Correlation of Tear Inflammatory Cytokines and Matrix Metalloproteinases with Four Dry Eye Diagnostic Tests METHODS. Reagents.

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1 Cornea Correlation of Tear Inflammatory Cytokines and Matrix Metalloproteinases with Four Dry Eye Diagnostic Tests Karl R. VanDerMeid, 1 Stephanie P. Su, 2 Keith W. Ward, 1 and Jin-Zhong Zhang 1 PURPOSE. Tear cytokines and matrix metalloproteinases (MMPs) can be extracted from the Schirmer strip. This study examined the extracted levels of tear cytokines and MMPs from Schirmer strips and potential correlation with Schirmer s test, tear breakup time (TBUT), tear osmolarity, and ocular surface disease index (OSDI). METHODS. Thirty healthy volunteers were clinically evaluated for known methods to diagnose dry eye disease, including Schirmer s test, tear osmolarity, OSDI, and TBUT. Tears were collected by Schirmer strips and proteins were extracted from the Schirmer strip in 0.5 M NaCl with 0.5% Tween 20 and analyzed using multiplex assay kits to examine cytokines or MMPs. Calculated cytokine and MMP concentrations for all samples were sorted into groups according to a positive or negative for each of the above-cited four dry eye diagnostic tests, individually and in combination. RESULTS. Five inflammatory cytokines (IL-1, -1, -6, -8, and TNF- ) and five MMPs (MMPs 1, 2, 7, 9, and 10) were extracted from clinical Schirmer strips. Schirmer strip measurement and tear osmolarity correlated well with increased concentrations of the inflammatory cytokines and MMPs, whereas TBUT and OSDI did not. CONCLUSIONS. Both the Schirmer s test and tear osmolarity may be more relevant to the clinician in the diagnosis of ocular surface diseases with an increased level of inflammatory mediators. (Invest Ophthalmol Vis Sci. 2012;53: ) DOI: /iovs Tear samples are being used with increasing frequency to detect biomarkers of normal and diseased states of the ocular surface, 1 3 including allergy 4 and dry eye. 5,6 Although use of ELISA is common, the use of multiplex bead, 7,8 multiarray, and proteomic 9 11 technology has enhanced the use of collected tears by allowing for analysis of small sample volumes while increasing the number of detectable targets. The most commonly reported method to collect tear samples for further analysis is by capillary tube In addition, it has been reported that cellulose acetate absorbent filters, 15 a cellulose ophthalmic sponge, 4 and a water eye wash 16 have been used to collect tear samples. Schirmer s test is routinely used for the clinical assessment of dry eye disease by measuring the tear volume, although it would be of added value if, rather than discarding the strips after the assessment, the tear components From the 1 Pharmaceutical R&D and 2 Research Clinic, Bausch & Lomb, Inc., Rochester, New York. Submitted for publication March 25, 2011; revised October 22, 2011 and January 27, 2012; accepted January 28, Disclosure: K.R. VanDerMeid, Bausch & Lomb, Inc. (E); S.P. Su, Bausch & Lomb, Inc. (E); K.W. Ward, Bausch & Lomb, Inc. (E); J.-Z. Zhang, Bausch & Lomb, Inc. (E) Corresponding author: Jin-Zhong Zhang, Bausch & Lomb, Inc., 1400 North Goodman Street, Rochester, NY 14609; jinzhong_zhang@bausch.com. could be eluted from these strips and used for the measurement of known biomarkers, such as cytokines and other inflammatory mediators. Cytokines, chemokines, and matrix metalloproteinases (MMPs) are strongly associated with ocular surface disease. Frequently, proinflammatory cytokines such as IL-1, IL-6, and TNF-, chemokines such as IL-8 and MCP-1, and MMP-9 are cited as biomarkers associated with ocular surface disease, 1,4,17,18 including dry eye. 19,20 The multianalyte profiling assay system (Luminex; Luminex Corp., Austin, TX) is a technology based on the principle of flow cytometry. The system allows one to simultaneously measure numerous analytes in a single microplate well, using very small sample volumes 7 while achieving excellent correlations to individual ELISAs for many cytokines. 21 This system has been successfully used to study the effect of anti-inflammatory agents on cytokine release profiles of cultured ocular cells. 22,23 Recently, the multianalyte profiling assay system (Luminex) has been used to measure the cytokine content in the tear samples collected with capillary tubes, and increased tear cytokine levels in patients with dry eye have been detected by this technology. 5 Schirmer s test, tear hyperosmolarity, and ocular surface disease index (OSDI) evaluation, along with tear film instability are among the diagnostic tests used by clinicians to evaluate patients for dry eye disease. 24 Although it has been demonstrated that inflammation is associated with dry eye disease, 5,6,25 it is unclear whether individual diagnostic tests can be used to distinguish inflammatory dry eye disease from conditions where inflammation plays a less important role. It would be of great benefit to the clinician to understand which tests may lead to a more accurate diagnosis of inflammatory dry eye. Results from the present study demonstrate that numerous cytokines and MMPs can be detected by a multianalyte profiling assay system (Luminex) from the tear samples collected using a Schirmer strip and the increased concentrations of proinflammatory cytokines and MMPs are associated with both Schirmer strip measurement and tear osmolarity. METHODS Reagents Schirmer diagnostic tear test strips (TearFlo; Contacare Ophthalmics and Diagnostics, Gujarat, India) have an inked ruler. Custom-designed human multiplex-cytokine and MMP kits were also used (Millipore, Billerica, MA), along with Tween 20 (Bio-Rad Laboratories, Hercules, CA) and sodium chloride (Sigma-Aldrich, St. Louis, MO). Subjects This study was approved by the Southwest Independent Institutional Review Board and was conducted in accordance with 21 Code of Federal Regulations Parts 50, 54 56, and 812, applicable Bausch & Lomb Standard Operating Procedures, and the Declaration of Helsinki. Thirty healthy volunteers were enrolled in this study and all subjects gave informed consent and were assessed for eligibility. Inclusion Investigative Ophthalmology & Visual Science, March 2012, Vol. 53, No Copyright 2012 The Association for Research in Vision and Ophthalmology, Inc.

2 IOVS, March 2012, Vol. 53, No. 3 Tear Cytokine and Dry Eye Dagnostic Tests 1513 criteria were as follows: must be 18 years of age or older and have full legal capacity to volunteer, must have no allergic conjunctivitis, must not be using any topical ocular medications, must have no contact lens wear or use of ophthalmic drops 8 hours before the study visit, must be willing and able to follow instructions, and must have signed a statement of informed consent. Exclusion criteria were: a core project team member, a Research Clinic employee, participation in a conflicting study, or considered by the Investigator not to be a suitable candidate for participation. Subject discontinuation criteria were: adverse effects, other ocular complications, subject noncompliance, subject request, or subject found to be ineligible during study participation. Each subject was clinically evaluated for dry eye, and the order of testing was: OSDI, tear breakup time (TBUT), tear osmolarity, and a 5-minute break was allowed before Schirmer s test. Tear Sample Collection The Investigator, with gloves, placed a Schirmer strip over the lid margin at the junction of the lateral and middle thirds of the lower eyelids and kept in place for 5 minutes while subjects closed their eyes without an anesthetic. The Schirmer strips were removed with gloves and tear volume (in millimeters) was recorded. Each Schirmer strip was placed into a sterile 2-mL centrifuge tube, stored on ice for 20 minutes to 1 hour, and then stored at 20 C until processed. Ocular Surface Disease Index The OSDI has previously been validated as a reliable method for measuring the severity of dry eye disease and correlates well with other questionnaires. 26 It is a relatively short questionnaire that provides a rapid assessment of the symptoms of ocular irritation consistent with dry eye disease and the impact on a patient s vision-related function. Tear Breakup Time and Tear Osmolarity Measurements A keratometer was used to assess noninvasive TBUT. The keratometer mires were projected onto the tear film. The time taken for the image to degrade after the last blink was recorded as the TBUT. Because the primary objectives of the study were tear cytokines and osmolarity, the TBUT was measured only once. Tear osmolarity was determined using a commercial osmometer (TearLab; OcuSense, Inc., San Diego, CA) according to the manufacturer s instructions. The osmometer was calibrated, followed by testing of normal and high osmolarity controls, before testing the first patient each day. The patient was seated with chin tilted upward and eyes directed toward the ceiling. The tip of the pen was positioned at the lateral extent of the lower eyelid and gently lowered until the tip touched the tear moisture between eyelid and eye. Extraction of Cytokines and MMPs from Schirmer Strips The extraction of cytokines and MMPs from Schirmer strips has been previously described. 27 In brief, the tubes containing a strip were warmed to ambient temperature and 200 L of extraction buffer (0.5 M NaCl with 0.5% Tween 20) 28 was added to each tube, incubated for 3 hours at ambient temperature on a rocker (VWR International, West Chester, PA), and stored on ice on completion. The strip was transferred to a 2-mL tube and residual liquid was removed by pinching the strip at the 25-mm mark in the sealed tube cap and centrifuged (Microfuge R; Beckman, Palo Alto, CA) at 1000 rpm for 10 seconds. This liquid was combined with stored elution buffer. TABLE 1. Clinical Subject Data Subject Age (y) Sex OSDI Severity Based on OSDI* SS Measurement Osmo TBUT OD OS OD OS OD OS 1 54 F F M F F F M F F F F F F M F F No data M M F M F F M F M F F M M M * OSDI scale: Normal (0) ; Mild (1) ; Moderate (2) ; Severe (3)

3 1514 VanDerMeid et al. IOVS, March 2012, Vol. 53, No. 3 Multiplex Cytokine Analysis Cytokine concentrations in the extracted tears were determined using a custom-designed five-plex cytokine/chemokine kit for IL-1, -1, -6, -8, and TNF- and were analyzed using multiplex bead technology (Luminex) 7,29 according to the manufacturer s instructions. Briefly, two 25- L aliquots of each extracted sample were transferred to a 96-well filter plate and incubated on a commercial titer plate shaker (Barnstead International, Dubuque, IA) with a 25- L aliquot of kit assay buffer and 25- L of antibody-coated capture beads overnight at 4 C. The plate was washed two times with kit wash buffer, liquid was vacuumed off, and beads were further incubated on a shaker with biotin-labeled anti-human cytokine antibodies for 2 hours at room temperature, followed by incubation with streptavidin-phycoerythrin for 30 minutes. Filter plates were washed twice, incubated with 150 L sheath fluid (Luminex), and incubated for 5 minutes on a shaker. Samples were analyzed (Luminex 200) and cytokine concentrations were determined using immunoassay workflow and analysis software (Statlia; Brendan Technologies, Inc., Carlsbad, CA). MMP levels in the extracted tear samples were determined using a five-plex MMP kit containing beads to detect MMP-1, -2, -7, -9, and -10. Analysis (Luminex) of two 25- L aliquots per sample was performed and analyzed as described for cytokines. The kit standards and low- and high-range quality controls were prepared and performed in duplicate according to the manufacturer s instructions. Both low- and high-range quality controls were compared with the ranges listed on the kit specification sheet, and the assay was validated if both were found to be in the expected range for each cytokine or MMP. Determination of pg/ml Measured in Tears Obtained via Schirmer Strips The amount of each cytokine or MMP measured in the tears eluted from Schirmer strips was expressed as total recovery in picograms per milliliter (pg/ml) as follows: First, the calculated sample (in pg/ml) was multiplied by the total extraction sample volume (0.2 ml) to give total picograms in the extracted sample. Final pg/ml based on Schirmer volume was calculated by dividing total picograms extracted by the calculated Schirmer strip volume (pg/ L) and multiplying by Cytokine and MMP Detection from Clinical Schirmer Strips Of the five cytokines tested in this study, four were detected in all 59 of the samples tested. TNF- was below the limit of detection for 50% of the samples. All five MMPs tested were detected in all the samples. No cytokine or MMP samples were above the upper limit of quantification for their respective assays. No significant differences were detected for all cytokines and MMPs by one-way ANOVA comparison of OD versus OS groups, and means and SE for these groups are shown in Figure 1. Correlation of Tear Cytokines and MMPs with Four Dry Eye Diagnostic Tests Use of the Spearman rank-order correlation determined that all cytokines (P for four of the five cytokines) and MMPs (P 0.001) increased as Schirmer strip measurement decreased, whereas no correlation was observed for TBUT or OSDI for any cytokine or MMP (Table 2). A positive correlation was determined between increased tear osmolarity and three of five cytokines tested (IL-1, IL-6, and TNF- ) and three of five MMPs tested (MMPs 2, 9, and 10) (Table 2). As an example, Figures 2 and 3 show the analysis results for IL-6 and MMP-9, respectively. DISCUSSION The diagnosis of dry eye disease currently includes symptom questionnaires such as OSDI and tests for tear film stability, reflex Statistical Analysis Each eye and corresponding Schirmer strip, TBUT, and osmolarity values collected from each subject were treated as an individual sample. Extracts from the Schirmer strip for each cytokine or MMP sample were reported as mean and SE. Comparison of each cytokine or MMP sorted by oculus dexter (OD, right eye) versus oculus sinister (OS, left eye) and analysis using a one-way ANOVA Tukey Kramer test (JMP 7 software; SAS Institute, Cary, NC) and P 0.05 was predetermined to be statistically significant. Further analyses were performed by treating each individual Schirmer strip measurement, tear osmolarity, TBUT, and OSDI as one set of independent variables and treating the corresponding cytokine or MMP concentration as the other independent variable not normally distributed. Under these conditions, nonparametric Spearman rank-order correlation was used to assess the correlation between each of the four evaluation measurements and the recovered cytokine or MMP using a commercial program (Sigmaplot; Systat Software, Inc., Chicago, IL). RESULTS Subject Data The age, sex, OSDI, Schirmer strip measurements (SS mm), tear osmolarity (Osmo), and TBUT collected for all subjects are shown in Table 1. Subjects were given a severity rating based on the OSDI score. FIGURE 1. No significant differences of cytokines and MMPs extracted from clinical Schirmer strips by comparison of OD versus OS groups. All data are expressed as mean SE, n 59 for all groups, except TNF-, for which n 29. OD (black bars) and OS (gray bars) were compared by one-way ANOVA Tukey Kramer test and the value of P 0.05 was determined to be significantly different.

4 IOVS, March 2012, Vol. 53, No. 3 Tear Cytokine and Dry Eye Dagnostic Tests 1515 TABLE 2. Spearman Correlation of Cytokines and MMPs with Four Clinical Evaluation Criteria OSDI SS Measurement Osmolarity TBUT Factor P P P P Cytokine IL IL IL IL TNF MMP MMP MMP MMP MMP MMP Schirmer strip measurement and tear osmolarity correlate well with inflammatory cytokines and MMPs, suggesting that tear osmolarity may be a relevant measurement in the diagnosis of ocular surface diseases with an increased level of inflammatory mediators., Spearman s correlation coefficient. Values of P 0.05 are statistically significant. tear flow, and tear osmolarity during the clinical evaluation of patients. However, the diagnosis of dry eye disease can be difficult because clinical symptoms and evaluation assessments do not always correlate well with the different forms of the disease Therefore, the diagnosis of dry eye continues with a broad array of tests and questionnaires as preferred by the clinician. In addition, lack of understanding of the primary cause of dry eye disease may hinder the clinician from prescribing a suitable treatment. Therefore, it would be an advantage to the clinician to better understand which diagnostic tests are associated with inflammatory dry eye disease since the treatments could be different. Aqueous-deficient dry eye (ADDE) characterized by reduced secretion of tears and evaporative dry eye (EDE), which is due to excessive evaporative water loss from the ocular surface, are FIGURE 2. IL-6 concentrations correlate with Schirmer strip volume and tear osmolarity but not TBUT and OSDI. Spearman rank-order correlation was used to determine the correlation (rho) between IL-6 concentration and four dry eye diagnostic tests. A value of P 0.05 was used to determine an association between the two variables. The solid line represents the linear regression through the points and the dashed lines represent 99% confidence intervals.

5 1516 VanDerMeid et al. IOVS, March 2012, Vol. 53, No. 3 FIGURE 3. MMP-9 concentrations correlate with Schirmer strip volume and tear osmolarity but not TBUT and OSDI. Spearman rank-order correlation was used to determine the correlation (rho) between MMP-9 concentration and four dry eye diagnostic tests. A value of P 0.05 was used to determine an association between the two variables. The solid line represents the linear regression through the points and the dashed lines represent 99% confidence intervals. the two major types of dry eye. 24,35 Although both types of dry eye are caused by different means, they initiate the disease by similar core mechanisms, characterized by, but not limited to, tear film instability, tear hyperosmolarity, and potential activation of inflammatory mediators. 24 Patients with ADDE are classified as either Sjögren syndrome (SS) or non-sjögren syndrome (NSS), and both are caused primarily as a result of lacrimal hyposecretion. 36,37 SS is characterized by autoimmune inflammation of the lacrimal glands, 38,39 whereas NSS is typically any lacrimal gland dysfunction not attributed to SS autoimmune disease. 24 Meibomian gland dysfunction (MGD) is cited as the most common cause of EDE and MGD can be initiated by a number of different mechanisms. 24 Recently, several methods used to collect tears from clinical subjects have been described, 4,8,15 and although each of these methods has its advantages, they would be an extra step in the clinical setting, costing additional time to both patient and clinician. Furthermore, because both the Schirmer strip and the other techniques removed tears from the ocular surface, if both were performed, either procedure followed by the other would compromise the second test. Therefore, the use of Schirmer strips for additional biomarker analysis, including cytokines, chemokines, and MMPs, could be of substantial benefit. Schirmer strips have been used to successfully recover single analytes including eotaxins, 28 cystatins, 40 secretory IgA, 41 and vitamin C. 42 Tear samples are an essential tool to understanding the molecular mechanisms behind dry eye disease and other ocular surface conditions, and recent reports include observations of increased inflammatory molecules in patients with keratoconjunctivitis, 17 keratoconus, 3 and conjunctivochalasis. 4 Yoon et al. 25 reported elevated levels of IL-6 and TNF- in the tears of patients suffering from ADDE compared with healthy volunteers. The multiplex (Luminex) platform is ideally suited for the detection of biomarkers from tear samples. 6,8,43 Recently, this technology was used to detect 25 cytokines, chemokines, and growth factors, including IL-1, -6, -8, and TNF-, in the tears of healthy subjects, 41 whereas elevated levels of these specific analytes were detected in the tears of seven patients with dry eye disease compared with healthy controls. 5 Further, Enriquez-de-Salamanca and colleagues 6 reported increases in five cytokines obtained from the tears of patients with mild to moderate evaporative dry eye disease. In these studies, tears were collected by capillary tubes. To date, a direct comparison of collection of tears by capillary tube versus Schirmer strip for the purpose of cytokine analysis has not been done. Stuchell et al. 44 compared the two collection techniques for the purpose of measuring lacrimal and serum proteins and found that concentrations of lysozyme and lactoferrin in samples collected by either method were not significantly different, whereas the concentration of albumin, IgG, and transferrin collected by the Schirmer filter paper technique was significantly higher than the concentration in tears collected by the capillary tube technique. Recently, several studies have been performed using capillary tube tear collection and Luminex technology for cytokine analysis. These studies demonstrated that concentrations for the control samples varied greatly for many cytokines. 5,6,19,45 The mean concentrations in pg/ml in the above four studies were 125, 150, 26.5, and for IL-6; 325, 500, 176, and 16,791 for IL-8; 45, no data; and for TNF-. The mean recoveries in pg/ml from our sample set for IL-6, IL-8, and TNF- were 386, 5234, and 41, respectively, suggesting that the cytokine recoveries using Schirmer strips are comparable to tear cytokine

6 IOVS, March 2012, Vol. 53, No. 3 Tear Cytokine and Dry Eye Dagnostic Tests 1517 concentrations using capillary tubes, although further studies directly comparing the two methods are warranted. In the present study, we report on five cytokines and five MMPs detected in the tear samples extracted from Schirmer strips. Of the five cytokines tested, all are considered to be proinflammatory and have been previously detected in tears 4,5,19,43 and four of five cytokines were extracted in all the clinical Schirmer strips. Of the five tested MMPs, MMP-9 has been cited frequently as an inflammatory marker found in tears, 3,4,17,46 whereas MMP-2 has been detected in the tear film during corneal wound healing. 47 It has been reported that there were no significant differences in the cytokine levels between OS and OD groups, 6 and our results shown in Figure 2 confirm this finding. Our results indicate a positive correlation between inflammatory cytokines and MMPs (MMP-9) with increasing osmolarity. The observation that tear film osmolarity may be a key marker of dry eye severity was recently suggested in a clinical study of over 300 subjects by Sullivan et al. 48 and it is also supported by the work from Suzuki et al. 49 Further, it has been reported that an osmolarity of 308 reported by Lemp et al. 50 and the milliosm/l (described in the TearLab user manual) is a reasonable cutoff number associated with dry eye. Unfortunately, because only 5 of 30 subjects had tear osmolarity readings 308 milliosm/l any conclusions associating osmolarity or cytokine concentrations to dry eye from our findings would be circumspect. For all the comparisons for OSDI and TBUT, no significant correlations were observed with any analyte. The TBUT result is somewhat contradictory to the findings of Yoon and colleagues 25 with an ADDE comparison group and Enriquez-de- Salamanca and colleagues 6 with an EDE group that indicated a decrease in inflammatory cytokine measurements with increasing TBUT. However, in both of these studies the comparison groups were both comprised of patients diagnosed with dry eye, whereas our group was not. Further, TBUT was measured only once in our study and was not an average of three separate measurements. Our findings are the first report describing inflammatory cytokines and MMPs extracted from Schirmer strip tear samples and correlation with tear osmolarity. Our findings also suggest that the Schirmer s test may be a useful marker for more severe forms of ocular conditions in which inflammation plays an important role. However, it should be pointed out that our subject pool did not reflect a dry eye population but more likely a healthy set. 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