The Comparison of Efficacies of Topical
`Corticosteroids and Nonsteroidal Anti-
`inflammatory Drops on Dry Eye Patients: A
`Clinical and Immunocytochemical Study
`
`AVNI MURAT AVUNDUK, MD, MUSTAFA CIHAT AVUNDUK, MD,
`EMILY D. VARNELL, BS, AND HERBERT E. KAUFMAN, MD
`
`● PURPOSE: To investigate whether conjunctival inflam-
`mation represents a primary event in the pathogenesis of
`keratoconjunctivitis sicca or whether it is a secondary
`inflammatory reaction caused by enhanced mechanical
`irritation as a result of surface dryness and whether
`anti-inflammatory drops (corticosteroids and nonsteroi-
`dal anti-inflammatory) have therapeutic effects and are
`similar.
`● DESIGN: Single-masked, randomized, prospective clin-
`ical trial.
`● METHODS: Thirty-two keratoconjuctivitis patients
`with or without Sjo¨gren syndrome were included in the
`study. The patients were randomized to three groups.
`Group 1 patients received a topical artificial tear substi-
`tute (ATS); group 2 received ATS plus nonsteroidal
`anti-inflammatory drops (NSAID); and group 3 received
`ATS plus topical corticosteroidal drops. The eye symp-
`tom severity scores, Schirmer test values, rose bengal and
`fluorescein staining scores were evaluated before treat-
`ment and 15 and 30 days after start of treatment.
`Impression cytology specimens were stained using immu-
`nohistochemical methods to detect the percentages of
`human leukocyte antigen II (HLA-DR) positive, Apo
`2.7 positive, and periodic acid–Schiff positive cells. Sta-
`tistical analyses were performed within and between
`groups.
`
`Accepted for publication March 14, 2003.
`InternetAdvance publication at ajo.com April 24, 2003.
`From the Louisiana State University, School of Medicine, LSU Eye
`Center, New Orleans, Louisiana (A.M.A., E.D.V., H.E.K.); and Selc¸uk
`University, School of Medicine, Department of Pathology, Konya,
`Turkey (M.C.A.).
`This study was supported in part by US Public Health Service Grant
`EY02377 (H.E.K.) from the National Eye Institute, National Institutes of
`Health, Bethesda, Maryland, and an unrestricted departmental grant from
`Research to Prevent Blindness, Inc., New York, New York.
`Inquiries to Avni Murat Avunduk, MD, KTU Lojmanlari No: 31/17
`61080, Trabzon, Turkey; fax: (⫹90) 462-3252270; e-mail: avunduk@
`ttnet.net.tr
`
`● RESULTS: Group 3 patients had significantly lower
`symptom severity scores, fluorescein and rose bengal
`staining, and HLA-DR positive cells on days 15 and 30
`compared with patients in other groups. They also had a
`significantly higher number of periodic acid–Schiff posi-
`tive (goblet) cells in their impression cytology specimens
`on days 15 and 30 compared with the other patients. On
`day 30, group 3 patients had significant differences
`compared with their baseline measurements in terms of
`above-mentioned parameters. However, we did not detect
`a significant effect of any treatment schedule on the
`Shirmer test value and the numbers of Apo 2.7 cells in
`impression cytology specimens.
`● CONCLUSIONS: Topical corticosteroids had a clearly
`beneficial effect both on the subjective and objective
`clinical parameters of moderate-to-severe dry eye pa-
`tients. These effects were associated with the reduction
`of inflammation markers of conjunctival epithelial cells.
`(Am J Ophthalmol 2003;136:593– 602. © 2003 by
`Elsevier Inc. All rights reserved.)
`
`A LTHOUGH THE IMMUNOPATHOLOGIC ANALYSIS OF
`
`the lacrimal gland has received considerable atten-
`tion,
`less work has been done on pathologic
`changes occurring in the ocular surface of patients with
`keratoconjunctivitis sicca (KCS). However, a strong ex-
`pression of human leukocyte antigen II (HLA-DR) anti-
`gens has been found in conjunctival epithelial cells of dry
`eye patients.1 A strong relationship also has been proposed
`between inflammatory pathways and apoptosis, which
`directly affects epithelial turnover.2 Overexpression of
`apoptotic markers was shown in impression cytology spec-
`imens from patients with KCS.3,4 The above findings
`support the immunopathogenesis of KCS. However, it is
`not clear that this inflammatory reaction represents a
`primary phenomenon. Conversely,
`it may result from
`chronic surface dryness and an increased friction between
`
`0002-9394/03/$30.00
`doi:10.1016/S0002-9394(03)00326-X
`
`© 2003 BY ELSEVIER INC. ALL RIGHTS RESERVED.
`
`593
`
`MYLAN - EXHIBIT 1060
`Mylan Pharmaceuticals Inc. et al. v. Allergan, Inc. - IPR2016-01127, -01128, -01129, -01130, -01131, & -01132
`
`

`

`palpebral and bulbar conjunctiva because of tear defi-
`ciency.
`Evidence of inflammatory processes in the pathogenesis
`of KCS led to the development of cyclosporine A (CsA) as
`a first attempt to treat this condition therapeutically. The
`proposed mechanisms of CsA are twofold: immunomodu-
`lation and anti-inflammation. Immunomodulatory activity
`of CsA is achieved by its selective inhibition of the signal
`transduction cascade of inflammatory cytokines such as
`interleukin 2 and an eventual prevention of the autoim-
`mune response.5 The anti-inflammatory effect of CsA is
`through its inhibition of phosphatases.6 Topical CsA
`treatment of dry eye patients has been reported to be
`clinically effective7,8 and to reduce the numbers of acti-
`vated lymphocytes and immune-related markers within the
`conjunctiva.9,10 The efficacy of topical CsA in dry eye
`patients suggested that other immunosuppressive or anti-
`inflammatory drops would have a similar or greater bene-
`ficial effect in KCS patients.
`The aims of this study are twofold: (1) to investigate
`whether conjunctival inflammation represents a primary
`event in the pathogenesis of KCS or whether it is a
`secondary inflammatory reaction caused by enhanced me-
`chanical irritation as a result of surface dryness; and (2) to
`investigate whether other anti-inflammatory drops (corti-
`costeroids and nonsteroidal anti-inflammatory) have sim-
`ilar therapeutic effects.
`
`METHODS
`
`THE STUDY WAS COMMENCED AS A SINGLE-SITE, PROSPEC-
`tive, randomized, and single-masked clinical trial. In-
`formed consent was obtained from all patients, and the
`research was begun after obtaining approval
`from the
`Institutional Review Board of the Louisiana State Univer-
`sity Health Sciences Center. The research was carried out
`according to the tenets of the Declaration of Helsinki. All
`study medications were dispensed in coded bottles. The
`examiner (A.M.A.) was masked as to the medication used
`by the patients. Thirty-two KCS patients with or without
`Sjo¨gren syndrome were included in the study. All patients
`were at least 21 years of age. Inclusion criteria for patients
`included Schirmer test (without anesthesia) of 7 mm in 5
`minutes or less in at least one eye; mild superficial punctate
`keratitis defined as a corneal punctate fluorescein score of
`⫹1 in either eye (scale 0 [none]–3 [severe]); and one or
`more moderate dry eye related symptom including itching,
`burning, blurred vision, foreign body sensation, dryness,
`photophobia, and soreness or pain.
`Patients were excluded from the study if they had eye
`injury, infection, nondry eye ocular inflammation, trauma,
`or surgery within the previous 6 months; received concur-
`rent treatment that could interfere with the interpretation
`of the study results (systemic corticosteroids, immunosup-
`pressive therapy, and so on); had an uncontrolled disease
`
`or significant illness; or were pregnant or lactating. Post-
`menopausal patients who were on hormonal replacement
`therapy were also excluded. Before initialization of the
`study, the patients were instructed not to use any topical or
`systemic medication for at least 1 week.
`On day 0, Schirmer test, tear breakup time, fluorescein
`and rose bengal staining examinations were performed on
`both eyes of all patients. The Schirmer test was performed
`after corneal staining, because it may affect the staining
`pattern of the cornea with either fluorescein or rose bengal.
`We used the previously described scoring system for rose
`bengal and fluorescein staining.11 We also obtained symp-
`tom severity scores from patients12 who were instructed to
`grade their symptoms averaging the symptom severities for
`both eyes. The results of the Schirmer test and rose bengal
`and fluorescein staining scores were evaluated separately
`from right and left eyes.
`Impression cytology specimens were obtained from the
`right eyes of all patients on day 0, day 15, and day 30 to
`avoid statistical comparison between different eyes in
`different periods. For this purpose, Whatman nitrocellulose
`filter paper (cat no: 7195004, Whatman Int. Ltd., Maid-
`stone, UK) was used. The paper was cut into strips of
`approximately 6 ⫻ 15 mm, which were pressed against the
`temporal bulbar conjunctiva for 5 seconds and removed.
`Patients were randomized to three groups according to a
`computerized list generated by the LSU Eye Center bio-
`statistician. Group 1 patients were treated with a preser-
`vative-free topical artificial tear substitute (ATS; Refresh,
`Allergan Inc., Irvine, California, USA) four times a day in
`both eyes. Group 1 patients did not receive any medication
`besides Refresh. Groups 2 patients were treated with
`topical nonsteroidal anti-inflammatory drug (NSAID) eye
`drops, flurbiprofen (Ocufen, Allergan Inc. Irvine, Califor-
`nia, USA) four times a day plus ATS four to eight times a
`day in both eyes. Group 3 patients were treated with
`topical corticosteroidal drops (TSD) FML (Allergan Inc.,
`Irvine, California, USA) four times a day plus ATS four to
`eight times a day in both eyes. The patients were in-
`structed to discuss their medications only with the study
`coordinator and not with the examiner.
`The impression cytology sample strips were cut into
`three pieces: one piece was used for periodic acid–Schiff
`(PAS) staining; one was used for HLA-DR (Monoclonal
`Mouse Anti Human HLA-DR, Alpha-Chain Clone TAL
`1B5 Code No. M0746 Lot068 DAKO); and the other for
`Apo 2.7 (Monoclonal Antibody Apo 2.7 Cat. No: 2087
`Immunotech) staining. The strip samples were placed
`separately cell side down on gelatin-coated slides. The
`slides, with adherent nitrocellulose strips, were air dried
`thoroughly at room temperature, placed in 100% metha-
`nol, and washed repeatedly with methanol until the
`nitrocellulose totally dissolved. The slides were then
`wrapped in waxed papers and mailed to the laboratory in
`suitable containers. The immunohistochemistry tech-
`niques reported by Bales and Durfeen13 were used.
`
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`FIGURE 1. An impression cytology specimen from a patient
`with pretreatment period. The monoclonal antibody (anti hu-
`man HLA-DR) stains cell membranes in a granular manner, as
`evidenced by this photomicrograph. Arrows indicate the cells
`stained positively (bar ⴝ 25 ␮).
`
`Negative and positive control slides were run with each
`sample. The staining pattern of goblet cells of colon
`mucous epithelium was used as control to PAS staining.
`Similarly, the staining pattern of human tonsil specimen
`was used as control to HLA-DR and the staining pattern of
`the basal cell layers of squamous epithelium was used as
`control to Apo 2.7 staining. The slides were viewed and
`photographed using a Zeiss Photomicroscope III (Zeiss,
`Oberkochen, Germany). To standardize data collection,
`four fields on each sample were viewed using a 25⫻
`objective and photographed. Cell counts were carried out
`on coded slides to avoid bias. In masked fashion, two
`examiners quantified the pattern of staining photographi-
`cally (Figures 1 and 2). Percentages of reactive cells were
`determined by counting at least 200 cells in each speci-
`men. Damaged cells were not considered.
`Statistical analyses were performed using SPSS for
`Windows (Version 6.0 and 10.0, SPSS Inc., Chicago,
`Illinois, USA). The first comparison between and within
`groups on different examination days was analyzed using a
`two-way analysis of variance (ANOVA) test. Examination
`days and groups were chosen as independent variables or
`factors, and the other parameters were chosen as depen-
`dent variables. Within groups, changes from baseline were
`evaluated with a one-way ANOVA test (day was the
`independent variable) using the Tukey post-hoc test. The
`comparisons between groups on different examination
`points were also analyzed with a one-way ANOVA test
`using the Tukey post-hoc test (group was an independent
`variable). P ⫽ .05 was considered significant for all effects.
`The null hypothesis was that there was no difference
`among the treatment groups with regard to changes from
`baseline values. The alternate hypothesis was that there
`was a change.
`
`FIGURE 2. An impression cytology specimen stained with
`antihuman Apo 2.7 after treatment with nonsteroidal anti-
`inflammatory drops plus artificial tear substitute. Apo 2.7 is a
`specific apoptosis marker, and anti-Apo 2.7 antibody stains cell
`membranes of reactive cells. Arrows indicate positive cells (bar
`ⴝ 25 ␮).
`
`Power was calculated to detect an among-group differ-
`ence in change from baseline in symptom severity scores
`on day 30. The power of the study was calculated accord-
`ing to the given formula.14 For a sample size of 11 patients,
`the power to calculate 1 grade difference was 0.64.
`
`RESULTS
`
`A TOTAL OF 32 PATIENTS WERE ENROLLED IN THE STUDY.
`Four patients were discontinued for administrative reasons.
`Eight patients in group 1, nine patients in group 2, and 11
`patients in group 3 concluded the whole study period.
`None of the above discontinued patients reported adverse
`effects that could be related to the medications used in this
`study.
`Group 1 contained five female and three male patients
`(mean age, 51.2 ⫾ 12.4 SD). Five female and four male
`patients constituted group 2 (mean age, 46.67 ⫾ 8.66 SD).
`Group 3 comprised seven female and four male patients
`(mean age, 57.6 ⫾ 12.4 SD).
`At the beginning of the study (day 0), no significant
`difference was detected between groups in terms of any
`parameters studied.
`We did not observe any complication that could be
`linked to the study medications during treatment period.
`Significant differences between groups, days, and mean
`effect were observed in terms of symptom severity scores
`(mean effect, P ⫽ .01; groups, P ⫽ .01; days, P ⫽ .09;
`two-way ANOVA). Group 3 patients had significantly less
`symptom severity scores both on days 15 and 30 compared
`with groups 1 and 2 patients (P ⫽ .02 and P ⫽ .03 for day
`15 comparisons, and P ⫽ .03 and P ⫽ .03 for day 30
`comparisons). When comparisons were made between the
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`FIGURE 3. Change from baseline in symptom severity scores. *Significantly different from baseline value (all P ⴝ .000 both for
`days 15 and 30; P ⴝ .003 on day 15 for ATSⴙTSD treated group, and P ⴝ .002 on day 30 of ATSⴙTSD treated group). ATS ⴝ
`artificial tear substitute; NSAIDs ⴝ nonsteroidal anti-inflammatory drops; TSD ⴝ topical corticosteroidal drops.
`
`treatment days in particular groups, no significant differ-
`ence was found in groups 1 and 2 patients. However, a
`significantly less symptom severity score was detected on
`day 30 compared with day 0 in group 3 patients (P ⫽ .02).
`Comparison between days 0 and 15 also gave a significant
`result (P ⫽ .03). Other comparisons within and between
`groups did not differ significantly (Figure 3).
`Similarly, when rose bengal staining of right eyes was
`compared between groups, significant differences were
`obtained in terms of main effect (P ⫽ .03) and group
`comparison (P ⫽ .04), but no significant difference was
`observed in terms of day comparison (P ⫽ .07) (two-way
`ANOVA). Although there was no significant difference
`observed between groups on day 15, group 3 patients had
`significantly lower rose bengal staining scores compared
`with group 2 patients on day 30 (P ⫽ .046), but compar-
`ison between groups 2 and 3 did not show a significant
`difference. In group 3 patients, the mean rose bengal
`staining score on day 30 was significantly lower than that
`of day 0 (P ⫽ .01), and the mean score on day 15 was also
`lower than that on day 0 (P ⫽ .02). However, comparison
`between days 15 and 30 was not significantly different.
`
`There was a significant difference between days 0 and 15 in
`group 2 patients (P ⫽ .007). However, other comparisons
`within and between groups did not give any significant
`difference (Figure 4). Left eye comparisons between groups
`gave similar results.
`Comparison between groups in terms of fluorescein
`staining patterns of right eyes was significantly different in
`terms of groups (P ⫽ .019), but no significant difference
`was observed in terms of days (P ⫽ .074) or main effect (P
`⫽ .092). Group 3 patients had a significantly lower
`fluorescein staining score compared with group 2 patients
`on day 30 (P ⫽ .017). In group 2 patients, the fluorescein
`staining score was significantly lower on day 15 compared
`with day 0 (P ⫽ .017). Similarly, comparisons between
`days 0 to 15 and 0 to 30 gave significant results in group 3
`patients (P ⫽ .018 and 0.016, respectively). Other com-
`parisons within and between groups did not differ signifi-
`cantly (Figure 5). Comparison between left eyes among
`fluorescein staining pattern gave similar results.
`In impression cytology examinations, comparison be-
`tween groups in terms of PAS staining gave a significant
`result among group comparison (P ⫽ .003) and main effect
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`FIGURE 4. Change from baseline in rose bengal staining of right eye. *Significantly different from baseline value (P ⴝ .007 on day
`15 of ATSⴙNSAIDs group; P ⴝ .02 on day 15 of ATSⴙTSD group; P ⴝ .01 on day 30 of ATSⴙTSD group). ATS ⴝ artificial
`tear substitute; NSAIDs ⴝ nonsteroidal anti-inflammatory drops; TSD ⴝ topical corticosteroidal drops.
`
`(P ⫽ .012), but no significant difference could be observed
`between day comparisons. On day 15, group 3 patients had
`a significantly greater number of PAS⫹ cells compared
`with both groups 1 and 2 patients (P ⫽ .034 and P ⫽ .028,
`respectively). Similar results were obtained on day 30
`comparisons. On day 30, the mean percentage of PAS⫹
`cells in group 3 patients was significantly higher than in
`both groups 1 and 2 patients (P ⫽ .000 and P ⫽ .001,
`respectively). In group 3 patients, the mean percentage of
`PAS⫹ cell was significantly higher on day 30 than both on
`days 0 and 15 patients (P ⫽ .01, P ⫽ .02). Other
`comparisons within and between groups were not signifi-
`cant (Figure 6).
`Human leukocyte antigen (HLA) staining comparison
`between groups also revealed significant differences in terms
`of group comparison (P ⫽ .046), but no significant difference
`could be detected in terms of either days or main effect. On
`day 15, group 3 patients had significantly lower HLA⫹ cells
`
`compared with both groups 1 and 2 patients (P ⫽ .032 and P
`⫽ .042, respectively). On day 30, group 3 patients had a
`significantly less HLA-DR⫹ cells compared with groups 1
`and 2 (P ⫽ .024 and P ⫽ .033, respectively). In group 3
`patients, mean HLA-DR⫹ cells on day 30 were significantly
`lower than that in both groups 1 and 2 on days 0 and 15 (P
`⫽ .042 and P ⫽ .038, respectively). Other comparisons
`within and between groups did not reveal any significant
`difference (Figure 7).
`No significant differences at any examination point (Figure
`8) were found with Schirmer test values and the percentage
`of Apo 2.7⫹ cells in impression cytology specimens.
`
`DISCUSSION
`
`THE MOST IMPORTANT RESULTS OF THIS STUDY WERE
`that treatment with TSD significantly improved the ocular
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`FIGURE 5. Change from baseline in fluorescein staining of right eye. *Significantly different from baseline value (P ⴝ .017 on day
`15 of ATSⴙNSAIDs group; P ⴝ .018 on day 15 of ATSⴙTSD group; P ⴝ .016 on day 30 of ATSⴙTSD group). ATS ⴝ artificial
`tear substitute; NSAIDs ⴝ nonsteroidal anti-inflammatory drops; TSD ⴝ topical corticosteroidal drops.
`
`signs and symptoms of moderate-to-severe dry eye patients,
`and these improvements were associated with reduction of
`HLA-DR⫹ cells and an increase of PAS⫹ cells in con-
`junctival impression cytology specimens. However, ATS
`alone or ATS plus NSAID did not change these parame-
`ters. Keratoconjunctivitis sicca is an autoimmune disease
`that involves not only the lacrimal gland but also the
`whole ocular surface. Increased conjunctival inflammation
`has been reported before in KCS patients.1,15 Brignole and
`coworkers found that conjunctival cells from patients with
`dry eye with moderate-to-severe KCS, with or without
`Sjo¨gren Syndrome, overexpressed inflammatory markers.16
`However, whether inflammation is a primary phenomenon
`in KCS or is the consequence of repetitive abrasion of the
`corneal surface after tear film deficiency is not clear. It
`causes chronic inflammation, lymphocytic infiltrates, and
`apoptosis of ocular epithelial cells that could hypotheti-
`
`cally result from chronic ocular surface dryness and epi-
`thelial cell degeneration caused by increased friction
`between ocular surfaces.10 Our results presented here are
`somewhat contradictory to this hypothesis. This study
`provides evidence that conjunctival inflammation plays a
`primary role in the pathogenesis of KCS, because treat-
`ment with ATS and ATS plus NSAID drops did not
`change HLA-DR expression in conjunctival cells, but
`treatment with corticosteroids significantly reduced these
`markers in conjunctival epithelial cells.
`Our study provides evidence that TSD treatment im-
`proved clinical signs and symptoms, but topical ATS plus
`NSAID and ATS alone had no effect on these subjective
`and objective clinical parameters. The beneficial effect of
`corticosteroidal drops in the treatment of KCS has been
`reported before. Marsh and Pflugfelder17 treated 21 severe
`KCS patients with Sjo¨gren syndrome with nonpreserved
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`FIGURE 6. Change from baseline in PASⴙ cells. *Significantly different from baseline value (P ⴝ .01). ATS ⴝ artificial tear
`substitute; NSAID ⴝ nonsteroidal anti-inflammatory drops; PAS ⴝ periodic acid–Schiff; TSD ⴝ topical corticosteroidal drops.
`
`topical methylprednisolone and observed impressive im-
`provement in clinical parameters. However, they advo-
`cated using this treatment as “pulse therapy” because they
`observed significant side effects of corticosteroids after
`long-term therapy. We did not observe any TSD-related
`complication during the study period, but using corticoste-
`roids was
`relatively weak compared with methypred-
`nisolone, and the duration of the therapy was short. Thus,
`it is possible to see corticosteroidal side effects in the
`longer therapy with TSD. Sainz de la Maza Serra and his
`coworkers treated 15 severe KCS patients with unpre-
`served TSD before punctual occlusion and compared the
`clinical results with the patients who did not receive any
`pretreatment before punctual occlusion.18 They reported
`that 2 weeks of therapy with unpreserved TSD significantly
`improved clinical parameters in severe KCS. Treating the
`patients with ocular surface inflammation with nonpre-
`served methylprednisolone drops was reported to avert
`delayed tear clearance.19 In our study, the clinical im-
`provement observed after TSD plus ATS treatment was
`associated with reduction of the number of HLA-DR⫹
`cells and an increase of goblet cell numbers in the
`
`impression cytology specimens. HLA-DR is a major im-
`mune-related marker normally expressed by immunocom-
`ponent cells, that has been shown to be upregulated in
`epithelial cells in cases of autoimmune and inflammatory
`disorders, and in KCS, conjunctival cells overexpress this
`marker.15 Part of the beneficial effect of TSD on dry eye
`patients might be a result of the reduction in HLA-DR⫹
`conjunctival cells. The decreasing effect of corticosteroids
`on HLA-DR expression of human epithelial cells has been
`reported before.16 The mechanism of this reduction may
`involve several possibilities. As HLA-DR expression in
`epithelial cells is stimulated by various cytokines, such as
`interferon-␥ (I-␥) and tumor necrosis factor-␣ (TNF-␣),
`which could be synthesized in the ocular surface and by
`infiltrating
`lymphocytes, TSD might
`downregulate
`HLA-DR expression by decreasing these cytokine produc-
`tions. The reductive effects of corticosteroids on TNF-␣
`and I-␥ are well known.17 Conversely, because corticoste-
`roids
`decrease
`intercellular
`adhesion molecule-1
`(ICAM-1) activation,18,19 TSD may display its beneficial
`effect on the ICAM-1 system. ICAM-1 plays an important
`role in cell-to-cell
`interactions and cell migration of
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`FIGURE 7. Change from baseline in HLA-DRⴙ cells. *Significantly different from baseline value (P ⴝ .042). ATS ⴝ artificial
`tear substitute; NSAID ⴝ nonsteroidal anti-inflammatory drops; TSD ⴝ topical corticosteroidal drops.
`
`lymphocytes into the surrounding tissues such as the
`conujunctival epithelium and substantia propria.20 We
`failed to find any effect of NSAID on HLA-DR expression
`of the conjunctival cells. However, this finding did not
`seem to be surprising, especially considering the above
`possible mechanisms of effect, because NSAID have no
`effect on cytokine production.21 The decrease of HLA-
`DR⫹ cells in conjunctival epithelial cells of dry eye
`patients (Sjo¨gren and non-Sjo¨gren) had been reported
`before after topical CsA treatment.9,10 Fukagawa and
`associates speculated that chronic stimulation from envi-
`ronmental challenges (contact lens, low humidity, wind,
`and so on) on the ocular surface interferes with the
`neuronal transmission to the lacrimal glands, resulting in
`the activation of vigilant trafficking lymphocytes and sub-
`sequent production of proinflammatory cytokines, promoting
`the autoimmune response.22 Therefore, CsA and TSD ther-
`apy are rational approaches for treating immune-based in-
`flammation in KCS.
`Another important finding of the current study is an
`increase of goblet cell numbers in impression cytology
`
`specimens of dry eye patients after treatment with TSD, as
`evidenced by a significant increase of PAS⫹ cells. Such an
`effect was demonstrated previously following treatment of
`non-Sjo¨gren syndrome patients with CsA ophthalmic
`emulsion.23 The authors speculated that reducing inflam-
`mation with topical CsA treatment might have a prolifer-
`ating effect on goblet cells. It is logical to think the same
`mechanism may be valid for TSD treatment.
`An interesting result was obtained in terms of apoptotic
`cell numbers that were investigated by Apo 2.7 expression
`in impression cytology specimens. Topical TSD treatment
`had no effect on the numbers of apoptotic cells. The same
`result has been reported before with topical CsA treat-
`ment.10 The authors speculated that increased apoptosis
`after CsA treatment might represent a very early normal-
`ization of epithelial cell differentiation from a severely
`affected state. Although this mechanism may operate for
`TSD treatment, another explanation is also present. Both
`corticosteroids and CsA have an inducing effect on apo-
`ptosis.24,25 Thus, an expected decrease in apoptosis after
`treatment with either CsA or TSD (because of reduction
`
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`FIGURE 8. Change from baseline in Apo 2.7ⴙ cells. Any significant difference is not present in any group. ATS ⴝ artificial tear
`substitute; NSAIDs ⴝ nonsteroidal anti-inflammatory drops; TSD ⴝ topical corticosteroidal drops.
`
`of immune-based inflammation in conjunctiva of dry eye
`patients) and direct stimulation of apoptosis by the drugs
`may negate each other.
`Commercial TSD and NSAID used in the study (Oc-
`ufen and FML) contain mercurial preservatives that could
`have confounded surface inflammation; however, this fac-
`tor was unlikely to affect the comparison between two
`groups because both drops contain preservatives.
`The results of the study implied that TSDs were more
`effective than topical NSAIDs or ATS in reducing the
`ocular surface inflammation in KCS patients. Topical
`steroids had a clear beneficial effect both on the subjective
`and objective clinical parameters of moderate-to-severe
`dry eye patients. These effects were associated with the
`reduction of inflammation markers of conjunctival epithe-
`lial cells. Based on the data provided, it may be speculated
`that conjunctival inflammation is a primary event in the
`pathogenesis of KCS rather than a secondary finding,
`because the decreasing surface friction by ATS did not
`provide any beneficial effect. We think that TSD might be
`a good alternative to topical CsA therapy in the treatment
`of dry eye disease. A comparison of the two drugs would
`clarify this hypothesis.
`
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