PUNCTAL OCCLUSION IMPACT ON TEAR FILM IMMUNO-REGULATORY PROTEINS MEASURED BY CYTOMETRIC BEAD-BASED ASSAY MARTIN W. LAFRANCE

Transcription

1 PUNCTAL OCCLUSION IMPACT ON TEAR FILM IMMUNO-REGULATORY PROTEINS MEASURED BY CYTOMETRIC BEAD-BASED ASSAY by MARTIN W. LAFRANCE RODERICK J. FULLARD, COMMITTEE CHAIR DAWN K. DECARLO JOHN F. KEARNEY KENT T. KEYSER DAVID R. WHIKEHART A DISSERTATION Submitted to the graduate faculty of The University of Alabama at Birmingham, in partial fulfillment of the requirements for the degree of Doctor of Philosophy BIRMINGHAM, ALABAMA 2008

2 Copyright by Martin W. LaFrance 2008

3 PUNCTAL OCCLUSION IMPACT ON TEAR FILM IMMUNO-REGULATORY PROTEINS MEASURED BY CYTOMETRIC BEAD-BASED ASSAY MARTIN W. LAFRANCE VISION SCIENCE GRADUATE PROGRAM ABSTRACT PURPOSE: This study compares vendor reagents used for cytometric bead-based assay (CBA) of cytokines in human tears and validates a protocol for use in evaluating punctal occlusion, a treatment for keratoconjunctivitis sicca. It has been proposed that punctal occlusion may alter tear cytokine concentrations based on the facts that: (1) tear clearance, a measure of the balance between tear production and elimination, declines following occlusion and (2) reduced tear clearance and increased levels of pro-inflammatory tear cytokines are correlated in dry eye patients. This study compares tear clearance values and the level of 24 tear cytokines measured by CBA following temporary unilateral punctal occlusion. METHODS: Multiple reagent kits from three vendors were compared in parallel. Vendor protocols were modified to accommodate sample volumes and performance measures of precision, recovery, linearity, sample stability, and tear collection effects were evaluated. Twenty-three subjects received superior and inferior unilateral intracanalicular punctal occlusion with six-month dissolvable plugs. Nonstimulated tear samples were collected from both eyes by micropipette (stored at -80 C) and tear clearance was measured using the Standardized Visual Scale Test prior to occlusion and again at 1, 4, and 7-weeks post occlusion. Tear cytokine concentrations were determined using an optimized CBA. RESULTS: Acceptable assay performance measures were observed iii

4 for the majority of cytokines, and sample attributes were favorable for tear CBA analysis. No between eye differences in tear clearance values were found prior to occlusion by the Standardized Visual Scale test (p = 0.23). Following occlusion, reduced tear clearance was observed for the occluded eye at week 1 (p = 0.02), week 4 (p = 0.01), and week 7 (p < ). For the tear cytokines assayed, no statistically significant differences were observed, nor was there a statistically significant interaction with time. CONCLUSION: Tear cytokines can be reliably measured by CBA using an optimized protocol. Concentrations remain stable during periods consistent with sample collection. Unilateral punctal occlusion reduces tear clearance when compared to the fellow non-occluded eye in normal subjects. However, this does not alter the concentration of tear cytokines compared to pre-occlusion levels or when compared to the fellow non-occluded eyes. iv

5 ACKNOWLEDGEMENTS I am indebted to my advisor, Dr. Roderick Fullard, for his inspiration and insight. Through late night assays to early morning discussions, I am forever thankful for the guidance and direction provided. Dr. Fullard leaves an indelible mark of excellence on the School of Optometry and Vision Science graduate programs. It has been an honor to work alongside an individual of his caliber. I am thankful for the unwavering support of my wife Kelli, and children Amy and Matthew. They are my greatest blessing and to whom I owe all. They have shared the sacrifice of time, through weekends and evenings devoted to coursework and this project. It is my hope that Amy and Matthew travel a similar road in the future. I thank my committee members for devoting valued time and resources to this project. This dissertation is a reflection of their wise input and guidance. I thank the United States Air Force for the financial support and opportunity to complete this program. It is an honor to be a part of an institution whose core values: integrity, selfless service, and respect, bring pride to the uniform and all who serve. I thank the following entities for providing additional support to this project: The University of Alabama at Birmingham School of Optometry (UABSO) Clinical Research Advisory Council, The UABSO Vision Science Research Center Molecular Core, the Minnie F. Turner Memorial Fund for Impaired Vision Research, Lacrimedics Incorporated, and the American Academy of Optometry. v

6 TABLE OF CONTENTS Page ABSTRACT... iii ACKNOWLEDGEMENTS...v LIST OF TABLES... ix LIST OF FIGURES... xi LIST OF ABBREVIATIONS... xviii INTRODUCTION...1 BACKGROUND AND SIGNIFICANCE...4 Normal Ocular Surface Homeostasis...4 Classification and Pathogenesis...7 Immune Response...10 Diagnostic Tests...15 Cytometric Bead-Based Assays...17 Punctal Occlusion...19 AIMS AND RATIONALE...23 Aim One Protocol Development...23 Aim Two Preliminary Studies...23 Aim Three Punctal Occlusion...24 Aim Four Future Contributions...25 Study Overview...25 RESEARCH DESIGN CONSIDERATIONS...27 Cytometric Bead-Based Assay - Instrument Reliability...27 Cytometric Bead-Based Assay - Measurement Reliability...27 Tear Sample Analysis Proof of Concept...31 Diagnostic Test Correlation...32 Non-Test Factors...35 vi

7 Sample Size - Power Analysis...38 PILOT STUDIES...42 METHODS...46 Subjects...46 Statistical Methods...47 Tear Sample Collection...47 Acquisition Instrument and Software...48 General Cytometric Bead-Based Assay Protocol...49 Developmental Protocol Properties, Precision, and Concentration Differences...50 Initial Comparative Assays...50 Second Comparative Assays...51 Sample Matrix Diluent Comparison...54 Preliminary Studies...55 Recovery...55 Linearity of Dilution...56 Sample Stability...56 Stimulus Effects...57 Time and Laterality Comparisons...58 Punctal Occlusion...59 Occlusion Procedure...59 Biomicroscopy...60 Non-Invasive Tear Breakup Time...60 Schirmer-I Testing...61 Tear Fluorescein Clearance...61 Fluorescein Staining...62 Lissamine Green Staining...62 Tear Assay...62 RESULTS...64 Developmental Protocol Properties, Precision, and Concentration Differences...64 Initial Comparative Assays...64 Second Comparative Assays...68 Sample Matrix Diluent Comparison...70 Preliminary Studies...72 Recovery...72 Linearity of Dilution...76 Sample Stability Stimulus Effects Time and Laterality Comparisons Punctal Occlusion vii

8 Biomicroscopy Non-Invasive Tear Breakup Time Schirmer-I Testing Tear Fluorescein Clearance Fluorescein Staining Lissamine Green Staining Tear Assay DISCUSSION Protocol Development Preliminary Studies Effects of Unilateral Punctal Occlusion Future Directions CONCLUSION LIST OF REFERENCES APPENDICES: A Materials For Cytometric Bead-Based Assays B Protocols X and X Approval C Assurances D Example Reference Standard Curves E Standardized Visual Scale Test for Fluorescein Clearance F Oxford Grading Scale G Resources and Environment H Budget and Justification I Previously Published Human Tear Cytokine Concentrations viii

9 LIST OF TABLES Table Page 1 Summary of Cytokines Evaluated, T H bias, and Principal Activities Pilot Studies Assays and Modifications of Published Protocols Initial Comparative Assays and Modifications of Published Protocols Second Comparative Assays: Sample Storage, Application, and Tear Volumes Second Comparative Assays and Modifications of Published Protocols Influence of Sample Storage Conditions and Matrix Composition on Assay Precision for Optimal Tear Assay Kit Intra-Assay Coefficients of Variation: First Comparison Comparison of Mean Cytokine Concentrations between Kits from Paired Nonstimulated Tear Samples Intra-Assay Coefficients of Variation: Second Comparison Sample Storage and Matrix Composition Influence on Assay Precision Percent Recovery for Three Levels of Spiked Nonstimulated Tear Sample Standards-Recovery for the Spike-Recovery Assay of Nonstimulated Tear Samples Linearity of Dilution: Precision by Sample Volume Linearity of Dilution: Cytokine Concentration-Volume Correlation ix

10 Table Page 15 Sample stability Expressed as Coefficient of Variation Significance of Nonstimulated versus Stimulated Tear Cytokine Concentrations Occluded versus Contralateral Eye Control Non-Invasive Tear Breakup Times Occluded versus Contralateral Eye Control Schirmer-I Measurements Occluded versus Contralateral Eye Control Standardized Visual Scale Test Measurements Occluded versus Contralateral Eye Control Corrected Standardized Visual Scale Test Measurements Occluded versus Contralateral Eye Control Ocular Surface Fluorescein Staining Scores Occluded versus Contralateral Eye Control Ocular Surface Lissamine Green Staining Scores Decomposition of General Linear Model Analysis with Significance for Factors Treatment and Time x

11 LIST OF FIGURES Figure Page 1 Lacrimal functional unit feedback loop Comparison of mean cytokine concentrations assayed with Linco and BioRad kits Comparison of mean cytokine concentrations assayed with Upstate and BioRad kits Comparison of mean cytokine concentrations assayed with Upstate and BioRad kits IL-6 linearity of dilution determined using a BioRad 6-plex Assay IL-7 linearity of dilution determined using a BioRad 6-plex Assay IL-8 linearity of dilution determined using a BioRad 6-plex Assay IL-10 linearity of dilution determined using a BioRad 6-plex Assay Eotaxin linearity of dilution determined using a BioRad 6-plex Assay MCP-1 linearity of dilution determined using a BioRad 6-plex Assay IL-1β linearity of dilution determined using a BioRad 27-plex Assay...82 xi

12 Figure Page 12 IL-1ra linearity of dilution determined using a BioRad 27-plex Assay IL-2 linearity of dilution determined using a BioRad 27-plex Assay IL-4 linearity of dilution determined using a BioRad 27-plex Assay IL-5 linearity of dilution determined using a BioRad 27-plex Assay IL-6 linearity of dilution determined using a BioRad 27-plex Assay IL-7 linearity of dilution determined using a BioRad 27-plex Assay IL-8 linearity of dilution determined using a BioRad 27-plex Assay IL-9 linearity of dilution determined using a BioRad 27-plex Assay IL-10 linearity of dilution determined using a BioRad 27-plex Assay IL-12p70 linearity of dilution determined using a BioRad 27-plex Assay IL-13 linearity of dilution determined using a BioRad 27-plex Assay IL-17 linearity of dilution determined using a BioRad 27-plex Assay IP-10 linearity of dilution determined using a BioRad 27-plex Assay Eotaxin linearity of dilution determined using a BioRad 27-plex Assay G-CSF linearity of dilution determined using a BioRad 27-plex Assay...97 xii

13 Figure Page 27 GM-CSF linearity of dilution determined using a BioRad 27-plex Assay INFγ linearity of dilution determined using a BioRad 27-plex Assay MIP-1α linearity of dilution determined using a BioRad 27-plex Assay MIP-1β linearity of dilution determined using a BioRad 27-plex Assay PDGF-BB linearity of dilution determined using a BioRad 27-plex Assay RANTES linearity of dilution determined using a BioRad 27-plex Assay VEGF linearity of dilution determined using a BioRad 27-plex Assay Nonstimulated tear sample stability expressed as means and standard errors for IL-1β, MIP-1α, IL-4, IL-10, and IL Nonstimulated tear sample stability expressed as means and standard errors for MIP-1β, IL-6, G-CSF, RANTES, and PDGF-BB Nonstimulated tear sample stability expressed as means and standard errors for IL-13, IL-2, IL-9, IL-12p70, IL Nonstimulated tear sample stability expressed as means and standard errors for IFNγ, VEGF, IL-1ra, IP Nonstimulated versus stimulated tear concentrations of IL-1β, IL-15, MIP-1α, IL-4, IL-10, and IL Nonstimulated versus stimulated tear concentrations of MIP-1β, IL-6, G-CSF, RANTES, PDGF-ββ, and IL Nonstimulated versus stimulated tear concentrations of IL-2, IL-9, IL-12p70, MCP-1, IL Nonstimulated versus stimulated tear concentrations of IL-8, GM-CSF, Eotaxin, IL-7, TNFα xiii

14 Figure Page 42 Nonstimulated versus stimulated tear concentrations of IFNg, VEGF, IL-1ra, IP Correlation of Mean Cytokine Concentrations for Nonstimulated and Stimulated Tear Samples Effect of collection period on the difference between nonstimulated and stimulated tear concentrations of IL-1β, MIP-1α, IL-4, IL-10, and IL Effect of collection period on the difference between nonstimulated and stimulated tear concentrations of MIP-1β, IL-6, G-CSF, RANTES, and PDGF-BB Effect of collection period on the difference between nonstimulated and stimulated tear concentrations of IL-13, IL-2, IL-9, IL-12p70, and IL Effect of collection period on the difference between nonstimulated and stimulated tear concentrations of IL-8, GM-CSF, Eotaxin, and IL Affect of collection period on the difference between nonstimulated and stimulated tear concentrations of IFNγ, VEGF, IL-1ra, and IP Effect of sustained stimulus on tear cytokine concentrations Effect of self versus investigator collection method on tear cytokine concentrations Interval and contralateral eye comparison of IL-1β concentration in tears Interval and contralateral eye comparison of IL-1ra concentration in tears Interval and contralateral eye comparison of IL-2 concentration in tears Interval and contralateral eye comparison of IL-5 concentration in tears xiv

15 Figure Page 55 Interval and contralateral eye comparison of IL-6 concentration in tears Interval and contralateral eye comparison of IL-7 concentration in tears Interval and contralateral eye comparison of IL-8 concentration in tears Interval and contralateral eye comparison of IL-10 concentration in tears Interval and contralateral eye comparison of IL-12p70 concentration in tears Interval and contralateral eye comparison of IL-13 concentration in tears Interval and contralateral eye comparison of IL-15 concentration in tears Interval and contralateral eye comparison of IL-17 concentration in tears Interval and contralateral eye comparison of Eotaxin concentration in tears Interval and contralateral eye comparison of FGF-basic concentration in tears Interval and contralateral eye comparison of G-CSF concentration in tears Interval and contralateral eye comparison of GM-CSF concentration in tears Interval and contralateral eye comparison of IFNγ concentration in tears Interval and contralateral eye comparison of IP-10 concentration in tears Interval and contralateral eye comparison of MCP-1 concentration in tears xv

16 Figure Page 70 Interval and contralateral eye comparison of MIP-1α concentration in tears Interval and contralateral eye comparison of MIP-1β concentration in tears Interval and contralateral eye comparison of PDGF-BB concentration in tears Interval and contralateral eye comparison of RANTES concentration in tears Interval and contralateral eye comparison of VEGF concentration in tears Occluded versus contralateral eye control comparison of IL-1β concentration in tears Occluded versus contralateral eye control comparison of IL-1ra concentration in tears Occluded versus contralateral eye control comparison of IL-2 concentration in tears Occluded versus contralateral eye control comparison of IL-4 concentration in tears Occluded versus contralateral eye control comparison of IL-5 concentration in tears Occluded versus contralateral eye control comparison of IL-6 concentration in tears Occluded versus contralateral eye control comparison of IL-7 concentration in tears Occluded versus contralateral eye control comparison of IL-8 concentration in tears Occluded versus contralateral eye control comparison of IL-9 concentration in tears Occluded versus contralateral eye control comparison of IL-10 concentration in tears xvi

17 Figure Page 85 Occluded versus contralateral eye control comparison of IL-12p70 concentration in tears Occluded versus contralateral eye control comparison of IL-13 concentration in tears Occluded versus contralateral eye control comparison of IL-17 concentration in tears Occluded versus contralateral eye control comparison of Eotaxin concentration in tears Occluded versus contralateral eye control comparison of G-CSF concentration in tears Occluded versus contralateral eye control comparison of GM-CSF concentration in tears Occluded versus contralateral eye control comparison of IFNγ concentration in tears Occluded versus contralateral eye control comparison of IP-10 concentration in tears Occluded versus contralateral eye control comparison of MCP-1 concentration in tears Occluded versus contralateral eye control comparison of MIP-1α concentration in tears Occluded versus contralateral eye control comparison of MIP-1β concentration in tears Occluded versus contralateral eye control comparison of PDGF-BB concentration in tears Occluded versus contralateral eye control comparison of RANTES concentration in tears Occluded versus contralateral eye control comparison of VEGF concentration in tears xvii

18 LIST OF ABBREVIATIONS ANOVA CBA CD CV DNA EGF ELISA FGF FTBUT G-CSF GM-CSF IL-1α IL-1β IL-1ra Analysis of Variance Cytometric Bead-Based Assay Clusters-of-Differentiation Coefficients of Variation Deoxyribonucleic Acid Epidermal Growth Factor Enzyme Linked Immunosorbant Assay Fibroblast Growth Factor Fluorescein Tear Breakup Time Granulocyte-Colony Stimulating Factor Granulocyte Macrophage Colony Stimulating Factor Interleukin 1 alpha Interleukin 1 beta Interleukin 1 receptor antagonist IL-2 Interleukin 2 IL-4 Interleukin 4 IL-5 Interleukin 5 IL-6 Interleukin 6 xviii

19 IL-7 Interleukin 7 IL-8 Interleukin 8 IL-9 Interleukin 9 IL-10 Interleukin 10 IL-12(p40)/(p70) Interleukin 12 (p40)/(p70) IL-13 Interleukin 13 IL-15 Interleukin 15 IL-17 Interleukin 17 INF-γ Interferon-gamma IP-10 Interferon-Inducible Protein 10 KCS LTβ4 MCP-1 MHC MIP-1α MIP-1β NITBUT OSDI PAF PDGF bb PE RANTES RNA Keratoconjunctivitis sicca Lymphotoxin-beta Monocyte Chemoattractant Protein-1 Major Histocompatibility Complex Macrophage Inflammatory Protein-1 alpha Macrophage Inflammatory Protein-1 beta Noninvasive Tear Breakup Time Ocular Surface Disease Index Platelet Activating Factor Platelet-Derived Growth Factor-bb Phycoerythrin Regulated on Activation, Normal T-cell Expressed and Secreted Ribonucleic Acid xix

20 SVST TGF-β T H TNF-α VEGF Standardized Visual Scale Test Transforming Growth Factor-beta Helper-T Lymphocyte Tumor Necrosis Factor-Alpha Vascular Endothelial Growth Factor xx

21 INTRODUCTION Keratoconjunctivitis Sicca (KCS) or, more commonly, dry eye is a disorder of one or more physiologic processes involved in maintaining ocular surface health and homeostasis. It manifests as deficiencies of the tear film driven by an alteration of tear film quality, quantity, and or excessive evaporation [1, 2]. It may present as symptoms only, in conjunction with localized ocular surface changes, or as part of an array of findings associated with systemic disorders [3]. It is estimated 30 million individuals in the United States are afflicted by KCS [4, 5] and the economic burden and quality of life impact can be significant [6-8]. Keratoconjunctivitis sicca traditionally results in reduction of secreted tear film components and increased ocular surface inflammation [9-11]. The events triggering an inflammatory response are diverse and often elusive, and result in pathologic changes to the ocular surface characterized by corneal and conjunctival ophthalmic dye staining, diminished tear break-up time, and a wide range of patient symptoms. Rarely, KCS can lead to recurrent corneal erosions with corneal edema and progression to sight threatening tissue destruction. Corneal scarring of the visual axis and loss of vision are potential sequelae [3, 12]. Historical treatment of KCS has been palliative, focusing primarily on alleviating patient symptoms through tear film augmentation [13, 14]. Widely accepted approaches to augmentation include artificial tears of varying composition, tear preservation with Page 1 of 255

22 punctal occlusion, and pharmacological stimulation of the lacrimal gland via secretagogues. As understanding of the pathophysiologic basis of KCS has evolved, treatment now includes anti-inflammatory therapies such as topical Cylcosporin-A and ophthalmic steroids in advanced cases [13-15]. Presently these agents are employed when patient symptoms cannot be controlled by tear film augmentation and ocular surface structures demonstrate advanced signs of inflammation. However, the optimal point for intervention with anti-inflammatory agents in the treatment regimen of KCS remains unclear. A promising method to characterize the relationship between KCS and ocular surface health is to investigate the concentrations of multiple tear proteins which function as mediators of immunity and serve as objective markers of ocular surface health [16]. Conventional enzyme-linked immunoassays (ELISA) of tears provide the high sensitivity and specificity required for this type of protein analysis [17], but is limited to the assay of a relatively small number of proteins in a single non-stimulated tear sample. Using stimulus to increase tear sample volume, and thus assay more proteins, is questionable because protein concentrations change between non-stimulated and stimulated tears [18]. Cytometric bead-based assay (CBA) is a novel ELISA-based methodology that circumvents sample volume limitations by allowing the simultaneous assay of multiple proteins in a single, small, tear sample. This technology therefore has the potential to take us beyond standard clinical tests and to provide a truly objective, highly sensitive, measure of ocular surface changes [19]. This study examines the impact of a treatment for KCS, punctal occlusion. According to some, punctal occlusion alters tear protein concentrations which may promote inflammation [20-22]. This study is the first to optimize a CBA protocol tailored for Page 2 of 255

23 KCS and specifically examine the impact of a treatment option utilized in the management of KCS. Page 3 of 255

24 BACKGROUND AND SIGNIFICANCE Normal Ocular Surface Homeostasis The health and of the eye is maintained through interaction of ocular surface structures, nervous system integration, secretory glands, endocrine influence, and immune oversight. This integrated anatomy and physiology is detailed in what has been described as the lacrimal functional unit (Figure 1) [9]. The lacrimal functional unit is a feedback loop which consists of the links between and structures of the ocular surface (cornea, conjunctiva, and lacrimal drainage apparatus), it s sensory afferent nerves, central brainstem, efferent nerves to tear secretory glands, the secretory glands themselves, and the tear fluid [1, 9, 23-26]. Ocular Surface Corneal Conjunctiva Lacrimal drainage Neural Afferent N. Brainstem Efferent N. Tear Production Secretory Glands Tears Figure 1. Lacrimal functional unit feedback loop Page 4 of 255

25 Ocular surface structures contribute to the lacrimal functional unit through tear film support and signaling of ocular surface status [26-28]. The corneal nerves, comprising the body s greatest density of sensory receptors, mediate signaling and neuropeptide release and are central to maintaining ocular surface health and drive lacrimal gland secretion [23, 29, 30]. Unique sensory afferent signals monitoring corneal moisture status are transmitted by branches of the trigeminal nerve and processed by trigeminal subnuclei [31]. Central processing occurs with sympathetic and parasympathetic efferent signal genesis in the sympathetic preganglionic neurons of the spinal cord or pons respectively. Both autonomic pathways project to the lacrimal gland with sympathetic neurons projecting via the superior cervical ganglia and parasympathetic neurons projecting via the facial nerve. Both sympathetic and parasympathetic projections innervate lacrimal gland vasculature. In addition, parasympathetic projections innervate and control lacrimal and accessory gland acinar cell secretions. The main lacrimal gland, located superior temporal to the globe, along with accessory lacrimal glands in the superior bulbar conjunctiva and upper lid secrete the aqueous component of the tear film. Innervation and hormonal factors converge to influence these glands, with both essential for normal function [32, 33]. The tear fluid completes the loop for the lacrimal functional unit. It is an admixture of a distal meibomian gland derived lipid layer and proximal bi-laminar lacrimal gland derived aqueous and conjunctival goblet cell derived mucin layer [34, 35]. The tear film contains numerous constituents such as stabilizing agents, anti-bacterial proteins, and immuno-proteins commonly referred to as cytokines (discussed in greater detail be- Page 5 of 255

26 low) that closely influence the activity of ocular surface cells and sensory stimulation [36-38]. The lacrimal functional unit is intimately dependent on endocrine influences, specifically androgens like testosterone, to maintain proper function [33, 39]. Androgens regulate lacrimal gland and ocular surface cell function by controlling production of two paracrine factors that in turn regulate the balance of T-helper and cytotoxic T lymphocytes within these structures. These lymphocytes are involved in the removal of cell proteins, potential auto-antigens, that are secreted by surface and lacrimal epithelial cells into lymphatic spaces [40]. Meibomian gland function and lipid composition are also modulated by circulating androgen levels [41]. The immune system sustains normal ocular surface function by preventing or limiting pathogenesis. It prevents access and/or removes invading antigens, a process mediated by a complex network of specialized structures, cells, and molecules that provide nonspecific (innate) and specific (adaptive) protection [42]. The adaptive immune response is of particular interest due to its implication in ocular surface disease to include KCS [43, 44]. Facets of adaptive immune response activation include antigen processing and presentation by antigen presenting cells, specific lymphocyte recognition and activation, cellular and humoral responses, and targeted antigenic destruction or elimination [45]. Normal physiologic interaction of lacrimal functional unit structures and immune surveillance are required to ensure asymptomatic ocular function and sight preservation. Page 6 of 255

27 Classification and Pathogenesis A comprehensive effort to define and categorize KCS was made by the National Eye Institute s workgroup on clinical trials in dry eye in 1995 [1]. It was revised in 2007 and remains in predominant use today [2]. This group divided KCS into two broad categories, aqueous tear deficient and evaporative. Aqueous tear deficient KCS encompasses factors leading to the reduction of lacrimal gland secretion and is further subdivided into two categories, Sjogren s syndrome and Non-Sjogren s tear deficiency. Sjogren s syndrome tear deficiency may be further subdivided into primary and secondary. Primary Sjogren s syndrome patients manifest, along with aqueous deficiency, dry mouth and positive tests for autoantibodies. Secondary Sjogren s s syndrome patients possess the features of Primary Sjogren s along with overt systemic findings of autoimmune disease such as rheumatoid arthritis, lupus erythematosus, and diabetes. Non-Sjogren s aqueous tear deficient KCS is characterized by reduced secretion from loss of lacrimal gland function. This encompasses the largest proportion of those with KCS and may be congenital, age-related, or follow glandular inflammation secondary to trauma, infection, or loss of innervation. There are three possible causes of evaporative KCS: (i) alteration of the ocular surface exposure time by reduced blink rate or physical defect of lid and/or ocular surface structures, (ii) alteration of lipid contributions to the tear film, or (iii) alteration of tear film adhesion processes. All three destabilize the tear film and lead to premature tear film breakup. Alteration of the blink rate or physical defect of the lid and/or ocular surface increases ocular surface exposure time so that it exceeds the period of tear film stability. Page 7 of 255

28 Alterations of the lipid component may occur via contamination by skin lipids, meibomian gland hyper- or hypo-secretion, and/or alteration of the ratio of lipid components produced by the meibomian gland. Features of altered tear film lipid production have been characterized as meibomian gland disease with further divisions based on overt inflammatory components [46]. Tear film adhesion, supported by interaction of surface cell glycoprotein extensions and secretion; provide a hydrophilic environment for tear aqueous component adhesion. Alteration or reduction of these proteins also contributes to accelerated tear breakup promoting evaporation [27]. KCS classification is directed toward promoting tailored treatment, yet an allinclusive classification scheme remains elusive. Classification is limited by the dynamic nature of lacrimal functional unit interactions. A deviation in normal function for most structures appears to serve as a trigger for disseminating an inflammatory response. This domino effect of pathogenesis is well documented. Primary damage to corneal sensory nerves, as seen with refractive surgery, can result in excessive stimulation with subsequent down regulation of the neural signaling response necessary for both normal lacrimal gland function and neurotrophic effects on the ocular surface [23, 25, 47, 48]. Ablation of sensory and or parasympathetic input to the lacrimal gland, as observed with trauma or neuropathies, can result in lacrimal gland dysfunction with alterations in tear protein content and reduced secretory rates [32, 49]. Likewise, alterations in circulating androgen levels disrupts the balance of paracrine factors that in turn alter the ratio of Helper-T and Cytotoxic-T lymphocytes associated with the ocular surface in favor of heightened cytotoxic lymphocyte activity [40]. This results in up regulated inflammation at the ocular surface and subsequent reduction in lacrimal gland secretory rates. In addi- Page 8 of 255

29 tion, abnormally low androgen levels also alter meibomian gland secretions and disrupt tear film stability [41]. Contact lenses [50] and environmental factors [51-53] are known to alter tear film stability and induce ocular surface inflammation. Proposed deviations in immune privilege mediated by lacrimal drainage has been proposed as yet another contributor to KCS [26]. Immune privilege is a process that restricts immune system recognition of foreign antigens in some tissues by blocking activation of destructive inflammatory responses. This could conceivably alter the balance of lymphocytes at the ocular surface in favor of a pro-inflammatory state. Increased tear osmolarity, possibly precipitated by these or other factors, impacts corneal nerve activity and is centrally implicated in the pathogenesis of KCS [2, 36, 54]. Tear hyperosmolarity is established as a key factor initiating ocular surface inflammation, driving symptoms and compensatory events. Classification is further limited by discordance with diagnostic tests and symptomology [55-57], lack of uniformity in diagnostic approaches [6], and treatment. For example, aqueous tear deficiency and evaporative forms of KCS reveal distinct differences among diagnostic tests [46] yet the conditions are frequently treated in the same way. Classification also fails to address confounding variables such as medications or the fact that multiple triggers for KCS may coexist. Regardless of initial insult to the lacrimal functional unit, inflammation becomes the hallmark of KCS. Stimulation and expression of cell signaling molecules [43, 44] leads to an accumulation in pro-inflammatory cytokines in tear fluid which accelerates the inflammatory response [58, 59]. This establishes a cycle of chronicity sustaining recruitment of inflammatory cells at the ocular surface [60-63]. Recognizing that inflam- Page 9 of 255

30 mation is a hallmark of KCS, developing a better method of characterizing KCS based on inflammatory markers is essential. Immune Response The chronic inflammation of KCS is characterized by infiltration of ocular surface structures by cytotoxic lymphocytes. Lymphocyte classification and function provides insight into the type of immune response affecting the ocular surface and relationship to immune-related proteins mediating the response. Lymphocyte formation represents one of the terminal branches for bone-marrow derived stem cell differentiation. Lymphocytes are responsible for the specific recognition of antigens and for immunologic memory unique to adaptive immunity. Lymphocytes are broadly categorized as bone-marrow derived B-lymphocytes (10-15% of circulating lymphocytes), thymus derived T- lymphocytes (75%) and null cells (remainder; to include natural killer cells) [45]. B- lymphocytes primarily function in immunoglobulin synthesis and represent the humoral immune response. T-lymphocytes (along with natural killer cells) execute the cellmediated immune response and are further subdivided into Cytotoxic-T, Helper-T (T H ), and Regulatory-T cells. Each lymphocyte has unique cell surface molecules, clusters-ofdifferentiation (CD), that signals identity and activity. The CD markers for Cytotoxic-T and Helper-T are CD-8 and CD-4 respectively. Cytotoxic-T lymphocytes act by directly killing infected host cells that express unique surface proteins (major histocompatibility complex class I / MHC-I) in conjunction with foreign antigen. MHC proteins, found on the cell surface of all host cells, identify specific host cell subgroups (MHC I-III) and fa- Page 10 of 255

31 cilitate self-non-self recognition by other cells (i.e. lymphocytes). MHC proteins, when bound with antigen fragments, present these to lymphocytes initiating a response. Helper-T lymphocytes are the coordinators of adaptive immunity and are essential for humoral and/or cellular branch activation [45, 64-67]. Detection of processed antigen in conjunction with MHC-II bearing cells by Helper T lymphocytes and their subsequent activation represent the critical events in adaptive immunity. To that end Helper-T lymphocytes have been further subdivided into several populations based on their unique activities and immune-related protein expression patterns. Of these, the Helper-T subtypes- I and II (T H -1, T H -2) are of central interest and have classically characterized the divergence between allergic and KCS related ocular surface disease [43]. Orchestration and bias toward a particular type of immune response is mediated by immuno-proteins: cytokines, chemokines, and growth factors [68, 69]. These proteins form the basis of a complicated immunologic communication network. Cytokines modulate the growth, differentiation, and function of various target cells and are released from diverse cell types. Their functions can be interdependent and many cytokines aid or negate the effects of other cytokines. Cytokines exhibit both redundancy and pluripotency, mimicking functions and activation of specific behavior in some cell types while inhibiting that behavior in other cell types. Cytokines differ from hormones and growth factors by their non-constitutive short-lived secretion and short half-life of activity [68, 70]. These attributes generally limit cytokines to short distance autocrine or paracrine activities. Chemokines are a group of cytokines with similar properties in source and target cell diversity but also demonstrate prominent chemotactic activities, recruiting or ex- Page 11 of 255

32 cluding other immunologic cells from a site of inflammation [69]. Collectively cytokines, chemokines, and immuno-active growth factors are referred to as cytokines. The sources of cytokines are diverse; however, T H cells, dendritic cells and macrophages are the primary producers and regulators of these proteins. The cytokine patterns for T H -1 lymphocytes include development in the presence of interleukin (IL)-12 secreted from activated antigen presenting cells. T H -1 cells in turn express IL-2, interferon-gamma (IFN-γ), and tumor neucrosis factor-beta (TNF-β) but not IL-4 and IL-5 [71] (all cytokines) directing cell-mediated cytotoxic and delayed hypersensitivity reactions typical of chronic inflammation and KCS. T H -2 cells develop in the presence of IL-4 and secrete IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13 but not IFN-γ and TNF-β. T H -2 cells direct allergic/immediate hypersensitivity and humoral immune responses [64]. Beyond characterizing the type of adaptive response, cytokines can be functionally organized into groups according to major activities. Elevated IL-1, IL-6, and TNF-α are associated with acute inflammation. Those mediating chronic inflammation include IFN-γ and TNF-α. Cytokines mediating innate immunity include IL-1, IL-6, IL-12, IFNα, and IFN-β. Those primarily involved in supporting allergic inflammation are IL-4, IL- 5, and IL-13. Cytokines controlling lymphocyte regulatory activity include IL-2, IL-4, IL-5, IL-6, and IL-10, IL-12, IL-13, IFN-γ, and TNF-β. Those involved in the formation of blood cells: IL-3, IL-7, and granulocyte macrophage colony stimulating factor (GM- CSF). For a summary of cytokines discussed, T H bias, and principle activities, see Table 1 below. Page 12 of 255

33 Table 1 Summary of Cytokines Evaluated, T H bias, and Principal Activities IL-Iβ IL-1αR IL.-2 IL-3 IL-4 IL-5 IL-6 IL-7 IL-8 IL-9 IL-10 ll-12 (p70) IL-12 (p40) IL-13 Polarized T H -1 Principal T H -1 Polarized T H -1 Principle T H -2 Polarized T H -2 Polarized T H -2 Polarized T H -2 Polarized T H -2 Polarized T H -1 Polarized T H -1 Polarized T H -2 Cytokine T H -bias Principle Activities [45, 67-70, 72, 73] IL-lα Polarized T H -1 Membrane bound IL-1; early phase induction of inflammatory response following infection, up-regulation of pro-inflammatory cytokines and cell adhesion molecule expression; endogenous pyrogen. Stored in corneal epithelial cells; released with cell membrane rupture. Secretory IL-1 IL-1α antagonist; secreted by corneal epithelial cells; modulates IL-1α Activation-growth-differentiation of T-cells; induces TNF-β and IFN-γ expression Hematopoietic growth factor; acts with GM-CSF to promote multi-lineage lymphocyte colony development. Suppresses pro-inflammatory IL-1 and TNF-α production; modulates their response; Induces IgE synthesis Chemotaxis/activation of eosinophils, activation of basophiles, B cell differentiation. Pleotropic (pro- and anti-inflammatory); induction/control of acute phase hepatocyte proteins; induces B cell Ig production. Produced by keratocytes acting synergistically with IL-1α Early B and T cell growth factor Early phase leukocyte chemokine; produced by macrophages and vascular endothelium among others, recruits/activates neutrophils. Produced by infected keratocytes and corneal epithelium exposed to calcitonin gene-related peptide and substance P from sensory nerves Promotes growth/enhances activity of T-cells, mast cells, erythroid proginators; enhances IgG & IgE production with IL-4; role in hippocampal progenitor differentiation Pleotropic: inhibits IFN-γ production by NK and other proinflammatory cytokines by monocyte/macrophages; promotes mast and B cell proliferation/ig production, Bioactive heterodimer [p(35) and p(40) subunits]; regulates T and B cell, central to host defense from intracellular pathogens Bioactivity linked to this subunit; antagonizes IL-4 in T H bias in early phase T cell activation. Anti-inflammatory; down regulates IL-1a, IL-1b, IL-6, IL-8, IL- 10, IL-12, MIP-1α, GM-CSF, granulocyte CSF (G-CSF), IFN-α, and TNF-α, alters monocyte phenotypic expression; induces IgE response Page 13 of 255

34 Table 1 (continued) Summary of Cytokines Evaluated, T H bias, and Principal Activities Cytokine T H -bias Principle Activities [45, 67-70, 72, 73] IL-15 Polarized T H -1 Activation-growth-differentiation of NK and T-cells; induces TNF-α and IFN-γ expression IL-17 Pro-inflammatory; exclusive helper-t cell activator; promotes production of IL-6 and IL-8 IP-10 Defensin-like chemokine (for monocytes and T-cells); promotes T cell adhesion to endothelium; direct antiviral effects at ocular surface [74] TNF-α Polarized T H -1 Potent paracrine and endocrine mediator of inflammatory response; activates leukocytes, endothelial cells, others; endogenous pyrogen, induces shock-like symptoms G-CSF Polarized T H -2 GM-CSF Polarized T H -2 IFN-γ: Principle T H -1 Activates/enhances neutrophils, granulocytes and precursors; stimulates production of TNF-α and IL-1α Activates/enhances function of hematopoietic proginators, T-, monocytes, neutrophils, eosinophils Earliest detectable cytokine with protein antigen; activates many leukocytes, endothelial cells; up-regulates MHC I & II expression, increased macrophage & APC activity; down-regulates IL-4 directed Th2 cell growth Eotaxin MCP-1 MIP-lα Basic FGF VEGF PDGF- BB RANTES Polarized T H -2 Chemokine: indicative of chronic disease; eosinophils/initial T H - 2 cell recruitment; contributes to mast cell recruitment Monocyte chemotactic factor; induces macrophage infiltration; regulates monocyte cytokine production and macrophage adhesion molecule expression; enhanced during leukocyte associated inflammation Macrophage inflammatory protein, monocytes, T cells, neutrophils and eosinophils chemotactic protein; increases with infection/endotoxin Stimulates angiogenesis; promotes neuron regeneration and survival Stimulates angiogenesis; promotes/sustains endothelial cells; promotes vascular permeability Activates/chemotactic for glial, endothelial, vascular smooth muscle, fibroblast cells among others, inhibits NK cells Regulation on Activation Normal T-cell Expressed and Secreted; chemotactic for monocytes, CD4 specific T; eosinophils, basophils; enhances histamine release. Page 14 of 255

35 The diverse function of cytokines underscores the limitations inherent in measuring the level of only one in a sample. T-lymphocytes predominantly show a striking dichotomy between a T H- 1 and T H- 2 responses following most persistent immunologic challenges and the T H- 1/ H- 2 classification is an important functional division [71]. However, the classification of ocular surface disease based on T H bias has become more complex with expanding discovery of new T-lymphocyte subpopulations, cross over response patterns, and improved understanding in cytokine interactions [43, 44]. Cytokine concentrations may reflect a continuum in T H bias or activity of discrete subsets of T-cell activity. Therefore, it is not only the presence, but relative concentration differences of multiple cytokines that is necessary to reflect the depth and dynamic range of immunologic status [64, 75]. The relative concentrations of cytokines in tears provide an indication of functional immune status for the ocular surface. Diagnostic Tests Tests traditionally used to differentiate KCS from other ocular surface conditions demonstrate wide ranges of specificity and sensitivity [76]. Clinically the preferred methods are use of a symptoms-based questionnaire, measures of tear stability, tear production and elimination, and ocular surface appearance aided by the use of dyes [77]. There is significant lack of agreement between signs and symptoms when employing clinical tests [57, 78, 79]. Laboratory tests include measures of ocular surface cell density and morphology via impression cytology, and genomic and proteomic analysis of ocular tissue or tear samples. Page 15 of 255

36 The least invasive approach for laboratory analysis is tear assay. Tears serve as a global indicator of ocular surface health and are well suited for proteomic analysis. Using traditional ELISA techniques a broad range of cytokines and growth factors have been measured in tear fluid to include: IL-1α, IL-1β, IL-2, IL-2R, IL-5, IL-6, IL-8, IL-10, IFN-γ, GM-CSF, TNF-α, TGF-β, EGF, PDGF-BB, monocyte chemotactic protein 1 (MCP-1), lymphotoxin-beta (LTβ 4 ), and platelet activating factor (PAF) [15, 36, 63, 80-87]. However, tear sample volume constraints limit the number of cytokines (two or three serially) that can be assayed by ELISA from a single tear sample [84] To overcome this limitation many ELISA based study designs have used pooled tear samples; however, this masks individual subject differences. Thus, the conclusions that can be drawn from tear ELISA studies are limited to just a portion of the complex immune signaling response. Apart from the inherent problem of tear sample size restrictions, varying protein concentration with stimulus is another potential limitation [18, 83, 88]. To understand the immunologic status of the ocular surface, it is desirable to simultaneously quantitate concentrations of multiple cytokines from a single, relatively small sample collected under controlled stimulus conditions as quickly as possible. Cytometric bead-based assays are a relatively new laboratory test that have the potential to overcome these technical limitations and bridge the disconnect between signs, symptoms, and ocular surface damage [19, 89, 90]. Page 16 of 255

37 Cytometric Bead-Based Assays Cytometric bead-based assay technology, pioneered in the 1970s [91], achieved mainstream research application around 2000 [92-96]. It pairs the specificity of sandwich ELISA with the dynamic properties of flow cytometry. It is used to measure the concentrations of unknown analytes within a biological sample (serum, tears, saliva, and cell lysates). Unknown analytes of interest can be proteins, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Cytometric bead-based assays, using a Luminex instrument (Austin, TX), rely on multiple populations (plexs) of uniform size (5.6 micrometer diameter) polystyrene beads designed to pass through a multi-laser flow cytometer. Each bead population is internally labeled with a ratio of two fluorophores that, when excited by a 635nm classification laser, emit light at 658 and 712 nm. Each polystyrene bead possesses surface carboxyl groups to which specific monoclonal capture antibodies, targeting specific analytes (proteins, DNA, RNA), are covalently bound creating a capture bead. The internal fluorophor or ratios uniquely classifies/identifies beads for specific target analytes, allowing multiple analyte specific capture bead populations to be mixed or multiplexed. The multiplexed capture bead populations, when mixed with unknown sample containing target analytes, bind the unknown analytes to the capture bead antibodies initiating a unique sandwich ELISA. Phycoerythrin (PE)-conjugated target analyte specific detection antibodies are added to the assay to complete the sandwich for each capture bead population. PE, when excited by a 532 nm reporter laser, emits fluorescent light at 578 nm. During analysis, beads pass through a flow column and are excited by the classification laser and detected by temperature compensated avalanche photo diodes, confirm- Page 17 of 255

38 ing the presence of a single bead and classifying the bead (identifying target analyte) based on its internal fluorophor emission ratio. A second reporter laser directed at the column excites calibration standards or sample analyte bound PE. A photomultiplier tube with bandwidth detection of nm reports the emission, generating a fluorescent intensity histogram. The background fluorescence generated from the flow column in the absence of a bead is subtracted and the difference in reporter signal (bead associated less background fluorescence) indirectly quantifies bead-bound target analyte [97, 98]. Typically, 100 beads per target analyte are assayed from each sample. The number of capture bead populations multiplexed typically exceeds ten, thus thousands of beads are detected and characterized in the flow column for each unknown sample. The final concentration values of analytes in a sample are interpolated from calibrated standards curves transformed using a four or five parameter regression analysis. The PE generated median fluorescent intensity is the predictor variable in determining the concentration of unknowns interpolated from standards. The advent of cytometric bead based assays with multiplexing capability has increased understanding of the immunologic response [19, ]. This technology can simultaneously measure multiple (currently up to 100) target analytes (proteins, RNA, DNA) from a single small volume sample (volumes too small for traditional immunoassays) with high sensitivity and reproducibility. Bead based assays yield a wide detection range (at least three orders of magnitude) in fluorescent signaling [19] and is well suited to clarify changes observed with KCS pathogenesis and measure treatment efficacy. Page 18 of 255