In your ongoing fight against dry eye, the mucus layer, suggested by some research groups to be the thinnest layer of the tear film, may eventually loom large. This layer is vital to a stable ocular surface, since it allows the tear film to actually adhere to the eye. Because of this layer's unique role, its primary components, mucins, are attracting increased attention from researchers. In this article, we'll explain the mucins' role in ocular protection, and look at new agents researchers are developing to help clinicians increase the effectiveness of the mucus layer by way of increased mucin production and/or integrity.

What Are Mucins?
Mucins are high-molecular weight glycoproteins with a protein backbone and a high carbohydrate content. In addition to contributing to the mucus layer, the mucins themselves form the glycocalyx, a scaffold-like structure that helps contribute to cell adhesion.¹ They're also a defense against ocular surface damage.

One important role of mucins is in making the tear film hydrophilic. This stabilizes the tear film and decreases its surface tension, allowing the aqueous layer to spread evenly over the surface of the eye. Without this layer, tears wouldn't adhere to the surface, making it susceptible to damage.^2-3

A mucin deficiency can lead to extensive fluorescein staining such as that seen in this dry eye patient.

There are two primary types of mucins that are produced within the body: membrane-spanning and secreted. The membrane-spanning mucins are embedded in the lipid bilayer of the cells, while the secreted mucins are released into the extracellular environment. Some think that the membrane-spanning mucins, expressed by the corneal and conjunctival non-goblet epithelial cells, help to spread the secreted mucins produced by goblet cells across the ocular surface. These two types of mucins work together for the formation of a viable tear film.⁴ Note that mucins in the secreted category are divided into gel-forming and soluble varieties.¹

Where Are Mucins Produced?
Mucins found on the ocular surface are primarily produced by goblet cells, apical cells of the conjunctiva and cornea and the lacrimal gland. The body produces many types of mucins, but not all of them are found on the ocular surface (See Table 1). Goblet cells primarily produce the gel-forming secretory mucin MUC5AC, the only gel-forming mucin of the ocular surface to be identified. Secretion typically occurs in response to a stimulus, such as a foreign body on the ocular surface. Thus, parasympathetic nerves act on these cells to stimulate mucus secretion. To prevent overstimulation and depletion of mucin from these cells, the number of goblet cells that respond to a neural stimulus must be regulated.⁵

Table 1. Mucin Breakdown
Mucins	Where Produced	Function
MUC1	Expressed as transmembrane mucin by cells of the corneal and conjunctival epithelial stratified squamous cells.	Acts as  a disadhesive.  It may prevent cells of the lid epithelium and inflammatory cells, as well as preventing debris and other pathogens from adhering to the corneal surface.  It may also help facilitate the spread of gel-forming mucins from the goblet cells.
MUC2	Expressed in the trachea, bronchi of the lungs, stomach and endocervix.  Not a major mucin on the ocular surface.	Secreted as a gel-forming mucin.
MUC3	Expressed in the colon.  Not yet detected on the ocular surface.
MUC4	Widely expressed in the body.  It is expressed by simple, stratified, squamous and non-keratinized epithelial cells.  Expressed by cells of the conjunctival epithelium.	Little is known about the function of this mucin.  Researchers are attempting to determine if it is secreted, membrane-spanning, gel-forming or soluble.
MUC5AC	Expressed in the trachea, bronchi of the lungs, stomach, endocervix and conjunctival epithelium.  Goblet cells of the conjunctiva secrete this mucin.	This is a secreted gel-forming mucin. It is currently thought that this may be the major structural mucin of the tear film mucus. To date, this is the only gel-forming mucin found on the surface epithelium of the eye.
MUC5B	Expressed in trachea, bronchi of the lungs, colon and endocervix.  Not detected on the eye.	Secreted gel-forming mucin.
MUC6	Expressed in the stomach and endocervix.  Not detected on the eye.	Secreted gel-forming mucin.
MUC7	Expressed in both the conjunctiva and lacrimal glands.	Small, secreted, monomeric, non-gel-forming mucin.  Thought to be bactericidal and a component of the tear film, though this hasn't been verified.
MUC8	Expressed in the reproductive tract and bronchial epithelium.  Not detected on the eye.

Meanwhile, the apical cells of the conjunctiva and cornea produce the membrane-bound mucins MUC1, MUC2 and MUC4. These mucins primarily form the glycocalyx that protects the ocular surface. Finally, the lacrimal gland produces MUC7, a soluble secreted mucin that forms a part of the mucus layer.¹This mucin is also found in the aqueous layer.

Mucin and Dry Eye
Decreased mucin levels have been found to be associated with dry eye. In dry-eye patients, the decreased levels of mucin are correlated with a decreased density of goblet cells. As the dry eye progresses, squamous metaplasia of the conjunctival cells occurs. The epithelial cells flatten and increase in area and the goblet cell density decreases.⁶ It's been found that vitamin A deficiencies, topical medications, excessive dosing with drops containing preservatives and cicatrizing conjunctival disorders can all damage goblet cells and the ocular surface. It's important to note that vitamin A is essential for maintaining goblet cells and mucins on the ocular surface.⁴

In addition, it's been reported that transmembrane mucins found on the ocular surface are also altered. The pattern in which the mucins are present on the surface differs significantly between normal patients and those with dry eye. Further, researchers have proposed that the transmembrane mucins, such as MUC1, produced in dry-eye patients are not glycosylated in the same way that mucins of normal patients are. The altered structure potentially changes their functioning on the ocular surface and can further the development of dry eye.⁴ 
The mucous found in the eye also differs between conditions. There is the stringy thin translucent mucin of dry eye, the globular and crusting mucous of infection, and the thick, tenacious, stretchy, mucous strands of vernal conjunctivitis.

Measuring Mucins
There are several ways to investigate mucin levels on the eye and in dry eye itself. One technique is called impression cytology, a noninvasive, highly repeatable test that can be used to look at cellular characteristics in conjunction with the components of the tear film.

Impression cytology involves placing small cellulose discs on the ocular surface. The filter paper is then carefully removed, taking with it a thin layer of superficial conjunctival cells and goblet cells, as well as the tear film and the conjunctival mucus network that lies next to the goblet cells.⁷ These samples can then be analyzed for various parameters, including goblet cell density, which relates to the amount of mucin secreted onto the ocular surface. In this case, anti-M1 monoclonal antibodies can be used to adhere to the peptidic core of the mucin molecules, allowing the goblet cells of the conjunctival surface to be detected and quantified.⁷

Further, flow cytometry can be performed from the impression cytology samples. This is performed by first extracting the cells from the filter paper through gentle agitation, which is then followed by centrifugation. The cells are then mixed with antibodies specific to the molecule in question, followed by an immunofluorescent antibody specific to the initial antibody. Then the sample is run through the flow cytometry machine. The machine quantifies the number of cells that have bound each immunofluorescent antibody, thus indicating the number of cells displaying the molecules in question.⁸

In the case of mucins themselves, rather than cells, several interesting studies have been conducted. One study investigated the production of mucin on the ocular surface by tagging mucin mRNA molecules obtained through impression cytology. These levels were quantified for normal patients, thus showing that the combination of impression cytology and flow cytometry is a viable means for quantifying the amount of mucins produced on the ocular surface.⁹

A third technique for examining mucin levels that was recently developed involves the use of Schirmer strips to collect tear film samples. The tear samples are then exposed to antibodies specific to the mucin in question. This method was used by Hongcheng Zhao, MD, and his co-workers at the Department of Ophthalmology and Visual Sciences at Kentucky's University Of Louisville, in their investigation of the MUC5AC protein.¹⁰ Through the use of this technique, they were able to determine that dry eye patients showed reduced concentrations of MUC5AC in their tear film when compared to normal patients.
 
Current Therapies
Dry eye, unfortunately, is difficult to treat. The problem lies in the lack of currently approved agents that increase tear secretion from the ocular surface epithelia or the orbital (tear) glands. Clinicians use various ointments, tear substitutes and punctal plugs to help alleviate the symptoms of dry eye, but these don't really address the cause of the problem. To get at the cause, researchers have focused on means to help stimulate tear secretion. Two of these therapies are Inspire's P2Y2 agonist and Alcon's 15-HETE. A third, Milcin, supplements the tear film by mimicking mucins.

 • Inspire's P2Y2 agonist. It's been shown that administering nucleotides that stimulate P2Y2 receptors on the ocular surface in animals causes increased mucin and chloride secretion, in addition to increased fluid transport into tears.¹¹ The consensus is that Inspire's new P2Y2 agonist acts similarly to the nucleotides, increasing mucin secretion. Many hope that similar results will be seen in humans, potentially leading to a very effective treatment for dry eye. (For a more in-depth discussion of P2Y2 and other secretagogues, please refer to the November 2002 installment of Therapeutic Topics.) 

 • Alcon's 15(S)-HETE. Along similar lines to Inspire's agent, Alcon has evaluated the mucin secretagogue 15(S)-HETE (hydroxyeicosatetraenoic acid). 15(S)-HETE is a fatty acid that occurs naturally within the body. The company hopes that administration of this compound will stimulate the production of mucins by cells of the ocular surface. Studies investigating the product on desiccated rabbit eyes significantly decreased ocular injury by increasing the mucin layer on the ocular surface.¹²

 • Vista Scientific's Milcin. This is another medication currently being tested for use in dry-eye patients. Milcin acts as a supplement to the tear film by becoming incorporated into the mucin layer and mimicking the secreted mucins produced within the eye. This agent differs from the others in that it doesn't attempt to actively promote mucin secretion.

Dry eye is a painful and irritating disease, and, since there are currently no approved treatments, present research is strongly focused on it. As researchers categorize the causes of dry eye, the mucin layer may emerge as one of the key players in the quest for a good treatment. 
 
Dr. Abelson, an associate clinical professor of ophthalmology at Harvard Medical School and senior clinical scientist at Schepens Eye Research Institute, consults in ophthalmic pharmaceuticals. Dr. Dartt is an associate professor of ophthalmology at Harvard Medical School, and is also a senior scientist at Schepens. Ms. Humphrey is a research associate at Ophthalmic Research Associates in North Andover.

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2. Gibson IK, Kunert KS, Argueso P. Regulation of mucin gene expression in the ocular surface epithelia. Cornea 2000;19:6:S97.
3. Rolando M, Zierhut M. The ocular surface and tear film and their dysfunction in Dry Eye Disease. Surv of Ophthalmol 2001;45:S203-S210.
4. Danjo Y, Watanabe H, Tisdale A, Georgoe M, Tsumura T, Abelson M, Gipson I. Alteration of mucin in human conjunctival epithelia in dry eye. Invest Ophthalmol &Vis Sci 1998;39:13:2602-2609.
5. Diebold Y, Rios JD, Hodges RR, Rawe I, Dartt DA. Presence of nerves and their receptors in mouse and human conjunctival goblet cells. Invest Ophthalmol &Vis Sci 2001;42:10:2270-2282.
6. Gilbard, JP. Dry-Eye Disorders. In: Albert M, Jakobiec F, Azar D, Gragoudas E, Power S, and Robinson N., eds. Principles and Practice of Ophthalmology: Second Edition, vol. 2. Philadelphia: W.B. Saunders Company, 2000:982-1001.
7.Garcher C, Bron A, Baudouin C, Bildstein L, Bara J. CA 19-9 ELISA test: a new method for studying mucus changes in tears. Br J Ophthalmol 1998;82:88-90.
8. Pisella PJ, Brignole F, Debbasch C, Pharm D, Lozato PA, Creqzot-Garcher C, Bara J, Saiag P, Warnet JM, Baudouin C. Flow cytometric analysis of conjunctival epithelium in ocular rosacea and keratoconjunctivitis sicca. Ophthalmology 2000;107:10:1841-1849.
9. Corrales RM, Calonge M, Chaves FJ, Saez V, Herreras JM, Diebold Y. Ocular mucin gene expression in conjunctival impression cytology samples from healthy donors. 2001 ARVO poster presentation, abstract #2613.
10. Zhao H, Jumblatt JE, Wood TO, Jumblatt MM. Quantification of MUC5AC protein in human tears. Cornea 2001;20:8:873-7.
11. Jumblatt JE, Jumblatt MM. Regulation of ocular mucin secretion by P2Y2 nucleotide receptors in rabbit and human conjunctiva. Exp Eye Res 1998;67:341-346.
12. Gamache D, et al. Preservation of ocorneal integrity by the mucin secretagogue 15(S)-HETE in a rabbit model of desiccation-induced dry eye. Cornea 2000;19:6:S88.
13. Gipson I, Inatomi T. Cellular origin of mucins of the ocular surface tear film. In: Sullivan DA, Dartt DA, Meneray MA, eds. Lacrimal Gland, Tear Film and Dry Eye Syndromes 2. New York: Plenum Press, 1998. Pages 221 - 228
14. Dartt, DA, Sullivan DA. Wetting of the Ocular Surface. In: Albert M, Jakobiec F, eds. Principles and Practice of Ophthalmology: 2nd Edition, vol. 2. Philadelphia: W. B. Saunders Company, 2000:960-981.

Suggested Reading:
1. Abelson M, Nally L, Nichols L, Chapin M. What we learned at the ARVO meeting. Review of Ophthalmology 2002;7:84-86.
2. Abelson M, Nally L, Ousler G. Dry Eye, Today and Tomorrow. Review of Ophthalmology 2000;4:132-134.
3. Abelson M, Ousler G, Nally L. The latest word on Dry Eye. Review of Ophthalmology 2001;3:86 – 89.
4. D'Cruz OJ, Dunn TS, Pichan P, Hass GG Jr, Sachdev GP. Antigenic cross-reactivity of human tracheal mucin with human sperm and trophoblasts correlates with the expression of mucin 8 gene messenger ribonucleic acid in reproductive tract tissues. Fertil Steril 1996;66:2:316-26.
5. Holly FJ, Lem MA. Tear physiology and dry eyes. Survey of Ophthalmology 1977;22:2:69-87.
6. Lopez-Ferrer A, Curull V, Barranco C, Garrido M, Lloreta J, Real FX, deBolos C. Mucins as differentiation markers in bronchial epithelium. Squamous cell carcinoma and adenocarcinoma display similar expression patterns. Am J Respir Cell Mol Biol 2001;24:1:22-29.