In 2007, the Report of the International Dry Eye Workshop (DEWS) defined dry eye as “a multifactorial disease of the tears and ocular surface that results in symptoms of discomfort, visual disturbance, and tear film instability, with potential damage to the ocular surface. It is accompanied by increased osmolarity of the tear film and inflammation of the ocular surface.” The comprehensive DEWS report summarizes the current knowledge of the disease, its classification, symptoms and signs, epidemiology, diagnostic methods, and management and therapy. Research into dry eye is also summarized, and a hypothesis as to the mechanisms of dry eye is put forward ( Figure 18.1 ). As indicated in Figure 18.1 , mucin loss from the tear film and the ocular surface glycocalyx is proposed to be a core mechanism of the disease. This chapter will describe current understandings of the ocular surface and tear mucins – focusing on their character, origins, and alterations in drying eye diseases – as well as therapeutics targeted toward mucin restoration.
The Ocular Surface System
The tear film is essential for vision, functioning to maintain hydration and lubrication of the surface of the eye and to provide the major refractive surface for the visual system. The tear film is composed of many products, all produced by the epithelia of the Ocular Surface System ; these include water, protective antimicrobials, cytokines, lipids, and mucins ( Box 18.1 ).
The Ocular Surface System is responsible for producing and maintaining the all-important tear film on the surface of the cornea. The tear film is essential for vision, functioning to maintain hydration and lubrication of the surface of the eye, and to provide the major refractive surface for the visual system. All components of the system are integrated functionally by continuity of the epithelia, by innervation from the trigeminal nerves, and by the endocrine, vascular, and immune systems
The Ocular Surface System is responsible for producing and maintaining the all-important tear film on the surface of the cornea ( Figure 18.2 ). The system includes the surface or glandular epithelia of the cornea, conjunctiva, lacrimal glands, accessory lacrimal glands and meibomian glands, the nasolacrimal duct, as well as the eyelashes, with their associated glands of Moll and Zeis. Also included are the extracellular matrices and their resident cells, which underlie the epithelia, the vasculature, and migrating immune system cells, which survey the tissues. All components of the system are integrated functionally by continuity of the epithelia, by innervation from the trigeminal nerves, and by the endocrine, vascular, and immune systems.
The rationale for the concept of “the Ocular Surface System” is that all regions of the epithelia are derived from the surface ectoderm, that each region is continuous with another, and that all regions produce components of the tear film. For example, the hydrophilic mucins, responsible for holding tears on the surface of the eye, are products of the conjunctival and corneal epithelia. Water and protective proteins are secreted by the epithelia of the lacrimal and accessory lacrimal glands, and the superficial lipid layer, which prevents tear evaporation, is provided by the epithelia of the meibomian glands. When one or more components of the system is defective, the normal function of the ocular surface tear film is disrupted, which can result in chronic dry-eye disease and, at end stage, loss of the tear film with keratinization of the epithelia.
The early hypothesis of tear film structure separated the several secreted components into different layers: the lipid, aqueous, and mucin layers. While it is known that the superficial lipid layer, a product of the meibomian glands, is a distinct layer at the tear surface, recent data suggest that the aqueous tears are a mixture of lacrimal fluid and soluble mucins, without a distinct mucin layer ( Figure 18.3 ). The interface between the tear film and the corneal and conjunctival epithelia is composed of the hydrophilic and heavily glycosylated glycocalyx, a major component of which is membrane- or cell surface-associated mucins (MAMs).
Mucins expressed by the ocular surface epithelium
Mucins are high-molecular-weight glycoproteins. The common features of the 20 mucin gene products known to date are: (1) the presence of tandem repeats of amino acids, in their protein backbone, that have high levels of serine, threonine, and proline – with the serines and threonines being sites for O-glycan attachment; and (2) a major portion of mass of mucin molecules being made up of O-linked carbohydrate ( Box 18.2 ). The heavy glycosylation of mucins gives molecules of this class their hydrophilic, lubricating character.
The high-molecular-weight glycoproteins, known as mucins, share two common features: (1) the presence of tandem repeats of amino acids in their protein backbone that have high levels of serine, threonine, and proline – with the serines and threonines being sites for O-glycan attachment; and (2) a mass primarily made up of O-linked carbohydrates. Two types of mucins have been identified: secreted, and membrane-associated or membrane-spanning. Of the 20 mucins identified to date, 7 have been described as secreted and 10 as membrane-associated; several mucins remain uncharacterized as to type. Mucins are named in order of their characterization – MUC1, MUC2, etc. The ocular surface epithelia express mucins of both types. The major mucin of the conjunctival goblet cell is the secreted mucin MUC5AC. Three major membrane mucins expressed by the ocular surface epithelia include MUCs 1, 4, and 16
As indicated above, two types of mucins have been identified: secreted and cell-associated or membrane spanning. To date, 7 mucins have been described as secreted mucins and 10 as membrane-associated; several mucins remain uncharacterized as to type. Human mucins have been designated in order of discovery as MUC1, -2, -3, etc., with mouse homologs designated Muc1, -2, etc.
Of the secreted mucins, two types have been characterized: the so-called large, gel-forming mucins and the small, soluble mucins (one of each type is expressed by the ocular surface epithelia). The large, gel-forming mucins include MUCs 2, 5AC, 5B, 6, and 19, and the smaller, soluble mucins MUCs 7 and 9.
The five large mucins are termed gel-forming because they are responsible for the rheological properties of mucus. They share common structural motifs, including cysteine-rich, von Willebrand factor-like D domains at the amino terminal and carboxy termini that allow intermolecular associations among mucins of the same gene product. They are encoded by the largest genes known (15.7–17 kb), and their deduced proteins are at least 600 kDa. These gel-forming mucins are expressed by the goblet cells of the conjunctival, respiratory, gastrointestinal, and endocervical epithelia. However, there is a tissue- and cell-specific pattern of expression of specific mucins. The major mucin of this class expressed by the goblet cells of the conjunctiva is MUC5AC ( Figure 18.4 ). For a complete description of the structure of MUC5AC, see Gipson and Argüeso and Gipson.
The second category of secreted mucins, small soluble mucins, includes MUC7 and MUC9, which are present predominantly as monomeric species and lack cysteine-rich D domains. MUC7 is produced by lacrimal gland epithelia, but MUC7 is not present in the tear film.
The 10 mucins that have been categorized as cell MAMs include MUCs 1, 3A, 3B, 4, 12, 13, 15 16, 17, and 20. Mucins of this type are present on all the wet-surfaced epithelia of the body. All MAMs have a short cytoplasmic tail, and the majority have a large, extended extracellular domain, also known as the ectodomain, which is formed by heavily O-glycosylated tandem repeats of amino acids. The ectodomains may extend 200–500 nm from the cell surface and comprise a major portion of the glycocalyx. The ectodomain functions as a protective, disadhesive surface, preventing cell and pathogen adherence. Ectodomains of MAMs are found in fluids at the surface of wet-surfaced, mucosal epithelia, including the tear film. The ectodomains are proteolytically cleaved or released from the apical membranes, giving rise to the soluble form, or in some instances (particularly MUC1), the soluble form may be a result of splice variants that lack the membrane-spanning domain.
MUC1, MUC4, and MUC16 are MAMs that are expressed by ocular surface epithelia ( Figure 18.5 ). MUC1 mRNA is expressed by all the epithelia of the Ocular Surface System. The protein is expressed in apical surface cells of the corneal epithelium and in apical and subapical cells of the conjunctival epithelium. MUC4 protein is most prevalent in conjunctival epithelium with a diminished amount in the cornea. MUC16 protein is present in apical cells of corneal epithelia and in apical and subapical cells of conjunctival epithelia. The membrane mucins, MUCs 1, 4, and 16, are considered to be multifunctional molecules, with the glycosylated region of their ectodomain serving to prevent adhesion of cells/pathogens, and their juxtamembrane region and cytoplasmic tail serving additional functions. In studies of nonocular epithelia, MUC1 has signaling capabilities through its cytoplasmic tail, and MUC4 has been shown to have an epidermal growth factor (EGF)-like domain present extracellularly near its membrane-spanning domain. Studies of MUC16 function in human corneal epithelial cells have shown that it provides a disadhesive, protective barrier to the epithelial membrane, since, in siRNA knockdown experiments, the binding of Staphylococcus aureus was significantly increased with MUC16 suppression. Knockdown of MUC16 also allows penetrance of rose Bengal dye, a dye that is used in the diagnosis of dry eye. MUC16 is especially prevalent on the tips of microplicae at the tear film interface, and its cytoplasmic tail is linked to the actin cytoskeleton through a class of linker molecules known as ERMs ( Figure 18.6 ).