Conjunctival Epithelial Cell Expression
`of Interleukins and Inflammatory Markers
`in Glaucoma Patients Treated over the
`Long Term
`
`Christophe Baudouin, MD, PhD,1,2 Pascale Hamard, MD, PhD,1,2 Hong Liang, MD,2
`Catherine Creuzot-Garcher, MD, PhD,3 Larry Bensoussan, MD,1 Françoise Brignole, MD, PhD2,4
`
`Purpose: To compare the conjunctival epithelial cell expressions of inflammatory cytokines in normal
`subjects and in glaucoma patients treated over the long term.
`Design: Case– control study.
`Participants: A total of 69 glaucoma patients treated over the long term and 15 normal subjects with no
`ocular abnormality or topical treatment.
`Methods: Amongst the 69 glaucoma patients, 27 were treated with preserved ␤-blockers, 24 with unpre-
`served 0.5% timolol, and the other 18 patients with an association of ⱖ2 preserved drugs. All patients were
`treated for more than 1 year with the same treatment, with no significant differences between groups for mean
`ages and durations of treatment at the time of the study. Impression cytology specimens were taken and
`processed for immunofluorescence techniques. Conjunctival cell expressions of HLA DR, as a standard for
`inflammatory level, and the interleukins IL-6, IL-8, and IL-10 were obtained and quantified using flow cytometry.
`Main Outcome Measures:
`Immune markers and proinflammatory cytokines in impression cytology
`specimens.
`Results: We found a significantly increased expression of all immunoinflammatory markers and mediators in
`the conjunctival epithelium of glaucoma patients compared with normal eyes. Human leukocyte antigen DR was
`significantly higher in the 2 groups receiving preserved drugs than in the unpreserved timolol group. The 3
`interleukins were similarly overexpressed in all glaucoma groups, with no significant between-groups differences
`except for the expression level of IL-8, which was significantly higher in the multitreatment group than in the
`preservative-free one.
`Conclusions: The present study confirms the increased expression of immunoinflammatory markers by the
`conjunctival epithelium of glaucoma patients treated over the long term. The development of nontoxic preser-
`vatives or preservative-free solutions is therefore of great interest. Ophthalmology 2004;111:2186 –2192 © 2004
`by the American Academy of Ophthalmology.
`
`There is now growing evidence from experimental and
`clinical studies that the long-term use of antiglaucoma drugs
`may induce ocular surface changes, causing ocular discom-
`fort upon instillation,1 tear film instability,2 conjunctival
`inflammation,3 subconjunctival fibrosis,4 conjunctival epi-
`thelium apoptosis,5 corneal surface impairment,1,6 and po-
`tential risk for failure of further glaucoma surgery.7,8 Sub-
`
`inflammation has also been described with
`clinical
`significant infiltration of the conjunctival epithelium and
`substantia propria by inflammatory cells,3,9,10 as has con-
`junctival epithelial cell expression of inflammatory mark-
`ers,11,12 in patients receiving antiglaucoma treatments for
`long periods. However, the respective roles of the active
`compound and the preservative in inducing toxic and/or
`
`Originally received: March 17, 2004.
`Manuscript no. 240204.
`Accepted: June 16, 2004.
`1 Departments of Ophthalmology, Quinze-Vingts National Ophthalmology
`Hospital and Ambroise Paré Hospital, APHP, Paris-Ouest School of Med-
`icine, University of Versailles, Paris, France.
`2 INSERM U598, Cordeliers Biomedical Institute, Paris, France.
`3 Department of Ophthalmology, University Hospital of Dijon, Burgundi
`University, Dijon, France.
`4 Department of Toxicology, Faculty of Biological and Pharmacological
`Sciences, University Paris 5 René Descartes, Paris, France.
`
`2186
`
`© 2004 by the American Academy of Ophthalmology
`Published by Elsevier Inc.
`
`Presented at: American Academy of Ophthalmology annual meeting, No-
`vember 17, 2003; Anaheim, California.
`This study was supported by University Paris 6, Paris, France (grant no.:
`EA3123).
`Correspondence to Christophe Baudouin, MD, PhD, Quinze-Vingts Na-
`tional Ophthalmology Hospital, 28 rue de Charenton, 75012 Paris, France.
`E-mail: baudouin@quinze-vingts.fr.
`
`ISSN 0161-6420/04/$–see front matter
`doi:10.1016/j.ophtha.2004.06.023
`
`ALCON 2013
`Apotex Corp. v. Alcon Research, Ltd.
`Case IPR2013-00428
`
`

`

`Baudouin et al
`
`䡠 Conjunctival Cytokines and Antiglaucoma Drugs
`
`Table 1. Demographic Characteristics of Normal Subjects and the 3 Groups of Glaucoma Patients
`
`n (eyes)
`Age (yrs)
`Mean ⫾ SD
`Range
`Gender
`Male
`Female
`Type of treatment
`
`Preservative-Free
`Timolol
`
`24
`
`62.7 ⫾ 15.5
`45–84
`
`10 (42%)
`14 (58%)
`0.5% timolol
`
`Preserved
`Timolol
`
`27
`
`60.0 ⫾ 7.5
`46–72
`
`12 (44%)
`13 (56%)
`0.5% timolol
`
`Multitreatments
`
`18
`
`56.6 ⫾ 16.9
`28–79
`
`10 (55%)
`8 (45%)
`0.5% timolol (n ⫽ 18)*
`Prostaglandin (n ⫽ 11)*
`CAI (n ⫽ 9)*
`␣-2 agonist (n ⫽ 5)*
`
`Normal
`Subjects
`
`15
`
`49.3 ⫾ 20.7
`37–66
`
`7 (46%)
`8 (54%)
`NA
`
`Total duration of treatment (yrs)
`Mean ⫾ SD
`Range
`
`4.7 ⫾ 2.5
`2–7
`
`4.6 ⫾ 2.8
`1–10
`
`5.9 ⫾ 6.6
`2–24
`
`NA
`NA
`
`CAI ⫽ carbonic anhydrase inhibitor; NA ⫽ not applicable; SD ⫽ standard deviation.
`No statistical difference between each group for age and duration of treatment (Mann–Whitney U test).
`*Eleven patients had 2 medications; 7 received 3 different drugs.
`
`proinflammatory effects of antiglaucoma ophthalmic solu-
`tions are still being debated. The most frequently used
`preservative, benzalkonium chloride, has widely demon-
`strated its toxic effects in laboratory,13,14 experimen-
`tal,9,15,16 and clinical studies.1,17,18 As a quaternary ammo-
`nium, this compound is much more than an excipient and
`has been shown to cause directly or has shown some evi-
`dence of causing tear film instability,2 loss of goblet
`cells,17,19 conjunctival squamous metaplasia and apopto-
`sis,5,20 disruption of the corneal epithelium barrier,21 severe
`loss of endothelial cells if accidentally introduced in the
`anterior chamber,22 and even blood–aqueous barrier disrup-
`tion in the early phase of pseudophakia,23 leading to the new
`concept of pseudophakic preservative maculopathy.24
`In a previous series of reports on the ocular surface, our
`group developed new objective and reliable techniques for
`exploring the conjunctival epithelium and assessing the
`inflammatory and apoptotic status of conjunctival cells by
`using flow cytometry in impression cytology speci-
`mens.25–27 In a recent article,12 we used this technique in
`patients who had received antiglaucoma drugs over an ex-
`tended period and demonstrated for the first time in a
`case– control study that patients treated with preserved
`␤-blockers exhibited high levels of HLA DR class II anti-
`gens and intercellular adhesion molecule (ICAM) 1, as well
`as a dramatic decrease in MUC-5AC– expressing cells,
`whereas eyes receiving unpreserved drugs showed almost
`normal patterns of inflammation and goblet cell density.
`Little is known, however, about the inflammatory pathways
`and mediators involved in ocular surface inflammation re-
`lated to toxic side effects of ophthalmic drugs and preser-
`vatives. In the present case– control study, we used the flow
`cytometry technique to assess the expression of 3 major
`interleukins involved in inflammation and inflammatory cell
`recruitment: IL-6, IL-8, and IL-10. As the overexpression of
`these 3 interleukins has previously been described in differ-
`ent ocular surface diseases,28 –30 our aims were to validate
`the technique of flow cytometry in impression cytology for
`
`these intracellular markers, investigate interleukin expres-
`sion by conjunctival epithelial cells in normal eyes and
`glaucoma patients, and compare their expression in differ-
`ent groups of patients receiving preserved or unpreserved
`drugs.
`
`Patients and Methods
`
`Patients
`A total of 69 patients with glaucoma and 15 normal subjects were
`included in this case– control study conducted in compliance with
`the Declaration of Helsinki (Scotland amendment, 2000). All
`glaucoma patients had chronic primary open-angle glaucoma but
`no other ocular disease, as assessed after a complete ocular exam-
`ination. They were treated for at least 1 year with the same
`treatment, 27 patients with preserved 0.5% timolol (containing
`0.01% benzalkonium chloride as a preservative), 24 patients with
`unpreserved 0.5% timolol, and the other 18 patients with an
`association of ⱖ2 preserved drugs (Table 1). In the first 2 groups,
`no patient had received other antiglaucoma drugs before the ones
`investigated in this study or a concomitant treatment during the last
`6 months before the time of conjunctival imprint collection. There
`was no significant difference for age and total treatment durations
`between all groups of glaucoma patients and normal subjects. The
`multitherapy group had a longer though not significantly different
`history of glaucoma treatment and served as a control group with
`multiple and long-term glaucoma treatments. In this group, treat-
`ments consisted of a preserved ␤-blocker in all eyes associated
`with a prostaglandin analog (11 eyes), a carbonic anhydrase in-
`hibitor (9 eyes), or an ␣-2 agonist (5 eyes), 11 eyes receiving 2
`drugs and 7 eyes 3 drugs. At the time of specimen collection,
`patients did not show any clinical evidence of intolerance to the
`administered drugs or ocular surface impairment. Patients with
`other diseases such as ocular or systemic inflammatory disorders
`or contact lens wear that might affect the conjunctival epithelium
`were excluded from the study. Fifteen normal subjects who had no
`history of ocular disease or clinical ophthalmic abnormality and
`who had not received any topical treatment for at least 6 months
`were also investigated, after approval by the ethics committee of
`
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`

`Ophthalmology Volume 111, Number 12, December 2004
`
`Dijon University, France. The ethics committee of University Paris
`6 had previously indicated that
`the exploration of the ocular
`surface of glaucoma patients using impression cytology collection
`did not require specific approval. Nevertheless, all patients had
`received specific explanations of impression cytology and gave
`informed consent for the procedure.
`
`Impression Cytology Specimens
`One eye was randomly selected for impression cytology analyses.
`Specimens were collected in the superior bulbar conjunctiva using
`0.20-␮m polyether sulfone filters (13 mm in diameter; Supor
`Membranes, Gelman Sciences, Ann Arbor, MI), according to
`previously described techniques.12,27 After instillation of 1 drop of
`0.04% oxybuprocaine, 2 pieces of filters, each 13 ⫻ 6.5 mm, were
`applied to the superior and superotemporal bulbar conjunctiva
`without the exertion of any pressure. Specimens were collected at
`least 15 minutes after the use of fluorescein eyedrops, to avoid
`interference with immunofluorescence analyses. Membranes were
`immediately suspended and fixed in cold phosphate-buffered sa-
`line (PBS; pH 7.4, at 4° C) containing 0.05% paraformaldehyde.
`Conjunctival cells were further extracted by gentle agitation and
`then centrifuged at 1600 rpm for 5 minutes before processing for
`flow cytometry.
`
`Immunofluorescence Procedure
`Four antibodies and 3 isotypic negative controls were used in this
`study. To determine the inflammatory profile of impression cytol-
`ogy specimens, indirect immunofluorescence was performed with
`mouse immunoglobulin G1 (IgG1) anti–HLA DR ␣ chain (clone
`TAL.1B5, 50 ␮g/ml, Dako SA, Copenhagen, Denmark) as the
`primary monoclonal antibody. The secondary antibody was fluo-
`rescein isothiocyanate– conjugated goat antimouse immunoglobu-
`lins (Dako). A nonimmune mouse IgG1 (Dako) was used as a
`negative isotypic control. For intracellular interleukin assessment,
`after a first step of cell permeabilization with 1% saponin for 5
`minutes (Sigma Chemical Co., St. Louis, MO), phycoerythrin-
`conjugated anti–IL-6 mouse IgG2b, anti–IL-8 rat IgG2a, and anti–
`IL-10 rat IgG2a (BD Biosciences, San Diego, CA) were used in
`direct
`immunofluorescence procedures, with their
`respective
`mouse and rat isotypic negative controls (BD Biosciences). All
`antibodies were diluted in 1% bovine serum albumin– containing
`PBS to obtain a final dilution of 1:50, following the manufacturer’s
`instructions. After 30 minutes of incubation with monoclonal
`antibodies, cell suspensions were washed in PBS, centrifuged for
`5 minutes (1600 rpm), and resuspended in 500 ␮l of PBS before
`flow cytometry analysis. For HLA DR assessment, cells were
`additionally incubated for 30 minutes with the secondary anti-
`mouse immunoglobulins in a 1:50 dilution, centrifuged in PBS
`(1600 rpm, 5 minutes), and resuspended in 500 ␮l of PBS for flow
`cytometry processing, performed with an Epics-XL (Beckman
`Coulter, Miami, FL).
`
`Flow Cytometry Analysis
`Cells were analyzed from a 2-parameter histogram showing side
`scatter (cell size) versus forward scatter (cell granulometry) in
`logarithmic and linear modes, respectively. For each antibody, at
`least 1000 conjunctival cells were gated to give a logarithmic
`fluorescence histogram showing the number of cells as a function
`of fluorescence intensities. A cursor was first set at the highest
`fluorescence level obtained for each isotypic negative control,
`which determined the limit of background fluorescence and the
`threshold of positivity for the tested antibodies. Results were
`obtained in percentages of positive cells and in mean fluorescence
`
`2188
`
`intensities. Mean fluorescence intensity levels of HLA DR and the
`3 interleukins tested were further quantified by the Quantitative
`Indirect Fluorescence Intensity (Dako) and Dakofluorosphere
`(Dako) methods, respectively, for indirect and direct immunoflu-
`orescence techniques. Calibration curves were then obtained, giv-
`ing mean fluorescence intensities versus the number of molecules
`of bound antibody, thus defining arbitrary units of fluorescence
`(AUFs). The actual number of AUFs for a specific marker was
`then obtained by subtraction of that found for the corresponding
`isotypic negative control. All flow cytometric analyses were per-
`formed in a masked manner for treatment groups and patient
`characteristics. Statistical comparisons were performed using the
`nonparametric Mann–Whitney U test.31
`
`Results
`
`The mean percentage of HLA DR–positive conjunctival cells (Fig
`1A) was higher in both the preserved and the multitreatment
`groups (46.4% and 63.4%, respectively) than in the control group
`(13.1%; P⬍0.001 for both groups) and the eyes receiving preser-
`vative-free timolol (19.3%; Pⱕ0.03 for both groups). No differ-
`ence was found between the control and preservative-free groups.
`Although the mean percentage of HLA DR–positive cells was
`higher in the multitreatment group than in the preservative group,
`the difference did not reach significance. The mean levels of HLA
`DR expression given in AUFs showed similar results (Fig 1B),
`with values in the preservative treatment and multitreatment
`groups (47 784 and 77 406 AUFs, respectively) significantly
`higher than those in the control group (5119 AUFs; P⬍0.01 for
`both groups) and the eyes not treated with preservatives (29 480
`AUFs; Pⱕ0.03 for both groups). There was also a significant
`difference between the control and preservative-free groups
`(P ⫽ 0.035).
`The result of IL-6 expression in percentage of positive cells
`(Fig 2A) showed a significant difference between the control group
`and the other 3 groups (P⬍0.001), without a significant difference
`between the 3 glaucoma groups. Similarly, IL-6 intracellular ex-
`pression levels assessed in AUFs (Fig 2B) showed a highly sig-
`nificant increase in the 3 glaucoma groups as compared with
`control normal eyes (P⬍0.001), with no significant difference
`between the 3 groups receiving antiglaucoma treatments, despite a
`slight nonsignificant increase in the multitreatment group.
`The preservative-free, preservative-containing, and multitreat-
`ment groups also had IL-8 expression significantly higher than that
`of the control group (Fig 3), both in percentage of positive cells
`(P⬍0.001 for the 3 groups) and in levels of expression (P⬍0.001
`for the 2 preservative-containing groups; P ⫽ 0.01 for the preser-
`vative-free group). Arbitrary unit of fluorescence results, however,
`showed higher levels in the multitreatment group than in the
`preservative-free one (P ⫽ 0.03).
`The mean percentage of IL-10 –positive conjunctival cells (Fig
`4) was higher in the preservative and multitreatment groups than in
`the control group (Pⱕ0.006 for both comparisons), but not in the
`preservative-free group. Levels of intracellular expression simi-
`larly reached a significant difference between the 2 preservative-
`containing groups and normal eyes (P⬍0.05 for both groups).
`
`Discussion
`
`In previous studies on ocular surface immunopathology,
`some evidence has suggested that conjunctival epithelial
`cells may play an active role in ocular inflammation.28 –30
`Conjunctival epithelial cells have therefore previously been
`
`

`

`Baudouin et al
`
`䡠 Conjunctival Cytokines and Antiglaucoma Drugs
`
`Figure 1. Results of flow cytometry in impression cytology specimens (means and standard errors). A, Percentage of HLA DR–positive cells in normal
`eyes and glaucomatous patients. §Pⱕ0.03, compared with the preservative-free group. **P⬍0.001, compared with controls. B, Level of expression,
`expressed in arbitrary units of fluorescence (AUF). §Pⱕ0.03, compared with the preservative-free group. *P ⫽ 0.035, compared with controls. **P⬍0.01,
`compared with controls.
`
`shown to express immune-related markers29,32 and to be a
`possible source for proinflammatory cytokines.33,34 Normal
`conjunctival epithelial cells express mRNA for IL-1, IL-6,
`IL-8, tumor necrosis factor ␣ (TNF-␣), and regulated-on-
`activation normal T-cell expressed and secreted (RANTES)
`protein.35,36 However, ICAM-1 and HLA DR are not ex-
`pressed by normal conjunctival epithelial cells,26,27,36 but
`are induced to be expressed at high levels on conjunctival
`epithelial cells in inflammatory conditions.29,37 In the
`present study, we used a reliable and objective technique of
`flow cytometry in impression cytology to investigate the
`expression of 3 major interleukins by conjunctival cells. We
`also analyzed HLA DR expression as a hallmark for con-
`junctival inflammatory status and confirmed the overexpres-
`sion of this immune marker in glaucoma patients receiving
`long-term treatments, consistent with a previous similarly
`conducted case– control study.12 Our results also confirmed
`that preserved drugs, especially when ⱖ2 drugs are associ-
`ated, induced significantly higher HLA DR expression than
`did unpreserved ␤-blockers. Human leukocyte antigen DR
`expression was slightly more elevated in the preservative-
`
`free group than in the nontreated eyes, which suggests a low
`level of subclinical inflammation induced by topical treat-
`ment, even though the active compound timolol was found
`in previous in vitro studies not or almost not to be toxic.15
`The mechanisms by which topical treatments may induce
`HLA DR expression are not fully determined, but it is likely
`that in the ocular surface proapoptotic and proinflammatory
`cytokines, including interferon ␥and TNF-␣, may stimulate
`common pathways.26
`The conjunctival profile of interleukin expression, how-
`ever, differed from those of HLA DR and ICAM-1.12 Ex-
`cept for an increase in IL-8’s level of expression (Fig 3B) in
`the preservative-containing groups as compared with the
`preservative-free group, IL-6, IL-8, and IL-10 were simi-
`larly overexpressed in all glaucoma groups, whatever the
`type of treatment received. This would suggest that inter-
`leukin expression by conjunctival cells is dependent upon
`immune mechanisms and pathways other than those influ-
`encing adhesion molecules and class II antigens.
`As HLA DR, IL-6 is known to be secreted in response to
`TNF-␣. Although IL-6 can be found in the normal eye, it is
`
`Figure 2. Expression of interleukin 6 (IL-6) by conjunctival cells. A, Percentage of positive cells. B, Levels of intracellular expression. AUF ⫽ arbitrary
`units of fluorescence. **P⬍0.001, compared with controls.
`
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`

`Ophthalmology Volume 111, Number 12, December 2004
`
`Figure 3. Expression of interleukin 8 (IL-8) in the conjunctival epithelium. A, Percentage of positive cells. B, Levels of intracellular expression. AUF
`⫽ arbitrary units of fluorescence. *P⬍0.01, compared with controls. **P⬍0.001, compared with controls. §P ⫽ 0.03, compared with the preservative-free
`group.
`
`generally considered to be an inflammatory cytokine. Jones
`et al35 and Pflugfelder et al38 observed a very high expres-
`sion of mRNA encoding IL-6 and IL-8, as well as HLA DR,
`ICAM-1, TNF-␣, IL-1␣, IL-1␤, and transforming growth
`factor ␤1, in the conjunctival epithelium of Sjögren’s syn-
`drome eyes as compared with normal eyes using the reverse
`transcriptase–polymerase chain reaction method in impres-
`sion cytology. Human leukocyte antigen DR and IL-6 gene
`expressions were also found at higher levels in Sjögren’s
`syndrome conjunctival cells than in normal conjunctiva.37
`Interleukin 6 and IL-8 have been shown to be present in
`normal tears39 and to be expressed abundantly in pterygium
`epithelium.40 In this latter study, mRNAs and proteins were
`stimulated by ultraviolet radiation in a time- and dose-
`dependent manner, thus showing the influence of external
`noninflammatory stimulations on these cytokines.40 Other
`models of ocular surface diseases, such as corneal and
`limbal debridement or vernal keratoconjunctivitis, demon-
`strated an overexpression of proinflammatory cytokines and
`markers, including IL-6 or ICAM-1.41,42
`In addition to IL-6, we found overexpression of 2 other
`
`major interleukins, IL-8 and IL-10. Interleukin 8 has pow-
`erful chemotactic effects on T lymphocytes and neutrophils
`and may lead to inflammatory reactions. Little is known
`about IL-8’s role in ocular surface diseases, but significantly
`increased levels were found in the conjunctival epithelium
`of Sjögren’s syndrome patients36 and in chronic allergic
`disorders,37,43,44 as compared with normal eyes. Interleukin
`10, a cytokine of the Th2 profile, mainly acts as an inhibi-
`tory factor to restrain proinflammatory cytokines produced
`by inflammatory cells.45 In ocular surface tissues, T cells
`found in atopic keratoconjunctivitis were shown to produce
`increased levels of IL-10 in comparison with those from
`vernal keratoconjunctivitis and giant papillary conjunctivitis
`groups.46
`In our study, not only the expression of HLA DR and
`inflammatory cytokines (IL-6, IL-8) but also expression of
`the theoretically inhibitory factor IL-10 were higher in
`preserved and multitreatment groups than in the control
`group composed of normal untreated eyes. Interleukin ex-
`pression could be hypothesized as a global reaction to toxic
`drugs administered for long periods of time to the ocular
`
`Figure 4. Expression of interleukin 10 (IL-10) in the conjunctival epithelium. A, Percentage of positive cells. B, Levels of intracellular expression. AUF
`⫽ arbitrary units of fluorescence. *P⬍0.05, compared with normal eyes. **Pⱕ0.006, compared with normal eyes.
`
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`Baudouin et al
`
`䡠 Conjunctival Cytokines and Antiglaucoma Drugs
`
`surface. However, the conjunctival profiles of HLA DR and
`the 3 interleukins seemed to differ, and further studies will
`be necessary better to understand the roles of immune
`markers and chemotactic, proinflammatory, or inhibitory
`cytokines in conjunctival
`immunopathology. Indeed,
`in
`vitro studies in conjunctival epithelial cells showed that
`release of proinflammatory cytokines may be stimulated by
`different mediators, as TNF-␣ and IL-1␤ are potent stimu-
`lators of IL-8, but not of ICAM-1 and HLA DR, whereas
`interferon ␥ has the inverse profile.32,47 Histamine also
`stimulates the secretion of IL-6 and IL-8 by conjunctival
`epithelial cells in a dose- and time-dependent manner,35
`which confirms the role of the conjunctival epithelium in
`inflammatory and/or allergic disorders and illustrates the
`complex pathways leading to cytokine expression in the
`ocular surface.
`An explanation for our results in glaucoma patients could
`therefore be that IL-6, IL-8 and IL-10 are highly sensitive to
`any proinflammatory stimulation, including the weakly toxic
`preservative-free ␤-blockers, whereas HLA DR and ICAM-1
`may require higher levels of stimulation. Indeed, it has been
`shown that closed-eye tears contain high levels of IL-6 and
`IL-8 in comparison with open-eye tears,48 which may demon-
`strate that these cytokines can be easily overexpressed in
`pathophysiological conditions. It is also possible, however, that
`these interleukins are released after inflammatory or toxic
`challenges in a soluble form in the tear flow and that the high
`levels of expression in the subcellular compartment do not
`reflect the actual amounts of cytokines present in tears and
`globally synthesized by conjunctival cells. Further studies
`combining cellular and tear concentrations would be of interest
`to try to discriminate between the different groups of glaucoma
`treatments, especially in differentiating preservative-free and
`preservative-containing topical drugs.
`Nevertheless, subclinical inflammation may raise further
`serious ocular surface issues in glaucoma patients, espe-
`cially at the time of filtering surgery.9 Care should be taken
`to avoid preservatives in a long-term use as much as pos-
`sible and to limit their concentration, as their toxicity is
`dose- and time-dependent,15 or to develop new nontoxic
`preservatives49 capable of reducing allergic reactions and
`improving ocular surface tolerance.
`
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