The Tear Film



The Tear Film


Erin Peters

Kathryn Colby



INTRODUCTION

The tear film is an exceedingly complex structure, the intricacies of which are not yet completely understood. There has been tremendous growth in our understanding of tear film biology over the last decade. Gone are the days when we viewed the tear film as a simple structure composed of segregated layers of mucus, water and electrolytes, and lipid. Now we know that many of the tear film components interact to create a hydrated gel that allows the tear film to accomplish its multiple functions. This chapter reviews the current understanding of the structure of the tear film and will introduce the concept of the integrated lacrimal functional unit as a key component of the healthy ocular surface. Currently available techniques to evaluate tear film function and stability are discussed. Finally, current theories of dry eye disease are reviewed to add insight into the biology of the normal tear film.


TEAR FILM STRUCTURE

The tear film is responsible for providing a smooth refractive surface for clear vision, maintaining the health of corneal and conjunctival epithelia, and acting as the first line of defense against microbial infections. Tear film composition is dynamic and in a constant state of flux, responding to environmental conditions in order to maintain ocular surface homeostasis. Traditionally, the tear film was described as being composed of three separate and distinct layers: mucin, aqueous, and lipid. However, new studies suggest that mixing between the mucin and aqueous layer occurs, creating a gradient of decreasing mucin concentration into the aqueous layer. This aqueous-mucin layer forms a hydrated gel (Fig. 1) with complex biology, which is then covered by the lipid layer, which has its own highly ordered structure. For the sake of simplicity, the mucin, aqueous, and lipid layers are considered separately here.






Fig. 1. Diagram of the tear film hydrated gel. Membrane-associated mucins on the microplicae of the epithelium form the glycocalyx. Secretory mucins admix with the aqueous layer containing antimicrobial factors, such as lysozyme, and immunoglobulins secreted by the lacrimal gland. The anterior lipid layer provides stability by interacting with the mucin-aqueous phase, and is itself composed of polar and nonpolar phases. (From Gipson IK, Argueso P. The role of mucins in the function of the corneal and conjunctival epithelia. Int Rev Cytol 231:1-49, 2003, with permission.)


MUCIN LAYER

Ocular mucus is composed of mucin, immunoglobulins, urea, salts, glucose, leukocytes, cellular debris, and enzymes.1 Mucins are high molecular weight glycoproteins that are heavily glycosylated: 50% to 80% of their mass can be attributed to their carbohydrate side chains.2 Tandem repeats of amino acids rich in serine and threonine found in their protein backbone serve as sites for O-type glycosylation.3,4 Heavy glycosylation imparts an overall negative charge to the glycoprotein, making the mucins highly hydrophilic and able to admix with the aqueous layer and maintain water on the surface of the eye.

Mucins are classified as either membrane-associated or secretory. Secretory mucins are further divided into two groups: large gel-forming mucins or small soluble mucins.

Membrane-associated mucins form the glycocalyx, a dense barrier to pathogen penetrance, at the epithelial cell–tear film interface.2 The membrane-associated mucins contain a hydrophobic transmembrane domain that anchors the mucin on the apical surface of the epithelial cells, a short cytoplasmic tail that extends into the cytoplasm, and an extracellular domain that reaches into the tear film.5

Estimated to extend 200 to 500 nm from the cell surface, the extracellular, highly glycosylated tandem repeat domains, also called ectodomains, function as a disadhesive preventing cell–cell and cell–matrix interactions.2,6 This property provides a lubricating surface that allows lid epithelia to glide over the corneal and conjunctival epithelia without adherence.7

The cytoplasmic tail of the membrane-associated mucin is thought to affect epithelial activity by interacting with cytoplasmic proteins and facilitating signal transduction.2 The short cytoplasmic domains are also reported to be associated with the actin cytoskeleton,8,9 which helps support the microplicae structure.2 The presence of epidermal growth factor (EGF)-like domains on the membrane associated mucins suggests a potential role in the regulation of epithelial cell growth.9,10,11,12,13,14

Soluble forms of the membrane-associated mucins have also been identified in the tear film, although the exact mechanisms for this occurrence are still unknown.2,13,15 Possible mechanisms include: cleavage and release of the extracellular domain into the tear film in a process termed ectodomain shedding; posttranslational processing of the mucin into two subunits where one subunit remains anchored in the plasma membrane, and the other soluble subunit is packaged in secretory granules and released into the tear film; or as some data suggest, mucin shedding from the cell surface over time leaves the oldest cells without microplicae and membrane associated mucins. The oldest cells lose their disadhesive character with the loss of the mucin, which results in the adherence of the old cells to the mucus of the tear film and their removal via the nasolacrimal duct.

Secreted mucins move easily over the mucins composing the glycocalyx because of the repulsive forces between them, which result from their anionic character.7 Secretory mucins act as a “cleaning crew,” moving through the tear fluid and collecting debris that can then be removed via the nasolacrimal duct during blinking.7 The secreted mucins are classified as either gel-forming or soluble. The large gel-forming mucins are probably the largest glycoproteins known based on their high molecular weight and are considered gel forming because they are responsible for the rheological properties of mucus.2,16 The small soluble mucins lack cysteine-rich D domains and are present as monomeric species.2

The majority of ocular mucins are secreted by the conjunctival goblet cells;1 however, ocular mucins are also produced by the stratified squamous epithelium of the cornea and conjunctiva, and new evidence suggests that the lacrimal gland also contributes to mucin production.17,18,19 Corneal and conjunctival stratified squamous cells contain the membrane-associated mucins MUC1, MUC4, and MUC16.20,21,22 MUC1 is a likely candidate for the glycocalyx as it is present in the apical cell membranes of the superficial ocular surface cells.23,24 Soluble forms of MUC1, MUC4, and MUC16 have also been detected in the tears2 and MUC4 has been detected in the lacrimal gland.18 MUC2, a gel-forming secretory mucin, and MUC7, a soluble secretory mucin, have been identified in tears and are both present in the conjunctiva, although the exact cellular source in the conjunctiva is unknown.17,19 MUC7 is also secreted by the lacrimal gland.19 MUC5AC, a large gel-forming mucin, is expressed by the goblet cells of the conjunctival epithelium and has been identified in tears in some individuals.25 MUC5AC is the major mucin present at the ocular surface providing the scaffolding of the mucus layer.2,17,20 Mucin secretion by the corneal and conjunctival stratified squamous epithelial cells is not as well studied as mucin secretion by conjunctival goblet cells, which is discussed later in the section on the lacrimal functional unit.


AQUEOUS LAYER

The middle aqueous layer of the tear film consists of water, electrolytes, proteins, peptide growth factors, immunoglobulins, cytokines, vitamins, antimicrobials, and hormones secreted by the lacrimal glands (Table 1).26,27








Table 1. The normal tear film has numerous constituents




























































































Tear Constituents
Water
Electrolytes
Na+
K+
Mg2+
Ca2+
Cl
HCO3
PO43-
Proteins
Albumin
β-Lysin
Ceruloplasmin
Complement
Defensins
Group II phospholipase A2
Histamine
Interferon
Lactoferrin
Lipocalin
Lysozyme
Matrix metalloproteinases
Plasminogen activator
Prostaglandins
Proteases
Transferrin
Peptide growth factors
Epidermal growth factor (EGF)
Hepatocyte growth factor (HGF)
Transforming growth factor-β (TGF-β)
Immunoglobulins
IgA
IgG
IgD
IgE
IgM
sIgA (2 molecules of IgA joined by SC)
Cytokines
IL-1α
IL-1β
IL-6
Vitamins
Lipids
Mucins

Electrolytes present in the tear film include sodium, potassium, magnesium, calcium, chloride, bicarbonate, and phosphate ions.27 The electrolytes are responsible for the osmolarity of tears, acting as a buffer to maintain a constant pH and contribute to maintaining epithelial integrity of the ocular surface.28,29 An increase in osmolarity of the aqueous layer is a global feature of dry eye syndrome and damages the ocular surface directly and indirectly by triggering inflammation.27,30

Proteins found in human tears are species-specific. More than 60 proteins have been identified in human tears including albumin, immunoglobulins, metal-carrying proteins, complement, histamine, plasminogen activator, prostaglandins, proteases, and antimicrobials.31 Considering that the thin nonkeratinzed epithelium and abundant blood supply of the conjunctiva make the conjunctiva an ideal entrance for infectious agents, it is imperative that the ocular surface have a strong defense system to protect against invading microorganisms. The primary defense system of the ocular surface is composed of the nonspecific immunity conferred by lysozyme, lactoferrin, β-lysin, complement, defensins, and group II phospholipase A2 and the specific immunity of antibodies, such as secretory immunoglobulin A (sIgA).27 In aqueous-deficient dry eye syndrome, the concentration of lysozyme, lactoferrin, lipocalin, and sIgA are reduced, compromising the integrity of the defense system, which may make the ocular surface more susceptible to infection, in addition to the symptoms of dry eye.32,33

Lysozyme, lactoferrin, and lipocalin are regulated antimicrobial proteins, secreted in response to an intracellular stimulus with a rate of secretion that approximately matches the rate of tear flow. Therefore, their concentration remains relatively constant with various flow rates.34,35,36 Lysozyme, an antimicrobial enzyme, lyses bacterial cell walls in the same manner as penicillin. It is one of the most important protein components in the tear film and acts synergistically with β-lysin, an enzyme that attacks bacterial cell membranes. Lactoferrin, a metal-binding protein, also has antimicrobial properties and may enhance antibody activity against certain microorganisms. Lipocalin, a lipid-binding protein, scavenges potentially harmful hydrophobic molecules and has recently been shown to inhibit bacterial and fungal infections through sequestering microbial siderophores.37 Further evidence suggests that lipocalin may contribute to a stable tear film by interacting with meibomian lipids and delivering them to the aqueous layer, and when complexed with other tear components, may be responsible for the high viscosity and low surface tension of tears.38,39,40 Immunoglobulins are constitutively produced and transported to the tear film from the conjunctiva. Thus, reflex tearing reduces the concentration of immunoglobulins and a reduction in tear flow increases their concentration.36 An example of one such immunoglobulin is sIgA. sIgA is formed by two molecules of immunoglobulin A (IgA) produced by plasma cells in the adenoid layer of the conjunctiva being joined by secretory component (SC), a protein molecule produced by the conjunctival epithelium. sIgA is produced at a rate dependent on its rate of synthesis, which is regulated by its local endocrine environment.34,35,41

In nonstimulated tears, a small proportion of proteins are derived through the leakage of plasma components through ocular surface capillaries.27 These components include albumin; IgG; ceruloplasmin, a copper-carrying protein with oxidizing potential; transferrin, an iron-carrying protein; and monomeric IgA.27 Their concentrations increase in dry eye syndrome resulting from decreased tear volume and leakage from chronically inflamed surface capillaries.27 Peptide growth factors (such as EGF, transforming growth factor-β (TGF-β) and hepatocyte growth factor [HGF]), and vitamin A act via autocrine and paracrine mechanisms to regulate epithelial proliferation, motility, and differentiation. Peptide growth factors are also involved in corneal wound healing and immune modulation.42,43 Evidence suggests that the concentration of EGF is decreased in dry eye syndrome, and it seems reasonable to suppose that the other growth factors secreted by the lacrimal glands are similarly affected.27,44


LIPID LAYER

The anterior layer of the tear film is composed of meibomian oil secreted by the meibomian glands and is the major barrier to evaporation from the ocular surface.45,46 The lipid layer is also responsible for providing stability to the tear film through interaction with the aqueous-mucin phase, providing a smooth optical surface for the cornea, and acting as a barrier against foreign particles.38 McCulley and Shine47 have proposed that the anterior lipid layer is composed of two phases: a polar surfactant phase overlaid by a nonpolar phase. The polar lipid phase, primarily composed of phospholipids and glycolipids, is multifunctional. The highly structured polar lipid layer acts as a surfactant between the hydrophilic aqueous mucin layer and the thick, nonpolar lipid layer; facilitates interaction with the aqueous–mucin layer; provides a barrier; and offers a structural component on which the nonpolar phase depends.47,48 The nonpolar phase (mainly composed of wax, cholesterol esters, and triglycerides) provides the air-tear film interface and is responsible for retarding evaporation.47

A normal tear film lipid layer is able to reduce evaporation by approximately 90% to 95%.49,50,51 The rate of evaporation is affected by the thickness of the lipid layer, and it has been postulated that a decrease in thickness may cause evaporative dry eye.45 In fact, mild to moderate dry eye states exhibit a lack of confluence in the tear film lipid layer.52 Lipid composition may also affect evaporation because it has been reported that the phospholipid content of meibomian oil is decreased in patients with dry eye and meibomian lipid composition is altered in anterior and posterior blepharitis, although the exact mechanisms have yet to be elucidated.48,53,54,55,56,57,58 The melting range of meibomian oil is dependent on lipid composition and may be lowered by the presence of branched and unsaturated fatty acids and alcohols.38 The low melting range of meibomian oil (19.5°C to 32.0°C), attributed to the variety of lipids present in the meibomian oil, facilitates meibomian delivery and contributes to tear film stability as the surface corneal temperature (32°C) is close to the upper limit of the melting range, allowing the lipid to exist in a relatively solid state.45

Meibomian oil secretion is a continuous process, occurring 24 hours per day during waking and sleeping hours, and is aided by blink action.45 The rate of secretion is controlled by neural, hormonal, and vascular influences.38 The lid margin reservoir of oil in an adult male has been estimated to contain approximately 300 μg of meibomian oil, which is more than adequate to refresh the lipid layer after each blink considering that the preocular tear film holds 9 μg of lipid.59,60 The casual oil level (or resting level) in the marginal lid reservoir is highest just after waking and lower late morning and at the end of the day, suggesting that oil retained during sleep is discharged when blinking is resumed after waking.59,61 Forced blinking, as well as deliberate expression of meibomian oil, has been shown to increase the thickness of the tear film lipid layer;62,63 however, evidence also shows that oil delivery can be maintained in the absence of blinking, implying that blinking is not essential for delivery. Studies show a rise in the casual level of lipid on the lid margin with age in both genders; however, lower rises in levels in women before menopause suggests a hormonal influence on meibomian secretion.23


LACRIMAL FUNCTIONAL UNIT

In order to maintain ocular surface homeostasis, the appropriate release in quantity and composition of tear film components must occur in response to various stimuli. Secretion of tear film components is coordinated and controlled by the lacrimal functional unit described by Stern et al.64 The components of the functional unit act in tandem to maintain the integrity of the normal ocular surface; when any component of the unit is compromised, normal lacrimal support of the ocular surface is impaired, resulting in ocular pathology.64

The lacrimal functional unit is composed of the ocular surface tissues (cornea and conjunctiva, including goblet cells and meibomian glands), the lacrimal glands (main and accessory [Wolfring and Krause]), and their interconnecting sensory (CN V) and autonomic (CN VII) innervation.64 Innervation occurs from the parasympathetic system (acetylcholine and vasoactive intestinal peptide [VIP] containing nerve terminals), sympathetic nerves (neuropeptide Y and norepinephrine), and sensory fibers from the trigeminal nerve (substance P and calcitonin gene-related peptide).65,66,67

Tear film secretion from the lacrimal functional unit is reflexive.68 The cornea has 60 times more nerve endings than dental pulp.69 The sensory nerves of the highly innervated cornea and conjunctiva form the afferent limb of a simple reflex arc that conducts stimuli from the external environment back to the central nervous system.70,71 The efferent limb of the reflex arc is composed of the sympathetic and parasympathetic nerves that innervate the ocular surface epithelia and tear-producing glands. These nerves are responsible for stimulating the conjunctival epithelial goblet cells, lacrimal glands, and meibomian glands to secrete their respective components (mucus, aqueous, and lipid, respectively) onto the ocular surface to provide protection from the original stimulus.71


GOBLET CELLS

In addition to the epithelial cells, goblet cells are the second cell type found in the conjunctival epithelium and are the main source of mucus secretion. Found singly or in clusters, goblet cells are interspersed among the stratified squamous cells of the conjunctival epithelium. Goblet cells contain secretory granules in the apical portion of the cell that contain the mucins and other glycoproteins that are secreted onto the mucus layer of the tear film. MUC5AC, the large gel-forming mucin that is the major mucin present at the ocular surface, has been identified to be associated with the goblet cells, but other mucins, such as MUC2 and MUC7, may also be associated.

Conjunctival goblet cells are secretory apocrine glands, meaning all or most of the contents of the secretory granules in each cell are secreted in response to a given stimulus. Thus, controlling the number of cells responding to the stimulus regulates secretion.71 Direct and indirect neural control of mucin secretion has been described.72 Activation of sensory nerves innervating the stratified squamous cells of the cornea and conjunctiva induces goblet cell secretion by stimulating a local reflex arc.65 Both sympathetic and parasympathetic nerves innervate the conjunctival goblet cells. However, only parasympathetic neurotransmitters, acetylcholine and VIP, have been shown to activate mucus synthesis and secretion by the goblet cells.71 Muscarinic receptors present on conjunctival goblet cells bind cholinergic agonists leading to activation of a specific signaling pathway, which results in an increase in intracellular Ca2+ concentration and goblet cell secretion.73,74,75 Although sympathetic nerves have not been shown to stimulate goblet cell secretion, α-adrenergic receptors are present on immature goblet cells and may participate by regulating mucin synthesis, goblet cell proliferation, or other processes.71,76

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Jul 11, 2016 | Posted by in OPHTHALMOLOGY | Comments Off on The Tear Film

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