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Effects of Cyclosporin A on Human Conjunctival Fibroblasts
Andrea Leonardi, MD;
Giuseppe DeFranchis, MD;
Iva A. Fregona, PhD;
Daniele Violato;
Mario Plebani, MD;
Antonio G. Secchi, MD
Arch Ophthalmol. 2001;119:1512-1517.
ABSTRACT
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Objective To evaluate the effects of cyclosporin A (CsA) on cytokine and/or collagen
production, cell growth, and apoptosis in conjunctival fibroblast cultures.
Methods Fibroblast cultures derived from normal subjects and patients with vernal
keratoconjunctivitis and pemphigoid were exposed to different concentrations
of CsA for either 24 hours or 30 days. The effects were evaluated by the colorimetric
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) test to
assess cell proliferation, and by the measurement of procollagen I (PIP) and
procollagen III (PIIIP) cytokines and total protein in culture medium. CsA-induced
apoptosis was assessed by fluorescence-activated cell sorter analysis.
Results After 24 hours of exposure to doses of CsA of more than 10 µg/mL,
cell proliferation and migration were significantly reduced. Cyclosporin A
reduced PIP and interleukin 1 (IL-1) production in a dose-dependent manner.
Interleukin 6 and IL-8 were increased by 10 µg/mL of CsA, whereas transforming
growth factor  , PIIIP, and total protein were unaffected. Cyclosporin
A exposure induced apoptosis in a time- and dose-dependent manner. Long-term
exposure to CsA reduced IL-6 but did not modify PIIIP production.
Conclusion Exposure to CsA directly modified fibroblast behavior.
Clinical Relevance Cyclosporin A ability to accelerate apoptosis in clinically fibrotic
tissues may prove to be therapeutic and useful in hyperproliferative conjunctival
disorders.
INTRODUCTION
CLINICAL TRIALS have suggested that topical cyclosporin A (CsA) is effective
and without serious adverse effects for the treatment of corneal graft failure
and immune-mediated ocular diseases such as vernal keratoconjunctivitis (VKC),
atopic keratoconjunctivitis, Sjögren syndrome, phlyctenular keratoconjunctivitis,
ocular pemphigoid, and corneal ulcers.1-8
Although these chronic inflammations have different immunopathogeneses, clinical
features, and evolution, some of these entities can lead to morphological
and functional modifications of the conjunctiva that are associated with different
degrees and sites of fibrosis, such as giant papillae, subconjunctival fibrosis,
and symblepharon.
Cyclosporine interferes with the antigen-induced phase of T-cell activation,
selectively inhibiting gene transcription for interleukin 2 (IL-), IL-3, IL-4,
and interferon  .9 Relatively little
is known with regard to the effects of CsA on nonhemopoietic cells. It is
very hydrophobic and readily diffuses into cell membranes. High and persistent
concentrations of CsA have been found in the cornea and sclera after a single
application of the 2% ocular solution.10 Cyclophilin,
a cytosolic protein capable of binding CsA, is known to be present in all
cell types, including fibroblasts.11 The CsA-cyclophilin
complex has been shown to inhibit the activity of the calcium/calmodulin-dependent
phosphatase calcineurin, but not the protein kinase C and A pathways.12
There is growing evidence that CsA is also able to affect the biological
functions of some nonimmune cell types, including endothelial cells, keratinocytes,
skin and gingival fibroblasts, and mast cells.13-16
Since CsA is already widely used topically in immune-mediated ocular conditions
in which not only T cells but also structural cells are activated, its effect
on conjunctival fibroblasts needs to be elucidated. The effect of CsA on fibroblasts
has been investigated mostly because of its adverse effect of gingival overgrowth.
Cyclosporin A has been shown in in vitro and in vivo experiments to either
stimulate or inhibit functioning of gingival fibroblasts.17-18
Whether these are direct effects of CsA on gingival fibroblast activity or
an effect on various cytokines and growth factors produced by inflammatory
cells remains to be clarified.19-20
A recent study showed that CsA reduced rabbit subconjunctival fibroblast proliferation
in a dose-dependent manner,21 indicating an
antiproliferative effect of this drug. Induction or modulation of apoptosis
may be one of the mechanisms of action of CsA on different cells. Fibroblast
apoptosis may be induced by nutritional and cytokine deprivation22
and by exposure to antimetabolitic agents such as mitomycin C.23
The aim of the present study was to evaluate in vitro the direct effects
of clinically relevant doses of CsA on conjunctival fibroblast functions by
assessing cell migration and proliferation, the production of procollagens
and cytokines, and the induction of apoptosis.
MATERIALS AND METHODS
CELL CULTURES
After informed consent was obtained, biopsy specimens were obtained
under topical anesthesia (4% oxybipuvocaine eye drops) from the upper tarsal
conjunctiva of 4 patients with VKC, from the lower tarsal conjunctiva of 2
patients with pemphigoid, and from the lower tarsal conjunctiva of 4 healthy
subjects after subconjunctival injection of 1% mepivacaine hydrochloride.
Clinical research followed the tenents of the Declaration of Helsinki. Biopsy
specimens were washed, cut into small pieces, and seeded in Nunclon Multidishes
(NUNC, Roskilde, Denmark) containing 100 µL of Ham's F12 medium (Sigma,
St Louis, Mo), supplemented with 10% fetal calf serum (Sigma) and antibiotics
(100 U/mL of penicillin, 100 µg/mL of streptomycin, and 2 mmol/L of
L-glutamine; Sigma). Tissues were incubated at 37°C in 5% carbon dioxide
in a humidified air atmosphere and fed daily. When cells began to form a monolayer,
tissue pieces were removed. Cells were fed with 500 µL of supplemented
medium twice a week. When fibroblast cultures reached confluence, they were
detached from the wells with trypsin and replated into 24-well plates (>95%
vitality). The fibroblasts were characterized morphologically, stained positively
with vimentine, and stained negatively with cytokeratines.
EXPERIMENTAL DESIGN
Third- to eighth-passage fibroblasts were used for the experiments.
Cyclosporin A was obtained from commercially available 5-mg/mL intravenous
preparations (Novartis, Basel, Switzerland) and added to the medium in the
following doses: 0, 0.001, 0.01, 0.1, 1.0, 10.0, 50.0, 100.0, or 1000.0 µg/mL.
In all the experiments, CsA was diluted with ethanol. Controls in each experiment
were cells treated with a medium containing the maximal ethanol concentration
used to solubilize and dilute CsA. Before each experiment, culture medium
was switched from 10% fetal calf serum to 0.4% fetal calf serum for 24 hours.
Then, cells were incubated in medium containing 0.4% serum and different concentrations
of CsA. The maximal concentration of CsA (1000 µg/mL) was found to be
toxic to cells and was studied no further.
To evaluate the effect of long-term exposure to CsA, fibroblasts at
confluence were maintained for 30 days with different concentrations of the
drug. Medium supplemented with CsA and 10% serum was changed at day 1, then
twice a week and collected for IL-6 and procollagen III (PIIIP) determination
at days 1, 3, 15, and 30. Cells were evaluated morphologically by inverted
microscope every day and for cell vitality at day 30.
Cyclosporin A concentrations in supernatants were evaluated after 24
hours of culture and found to correlate with expected values in culture medium.
PROLIFERATION ASSAY
To determine the proliferation rate of human conjunctival fibroblasts,
the colorimetric tetrazolium salt MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl
tetrazolium bromide; Sigma) test was performed. Two thousand cells per well
were seeded into 96-well tissue-culture plates and then treated with CsA as
previously described. After incubation times of 24, 48, and 72 hours, the
MTT test was performed. The optical density of each well was measured using
an automatic plate reader with a 560-nm test wavelength and a 690-nm reference
wavelength. In each experiment, 8 wells were used for the same concentration.
Experiments were repeated twice for each fibroblast line.
IN VITRO WOUND PRODUCTION MODEL
To assess cell migration, an in vitro wound model was used as previously
described.24 Briefly, in confluent cultures
in 35-mm dishes, a wound was produced with a 35-mm blade cut. A cotton swab
was used to scrape off the fibroblasts from one side of the blade, then the
wounded monolayer was washed twice with buffer. After wounding, fibroblast
cultures were treated with CsA as described above. Experiments were performed
3 times for each fibroblast population. After incubation, the supernatants
were removed, and the wounded cultures were fixed and stained with an ethanol
solution of 0.007% toluidine blue with a pH of 3.5 for 1 minute at room temperature.
At a magnification of x400, the total number of fibroblasts located
250 mm beyond the wound line was quantified in at least 5 different fields.
APOPTOSIS-SPECIFIC PROTEIN AND PROPIDIUM IODIDE
To determine any toxic or apoptotic effect of CsA, 12 000 cells
per well were seeded into 35-mm dishes and treated with CsA as described.
After a 24-hour incubation, cells were removed with trypsin, washed 3 times
with phosphate-buffered saline and resuspended in binding buffer. Five microliters
of fluorescein isothiocyanate–labeled annexin V (Kamiya, Biomedical
Company, Seattle, Wash) were added to cell suspensions, which were then incubated
for 10 minutes at room temperature, washed, and resuspended. Ten microliters
of propidium iodide stock solution (Becton & Dickinson, San José,
Calif) were then added. Fluorescence-activated cell sorter analysis was performed
using FACSCalibur (Becton & Dickinson).
As a positive control, cells were exposed for 5 minutes to 0.4 mg/mL
of mitomycin C, and maintained in 0.4% serum for 24 and 48 hours before analysis.23
Time-course experiments were also performed evaluating annexin V expression
after 1, 3, 6, 12, and 24 hours of exposure to 10 µg/mL of CsA.
PROCOLLAGEN, TOTAL PROTEIN, AND CYTOKINE PRODUCTION
In the study of CsA's effect on procollagen synthesis, 6250 cells per
well were seeded onto 24-well plates and treated with the same CsA concentrations
as described earlier. After an incubation time of 24 hours, the medium was
removed and stored at -20°C. Experiments were repeated twice for
each fibroblast line. The level of procollagen I (PIP) was determined using
an radioimmunoassay method (Orion Diagnostica, Espoo, Finland); PIIIP, using
a 2-stage sandwich assay (Cis Bio International, Gif-Sur-Yvette, France);
and total protein synthesis, by the Lowry method.25
An enzyme-linked immunoassay was used to measure levels of IL-1,
IL-8, transforming growth factor 1 (TGF-1) (BioSource, Nivelles,
Belgium), and IL-6 (DPC, Los Angeles, Calif). Samples were analyzed in duplicate.
The sensitivity of the assays was as follow: PIP, 25 µg/L; PIIIP, 0.1
U/mL; IL-1, 2 pg/mL; IL-6, 1 pg/mL; IL-8, 1 pg/mL; and TGF1, 1
pg/mL.
STATISTICS
Data are presented as the mean ± SD of the percent control values.
They were analyzed by analysis of variance (ANOVA) with a post hoc analysis
(Fisher protected least significant difference test). Differences among fibroblast
populations were calculated using the unpaired t
test.
RESULTS
EFFECTS OF CsA ON CELL GROWTH AND MIGRATION
Early passage fibroblasts from patients with pemphigoid and patients
with VKC showed a higher proliferation rate compared with cells derived from
normal subjects. Preliminary effects of CsA on fibroblast proliferation were
determined at 24, 48, and 72 hours. The most satisfactory results were obtained
with the 24-hour time point since toxic effects were noted at high doses of
CsA after 48- and 72-hour incubations. Thus, the subsequent experiments were
performed with the 24-hour incubation time.
With increasing doses of as high as 10 µg/mL of CsA per culture,
fibroblast growth remained unchanged. Above this concentration, a significant
reduction in cell growth was detected compared with that of the minimal concentration
of CsA (0.001 µg/mL) (ANOVA, P<.05). The
same behavior was observed for all fibroblast lines, both from normal subjects
and from those derived from vernal and pemphigoid patients. The concentration
required for 50% inhibition of cell growth was 61 µg/mL as interpolated
from the dose-inhibition curve (Figure 1).
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Figure 1. Effect of cyclosporin A (CsA)
on fibroblast growth after 24 hours of exposure. Data are expressed as mean
percent control levels (controls were cells exposed to the maximal concentration
of ethanol used to solubilize CsA). A dose-dependent, significant reduction
of cell growth was shown with increasing doses of CsA to more than 10 µg/mL.
ID50 indicates 50% inhibition of growth. Asterisks indicate P<.05 compared with the lowest concentration of CsA using analysis
of variance.
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With regard to CsA's effect on cell migration in the wounded fibroblast
monolayer, CsA doses of 10 µg/mL and higher significantly reduced the
number of cells behind the wound line when compared with controls (ANOVA, P<.05).
EFFECTS OF CsA ON ANNEXIN V EXPRESSION
Nontreated fibroblast lines cultured in 0.4% fetal calf serum for 24
hours showed a low basal expression of annexin V by fluorescence-activated
cell sorter analysis. This expression changed after treatment with CsA for
24 hours in the 3 cell lines derived from pathologic tissues. Annexin V expression
increased in a dose-dependent manner, with a maximal increase of 210%±27%
for control levels with 10 µg/mL of CsA (Figure 2). In all experiments, maximal propidium iodide levels (ie,
necrotic cells) were detected in cells treated with 50 µg/mL CsA. The
maximal increase of annexin V in the presence of 10 µg/mL of CsA was
detected after 24 hours (Figure 3).
Cells treated for 5 minutes with the positive control, 0.4 mg/mL of mitomycin
C, expressed 223% of annexine V above control levels at 24 hours, and 280%
above control levels at 48 hours.
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Figure 2. Expression of the apoptosis marker,
annexin V (AnV), and staining with the necrosis marker, propidium iodide (PI),
detected by fluorescence-activated cell sorter analysis in 3 fibroblast lines
exposed to cyclosporin A (CsA). Data are expressed as mean percent control
levels. A, Cyclosporin levels of 10 µg/mL maximally increased AnV, whereas
CsA levels of 50 µg/mL maximally increased PI. B, In one nontreated
culture, 14% of cells expressed AnV. C, Twenty-six percent of cells treated
with 10 µg/mL of CsA expressed AnV.
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Figure 3. Time course of annexin V (AnV)
expression after exposure to 10 µg/mL of cyclosporin A (CsA) for 1,
3, 6, 12, and 24 hours in 3 CsA-responder conjunctival fibroblast cultures.
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EFFECTS OF CsA ON PROCOLLAGENS AND CYTOKINE PRODUCTION
Culture medium of nonstimulated fibroblasts contained detectable amounts
of PIP and PIIIP. When CsA was added, PIP levels, expressed in nanograms per
microgram of total protein, progressively decreased in a dose-dependent manner
(Figure 4). Doses higher than 1
µg/mL of CsA significantly reduced PIP production compared with control
values (ANOVA, P<.05). Production of PIIIP and
total protein in culture medium did not change significantly. No significant
differences were observed among the different cell lines.
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Figure 4. Effect of cyclosporin A (CsA)
on procollagen I (PIP) production of fibroblast cultures at confluence. Data
are expressed as mean values of PIP per total protein detected in culture
medium. Asterisks indicate P<.05 compared with no CsA using
analysis of variance.
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Culture medium of nonstimulated fibroblasts contained detectable amounts
of IL-1, IL-6, IL-8, and TGF-1. With increasing doses of CsA,
IL-1 levels progressively decreased. At the highest doses of CsA (10 µg/mL
and 50 µg/mL), IL-1 levels were significantly lower than those found
to have the lowest concentration of CsA (0.01 µg/mL). Conversely, the
levels of IL-6 and IL-8 significantly increased with 50 µg/mL and 10
µg/mL of CsA, respectively, compared with those found to have the lowest
dose of CsA (0.01 µg/mL) (Figure 5). Levels of TGF-1 did not change significantly.
EFFECTS OF LONG-TERM EXPOSURE TO CsA
Fibroblasts at confluence in 10% serum maintained normal morphology
for all 30 days, with concentrations of CsA from 0.001 µg/mL to 10 µg/mL,
as well as controls exposed to medium supplemented with ethanol only. Cells
treated with 100 µg/mL CsA showed 50% vacuolization after 24 hours of
exposure and were completely dead at day 3. Cells exposed to 50 µg/mL
of CsA started to decline at day 2, 50% of these demonstrated vacuolization
at day 3, and all were completely dead at day 5. On day 30, cell vitality
was not affected by doses of CsA ranging from 0.001 µg/mL to 10 µg/mL.
Collagen production did not change within 30 days, while IL-6 levels were
reduced at day 15 and day 30, compared with levels determined at day 1 (ANOVA, P<.05) with CsA doses of 1 µg/mL and 10 µg/mg.
COMMENT
Fibroblast overgrowth, extracellular matrix deposition, and fibrosis
are some of the consequences of severe and protracted immunomediated keratoconjunctivitis.
Cyclosporin A has been used topically as an immunosuppressive agent for its
ability to reduce the cytokine expression from T cells, and thus, reduce local
inflammation. However, the effects of CsA on resident stromal conjunctival
cells and nonhemopoietic cells are less known. Kidney dysfunction and gingival
overgrowth are common adverse effects of the systemic use of CsA, probably
caused by a direct or indirect effect of the drug on renal proximal tubule
cells or renal cortical fibroblasts,26 and
on gingival fibroblasts.18-20
The potential of ocular topical CsA to modify conjunctival fibroblast metabolism,
and thus the fibrogenic phases of chronic keratoconjunctivitis, was investigated
in this study. Results demonstrated that CsA did influence conjunctival fibroblast
metabolism.
Previous pharmacokinetic studies in animals demonstrated persistent
levels, from 900 to 1400 ng/mL, in the cornea and sclera after a single application
of 2% CsA.10 In both dog and rabbit models,
single topical doses of CsA in a castor oil–water emulsion formulation
reached peak conjunctival concentrations within 1 hour and maintained high
concentrations for several hours.27 Considering
the highly lipophilic characteristics of CsA and its diffusion into cell membranes,
the doses used in the present experiments may have closely reproduced in vivo
conditions.
Conflicting data have been reported18-19
on the effects of CsA in gingival fibroblast cultures. CsA was shown to both
stimulate and inhibit PIP, IL-6, collagen I mRNA expression, and cell growth
in different gingival cell lines. This discrepancy may have resulted from
the use of different and high concentrations of serum. Serum not only contains
growth factors that may have muddled results, but also lipoproteins to which
CsA nonspecifically binds, thus modifying its availability. In the present
study, CsA was shown to have a direct effect on conjunctival fibroblast metabolism
by reducing cell proliferation rate and cell migration. Since fibroblasts
have been shown to be activated in immunomediated conjunctivitis, inducing
both tissue remodeling and scarring,28-29
these effects of CsA may have relevant clinical implications for the down-regulation
of conjunctival structural cells. These data agree with a previous finding
of CsA having inhibited rabbit subconjunctival fibroblast proliferation.22
Clinical1-8
and histopathological30-31 benefits
of local CsA administration have been suggested in several immunomediated
corneal and conjunctival diseases. The immunosuppressive effect of CsA may
be exerted through inhibition of cytokine and mitogen-induced gene expression.
In vitro studies have established that CsA affects the initial mitogen- or
antigen-induced phase of T-cell activation, selectively inhibiting the induction
of a small number of genes responsible for IL-2, IL-3, IL-4, INF- and
c-Myc, and several mitogen-induced genes.9, 32
In the present study, all conjunctival cell lines spontaneously produced and
released detectable amounts of IL-1, IL-6, IL-8, TGF-1, PIP, and
PIIIP. Exposure to CsA for 24 hours reduced the production of IL-1 and
procollagen I in a dose-dependent manner starting from 1 µg/mL of CsA.
Interleukin 6 and IL-8 concentrations were unchanged with CsA exposures of
up to 1 µg/mL, then were significantly increased with doses of CsA ranging
from 10 µg/mL to 50 µg/mL. This may be related simply to the toxic
activity of high doses of CsA, and it may explain the irritation reported
by some patients treated with topical CsA. This finding is in agreement with
the reported up-regulation of IL-6 expression by gingival fibroblasts exposed
to CsA.18 However, in cultures exposed to 1
µg/mL and 10 µg/mL of CsA for 30 days, IL-6 production was reduced
without affecting cell vitality and collagen production, confirming that doses
similar to those obtained with the current clinical use, can modulate fibroblast
activity.
Cyclosporin A has been shown to either promote or inhibit apoptosis
in a dose-dependent manner in different experimental models and cell lines.33-35 Apoptosis can be
triggered in conjunctival fibroblasts by topically applied cytotoxic drugs
such as mitomycin C.23 In dogs affected by
chronic idiopathic keratoconjunctivitis sicca, 0.2% topical CsA was shown
to stimulate infiltrating lymphocyte apoptosis and reduce epithelial cell
apoptosis,36 suggesting that CsA facilitates
the reestablishment of a normal apoptotic equilibrium. The induction or suppression
of apoptosis in different diseases and cells may depend on the cell circle
phase, cell type, or the presence of other factors. In the present study,
CsA's induction of apoptosis in 3 cell lines derived from pathologic tissues
with signs of scarring lends support to the therapeutic use of this drug in
hyperproliferative conjunctival disorders. Interestingly, the CsA concentration
that stimulated apoptosis was identical to that shown to inhibit cell proliferation
and increase IL-6 and IL-8 release. Toxic effects of CsA, shown by increased
propidium iodide captation, were found with doses equal to or greater than
50 µg/mL.
Studies using tissues from patients with pemphigoid and VKC reported
both an increased expression of fibrogenic cytokines and growth factors,37-38 and an increased proliferation rate
of cultured primary fibroblasts.28-29
The successful use of antimetabolitic agents for the management of pemphigoid39 and severe VKC40 provides
additional evidence that apoptosis is involved. Long-term management of VKC
with topical CsA is known to reduce conjunctival inflammation and the size
of limbal infiltrates and giant papillae. In the past, this last effect was
considered secondary to the immunosuppressive activity of CsA. From the present
study, within the limits of in vivo and in vitro comparisons, topical CsA
may be considered not only an immunosuppressive agent, but also a direct fibroblast
inhibitor that may also promote apoptosis.
AUTHOR INFORMATION
Accepted for publication April 11, 2001.
Corresponding author: Andrea Leonardi, MD, via Foscari 8, 35127 Padova,
Italy (e-mail: mdvol{at}tin.it).
From the Ophthalmology and Ocular Inflammation Unit, Department of
Neuroscience (Drs Leonardi, Fregona, and Secchi, and Mr Violato), and the
Department of Laboratory Medicine (Drs Leonardi, DeFranchis, and Plebani),
University of Padova, Padova, Italy. The authors have no financial interest
in any of the products or companies mentioned in this article.
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