Ординатура / Офтальмология / Английские материалы / Ocular Periphery and Disorders_Dartt, Bex, Amore_2011

Figure 4 Cartoon of the established receptors that stimulate electrolyte and fluid secretion in the stromal-to-tear direction under open-circuit conditions, which are analogous to the in vivo situation. Some transporters present in the epithelium have been omitted for clarity. The specific channel subtypes activated by calcium and cAMP remain unverified. Abbreviations: UTP (uridine 50-triphosphate); INS365 (a synthetic activator of the P2Y2 receptor); P2Y2 (a receptor subtype for the pyrimidine, UTP, that triggers calcium signaling via the phosphoinositide pathway); CLCA2 (a calcium-activated chloride channel subtype thought to be present in the conjunctiva); CFTR (cystic fibrosis transmembrane conductance regulator, which mediates a cAMP-gated chloride conductance via PKA); SK4 (a potassium channel subtype thought to be present in the conjunctiva that is activated by calcium, possibly via calmodulin, a calciumregulatory protein); KvLQT (a potassium channel protein subunit linked to cAMP-activated potassium conductances). The presence of the above channel subtypes has been suggested from the expression levels of message in gene microarray assays of human conjunctiva, but the operational existence of the putative channel proteins has not yet been confirmed in functional experiments.

sides to determine the diffusion of 3H2O, which is proportional to Jdw.

With method 2, the osmotic permeability coefficient, Pf , is expressed in cm s 1 and can be calculated from the following expression:

Pf ¼ Jv=A Vw DCs

where Jv (the measured net H2O flux) is expressed in cm3 s 1 and DCs is the difference in solute concentration (mol cm 3). For this approach, a two-compartment arrangement could also be used, along with a graduated capillary tube, or appropriate detection system, in order to directly measure Jv as a function of time.

Unidirectional fluxes of water determined with 3H2O (method 1) are usually large and similar in both directions. Thus, a small difference (the net volumetric flow, or Jv, which is detected directly with method 2) is difficult to detect by method 1 and is usually not calculated as a difference between two unidirectional fluxes. For example, in the case of the conjunctival epithelium, unidirectional water fluxes (Jdw) across the tissue are statistically identical in either direction, and have a magnitude60-fold larger than the reported values for the net flux

(Jv) of fluid secreted to the tear side by the isolated conjunctiva ( 4–6 ml h 1 cm 2, using method 2 (a volumetric approach). Because of this discrepancy in magnitude, it is unfeasible (if not impossible) to calculate Jv as the difference between the two, relatively large, unidirectional fluxes in the opposite directions. However, method 1 (a diffusional approach) is useful for determining the effects of agents or various experimental conditions on water permeability (Pdw); because although labeled water will cross cell membranes via all available pathways – lipid bilayer, aquaporins, and other channels, the measurements of Jdw, which reflect Pdw, change equally in both directions when an experimental maneuver changes the water permeability of the epithelium.

From diffusional water fluxes (Jdw) and mannitol fluxes it was determined that the conjunctival apical surface is highly permeable to water, and that the transepithelial water permeability (10 4 cm s 1) exceeded the paracellular permeability (10 6 cm s 1). A recently described element contributing to the water permeability of the apical surface is the water channel homolog known as aquaporin type 5 (AQP5). Generally, epithelia exhibit distinct AQPs in the apical and basolateral domains, and in the case of the conjunctiva, AQP3 is expressed in the lateral membranes. Together, AQP5 and AQP3 may be necessary in the conjunctiva for transepithelial fluid transport. AQP5 could serve as a potential target for pharmacological upregulation to enhance fluid secretion given that cAMP via PKA activity has been reported to increase the expression levels of this water channel at both transcriptional and posttranscriptional levels in other cell systems.

From measurements of Jv, a spontaneous fluid transport across the isolated conjunctival epithelium in the basolateral-to-apical direction has been described, a property consistent with the more dominant Cl secretory activity of the tissue. As noted, the reported fluid secretion rates were 4–6 ml h 1 cm 2. This flow was dependent upon transepithelial electrolyte transport given its abolition by ouabain, sensitivity to K+ channel blockade, and Cl dependency. In addition, in experiments that increased the Na+ absorptive activity by raising the glucose concentration (to 25 mM) of the apical bath, fluid transport was inhibited by 77%; an inhibition that did not occur with a similar concentration of mannitol. Studies found that the fluid transport rate was increased ( 50–100 %) by Cl secretogogues that included purinergic agonists acting via P2Y2 receptors. As purinergic agonists stimulate mucin secretion by conjunctival goblet cells, it seems plausible that the roles of epithelial Cl transport include the hydration of mucins upon release.

Overall, the conjunctival epithelium has sufficient water permeability and the transporters necessary to contribute significant fluid to the tear film ( 50 ml h 1 based upon its total surface area). This level of fluid flow is sufficiently large that it may represent a baseline tear

secretion beyond that contributed by the lacrimal gland, which mediates reflex tearing under neuronal control. It is not yet clear if the innervation of the conjunctiva directly regulates the rate of fluid transported across the conjunctiva in vivo. However, the transport systems of the conjunctiva can potentially be manipulated pharmacologically.

See also: Antigen-Presenting Cells in the Eye and Ocular Surface; Cornea Overview; Corneal Angiogenesis; Imaging of the Cornea; Stem Cells of the Ocular Surface.

Further Reading

Anderson, J. M. (2001). Molecular structure of tight junctions and their role in epithelial transport. News in Physiological Sciences 16: 126–130.

Bron, A., Tripathi, R., and Tripathi, B. (eds.) (1997). Wolff’s Anatomy of the Eye and Orbit, 8th edn. London: Chapman and Hall.

Candia, O. A. (2004). Electrolyte and fluid transport across corneal, conjunctival and lens epithelia. Experimental Eye Research 78: 527–535.

Dartt, D. A. (2004). Control of mucin production by ocular surface epithelial cells. Experimental Eye Research 78: 173–185.

Gipson, I. K. and Argu¨eso, P. (2003). Role of mucins in the function of the corneal and conjunctival epithelia. International Review of Cytology 231: 1–49.

Hosoya, K., Lee, V. H., and Kim, K. J. (2005). Roles of the conjunctiva in ocular drug delivery: A review of conjunctival transport mechanisms and their regulation. European Journal of Pharmaceutics and Biopharmaceutics 60: 227–240.

Kaufman, P. L. and Alm, A. (eds.) (2003). Adler’s Physiology of the Eye, Clinical Application, 10th edn. St. Louis, MS: Mosby.

Li, H., Sheppard, D. N., and Hug, M. J. (2004). Transepithelial electrical measurements with the Ussing chamber. Journal of Cystic Fibrosis 3(supplement 2): 123–126.

Nichols, K. K., Yerxa, B., and Kellerman, D. J. (2004). Diquafosol tetrasodium: A novel dry eye therapy. Expert Opinion on Investigational Drugs 13: 47–54.

Oen, H., Cheng, P., Turner, H. C., Alvarez, L. J., and Candia, O. A. (2006). Identification and localization of aquaporin 5 in the mammalian conjunctival epithelium. Experimental Eye Research 83: 995–998.

Tiffany, J. M. (2008). The normal tear film. Developments in Ophthalmology 41: 1–20.

Ussing, H. H. (1949). Transport of ions across cellular membranes.

Physiological Reviews 29: 127–155.

Wei, Z. G., Sun, T. T., and Lavker, R. M. (1996). Rabbit conjunctival and corneal epithelial cells belong to two separate lineages. Investigative Ophthalmology and Visual Science 37: 523–533.

Glossary

Dry eye – A multifactorial disorder of the tear film characterized by either decreased tear production or increased tear evaporation.

Glycosylation – The process by which a carbohydrate is added to protein.

Goblet cells – Specialized epithelial cells that secrete mucins.

Mucins – Large, highly glycosylated proteins. Signal transduction – The process by which a cell converts a signal or stimulus from outside the cell into a functional change.

Goblet cells are columnar epithelial cells that synthesize and secrete mucins, for example, the gel-forming mucin MUC5AC. These cells were originally termed goblet cells because of their distinctive goblet-like shape (Figure 1). The basal portion is narrow and shaped like the stem of a goblet containing the nucleus and organelles, while the apical portion of the cell is shaped like a cup due to the presence of numerous secretory granules. Goblet cells are found in all wet-surfaced epithelia such as the respiratory, gastrointestinal, and reproductive tracts, and the conjunctiva and are surrounded by stratified squamous epithelial cells. Goblet cells of the respiratory, gastrointestinal, and reproductive tracts have been extensively studied with regard to secretion and proliferation in healthy and diseased states. Conjunctival goblet cells have not been studied as extensively, but much information is available. The purpose of this article is to examine the current knowledge of conjunctival goblet cells.

Goblet Cell Development

In humans, conjunctival goblet cells begin to appear in the eighth to ninth week of gestation in the region of the lid margin. By the 11th to 12th week, mature goblet cells can be seen containing secretory granules in the palpebral conjunctiva. Goblet cells appear in the bulbar conjunctiva around the 20th week. In the developing chick, goblet cells appear 2 days after hatching in the fornix and 3 days after hatching in the palpebral and bulbar conjunctivae. In rats, messenger ribonucleic acid (mRNA) expression for the conjunctival goblet cell-specific mucin, MUC5AC, first appears 1 day after birth, before eyelid

opening. This expression was detected in cells in the fornix region. By day 13 (before eyelid opening), a few round-shaped cells in the fornix expressed cytokeratin 7 (a cytokeratin expressed in goblet cells of adult rats) and were positive for alcian blue/periodic acid Schiff ’s (AB/PAS) reagent, a stain that that binds to sialomucins synthesized and secreted by the goblet cells. These cells also bind Ulex europaeus agglutinin I (UEA-I), which is a lectin that binds specific glycoprotein moieties present in adult goblet cells of the rat. In humans, the lectin Helix pomatia agglutinin (HPA) specifically binds to the goblet cell secretory products. On day 17, single and small clusters of these cells were seen distributed in the fornix and palpebral regions. Goblet cell clusters were seen by day 60. In addition to the appearance of the secretory product, by day 17, immunofluorescence microscopy showed nerves surrounding the basolateral portion of goblet cell clusters and the muscarinic receptor subtypes M2 and M3 and b1- and b2-adrenergic receptors were located on the goblet cells. The presence of nerves and neurotransmitter receptors on goblet cells around the time of eyelid opening (12–15 days after birth) implies that mucin secretion is regulated as the eyes open (Figure 2).

There is considerable evidence that stem cells of the conjunctival epithelium, including goblet cells, are distinct from the stem cells of the cornea. This is despite the fact that conjunctival cells can, in the case of a corneal wound involving the limbus, rapidly migrate over the wound and eventually form an epithelium similar to the normal cornea that does not contain goblet cells. However, when bulbar, fornical, and palphebral conjunctivae were grown separately, each with 3T3 feeder cells, they did not express the cornea-specific cytokeratin pair of K3/K12. In addition, when cultured conjunctival cells were injected into athymic mice, the epithelial cyst formed contained epithelial and goblet cells. Given these data, it is thought that conjunctival stem cells are different from stem cells of the cornea.

It is well established that corneal stem cells reside in the limbus, the area between the cornea and bulbar conjunctiva. The location of conjunctival stem cells is not as clear. In the mouse and rabbit, it is believed that the stem cells reside in the fornix based on the fact that slowcycling cells are clustered in the fornix, and these cells do not incorporate tritiated thymidine or bromodeoxyuradine that label dividing cells. In addition, cells in the fornix have the highest rate of proliferation in vitro.

Cells grown from the bulbar conjunctiva and fornix had the same proliferative capacity as cells grown from

Figure 1 An electron micrograph of rat conjunctival goblet cells. Numerous secretory vesicles can be seen in the apical portion of the cells, while nuclei can be seen in the apical portion. Magnification 6000. Reprinted from Dartt, D. A. Regulation of mucin and fluid secretion by conjunctival epithelial cells.

Progress in Retinal and Eye Research 21: 555–576. With kind permission of Elsevier.

Figure 2 Co-localization of the M3 muscarinic receptor with goblet cells from the developing rat conjunctiva. Sections from a 17-day-old rat conjunctiva were incubated with an antibody against M3 muscarinic receptor (shown in green) and the lectin UEA-I conjugated to rhodamine (shown in red) which is specific for rat goblet cells. Goblet cells were visualized with differential interference contrast microscopy. Arrows indicate the presence of M3 muscarinic receptor subjacent to the secretory vesicles (shown in red). Magnification 200.

the corneal limbus. Cells from all areas of the fornix and bulbar conjunctiva were capable of undergoing 80–100 cell divisions before reaching senescence, similar to limbal cells. Interestingly, goblet cells were present during the entire life span of the cultures of fornix and bulbar conjunctivae and the number of goblet cells increased during cultivation of the cell cultures. As goblet cells were present during the entire culture time, it seems likely that conjunctival stem cells are bipotent, that is, capable of differentiating into either goblet or stratified squamous cells. It is not known what causes the stem cell to differentiate

into either cell type. Possible explanations include genetic programming as it was demonstrated that conjunctival cells with high proliferative capacity differentiate into goblet cells at specific times in their cell cycle of duplication. The first goblet cells appear after a cell has achieved 45–50 doublings and, subsequently, another 10–20 doublings before senescence occurs. It is also possible that environmental effectors such as cytokines or growth factors play a role in the differentiation of a goblet cell.

The number of goblet cells in the adult conjunctiva varies depending upon the location in the conjunctiva and species. For example, in the rabbit: (1) the tarsal conjunctiva contains the highest number of goblet cells while the bulbar conjunctiva contains the least, (2) the goblet cells of the tarsal conjunctiva were larger than those present in the bulbar conjunctiva, and (3) conjunctival goblet cells appeared as single cells interspersed throughout the stratified squamous epithelial cells. In contrast, in the rat: (1) the fornix contained the most goblet cells while very few were seen in the bulbar and limbal conjunctivae and (2) goblet cells appear as clusters with as many as 10 goblet cells present in some clusters in the fornix (Figure 3). Goblet cells in the human conjunctiva tend to appear as single cells (Figure 4), similar to the rabbit, though in certain areas, the density of goblet cells is high enough such that they appear to be clustered. The highest goblet cell density in human conjunctiva is in the inferior palpebral conjunctiva. It has been proposed that clusters of goblet cells, such as those that occur in rats, arise from the fact that goblet cells divide several times before senescence giving rise to the clusters. In contrast, in species such as rabbit and human, where goblet cells occur as single cells in the conjunctiva, differentiation into goblet cells occurs after the cell has undergone terminal differentiation.

Function of Conjunctival Goblet Cells

The tear film is a thin layer of fluid that covers the ocular surface. Tears are secreted in response to decreased humidity, bright light, mechanical stimulation, bacterial and viral pathogens, and other environmental factors. The tear film is a stratified fluid layer consisting of three layers:

(1) the outermost, which is a lipid layer secreted by meibomian glands of the upper and lower eyelids and is thought to be a barrier to evaporation; (2) the middle, which is an aqueous layer secreted essentially by the main and accessory lacrimal glands and contains water, electrolytes, and proteins such as growth factors and antibacterial proteins necessary for the health of the ocular surface; and (3) the innermost, which is a mucin layer, containing mucins that are not only secreted mainly by the conjunctival goblet cells, but also the stratified squamous cells of the cornea and conjunctiva. The mucin layer moves freely over the ocular surface toward the nasolacrimal

duct with the blink providing the mechanism of movement. As it moves, it traps debris and pathogens.

The function of conjunctival goblet cells is to synthesize and secrete mucins onto the ocular surface. Mucins are large, highly glycosylated proteins containing tandem repeats of amino acids that are rich in serine and threonine. Owing to the large amount of glycosylation, mucins are highly negatively charged molecules that are believed to be a barrier to pathogens. The glycosylation moieties are quickly hydrated upon exiting the cell, causing them to swell. Thus, mucins provide lubrication, water retention, and a barrier to infectious agents. Members of the mucin family can be subdivided based on whether they are secreted from the cell (secretory) or remain associated with the plasma membrane (membrane associated). While other members of the mucin family, namely MUC1, MUC2, MUC4, and MUC16, are present in the conjunctiva, only the gel-forming, secretory mucin MUC5AC has been identified in conjunctival goblet cells.

MUC5AC is a large gel-forming mucin that is closely related to the other gel-forming mucins MUC2, MUC5B, and MUC6. It contains four cysteine-rich domains (D domains), a tandom repeat region that can be duplicated 17–124 times, as well as a cysteine knot along with an additional cysteine region. The D domains, based on their large number of cysteines, form disulfide bridges with other MUC5AC molecules to form a large gel-like association of mucin molecules (Figure 5).

Control of Goblet Cell Proliferation and

Mucin Secretion

It is vital for the mucin in the tear film be of sufficient quantity and quality. The quantity of mucin depends on the: (1) number of goblet cells present (proliferation or differentation), (2) amount of mucin synthesized and

stored in secretory granules, (3) rate of mucin secretion, and (4) rate of mucin degradation. There are no studies on the rate of mucin synthesis or mucin degradation.

Goblet Cell Proliferation

With the development of a method to culture conjunctival goblet cells, studies of goblet cell proliferation are now underway. One important stimulus of goblet cell proliferation is the epidermal growth factor (EGF) family as measured by both a cell proliferation assay kit and by immunoflouresence microscopy using the an antibody against Ki-67, a protein known to be present in cells that have entered the cell cycle. EGF, transforming growth factor a (TGFa), heparin-binding EGF (HB-EGF), and heregulin are present in rat conjunctiva as well as cultured rat goblet cells as determined by reverse transcriptase polymerase chain reaction (RT-PCR), Western blot analysis, and immunofluorescence microscopy. EGF, TGFa,

Muc5AC

D domain

Cysteine domain

Repetitive domain

Figure 5 A schematic of MUC5AC.

and HB-EGF increased goblet cell proliferation through activation of the EGF receptor. There are four types of EGF receptors, namely ErbB1 (the EGF receptor), ErbB2 (HER), ErbB3, and ErbB4. Each receptor binds to specific members of the EGF family. Activated EGF receptors form homoor heterodimers and then recruit adaptor molecules including Shc/Grb2, phosphoinositide-3 kinase (PI-3K), phospholipase C (PLC)g, p38 mitogen-activated protein kinase (MAPK), and c-jun NH (2)-terminal kinase ( JNK). Each of these kinases initiates a cascade of kinases leading to a cellular response. The downstream kinase activated by Shc/Grb2 is the extracellular-related kinase 1/2 (ERK1/2); the downstream kinase activated by PI-3K is AKT; the downstream kinase activated by PLCg is protein kinase C (PKC); and the downstream kinase activated by JNK is c-jun (Figure 6).

Further investigation of the role of ERK in EGFstimulated conjunctival goblet cell proliferation demonstrated that EGF increased the number of cells expressing ERK in their nucleus in a biphasic manner. Under basal conditions, ERK is present in the cytosol. Upon stimulation, ERK translocates to the nucleus where it phosphorylates proteins necessary for proliferation. The first, major peak occurs 1 min after the addition of EGF to cultured rat goblet cells. The number of goblet cells with ERK present in the nucleus returned to basal levels before increasing again approximately 18 h after the addition of EGF. The second peak corresponded with the appearance

of Ki-67, indicating that goblet cells were starting to proliferate. The ERK inhibitor U0126, added 20 min prior to EGF, inhibited both peaks of the translocation of ERK as well as goblet cell proliferation. Interestingly, inhibition of the second peak of ERK translocation with U0126 also prevented EGF-stimulated proliferation. In addition to the activation of ERK, EGF also activates PKC isoforms -a and -e to, in turn, stimulate proliferation.

Most proliferation studies to date have been performed on cultured goblet cells from rat. Several studies have examined the proliferation of human goblet cells in comparison to rat cells, and demonstrated that human goblet cells respond similarly to EGF. For example, EGF stimulates human goblet cell proliferation in a similar timeand concentration dependency. In both cell types, EGF activates ERK and PKC to stimulate proliferation. Due to the difficulty of obtaining a human conjunctiva and culturing human goblet cells, rat goblet cells are an excellent model for studying human goblet cell proliferation.

Goblet Cell Mucin Secretion

Goblet cell secretion occurs through an apocrine mechanism. In this mechanism, most or all the secretory vesicles fuse with one another upon stimulation and subsequently with the apical membrane releasing the mucin into the extracellular space. Therefore, the amount of mucin released is dependent upon the number of cells responding to a given stimulus. A strong stimulus would involve responses from more goblet cells than a weak stimulus. Taking advantage of this property, conjunctival samples can be treated with histochemical stains that recognize mucins in goblet cells, and the number of goblet cells can be counted. A decrease in the number of goblet cells would indicate an increase in secretion.

Another method to measure conjunctival goblet cell secretion is based on the fact that the lectins UEA-I and HPA bind to specific carbohydrate residues found in MUC5AC, depending on the species. The amount of secreted mucin can be determined by an enzyme-linked lectin assay (ELLA) and Western blot or dot-blot analyses.

Using the histochemical method of staining for mucins in a conjunctival button, it was demonstrated, for the first time, that goblet cell mucin secretion is neurally mediated, as a wound to the central cornea induced goblet cell mucin secretion. Sensory nerves in the cornea were activated by the wound causing a neural reflex arc in which the parasympathetic nerves that surround the goblet cells released their neurotransmitters to stimulate mucin secretion.

These results were confirmed when parasympathetic nerves containing the neuropeptide vasoactive intestinal peptide (VIP) were demonstrated to be present in the conjunctiva surrounding the goblet cells subjacent to the secretory granules. Addition of exogenous VIP or

the cholinergic agonist carbachol (an analog of the parasympathetic neurotransmitter acetylcholine) stimulated goblet cell secretion. Other compounds which have been shown to stimulate conjunctival mucin secretion include activators of the purninergic receptor subtype P2Y2, such as ATP, UTP, and INS365, the neurotrophins nerve growth factor, and bone-derived neurotrophic factor, and the drug OPC-12759, an antigastric ulcer drug. Interestingly, no sympathetic neurotransmitter has been shown to stimulate goblet cell secretion despite the presence of the receptors for these neurotransmitters on goblet cells.

The signal transduction pathways utilized by cholinergic agonists have been well studied. Cholinergic agonists bind to M2 and M3 muscarinic receptors which are present on conjunctival goblet cells. Classically, these receptors activate PLC, which hydrolyzes phosphatidylinositolbisphosphate into 1,4,5-inositol trisphosphate (IP3) and diacylglycerol (DAG) (Figure 7). IP3 induces the release of Ca2þ from the endoplasmic reticulum into the cytosol. It is not known if this occurs in goblet cells; however, what is known is that an increase in intracellular Ca2þ alone is sufficient to cause mucin secretion from conjunctival goblet cells. Calcium can also activate Ca2þ-/calmodulin- dependent protein kinase. However, inhibitors of Ca2þ/ calmodulin protein kinase do not have any effect on cholinergic agonist-stimulated mucin secretion.

DAG is a phospholipid activator that along with Ca2þ activates PKC. Though a direct role for PKC involvement in conjunctival goblet cell mucin secretion has not been demonstrated, activators of PKC (phorbol esters) do stimulate secretion.

In addition to the Ca2þ pathway, cholinergic agonists in goblet cells activate a second pathway leading to protein secretion. This pathway involves the transactivation of the EGF receptor through the stimulation of the focal adhesion kinase Pyk2. Pyk2, in turn, activates the nonreceptor tyrosine kinase p60Src. Pyk2 and p60Src are activated in goblet cells by Ca2þ and PKC as the addition of either a calcium ionophore or a phorbol ester increases phosphorylation (activation) of Pyk2 and p60Src and the use of PKC inhibitors inhibits cholinergic agonist-stimulated Pyk2 and p60Src phosphorylation (Figure 7). This again implies that PKC plays a vital role in conjunctival goblet cell secretion. The Pyk2/p60Src complex then transactivates the EGF receptor, recruiting the adaptor molecules Shc, Grb2, and the Ras guanine nucleotide exchange factor Sos. Sos binds to and activates the low molecular weight guanosine triphosphate (GTP)ase, Ras, causing the exchange of guanosine diphosphate (GDP) for GTP to activate Ras. Ras initiates another kinase cascade of Raf (also known as MAPK kinase kinase), mitogen-activated kinase kinase (MEK) (MAPK kinase), and ERK 1/2 (p44/p42 MAPK). Upon stimulation by cholinergic agonists, ERK activates proteins in the cytosol, leading to secretion. Inhibition of ERK inhibits cholinergic agonist-stimulated mucin secretion (Figure 7).

In contrast, when ERK is activated by exogenous EGF, ERK 1/2 translocates to the nucleus where it stimulates proliferation.

The signaling pathways of the other compounds that stimulate mucin secretion are not well studied. Receptors for VIP (VPAC1 and VPAC2) are present on goblet cells both in vivo and in vitro. It is not known if exogenous VIP (which causes mucin secretion) acts through these receptors. As it is well established in other tissues that VIP increases the intracellular cyclic adenosine monophosphate (cAMP) concentration as well as the intracellular [Ca2þ], it is likely that a similar mechanism occurs in goblet cells.

It is not know if the EGF family stimulates mucin secretion. As these growth factors activate ERK, which plays an important role in both secretion and proliferation, it is likely that they do. It is interesting that many of the same proteins and kinases are used by EGF to stimulate proliferation and by cholinergic agonists to stimulate mucin secretion. ERK1/2 is known to phosphorylate over 100 proteins. In order to confer specificity, ERK1/2, as is the case for many signaling molecules, is organized through the use of scaffold proteins. These proteins bind to two or more components of a signaling pathway in close proximity to assist with their interactions. Scaffolding proteins also target signaling molecules to particular areas of the cell in order to phosphorylate specific substrates. They can prevent cross talk between pathways. As the scaffolding proteins are also regulated, their stability can affect the duration of the signal. Several different scaffolding proteins have been identified in mammalian cells including kinase suppressor of Ras, MEK partner-1,

Morg1, IQGAP1, and b-arrestins 1 and 2. Not surprisingly, scaffolding proteins are required for the translocation of ERK1/2 to the nucleus and other proteins are responsible for maintaining ERK1/2 in the cytosol. In fact, ERK1/2 translocation to the nucleus requires phosphorylation by MEK, which causes conformational changes in ERK1/2, allowing for the formation of ERK dimers. This dimerization facilitates the translocation of phosphorylated ERK1/2 into the nucleus. In contrast, PEA-15, a small phosphoprotein, has been shown to anchor ERK1/2 in the cytosol.

Clinical Implications of Mucin Deficiency on the Ocular Surface

The presence of goblet cells in the conjunctiva is of great importance to the health of the ocular surface. Dry eye, which is characterized by a deficient tear film or excessive evaporation, is a multifactorial disorder that causes damage to the ocular surface. One characteristic of dry eye is a decrease in the number of mucin-containing goblet cells. All in vivo studies to date identify goblet cells by their secretory product, either by staining with AB/PAS or by the presence of MUC5AC within the cell, and should be termed filled goblet cells. Thus, a decrease in the number of filled goblet cells in the conjunctival tissue indicates that goblet cells have secreted. Under chronic conditions, the decrease in filled goblet cells could indicate repeated stimulation such that mucin synthesis is unable to keep pace with secretion or indicate a loss of goblet cells through either a decrease in goblet cell proliferation,

dedifferentiation, or an increase in goblet cell death. It is difficult to know whether the loss of goblet cells is a cause of the disease or a result of it. Indeed, the mechanism by which goblet cells are lost from the conjunctiva has not been studied. In animal models of dry eye, proteins involved in apoptosis have been shown to be upregulated in conjunctival epithelial cells, though the effects on the goblet cells themselves were not clear. The loss of goblet cells in dry eye patients warrants further examination.

The presence or absence of conjunctival goblet cells has been examined in different types of dry eye. These have been described below.

Sjo¨gren Syndrome

Sjo¨gren syndrome is an autoimmune disease characterized by profound lymphocytic infiltration of the lacrimal and salivary glands, resulting in dry eye and dry mouth. It is not known whether the changes seen in the conjunctiva are a result of the autoimmune disease itself or as a result of the change in tears due to lacrimal gland destruction. Though the changes in the conjunctiva are relatively mild compared to the destruction of the lacrimal glands, there is a decrease in the number of mucin-containing goblet cells in patients with Sjo¨gren syndrome, as determined by histochemical staining methods. In addition, the amount of MUC5AC mRNA is decreased in patients with Sjo¨gren syndrome and the amount of MUC5AC detected in tears of these patients is also decreased.

Vitamin A Deficiency

Vitamin A is vital for the health of the ocular surface as it is essential for the development of goblet cells in the conjunctiva. Vitamin A deficiency has been shown to cause keratinization and squamous metaplasia of the conjunctiva. The lack of goblet cells in the conjunctiva in vitamin-A-deficient patients has been documented through the use of impression cytology and histochemical staining methods. It has also been demonstrated that in vitamin-A-deficient rats, not only were there no mucincontaining goblet cells, but the mRNA for MUC5AC and MUC5AC protein also disappeared 20 weeks after the initiation of a vitamin-A-deficient diet. Reintroduction of vitamin A into the diet has been shown to increase the number of conjunctival mucin-containing goblet cells.

Topical Preservatives

Preservatives, such as benzalkonium chloride, are often found in artificial tears and medications such as those used to treat glaucoma and anti-inflammatory medications. These patients often report the signs and symptoms of dry

eye. Benzalkonium chloride has been shown to decrease the number of mucin-containing goblet cells present in the conjunctiva.

Ocular Cicatrical Pemphigoid

Mucous membrane pemphigoid is an autoimmune disease that is characterized by blisters in the mucous membranes of the body including the mouth, nose, trachea, and conjunctiva. When the conjunctiva is involved, it is known as ocular cicatrical pemphigoid (OCP). This condition causes chronic conjunctivitis and, eventually, complete keratinization of the conjunctiva, resulting in severe dry eye. It has been observed that the mucin-containing goblet cell number is reduced in the later stages of the disease, likely as a result of the keratinization. Interestingly, it has been shown that the expression of a specific glycosyltransferase present exclusively in goblet cells in normal human conjunctiva is altered in late stages of OCP. This enzyme, which is responsible for the glycosylation of mucins, is expressed in stratified squamous cells in areas of the conjunctiva that were nonkeritinized in OCP patients, and, as the disease progressed, the expression disappeared.

Laser-assisted In Situ Keratomileusis

Patients undergoing laser-assisted in situ keratomileusis (LASIK) often experience dry eye symptoms. These symptoms are usually temporary, but can develop into chronic dry eye. It is known that the number of mucincontaining goblet cells decreases significantly within 1 week postsurgery, but returns to preoperative levels by 3 months after surgery.

Diseases and disorders of goblet cells are a result not only of mucous underproduction, but also of mucous overproduction.

Ocular Allergies

The symptoms of allergic conjunctivitis include mucus production, ocular itching, foreign-body sensation, tearing, hyperemia, chemosis, and lid edema. Traditionally, allergic eye disease has been classified into: seasonal allergic conjunctivitis (SAC), perennial allergic conjunctivitis (PAC), vernal keratoconjunctivitis (VKC), atopic keratoconjunctivitis (AKC), and contact-lens-induced papillary conjunctivitis (CLPC). Each of the five categories of allergic conjunctivitis has a distinct pathology. In SAC and PAC, conjunctival inflammation is mild and of short duration. In VKC and AKC, conjunctival inflammation has an unclear history of exposure to allergen, is more severe, and lasts longer than SAC and PAC.

A common symptom of allergic conjunctivitis in the human is alteration in mucus production. One hypothesis as to how mucous production is altered in allergic conjunctivitis is that activation of sensory nerves in the cornea

and conjunctiva, manifested by itchiness, foreign-body sensation, and increased tearing, could occur. This, in turn, could activate the efferent parasympathetic and sympathetic nerves that surround conjunctival goblet cells to release the neurotransmitters acetylcholine and VIP, which are known to stimulate conjunctival goblet cell secretion. When goblet cells secrete, all secretory granules are released at once. Under these chronic conditions, the decrease in filled goblet cells could indicate repeated stimulation with mucin synthesis unable to keep pace with secretion. In support of this, a decrease in filled conjunctival goblet cells in VKC and AKC patients, respectively, compared to control subjects, has been noted. In addition, the amount of MUC5AC RNA in the conjunctiva was decreased in AKC and VKC patients versus control. The excess mucus seen on the conjunctiva in individuals with VKC is a stringy mucus and does not necessarily represent goblet cell MUC5AC, but could be stratified squamous cell membrane-spanning mucins, poorly hydrated mucins, or mutated mucin variations. Additional study of the role of goblet cell mucin secretion in different types of ocular allergy is warranted.

Results from animal models of allergic conjunctivitis, which most closely mimics SAC and PAC, found that the number of filled goblet cells decreased for 6 h after the final allergen challenge. Over 48 h, the number of filled goblet cells returned toward control values, indicating that goblet cell mucin secretion increased during delayed hypersensitivity, but that goblet cells refilled after allergen removal. MUC5AC RNA was also depleted by the final challenge and recovered at 6 h, indicating that the delayed hypersensitivity depletes MUC5AC RNA; however, it recovers quickly to begin synthesizing MUC5AC to refill the goblet cells.

Summary

Much information has been gathered regarding the role of conjunctival goblet cells in the health of the ocular surface. Goblet cell proliferation, mucin synthesis, and secretion control the amount of mucin present on the ocular surface. These processes are regulated as growth factors increase proliferation and neural stimulation causes mucin secretion. However, many questions remain. Are the three processes that regulate mucin amount coordinated so that

one stimulus activates all three? While some of the intracellular pathways leading to proliferation or secretion are known, it is not known if these pathways are altered in diseases. The life span of a conjunctival goblet cell as well as the mechanisms by which goblet cells are lost in disorders of the tear film are unknown. It is also not known if goblet cells are able to secrete mucins multiple times, perhaps refilling secretory granules, in response to either a sustained signal or multiple stimuli. Further research into these questions may lead to the development of treatments for many of the ocular surface diseases.

See also: Defense Mechanisms of Tears and Ocular Surface; Ocular Mucins; Overview of Electrolyte and Fluid Transport Across the Conjunctiva.

Further Reading

Dartt, D. A. (2004). Control of mucin production by ocular surface epithelial cells. Experimental Eye Research 78: 173–185.

Gipson, I. K. and Argueso, P. (2003). Role of mucins in the function of the corneal and conjunctival Epithelia. International Review of Cytology 231: 2–49.

Gipson, I. K., Hori, Y., and Argueso, P. (2004). Character of ocular surface mucins and their alteration in dry eye disease. Ocular Surgery 2: 131–148.

International Dry Eye Workshop (2007). The definition and classification of dry eye disease. Report of the definition and classification subcommittee of the International Dry Eye Workshop (2007). Ocular Surgery 5: 75–92.

Lavker, R. and Sun, T-T. (2003). Epithelial stem cells: The eye provides a vision. Eye 17: 937–942.

Pellegrini, G., Golisano, O., Paterna, P., et al. (1999). Location and clonal analysis of stem cells and their differentiated progeny in the human ocular surface. Journal of Cell Biology 145: 769–782.

Ramos, J. W. (2008) The regulation of extracellular signal-regulated kinase (ERK) in mammalian cells. International Journal of Biochemistry and Cell Biology, 40: 2707–2719.

Rios, J. D., Forde, K., Diebold, Y., et al. (2000). Development of conjunctival goblet cells and their neuroreceptor subtype expression. Investigative Ophthalmology and Visual Science

41: 2127–2137.

Sellheyer, K. and Spitznas, M. (1988). Ultrastructural observation on the development of the human conjunctival epithelium.

Graefe’s Archive of Clinical and Experimental Ophthalmology

226: 489–499.

Shapiro, M. S., Friend, J., and Thoft, R. A. (1981). Corneal reepithelialization from the conjunctiva. Investigative Ophthalmology and Visual Science 21: 135–142.

Shatos, M. A., Rios, J. D., Tepavcevic, V., Kanno, H., Hodges, R. R., and Dartt, D. A. (2001). Isolation, characterization, and propagation of rat conjunctival goblet cells in vitro. Investigative Ophthalmology and Visual Science 42: 1455–1464.