Tear film overview
The tear film overlays the ocular surface, which is comprised of the corneal and conjunctival epithelia and provides the interface between these epithelia and the external environment. The tear film is essential for the health and protection of the ocular surface and for clear vision, as the tear film is the first refractive surface of the eye.
Tears produced by the ocular surface epithelia and adnexa are distributed throughout the cul-de-sac. Using ocular coherence tomography, the thickness of the precorneal tear film was measured as 3.4 + 2.6 μm, agreeing with previous measurements using less accurate techniques.
The tear film is an exceedingly complex mixture of secretions from multiple tissues and epithelia ( Fig. 15.1 ) and consists of two layers ( Fig. 15.2 ). Not a layer, but a component that interacts with the tear film, is the glycocalyx, which extends from the superficial layer of the ocular surface epithelia. The first layer of the tear film consists of two components. The first is mucus that covers the glycocalyx and mixes with the aqueous components. The second layer is the outermost layer that contains lipids. Production and function of main tear film components are distinct and will be presented separately.
Tear secretion by all ocular adnexa and ocular surface epithelia must be coordinated. For the mucoaqueous layer, secretion is predominantly regulated by neural reflexes. Stimulation of sensory nerves in cornea and conjunctiva activates a complex neural reflex. Briefly, the ascending portion includes the corneal and conjunctival sensory nerves, the trigeminal ganglion, then the trigeminal brainstem complex nucleus, and the superior salivatory nucleus. The descending parasympathetic pathway runs from the parasympathetic nucleus of the facial nerve to the pterygopalatine ganglion, and the post-ganglionic parasympathetic nerves that travel to the lacrimal gland ( Fig. 15.3 ). The descending sympathetic pathway travels through the superior cervical ganglion and the post-ganglionic sympathetic nerves innervate the lacrimal gland. Activation of this reflex stimulates the lacrimal gland and conjunctival goblet cells to cause mucus, protein, and fluid secretion. For the lipid layer, the blink itself regulates release of presecreted meibomian gland lipids stored in the meibomian gland duct. When the eyelids retract, a thin film of lipid overspreads the underlying mucoaqueous layer.
Tear secretion is balanced by drainage and evaporation. Tears on the ocular surface are drained through lacrimal puncta into the lacrimal drainage system. Drainage of tears can be regulated by neural reflexes from the ocular surface that cause vasodilation and vasoconstriction of the cavernous sinus blood supply of the drainage duct ( Fig. 15.4 ). Both vasoconstriction and vasodilation cause a change in geometry of the lumen that decreases drainage. Evaporation depends on the amount of time the tear film is exposed between blinks and temperature, humidity, wind speed, and composition of the tear film. The remainder of the chapter focuses on regulation of tear secretion.
Glycocalyx
Structure
The glycocalyx is a network of polysaccharides that project from cellular surfaces. In corneal and conjunctival epithelia, the glycocalyx can be found on the apical portion of the microvilli that project from the apical plasma membrane of the superficial cell layer ( Fig. 15.2 ). Mucins are a critical component of the glycocalyx. Mucins consist of a protein core of amino acids linked by O -glycosylation to carbohydrate side-chains of varying length and complexity. Mucins are classified by the nomenclature MUC1–21 and are divided into secreted and membrane-spanning categories ( Fig. 15.5 ). Membrane-spanning mucins consist of a short intracellular tail, a membrane-spanning domain, and a large, extended extracellular domain that forms the glycocalyx. Secreted mucins are either gel-forming or small soluble. Gel-forming mucins are large molecules (20–40 million Da) secreted by exocytosis from goblet cells. Small soluble mucins are secreted by the lacrimal gland.
The ocular surface contains the membrane-spanning mucins MUC1, MUC4, and MUC16. These mucins are produced by stratified squamous cells of the cornea and conjunctiva and are stored in small, clear secretory vesicles in the cytoplasm ( Fig. 15.1 ). Fusion of secretory vesicles with the plasma membrane inserts these molecules into the plasma membrane. Mucin molecules are localized to the tips of the squamous cell microplicae and extend up to 500 nm above the plasma membrane. These mucins form a distinct glycocalyx by extending far above the other glycoconjugates such as the proteoglycans (heparan sulfate and chondroitin sulfate) and gangliosides that are richly present in the glycocalyx. In addition, the galactosyl residues on the mucins in the glycocalyx are crosslinked by the multimeric lectin galectin-3. This interaction is important for the maintenance of the epithelial barrier and the prevention of cellular damage.
There is limited information on the regulation of membrane-spanning mucin synthesis and secretion. Regulation of secretion would be by the regulation of insertion of mucins into plasma membranes or by control of ectodomain shedding, whereby matrix metalloproteinases (MMPs) cleave the mucin and release the extracellular, active domain of the protein into the extracellular space. In immortalized stratified corneal-limbal cells, tumor necrosis factor induced the ectodomain shedding of MUC1, MUC4, and MUC16, whereas MMP-7 and neutrophil elastase induced the shedding of MUC16 only. These compounds that caused shedding are elevated in the tears of dry eye patients and thus may cause the increase in Rose-Bengal staining found in dry eye and associated with loss of MUC16 ( Box 15.1 ).
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Dry eye is a multifactorial disease of the tears and ocular surface
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Dry eye is characterized by a loss of homeostasis of the tear film
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Dry eye is accompanied by ocular symptoms
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In dry eye, tear film instability and hyperosmolarity, ocular surface inflammation and damage, and neurosensory abnormalities play etiological roles
Function on the ocular surface
The membrane-spanning mucins function to hydrate the ocular surface and serve as a barrier to pathogens. Membrane-spanning mucins are considered to be dysadhesive, allowing the goblet cell mucus (see section Mucus production) to move over the ocular surface. The carbohydrate side-chains hold water at the surface of the apical cell membranes. The function of individual membrane-spanning mucins remains unclear.
Membrane-spanning mucins appear to be altered in dry eye. MUC16 protein levels were decreased in conjunctival epithelium and increased in tears of patients with Sjögren’s syndrome. , MUC1 splice variants also play a role in dry eye. Human cornea and conjunctiva contain five previously identified MUC1 splice variants and a new splice variant. These splice variants have unique changes that could affect their ectodomain shedding, signaling properties of the intracellular domain, and water retention, lubrication, and barrier properties of the extracellular domains. When the type of MUC1 splice variant was determined in dry eye patients (both evaporative dry eye and Sjögren’s syndrome), compared with control patients, there was a reduced frequency of MUC1/A variant and an increase in MUC1/B variant in the dry eye patients. ,
Mucous production
Structure
Mucus produced by the conjunctiva consists of MUC5AC, proteins, electrolytes, and water synthesized and secreted by the goblet and stratified squamous cells of the corneal and conjunctival epithelia. The backbone of mucus is the gel-forming mucin, MUC5AC, synthesized and secreted by conjunctival goblet cells. MUC5AC is encoded by one of the largest genes known, producing a protein of about 600 kDa. The protein backbone of MUC5AC consists of four D domains (cysteine-rich domains) ( Fig. 15.5 ) that flank a tandem repeat sequence in which amino acids in the protein backbone are O -glycosylated and linked to carbohydrate side-chains. The D domains provide sites for disulfide bonds crosslinking multiple MUC5AC molecules, which forms the framework of the mucus. Also contained in the mucous layer are shed ectodomains of membrane-spanning mucins, membrane-spanning mucins secreted by a soluble pathway, other proteins synthesized and secreted by goblet cells, and electrolytes and water secreted by goblet and stratified squamous cells.
Conjunctival goblet cells
Goblet cells are interspersed among stratified squamous cells of the conjunctiva (see Fig. 15.1 ). Goblet cells occur in clusters in rat and mouse, but singly in rabbit and humans. In all species studied, goblet cells are unevenly distributed over the conjunctiva.
Goblet cells are identified by the large accumulation of mucin granules in the apex (see Fig. 15.1 ). Secretory products can be visualized using Alcian blue–periodic acid stain, the lectins Ulex europaeus agglutinin I (UEA-I) or Helix pomatia agglutinin (HPA), or antibodies to MUC5AC. MUC5AC is synthesized in the endoplasmic reticulum, and carbohydrate side-chains are added in the Golgi apparatus. The mature proteins are stored in secretory granules. Upon stimulation, secretory granules fuse with each other and the apical membrane and release secretory product into the tear film. Upon cell stimulation the entire complement of granules is released. The amount of secretion is controlled by regulating the number of cells that are activated by a given stimulus.
Regulation of goblet cell mucin production
Overview
Mucin production is regulated by controlling the rate of mucin synthesis, rate of mucin secretion, and the number of goblet cells present in the conjunctiva. The rate of mucin synthesis has yet to be studied in conjunctival goblet cells. Thus, the remainder of this section focuses on regulation of secretion and proliferation. Under physiologic conditions in health, goblet cells secrete mucus in response to the challenges from the environment to maintain homeostasis. In disease, goblet cell mucous secretion can be overproduced, as in ocular allergy, or underproduced, as in dry eye. As goblet cell secretion is tightly regulated, goblet cells and their secretion can be returned to homeostasis.
Regulation of goblet cell secretion in health
Nerves are the primary regulators of conjunctival goblet cell secretion under physiologic conditions. The conjunctiva is innervated by afferent sensory nerves and efferent sympathetic and parasympathetic nerves. Sensory nerves end as free nerve endings between the stratified squamous cells. The parasympathetic and sympathetic nerve endings also surround the middle of the goblet cells at the level of the most basal secretory granules ( Fig. 15.6 ). Stimulation of the sensory nerves in the cornea by a neural reflex induces goblet cell secretion via the efferent nerves. Goblet cells have receptors for neurotransmitters from both parasympathetic and sympathetic nerves. Parasympathetic nerves release both acetylcholine (Ach) and vasoactive intestinal peptide (VIP). Muscarinic receptors of the muscarinic 3 acetylcholine receptor (M 3 AchR) and M 2 AchR subtypes are located on the goblet cells at the middle near the efferent nerve endings ( Fig. 15.6 ). , VIP receptors of the VIPAC2 receptor subtype are located in the same area as M 3 AchR. Sympathetic nerves release norepinephrine and NPY. Several subtypes of both α 1 – and β-adrenergic receptors are present on goblet cells. It appears that parasympathetic nerves using Ach and VIP are the primary stimuli of goblet cell secretion. The function of the sympathetic nerves remains unstudied.
Components of the signaling pathways used by Ach and VIP have been delineated. Cholinergic agonists use M 3 AchR and M 2 AchR to stimulate goblet cell secretion. Cholinergic agonists presumably use the G αq/11 subtype of G protein that activates phospholipase C to break down phosphatidylinositol 4,5 bisphosphate to produce inositol 1,4,5 trisphosphate (IP 3 ) and diacylglycerol. IP 3 would then release intracellular Ca 2+ by binding to its receptors on the endoplasmic reticulum. Based on a variety of experiments, cholinergic agonists are known to increase the intracellular [Ca 2+ ] to stimulate secretion. ,
The diacylglycerol released with IP 3 activates protein kinase C (PKC). Nine PKC isoforms are present in conjunctival goblet cells, and phorbol esters, activators of PKC isoforms, stimulate goblet cell secretion. Although a role for PKC isoforms in secretion could not be substantiated, it is likely that cholinergic agonists use PKC isoforms to stimulate secretion, as PKC inhibitors block a distal step in the signaling pathway, the activation of extracellular regulated kinase (ERK1/2) ( Fig. 15.7 ).
Cholinergic agonists are known to activate the epidermal growth factor (EGF) signaling pathway. In goblet cells, cholinergic agonists activate the nonreceptor tyrosine kinases Pyk2 and p60Src. These kinases transactivate (phosphorylate) the EGF receptor. This transactivation is usually mediated by ectodomain shedding by MMP, causing the release of the extracellular, active domain of one member of the EGF family of growth factors, but this ectodomain shedding has yet to be tested in goblet cells. The released growth factor would bind to the EGF receptor inducing two receptors to associate and be autophosphorylated. This attracts the adapter proteins Shc and Grb2 that are phosphorylated, activating the guanine nucleotide exchange factor, SOS, to increase Ras activity. Ras activates MAPK kinase kinase (Raf) that phosphorylates MAPK kinase (MEK) that phosphorylates ERK1/2. In both rat and human goblet cells, cholinergic agonists increase intracellular [Ca 2+ ] and activate PKC to stimulate goblet cell secretion by activating Pyk2 and p60Src to transactivate the EGF receptor, inducing the signaling cascade that activates extracellular signal-regulated kinase 1/2 (ERK1/2) ( Fig. 15.7 ). , ,
There are several non-neural agonists that stimulate goblet cell secretion. These agonists include the specialized proresolving mediators (SPMs) that are biosynthesized from omega-6 and omega-3 fatty acids. They also include ATP, which stimulates goblet cell secretion via purinergic receptors (P) of the P2X 7 and P2Y 2 subtypes, and the neurotrophin family of growth factors, of which nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) stimulate secretion. The SPMs include the lipoxins (LXA 4 ), D-series resolvins (RvD1–2), , E-series resolvins (RvE1), and maresins (MaR1). In conjunctival goblet cells, these mediators use specific G protein–coupled receptors; activate phospholipase C, D, and/or A 2 ; increase intracellular [Ca 2+ ]; activate PKA (selected mediators); transactivate epidermal growth factor receptor (EGFR) (selected mediators); and stimulate secretion.
The action of the purinergic receptor, P2X 7, was studied by patch-clamp analysis in freshly isolated tissue, whereas all the other agonists that were studied were by biochemical and cell biologic methods in cultured cells in health and disease.
Regulation of goblet cell secretion in disease
In health, goblet cell secretion is regulated by nerves and SPMs responding to the environment to secrete enough mucus to protect the ocular surface. Both a decrease and an increase in goblet cell secretion leads to disease. Diseases such as allergic conjunctivitis and dry eye increase and decrease secretion, respectively, with destabilization of the tear film causing symptoms in both. In allergy, autacoids such as histamine and proinflammatory mediators such as leukotrienes and prostaglandins, through their receptors present on goblet cells, causing an overproduction of mucus. SPMs can block this overproduction by phosphorylating the proinflammatory receptors and counter-regulating their actions. This returns the tear film back to homeostasis.
The P2X 7 receptor plays a role in dry eye disease. Patch-clamp analysis indicated that hyperosmolarity, as occurs in dry eye, activates the ATP-sensitive potassium channels (K ATP ), nonspecific cation channels (NSCs), voltage-gated calcium channels, and P2X 7 channels all found on goblet cells, and in that order. These channels, either by themselves or by interacting with efferent nerve neurotransmitters, cause mucous secretion through exocytosis and resolve dry eye by replenishing the mucous layer. Activation of the P2X 7 receptors by ATP released from dying cells can also cause goblet cell death that contributes to chronic dry eye. Thus, changing the balance of action of P2X 7 receptors can either resolve or exacerbate dry eye disease. The finding of voltage-sensitive channels in the goblet cells suggests that their role should be re-evaluated in the action of neural agonists and SPMs.
Regulation of goblet cell proliferation
The amount of goblet cell mucin on the ocular surface can also be modulated by controlling goblet cell proliferation. Serum, which contains a variety of growth factors, stimulates proliferation of cultured conjunctival goblet cells, both human and rat. EGF, transforming growth factor (TGFα), and heparin binding–EGF (HB-EGF), but not heregulin, stimulate proliferation. EGF, TGFα, and HB-EGF bind to the EGF receptor (EGFR or erb-B1), EGF binds to erb-B4, and heregulin binds to erb-B3 and erb-B4. That EGF, TGFα, and HB-EGF are equipotent in stimulating conjunctival goblet cell proliferation suggests that EGFR and erb-B4 are the primary receptors used.
EGF was used as the prototype of the EGF family. EGF increases the activation of EGFR in rat conjunctival goblet cells and activates ERK1/2 (also known as p44/p42 mitogen-activated protein kinase [MAPK]) ( Fig. 15.8 ) causing its biphasic translocation to the nucleus. The slower sustained second peak response is responsible for cell proliferation, but the role of the rapid transient first peak is not known. Activation of PKC isoforms also mediates EGF-stimulated goblet cell proliferation ( Fig. 15.8 ). In rat and human goblet cells, EGF uses PKCα and PKCε to stimulate conjunctival goblet cell proliferation.
The experiments on proliferation were performed in cell culture. These findings may not apply to goblet cells in vivo, because the conjunctival epithelium architecture, including polarized goblet cells linked with each other and stratified squamous cells by tight junctions, is lacking in culture. Experiments on cell proliferation in tissues are challenging but critical to perform.
Regulation of conjunctival electrolyte and water secretion
The electrolyte and water composition of the external environment has an important effect on release of mucins from secretory granules. Both the stratified squamous and goblet cells of the conjunctiva express ion and water transport proteins and appear to secrete electrolytes and water. Mucin, electrolyte, and water secretion may not be coordinated as the stimuli of mucin secretion appear to differ from those of electrolyte and water secretion.
The conjunctival epithelium secretes Cl − and absorbs Na + with the ratio of 1.5 to 1 causing a net secretion of fluid into the tear film. Interestingly, both absorption and secretion occur within the same cell in the conjunctiva. The ion transport proteins are distributed evenly in all areas of the conjunctiva, suggesting that the entire conjunctiva participates in fluid secretion.
The driving force for conjunctival fluid secretion is the basolaterally located Na + K + -ATPase (NKA) that extrudes three Na + ions for the influx of two K + ions and generates a negative intracellular voltage ( Fig. 15.9 ). Also on the basolateral side is the Na + -K + -2Cl − cotransporter (NKCC1). The Cl − /HCO 3 − exchangers take up Cl − into the cell. Cl − is secreted from the apical side of the cell through a variety of Cl − channels. K + is also secreted from the apical side with Na + moving through the paracellular pathway to the apical side. The net effect is isotonic secretion of Na + , K + , and Cl − into the tears.
