Cytokine changes in tears and relationship to contact lens discomfort

• Introduction
• Methods
• Results
• Discussion

Abstract

Purpose: To determine the reproducibility of a multiplex bead assay for measuring cytokines in tears and correlations between ocular discomfort with or without contact lens wear and the concentration of cytokines in tears.

Methods: Ninety participants (divided into two groups) were enrolled in this prospective study. They were asked to rate their ocular comfort and collect their tears in the morning and just before sleep for 10 days with or without contact lenses. The participants collected their tears using a glass microcapillary tube for both stages. Galyfilcon A lenses were worn on a daily disposable basis during the contact lens stage, and comfort scores and tears were collected before lens insertion and prior to lens removal at the end of the day. Tears were analyzed for cytokine concentrations using a 27-plex multibead assay. Correlations were sought between cytokine concentrations and comfort.

Results: There was a significant (p<0.022) decrease in ocular comfort over the day with or without lens wear. The magnitude of ocular discomfort was significantly greater (p<0.009) with lens wear. The concentrations of 12 cytokines differed significantly between the groups; thus, these cytokines were not analyzed further. For the remaining 15 cytokines, interleukin-8 (IL-8) was the only cytokine that changed in both groups during the day without (reduced by >-0.5 Log pg/ml, p<0.001) or with lens wear (reduced by >-0.2 Log pg/ml, p<0.001). The change in the vascular endothelial growth factor (VEGF) concentration only in tears was correlated to ocular comfort, but this was not changed by contact lens wear.

Conclusions: Ocular comfort during the day is magnified by contact lens wear. However, the increase in the change in comfort during lens wear was not associated with changes in 15 cytokines in the tear film.

The tear film is a dynamic fluid that contains various biological molecules, including proteins, lipids, and mucins. The human tear film has a complex multilayered fluid structure, consisting of an extensive aqueous layer situated between a mucin layer and a lipid layer. The function of the tear film is to hydrate and lubricate the ocular surface, to protect it from invading pathogens, and to provide a trophic environment for the corneal epithelium and a smooth optical surface for normal vision [1]. The aqueous component of tears comprises the major part by volume and is secreted by the lacrimal gland, accessory lacrimal glands as well as contribution from plasma leakage from conjunctival blood vessels. The aqueous layer contains electrolytes, minerals, enzymes, non-enzyme proteins, immunoglobulins, and growth factors. De Souza et al. [2] identified 491 proteins in the tear film of one individual, and Zhou et al. [3] identified 1,543 proteins in tears collected from four healthy non-contact lens wearers; 714 proteins were present in all samples.

Several studies have examined the tear film for changes in inflammatory mediators during dry-eye disease or contact lens-associated inflammation. Dry-eye disease is defined as being associated with inflammation of the ocular surface [4]. An increase in the concentration of several cytokines, including interleukin-1β (IL-1β), IL-5, IL-6, IL-8, interferon gamma (IFN-γ), and tumor necrosis factor-α (TNF-α), has been reported in the tears of people with dry-eye disease [5,6], and the IL-6 concentration correlates with corneal desiccation, surface damage, irritation, and symptom severity [6]. Studies have shown that contact lens wear can result in increases in the concentration of the cytokines IL-6, IL-8, TNF-α, and endothelial growth factor (EGF) in tears [7-11]. Furthermore, adverse events associated with infiltration of the cornea have been shown to result in increases in the IL-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF) concentrations in tears [12]. Interestingly, there have been no reports of associations between the concentration of cytokines in tears and dryness or discomfort symptoms with contact lens wear.

Contact lens wear can result in symptoms of dryness and discomfort. The prevalence of dryness and discomfort ranges from approximately 30% up to 70% for different contact lens–wearing populations [13-16]. Furthermore, dryness or discomfort appears to increase during the day [17]. This study was designed to measure changes in comfort during the day with or without contact lenses, to determine whether these changes correlated with the level of a range of cytokines in the tear film, and to determine whether a multiplex bead assay for cytokine analysis was reproducible when used with tears.

The current study aimed to demonstrate whether contact lens discomfort over the course of a day was associated with changes to inflammatory mediators in tears. Our results revealed two important findings, namely, that the decrease in ocular comfort associated with contact lens wear (over and above the decrease in ocular comfort that occurs normally during the day without lenses) is not related to changes in at least 15 cytokines, chemokines, and growth factors in tears and that the multiplex bead assay used in the current study is not reproducible for a set of 12 tear cytokines.

In the absence of contact lens wear, the ocular comfort response of both groups significantly decreased during the day by approximately the same amount (3–4 units). In the presence of contact lens wear, comfort decreased for both groups by a greater extent (6–10 units). These numbers correspond to the 7–8 point difference reported as the just-noticeable difference in ocular comfort responses for contact lens wearers [29]. The decrease in ocular comfort during contact lens wear has been shown in previous studies [17,30] as has the decrease in ocular comfort in the absence of lens wear [17]. The increase in ocular discomfort during contact lens wear has been postulated as one of the major reasons for contact lens drop-out [31].

The 27-plex kit reproducibly measured the concentrations of 15 cytokines in the two populations studied. Thus, the hypothesis that changes to ocular comfort with and without contact lens wear during the day could be correlated with the concentration of these cytokines in tears was tested. Interestingly, the majority of the cytokines in tears (G-CSF, IL-12p70, IL-13, IL-15, IL-1β, IL-1Ra, IL-4, IL-5, IL-6, IL-7, IL-8, MCP-1, MIP-1α, and TNF-α) were not related to changes in ocular comfort. Some cytokines changed in the tears of one population but not the other, or did not change at all. This emphasizes the need to test different populations using different batches of test kits. Even when the data were normalized by using concentrations of individual cytokines as a percentage of the total cytokine concentration, there were no correlations between these cytokines and comfort. The only correlation with comfort was for the VEGF concentration in tears, either the absolute concentration or the relative concentration. The VEGF concentration was high when there was a high level of discomfort. Interestingly, when there was a change in the concentration of cytokines during the day, the change was often of a smaller magnitude than without lens wear.

VEGF is a pleiotropic growth factor that can promote the growth of vascular endothelial cells, induce vascular leakage and vasodilation (among many other potential activities) [32]. A study by Papas et al. [33] demonstrated that in the absence of lens wear, limbal redness increases during sleep but does not change during the day. During wear of low oxygen transmissibility lenses, limbal redness increases dramatically after only 4 h and remains high [33]. In contrast, eyes wearing a highly oxygen transmissible contact lens showed almost the same response as those wearing no contact lens [33]. The current study used a relatively high oxygen permeable lens, Galyfilcon A. Despite the lack of redness seen clinically, it may be that VEGF is active during the day and during contact lens wear promoting sub-clinical vessel leakage that then leads to symptoms of ocular discomfort. The finding that ocular comfort during the day was associated with increased VEGF concentration in tears but was not modulated by contact lens wear implies that the increased discomfort seen during contact lens wear is not mediated by VEGF concentrations in tears. The fact that the group with <15 point decrease in ocular comfort showed no correlation with VEGF may imply that changes to VEGF in tears are secondary to ocular comfort changes and not a causative effect, as it might be expected that there would be a gradual increase in VEGF as the level of discomfort increased. The correlation between VEGF and ocular comfort may be simply that, a correlate and not a causative event.

Several studies have shown that cytokines can be found in tears during frank inflammation [34-36] and during dry-eye disease [5,37-42], which may also be an inflammatory disease. These have often used non-multiplex assays, or multiplex assays from other manufacturers. However, they also have not been tested for reproducibility between groups or kits. Assuming at least some of the mediators that have been assayed in these studies are reproducible, as some are in the current study, contact lens wear and associated ocular discomfort are not likely to be an inflammatory condition since the current study did not find evidence of an increase in cytokines in tears during contact lens wear. If there is any contribution of an inflammatory response, it is likely to be at levels much reduced compared to dry-eye or other inflammatory conditions. When there were differences in the concentrations of cytokines between the morning and evening in the absence of contact lens wear, when lenses were worn these differences were muted.

The current study used the Bio-Plex Human Cytokine 27-plex panel bead-assay kit. This antibody-based assay was designed to measure 27 cytokines and chemokines or growth factors, and has been optimized to work for serum, plasma, and tissue culture supernatants (see BioPlex Pro; accessed on November 22, 2013). This assay has been used previously to examine the concentration of cytokines in tears of normals and diabetic patients [43], and a similar multiplex kit from the same manufacturer was used to measure the concentration of 17 cytokines in the tears of patients with glaucoma and control individuals [44]. A study also used a single-plex assay from the same manufacturer to measure the IL-1β concentration in tears [45]. However, unlike the current study, none of these previous studies examined the reproducibility of the assay between groups of different participants. The finding in the current study that several cytokines did not give reproducible results using the 27-plex kit is important to keep in mind when reviewing those previous reports. Examinations of the reported cytokine concentrations in tears [46] and the effect of contact lens wear have demonstrated that the concentrations vary widely between studies [47], and this may be due to inherent deficiencies in the methods used to analyze the cytokines. However, without analysis of the reproducibility of the tests between subject groups, or between batches of tests, the reproducibility of other test kits is unknown. Using a different multiplex bead assay from Luminex (Austin, TX), Huang et al. [48] found good (≥0.70) interclass correlations between 18 tear proteins, including the cytokines IL-15, IL-1β, eotaxin, IL-17, MCP-1, IL-12p70, VEGF, IL-1α, IL-8, and Il-1Ra when the researchers tested the same participants on different days or between eyes. This differs in some respects with the analysis in the current study, although both studies found reproducible concentrations of IL-15, IL-1β, MCP-1, IL-12p70, IL-8, IL-1Ra, and VEGF. Unlike in the current study, there was relatively poor reproducibility for the IL-6 concentration in tears in the study by Huang et al. [48] Tears can contain substances that may interfere with enzyme-linked immunosorbent assays such as immunoglobulin components [49] or lysozyme [50], and these may also be interfering with some of the analytes used in different protocols or kits. A study used the same 27-plex kit as used in the current study [51] and reported that the concentration many cytokines in tears that were tested on two groups varied widely when different contact lens multipurpose disinfecting solutions were used. When the second group was retested for changes to tear cytokines using the same disinfecting solution as a previous group, apparent changes to tear cytokines concentrations were not seen with the second group. The authors [51] also measured comfort responses during lens wear with different lenses, but did not attempt to correlate comfort with cytokine concentrations. Perhaps the issue was that the kits could not reproducibly measure the level of cytokines in tears. The level of the cytokines RANTES (CCL5), Eotaxin (CCL11), GM-CSF, IFN-γ, IL-2, and IL-17 did not appear to be reproducible in the paper by Kalsow [51] or in the current study. Comparison of the data from the current study to that of Liu et al. from the same laboratory [43] is also illuminating. These two studies used identical multiplex assay kits from the same manufacturer (Bio-Rad). Liu et al. [43] reported low positive detection rates for IL-17 (20%), TNF-α (27%), and MIP1-α (60%). Although the present study is in agreement regarding the low positive detection rate of these cytokines, the present study also found many other cytokines, reported to be in most samples from the Liu study, at a much reduced positive rate. The reason for this discrepancy is unknown, but may be related to different populations, or the effect of tear storage (discussed in more detail below). However, the concentrations of all the reproducible cytokines in the current study were at approximately the same concentration in the study of Liu et al. [43], with the exception of G-CSF, which was approximately 7–8 times less concentrated in the present study. The study by Liu et al. [43] using the same multiplex kit demonstrated excellent recovery rates (68–113%) for spiked samples for most of the reproducible cytokines from the current study, with the exception of C-CSF which had a recovery rate of only 27% (they did not report recovery rates for TNF-α, IL-17, or MIP-1α) [43].

This study had several limitations. There is, of course, the possibility that participants induced reflex tearing on some occasions when they were collecting their tears. Although we did perform rigorous training in tear collection, and asked participants to immediately stop collection if they felt they were producing reflex tears, since the tears were collected by the participants in their own homes we could not control for this. As stated, the tears were analyzed for the secretory immunoglobulin A (sIgA) concentration in tears as the concentration of this protein is known to vary between reflex and basal tears [18]. Even if the concentration of cytokines was normalized to the concentration of sIgA, the cytokines that showed inconsistencies were still inconsistent. Thus, although reflex tearing may be a problem inherent in the study design, our best efforts to control for this did not seem to affect the problems encountered with concentrations of certain cytokines between groups. Tear samples were pooled over the 10 days of collection into those collected in the morning and those collected in the evening for each individual. The decision to pool tears collected in the morning or evening from individuals was taken so that the tears could also be used in other assays (not reported herein) that required more sample volume than the multiplex bead assay. This may have masked some variations that could have resulted in additional correlations to comfort. However, the fact that the change in comfort response for an individual during the day over the 10 days showed no significant differences suggest that a participant’s comfort response over the 10 days was consistent. Thus, we assumed that there should then be consistent changes in cytokine levels if they were related to the comfort response. This means that it would be expected that, if changes in cytokine concentrations are correlated with changes in comfort, the changes over the 10-day collection period should be similar, since the comfort change for an individual was similar.

The study examined comfort and changes in tears over several days, and thus, participants were required to store their tears in their home freezers. These may have been of the “frost-free” variety where freezers go through freeze–thaw cycles to reduce the buildup on ice. This may have affected the quality of the tears collected. However, since the tear cytokines IL-6, IL-12p70, IL-13, IL-15, IL-1β, IL-1Ra, IL-8, MCP-1, and VEGF were within the same concentration range as a previous study where tears were collected and stored at the clinic [43], and the IgA concentration was within the normal range expected, this may not a major concern. Nonetheless, the discrepancies between the percentage of samples positive for cytokines between the current study and a previous study from the same group where samples were collected in the clinic [43] may indicate some cause for concern. An alternative explanation for the differences between percentage positive samples is that the current study used predominantly Caucasian participants living in Australia, whereas the study by Liu et al. [43] used exclusively Chinese participants living in China. The IgA level detected in tears had a large range between participants (i.e., large standard deviation in the populations). This may imply that some participants collected reflex tears, but even when the concentration of cytokines was calculated per milligram of IgA, this did not produce new correlations with comfort scores. However, in future experiments it may be desirable to collect tears individually (avoiding pooling) and collecting in the clinic so that tears could be stored at −80 °C immediately to avoid any issues, if present, with collecting reflex tears and freeze–thawing of the samples during storage.

In conclusion, this study has shown that ocular comfort decreases during the day and that contact lens wear exacerbates this decrease in comfort. However, there was no evidence that this change in ocular comfort, or contact lens wear in general, was associated with a change in the concentrations of 15 cytokines, chemokines, or growth factors in tears. Contact lens wear might induce changes to levels of these inflammatory mediators in the ocular tissues that are not released into the tear film. However, it seems equally likely that contact lens wear is not associated with increases in these mediators, and thus not associated with the production of inflammation. Even when cytokine levels increased during the day, the difference in concentration (percentage) of the cytokines between morning and evening tears was less than when lenses were worn. In addition, this study emphasizes the need to perform tests on the reproducibility of methods for measuring cytokine concentrations, and possibly other proteins, in tears.