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Original Paper Ophthalmic Res 2008;40:315–321 DOI: 10.1159/000150445 Received: July 19, 2007 Accepted after revision: October 23, 2007 Published online: August 7, 2008 Inflammatory Markers in the Tears of Patients with Ocular Surface Disease Arantxa Acera a Germán Rocha d Elena Vecino a Isabel Lema b Juan A. Durán a, c a Departamento de Oftalmología, Facultad de Medicina, Universidad del País Vasco, Leioa, b Instituto Galego de Oftalmoloxía, Universidad de Santiago de Compostela, Santiago de Compostela, c Instituto Clínico-Quirúrgico de Oftalmología, Bilbao, España; d Universidad Católica, Santiago, Chile Key Words Tear fluid Interleukins Matrix metalloproteinase Ocular surface diseases IL-6 were only found to be overexpressed in conjunctivochalasis. These findings illustrate the selective implication of different molecules in each disorder. Copyright © 2008 S. Karger AG, Basel Abstract Purpose: To determine the concentration of interleukins (IL1 and -6) and matrix metalloproteinase 9 (pro-MMP-9) in the tears of patients with different ocular surface diseases and to examine the possible relationship between the disorders and molecular inflammation. Methods: 77 patients diagnosed as having different ocular surface disorders and 18 normal control subjects were studied. Patients were routinely examined and separated into 5 groups: (1) control, (2) blepharitis, (3) ocular allergic disease, (4) dry eye and (5) conjunctivochalasis. Ten microliters of tears were collected by a Weck cell sponge. The concentrations of IL-1, IL-6 and proMMP-9 were measured by enzyme-linked immunosorbent assay, and the MMP-9 activity was evaluated with gelatin zymography. Results: Levels of IL-1 and IL-6 in tear fluid were significantly higher in conjunctivochalasis (p = 0.0062 and p = 0.0134) than in the control group. Pro-MMP-9 levels were significantly elevated in blepharitis (p = 0.013), in allergic eye disease, in dry eye and in conjunctivochalasis (all p ! 0.001), in comparison to controls. Conclusions: Pro-MMP-9 levels in tears are elevated in all of the studied pathologies especially in ocular allergy and conjunctivochalasis. However, IL-1 and © 2008 S. Karger AG, Basel 0030–3747/08/0406–0315$24.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com Accessible online at: www.karger.com/ore Introduction The cornea, conjunctiva and intervening transition area comprise the tissues at the ocular surface. All three are covered by a stratified, squamous, nonkeratinizing epithelium at the surface of the eye. Functionally, all three regions of the epithelium support the tear film which plays a pivotal role in providing a smooth refracting surface for optimal visual acuity and in maintaining a healthy, well-differentiated ocular surface [1]. A number of chronic diseases of the ocular surface give rise to symptoms known as chronic ocular discomfort. They account for a large amount of ophthalmic consultations and the specific diagnosis is not always straightforward. Moreover, symptoms are not always consistent with clinical signs and the associated pathogenic mechanisms are not clearly understood. Blepharitis (lid margin inflammation), allergic eye disease, hyposecretive dry Presented at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Fla., USA, May 4, 2006. Juan A. Durán, MD, PhD Instituto Clínico-Quirúrgico de Oftalmología Virgen de Begoña 34 ES–48006 Bilbao (Spain) Tel. +34 94 473 3545, E-Mail [email protected] eye and conjunctivochalasis are among the most frequent ocular surface disorders. It has been demonstrated that inflammation plays an essential role in the pathogenesis of these conditions. In recent years, many studies of the biochemical changes occurring in tears have been published. Smith et al. [2] observed a correlation between tear metalloproteinase (MMP) content and clinical evidence of disease progression. Meller et al. [3] reported that inflammatory cytokines such as interleukin (IL) 1 and tumor necrosis factor (TNF) , which can be derived from the ocular surface and tears, may be responsible for the increased expression of MMPs in conjunctivochalasis fibroblasts. This suggests that ocular inflammation might be one important denominator in the pathogenesis of conjunctivochalasis [3]. Solomon et al. [4] studied the expression of the pro- and anti-inflammatory forms of IL-1 in the tear and conjunctival epithelium of normal eyes and those with dry-eye disease and concluded that dry-eye disease is accompanied by an increase in the proinflammatory forms of IL-1 (IL-1 and mature IL-1). IL-1 is an important mediator of inflammation and immunity. Both proinflammatory forms of IL-1 (IL-1 and IL-1) are multifunctional cytokines which in general produce similar biological effects. IL-1 is a potent inducer of other inflammatory cytokines such as IL-6, IL-8 and TNF-, and it can stimulate the production of MMP enzymes [4–6]. IL-6 is a pleiotropic proinflammatory cytokine synthesized by various cells, such as fibroblasts, endothelial cells and keratinocytes, in response to numerous cytokines including TNF- and IL-1. IL-6 can also induce the expression of MMPs [7, 8]. There is much evidence to suggest that MMPs play a vital role in several physiological and pathological processes. They participate in extracellular matrix (ECM) remodeling after wounding of the corneal surface and have been implicated in the pathogenesis of sterile corneal ulceration, dry eye and other ocular diseases [9]. Gelatinase B (MMP-9) and gelatinase A (MMP-2) are the primary matrix-degrading enzymes produced by the corneal epithelium and fibroblasts. MMP-9 has been found to be of central importance in catalyzing the cleavage of epithelial basement membrane components [10, 11]. The aim of this research was to determine the concentration of IL-1, IL-6 and pro-MMP-9 in the tears of patients with different ocular surface diseases employing the enzyme-linked immunosorbent assay (ELISA) and to analyze the possible relationship between the disorder and molecular inflammation. 316 Ophthalmic Res 2008;40:315–321 Methods Patients We designed a prospective, case-controlled study, in which 77 patients with different ocular surface disorders and 18 normal subjects (controls) were enrolled. Patients and normal subjects were recruited from the Cornea and Ocular Surface Unit, Instituto Clínico Quirúrgico de Oftalmología, Bilbao, Vizcaya, Spain. The collection of tears was performed from 13 chronic blepharitis patients whose mean age was 44 8 9.8 years (10 women, 3 men). The second group comprised 19 patients diagnosed for allergic eye disease with a mean age of 43 8 12.7 years (13 women, 6 men). All patients were diagnosed as having chronic allergic conjunctivitis. Diagnosis was based on symptoms and on their medical history. The third group comprised 20 hyposecretive dry eye patients, whose mean age was 56 8 12.63 years (13 women, 7 men). Dry eye patients included in the study had moderate dry eye with typical symptoms including Schirmer test (with anesthesia) values below 5 mm, and low tear meniscus and fluorescein and/or rose Bengal staining (between 1+ and 3+). Patients diagnosed as having Sjögren syndrome were not included in the study. The last group comprised 25 patients with conjunctivochalasis disease manifesting itself at the lower tear meniscus. Diagnosis was based on fluorescein- and rose-Bengal-assisted slitlamp examination. The mean age was 62 8 7.1 years (11 women, 14 men). We collected tear samples from 18 healthy individuals whose mean age was 40 8 12.21 years (11 women, 7 men) and who were not suffering from any ocular disease (no allergic or atopic history). The most symptomatic eye from each patient was analyzed. The exclusion criteria were the presence or history of any systemic or ocular disorder or condition (including ocular surgery, trauma and disease) that could possibly interfere with the interpretation of the results. Current or recent use of topical ophthalmic or systemic medications that could affect the pathology condition and patients wearing contact lenses were also excluded from this study. Tear Collection This research was conducted by medically qualified personnel after approval by the Instituto Clínico Quirúrgico de Oftalmología Ethical Committee and in strict accordance with the tenets of the Declaration of Helsinki. Informed consent was obtained from all patients after the nature and possible consequences of the study had been explained. For all experiments, tears were collected from the inferior lateral tear meniscus, minimizing irritation of the ocular surface or lid margin. Anesthetic drops were not instilled. We obtained the tear sample using a polyvinyl acetal surgical sponge (Merocel, ref. 0525, Oasis, Glendora, Calif., USA). After collection, the sponge was introduced into a 0.5-ml tube (Eppendorf, Fremont, Calif., USA), and the tear fluid was subsequently recovered by centrifugation at 13,000 rpm at 4 ° C for 15 min. The tears used for protein quantification were immediately placed on ice !1 h before freezing and storage at –80 ° C until use. We obtained small volumes of tear samples (10 l) and all the sample was used to quantify the three markers by ELISA. Acera /Rocha /Vecino /Lema /Durán Gelatinolytic Activity of MMP-9 Assay (Zymography) SDS-gelatin polyacrylamide gel electrophoresis (zymography) affects in situ activation of gelatinolytic proteases and was used to separate and visualize the gelatinase activities present in tear samples [3, 12, 13]. All samples were analyzed without heating or reduction. The samples contained the same volume of tears, but not necessarily the same protein content. High-molecular-weight marker proteins (Sigma Chemical Co.) were routinely included in each gel (120 g/well), and electrophoresis was performed at 75 V for approximately 2.5 h. The gels were then removed from their casts, washed and incubated sequentially at 37 ° C in Triton-X100 (30 min), 0.05 M Tris-HCl and 5 mM CaCl2. After this incubation, the gel was briefly rinsed in distilled water and stained with 0.25 % Coomassie brilliant blue for 1 h. The gel was destained with 7% acetic acid. Gelatinase activity in the gel was visible as a clear area in the blue background, indicating an area where the gelatin had been digested. Statistical Analysis The Mann-Whitney U test was used for statistical comparisons between groups. Data are expressed as means 8 SD and the differences were considered statistically significant at p ! 0.05. 300 * IL-1 (pg/ml) 250 200 150 100 50 0 C BL AD DE CCH Fig. 1. The concentration of IL-1 in tear samples from control sub- jects (C) and patients with chronic blepharitis (BL), allergic eye disease (AD), hyposecretive dry eye (DE) and conjunctivochalasis (CCH), measured by the ELISA technique. * p ! 0.05 compared with control subjects. Conjunctivochalasis is the only studied ocular surface disease characterized by a massive increase in IL-1 levels. * 140 120 IL-6 (pg/ml) Enzyme-Linked Immunosorbent Assay The tear samples collected to measure the levels of proinflammatory molecules were obtained from different patients with the same pathologies, but we used one ELISA kit per molecule. Double-sandwich ELISAs for human IL-1 (17 kDa), IL-6 (28 kDa) and pro-MMP-9 (92 kDa) were performed with commercial kits (Calbiochem, Darmstadt, Germany), according to the manufacturer’s protocol. In brief, 50 l of assay buffer and 50 l of standard dilutions of recombinant human (IL-1, IL-6 and proMMP-9) proteins, and 10 l of tears from each patient were dispensed into a 96-well microtiter plate coated with the relevant monoclonal antibodies. The plate was sealed and incubated at room temperature for 2 h. After that, the plate was washed 4 times. Then, 200 l of the corresponding rabbit secondary antiserum conjugated with horseradish peroxidase was added into each well and incubated at room temperature for 2 h. Aliquots of 200 l of the color reagent 3,3,5,5-tetramethylbenzidine were then applied for 20–30 min to develop a blue color, and the reaction was stopped by adding 50 l of 1 M H2SO4. Absorbance was read at 450 nm by an automatic plate reader. Accurate sample concentration was calculated from the linear correlation made with standard antigen versus absorbency. 100 80 60 40 * 20 0 C BL AD DE CCH Fig. 2. The concentration of IL-6 in tear samples from control subjects (C) and patients with chronic blepharitis (BL), allergic eye disease (AD), hyposecretive dry eye (DE) and conjunctivochalasis (CCH), measured by the ELISA technique. Elevated levels of this cytokine are specifically associated with both blepharitis and conjunctivochalasis. * p ! 0.05 compared with control subjects. The results show increased levels of IL-6 in allergic disease, but this increase was not statistically significant. This apparent contradiction could be resolved by a large cohort study. No statistically significant age- or gender-related differences were detected between patients with different ocular disorders and control subjects. The cytokines analyzed were directly related to the MMP-9 enzyme and ocular surface inflammation. The mean level of IL-1 in tear fluid was significantly higher in conjunctivochalasis (4.2-fold; 211.45 8 58.5 pg/ ml; p = 0.0062) than in the control group (49.6 8 26.99 pg/ml). However, there was no significant difference in the concentration of IL-1 in blepharitis (36.54 8 1.28 pg/ml; p = 0.6547), allergic eye disease (14.5 8 14.48 pg/ ml; p = 0.3272) or dry eye (32.57 8 2.38 pg/ml; p = 0.6242) compared to controls (fig. 1). Mean levels of IL-6 in tear fluid were significantly higher in conjunctivochalasis (6.3-fold; 51.78 8 81.53 pg/ ml; p = 0.0134) and blepharitis (2.02-fold; 16.53 8 1.26 Inflammatory Markers in the Tears Ophthalmic Res 2008;40:315–321 Results 317 Pro-MMP-9 (ng/ml) 250 * * 200 * 150 * 100 50 0 C BL AD DE CCH Fig. 3. The concentration of pro-MMP-9 in tear samples from control subjects (C) and patients with chronic blepharitis (BL), allergic eye disease (AD), hyposecretive dry eye (DE) and conjunctivochalasis (CCH), measured by ELISA. Elevated levels of this enzyme are found in all 4 ocular pathologies. * p ! 0.05 compared with control subjects. C CCH DE MMP-9 (92 kDa) MMP-2 (72 kDa) a AD AD BL BL DE DE C Discussion MMP-9 (92 kDa) MMP-2 (72 kDa) b Fig. 4. a Gelatin zymogram of tear samples from a control subject (C), a patient with conjunctivochalasis (CCH) and a patient with hyposecretive dry eye (DE). The 92-kDa pro-MMP-9 and 72-kDa pro-MMP-2 bands are indicated with arrows. b Gelatin zymogram of tears from a control, 2 patients with allergic eye disease (AD), 2 patients with blepharitis (BL) and 2 patients with dry eye. pg/ml; p = 0.0323) than in the control group (8.15 8 2.73 pg/ml). However, there was no significant difference in the concentration of IL-6 in dry eye (16.52 8 10.58; p = 0.0814) or allergic eye disease (33.11 8 8.66 pg/ml; p = 0.5508) compared to controls (fig. 2). 318 The concentration of pro-MMP-9 (92 kDa) was measured by ELISA in the samples of tear film from controls and patients with blepharitis, dry eye, conjunctivochalasis and allergic eye disease. Pro-MMP-9 levels were significantly elevated in blepharitis (2.5-fold; 58.56 8 30.1 ng/ml; p = 0.013), in allergic eye disease (5.6-fold; 132.33 8 77.99 pg/ml; p ! 0.001), in dry eye (4.1-fold; 97.25 8 49.5 ng/ml; p ! 0.001) and in conjunctivochalasis (5.3fold; 126.40 8 101.97 ng/ml; p ! 0.001), in comparison to controls (23.61 8 17.4 ng/ml). Thus, this proteolytic enzyme exhibited increased levels in all pathological groups (fig. 3). Minimal 92-kDa pro-MMP-9 activity was observed by gelatin zymography of tear fluid samples taken from 2 normal control subjects (representative samples shown in fig. 4a, b). Higher levels of pro-MMP-9 were found in tear fluid samples taken from patients with blepharitis, allergic eye disease, dry eye and conjunctivochalasis (fig. 4a, b). The strongest bands were observed in conjunctivochalasis and allergic eye disease, in keeping with the ELISA results of figure 3. In fact, a general correlation between the quantitative data measured by ELISA and the gelatinolytic activity revealed by zymography can be observed (order of enzyme activities: conjunctivochalasis 1 allergic eye disease 1 dry eye 1 blepharitis). Ophthalmic Res 2008;40:315–321 We characterized 4 inflammatory-type ocular surface disorders in terms of the concentration of the proinflammatory molecules IL-1, IL-6 and pro-MMP-9 present in tears, with the aim to analyze the implication of these proteins in each disorder. In addition to pro-MMP-9, that is the inactive form of MMP-9, we also studied its active form by zymography. We found that these different molecules are selectively implicated in each type of inflammation, raising the possibility of a biochemical diagnosis of ocular surface disorders in the future. In patients with blepharitis, the concentration of proMMP-9 with respect to control was significantly elevated; however, this increase was smaller than those differences found in the other studied pathologies. Blepharitis is characterized by tear film instability, which leads to a reduction of nonpolar lipids, fundamentally sphingomyelin [14, 15], in turn, provoking conjunctival inflammation and tear evaporation. Evaporative dry eye which is associated with blepharitis may be the cause of the increase in the concentration of ECM degrading enzymes, Acera /Rocha /Vecino /Lema /Durán since in this disorder the other analyzed molecules do not appear to play a relevant role. Nevertheless, lid margin inflammation is associated with the liberation of large quantities of irritating lipids by the meibomian glands [16]. We found a significant increase in pro-MMP-9 levels in patients with allergic eye disease with respect to control subjects. This finding may be due to an increased production of collagen in allergic processes. In fact, as reported Leonardi et al. [17], histamine, as well as the majority of allergic mediators, can increase collagen production by conjunctival fibroblasts. Although the IL-6 level in the tears of patients with allergic eye disease was increased compared with the controls (33.11 8 8.66 vs. 8.15 8 2.73 pg/ml), it was not statistically significant. In hyposecretory dry eye, which is characterized by reduced tear production and a decreased tear clearance, pro-MMP-9 enzyme is accumulated and activated subsequently in the tears. In addition, the accumulation of other substances, such as the zonula occludens protein 1 and occludin binding protein, which are components of the basal membrane, acts as a stimulus for MMP-9 overexpression [18, 19]. Moreover, tears from dry eyes exhibit an increase in levels of inflammatory cytokines such as IL-1 and a reduction in anti-inflammatory factors such as lactoferrin [4]. Pflugfelder et al. [20] observed increased levels of IL-1 and MMP-9 in cultures of human corneal epithelium, concluding that this cytokine was the most potent inducer of MMP-9 mRNA synthesis, leading to an increase in pro-MMP-9 synthesis by the corneal epithelium and activated MMP-9 in the tear. Our results show a substantial increase in pro-MMP-9 levels in the tears of patients with hyposecretory dry eye. However, in the present study, the concentration of IL-1 was found to be similar to that of the control group. This finding may be due to the increase in the concentration of this enzyme at an intracellular level, whereas we measured levels of this interleukin in tears. Our results also corroborate those of Narayanan et al. [21], who did not find significant differences in the tears of patients with moderate dry eye with respect to control. We included only patients with hyposecretive dry eye not associated with Sjögren syndrome. Consequently, IL-1 may not be a good marker of inflammation in the tears of individuals with hyposecretory dry eye without Sjögren syndrome. Nevertheless, this conclusion should not be extrapolated to Sjögren-type dry eye, since in this case, IL-1 has been claimed to play a role as an inducer of inflammation [4]. It appears that in dry eye there is an increased production of proinflammatory cytokines such as IL-1 and TNF- in the conjunctiva [4] and IL-6 in the tears [22]. Although our results show also an increase in IL-6, it was not statistically significant. This apparent contradiction can be explained by the high range in the results observed in all the three studies. Besides, patients in our study did not suffer from Sjögren syndrome as did some of the patients from the previous studies. The results presented recently at the International Dry Eye Workshop (2007) show that levels of IL-1, TNF-, IL-8 and IL-6 are increased in Sjögren syndrome and in rosacea dry eye, but not in undifferentiated keratoconjunctivitis sicca or in non-Sjögren dry eye, as measured in tears [23]. One intrinsic limitation of tear analysis is the potential variability of the results in samples obtained from disorders that may fluctuate even during the day. In all of our patients we took the tear samples in the morning (9.30 to 11.30 a.m.). Also, as discussed later, the method for the tear uptake may influence the quality of the sample. In any case, we consider that the role of these cytokines as dry eye markers has to be reevaluated. Our most surprising result is the significant increase in the concentration of IL-1, IL-6 and pro-MMP-9 in tears from patients with conjunctivochalasis. An alteration in the ECM of the inferior bulbar conjunctiva has been proposed to occur in conjunctivochalasis, which would provoke the degeneration of the elastic fibers but not their fragmentation [24]. The redundant conjunctiva which appears in conjunctivochalasis suggests that ECM degradation enzymes may contribute to the pathogenesis of conjunctivochalasis. We found overexpression of proMMP-9 which could be involved in the degeneration of gelatin, type I and IV collagen of the basal membrane and of elastin. As mentioned previously, pro-MMP-9 synthesis is regulated by proinflammatory cytokines such as IL1, which in turn regulate the expression of other cytokines such as IL-6. The high concentrations which we observed may be due to the accumulation of these proinflammatory markers in tears due to the delay in tear clearing which occurs in conjunctivochalasis, which in turn translates into a worsening of ocular irritation. Together, these results strongly corroborate the inflammatory theory of the pathogenesis of conjunctivochalasis [3, 25]. Tear sampling, which is crucial in the study methodology, has its difficulties and limitations. We have used for tear sampling a Weck cell sponge (Merocel sponge, polyvinyl acetal, ref. 0525, Oasis) which enables optimal tear volumes to be obtained in a rapid and simple manner. In Inflammatory Markers in the Tears Ophthalmic Res 2008;40:315–321 319 contrast to the method of tear extraction using capillaries (which requires 1 min/l of sample), this method is less traumatic for the patient and requires less time for the same amount of tear fluid. Consequently, the method involves less alteration to the relative viscosity of the tear [13]. A theoretical advantage of the capillaries compared to the other methods is that it can collect the pure tear. Nevertheless, López-Cisternas et al. [26] have demonstrated that the results of the electrophoretic protein profiles of tear samples were indistinguishable between polyurethane foam cylinders (similar material to polyvinyl acetal) and to the one obtained with capillaries. Other available methods include Schirmer strips [27] or polyester rods [13], but the former requires the addition of external agents such as buffer solutions. Thus, the method we report here does not alter the sample either quantitatively or qualitatively. We have made previous studies comparing the absorption and release rate of different materials (i.e. polyvinyl acetal and cellulose). The selected material was the one that allowed us taking a relatively large quantity of tears and releasing it practically in its totality. We cannot exclude the adherence of certain molecules to the Merocel sponge. However, this would affect in a similar way all the tear samples so that it would not cause any alterations to the measured concentrations. In conclusion, elevated pro-MMP-9 levels in tears appear to be a pan-marker for the 4 studied ocular surface disorders, especially allergic eye disease and conjunctivochalasis. Increased IL-6 levels in the absence of IL-1 were found to be associated with blepharitis, whereas elevated IL-6 and IL-1 were characteristic of conjunctivochalasis. These findings illustrate the selective implication of different molecules in each type of inflammation, bringing us closer to a biochemical diagnosis of ocular surface disorders in the future. 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