ml.
TPA and Staurosporine Treatments
TPA, similar to other tumor-promoting phorbol esters, at first
activates, but then depletes cells of PKC during prolonged treatment.
Cells were treated for 3 hours with 10 ng/ml TPA (PKC-stimulating
action) and 50 µM staurosporine (a PKC inhibitor), followed by a
300-U/ml IFN treatment for 72 hours. The effects of PKC depletion
were studied after a 24-hour application of 10 ng/ml TPA, followed by a
300-U/ml IFN treatment for 72 hours. After these periods,
expressions of Fas, HLA-DR, and ICAM-1 were studied by flow cytometry.
Cell Viability and Cell Number Assays
Assays were conducted using 96-well microtiter plates. At 24, 48,
and 72 hours of IFN treatment, cell viability was assessed with MTT
assay, as described previously.52
MTT is bioreduced in
metabolically active cells into a colored formazan product insoluble in
tissue culture medium. At times indicated previously, 5 mg/ml MTT
solution was added to the culture medium (10 µl per 100 µl of
medium), and plates were incubated at 37°C for 4 hours. After this
period, the liquid was carefully discarded. Acid-isopropanol (0.04 N
HCl in isopropanol) was added (100 µl/well) and mixed thoroughly to
dissolve all formazan crystals. Plates were then rapidly read on an
enzyme-linked immunosorbent assay (ELISA) multiwell plate reader (iEMS
Reader; Labsystems, Helsinki, Finland) at 570nm.
To determine relative cell number, cells were stained with crystal
violet at 24, 48, and 72 hours of IFN treatment, as described
previously.53
Briefly, the cells were rinsed twice with
sterile phosphate-buffered saline (PBS; pH 7.4) and then fixed in 70%
cold ethanol for 10 minutes at room temperature; 100 µl/well of 0.5%
crystal violet solution was added. The relative cell number was
determined by eluting the dye from stained cells with 33% acetic acid,
and absorbance was measured at 540 nm on an ELISA multiwell reader.
In both experiments, absorbance was expressed as a percentage of control values. The background absorbance was determined on wells without cells, but containing the dye solution. At each time point, cell viability or relative cell number values were the mean of three to six determinations.
Nuclear DNA Isolation and Electrophoresis
After 72 hours of treatment with 300 U/ml IFN, DNA was isolated
from adherent cells cultured in 75-cm2 flasks by
a proteinase K–phenol method, as previously described.54
DNA samples were treated with 50 µg/ml DNase-free RNase, extracted
twice with phenol/chloroform, precipitated with ethanol, and dissolved
in 10 mM Tris-HCl (pH 7.6) and 1 mM EDTA. DNA samples (10 µg) were
fractionated by electrophoresis on 1% agarose gels and visualized by
staining with ethidium bromide (0.5 µg/ml).
Morphologic Procedures
Phase-Contrast Microscopy.
Treated cells were observed after 24, 48, and 72 hours of treatment
with 30 and 300 U/ml IFN.
Nuclear Staining.
Cells were processed for DAPI staining after 48 and 72 hours of 30- and
300-U/ml IFN treatment. Cells cultured on chamber slides and
supernatants were rinsed twice with PBS, fixed, and permeabilized for
10 minutes in ice-cold 70% ethanol, then washed in PBS and stained
with DAPI at a concentration of 0.5 mg/ml for 5 minutes at room
temperature. After staining, the slides were washed extensively and
mounted (Quantafluor Mounting Medium; Kallestad, Chaska, MN) before
examination. A Leica DML light microscope (Leica, Heildelberg,
Germany) was used for visualization. Morphologic analysis was performed
in a masked manner by the same investigator during the whole
experimental procedure.
Transmission Electron Microscopy.
After 72 hours of 300-U/ml IFN treatment, cells cultured in
75-cm2 flasks were harvested in PBS by gentle
scraping and pelleted by centrifugation. The cells were fixed in 2.5%
buffered glutaraldehyde for 1 hour at 4°C, rinsed in PBS, postfixed
with 1% osmium tetroxide for 2 hours at room temperature, and then
dehydrated in a graded ethanol series, followed by embedding of the
cell pellets in Epon 812. Ultrathin sections were stained with uranyl
acetate and lead citrate and examined with an electron microscope
(JEOL, Tokyo, Japan) operating at 80 kV.
Flow Cytometry
All measurements were performed on a flow cytometer(FACScan;
Becton Dickinson, Mountain View, CA) equipped with an argon laser
emitting at 488 nm, using software (Lysis II, Becton,
Dickinson) for data analysis. Forward-scatter and side-scatter,
FITC fluorescence (FL1, 525 nm band pass), and propidium iodide
fluorescence (FL3, 630 nm band pass) were measured. At least 10,000
events were collected per sample. The flow cytometry data were reported
as mean fluorescence intensities.
Expression of Inflammation and Apoptosis-Related Proteins.
Besides two principal tested IFN concentrations (30 and 300 U/ml),
we introduced an intermediate concentration at 150 U/ml to study the
dose-dependence of protein expressions.
For flow cytometric analysis of Fas, HLA-DR, and ICAM-1 expression,
cells were harvested with trypsin-EDTA, pelleted, washed twice in PBS,
and incubated for 30 minutes with FITC-conjugated anti-Fas,
FITC-conjugated anti-HLA-DR, or purified anti-ICAM-1 antibodies, with
FITC-conjugated and purified mouse IgG1 as negative controls. After
incubation with anti-ICAM-1 antibody and purified mouse IgG1, cells
were washed twice and pelleted, and a secondary antibody,
FITC-conjugated goat anti-mouse immunoglobulin, was applied for 30
minutes. For p53 and bax labeling, cells were fixed and permeabilized
for 5 minutes with 1% paraformaldehyde in PBS, followed by 100% cold
methanol (10 minutes at -20°C).55
Then labeling was
performed with FITC-conjugated anti-p53 antibody and FITC-conjugated
mouse IgG2 as a negative control. Bax labeling was performed with
purified anti-Bax antibody and FITC-conjugated goat anti-mouse
antibody. Purified mouse IgG2 was used as a negative control. The
results are presented in flow cytometric tracings, graphs, or bar
charts.
DNA Content Analysis.
After 72 hours of 300-U/ml IFN treatment, cells were trypsinized,
washed with cold PBS, and fixed with 70% ethanol in PBS at -20°C.
After 12 hours, samples were washed with cold PBS and stained (DNA Prep
Stain), containing propidium iodide and RNase III-A, for 30 minutes at
room temperature, according to the manufacturer’s instructions, then
stored in the dark before analysis (within 24 hours) with the flow
cytometer.
Gel Electrophoresis and Western Blot Analysis
Cytosol and nuclei-containing cell extracts were prepared by
lysing cells at 4°C in hypotonic buffer (10 mM HEPES-KOH [pH 7.9],
1.5 mM MgCl2, 10 mM KCl, 1 mM spermidine, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml
aprotinin, 1 µg/ml pepstatin, and 1 µg/ml leupeptin) for 10 minutes
on ice. Lysates were centrifuged for 30 seconds at 500g, and
the cytosol-containing supernatants were separated from the
nuclei-containing pellets. The pellets were resuspended in high-salt
buffer (hypotonic buffer with 20% glycerol and 400 mM NaCl) for 30
minutes on ice and then centrifuged for 2 minutes at
18,000g. The nuclei-containing supernatant was transferred
into tubes (Ependorf, Fremont, CA). The cytosol- and nuclei-containing
samples were fractionated (30 µg protein per lane) by sodium dodecyl
sulfate–polyacrylamide gel electrophoresis and transferred to
nitrocellulose (Protran BA 83; Schleicher & Schuell, Dassel, Germany)
by a semidry transfer procedure (Trans-Blot SD, Bio-Rad, Ivry sur
Seine, France). The nitrocellulose membranes were then incubated for 1
hour in blocking buffer (PBS, containing 0.1% Tween 20 and 5% nonfat
milk powder), rinsed, and incubated for 1 hour with specific antibodies
(anti-STAT1, anti-PARP, anti-bax, anti-Bcl-2, and anti-p53), used as
recommended by the suppliers. Blots were developed using enhanced
chemiluminescence reagent (ECL; Amersham, Arlington Heights, IL).
Statistical Analysis
Flow cytometric results were calculated as arithmetic
means ± SD, and significance values were calculated by means of
the unpaired Student’s t-test with P <
0.05 regarded as significant. Results of colorimetric assays were
calculated as arithmetic means ± SEM, and significance values
were calculated by means of the two-way analysis of variance with
P < 0.05 regarded as significant. All experiments in
this study were performed at least in duplicate.
Results
Cell Viability and Relative Cell Number Assays
Cell viability significantly decreased after 48 hours of treatment
with 300 U/ml IFN (P < 0.01), but it was not
modified by 30 U/ml IFN at any tested time point (Fig. 1
A). A significant decrease (P < 0.01) in relative cell
number was observed after 72 hours of treatment with 300 U/ml IFN
(Fig. 1B)
.
|
after 48 hours of treatment, and this number increased with
time of treatment. Density of adherent cells was decreased when
compared with control (Fig. 2)
. After DAPI staining, the supernatant cells and most of the adherent
cells showed chromatin condensation and fragmentation (Fig. 3)
. Characteristic apoptotic morphology of 300 U/ml IFN-treated cells
with nuclear and cytoplasmic alterations was confirmed by electron
microscopy (Fig. 4)
.
|
|
|
, confirming the
presence of an apoptotic process (Fig. 5)
.
|
, but the level of expression did not vary with
time or with concentrations of IFN. Immunoblotting of PARP showed
the increase of quantity of the native form of 116 kDa in nuclear cell
extracts after 24 hours of treatment with 300 U/ml IFN and its
proteolytic cleavage and the presence of an 85-kDa fragment after 48
hours, but not after 24 hours of treatment with 300 U/ml IFN (Fig. 7) . This cleavage signified the presence of an apoptotic process. As
shown by western blot and flow cytometry, bax was slightly expressed in
untreated cells, was upregulated after 48 hours of treatment with 300
U/ml IFN, and was increased further after 72 hours of treatment
(Figs. 8
A, 8B). Western blot analysis showed no modification of Bcl-2 expression
after IFN treatment at all tested concentrations (Fig. 9
A). As shown by western blot and flow cytometry, the expression of p53
was not modified by treatment with IFN at 30, 150, and 300 U/ml
(Figs. 9B
9C)
.
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|
concentrations after
24 hours of treatment. (Fig. 10
A, 10B). HLA-DR was negative at the basal level and became positive
after 48 hours of treatment (Figs. 11
A, 11B). Fas was expressed at a low level in nontreated cells. The
upregulation of expression was observed with all tested concentrations
of IFN after 24 hours of treatment (Fig. 12) . The intensity of expression of these three proteins increased with
time of treatment and concentrations of IFN.
|
|
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treatment, staurosporine and PKC depletion
potentiated an IFN-induced increase in Fas (Fig. 13B)
, ICAM-1 (Fig. 14B)
, and HLA-DR (Fig. 15)
expressions.
|
|
|
treatment, TPA induced
reduction in expressions of Fas (Fig. 13B)
and HLA-DR (Fig. 15)
, when
compared with cells treated with 300 U/ml IFN alone, but potentiated
IFN-induced upregulation of ICAM-1 (Fig. 14B)
.
Therefore, all results considered, the PKC stimulator TPA had a
negative effect on HLA-DR and Fas expression. The PKC inhibitor
staurosporine and PKC-depleting treatment enhanced the expression of
Fas, when applied alone, and potentiated IFN-induced stimulation of
HLA-DR and Fas. PKC inhibitors and stimulator, all enhanced the basal
expression of ICAM-1 and IFN-induced upregulation of this protein.
Cell Cycle Alterations
DNA content analysis showed alterations of cell cycle after 48
hours of treatment with 300 U/ml IFN. A significant reduction in
number of cells in the S–M phase was observed, and this reduction
amplified with time of treatment, as shown in Figure 16
. There was no modification in cell cycle in 30 U/ml IFN-treated
cells (data not shown).
|
Our data show that in a human conjunctival cell line IFN at 300
U/ml induced both apoptosis and expression of inflammatory markers.
Furthermore, we observed cell cycle alterations involving a reduction
in number of cells in the S–M proliferative phase and a consequent
cell growth arrest. Cell cycle alterations and IFN inhibition of the
G1 transit into the S phase has been reported in some in vitro and in
vivo models.56
57
Besides the determinant action of
STAT1,17
58
growth inhibition can involve other parallel
intracellular steps. Some of them are reported in literature, such as
downregulation of c-myc59
ori cyclin
A,60
hypophosphorylation of the retinoblastoma gene
product,60
and inhibition of cyclin-dependent kinase 2
(CDK2).61
IFN-induced apoptosis has been shown recently in some tumor cell
lines.18
19
20
Similar to cell growth arrest, the role of
STAT1 also seems to be crucial in this IFN-mediated process and in
apoptosis induced by other proapoptotic stimuli such as actinomycin D
or TNF.62
In fact, recent data show that TNF-treated
U3A cells deficient in STAT1 cannot undergo apoptosis because of the
absence of constitutive expression of caspases.62
Therefore, STAT1 seems to play the role of a proapoptotic mediator, per
se.63
64
Additionally, some STAT1-transactivated gene
products such as Bcl-2 family members (proapoptotic bax, bak) or the
Fas receptor and STAT-induced growth arrest can also participate in a
cell’s increased susceptibility to programmed cell death after IFN
treatment.19
65
In our model, upregulation of Fas;
alteration of the balance between anti- and proapoptotic factors Bcl-2
and bax (Bcl-2 was not modified, whereas bax expression was stimulated
by IFN treatment); activation of caspases, evidenced indirectly by
cleavage of their substrate PARP; and cell growth arrest could explain,
at least in part, the proapoptotic action of IFN. That we observed
upregulation of STAT1 with all tested concentrations of IFN does not
contradict the hypothesis of its important role in the induction of
apoptosis (observed only after 300-U/ml IFN treatment). In fact,
western blot analysis is only a semiquantitative method, and small
differences of protein levels cannot be detected. We did not test the
activity and DNA binding capacity of STAT1, however, which could be
decisive in induced effects. Moreover, it cannot be excluded that in
our system, IFN-induced growth arrest and apoptosis result from the
combination of activation of STAT1 and modification of other
transduction factor activity, such as that of IFN-regulating factor 1
or NF-B, which can be modulated by IFN treatment. Moreover, as in
the case of other intracellular molecules, the apoptotic process can
lead to the cleavage of STAT1 by caspases,66
which could
explain the absence of increase in apoptosis after 300-U/ml IFN
treatment.
In our model, cell exposure to the proapoptotic agent IFN induced
the early activation of the DNA repair enzyme PARP, evidenced by the
increase in PARP in nuclear cell extracts after 24 hours of treatment
with IFN. The later proteolytic cleavage of PARP occurred only after
48 hours of treatment, signaled by the appearance of an 85-kDa
fragment. The role of the activation of PARP during the apoptotic
process still remains unclear. PARP inhibition in human T cells or
lymphoblasts decreases apoptosis induced by different triggers such as
alkylating agents.67
68
In contrast, PARP-deficient
primary bone marrow cells are extremely sensitive to apoptosis induced
by DNA-damaging stimuli. After transfection of these PARP -/- cells,
the expression of the wild-type PARP or of an uncleavable PARP mutant
significantly delays cell death.69
Furthermore, a product
of PARP cleavage was suggested to bind irreversibly to broken DNA ends,
blocking the access of repair enzymes to DNA strand
breaks.70
Thus, PARP activation seems to be induced to
protect the cell from DNA damage, but the cleavage of the increased
amount of PARP accelerates the apoptotic process and leads to its
irreversibility. In opposition, the susceptibility to apoptosis of
PARP-deficient mouse thymocytes, hepatocytes, or neurons is not
modified in comparison with the wild-type cells, and PARP activation in
these systems seems not to be crucial during programmed cell
death.71
Thus, further investigations involving PARP
inhibitors are necessary to increase understanding of the role of PARP
in our model of Chang conjunctival cells.
Although cell cycle arrest was observed in our model, IFN-induced
apoptosis seemed to be p53-independent. In fact, p53 did not vary (in
western blot and flow cytometry analysis) after 72 hours of 300-U/ml
IFN treatment, whereas cells underwent apoptosis. Similarly,
IFN-induced apoptosis was reported to be p53-independent in other
epithelial cell systems.18
65
The signal transduction pathways of IFN inflammatory effects
(upregulation of MHC II and ICAM-1) vary between different cell
systems. The role of PKC in generation of these changes seems important
but rather controversial and seems also to depend closely on the cell
type.16
30
33
72
73
IFN was shown to induce activation
and membrane translocation of PKC in several in vitro cellular systems
such as murine macrophages, human glioma and retinoblastoma cell lines,
rat astrocytes, and other models.26
31
74
75
PKC
activation has often been reported as an inducer of
ICAM-1,23
32
72
whereas its effects on HLA-DR are less
constant.33
Furthermore, PKC is reported to stimulate Fas
expression in several murine and human tumor cell lines (especially in
lymphocyte-derived cell lines), most likely by activation of the newly
identified Fas-regulatory genes, the IPL gene,
the murine TDAG51, or its human homologue
TSSC3.34
76
77
Therefore, we investigated
whether the PKC pathway may participate in IFN-stimulatory effects
on ICAM-1, HLA-DR, and Fas expression in the conjunctival cell line.
IFN alone induced a very rapid increase of ICAM-1 expression in a
dose- and time-dependent manner. As expected, the application of 10
ng/ml TPA, a PKC activator, elicited an increase of the steady state
ICAM-1 level and significantly enhanced IFN stimulation of ICAM-1.
However, surprisingly, the PKC inhibitor staurosporine and the
PKC-depleting treatment not only had no negative effect on the
IFN-mediated response but also enhanced IFN-induced upregulation
of ICAM-1. Thus, our first conclusion was that as far as ICAM-1 was
concerned, the IFN signal was independent of PKC activation and that
the cytokine probably acts by stimulation of other intracellular
secondary messengers. Recent data have shown that IFN-induced
expression of ICAM-1 relies on a tyrosine kinase–dependent mechanism
distinct from the PKC pathway activated by TPA.72
Moreover, the ICAM-1 gene promoter possesses several consensus sites
that are important for regulating gene expression, such as phorbol
ester–responsive element (implied in the interaction with TPA),
IFN-responsive element (gamma-activating sequence GAS), and NF-B
motif, which enable concurrent intervention of multiple modulators of
ICAM-1 expression.78
79
In fact, IFN-dependent
transcriptional factor STAT1 and its interaction with the sequence GAS
seems to play an important role in ICAM-1 expression in epithelial cell
models in vitro, because STAT1-dominant negative mutants or a
STAT1-deficient cell line U3A fail to upregulate ICAM-1 after IFN
stimulation.23
Our subsequent investigations concerned Fas and HLA-DR. In our
cell line Fas was expressed at low constitutive levels, and
IFN-induced upregulation was observed after 24 hours of treatment.
HLA-DR was constitutively negative, and IFN stimulated its
expression in a dose- and time-dependent manner, with kinetics less
rapid than that of Fas or ICAM-1. The PKC inhibitor staurosporine and
PKC depletion both enhanced IFN-induced HLA-DR and Fas upregulation,
whereas TPA, a PKC activator, had an antagonist effect and tended to
reduce IFN-stimulatory effects. Thus, the PKC activation was
inhibitory for HLA-DR and Fas induction in our system. IFN-positive
effects on HLA-DR and Fas expressions in our cell model confirmed that
the PKC pathway, even if present, was not predominant after IFN
stimulation. Consequently, we conclude that transduction pathways other
than those that are PKC-related, probably those of tyrosine kinases and
STAT1, controlled the effects of IFN in our system. This conclusion
is in agreement with some recent reports of IFN’s effects
independent of PKC in other epithelial cell lines such as the bronchial
epithelial cell line NCl-H292.72
Thus, in our in vitro model of conjunctival cells, Fas, HLA-DR, and
ICAM-1 were all upregulated by the Th1 cytokine IFN. This
observation coincides with the close correlation of intensities of
expressions of these proteins in ocular surface inflammatory disorders.
In fact, some recent data concerned concomitant and correlated
expressions of Fas, HLA-DR, and ICAM-1 in conjunctival cells in
Sjögren’s syndrome and in lacrimal gland acinar cells in
Sjögren-like animal models.44
80
81
Fas and HLA-DR
were also shown to be simultaneously overexpressed in conjunctival
epithelium in lens wear–induced inflammatory disorders and in patients
undergoing topical long-term antiglaucoma treatments containing
preservatives.44
Because the human continuous conjunctival
cell line that we used in this study differs in its characteristics
from normal epithelium, further investigations in vivo or in
first-passage culture of human conjunctival epithelium could be
required to confirm our findings and to allow better extrapolation to
these pathologic states.82
However, the coexistence of apoptosis and inflammation is one of the principal features of Sjögren’s dry eye syndrome, and both processes are improved by cyclosporin A treatment.46 That apoptosis and inflammation are possibly mediated by the same stimuli and transduction pathways opens up new prospects with on the therapeutics of some ocular diseases. Pharmacologic research on antiapoptotic drugs such as caspase inhibitors, very promising in other diseases such as liver or heart failures, in the future also may resolve ocular surface and lacrimal gland inflammatory disorders.
Acknowledgements
The authors thank Annie–France Bringuier and Alain Moreau for their excellent technical assistance.
Footnotes
Submitted for publication December 21, 1998; revised March 29, 1999; accepted April 12, 1999.
Proprietary interest category: N.
Corresponding author: Christophe Baudouin, Service d’Ophtalmologie, Ambroise Paré Hospital, 9 Avenue Charles de Gaulle, 92104 Boulogne Cedex France. E-mail: arepo@worldnet.fr
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