Cornea 21(Suppl. 1): S17S22, 2002.

2002 Lippincott Williams & Wilkins, Inc., Philadelphia

Significance of Mucin on the Ocular Surface

Hitoshi Watanabe, M.D., Ph.D.

Purpose. To review the significance of mucin in the tear film and the ocular surface epithelium. Methods. Summary of the information on how mucin derived from the corneal and conjunctival epithelia and from goblet cells plays a role in the stability of the tear film over the ocular surface. The change in mucin expression derived from the ocular surface epithelium is also discussed with reference to ocular surface disease. Results. The corneal and conjunctival epithelia produce transmembrane mucins such as MUC1, MUC2, and MUC4. In contrast, goblet cells produce the gelforming secretory mucin, MUC5AC. The lacrimal gland produces MUC7. On the ocular surface, cooperation between transmembrane mucin and secretory mucin is necessary for the stability of the tear film. The expression of mucin from the ocular surface epithelium is coordinated from the time of eyelid opening and is altered in conditions such as squamous metaplasia and dry eye. This alteration may result in instability of the tear film. Conclusion. The induction of mucin from the ocular surface may facilitate the stability of the tear film, and increased knowledge may lead to the development of a new modality for the treatment of dry eye. Key Words: MucinTear filmOcular surfaceCornea Conjunctiva.

MUC 1, 2, and 7 cDNAs have been completely sequenced, while the structures of MUC 3, 4, 5AC, 5B, 6, and 8 have been partially determined. The number of tandem repeats per mucin molecule can vary in each individual. Each mucin has a unique set of tandem repeats, and the only common feature between mucins is that they have a rich serine or threonine content, providing sites for O-glycosidic linkage of carbohydrates. Based on the full or partial cloning of mucin cDNA, human mucins are categorized into two types: transmembrane and secretory.23 Secretory mucins are further subdivided into gel-forming and soluble types.

MUCIN EXPRESSED BY THE OCULAR SURFACE EPITHELIUM AND TEAR FILM

On the ocular surface, mucin is well known to be secreted from goblet cells in the conjunctiva. This mucin is the main component of the innermost layer of the tear film. However, in addition to the mucin derived from the goblet cell, the corneal and conjunctival epithelia have recently been reported to produce mucin,14,15 in particular the transmembrane mucin, MUC1.16 MUC1 mRNA has been detected in all cell layers of the corneal epithelium, and immunohistochemical analysis has shown that MUC1 protein was present in the apical membranes of the superficial cells of the cornea and conjunctiva (Fig. 2). The role of MUC1 is not known, but it may facilitate the spread of gel-forming mucin from the goblet cells. However, MUC1 knockout mice do not apparently show any instability of the tear film.17,18 In contrast, MUC1 has been shown to play a protective role against the adherence of pathogens.17 There remains, however, debate on this issue,18 and further examination is required. MUC2 has been reported to be produced in the corneal and conjunctival epithelia.19,20 However, the amount of MUC2 is small, and this mucin is not recognized to play an important role in the ocular surface epithelium. MUC4 is expressed in the conjunctival epithelium.19,21 The corneal epithelium also expresses MUC4 in humans. Although MUC4 is present in the cornea, it would be localized in the limbal region and not in the central portion of the cornea.19,21 In situ hybridization studies have shown that the conjunctival epithelium expresses MUC4 mRNA in all layers.21 The goblet cells in the conjunctiva express gel-forming secretory mucin, MUC5AC20,21 (Fig. 3). In addition to these mucins, lacrimal glands have been shown to produce soluble MUC7 (M.M. Jumblatt, personal communication, 2000). MUC7 has not been shown to be present in the tear film. However, MUC7 is supposedly present in the tear film. Overall, there are at least four types of mucin in the ocular surface epithelium and tear film (Fig. 4).

Mucin is a high-molecular-weight glycoprotein with a high proportion of carbohydrate content.1 The structure of mucin resembles a test tube brush (Fig. 1). The carbohydrate is linked by Oglycosidic bonds to serine or threonine residues that are present in the protein core. Although the core protein of mucin appears to be identical between the tissues, the carbohydrate portion varies according to the organ, the individual, and other factors. Therefore, it is difficult to classify mucin based on the carbohydrate portion. Recently, however, it has been shown that the protein core of mucin contains a tandem repeat of a certain number of amino acids, and this forms the basis for classification.2 To date, human genome mapping has identified 13 human mucin genes (MUC1 MUC12), numbered in the order in which their cloning was reported. The human mucin genes and some of their characteristics are shown in Table 1.313

Submitted September 24, 2001. Accepted October 3, 2001. From the Department of Ophthalmology, Osaka University Medical School, Suita, Japan. This work was supported in part by grant-in-aid 1038769 for scientific research from the Japanese Ministry of Education, Science, Sports, and Culture and by a grant for scientific research from Osaka Eye Bank Association Fund. Address correspondence and reprint requests to Dr. H. Watanabe, Osaka University Medical School, Department of Ophthalmology, Room E7, 2-2 Yamadaoka, Suita, 565-0871, Japan; E-mail: watanabe@ophthal.med. osaka-u.ac.jp

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FIG. 1. Structure of mucin. The sugar portion binds to the protein core through serine or threonine residues in the protein core.

ADDITIONAL MUCINS EXPRESSED IN THE OCULAR SURFACE EPITHELIUM

It is possible that additional mucins are present in the ocular surface epithelium. Mucin is recognized to play a role in the maintenance of the tear film, but because it has not been clarified which mucin is important for the tear film spread, other mucins than MUC1 and MUC4 may be involved. To date, there are several candidates for this, although they have not been fully characterized. In the rat, Gipson et al. have reported that a highly glycosylated high-molecular-weight glycoprotein recognized by monoclonal antibody R339 is present along the glycocalyx of the apical membrane of the apical cells (Fig. 5).22 This glycoprotein was particularly prominent at the tip of the microplicae (Fig. 6) and was also found in the small vesicles in the cytoplasm of two or three cell layers of subapical cells of the corneal and conjunctival epithelium. Biochemical analysis showed that this molecule has the classic characteristics of a mucin; 60% of the glycoprotein is carbohydrate and it has predominantly O-linked sugars.14 Moreover, the protein part contains high percentages of serine and threonine.14 The comparable mucin in the human ocular surface epithelium is the glycoprotein recognized by monoclonal antibody H185.15 This glycoprotein has a high molecular weight and is highly glycosylated. Moreover, the glycoprotein is O-linked. The core protein structure has not been determined, and the glycoprotein cannot be defined as a mucin, but the available data suggest that the glycoprotein recognized by H185 is a mucin-like glycoprotein. It is present in the glycocalyx of the apical membrane of apical cells of the cornea and the conjunctiva (Fig. 7), and is prominent at the tips of the microvilli and microplicae. This mucin-like glycoproTABLE 1. Human mucin gene
Amino acids Chromosomal in tandem mapping repeat Ocular surface 1q21q24 11p15 7 3 11p15 11p15 11p15 4 12 1p13 11p14.3 7q22 7q22 20 23 17 16 8 29 169 23 13/41 15 28 28 Cornea Conjunctiva Cornea Conjunctiva Conjunctiva Cornea Goblet cells Lacrimal gland Not detected Not detected Not detected Not detected Not detected

FIG. 2. Presence of MUC1 in the corneal and conjunctival epithelium. A: MUC1 protein in the apical membrane of the superficial cell of the corneal epithelium. B: Phase contrast of Figure 2A. Reproduced from Inatomi et al.16 courtesy of Investigative Ophthalmology and Visual Science.

tein is also present in the cells below the apical cells of the cornea, along the small vesicle in the cytoplasm of the subapical cells.15 A definitive comparison between this mucin-like glycoprotein and MUC1 has not been completed, but biochemical analysis suggests that it is different from MUC1. Detailed characterization awaits further investigation.

SIGNIFICANCE OF MUCINS ON THE OCULAR SURFACE

The most important role of mucin is in stabilizing the tear film. The findings to date suggest that the mucin from goblet cells is a

Mucin gene MUC1 MUC2 MUC3 MUC4 MUC5AC MUC5B MUC6 MUC7 MUC8 MUC9 MUC10 MUC11 MUC12

Type if sequence verified Transmembrane Gelforming/secretory Transmembrane Gelforming/secretory Gelforming/secretory Gelforming/secretory Soluble, monomer/ secretory

FIG. 3. MUC5AC expression in the conjunctiva. A: MUC5AC mRNA was observed in goblet cells in the conjunctiva. B: High magnification of the photograph shows MUC5AC mRNA was intense in the goblet cell. C: Sense riboprobe showed no binding in the goblet cells. Reproduced from Inatomi et al.21 courtesy of Investigative Ophthalmology and Visual Science.

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FIG. 4. Structure of the tear film and the origin of mucin in the tear film and the ocular surface epithelium.

gel-forming mucin that is the main component of the mucous layer, the innermost layer of the tear film. The mucous layer spreads over the glycocalyx containing the mucin derived from the corneal epithelium and the conjunctival epithelium. These two types of mucin, the transmembrane and the gel-forming secretory mucin, facilitate generation of the overlying aqueous layer of the tear film. In this way, transmembrane mucin derived from the ocular surface epithelium and the gel-forming mucin from the goblet cells are necessary for the spread of the tear film. There is no direct evidence to show that the mucin produced by the ocular surface epithelium is important for the spread of the tear film. However, clinical data suggest a significant role for this mucin in the spread of the tear film. Anterior specular microscopy (DR-1, Kowa Co., Tokyo, Japan) is used to detect dry eye.23 The technique shows the pattern of the lipid layer of the tear film, which is helpful in determining whether the patient has dry eye. The microscopy shows not only the pattern of the lipid layer, but also shows how the tear film spreads over the cornea. In normal circumstances, the lipid layer spreads from the lower to the upper part of the cornea just after lid opening. Usually, the goblet cell

FIG. 6. Immunoelectron microscopic observation of mucin expression recognized by R339 monoclonal antibody in the ocular surface epithelium. A: Immunoelectron microscopy showed the mucin was observed along the apical membrane of the apical cell. Arrows indicate the membrane of the abutting subapical cell. B: High magnification electron micrograph shows that the mucin is prominent at the microplicae in the superficial cell. C: The mucin was observed along the cytoplasmic vesicles. Reproduced from Gipson et al.22 courtesy of Investigative Ophthalmology and Visual Science.

derived mucin spreads over the glycocalyx containing the ocular surface epithelium-derived mucin. The spread of the goblet cell derived gel-forming secretory mucin over the ocular surface epithelium facilitates the spread of the aqueous and lipid layer of the tear film. When we examined patients with corneal ulcer resulting from debridement of the epithelium caused by a foreign body in the cornea using anterior specular microscopy, we observed that the lipid layer moved similarly to the normal stream in the intact

FIG. 5. Mucin expression recognized by R339 monoclonal antibody in the rat ocular surface epithelium. A: Immunofluorescence study has shown that R339 binding was observed on the several superficial layers of the corneal epithelium. B: R339 binding was also observed in the goblet cells and in the several superficial layers of the conjunctival epithelium. Reproduced from Gipson et al.22 courtesy of Investigative Ophthalmology and Visual Science.

FIG. 7. Expression of the mucinlike glycoprotein recognized by H185 monoclonal antibody in normal subjects. A: Light micrograph shows a corneal section. B: Immunofluorescence micrograph showed that the mucinlike glycoprotein recognized by H185 was localized to only flattened superficial cells. Arrows indicate the basal cell of the corneal epithelium. C: The mucinlike glycoprotein was also observed in the apical cells of the conjunctival epithelium and the goblet cells. Inset indicates abrupt ending of H185 binding at the lid edge in the conjunctiva. Reproduced from Watanabe et al.15 courtesy of Investigative Ophthalmology and Visual Science.

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FIG. 8. Schema of the tear spread over the ocular surface. The lipid layer interference pattern shows that the tear moves from the lower to the upper part of the cornea, but it splits at the lower edge of the corneal ulcer.

FIG. 10. The expression of H185-specific mucin in the ocular surface epithelium in normal individuals. A: Normal superficial cells of the conjunctival epithelium show a cobblestone appearance. Arrow indicates goblet cells stained by periodic acid-Schiff. B: The H185specific mucin expression in each normal cell is graded as high (X), medium (Y), or low (Z). The pattern of H185-specific mucin in the conjunctiva shows a mosaic pattern. Reproduced from Watanabe H, Maeda N, Kiritoshi A, et al. Expression of a mucin-like glycoprotein produced by ocular surface epithelium in normal and keratinized cells. Am J Ophthalmol 1997;124:753,27 with permission from Elsevier Science.

epithelium of the cornea, but that it divided at the edge of the corneal ulcer and moved to surround the ulcer site (Fig. 8). In these patients, the number of goblet cells was normal on impression cytology, suggesting that the gel-forming mucin was similar to normal under these circumstances. However, transmembrane mucin produced by the corneal epithelium was absent in the lesion of the corneal ulcer. As mentioned above, transmembrane mucin and gel-forming mucin are important for the spread of the tear film.

Thus, the tear film cannot spread over the ulcer site because the goblet cell mucin cannot spread over the site in the absence of transmembrane mucin. This observation suggests that transmembrane mucin expression in the ocular surface epithelium is necessary for the spread of the tear film.

DEVELOPMENTAL EXPRESSION OF MUCIN IN OCULAR SURFACE EPITHELIUM

What happens to mucin produced by the ocular surface during development? The mucin produced by the corneal and conjunctival epithelia has been investigated in developing rats using antibody R339.24 The mucin-like glycoprotein recognized by R339 is present in adults in the apical membrane of the superficial cells of the entire ocular surface epithelium. Newborn rats are born with

FIG. 9. The appearance of R339 specific mucin in the developing rat. A: After eyelid opening, the mucin is present all along the ocular surface, the corneal and conjunctival epithelium. B: While the eyelid is closed, the mucin was only localized in the palpebral conjunctiva at the lid edge. Reproduced from Watanabe et al.24 with permission of Investigative Ophthalmology and Visual Science.

FIG. 11. The expression of H185-specific mucin in superior limbic keratoconjunctivitis. A: Localized inflammation was observed in the superior limbic conjunctiva. B: Squamous metaplasia also was observed in the superior conjunctiva of superior limbic keratoconjunctivitis. C: H185-specific mucin was not observed in the conjunctival superficial cells with squamous metaplasia in superior limbic keratoconjunctivitis. Reproduced from Watanabe H, Maeda N, Kiritoshi A, et al. Expression of a mucin-like glycoprotein produced by ocular surface epithelium in normal and keratinized cells. Am J Ophthalmol 1997;124:754,27 with permission from Elsevier Science.

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their lids closed and open their eyes approximately 12 to 15 days after birth. When the eyelids are closed, the mucin-like glycoprotein is localized in the palpebral conjunctiva near the lid fusion. The glycoprotein is not found in the corneal epithelium while the eyelid remains closed. When the eyelid opens, the mucin-like glycoprotein expression in the ocular surface epithelium rapidly extends to all apical squamous epithelium of the entire ocular surface epithelium including that of the cornea (Fig. 9). These phenomena have been confirmed by subsequent experiments in which artificial eyelid opening induced rapid appearance of the mucin-like glycoprotein. This observation corresponds closely to previous reports. Hazlett et al. have shown that mucin levels are not appreciable before eyelid opening.25 In contrast, a heavier surface coat presumed to be mucin is present after eyelid opening.25 These data suggest that mucin derived from the ocular surface epithelium is expressed along the ocular surface epithelium after eyelid opening. With respect to the mucin derived from goblet cells in the rat, it has been shown that Muc5AC was first found in a few cells near the fornix 7 days after birth.26 By 14 days after birth, many goblet cells showed rMuc5AC expression not only in the fornix region, but also in the palpebral and bulbar conjunctiva. These two developmental experiments clearly showed that transmembrane mucin and gel-forming mucin work together to achieve tear spreading after lid opening, although the two types appear at different times.

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served in the conjunctiva of normal eyes, but as dry eye progressed, the pattern was gradually lost and replaced by a starry sky pattern in which a lack of apical cell binding (dark areas) and increased binding to goblet cells (light disks) was observed (data not shown). Therefore, the expression of the mucin-like glycoprotein produced by the ocular surface epithelium is diminished or absent in dry eye as well as superior limbic keratoconjunctivitis.

HOW TO DETECT MUCIN IN THE OCULAR SURFACE EPITHELIUM

There is no direct method to detect mucin over the ocular surface epithelium. However, rose bengal staining can be used in the clinical setting as an indirect method. As Tseng et al. showed,32,33 gel-forming mucin can form a diffusion barrier to rose bengal staining of the ocular surface epithelium under the condition that transmembrane mucin is present. Rose bengal staining does not stain the cell in vivo because these two kinds of mucin harmonize in vivo. In contrast, in vitro, the monolayer of the epithelial cell does not express the transmembrane mucin and gel-forming mucin cannot overlie the transmembrane mucin. In such cases, rose bengal actually stains the cell. Therefore, rose bengal staining indirectly shows whether both gel-forming mucin and transmembrane mucin are intact.

NORMAL EXPRESSION OF MUCIN IN THE OCULAR SURFACE EPITHELIUM

It has been reported that MUC1 is expressed in the superficial cells of the corneal epithelium and conjunctival epithelium.16 However, the pattern of expression in all the superficial cells has not been shown. We investigated the expression of the mucinlike glycoprotein recognized by H185 in each of the superficial cells.27 Our results showed that expression varied from cell to cell, allowing individual cells to be distinguished easily. The expression was graded as high, medium, and low (Fig. 10), and this produced a mosaic pattern. We hypothesized that this pattern could be explained as follows. The mucin-like glycoprotein was expressed at the tip of the microvilli and microplicae of the apical surface of the cornea and conjunctiva. The apical surface epithelia are divided into three types of cell: dark, medium, and light, according to the degree of scattering they effect during scanning electron microscopy.2830 The lighter cells contain a greater density of microvilli, and darker ones contain a lower density. Therefore, the mucin expression in the superficial cells varies according to the density of the microvilli and microplicae. The superficial cells with the fewer microvilli are thought to be the oldest cells at the ocular surface. The superior conjunctiva in patients with superior limbic keratoconjunctivitis shows squamous metaplasia. We found that the mucin-like glycoprotein expression is absent or remarkably reduced in those cells obtained from the superior conjunctiva of these patients27 (Fig. 11). These data suggest that the keratinized apical cells of the ocular surface epithelium are altered in appearance and lack the normal mosaic pattern of expression of the mucin produced by the ocular surface epithelium. This alteration of the mucin-like glycoprotein expression was restored with normalization of the cells.27 We further investigated the expression of the mucin-like glycoprotein in patients with dry eye.31 The mosaic pattern was ob-

INDUCTION OF MUCIN FOR THE TREATMENT OF DRY EYE

Transmembrane mucin and gel-forming mucin in the ocular surface are thought to be essential for maintenance of the tear film. In dry eye, the expression of transmembrane mucin in the ocular surface epithelium has been reported to be altered.31 This change in expression may result from dry eye, but in cases of dry eye caused by mucin deficiency, it is possible that the induction of mucin from ocular surface epithelium could be effective as treatment. It is important to investigate what could induce the expression of transmembrane mucin and gel-forming mucin. If certain factors or cytokines could be confirmed to induce these mucins, this could lead to the development of a drug for the treatment of dry eye. Further investigation is expected.

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