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Comparative Analysis of Tear Film Levels of Inflammatory Mediators in Contact Lens Users a

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Cansu Yüksel Elgin , Güzin İskeleli , Serap Talaz & Sibel Akyol

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Department of Ophthalmology, Cerrahpasa School of Medicine, Istanbul University, Istanbul, Turkey, b

Department of Ophthalmology, MD Birinci Eye Hospital, Istanbul, Turkey, and

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Department of Physiology, Cerrahpasa School of Medicine, Istanbul University, Istanbul, Turkey Published online: 16 Jun 2015.

Click for updates To cite this article: Cansu Yüksel Elgin, Güzin İskeleli, Serap Talaz & Sibel Akyol (2015): Comparative Analysis of Tear Film Levels of Inflammatory Mediators in Contact Lens Users, Current Eye Research To link to this article: http://dx.doi.org/10.3109/02713683.2015.1037001

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Current Eye Research, Early Online, 1–7, 2015 ! Informa Healthcare USA, Inc. ISSN: 0271-3683 print / 1460-2202 online DOI: 10.3109/02713683.2015.1037001

ORIGINAL ARTICLE

Comparative Analysis of Tear Film Levels of Inflammatory Mediators in Contact Lens Users 1 _ Cansu Yu¨ksel Elgin1, Gu¨zin Iskeleli , Serap Talaz2, and Sibel Akyol3 1

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Department of Ophthalmology, Cerrahpasa School of Medicine, Istanbul University, Istanbul, Turkey, Department of Ophthalmology, MD Birinci Eye Hospital, Istanbul, Turkey, and 3Department of Physiology, Cerrahpasa School of Medicine, Istanbul University, Istanbul, Turkey

ABSTRACT Purpose: To compare tear films levels of various inflammatory cytokines in asymptomatic contact lens (CL) users. CL users of rigid gas-permeable CLs (RGPCL) (group 1) or silicone hydrogel CLs (SiHCL) (group 2) were compared with non-CL-using healthy subjects (group 3). Materials and Methods: Tear samples were collected from subjects in each group after ensuring that there were no complications secondary to CL wear in the CL-wearing participants. Tear-film levels of interleukins (ILs)-1b, -6, and -8; granulocyte-macrophage colony-stimulating factor (GM-CSF) (using the Luminex method); and leukotriene B4 (LTB4) (using the ELISA method) were determined. Cytokine levels were compared among the three groups using analysis-of-variance (ANOVA) and Kruskall–Wallis tests. Results: There were significant differences in concentrations of IL-1b, GM-CSF and LTB4 among the three groups (p = 0.002, p = 0.021 and p = 0.009, respectively), as shown by the Kruskall–Wallis test comparing all three groups for the three cytokines. There were no significant differences for IL-6 and IL-8 (p = 0.079 and 0.094, respectively) when all three groups were compared. Conclusions: There were substantial statistically significant differences between RGPCL users, SiHCL users and control subjects in levels of tear film cytokines. Although CL users were asymptomatic, changes in tear-film levels of several important inflammatory mediators revealed that a chronic inflammatory process occurs during CL wear. Keywords: Chronic inflammatory process, contact lens use, rigid gas permeable contact lenses, silicone hydrogel contact lenses, tear film inflammatory mediators

INTRODUCTION

Cytokines play important roles in host defense and inflammation by coordinating several functions in immunological cells. Cytokines (i) regulate the release of forerunner inflammatory materials and mediators by affecting resting cells and (ii) play an important role in regional control of inflammation.4 The cytokines secreted from various cell types play important roles in cell activation, growth and differentiation; intercellular communication; and recruitment of various inflammatory cells to the inflamed area. Furthermore, cytokines act as an early warning system that initiates and enhances the inflammatory response of mucosa.5 Consequently, although some of these mediators exist normally in the tear film, others

Due to widespread contact lens (CL) use for correction of refractive errors, the effect of CLs on the ocular surface has become a highly popular research topic. CL wear is associated with significant physiologic changes in (i) corneal metabolism, (ii) tear composition and turnover and (iii) oxygen and carbon dioxide levels within the cornea.1 Moreover, CL surfaces (hydrophobic or hydrophilic) are especially prone to bacterial adhesion.2 This is of major importance since, under stress, the cornea is unable to remove pathogenic bacteria, creating an increased risk for infection and inflammation.3

Received 11 September 2014; revised 20 March 2015; accepted 29 March 2015; published online 16 June 2015 Correspondence: Cansu Yu¨ksel Elgin, Department of Ophthalmology, Istanbul University, Cerrahpasa Medical School, Istanbul, Turkey. E-mail: [email protected]

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emerge immediately following injury or immunological reactions.4,6 We investigated the effect of different CL materials on cytokine levels in tears of CL users. We examined and compared cytokine levels in the tear films of rigid gas-permeable CL (RGPCL) users (group 1), frequentreplacement daily-wear silicone hydrogel CL (SiHCL) users (group 2) and non-CL-using healthy subjects (group 3). Levels of five inflammatory mediators – interleukin (ILs)-1b, -6 and -8; granulocyte-macrophage colony stimulating factor (GM-CSF); and leukotriene B4 (LTB4) were compared. In recent years, SiHCLs have been used more frequently than RGPCLs, owing to their higher levels of oxygen permeability, elimination of hypoxia and enhanced bio-compatibility. Additionally, RGPCLs are often reported by patients as being less comfortable.7,8 However, the high level of oxygen permeability of RGPCLs [compared to polymethyl methacrylate (PMMA) lenses, but not SiHCLs], relative resistance to bacterial adhesion and provision of better visual quality with corneal astigmatisms, have instigated the frequent use of RGPCLs in our clinic. Considering their frequency of use and the fact that both RGPCLs and SiHCLs provide high levels of oxygen permeability (and, thereby, reduced hypoxia-based complications), we chose to investigate the effects of both CLs on the ocular surface.

MATERIALS AND METHODS Subjects and Lenses This study was approved by the Board of Ethics of Istanbul University, Istanbul, Turkey. Written informed consents were received from all participants. We investigated 27 permanent CL users (n = 27 eyes). Duration of CL wear was 42 years for CL participants. All participants had biomicroscopic and retinal examinations within normal limits, and no subject had any eye pathology (with the exception of refractive issues). Group 1 consisted of nine asymptomatic RGPCL users (five men and four women), mean age of 25.67 ± 7.75 years and average duration of CL wear of 37.55 ± 17.43 months. All subjects in group 1 were

using CLs made from fluoro silicone methacrylate co-polymers (Wo¨hlk A90, Carl Zeiss, Go¨ttingen, Germany). Group 2 consisted of nine asymptomatic SiHCL users (five men and four women), mean age of 24.78 ± 5.95 years and average duration CL wear of 35.56 ± 19.65 months. All CLs in group 2 were made of balafilcon A (Purevision, Bausch & Lomb, Rochester, NY). Water content, permeability of CL material (DK value) and central CL thickness are presented in Table 1. Group 3 subjects consisted of nine non-CL-wearing healthy subjects (five men and four women), mean age of 25.75 ± 8.24 years.

Procedures Best-corrected visual acuity (BCVA) assessment, biomicroscopic examination and fundus analysis were performed for each eye (left). Tears were collected from groups 1 and 2 thirty seconds after the removal of CLs to avoid irritation factors and reflex tear secretion. Tears were collected in capillary tubes, as described previously.9 A previous study indicated that levels of ILs-1b, -6, and -8 are significantly lower in reflex tears compared to basal tears.10 Considering this, we collected a total of 25 mL of tears from each subject (7–10 mL per collection) on three consecutive days to avoid any potential irritation in the eye during tear collection and to avoid reflex tearing. For statistical analysis, we used the average of three collections per subject. Tear collection with glass capillaries has been reported to be useful in overcoming reflex tears.11 The cornea was not anesthetized during tear collection, and touching the edge of eyelid, corneal surface and conjunctiva was avoided. Tear samples were routinely collected between 2.00 and 4.00 pm, anticipating that cytokine levels change during the day.12 Tears from left eyes only were collected using a 10 mL silica capillary tube inserted into the lower fornix. Tear samples were then transferred to 0.5 mL Eppendorf tubes and kept frozen at 70  C until assessment.

Instrumentation ILs-1b, -6, and -8; and GM-CSF cytokine levels were measured using LuminexÕ 100 TM IS (NASDAQ:

TABLE 1. Characteristics of CL groups. Group Group 1 (RGPCL) Group 2 (SiHCL)

Eyes (n)

CL material

Water content (%)

Diffusion-solubility (DK/t)

Central CL thickness ( 3.00) (mm)

9 9

Fluoro-silicone methacrylate co-polymer Balafilcon A

– 36

90 101

0.09

CL, contact lens; RGPCL, rigid, gas-permeable contact lens; SiHCL, silicone hydrogel contact lens; DK/t, permeability of contact lens material. Current Eye Research

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LMNX 2005) with the procartaÕ cytokine measurement kit (AffymetrixÕ Santa Clara, CA). LTB4 levels were measured using the ELISA BIO-TEKÕ ELX 800 (2002), with an Immunoassay kit (Correlate-E1AÕ , Winooski, VT).

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Statistical Analyses Once datasets were constructed, an unpaired mean comparison t-test was used (Stata version 12, StataCorp, College Station, TX) when comparing two groups. A p50.05 was considered to be statistically significant. When three groups were compared, analysis of variance (ANOVA) and non-parametric Kruskall–Wallis tests were used, as the concentrations of several cytokines were non-normally distributed (ILs-6 and -8 did not pass the Shapiro–Wilk and Skewness/Kurtosis tests for normality, whereas the levels of the other cytokines and LTB4 did). Results of both ANOVA and Kruskall–Wallis tests are reported for comprehensiveness

FIGURE 1. Tear film levels of IL-1b in the three groups (pg/ml; mean ± SD).

RESULTS There were no differences in age or gender ratios between the three groups, and no statistically significant differences were observed in the duration of CL wear between the two study groups. Mean tear film concentrations of IL-1b differed significantly between groups (48.17 ± 10.83, 69.28 ± 9.05 and 58.32 ± 3.61 pg/ml for groups 1, 2 and 3, respectively). In particular, the mean levels of IL-1b were significantly greater in group 2 than in groups 1 and 3 (p = 0.001 and p = 0.010, respectively). The mean tear film level of IL-1b in group 3 was also significantly higher than in group 1 (p = 0.051). Finally, ANOVA and the Kruskall–Wallis test indicated statistically significant differences in group means (p = 0.002 for both) (Figure 1). Mean tear film concentrations of IL-6 were 2,643.7 ± 259.49, 2,387.43 ± 58.74 and 2634.49 ± 251.99 pg/ml of groups 1, 2 and 3, respectively. However, the highest IL-6 value (group 1) was not significantly higher than that of group 2 (p = 0.952). Moreover, IL-6 values of both groups 1 and 3 were significantly higher than that of group 2 (p = 0.021, for both). Finally, while ANOVA revealed a significant difference in group means (p = 0.044), the Kruskall–Wallis test did not (p = 0.079) (Figure 2). As the distribution of IL-6 was found to be nonnormal by the Shapiro–Wilk and Skewness/Kurtosis tests, we considered the Kruskall–Wallis test to be more appropriate. Mean tear film levels of IL-8 were 835.32 ± 93.94, 733.01 ± 44.85 and 729.11 ± 46.26 pg/ml of groups 1, 2 and 3, respectively. The mean IL-8 level of group 1 !

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FIGURE 2 Tear film levels of IL-6 in the three groups (pg/ml; mean ± SD).

FIGURE 3 Tear film levels of IL-8 in the three groups (pg/ml; mean ± SD).

was significantly higher than that of groups 2 and 3 (p = 0.033, for both). However, there was no statistically significant difference between groups 2 and 3 (p = 0.886). While ANOVA revealed a significant difference in group means (p = 0.022), the Kruskall– Wallis test did not (p = 0.094) (Figure 3). As the distribution of IL8 was shown to be non-normal by the Shapiro–Wilk and Skewness/Kurtosis tests, we considered the Kruskall–Wallis test to be more appropriate.

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DISCUSSION

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FIGURE 4. Tear film levels of GM-CSF in the three groups (pg/ml; mean ± SD).

FIGURE 5. Tear film levels of LTB4 in the three groups (pg/ml; mean ± SD).

Mean tear film values of GM-CSF were 1122.91 ± 65.26, 1026.52 ± 53.82 and 1144.43 ± 49.78 pg/ml for groups 1, 2 and 3, respectively. The GM-CSF value for group 3 was significantly higher than that for group 2 (p = 0.003). There were no statistically significant differences between GMCSF values for groups 1 and 3 (p = 0.547), but there was a significant difference between groups 2 and 3 (p = 0.021). Finally, both ANOVA and Kruskall–Wallis tests revealed a significant difference between group means (p = 0.013 and p = 0.021, respectively) (Figure 4). Mean tear film values of LTB4 were 18.95 ± 9.18, 10.00 ± 0.75 and 9.9 ± 1.93 pg/ml for groups 1, 2 and 3, respectively. The mean LTB4 value was highest in group 1, and was significantly higher than those of groups 2 and 3 (p = 0.030 and p = 0.041, respectively). However, there was no significant difference between groups 2 and 3 (p = 0.921). Finally, both ANOVA and Kruskall–Wallis tests revealed a significant difference in group means (p = 0.009, for both) (Figure 5).

Comparisons of cytokine levels between study groups showed mean levels of IL-6, IL-8, GM-CSF and LTB4 to be higher in group 1 than in group 2, whereas the opposite was true for IL-1b levels. Mean IL-8 and LTB-4 levels for group 3 were similar to those of group 2. However, IL-1b, IL-6 and GM-CSF levels in group 3 were significantly greater than those in group 2. Epithelial cells of various tissues and organs respond to local inflammatory stimuli produced by the synthesis and secretion of cytokines.13 In ocular tissues, cultured human corneal cells secrete IL-6, IL-8, GM-CSF and/or LTB4 following stimulation.14–17 Furthermore, cultured human conjunctival epithelial cells produce pro-inflammatory cytokines, e.g. IL-6, IL-8 and GM-CSF, following stimulation.16 There is also evidence for the presence of IL-1b, IL-6, IL-8 and GM-CSF in basal tears of healthy subjects.12 IL-1b plays several important roles in ocular tissues. IL-1b activates matrix metalloproteinase (MMP) family enzymes in corneal stromal cells18 and increases centripetal movement of Langerhans cells.19 In addition, it has been hypothesized that IL1b serves as a mediator for keratinocyte apoptosis,20 not only by initiating damage, but also in aiding recovery by increasing the production of keratinocyte and hepatocyte growth factors.20,21 These latter two growth factors are strong paracrine mitogens for corneal epithelial cells.22,23 IL-1b has a powerful effect on other cytokines and receptor transcription factors in corneal and limbal fibroblasts in vitro.24 Several studies did not report IL-1b as being present in healthy eyes,3,4 while other studies, in line with ours, presented evidence of its existence.10,19 Increased secretion of IL-1b by epithelial cells may stimulate cellular and humoral immune systems for short periods, in response to the presence of pathogens. The fact that IL-1b levels in group 2 were high might indicate strong inflammatory response and their association with increased antibody production by accelerating proliferation of B-lymphocyte. IL-6 is synthesized by mononuclear cells, T- and B-lymphocytes, fibroblasts, endothelial cells, keratinocytes, hepatocytes and bone marrow cells.25 IL-6 in tears increases Ig-A production in B-cells,26 in addition to its other important functions, i.e. downregulation of IL-1b and TNF-a production; delaying apoptosis of polymorphonuclear leukocytes; inhibiting inflammatory responses during closed eyes with CLs; transformation of B-lymphocytes into mature plasma cells; T-cell activation, growth and differentiation; and limiting active pro-inflammatory cascades.27,28 It is also maintained that IL-6 concentrations increase in anterior segment eye diseases, ocular surface diseases and post-operative anterior segment pathologies.29,30 IL-6 is also an early indicator of various inflammatory reactions.31 Current Eye Research

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Tear Film Levels in CL Users One study showed that there was a statistically significant increase in tear film concentrations of IL-6 following a 2-week use of senofilcon-A silicon hydrogel CLs.32 Another study asserted that IL6 is absent in non-CL wearers, although it could be present in basal tears of asymptomatic CL users.33 It has also been shown that IL-6 levels become negligible in asymptomatic CL users shortly after stopping CL use. Since elevated levels of IL-6 are clinical markers for adverse responses associated with CL use,34 it may be concluded that CLs can act to stimulate IL-6 production. With respect to IL-6, many of our results are not in agreement with those of the aforementioned studies. We observed relatively high levels of IL-6 in tears, even in group 3 – non-CL users. One of the important functions of IL-6 is to suppress the production of IL-1 and TNF-a. In our study, even though IL-6 levels in group 1 were higher than that in group 2, IL-1b levels in group 1 were significantly lower than that in group 2. Thus, IL-6 might suppress IL-1b secretion. Furthermore, high levels of IL-6 in groups 1 and 3 suggest an active role of IL-6 in limiting pro-inflammatory responses and increasing antibody synthesis for humoral protection. IL-8 is a potential neutrophil chemotactic/activation factor. It is a primer inflammatory cytokine secreted in response to pro-inflammatory stimuli from many cell types, including monocytes, macrophages, T-cells, neutrophils, fibroblasts and endothelial cells.35 IL-8 is also secreted from retinal and corneal epithelial cells.34 A previous study found IL-8 concentrations to be significantly higher in RGPCL users than in non-CL or SiHCL users.36 In line with other studies, we found significantly higher tear levels of IL-8 in group 1 than in groups 2 and 3.2,37 Some studies showed that RGPCL users are less exposed to infections compared with soft CL users,35,37 a finding perhaps associated with higher IL-8 values in RGPCL users (group 1). GM-CSF is an important colony-stimulating factor, supporting maturation of dendritic cells, neutrophils and macrophages.28 GM-CSF supports erythrocyte and thrombocyte production, with synergist effects on other CSFs.38 Further, GM-CSF is an activation factor for mature neutrophils and mononuclear phagocytic cells. It also plays an important role in allergic immunity by suppressing eosinophil apoptosis and extending the lifespan of eosinophils in areas of inflammation. GM-CSF activates mature eosinophils in their degranulation, cytotoxicity and responses to chemoattractants.28,39 In our study, that fact that GM-CSF levels in groups 1 and 3 were similar (with levels in group 2 being significantly lower) may be due to a late pro-inflammatory response or to the inflammatory response being under suppression in group 2. Moreover, the !

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GM-CSF value of group 2 was significantly lower than that of control 3, suggesting that regulatory T-cells are responsible for this difference. In the eye, LTB4 is typically secreted following inflammation. Lipid inflammatory mediators may be secreted from the cornea, in connection with damage and infection.36 Leukotrienes participate in inflammatory responses via local or systemic effects. LTB4 has a chemotactic effect on leukocytes and neutrophils, increasing endothelial adhesion of leukocytes and migration of leukocytes to extravascular areas. We found LTB4 levels to be significantly higher in group 1 compared with groups 2 and 3. Considering that LTB4 can induce IL-6 synthesis by human blood monocytes through transcriptional activation of the IL-6 gene,40 it was not surprising that there were high levels of LTB4 and IL-6 in group 1. Since IL-6 is both pro- and anti-inflammatory, high levels of IL-6 could limit activation of LTB4. Such a reciprocal interaction would affect both initiation and control of inflammation. LTB4 is also associated with cell membrane arachidonic acids. Therefore, the high level of LTB4 in group 1 might a consequence of direct physical erosion of the cornel surface by a rigid material. Tear secretion of IL-6 and IL-8 in anterior segments increases under various conditions and, consequently, may be used to herald several early inflammatory responses.30 Increased concentrations of chemoattractants in tears are associated with increased numbers of polymorphonuclear leukocytes (PMN).41,42 Considering the rise in IL8 and LTB4 levels in group 1, rapid immune responses would lead to cytokine activation as an infection-reducing defense mechanism. Suppressed IL-6 and GM-CSF levels in group 2 might be due to increase in inflammatory reactions. Finally, the facts that IL-6 and GM-CSF levels in group 2, and IL-1b levels in group 1, were significantly lower than those of group 3, suggest that regulatory T-cells are associated with immunosuppressive mechanisms. Although our CL users were asymptomatic, changes in levels of several tear film inflammatory mediators indicate the presence of chronic inflammatory processes during CL wear. In our study, we analyzed five inflammatory mediators to understand the relationship between the inflammatory process and CL types. Future research efforts should extend this analysis by utilizing richer datasets, incorporating additional mediator types, including regulatory T-cell mechanisms. We also acknowledge the fact that our sample sizes may be considered statistically small. However, our sizes were the maximum number achievable for this research, which required duration of CL wear to exceed 2 years for each study subject. We intend to work with larger samples in our future studies.

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DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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