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1 From the Laboratory of Immunohematology and the 2 Department of Ophthalmology, Ambroise Paré Hospital, APHP, University of Paris-V René Descartes, Boulogne, France.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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METHODS. Patients with moderate to severe KCS included in a large European multicenter clinical trial (Cyclosporin Dry Eye Study, Allergan, Irvine, CA) underwent collection of IC specimens at baseline, month 3, and month 6. For 6 months, they randomly received 0.05% or 0.1% cyclosporin A or vehicle. Specimens were processed and analyzed in a masked manner by flow cytometry, using monoclonal antibodies directed to HLA DR, CD40, CD40 ligand, Fas, and the apoptotic marker APO2.7. Percentages of positive cells were calculated and levels of expression quantified after conversion into standardized units of fluorescence.
RESULTS. One hundred fifty-eight patients had at least two IC specimens available for flow cytometry analysis. HLA DR expression, both in percentage of positive cells and level of expression, was highly significantly reduced after 0.05% and 0.1% cyclosporin A treatment at months 3 and 6 compared with baseline values, whereas vehicle did not induce any change in HLA DR expression over time. The 0.05% and 0.1% cyclosporin emulsions were significantly more effective than the vehicle in reducing HLA DR at months 3 and 6 (0.05%), and at month 6 (0.1%). CD40 expression was significantly reduced at month 3 and partially at month 6, compared with baseline, with no reduction in patients who received the vehicle. CD40 ligand expression also decreased at months 3 and 6 in patients taking both concentrations of cyclosporin A. APO2.7 expression was significantly increased in all three groups, whereas percentage of Fas-positive cells decreased only in patients treated with 0.05% cyclosporin A at months 3 and 6.
CONCLUSIONS. Flow cytometry provided an objective technique to monitor the effects of topical cyclosporin A on immune- and apoptosis-related markers in the conjunctival epithelium of patients with KCS enrolled in a large multicenter trial. Topical cyclosporin A strikingly reduced HLA DR and to a lesser extent, other inflammatory and apoptotic markers, whereas the vehicle, used as a control tear substitute, had almost no effect. This study confirms that cyclosporin A may be efficient in reducing conjunctival inflammation in moderate to severe KCS and is consistent with clinical results in this indication.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Treatments of the most severe forms of KCS with steroids10 and topical cyclosporin A (CsA) have therefore been successfully attempted, first in Sjögren’s syndrome animal models of mouse11 and dog12 13 and then in human clinical trials.14 15 16 17 Topical CsA has been shown in animal models to be effective in reducing lymphocytic infiltration of the lacrimal glands and conjunctiva, both in the epithelium and substantia propria, and in reversing the abnormal apoptotic imbalance of the lacrimal glands in KCS (i.e., decreased lymphocyte apoptosis and increased epithelial apoptosis).13 CsA thus appears to be an efficacious drug capable of reducing inflammatory infiltrates, modulating immune reactions and apoptotic pathways, and even increasing tear fluid secretion.18 19
Large phase 2 and 3 clinical trials with topical emulsions of CsA have been undertaken in patients with moderate to severe KCS16 17 and have shown significant efficacy of topical CsA in this indication. Recently therefore, a large European multicenter clinical trial has been conducted in a similar complementary way to evaluate efficacy and safety of topical CsA in the treatment of moderate to severe KCS. As a part of this clinical study, impression cytology (IC) specimens were taken in a large series of patients as a tertiary complementary test, to investigate the expression of immune-related markers by the conjunctival epithelium, confirm the presence of inflammation in KCS, and monitor these markers throughout a 6-month treatment with CsA. We used a previously validated method of flow cytometry in IC8 20 to quantify the percentage of conjunctival cells expressing various inflammation- and apoptosis-related antigens, HLA DR class II antigens, CD40, CD40 ligand, Fas, and the apoptotic marker APO2.7, and to objectively measure their levels of expression. Results at baseline, before the randomized treatment, were recently published and showed highest levels of HLA DR expression and to a lesser extent of the other markers in the conjunctival epithelium in KCS, both in Sjögren’s and non-Sjögren’s syndrome eyes.21 We hereby present the effects on these markers of two concentrations of CsA topical emulsions and of their vehicle, using flow cytometry in IC specimens repeatedly obtained during a 6-month treatment.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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At selected centers, as a part of the whole study, IC specimens were collected at day 0, from the worse eye, defined as the one showing the highest degree of corneal staining, or the lowest Schirmer’s test when both eyes had the same corneal staining scores. If the two criteria were equal in both eyes, the right eye was chosen for IC. The same eye was used throughout the study. Patients providing samples for this study were recruited in 28 centers from four countries in Europe. Immediately after collection, specimens were shipped to the Department of Immunohematology, Ambroise Paré Hospital, Boulogne, France, for processing and analyses in a centralized procedure.
The laboratory work for this study as well as the study protocol were conducted in compliance with the Ethics Committee (CCPPRB) at the Ambroise Paré Hospital and the relevant ethics committees in each of the participating countries. Additional specific written informed consent was obtained to collect repeated conjunctival impressions. This study was conducted in compliance with the Declaration of Helsinki, South Africa amendment, 1996.
Experimental Procedures
IC specimens were obtained with patients under topical
anesthesia (0.04% oxybuprocaine), using 0.20-µm polyethersulfone
filters (Supor; Gelman Sciences, Ann Arbor, MI), applied on the
superior and superotemporal bulbar conjunctiva, according to previously
published procedures.8
20
21
Specimens were collected at
least 15 minutes after instillation of the last staining eye drop
(i.e., fluorescein and lissamine green), to avoid any interference with
immunofluorescence (IF) analyses. After collection, membranes were
immediately dipped into tubes containing 1.5 ml of cold
phosphate-buffered saline (PBS) with fixative (0.05% paraformaldehyde,
prepared monthly and sent regularly from the central laboratory to the
centers). Tubes were to be kept at or below 4°C before impression
collection and sent within 2 days to the Department of
Immunohematology, Ambroise Paré Hospital, in cold-conditioned
containers. Cells were extracted by gentle agitation for 30 minutes and
centrifuged (1600 rpm, 5 minutes). The cells were then counted in a
Malassez cell before processing for flow cytometry, according to
previously validated methods.8
20
21
Five different monoclonal antibodies and two corresponding negative
controls were used in this study. Mouse IgG1 anti-HLA DR 
chain
(clone TAL.1B5, 50 µg/ml, Dako, Copenhagen, Denmark), mouse IgG1
anti-CD40 (clone MAB89, 1 mg/ml, Immunotech, Marseille, France), and
mouse IgG1 anti-CD40 ligand (clone TRAP1, 1 mg/ml, Immunotech) were
used in indirect IF procedures. Fluorescein isothiocyanate
(FITC)–conjugated goat anti-mouse immunoglobulins were used as the
secondary antibody (Dako) and nonimmune mouse IgG1 (Dako) as a negative
isotypic control. FITC-conjugated mouse IgG1 anti-human Fas/CD95 (clone
UB2, 1 mg/ml, Immunotech) and phycoerythrin (PE)-conjugated mouse IgG1
anti-human Apo 2.7 (clone UB2, 1 mg/ml, Immunotech), as an apoptosis
marker,22
were used in a direct IF technique. The
FITC-PE–conjugated nonimmune mouse IgG1 (Immunotech) was used as a
negative isotypic control for the direct IF procedure. Antibodies were
used in a 1:50 dilution in PBS containing 1% bovine serum albumin.
After 30 minutes of incubation, cells were washed in PBS by a 5-minute
centrifugation and, for indirect IF procedures, were reacted with the
secondary anti-mouse immunoglobulins in a 1:50 dilution, for 30
minutes. After incubation, cells were centrifuged in PBS (1600 rpm, 5
minutes), resuspended in 100 µl of PBS, and analyzed on a flow
cytometer (FACScan; Becton Dickinson) according to previously validated
methods.8
20
21
The same flow cytometer was used during
the study.
All specimens were analyzed in a masked manner. For each marker, at least 1,000 cells were analyzed, and specimens with fewer than 10,000 cells were therefore discarded. The percentages of positive cells were obtained from logarithmic cytograms of mean fluorescence intensities, by comparison with the negative isotypic control. Fluorescence intensities were further quantified by using calibrated fluorospheres to translate the mean fluorescence of each sample into standardized arbitrary fluorescence units (AUF). A calibration curve was therefore established during each flow cytometric procedure by using four different beads (Immunobrite; Coulter, Hialeah, FL) with standardized fluorescence intensities.21 23 The actual AUF value was obtained by subtracting the isotypic negative control from the total AUF calculated for each marker.
Statistical Analyses
For both the percentage of positive cells and the AUF analyses,
a nonparametric method was used because of the high variability of the
data. A Kruskal–Wallis test was used to compare differences in change
from baseline among treatment groups. If the test for among-group
differences was significant (P < 0.05), then all three
pairwise comparisons were performed using a Wilcoxon rank sum test.
Within-group changes from baseline were analyzed by the Wilcoxon
signed-rank test. Statistical analysis software (SAS, ver. 6.12 for
UNIX; SAS Institute, Cary, NC) was used for computation and analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Levels of expression of HLA DR expressed in AUF confirmed the striking decrease of this marker with the CsA treatment. Mean expression of HLA DR decreased from 101,004 ± 117,356 AUF (mean ± SD) at baseline to 41,557 ± 44,842 AUF (P < 0.001) and 39,748 ± 38,495 AUF (P < 0.001) at months 3 and 6, respectively, for patients receiving 0.05% CsA, and from 124,883 ± 188,883 AUF to 61,949 ± 72,717 AUF (nonsignificant) and 38,492 ± 39,054 AUF (P < 0.001) for 0.1% CsA. Patients receiving vehicle did not show any significant difference from baseline (mean changes, -13,140 AUF and -16,387 AUF, respectively, at months 3 and 6, nonsignificant). Again, a significant difference was found between 0.05% CsA and vehicle at months 3 (P = 0.009) and 6 (P = 0.022) between 0.05% and 0.1% CsA at month 3 (P = 0.013) and between 0.1% CsA and vehicle (P = 0.011) at month 6.
The expression of the other markers also showed significant changes from baseline in the CsA-treated groups, whereas the vehicle had no or poor effect. Percentage of CD40-positive cells (Fig. 2) significantly decreased in those patients treated with 0.05% CsA at month 3 (P = 0.003) and in the 0.1% CsA group at months 3 (P < 0.001) and 6 (P < 0.001). The levels of CD40 expression significantly decreased at month 3 with 0.05% CsA treatment (P = 0.049). Treatment with vehicle slightly decreased CD40 expression but did not change significantly either the percentages of positive cells and levels of expression.
Percentages of cells positive to CD40 ligand (Fig. 3) decreased in all three groups, and differences from baseline reached significance, with 0.05% CsA treatment at month 6 (P = 0.047), 0.1% CsA at months 3 and 6 (P = 0.001 and P < 0.001, respectively) and vehicle at months 3 and 6 (P = 0.020 and P < 0.001, respectively). However, CD40 ligand expression remained low in all groups. Mean percentages of cells expressing this marker ranged from 7% to 10% at baseline, 0.4% to 5% at month 3, and 1.5% to 2.9% at month 6. Levels of expression of CD40 ligand also remained low but significantly decreased only in the CsA groups, whereas levels of expression in the vehicle group did not significantly decrease. Differences were thus significant at months 3 and 6 for patients treated with 0.05% CsA (P = 0.007 and P = 0.008, respectively) and 0.1% CsA (P < 0.001 and P = 0.044, respectively).
The apoptotic marker APO2.7 (Fig. 4) paradoxically showed an increase of expression in the three groups in percentages of positive cells, at the two time points, excepted for vehicle at month 3, but in levels of expression a difference was only found in patients treated with 0.05% CsA at months 3 (P < 0.001) and 6 (P = 0.001), and with 0.1% CsA at month 3 (P = 0.043). Fas (Fig. 5) showed only a significant decrease of percentage of positive cells in the 0.05% CsA group at months 3 (P = 0.046) and 6 (P = 0.001), whereas levels of expression did not significantly change in any group.
For all markers, additional statistical analyses in stratified groups of patients with and without Sjögren’s syndrome were performed, to detect eventual differences in efficacy of CsA treatment in either subgroup. At baseline, 41% of patients were found to have Sjögren’s syndrome according to clinical and biologic criteria given in detail in our previous study at baseline.21 Although the number of patients decreased in the stratified analyses, similar results and tendencies were found in the two subpopulations of Sjögren and non-Sjögren’s syndrome, which did not statistically differ from each other at any time and for any marker, as well as from the overall population (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In our previous study using this technique of flow cytometry,20 21 the gating process on the flow cytometer clearly showed a dense homogenous population of conjunctival cells that was mainly composed of epithelial cells with rare smaller inflammatory cells. Although interferences between expression of HLA DR by the two populations could not be totally excluded, it is therefore most unlikely that the eventual decrease of inflammatory cells alone could influence levels of HLA DR expression measured in the whole cell population. Nevertheless, the lipid vehicle, despite potential therapeutic effects as a viscous tear substitute, showed no effect on HLA DR expression. Although decreasing slightly, neither percentage of HLA DR expressing cells nor objectively quantified levels of expression significantly changed.
HLA DR is a major immune-related marker normally expressed by
immunocompetent cells, which has been shown to be upregulated in
epithelial cells in cases of autoimmune and inflammatory disorders. In
KCS, conjunctival and lacrimal acinar cells overexpress this marker at
very high levels, especially, but not only, in Sjögren’s
syndrome.1
2
5
In this study similar results were obtained
for eyes with and without Sjögren’s syndrome, both at baseline
and with CsA treatment, which confirms that class II antigen expression
is not a specific consequence of an autoimmune disease. HLA DR
expression by conjunctival cells is dependent on various cytokines,
such as interferon (IFN)-
or tumor necrosis factor
(TNF)-.5
24
IFN- induces class II antigen expression
together with Fas and CD40 overexpression and apoptosis in conjunctival
epithelial cells in a dose- and time-dependent
manner.20
24
Many inflammatory cytokines are involved in
ocular surface diseases, and IFN- is therefore a good candidate for
some of the major mechanisms found in ocular structures in KCS,
especially inflammation and apoptosis.9
13
It remains to
be determined whether conjunctival epithelial cells acquire
antigen-presenting properties when they abnormally express class II
antigens, as do corneal epithelial25
and lacrimal acinar
cells.3
Nevertheless, class II–expressing epithelial
cells after stimulation by inflammatory cytokines could participate in
immune reactions and/or recruitment of inflammatory
cells.5
The other markers we tested also showed significant changes with CsA treatment. CD40 and CD40 ligand significantly decreased in the CsA groups, whereas the vehicle had no effect for CD40. CD40 belongs to the TNF receptor family and is involved in regulation of immune response and apoptosis.26 27 It is normally expressed by conjunctival epithelial cells and has been shown to be upregulated during inflammatory processes of the conjunctiva, including dry eye.28 In the present clinical trial on moderate to severe dry eye syndrome, baseline levels were significantly higher in KCS eyes than in normal ones, and in eyes with Sjögren’s syndrome compared with those without. Moreover, both CD40 and CD40 ligand expressions were found to be significantly correlated with HLA DR levels, thus confirming their involvement in the inflammatory process in the ocular surface.21
In contrast, Fas, although previously found at higher levels in dry eyes than in normal ones,21 seemed to be poorly influenced by CsA treatment in this study, in that a percentage of positive cells significantly decreased only in the 0.05% CsA group at months 3 and 6. This could be explained by the wide SDs and the low levels of expressions obtained by using direct immunofluorescence procedures, compared with indirect ones that amplify the detection process. However, possible persistence in the conjunctival epithelium of Fas-positive lymphocytes that would be inactivated by topical CsA could not be definitively eliminated.
More paradoxical were the findings of increased APO2.7 expression, both in percentages of positive cells and in levels of staining. Treatment with CsA at both concentrations significantly increased APO2.7 at months 3 and 6, and even the vehicle increased the percentage of positive cells. According to previous work performed in conjunctival cells with this apoptotic marker20 and the description of apoptotic epithelial cells in lacrimal glands of KCS models,21 we would have expected a greater level of expression of APO2.7 at baseline and a lower number of apoptotic cells after CsA treatment. At baseline we did not find any difference between normal and KCS-affected eyes, in contrast with a study previously conducted in a patient group with less severe dry eye.20 A toxic effect of the lipid vehicle cannot be eliminated that may explain these results. In addition, the accurate significance of APO2.7 cannot be ascertained, because differences between TUNEL- and APO2.7-positive apoptotic cells may exist. However, it could also be hypothesized that the increase of the early marker of apoptosis APO2.721 after CsA treatment may indicate a form of regulation of a very severely impaired epithelium. In severe KCS, and especially in Sjögren’s syndrome, conjunctival epithelium has been shown to exhibit increased numbers of S-phase cells with a loss of normal epithelial differentiation.4 The epithelium may therefore become hyperplastic under permanent cytokine stimulation and after decreased cell maturation. Increased APO2.7 expression observed with CsA, and to a lesser extent with the vehicle, could thus in such severely affected epithelial cells reflect an initial step of normalization of epithelial differentiation by elimination of the hyperplastic epithelial layers. Further long-term monitoring of this marker with CsA treatment as well as with various tear substitutes could thus provide additional interesting information.
Nevertheless, CsA appeared to be an effective drug in reducing inflammatory markers in conjunctival cells, thus confirming results in animal models of KCS.11 12 13 18 19 A clinical study conducted with a similar design in the treatment of KCS with CsA also showed a significant decrease of infiltrating HLA DR-positive cells with CsA in conjunctival biopsy specimens, both in the epithelium and substantia propria.29 CsA has also shown to be efficient in atopic keratoconjunctivitis in reducing HLA DR-positive infiltrates, which confirms these results.30 CsA could act as an anti-inflammatory, immunosuppressive drug and possibly a modulator of apoptosis.9 13 CsA has been shown to inhibit phosphatase and more specifically calcineurin, but also to stimulate substance P release and therefore has an effect on neurotransmitter regulation, with positive properties for tear fluid secretion as a consequence.19
Although obtained by indirect techniques designed to evaluate expression of biological markers by conjunctival cells, our results strongly support the role of CsA as a potent regulator of the ocular surface in KCS. Clinical studies have been performed with topical CsA in KCS eyes either in former14 15 or currently used16 17 ophthalmic formulations. Clinical improvement, especially on corneal staining as a main outcome for ocular surface impairment, and good overall safety were consistently observed. Recent phase 2 and 3 studies, respectively conducted for 12 weeks and 6 months in 162 and 877 patients with KCS,16 17 showed significant improvements in both objective and subjective measures with 0.05% and 0.1% CsA ophthalmic emulsions. Results of these large multicenter placebo-controlled clinical trials in KCS are providing important information, both for the pathogenesis of this very complex disease and for therapeutic issues, until now limited to the repeated instillation of poorly effective tear substitutes.
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Acknowledgements |
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Footnotes |
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Commercial relationships policy: C (CB); N (all others).
Corresponding author: Christophe Baudouin, Ambroise Paré Hospital, Department of Ophthalmology, 9 avenue Charles de Gaulle, 92104 Boulogne/Seine, France. arepo{at}worldnet.fr
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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