Current Eye Research, 2015; 40(1): 40–47 ! Informa Healthcare USA, Inc. ISSN: 0271-3683 print / 1460-2202 online DOI: 10.3109/02713683.2014.915574
ORIGINAL ARTICLE
Increased Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) Expression in the Conjunctival Epithelium Exposed to Antiglaucoma Treatments
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Antoine Labbe´1,2,3,4,5*, Eric Gabison3,4,5,6,7*, Franc¸oise Brignole-Baudouin3,4,5,8, Luisa Riancho3,4,5, Suzanne Menashi6, and Christophe Baudouin1,2,3,4,5 1
Department of Ophthalmology, Quinze-Vingts National Ophthalmology Hospital, Paris and Ambroise Pare´ Hospital, AP-HP, Boulogne-Billancourt, France, 2Centre Hospitalier National d’Ophtalmologie des Quinze-Vingts, INSERM-DHOS CIC 503, Paris, France, 3INSERM, U968, Paris, France, 4Institut de la Vision, UPMC Univ Paris 06, UMR_S 968, Paris, France, 5CNRS, UMR_7210, Paris, France, 6CRRET, (EAC CNRS 7149), UPEC, Universite´ PARIS EST, Cre´teil, France, 7Rothschild Ophthalmologic Foundation, Bichat Hospital, AP-HP, Paris, France, and 8Faculte´ des Sciences Pharmaceutiques et Biologiques, Sorbonne Paris Cite´ University, Paris, France
ABSTRACT Objective: To analyze the effect of preserved antiglaucoma eye drops on the expression of extracellular matrix (ECM) metalloproteinase inducer (EMMPRIN) in conjunctival epithelial cells. Methods: A total of 18 patients treated for primary open-angle glaucoma with benzalkonium chloride (BAK) preserved eye drops and eight age-matched controls were included in this study. Glaucoma patients were divided into two groups according to their daily exposure to BAK: high-exposure (HE) group and low-exposure (LE) group. HLA-DR and EMMPRIN were quantified on conjunctival impression cytology specimens using flow cytometry. In parallel, IOBA-NHC conjunctival epithelial cells were exposed to different BAK concentrations, in the presence or absence of cyclosporine A (CsA), and their total and surface expressions of EMMPRIN were assessed by flow cytometry and results are given in relative fluorescence intensities (RFIs). Results: Compared to the control group (1.71 ± 0.39 RFI), EMMPRIN was significantly increased in the HE (4.19 ± 1.50 RFI, p50.001) and LE groups (2.55 ± 0.40 RFI, p = 0.029). Similar increase was observed in HLA-DR expression in the HE (4.58 ± 1.38 RFI, p50.001) and LE groups (2.52 ± 0.47 RFI, p = 0.046) as compared to control subjects (1.75 ± 0.27 RFI). Across all subjects enrolled in the study, there was a significant correlation between HLA-DR and EMMPRIN (R2 = 0.875, p50.0001). IOBA-NHC cells exposed to BAK presented a significant increase in EMMPRIN, which was proportional to the concentration of BAK. The surface expression of EMMPRIN was inhibited by CsA. Conclusions: The increased expression of EMMPRIN in patients topically treated with multiple antiglaucoma BAK-preserved eye drops suggests a matrix metalloproteinase-related modification of conjunctival ECM remodeling. In vitro results suggest that CsA has the potential to limit BAK effects on EMMPRIN. Keywords: Antiglaucoma treatment, benzalkonium chloride, conjunctiva, EMMPRIN, preservatives
*These authors contributed equally to this work. Received 18 November 2013; revised 27 March 2014; accepted 8 April 2014; published online 14 May 2014 Correspondence: Antoine Labbe´, MD, Service d’Ophtalmologie III, C.H.N.O. des Quinze-Vingts, 28 rue de Charenton, 75012 Paris, France. E-mail: [email protected]
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EMMPRIN Conjunctival Expression in Glaucoma
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INTRODUCTION Most patients with chronic glaucoma or ocular hypertension (OHT) are treated medically by the use of topical intraocular pressure (IOP)-lowering drugs. However, chronic use of these drugs is known to cause significant changes on the ocular surface.1 In conjunctival biopsy specimens, the long-term use of antiglaucoma eye drops was shown to result in a significant decrease in the number of goblet cells and an increase in the number of macrophages, lymphocytes, mast cells and fibroblasts.2 An increase in subepithelial collagen deposition was also demonstrated.3 Similarly, several studies using impression cytology (IC) have demonstrated an increased expression of inflammatory markers by conjunctival epithelial cells of patients receiving antiglaucoma eye drops.4–8 These ocular surface side effects have a direct impact on patient quality of life, which may limit patient adherence to the glaucoma or OHT treatments and eventually impair the success of glaucoma filtering surgery. Indeed, by stimulating inflammatory cells and fibroblasts in the conjunctiva, long-term topical antiglaucoma therapy has been identified as a significant risk factor for filtration surgery failure.9 Several studies have shown that preservatives, such as benzalkonium chloride (BAK), which are used to preserve stability and/or sterility of active drugs in multidose ophthalmic preparations, could be at least, in part, responsible for these ocular surface side effects.10 Matrix metalloproteinases (MMP) are a family of proteolytic enzymes catalyzing the degradation of extracellular matrix (ECM). During wound healing, MMP and their inhibitors, tissue inhibitors of metalloproteinases (TIMP), play a central role in ECM remodeling, cell–matrix interactions, inflammatory cell recruitment, cytokine activation and regulation of angiogenesis.11,12 Deregulation of MMP and TIMP was suggested to be associated with abnormal ECM turnover in numerous diseases including fibrotic disorders.13 MMP are tightly regulated by complex mechanisms that include cytokines and growth factors, cell-matrix and cell–cell interactions. ECM metalloproteinase inducer (EMMPRIN/CD147), a highly glycosylated cell surface transmembrane protein, was originally identified on tumor cell surfaces as an inducer of MMP production in neighboring fibroblasts.14 The presence and modulation of EMMPRIN in a variety of tissues associated with increased MMP expression suggests that this EMMPRIN-mediated MMP induction could also be a common mechanism in non-tumoral physiological and/or pathological situations.14 We and others have already described the presence of EMMPRIN in ocular tissues including conjunctival epithelium, corneal epithelium, endothelium and stromal keratocytes.11,15 It was also shown to be !
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involved in corneal wound healing and corneal ulcers11 and more recently in dry eye.16 The objective of this study was to analyze the expression of EMMPRIN by conjunctival epithelial cells ex vivo, in patients treated for glaucoma with BAK-preserved eye drops and in vitro, under the influence of BAK and cyclosporine.
PATIENTS AND METHODS Ex Vivo Study Patients A total of 18 patients treated for primary open-angle glaucoma (POAG) and eight control subjects were included in this study. The study was conducted at the Center for Clinical Investigations (INSERM CIC 503) of the Quinze-Vingts National Ophthalmology Hospital, Paris, France, with the approval of the Institutional Review Board of Saint-Antoine University Hospital (CPP-Ile de France 5, number 10793) and in compliance with the declaration of Helsinki. All glaucoma patients had POAG without known ocular surface disease prior to the use of antiglaucoma eye drops. Patients were subjected for at least six months to the same treatment in one or both eyes. Antiglaucoma eye drops were prostaglandin analogs, beta-blockers, alpha-agonists and carbonic anhydrase inhibitors, used separately or in fixed combinations. All commercially available preparations of these antiglaucoma drugs contained BAK with concentration ranging from 0.02% to 0.005%. Glaucoma patients were divided into two groups according to the number of instillations of BAKpreserved eye drops they received everyday: the highexposure (HE) group (nine patients) received from three to five instillations of BAK-preserved eye drops per day; the low-exposure (LE) group (nine patients) were treated with only one or two instillations of BAK-preserved eye drops per day. Eight age-matched subjects who had no history of ocular disease or clinical ophthalmic abnormality as assessed by slitlamp examination, and who had not received any topical treatment for at least six months, were included as controls. One eye of each patient was evaluated. IC Collection Specimens were collected in the superior bulbar conjunctiva using 0.20 -mm polyether sulfone filters (Supor Membranes; Gelman Sciences, Ann Arbor, MI) after instillation of one drop of 0.04% oxybuprocaine (MSD-Chibret, Paris, France).4,17 At least 15 min after fluorescein instillation for ocular surface assessment, two pieces of filters, measuring 13 6.5 mm, were applied gently on the conjunctiva in two neighboring areas covered by the upper eyelid to collect a
42 A. Labbe´ et al.
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homogeneous population of superficial conjunctival cells. The membranes were then immediately fixed in cold phosphate buffered saline (PBS) (pH 7.4; 4 C) containing 0.05% paraformaldehyde. Flow Cytometry Analysis Conjunctival cells were extracted from their support by mechanical agitation and then centrifuged at 2000 rpm for 5 min before immunofluorescence and flow cytometry analysis.18 An indirect immunofluorescence technique was used to evaluate the expression of HLA-DR and EMMPRIN by conjunctival epithelial cells. Mouse anti-HLA-DR antibody (Dako, Glostrup, Denmark) and goat anti-EMMPRIN antibody (Santa Cruz Biotechnology, Santa Cruz, CA) were used at 1:50 and 1:100 dilutions, respectively, in PBS containing 1% bovine serum albumin (Sigma-Aldrich, St Louis, MO). Both primary antibodies were incubated with conjunctival cells for 45 min in the dark at room temperature and were washed in PBS by a 5 min centrifugation. Cells were then incubated with a 1:50 dilution of a fluorescein isothiocyanate (FITC)conjugated goat anti-mouse antibody (Dako) or a FITC-conjugated rabbit anti-goat antibody (Dako).
Non-immune mouse (Dako) and goat (RD Biotech, Besanc¸on, France) corresponding antibodies were, respectively, used as negative isotypic controls. After incubation, cells were washed in PBS by a 5-min centrifugation and suspended in 250 ml of PBS before analysis with a flow cytometer (FC500-CXP, Beckman Coulter, Miami, FL) according to previously validated methods.17,19 All specimens were analyzed in a masked manner with respect to patient characteristics. For each antibody, a sample of at least 2000 conjunctival cells was analyzed (Figure 1). The results were expressed as relative fluorescence intensity (RFI) corresponding to the ratio of the mean fluorescence intensity (MFI) obtained for the specific antibody to the MFI obtained for the isotypic control.
In Vitro Study Conjunctival Cell Line and Treatments The IOBA-NHC cells, established from a spontaneously immortalized conjunctival epithelial cell line, were cultured under standard conditions (humidified atmosphere of 5% CO2 at 37 C) in DMEM/F12
FIGURE 1 Flow cytometry images of a typical case of impression cytology specimen (A and B) and of conjunctival IOBA cells after incubation with BAK 106% (C and D). (A and C) Conjunctival cells were gated from a biparametric diagram showing cell size in linear mode versus cell structure in logarithmic mode, in order to avoid debris (d) and cell aggregates (a). (B and D) Fluorescence histograms obtained from the conjunctival cell population gated in the biparametric diagram. Shown in grey line is the isotypicmatched negative control and in black line, the EMMPRIN expression. Current Eye Research
EMMPRIN Conjunctival Expression in Glaucoma
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supplemented with 1 mg/ml bovine pancreas insulin, 2 ng/ml mouse epidermal growth factor, 0.1 mg/ml cholera toxin, 5 mg/ml hydrocortisone, 10% fetal bovine serum, 50 UI/ml penicillin and 50 UI/ml streptomycin, as previously described.20 Cells from passages 5–20 were seeded into six-well culture plates (Corning, Schiphol-Rijk, The Netherlands) and subconfluent cells (approximately 70%) were exposed for 24 h to BAK solutions at 105%, 106% and 107% in PBS. Similar BAK treatment was also performed after a prior 24-h incubation of cells with cyclosporine A (CsA) (Sigma-Aldrich) at 5 mg/ml. Cells were then detached by a 10-min incubation with 1 mM ethylenediaminetetraacetic acid, collected and suspended in 1 ml of paraformaldehyde 0.5% in PBS.20 Flow Cytometry Analysis of Cultured Conjunctival Cells To analyze the total expression of EMMPRIN (surface and intracellular), cells were first permeabilized with 0.1% saponin for 5 min before incubation with the first antibody. The surface expression of EMMPRIN was examined by performing similar experiments without prior permeabilization. For each antibody, samples of at least 2000 conjunctival cells were analyzed (Figure 1). The results were expressed in RFI. Each experiment involving cell stimulation and immunostaining was independently performed in triplicate.
Statistical Analysis Statistical values of RFI were calculated in each group and for each marker and were presented as mean ± standard deviation. Kruskal–Wallis test was used to compare between groups. The correlation between HLA-DR and EMMPRIN was analyzed using Pearson’s correlation coefficient. Probability values less than 0.05 were considered significant. All statistical analyses were performed using XLSTAT 2011Õ (Addinsoft, Paris, France).
RESULTS Ex Vivo Study IC was performed in patients treated for glaucoma with BAK-preserved eye drops in order to analyze ex vivo the expression of EMMPRIN by conjunctival epithelial cells. Mean age of patients was 63.33 ± 12.74 years in the HE group (five women and four men), 61.3 ± 15.38 years in the LE group (five women and four men) and 67.12 ± 13.67 years in the control group (five women and three men). There was no statistical difference in terms of age and sex ratio between the three groups !
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FIGURE 2 Relative fluorescence intensities (RFIs) of EMMPRIN and HLA-DR on conjunctival epithelial cells in the highexposure (HE) group, low-exposure (LE) group and control subjects. Error bars represent standard deviation. *compared to the LE group, p = 0.029; **compared to the control group, p50.001; #compared to the LE group, p = 0.01; ##compared to the control group, p50.001; +compared to the control group, p = 0.019; and ++compared to the control group, p = 0.046.
(p40.05). Mean number of BAK-preserved eye drops instillation was 3.55 ± 0.72 in the HE group and 1.44 ± 0.52 in the LE group. Mean duration of treatment was 47.11 ± 40.63 months (12–120 months) and 27.7 ± 26.02 months (7–66 months) in the HE and LE groups, respectively. In IC specimens, RFI of the inflammatory marker HLA-DR was significantly raised in the HE group (4.58 ± 1.38) compared to the LE group (2.52 ± 0.47, p = 0.01) and control subjects (1.75 ± 0.28, p50.001). The difference between control subjects and the LE group was also significant (p = 0.046) (Figure 2). Similarly, the RFI of EMMPRIN was significantly increased in the HE group (4.18 ± 1.49) compared to the LE (2.55 ± 0.41, p = 0.029) and the control groups (1.84 ± 0.54, p50.001). As observed for HLA-DR, the difference between control subjects and the LE group was also significant (p = 0.019) (Figure 2). Across all subjects enrolled in the study, there was a significant correlation between conjunctival cells fluorescence levels of HLA-DR and EMMPRIN (R2 = 0.875, p50.0001) (Figure 3).
In Vitro Study Conjunctival cells exposed to BAK 105% showed a greater increase in the RFI for the total EMMPRIN expression (3.67 ± 1.05) as compared to cells receiving BAK 107% (1.77 ± 0.21, p = 0.003) or the control group (1.55 ± 0.11; p50.0001). Similarly, cells stimulated with BAK 106% (2.49 ± 0.64) had a significant RFI increase
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44 A. Labbe´ et al.
FIGURE 3. EMMPRIN and HLA-DR expressions on conjunctival epithelial cells in each patient, given in relative fluorescence intensities (RFIs). A statistically significant correlation was found between EMMPRIN and HLA-DR expressions (R2 = 0.875; p50.0001).
of the total EMMPRIN expression as compared to the control group (p = 0.023). No change was observed after BAK 107% stimulation. Similar results were observed for the surface expression of EMMPRIN with a significant RFI increase of EMMPRIN in conjunctival cells stimulated with BAK 105% (2.62 ± 1.23, p50.0001 compared to BAK 107% (1.10 ± 0.06) and p = 0.012 compared to the control group (1.05 ± 0.05)) and BAK 106% (1.54 ± 0.12, p = 0.033 compared to the control group) (Figure 4). No difference in the surface expression of EMMPRIN was observed after BAK 107% stimulation. When conjunctival cells were treated with CsA prior to BAK exposure, a significant RFI increase of the total expression of EMMPRIN was observed after BAK 105% (2.55 ± 1.03) and BAK 106% stimulation (1.81 ± 0.18) compared to cells exposed to PBS (1.04 ± 0.04, p = 0.032 and p = 0.040 compared to the BAK 105% and BAK 106% groups, respectively). Importantly, CsA suppressed the increase in the surface expression of EMMPRIN by BAK whatever the concentration used, as no significant difference in the surface expression of EMMPRIN was observed between the different groups (p40.05 for all comparisons) (Figure 4). The surface expression of EMMPRIN was also significantly lower after BAK 105% and BAK 106% exposure in the groups treated with CsA as compared to cells not treated with CsA (p = 0.007 and p = 0.024, respectively) (Figure 4).
FIGURE 4 Total and surface expression in relative fluorescence intensity (RFI) of EMMPRIN by conjunctival epithelial cells in vitro after 24 h of BAK stimulation. Error bars represent standard deviation. Without cyclosporin A (CsA) treatment: *compared to the control group, p50.0001; **compared to BAK 107% group, p = 0.003; #compared to the control group, p50.0001; ##compared to BAK 107% group, p = 0.012; +compared to the control group, p = 0.023; and ++compared to the control group, p = 0.033. After CsA treatment: xcompared to the control group, p = 0.032; xxcompared to the control group, p = 0.040. zcompared to BAK 106% group without CsA, p = 0.007; and zzcompared to BAK 105% group without CsA, p = 0.024.
Current Eye Research
EMMPRIN Conjunctival Expression in Glaucoma
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DISCUSSION Numerous studies have demonstrated that antiglaucoma topical medications and their preservatives induce significant histopathologic and inflammatory changes in the ocular surface.1,10 In agreement with previous reports, we describe in this study an increased expression of the major histocompatibility complex class II antigen HLA-DR, often used as a marker of inflammation, on conjunctival epithelial cells in patients chronically treated with multiple BAK-preserved antiglaucoma eye drops, hence confirming the presence of conjunctival inflammation.4–6,8,17,18 This increase in HLA-DR expression was highly correlated (R2 = 0.875) with the increase in EMMPRIN membrane expression. As EMMPRIN was shown to be able to stimulate the production of several MMP (MMP-1, MMP-2, MMP-3 and MMP-9) in pathological situations including corneal ulcers21 and dry eye; 16our results suggest an MMP-mediated modification of the conjunctival ECM remodeling in patients topically treated for glaucoma or OHT with high exposure to BAK. Conjunctival wound healing is a major determinant for the success of glaucoma filtering surgery, and glaucoma topical treatments are well-known risk factors for surgery failure by subconjunctival fibrosis.1,9,10 Conjunctival morphological tissue changes induced by the use of antiglaucoma eye drops, and their preservatives are associated with the expression of inflammatory markers, free radical production and deregulation of MMP (MMP-1, MMP-3 and MMP-9) and TIMPs (TIMP-1, TIMP–2 and TIMP-3) expression.1,22–25 Although the mechanism by which these factors lead to fibrosis is not fully understood, MMP are known to play a central role in wound healing, and their overexpression in ocular tissues has already been associated with excessive scarring in the eye.26,27 Furthermore, Wong et al. demonstrated that the healing process following glaucoma surgery could be modulated by inhibiting the effects of MMP.28,29 In this study, we observed an overexpression of EMMPRIN, an MMP inducer, on the conjunctival epithelial cells of patients using BAKpreserved eye drops. Consistently, on conjunctival biopsies from patients treated with preserved latanoprost, Terai et al.25 showed an upregulation of MMP-1 and MMP-3 in both conjunctival epithelial cells and subepithelial stromal cells, which was associated with a decreased stromal collagen density. Similarly, Honda et al. observed an increased expression of MMP-1 and MMP-9 in conjunctival epithelial cells as well as in tears.24 However, on Tenon’s capsule fibroblasts obtained from patients under multiple antiglaucoma treatments, Leng et al. have shown a down regulation of MMP-9 and MMP-3, which was associated with a higher !
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expression of transforming growth factor (TGF)-b.22 This difference may be due to the fact that superficial epithelial cells, unlike fibroblasts, are in direct contact with the eye drops. It may also be influenced by the type of antiglaucoma eye drops and preservatives used, as two previous studies, one in humans25 and one on a rat model,30 showed different MMP responses to the different type of eye drops. However, as no control group using unpreserved eye drops was evaluated in this study, we are not able, at present, to differentiate the effects of BAK from the active compounds. Similarly, as patients were using different types of IOPlowering medications with multiple combinations possible, it was not possible to perform subgroup analysis. Another limitation resides in the difference of the duration of treatments between the two groups (HE and LE). Nevertheless, Broadway et al. in a study evaluating conjunctival changes induced by antiglaucoma medications observed that both the number of medications and the duration of treatment of glaucoma had adverse effects on the conjunctival cell profile, patients with multiple drug therapy having the greatest changes.2 Similarly, in a recent study, we showed that the daily number of instillations of preserved eye drops could be a more important factor in ocular surface tissue changes induced by antiglaucoma medications than the duration of treatment.31 With these limitations in mind, our results suggest that, in addition to the inflammatory changes, an abnormal expression of MMP and their modulators, such as EMMPRIN, also contributes to the postoperative excessive scarring response. The clinical observations of Schwab et al. showing conjunctival fibrosis with foreshortening of the conjunctival fornix in glaucoma patients receiving topical medications is also in line with these findings.3 Although inflammatory cytokines have been considered as the main regulators of the wound healing process, it appears that interactions between these cytokines and other mediators may have a more important role in modulating the wound healing process than the inflammatory cytokines themselves.21 As an example, TGF-b and interleukin (IL)-1 are two cytokines produced during inflammatory reactions, with TGF-b being a potent stimulator of connective tissue formation,32 while IL-1 provokes tissue degradation through the upregulation of matrix degrading proteinases,33 suggesting that another level of regulation may exist. Interestingly, in the cornea, interactions between TGF-b, IL-1 and EMMPRIN have been demonstrated.21 The high correlation observed in this study between the conjunctival epithelial cells expression of HLA-DR and EMMPRIN suggests an association between inflammation and tissue remodeling in patients treated with antiglaucoma eye drops.
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46 A. Labbe´ et al. BAK, a nitrogenous cationic surface-acting agent belonging to the quaternary ammonium group, is the most commonly used preservative in ophthalmic preparations.10 Many studies have demonstrated that preservatives and BAK, in particular, are responsible, at least in part, for the inflammatory and toxic effects observed during long-term use of antiglaucoma eye drops.10 In a previous study, we demonstrated a dose-dependent toxicity of BAK in IOBA-NHC cells, supporting the use of this cell line for toxicological in vitro studies.20 Using the same model, we show that BAK could upregulate, in a dose-dependent manner, both total and membrane expression of EMMPRIN by epithelial conjunctival cells in vitro. Since BAK was shown to induce IL-1 and TNF in conjunctival and corneal cells in vitro34 and considering the known interactions between EMMPRIN and pro-inflammatory cytokines, these in vitro results emphasize the implications of BAK in the conjunctival activation induced by preserved antiglaucoma eye drops. It has been previously shown that the cell surface expression of EMMPRIN was significantly reduced by the cyclophilin-binding drug CsA.35 Yurchenko et al. demonstrated that cyclophilin 60 was involved in the cell trafficking of EMMPRIN36 as the treatment of tumor cells with CsA reduced cell surface expression of EMMPRIN without altering the total cellular level.35 In our toxicological model, CsA also downregulated the surface expression of EMMPRIN induced by BAK on epithelial conjunctival cells. As EMMPRIN is a transmembrane protein that exerts MMP-inducing effects by its extracellular portion, the blockage of EMMPRIN trafficking within conjunctival epithelial cells and consequently the cell membrane expression of EMMPRIN by CsA may represent an MMP inhibiting effect.37 Nevertheless, these results need to be confirmed on a stratified conjunctival epithelium and with different in vitro techniques evaluating, in particular, EMMPRIN mRNA levels. Interestingly, cyclosporin A was successfully tested in vitro for its antiproliferative properties on fibroblasts,38 and a randomized clinical trial evaluating the benefit of cyclosporin A in glaucoma filtering surgery have shown promising results.39 Hence, CsA, by inhibiting EMMPRIN cell membrane expression, may represent an interesting new option to modulate MMP expression in the ocular surface before surgery, particularly in patients using multiple preserved eye drops. Although the role of EMMPRIN in conjunctival changes induced by preserved eye drops, and the effects of CsA on its expression, should be further evaluated on better defined patients subpopulations and using other techniques, it opens new fields of research in order to increased long-term success of filtering surgery in particular after many years of antiglaucoma treatments.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This work was supported by the ‘‘Institut National de la Sante´ et de la Recherche Me´dicale’’ (INSERM) and Universite´ Pierre & Marie Curie, Paris, France.
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EMMPRIN Conjunctival Expression in Glaucoma 15. Maatta M, Tervahartiala T, Kaarniranta K, Tang Y, Yan L, Tuukkanen J, et al. Immunolocalization of EMMPRIN (CD147) in the human eye and detection of soluble form of EMMPRIN in ocular fluids. Curr Eye Res 2006;31:917–924. 16. Huet E, Vallee B, Delbe J, Mourah S, Prulie`re-Escabasse V, Tremouilleres M, et al. EMMPRIN modulates epithelial barrier function through a MMP-mediated occludin cleavage: implications in dry eye disease. Am J Pathol 2011;179: 1278–1286. 17. Baudouin C, Liang H, Hamard P, Riancho L, CreuzotGarcher C, Warnet JM, et al. The ocular surface of glaucoma patients treated over the long term expresses inflammatory markers related to both T-helper 1 and T-helper 2 pathways. Ophthalmology 2008;115:109–115. 18. Brignole-Baudouin F, Ott AC, Warnet JM, Baudouin C. Flow cytometry in conjunctival impression cytology: a new tool for exploring ocular surface pathologies. Exp Eye Res 2004;78:473–481. 19. Baudouin C, Liang H, Bremond-Gignac D, Hamard P, Hreiche R, Creuzot-Garcher C, et al. CCR 4 and CCR 5 expression in conjunctival specimens as differential markers of T(H)1/T(H)2 in ocular surface disorders. J Allergy Clin Immunol 2005;116:614–619. 20. Brasnu E, Brignole-Baudouin F, Riancho L, Warnet JM, Baudouin C. Comparative study on the cytotoxic effects of benzalkonium chloride on the Wong-Kilbourne derivative of Chang conjunctival and IOBA-NHC cell lines. Mol Vis 2008;14:394–402. 21. Gabison EE, Huet E, Baudouin C, Menashi S. Direct epithelial-stromal interaction in corneal wound healing: role of EMMPRIN/CD147 in MMPs induction and beyond. Prog Retin Eye Res 2009;28:19–33. 22. Leng F, Liu P, Li H, Zhang J. Long-term topical antiglaucoma medications cause enhanced Tenon’s capsule fibroblast proliferation and abnormal TGF-beta and MMP expressions: potential effects on glaucoma filtering surgery. Curr Eye Res 2011;36:301–309. 23. Lopilly Park HY, Kim JH, Lee KM, Park CK. Effect of prostaglandin analogues on tear proteomics and expression of cytokines and matrix metalloproteinases in the conjunctiva and cornea. Exp Eye Res 2012;94:13–21. 24. Honda N, Miyai T, Nejima R, Miyata K, Mimura T, Usui T, et al. Effect of latanoprost on the expression of matrix metalloproteinases and tissue inhibitor of metalloproteinase 1 on the ocular surface. Arch Ophthalmol 2010;128: 466–471. 25. Terai N, Schlotzer-Schrehardt U, Lampel J, Bo¨hm AG, Rummelt C, Schmidt E, et al. Effect of latanoprost and timolol on the histopathology of the human conjunctiva. Br J Ophthalmol 2009;93:219–224. 26. Di Girolamo N, Lloyd A, McCluskey P, Filipic M, Wakefield D. Increased expression of matrix
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metalloproteinases in vivo in scleritis tissue and in vitro in cultured human scleral fibroblasts. Am J Pathol 1997; 150:653–666. Azar DT, Pluznik D, Jain S, Khoury JM. Gelatinase B and A expression after laser in situ keratomileusis and photorefractive keratectomy. Arch Ophthalmol 1998;116: 1206–1208. Wong TT, Mead AL, Khaw PT. Matrix metalloproteinase inhibition modulates postoperative scarring after experimental glaucoma filtration surgery. Invest Ophthalmol Vis Sci 2003;44:1097–1103. Wong TT, Mead AL, Khaw PT. Prolonged antiscarring effects of ilomastat and MMC after experimental glaucoma filtration surgery. Invest Ophthalmol Vis Sci 2005;46: 2018–2022. Ito T, Ohguro H, Mamiya K, Ohguro I, Nakazawa M. Effects of antiglaucoma drops on MMP and TIMP balance in conjunctival and subconjunctival tissue. Invest Ophthalmol Vis Sci 2006;47:823–830. Labbe A, Terry O, Brasnu E, Van Went C, Baudouin C. Tear film osmolarity in patients treated for glaucoma or ocular hypertension. Cornea 2012;31:994–999. Khaw PT, Occleston NL, Schultz G, Gierson I, Sherwood MB, Larkin G. Activation and suppression of fibroblast function. Eye (Lond) 1994;8:188–195. Girard MT, Matsubara M, Fini ME. Transforming growth factor-beta and interleukin-1 modulate metalloproteinase expression by corneal stromal cells. Invest Ophthalmol Vis Sci 1991;32:2441–2454. Epstein SP, Chen D, Asbell PA. Evaluation of biomarkers of inflammation in response to benzalkonium chloride on corneal and conjunctival epithelial cells. J Ocul Pharmacol Ther 2009;25:415–424. Yurchenko V, Pushkarsky T, Li JH, Dai WW, Sherry B, Bukrinsky M. Regulation of CD147 cell surface expression: involvement of the proline residue in the CD147 transmembrane domain. J Biol Chem 2005;280:17013–17019. Pushkarsky T, Yurchenko V, Vanpouille C, Brichacek B, Vaisman I, Hatakeyama S, et al. Cell surface expression of CD147/EMMPRIN is regulated by cyclophilin 60. J Biol Chem 2005;280:27866–27871. Gabison EE, Hoang-Xuan T, Mauviel A, Menashi S. EMMPRIN/CD147, an MMP modulator in cancer, development and tissue repair. Biochimie 2005;87:361–368. Cristofanilli M, Pescosolido N, Risuleo G, Scarsella G. A murine cell culture model for post-trabeculectomy anfibrotic treatment: induction of apoptosis by cyclosporin. Acta Ophthalmol Scand 2001;79:309–312. Turacli E, Gunduz K, Aktan G, Tamer C. A comparative clinical trial of mitomycin C and cyclosporin A in trabeculectomy. Eur J Ophthalmol 1996;6:398–401.
ORIGINAL ARTICLE
Increased Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) Expression in the Conjunctival Epithelium Exposed to Antiglaucoma Treatments
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Antoine Labbe´1,2,3,4,5*, Eric Gabison3,4,5,6,7*, Franc¸oise Brignole-Baudouin3,4,5,8, Luisa Riancho3,4,5, Suzanne Menashi6, and Christophe Baudouin1,2,3,4,5 1
Department of Ophthalmology, Quinze-Vingts National Ophthalmology Hospital, Paris and Ambroise Pare´ Hospital, AP-HP, Boulogne-Billancourt, France, 2Centre Hospitalier National d’Ophtalmologie des Quinze-Vingts, INSERM-DHOS CIC 503, Paris, France, 3INSERM, U968, Paris, France, 4Institut de la Vision, UPMC Univ Paris 06, UMR_S 968, Paris, France, 5CNRS, UMR_7210, Paris, France, 6CRRET, (EAC CNRS 7149), UPEC, Universite´ PARIS EST, Cre´teil, France, 7Rothschild Ophthalmologic Foundation, Bichat Hospital, AP-HP, Paris, France, and 8Faculte´ des Sciences Pharmaceutiques et Biologiques, Sorbonne Paris Cite´ University, Paris, France
ABSTRACT Objective: To analyze the effect of preserved antiglaucoma eye drops on the expression of extracellular matrix (ECM) metalloproteinase inducer (EMMPRIN) in conjunctival epithelial cells. Methods: A total of 18 patients treated for primary open-angle glaucoma with benzalkonium chloride (BAK) preserved eye drops and eight age-matched controls were included in this study. Glaucoma patients were divided into two groups according to their daily exposure to BAK: high-exposure (HE) group and low-exposure (LE) group. HLA-DR and EMMPRIN were quantified on conjunctival impression cytology specimens using flow cytometry. In parallel, IOBA-NHC conjunctival epithelial cells were exposed to different BAK concentrations, in the presence or absence of cyclosporine A (CsA), and their total and surface expressions of EMMPRIN were assessed by flow cytometry and results are given in relative fluorescence intensities (RFIs). Results: Compared to the control group (1.71 ± 0.39 RFI), EMMPRIN was significantly increased in the HE (4.19 ± 1.50 RFI, p50.001) and LE groups (2.55 ± 0.40 RFI, p = 0.029). Similar increase was observed in HLA-DR expression in the HE (4.58 ± 1.38 RFI, p50.001) and LE groups (2.52 ± 0.47 RFI, p = 0.046) as compared to control subjects (1.75 ± 0.27 RFI). Across all subjects enrolled in the study, there was a significant correlation between HLA-DR and EMMPRIN (R2 = 0.875, p50.0001). IOBA-NHC cells exposed to BAK presented a significant increase in EMMPRIN, which was proportional to the concentration of BAK. The surface expression of EMMPRIN was inhibited by CsA. Conclusions: The increased expression of EMMPRIN in patients topically treated with multiple antiglaucoma BAK-preserved eye drops suggests a matrix metalloproteinase-related modification of conjunctival ECM remodeling. In vitro results suggest that CsA has the potential to limit BAK effects on EMMPRIN. Keywords: Antiglaucoma treatment, benzalkonium chloride, conjunctiva, EMMPRIN, preservatives
*These authors contributed equally to this work. Received 18 November 2013; revised 27 March 2014; accepted 8 April 2014; published online 14 May 2014 Correspondence: Antoine Labbe´, MD, Service d’Ophtalmologie III, C.H.N.O. des Quinze-Vingts, 28 rue de Charenton, 75012 Paris, France. E-mail: [email protected]
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EMMPRIN Conjunctival Expression in Glaucoma
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INTRODUCTION Most patients with chronic glaucoma or ocular hypertension (OHT) are treated medically by the use of topical intraocular pressure (IOP)-lowering drugs. However, chronic use of these drugs is known to cause significant changes on the ocular surface.1 In conjunctival biopsy specimens, the long-term use of antiglaucoma eye drops was shown to result in a significant decrease in the number of goblet cells and an increase in the number of macrophages, lymphocytes, mast cells and fibroblasts.2 An increase in subepithelial collagen deposition was also demonstrated.3 Similarly, several studies using impression cytology (IC) have demonstrated an increased expression of inflammatory markers by conjunctival epithelial cells of patients receiving antiglaucoma eye drops.4–8 These ocular surface side effects have a direct impact on patient quality of life, which may limit patient adherence to the glaucoma or OHT treatments and eventually impair the success of glaucoma filtering surgery. Indeed, by stimulating inflammatory cells and fibroblasts in the conjunctiva, long-term topical antiglaucoma therapy has been identified as a significant risk factor for filtration surgery failure.9 Several studies have shown that preservatives, such as benzalkonium chloride (BAK), which are used to preserve stability and/or sterility of active drugs in multidose ophthalmic preparations, could be at least, in part, responsible for these ocular surface side effects.10 Matrix metalloproteinases (MMP) are a family of proteolytic enzymes catalyzing the degradation of extracellular matrix (ECM). During wound healing, MMP and their inhibitors, tissue inhibitors of metalloproteinases (TIMP), play a central role in ECM remodeling, cell–matrix interactions, inflammatory cell recruitment, cytokine activation and regulation of angiogenesis.11,12 Deregulation of MMP and TIMP was suggested to be associated with abnormal ECM turnover in numerous diseases including fibrotic disorders.13 MMP are tightly regulated by complex mechanisms that include cytokines and growth factors, cell-matrix and cell–cell interactions. ECM metalloproteinase inducer (EMMPRIN/CD147), a highly glycosylated cell surface transmembrane protein, was originally identified on tumor cell surfaces as an inducer of MMP production in neighboring fibroblasts.14 The presence and modulation of EMMPRIN in a variety of tissues associated with increased MMP expression suggests that this EMMPRIN-mediated MMP induction could also be a common mechanism in non-tumoral physiological and/or pathological situations.14 We and others have already described the presence of EMMPRIN in ocular tissues including conjunctival epithelium, corneal epithelium, endothelium and stromal keratocytes.11,15 It was also shown to be !
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involved in corneal wound healing and corneal ulcers11 and more recently in dry eye.16 The objective of this study was to analyze the expression of EMMPRIN by conjunctival epithelial cells ex vivo, in patients treated for glaucoma with BAK-preserved eye drops and in vitro, under the influence of BAK and cyclosporine.
PATIENTS AND METHODS Ex Vivo Study Patients A total of 18 patients treated for primary open-angle glaucoma (POAG) and eight control subjects were included in this study. The study was conducted at the Center for Clinical Investigations (INSERM CIC 503) of the Quinze-Vingts National Ophthalmology Hospital, Paris, France, with the approval of the Institutional Review Board of Saint-Antoine University Hospital (CPP-Ile de France 5, number 10793) and in compliance with the declaration of Helsinki. All glaucoma patients had POAG without known ocular surface disease prior to the use of antiglaucoma eye drops. Patients were subjected for at least six months to the same treatment in one or both eyes. Antiglaucoma eye drops were prostaglandin analogs, beta-blockers, alpha-agonists and carbonic anhydrase inhibitors, used separately or in fixed combinations. All commercially available preparations of these antiglaucoma drugs contained BAK with concentration ranging from 0.02% to 0.005%. Glaucoma patients were divided into two groups according to the number of instillations of BAKpreserved eye drops they received everyday: the highexposure (HE) group (nine patients) received from three to five instillations of BAK-preserved eye drops per day; the low-exposure (LE) group (nine patients) were treated with only one or two instillations of BAK-preserved eye drops per day. Eight age-matched subjects who had no history of ocular disease or clinical ophthalmic abnormality as assessed by slitlamp examination, and who had not received any topical treatment for at least six months, were included as controls. One eye of each patient was evaluated. IC Collection Specimens were collected in the superior bulbar conjunctiva using 0.20 -mm polyether sulfone filters (Supor Membranes; Gelman Sciences, Ann Arbor, MI) after instillation of one drop of 0.04% oxybuprocaine (MSD-Chibret, Paris, France).4,17 At least 15 min after fluorescein instillation for ocular surface assessment, two pieces of filters, measuring 13 6.5 mm, were applied gently on the conjunctiva in two neighboring areas covered by the upper eyelid to collect a
42 A. Labbe´ et al.
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homogeneous population of superficial conjunctival cells. The membranes were then immediately fixed in cold phosphate buffered saline (PBS) (pH 7.4; 4 C) containing 0.05% paraformaldehyde. Flow Cytometry Analysis Conjunctival cells were extracted from their support by mechanical agitation and then centrifuged at 2000 rpm for 5 min before immunofluorescence and flow cytometry analysis.18 An indirect immunofluorescence technique was used to evaluate the expression of HLA-DR and EMMPRIN by conjunctival epithelial cells. Mouse anti-HLA-DR antibody (Dako, Glostrup, Denmark) and goat anti-EMMPRIN antibody (Santa Cruz Biotechnology, Santa Cruz, CA) were used at 1:50 and 1:100 dilutions, respectively, in PBS containing 1% bovine serum albumin (Sigma-Aldrich, St Louis, MO). Both primary antibodies were incubated with conjunctival cells for 45 min in the dark at room temperature and were washed in PBS by a 5 min centrifugation. Cells were then incubated with a 1:50 dilution of a fluorescein isothiocyanate (FITC)conjugated goat anti-mouse antibody (Dako) or a FITC-conjugated rabbit anti-goat antibody (Dako).
Non-immune mouse (Dako) and goat (RD Biotech, Besanc¸on, France) corresponding antibodies were, respectively, used as negative isotypic controls. After incubation, cells were washed in PBS by a 5-min centrifugation and suspended in 250 ml of PBS before analysis with a flow cytometer (FC500-CXP, Beckman Coulter, Miami, FL) according to previously validated methods.17,19 All specimens were analyzed in a masked manner with respect to patient characteristics. For each antibody, a sample of at least 2000 conjunctival cells was analyzed (Figure 1). The results were expressed as relative fluorescence intensity (RFI) corresponding to the ratio of the mean fluorescence intensity (MFI) obtained for the specific antibody to the MFI obtained for the isotypic control.
In Vitro Study Conjunctival Cell Line and Treatments The IOBA-NHC cells, established from a spontaneously immortalized conjunctival epithelial cell line, were cultured under standard conditions (humidified atmosphere of 5% CO2 at 37 C) in DMEM/F12
FIGURE 1 Flow cytometry images of a typical case of impression cytology specimen (A and B) and of conjunctival IOBA cells after incubation with BAK 106% (C and D). (A and C) Conjunctival cells were gated from a biparametric diagram showing cell size in linear mode versus cell structure in logarithmic mode, in order to avoid debris (d) and cell aggregates (a). (B and D) Fluorescence histograms obtained from the conjunctival cell population gated in the biparametric diagram. Shown in grey line is the isotypicmatched negative control and in black line, the EMMPRIN expression. Current Eye Research
EMMPRIN Conjunctival Expression in Glaucoma
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supplemented with 1 mg/ml bovine pancreas insulin, 2 ng/ml mouse epidermal growth factor, 0.1 mg/ml cholera toxin, 5 mg/ml hydrocortisone, 10% fetal bovine serum, 50 UI/ml penicillin and 50 UI/ml streptomycin, as previously described.20 Cells from passages 5–20 were seeded into six-well culture plates (Corning, Schiphol-Rijk, The Netherlands) and subconfluent cells (approximately 70%) were exposed for 24 h to BAK solutions at 105%, 106% and 107% in PBS. Similar BAK treatment was also performed after a prior 24-h incubation of cells with cyclosporine A (CsA) (Sigma-Aldrich) at 5 mg/ml. Cells were then detached by a 10-min incubation with 1 mM ethylenediaminetetraacetic acid, collected and suspended in 1 ml of paraformaldehyde 0.5% in PBS.20 Flow Cytometry Analysis of Cultured Conjunctival Cells To analyze the total expression of EMMPRIN (surface and intracellular), cells were first permeabilized with 0.1% saponin for 5 min before incubation with the first antibody. The surface expression of EMMPRIN was examined by performing similar experiments without prior permeabilization. For each antibody, samples of at least 2000 conjunctival cells were analyzed (Figure 1). The results were expressed in RFI. Each experiment involving cell stimulation and immunostaining was independently performed in triplicate.
Statistical Analysis Statistical values of RFI were calculated in each group and for each marker and were presented as mean ± standard deviation. Kruskal–Wallis test was used to compare between groups. The correlation between HLA-DR and EMMPRIN was analyzed using Pearson’s correlation coefficient. Probability values less than 0.05 were considered significant. All statistical analyses were performed using XLSTAT 2011Õ (Addinsoft, Paris, France).
RESULTS Ex Vivo Study IC was performed in patients treated for glaucoma with BAK-preserved eye drops in order to analyze ex vivo the expression of EMMPRIN by conjunctival epithelial cells. Mean age of patients was 63.33 ± 12.74 years in the HE group (five women and four men), 61.3 ± 15.38 years in the LE group (five women and four men) and 67.12 ± 13.67 years in the control group (five women and three men). There was no statistical difference in terms of age and sex ratio between the three groups !
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FIGURE 2 Relative fluorescence intensities (RFIs) of EMMPRIN and HLA-DR on conjunctival epithelial cells in the highexposure (HE) group, low-exposure (LE) group and control subjects. Error bars represent standard deviation. *compared to the LE group, p = 0.029; **compared to the control group, p50.001; #compared to the LE group, p = 0.01; ##compared to the control group, p50.001; +compared to the control group, p = 0.019; and ++compared to the control group, p = 0.046.
(p40.05). Mean number of BAK-preserved eye drops instillation was 3.55 ± 0.72 in the HE group and 1.44 ± 0.52 in the LE group. Mean duration of treatment was 47.11 ± 40.63 months (12–120 months) and 27.7 ± 26.02 months (7–66 months) in the HE and LE groups, respectively. In IC specimens, RFI of the inflammatory marker HLA-DR was significantly raised in the HE group (4.58 ± 1.38) compared to the LE group (2.52 ± 0.47, p = 0.01) and control subjects (1.75 ± 0.28, p50.001). The difference between control subjects and the LE group was also significant (p = 0.046) (Figure 2). Similarly, the RFI of EMMPRIN was significantly increased in the HE group (4.18 ± 1.49) compared to the LE (2.55 ± 0.41, p = 0.029) and the control groups (1.84 ± 0.54, p50.001). As observed for HLA-DR, the difference between control subjects and the LE group was also significant (p = 0.019) (Figure 2). Across all subjects enrolled in the study, there was a significant correlation between conjunctival cells fluorescence levels of HLA-DR and EMMPRIN (R2 = 0.875, p50.0001) (Figure 3).
In Vitro Study Conjunctival cells exposed to BAK 105% showed a greater increase in the RFI for the total EMMPRIN expression (3.67 ± 1.05) as compared to cells receiving BAK 107% (1.77 ± 0.21, p = 0.003) or the control group (1.55 ± 0.11; p50.0001). Similarly, cells stimulated with BAK 106% (2.49 ± 0.64) had a significant RFI increase
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44 A. Labbe´ et al.
FIGURE 3. EMMPRIN and HLA-DR expressions on conjunctival epithelial cells in each patient, given in relative fluorescence intensities (RFIs). A statistically significant correlation was found between EMMPRIN and HLA-DR expressions (R2 = 0.875; p50.0001).
of the total EMMPRIN expression as compared to the control group (p = 0.023). No change was observed after BAK 107% stimulation. Similar results were observed for the surface expression of EMMPRIN with a significant RFI increase of EMMPRIN in conjunctival cells stimulated with BAK 105% (2.62 ± 1.23, p50.0001 compared to BAK 107% (1.10 ± 0.06) and p = 0.012 compared to the control group (1.05 ± 0.05)) and BAK 106% (1.54 ± 0.12, p = 0.033 compared to the control group) (Figure 4). No difference in the surface expression of EMMPRIN was observed after BAK 107% stimulation. When conjunctival cells were treated with CsA prior to BAK exposure, a significant RFI increase of the total expression of EMMPRIN was observed after BAK 105% (2.55 ± 1.03) and BAK 106% stimulation (1.81 ± 0.18) compared to cells exposed to PBS (1.04 ± 0.04, p = 0.032 and p = 0.040 compared to the BAK 105% and BAK 106% groups, respectively). Importantly, CsA suppressed the increase in the surface expression of EMMPRIN by BAK whatever the concentration used, as no significant difference in the surface expression of EMMPRIN was observed between the different groups (p40.05 for all comparisons) (Figure 4). The surface expression of EMMPRIN was also significantly lower after BAK 105% and BAK 106% exposure in the groups treated with CsA as compared to cells not treated with CsA (p = 0.007 and p = 0.024, respectively) (Figure 4).
FIGURE 4 Total and surface expression in relative fluorescence intensity (RFI) of EMMPRIN by conjunctival epithelial cells in vitro after 24 h of BAK stimulation. Error bars represent standard deviation. Without cyclosporin A (CsA) treatment: *compared to the control group, p50.0001; **compared to BAK 107% group, p = 0.003; #compared to the control group, p50.0001; ##compared to BAK 107% group, p = 0.012; +compared to the control group, p = 0.023; and ++compared to the control group, p = 0.033. After CsA treatment: xcompared to the control group, p = 0.032; xxcompared to the control group, p = 0.040. zcompared to BAK 106% group without CsA, p = 0.007; and zzcompared to BAK 105% group without CsA, p = 0.024.
Current Eye Research
EMMPRIN Conjunctival Expression in Glaucoma
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DISCUSSION Numerous studies have demonstrated that antiglaucoma topical medications and their preservatives induce significant histopathologic and inflammatory changes in the ocular surface.1,10 In agreement with previous reports, we describe in this study an increased expression of the major histocompatibility complex class II antigen HLA-DR, often used as a marker of inflammation, on conjunctival epithelial cells in patients chronically treated with multiple BAK-preserved antiglaucoma eye drops, hence confirming the presence of conjunctival inflammation.4–6,8,17,18 This increase in HLA-DR expression was highly correlated (R2 = 0.875) with the increase in EMMPRIN membrane expression. As EMMPRIN was shown to be able to stimulate the production of several MMP (MMP-1, MMP-2, MMP-3 and MMP-9) in pathological situations including corneal ulcers21 and dry eye; 16our results suggest an MMP-mediated modification of the conjunctival ECM remodeling in patients topically treated for glaucoma or OHT with high exposure to BAK. Conjunctival wound healing is a major determinant for the success of glaucoma filtering surgery, and glaucoma topical treatments are well-known risk factors for surgery failure by subconjunctival fibrosis.1,9,10 Conjunctival morphological tissue changes induced by the use of antiglaucoma eye drops, and their preservatives are associated with the expression of inflammatory markers, free radical production and deregulation of MMP (MMP-1, MMP-3 and MMP-9) and TIMPs (TIMP-1, TIMP–2 and TIMP-3) expression.1,22–25 Although the mechanism by which these factors lead to fibrosis is not fully understood, MMP are known to play a central role in wound healing, and their overexpression in ocular tissues has already been associated with excessive scarring in the eye.26,27 Furthermore, Wong et al. demonstrated that the healing process following glaucoma surgery could be modulated by inhibiting the effects of MMP.28,29 In this study, we observed an overexpression of EMMPRIN, an MMP inducer, on the conjunctival epithelial cells of patients using BAKpreserved eye drops. Consistently, on conjunctival biopsies from patients treated with preserved latanoprost, Terai et al.25 showed an upregulation of MMP-1 and MMP-3 in both conjunctival epithelial cells and subepithelial stromal cells, which was associated with a decreased stromal collagen density. Similarly, Honda et al. observed an increased expression of MMP-1 and MMP-9 in conjunctival epithelial cells as well as in tears.24 However, on Tenon’s capsule fibroblasts obtained from patients under multiple antiglaucoma treatments, Leng et al. have shown a down regulation of MMP-9 and MMP-3, which was associated with a higher !
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expression of transforming growth factor (TGF)-b.22 This difference may be due to the fact that superficial epithelial cells, unlike fibroblasts, are in direct contact with the eye drops. It may also be influenced by the type of antiglaucoma eye drops and preservatives used, as two previous studies, one in humans25 and one on a rat model,30 showed different MMP responses to the different type of eye drops. However, as no control group using unpreserved eye drops was evaluated in this study, we are not able, at present, to differentiate the effects of BAK from the active compounds. Similarly, as patients were using different types of IOPlowering medications with multiple combinations possible, it was not possible to perform subgroup analysis. Another limitation resides in the difference of the duration of treatments between the two groups (HE and LE). Nevertheless, Broadway et al. in a study evaluating conjunctival changes induced by antiglaucoma medications observed that both the number of medications and the duration of treatment of glaucoma had adverse effects on the conjunctival cell profile, patients with multiple drug therapy having the greatest changes.2 Similarly, in a recent study, we showed that the daily number of instillations of preserved eye drops could be a more important factor in ocular surface tissue changes induced by antiglaucoma medications than the duration of treatment.31 With these limitations in mind, our results suggest that, in addition to the inflammatory changes, an abnormal expression of MMP and their modulators, such as EMMPRIN, also contributes to the postoperative excessive scarring response. The clinical observations of Schwab et al. showing conjunctival fibrosis with foreshortening of the conjunctival fornix in glaucoma patients receiving topical medications is also in line with these findings.3 Although inflammatory cytokines have been considered as the main regulators of the wound healing process, it appears that interactions between these cytokines and other mediators may have a more important role in modulating the wound healing process than the inflammatory cytokines themselves.21 As an example, TGF-b and interleukin (IL)-1 are two cytokines produced during inflammatory reactions, with TGF-b being a potent stimulator of connective tissue formation,32 while IL-1 provokes tissue degradation through the upregulation of matrix degrading proteinases,33 suggesting that another level of regulation may exist. Interestingly, in the cornea, interactions between TGF-b, IL-1 and EMMPRIN have been demonstrated.21 The high correlation observed in this study between the conjunctival epithelial cells expression of HLA-DR and EMMPRIN suggests an association between inflammation and tissue remodeling in patients treated with antiglaucoma eye drops.
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46 A. Labbe´ et al. BAK, a nitrogenous cationic surface-acting agent belonging to the quaternary ammonium group, is the most commonly used preservative in ophthalmic preparations.10 Many studies have demonstrated that preservatives and BAK, in particular, are responsible, at least in part, for the inflammatory and toxic effects observed during long-term use of antiglaucoma eye drops.10 In a previous study, we demonstrated a dose-dependent toxicity of BAK in IOBA-NHC cells, supporting the use of this cell line for toxicological in vitro studies.20 Using the same model, we show that BAK could upregulate, in a dose-dependent manner, both total and membrane expression of EMMPRIN by epithelial conjunctival cells in vitro. Since BAK was shown to induce IL-1 and TNF in conjunctival and corneal cells in vitro34 and considering the known interactions between EMMPRIN and pro-inflammatory cytokines, these in vitro results emphasize the implications of BAK in the conjunctival activation induced by preserved antiglaucoma eye drops. It has been previously shown that the cell surface expression of EMMPRIN was significantly reduced by the cyclophilin-binding drug CsA.35 Yurchenko et al. demonstrated that cyclophilin 60 was involved in the cell trafficking of EMMPRIN36 as the treatment of tumor cells with CsA reduced cell surface expression of EMMPRIN without altering the total cellular level.35 In our toxicological model, CsA also downregulated the surface expression of EMMPRIN induced by BAK on epithelial conjunctival cells. As EMMPRIN is a transmembrane protein that exerts MMP-inducing effects by its extracellular portion, the blockage of EMMPRIN trafficking within conjunctival epithelial cells and consequently the cell membrane expression of EMMPRIN by CsA may represent an MMP inhibiting effect.37 Nevertheless, these results need to be confirmed on a stratified conjunctival epithelium and with different in vitro techniques evaluating, in particular, EMMPRIN mRNA levels. Interestingly, cyclosporin A was successfully tested in vitro for its antiproliferative properties on fibroblasts,38 and a randomized clinical trial evaluating the benefit of cyclosporin A in glaucoma filtering surgery have shown promising results.39 Hence, CsA, by inhibiting EMMPRIN cell membrane expression, may represent an interesting new option to modulate MMP expression in the ocular surface before surgery, particularly in patients using multiple preserved eye drops. Although the role of EMMPRIN in conjunctival changes induced by preserved eye drops, and the effects of CsA on its expression, should be further evaluated on better defined patients subpopulations and using other techniques, it opens new fields of research in order to increased long-term success of filtering surgery in particular after many years of antiglaucoma treatments.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This work was supported by the ‘‘Institut National de la Sante´ et de la Recherche Me´dicale’’ (INSERM) and Universite´ Pierre & Marie Curie, Paris, France.
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