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1 From the Wound Healing Research Unit, Institute of Ophthalmology, London, United Kingdom; the 2 Glaucoma Unit, Moorfields Eye Hospital, London, United Kingdom; and the 3 Department of Clinical Immunology, Royal Free Hospital School of Medicine, London, United Kingdom.
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Abstract |
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METHODS. Bcl-2, Bax, Bcl-x, Fas (CD95) and tumor necrosis factor (TNF) receptor
expression was determined by flow cytometry in control and
mitomycin-C–treated Tenon fibroblasts. Fibroblast death was quantified
using a lactate dehydrogenase release assay. The effect of Fas and
TNF-receptor signaling was evaluated using Fas-specific antibodies and
soluble TNF-
.
RESULTS. Tenon fibroblasts constitutively express Bcl-2, Bax, and Bcl-x in culture. Mitomycin-C (0.4 mg/mL) induced a small but consistent increase in the expression of all three proteins. Tenon fibroblasts express low levels of Fas but are resistant to the effects of Fas-receptor ligation. Mitomycin-C (0.01–1.0 mg/mL) led to a significant increase in Fas expression at all concentrations tested (P < 0.01). Pretreatment with mitomycin-C (0.4 mg/mL) rendered fibroblasts susceptible to agonistic anti-Fas monoclonal IgM antibodies (50–500 ng/mL) and led to a further 50% reduction in viable fibroblasts at 48 hours, compared with mitomycin-C alone (P < 0.05). Antibodies that block the Fas receptor did not inhibit mitomycin-C–induced apoptosis.
CONCLUSIONS. Mitomycin-C alters apoptosis gene expression and primes fibroblasts to the effects of Fas receptor ligation. Factors other than the level of Fas receptor expression modulate the response to Fas receptor signaling. Determining the signals that regulate fibroblast apoptosis may help to refine therapeutic strategies for switching off the subconjunctival healing response and maintaining intraocular pressure control.
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Introduction |
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The ability of single, short, intraoperative applications of mitomycin-C to induce long-term inhibition of fibroblast proliferation is well established.4 5 Histologic analysis of mitomycin-C–treated tissue, however, has revealed largely acellular bleb tissue,6 7 suggesting that treatment not only inhibits fibroblast proliferation, but also induces fibroblast death. We have shown previously that clinically relevant treatments with mitomycin-C in vitro induce Tenon capsule fibroblast death by apoptosis.8 Fibroblast apoptosis heralds the termination of a physiological wound-healing response by converting active granulation tissue into relatively inactive scar.9 It is therefore possible that mitomycin-C mediates its long-term inhibition by inducing fibroblast apoptosis and prematurely switching off the healing response.8
The Bcl-2 family of proteins are located on the mitochondrial membrane and either promote (Bax, Bak, Bad) or inhibit apoptosis (Bcl-2, Bcl-XL) through regulating release of cytochrome c from the mitochondria into the cytoplasm.10 11 The Tumor necrosis family of proteins, including the tumor necrosis factor (TNF) receptors and Fas (CD95/APO-1) are located on the plasma membrane. Receptor activation triggers apoptosis in susceptible cells. Fas is an important member of the TNF family, in that receptor ligation mediates apoptotic death in a wide range of physiological and pathologic processes. The role of Fas in chemotherapy-induced apoptosis remains controversial. A number of cytotoxic drugs including mitomycin-C and 5-fluorouracil have been shown to upregulate the expression of cell surface Fas in tumor cell lines.12 13 14 In addition, manipulating Fas receptor signaling may modulate chemotherapy-induced apoptosis.15 16 The role of Fas receptor signaling with respect to mitomycin-C–mediated apoptosis in fibroblasts has not previously been reported.
The purpose of this study was to determine whether Tenon capsule fibroblasts express apoptosis related gene products in culture, ascertain the effect of mitomycin-C treatment on gene expression, and investigate the effect of modulating Fas receptor signaling in mitomycin-C–treated fibroblasts.
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Methods |
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Fas (CD 95/APO-1)-Sensitive Jurkat T-Cells
Fas sensitive human leukemic lymphoblastic Jurkat
T-cells (J6) were provided as a kind gift of Stan
Wickeramesinghe (Department of Hematology, Royal Free Hospital, London,
UK). Jurkat T-cells were maintained in RPMI and 10% FCS and
passaged with a split ratio of 1:20.
Antimetabolite Treatment
Tenon fibroblasts were seeded at concentration of
10,000 fibroblasts per well into 24-well plates (BD Biosciences) and
incubated overnight. The fibroblast monolayers were then washed and
covered with single applications of mitomycin-C (Kyowa Hakko Kogyo,
Ltd., Tokyo, Japan), as described previously.4
Unless
otherwise stated, the treatment time for all experiments was 5 minutes.
Antimetabolites were diluted in phosphate-buffered saline (PBS).
Control fibroblasts were treated with a 5-minute application of PBS.
After treatment, the monolayers were washed immediately three times
with 500 µL PBS and incubated in growth medium.
Analysis of Cell Death
Cell Morphology.
The number of viable fibroblasts was evaluated as previously
described,18
by counting the number of attached fibroblasts at x40 magnification using phase-contrast microscopy
(model OM2; Olympus, Tokyo, Japan). To quantify the proportion of cells
displaying apoptotic morphology, cytospins of attached cells and cells
in supernatant were prepared as previously described.8
Apoptotic cells were easily identified on the basis of nuclear morphology when examined by light microscopy. The percentage of
apoptosis was calculated from the ratio of apoptotic to viable
fibroblasts from randomly selected fields. At least 300 cells were
counted per cytospin at x40 magnification.
Lactate Dehydrogenase (LDH) Release Assays.
A lactate dehydrogenase release assay was also used to quantify cell
death. This commercially available assay quantifies lactate
dehydrogenase released by dead cells into the supernatant. Lactate
dehydrogenase is a stable cytoplasmic enzyme present in all cells. It
is rapidly released after damage to the plasma membrane. Lactate
dehydrogenase catalyzes the reduction of a colorless tetrazolium salt
to colored formazan, which absorbs a broad spectrum of light with
maximum absorbance at approximately 492 nm. Absorbance was measured
with a spectrophotometric microtiter plate reader (Titertek Plus; MTX
Lab Systems, Vienna, VA). Lactate dehydrogenase is present in
serum and can therefore be used only to assay cell death in serum-free
conditions.
Lactate dehydrogenase was measured in the supernatant of
mitomycin-C–treated Tenon capsule fibroblasts, using a cytotoxicity
detection kit (LDH; Roche Molecular Biochemicals, Philadelphia, PA),
according to the manufacturer’s guidelines. Briefly, human Tenon
capsule fibroblasts previously seeded into 24- or 48-well plates (BD
Biosciences) were treated with a single 5-minute application of
mitomycin-C or 5-fluorouracil, as described earlier; washed in PBS; and
incubated in 400 µL phenol red–free DMEM with 1% bovine serum
albumin (Sigma). After 48 hours’ incubation, 100 µL of supernatant
was extracted from each well and placed into separate wells of a
96-well plate (BD Biosciences). Catalyst solution (100 µL at 37°C)
was added to each well, followed by 15 minutes’ incubation. Absorbance
was measured by a microtiter plate reader with a 490- to 492-nm filter.
Background absorbance was measured with wells containing phenol
red–free DMEM only. Low-absorbance control cultures contained
untreated Tenon capsule fibroblasts and high-absorbance control
cultures equating to 100% apoptosis were obtained by lysing
fibroblasts with 100 µL 0.1% Triton-X added to 300 µL DMEM. The
percentage of apoptosis was calculated by
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Flow Cytometry.
Flow cytometric analysis was performed (FACStar-plus; BD Biosciences)
with a 100-mW 488-nm argon laser light source. Light was filtered with
an FL-1 filter at 520 ± 20 nm or an FL-2 filter at
580 ± 20 nm. Acquisition and analysis was performed on computer
(LYSIS II software; BD Biosciences).
Cell Preparation.
For fibroblast staining, unfixed monolayer cells were collected by
trypsinization and collected together with cells in supernatant and
wash solutions. The fibroblast suspension was centrifuged at 1000 rpm
for 5 minutes and resuspended in 100 µL PBS. Non–contact-dependent
cells were washed, centrifuged, and resuspended in 100 µL PBS.
Fas and Bcl-2 Expression by Direct Immunofluorescence.
This single-step staining procedure with fluorochrome-conjugated
antibody was used when possible. Anti Fas-FITC (UB2; 5 µL at a
concentration of 0.5 µg/mL; Immunotech, Marseille, France)
and anti-Bcl-2-FITC (5 µL at a concentration of 0.5 µg/mL; Dako,
Glostrup, Denmark) was added to 100,000 cells in 100 µL PBS, mixed
and then incubated for 15 minutes at room temperature. The cells were
then washed and fixed in 100 µL 1% paraformaldehyde and stored at
4°C in the dark.
Bax and Bcl-x Detection by Indirect Immunofluorescence.
In this two-step procedure, cells were labeled with the unconjugated
first-layer anti-Bax IgG2 (Immunotech) and anti-Bcl-xl/s
polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA),
washed, and subsequently labeled with a second-layer
fluorochrome-conjugated monoclonal antibody directed against the first
layer. The primary antibody was added for 30 minutes at room
temperature. In experiments in which a rabbit polyclonal primary layer
was used, background fluorescence was reduced by a 15-minute incubation
in RPMI containing FCS (10%). After application of the first layer,
fibroblasts were washed and resuspended in 100 µL PBS. Five
microliters of appropriate FITC- or phycoerythrin (PE)-conjugated
second-layer antibody diluted to 0.5 µg/mL in PBS was added for 30
minutes at room temperature. The cells were then washed, fixed in 100
µL 1% paraformaldehyde, and stored at 4°C in the dark.
Cytoplasmic Staining.
Direct or indirect staining techniques were used. Cells (100,000)
suspended in 100 µL PBS were incubated in a fixative (Permeafix;
Ortho Diagnostic Systems, Amersham, UK) diluted 1:1 in water for 45
minutes at room temperature. The cells were then washed and resuspended
in 100 µL PBS and labeled with 5 µL FITC- or PE-conjugated
antibody, or an equivalent volume of unconjugated first-layer antibody
for 30 minutes at room temperature. Cells were then washed and, when
indicated, resuspended in 100 µL PBS and 5 µL FITC- or
PE-conjugated second-layer antibody for 30 minutes at room temperature.
The cells were again washed and fixed in 100 µL 2% paraformaldehyde
in PBS and stored at 4°C.
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Control Experiments |
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Control Antibody.
For indirect immunofluorescence, an isotype control irrelevant
first-layer antibody was used, followed by the standard protocol for
the conjugated second-layer antibody. For one-step labeling with FITC-
or PE-conjugated antibodies, an irrelevant directly conjugated isotype
control was used (e.g., anti-CD 45 RO-FITC, Dako) for fibroblast
staining.
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Analysis |
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TopAbstractIntroductionMethodsControl ExperimentsAnalysisResultsDiscussionReferences |
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Results |
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Bcl-2 Family Expression
Tenon fibroblasts constitutively expressed Bcl-2 in
culture. The median fluorescence intensity (MFI ± SD, expressed
in intensity units) obtained from three separate experiments
performed in triplicate with anti-Bcl-2 FITC was 28.1 ± 2.9
compared with 7.9 ± 4.4, obtained with an
FITC-conjugated IgG isotype control antibody (statistically significant
at P = 0.004, paired t-test). To determine
the effect of mitomycin-C on Bcl-2 expression, fibroblasts were
examined 48 hours after a single 5-minute application of mitomycin-C
(0.4 mg/mL) or PBS (control). Mitomycin-C led to a small but consistent
increase in Bcl-2 expression. MFI for three experiments performed in
triplicate was 33.1 ± 5.8, compared with PBS-treated control
samples (23.1 ± 2.7; statistically significant at
P = 0.045, paired t-test; Fig. 2
, top panel).
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Fas and TNF-Receptor Expression
Tenon fibroblasts expressed low levels of Fas
constitutively in culture. The MFI obtained with anti-Fas FITC was
14.7 ± 3.4 compared with isotype control IgG FITC (7.9 ±
1.6; Fig. 3
; statistically significant at P = 0.004, paired
t-test). Fas receptor expression increased in a
dose-dependent manner after 5-minute exposures to mitomycin-C in doses
ranging between 0.01 and 1.0 mg/mL. The increase in Fas expression was
statistically significant at all concentrations tested, compared with
PBS-treated control samples (P < 0.01 for all
concentrations, ANOVA with Bonferroni post-test correction; Figs. 4A
4B
). Peak expression was seen after exposure to 0.4 mg/mL (Fig. 4C)
,
with an increase in mean expression of approximately 250%. Fas
expression decreased at 1.0 mg/mL.
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To determine the effect of Fas receptor activation in mitomycin-C–treated Tenon fibroblasts, medium containing anti-Fas IgM (CH11) was added immediately after single 5-minute applications of mitomycin-C (0.4 mg/mL) or PBS in the control fibroblasts. The number of viable fibroblasts was determined by phase-contrast microscopy. Mitomycin-C alone reduced the number of fibroblasts compared with saline-treated control cells by inhibiting proliferation and inducing cell death (Fig. 6A 6B) . In the mitomycin-C–treated fibroblasts, anti-Fas IgM (CH11) led to a median reduction of 43.6% (range, 33.5%–71.1%) in viable fibroblasts compared with mitomycin-C alone (Fig. 6B) . The reduction in fibroblasts was statistically significant for 50 and 500 ng/mL (P < 0.05; Mann-Whitney test). Because antibody-induced killing may have been a consequence of complement-mediated lysis, we repeated the experiment in serum-free conditions with an isotype control antibody that also binds to the fibroblast surface (anti-fibroblast surface antigen IB10; Sigma). In these experiments cell death was measured using the lactate dehydrogenase release assay. Anti-Fas IgM (CH11) led to an approximate 50% increase in fibroblast death compared with mitomycin-C treatment alone (P < 0.001, Mann-Whitney). The isotype control antibody, however, had no effect compared with mitomycin-C alone (Fig. 6C) . A low but consistent increase in cell death was observed in PBS-treated fibroblasts incubated with anti-Fas IgM (<5% at 48 hours).
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In further support of the nonparticipation of TNF-,
mitomycin-C–treated fibroblasts were incubated in growth medium
containing soluble TNF-. TNF- had no effect on
mitomycin-C–induced fibroblast death compared with mitomycin-C
treatment alone (Fig. 5C)
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Fas Receptor Blockade after Mitomycin-C
To evaluate the efficacy of the Fas-blocking antibody (anti-Fas
IgG; M3; Immunotech), Jurkat T-cells were pretreated with the blocking
antibody for 30 minutes before the addition of Fas-activating anti-Fas
IgM (CH11). Jurkat T-cell death was determined by cell morphology on
cytospin preparations. Anti-Fas (M3) inhibited apoptosis
(P < 0.01, Mann-Whitney), whereas the isotype control
(M33, Immunotech), which binds to the Fas receptor but has no effect on
signal transduction, had no effect on anti-Fas IgM–induced apoptosis
(Fig. 7A)
. To determine whether Fas receptor blockade affects
mitomycin-C–induced apoptosis, fibroblasts were incubated in anti-Fas
IgG (M3) at various concentrations for 30 minutes before treatment with
mitomycin-C (0.4 mg/mL), as previously described.21
After
treatment, the monolayers were irrigated and reincubated in
Fas-blocking IgG (M3) at the same concentrations. Apoptosis was
measured after 48 hours, using the lactate dehydrogenase release assay.
Anti-Fas M3 had no effect on mitomycin-C–induced apoptosis at the
concentrations tested (Fig. 7B)
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Discussion |
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Apoptosis is an active gene-directed mode of cell death that plays a critical role in the maintenance of homeostasis. It is activated by numerous triggers, including irreparable internal damage, deprivation of external survival factors, and conflicts in cell-cycle signals that both promote and inhibit cell division. In addition, in mammalian cells a receptor-based mechanism of "instructive" apoptosis has evolved, in which ligation of cell surface death receptors enables an organism to eliminate specific cells. Death receptors play a role in diverse processes, such as T-cell-mediated cytotoxicity,23 peripheral T-cell tolerance,20 and immune privilege, in which Fas-ligand expression by uveal tissue induces apoptosis in activated lymphocytes.24 The wound-healing response leads to a large expansion in the number of fibroblast that is restored at the end of the healing response by fibroblast apoptosis.9 25 26 The mechanisms that regulate fibroblast death remain poorly understood.
The Bcl-2 family of proteins regulate the susceptibility of a cell to apoptotic stimuli. The relative ratio of pro- to anti-apoptotic protein dimers regulates cytochrome c release and therefore sets the apoptotic threshold.27 Tenon fibroblasts constitutively express Bcl-2, Bcl-x, and Bax in culture. Mitomycin-C led to a small increase in protein expression of all the Bcl-2 family members tested, without an obvious shift to a proapoptotic phenotype. This was surprising, in light of the fact that these cells had been exposed to a potent apoptotic signal.
The cell surface death receptors Fas and the TNF receptor trigger apoptosis after binding their respective ligands. Increased death receptor expression primes cells to stimuli that signal through these receptors. In our experiments, although Tenon fibroblasts constitutively expressed Fas in culture, they did not undergo apoptosis after Fas receptor stimulation. Fas expression increased after mitomycin-C in a dose-dependent manner, up to a concentration of 0.4 mg/mL. Expression was decreased after treatment with 1.0 mg/mL, possibly reflecting the extremely potent apoptotic stimulus and the inability of severely disrupted fibroblasts to maintain protein production. Fas receptor ligation promotes mitomycin-C–induced fibroblast death, confirming that the upregulated receptor is functional. The observation that lower concentrations of mitomycin-C also upregulated Fas expression is exciting, because it suggests that the growth-arrested fibroblasts can be primed to secondary signals that activate the Fas receptor. This would have potential clinical value, because wound-healing responses slowed by the lower, safer intraoperative doses of mitomycin-C may be terminated by subsequent Fas receptor ligation. We found that Fas receptor ligation did not induce significant levels of apoptosis in fibroblasts treated with the lower sublethal concentrations of mitomycin-C. Regulatory mechanisms other than Fas receptor expression must therefore influence the outcome of Fas signaling. A potential regulatory candidate is the Fas ligand inhibitory protein (Flip),28 which inhibits downstream of the receptor. Our findings are in accord with others that increased Fas receptor expression does not necessarily render cells susceptible to Fas ligand–mediated apoptosis.28
Fas receptor blocking antibody (M3) did not inhibit mitomycin-C–induced apoptosis. We excluded the possibility that the antibody does not block Fas signal transduction by demonstrating that anti-Fas IgM–induced apoptosis in Jurkat T-cells was inhibited by the M3 antibody. There is conflicting evidence of the role of Fas in tumor cell death after exposure to chemotherapeutic agents. Fas signaling has been shown to be essential for bleomycin-induced apoptosis in hepatoma cells,21 as well as doxorubicin-induced apoptosis in human leukemia T-cell lines.15 The absolute requirement for Fas signaling in all chemotherapy-induced apoptosis has recently been brought to question, however, with evidence that a number of chemotherapeutic agents including doxorubicin can induce Fas-independent apoptosis.29 Our findings contribute to this, in that Tenon fibroblast apoptosis induced by mitomycin-C can proceed independently of Fas. The presence of a number of possible pathways that can induce apoptosis after severe cell damage has obvious selection advantage.
Accurate control of subconjunctival scar formation remains an elusive goal in our ability to achieve predictable lowering of intraocular pressure after glaucoma filtration surgery. The potent ability of single, short applications of mitomycin-C to induce apoptosis in fibroblasts may account for the long-term inhibition in scar formation. Devitalizing bleb tissue may also contribute to postoperative complications, however, such as bleb leak and susceptibility to infection. An understanding of the mechanisms that regulate fibroblast death in normal, abnormal, or drug-modified wound-healing responses should permit tighter control of the scarring response and optimize the effect of filtration surgery. In particular, the ability to prime a fibroblast to subsequent death signals provides an attractive approach to switching off the response once sufficient healing has occurred and a target intraocular pressure has been achieved.
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Footnotes |
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Supported by Wellcome Trust Grants 045202 (JGC) and 1055183 (LHC) and the Royal National Institute for the Blind (JTD).
Submitted for publication March 14, 2001; revised August 23, 2001; accepted September 10, 2001.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked
"advertisement" in accordance with 18 U.S.C. 
1734
solely to indicate this fact.
Corresponding author: Jonathan G. Crowston, Wound Healing Research Unit, Institute of Ophthalmology, Bath Street, London EC1V 9EL, UK; jcrowston{at}lineone.net
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