Mucins and TFF peptides of the tear film and lacrimal apparatus

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PROGRESS IN HISTOCHEMISTRY

AND CYTOCHEMISTRYProgress in Histochemistry and Cytochemistry 41 (2006) 1–53

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Mucins and TFF peptides of the tear film and

lacrimal apparatus

Friedrich P. Paulsena,�, Monica S. Berryb

aDepartment of Anatomy and Cell Biology, Martin Luther University Halle-Wittenberg,

Große Steinstr. 52 06097 Halle (Saale), GermanybAcademic Unit of Ophthalmology, University of Bristol, Lower Maudlin Street,

Bristol BS1 2LX, United Kingdom

Abstract

The three-dimensional organization of the tear film, which is produced and drained by the

different structures of the ocular adnexa, is essential for maintainance and protection of the

ocular surface. This is facilitated by a class of large, highly glycosylated, hydrophilic

glycoproteins, the mucins, which are usually expressed in association with a class of peptides

having a well-defined, structurally conserved trefoil domain, the mammalian trefoil factor

family (TFF) peptides. In this review, the latest information regarding mucin and TFF peptide

function and regulation in the human lacrimal system, the tear film and the ocular surface is

summarized with regard to mucous epithelia integrity, rheological and antimicrobial

properties of the tear film and tear outflow, age-related changes and certain disease states

such as dry eye, dacryostenosis and dacryolith formation.

r 2006 Elsevier GmbH. All rights reserved.

Keywords: Conjunctiva; Cornea; Dry eye; Lacrimal apparatus; Lacrimal gland; MUC; Mucin;

Nasolacrimal ducts; Ocular surface; Precorneal tear film; Preocular fluid; Rheology; Tear film;

Tear outflow; TFF peptides

see front matter r 2006 Elsevier GmbH. All rights reserved.

.proghi.2006.03.001

nding author. Tel.: +493455571707; fax: +493455571700.

dress: [email protected] (F.P. Paulsen).

www.elsevier.de/proghi

dx.doi.org/10.1016/j.proghi.2006.03.001

mailto:[email protected]

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F.P. Paulsen, M.S. Berry / Progress in Histochemistry and Cytochemistry 41 (2006) 1–532

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2. Anatomy and embryology of the lacrimal apparatus and

ocular surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

2.1. The lacrimal gland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

2.2. Eyelid and ocular surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

2.2.1. Eyelid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

2.2.2. Ocular surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

2.3. The nasolacrimal ducts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

3. The preocular tear film. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

4. Properties of mucins and TFF peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

4.1. Mucins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

4.1.1. Classification and general properties . . . . . . . . . . . . . . . . . . . . . . . 15

4.2. TFF peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

4.2.1. Classification and general properties . . . . . . . . . . . . . . . . . . . . . . . 21

5. Mucins and TFF peptides of the tear film . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

5.1. Mucins of the tear film . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

5.1.1. Membrane bound mucins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

5.1.2. Secreted mucins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

6. Mucins and TFF peptides of the lacrimal gland . . . . . . . . . . . . . . . . . . . . . . . . . . 25

6.1. Lacrimal gland mucins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

6.1.1. Membrane-anchored mucins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

6.1.2. Secreted mucins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

6.2. Accessory lacrimal gland mucins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

6.3. Relationship between lacrimal gland mucins and tear film . . . . . . . . . . . . . . 30

6.4. Function of lacrimal gland mucins in antimicrobial defence . . . . . . . . . . . . . 31

6.5. Lacrimal gland TFF peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

7. Age-related changes in the composition of lacrimal gland and tear film mucins . . . . 32

7.1. Age-related dry eye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

7.2. Age-related changes in lacrimal gland mucins . . . . . . . . . . . . . . . . . . . . . . . 32

7.3. Age-related changes in tear film mucins . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

7.4. Neuronal regulation of lacrimal gland and tear film mucins . . . . . . . . . . . . . 33

7.5. Hormonal regulation of lacrimal gland and tear film mucins . . . . . . . . . . . . 35

8. Interaction of mucins with TFF peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

9. Mucins and TFF peptides of the nasolacrimal ducts . . . . . . . . . . . . . . . . . . . . . . . 36

9.1. Efferent tear duct mucins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

9.1.1. Membrane-spanning mucins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

9.1.2. Secreted mucins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

9.2. TFF peptides of the efferent tear ducts . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

9.3. Comparison of efferent tear duct mucins and TFF peptides with mucins and

TFF peptides in the tear film. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

9.3.1. Mucins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

9.3.2. TFF peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

9.4. Relevance to tear outflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

9.5. Mucin changes in diseases of the nasolacrimal ducts . . . . . . . . . . . . . . . . . . 41

9.6. TFF peptide changes in diseases of the nasolacrimal ducts. . . . . . . . . . . . . . 41

10. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

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Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

1. Introduction

The unique composition of tear film enables it to maintain a smooth surface forlight refraction, lubricate the eyelids, lubricate the cornea and the conjunctiva,supply the cornea with nutrients and provide white blood cells with access to thecornea and conjunctiva, remove foreign materials from the cornea and conjunctiva,and defend the ocular surface against pathogens by means of both specific andnonspecific antibacterial substances.

The epithelial surface of the eye and its specialized glandular infoldings producethe components of the tear film, which include water, protective antimicrobials,cytokines, lipids as well as mucins and trefoil factor family (TFF) peptides.

Mucins and TFF peptides perform a number of essential functions which, collectively,provide protection of the ocular surface. Mucins are present both in the epithelialglycocalyx and in the tear fluid. They are hydrophilic and play a role in maintenance ofwater on the surface of the eye. Membrane-anchored mucins are part of the glycocalyx,together with a variety of other glycoconjugates, providing a continuous barrier acrossthe surface of the eye that prevents pathogen penetration. The membrane spanningmolecules have signalling capabilities that influence epithelial activity. The high molecularsecreted mucins and TFF peptides are responsible for the rheological properties of thetear film, enabling movement over the ocular surface by simultaneous attachment of thetear film. TFF peptides have many other physiological functions in addition to theirrheological properties, such as promotion of epithelial cell migration, antiapoptoticproperties, induction of cell scattering, epithelial restitution and neuropeptide functions(for a review, see Hoffmann et al., 2001; Hoffmann and Jagla, 2001).

Reduced production of ocular mucins and TFF peptides with altered rheologicalproperties is an important factor in the pathogenesis of dry eyes that arecharacterized by an inadequate ocular lubrication as a result of impaired lacrimalfunction, failure in the transfer of lacrimal fluid to the ocular surface epithelia and/orexcessive tear evaporation. Moreover, dry eye syndromes also include ocular surfaceepitheliopathy, tear hyperosmolality, an unstable preocular tear film, varying degreesof inflammation, and symptoms of ocular irritation (Lemp, 1995).

A variety of factors have been found to regulate mucin and TFF peptide geneexpression and production at the ocular surface, but the biochemical characteristicsof preocular mucins and TFF peptides that determine their physiological roles in theeye have yet to be identified. It should also be pointed out that other molecules suchas lipids, proteoglycans and DNA may contribute to the physical properties ofocular surface mucus, and perhaps more so in pathological conditions.

Great strides have been made in identifying and characterizing the majoroligomeric mucins and TFF peptides present in tears, the ocular surface and thelacrimal system over the past decade. In this, the collective physical properties ofmucins (i.e., length, charge density, macromolecular architecture, interaction and

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mutual influences) have been related to a specific set of rheological parameters forthe mucus gel present in tears and at the ocular surface (Ellingham et al., 1999; Berryet al., 2001, 2004a; Round et al., 2002, 2004; Brayshow et al., 2003, 2004). For thisarticle, we were given the task of reviewing our own contributions to this area. Wehave also suggested further avenues to investigation that will prove necessary to gaina better understanding of the biology and pathobiology of mucins and TFF peptidesof the ocular surface and lacrimal apparatus.

2. Anatomy and embryology of the lacrimal apparatus and

ocular surface

The ocular surface and its adnexa comprise the cornea, the conjunctiva with thebulbar, fornical and palpebral segments (Fig. 1), the main lacrimal gland, the glandsof the eye lids, i.e. the Meibomian, Moll and accessory lacrimal glands, and thenasolacrimal system (also termed nasolacrimal ducts or efferent tear ducts) with theupper and lower puncta, paired lacrimal canaliculi, lacrimal sac and nasolacrimalduct. The nasolacrimal ducts collect the tear fluid from the ocular surface andconvey it into the nasal cavity. All the other structures contribute to formationof the preocular tear film. The term ‘‘lacrimal apparatus’’ includes the lacrimalgland and the accessory lacrimal glands, which secrete a complex fluid (tears) andwhose excretory ducts convey fluid to the surface of the eye where the fluid formspart of the tear film, the paired lacrimal canaliculi, the lacrimal sac, and thenasolacrimal duct.

Fig. 1. Conjunctival anatomy. The conjunctiva consists of three segments: a bulbar (light

green), a fornical (middle green) and a palpebral (dark green) part.

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2.1. The lacrimal gland

The development of the main lacrimal gland is based on invaginations of theectoderm at the superolateral angles of the conjunctival sac that do not mature, inhuman, until about 6 weeks after birth. When mature, the gland is localizedanteriorly in the superolateral region of the orbit and is divided into two parts by thelevator palpebrae superioris muscle (Fig. 2). The orbital segment approximates thesize and shape of an almond and is lodged in the lacrimal fossa on the medial aspectof the zygomatic process of the frontal bone, just within the orbital margin. Thesmaller palpebral segment (nearly one-third the size of the orbital segment) is inferiorto the levator palpebrae superioris in the superolateral part of the eyelid. Infunctional terms, the lacrimal gland synthesizes, stores and secretes a bulk ofdifferent proteins and peptides, water, and electrolytes that are released into the tearsand, via the excretory duct system of the lacrimal gland, onto the surface of the eyeas part of the tear film. Similar to the salivary glands and the pancreas, the lacrimalgland is a multilobed, tubuloalveolar gland of the serous type (Fig. 3). Dischargefrom the tubules does not employ any characteristic excretory duct system(histological distinction from serous salivary glands) on its way into the interlobularexcretory ducts. In contrast to rats and rabbits, with only a single excretory duct, thehuman lacrimal gland features 8–10 main excretory ducts, which open onto thesurface of the eye in front of the lateral portion of the superior conjunctival fornix.In cross section, the secretory units of the gland show a ring-like structure, the acinus(Fig. 3). An acinus is composed of secretory cells, the acinar cells (shaped like abunch of grapes). Since several secretory units open into one secretory duct, the

Fig. 2. The lacrimal gland is localized anteriorly in the superolateral region of the orbit and is

divided into two parts by the levator palpebrae superioris muscle.

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Fig. 3. Histological section of the lacrimal gland. A serous gland of tubulo-acinar type

without a differentiated secretory duct system. The tubules have wide lumina. Arrows mark an

intralobular excretory duct; the arrowhead indicates an acinus. Azan staining, bar 27.5 mm.


lacrimal gland is said to be ‘‘branched.’’ Acinar cells comprise about 80% of themass of the lacrimal gland. The ductal cells of the excretory duct system possess theability to modify the secretory product of the acinar cells.

Lacrimal gland acinar cells are polarized secretory cells that secrete their proteinsunidirectionally. This functionality is facilitated by a dense ring of tight junctionsthat surround the cells at the lumen, dividing the plasma membrane into apical(luminal) and basolateral (blood-related) membranes. Receptors for neurotransmit-ters and neuropeptides are located at the basolateral membrane of the acinar cells.The lacrimal gland is densely innervated (for a review, see Hodges and Dartt, 2003)release of the transmitters and peptides takes place in close proximity to acinar cellmembranes. These basolateral membranes also contain several of the transportproteins and ionic channels required for electrolyte and water secretion (Hodges andDartt, 2003; Takata et al., 2004). Acinar cells feature a prominent endoplasmicreticulum and Golgi apparatus. Both of these organelles are situated, together withthe nucleus, mainly in the basal portion of the cells, whereas the apical portion of thecell contains numerous secretory granules containing proteins. It has beendemonstrated that acinar cells are influenced by various hormones, i.e. androgens,estrogens, glucocorticoids, mineralocorticoids, retinoic acid, prolactin, a-melano-cyte-stimulating hormone, adrenocorticotropic hormone, luteinizing hormone,follicle-stimulating hormone, growth hormone, thyroid-stimulating hormone,arginine, vasopressin, oxytocin, thyroxine, parathyroid hormone, insulin, glucagons,melatonin, human chorionic gonadotropin, cholecystokinin (for a review, seeSullivan, 2004) and, as recently demonstrated by our own unpublished results,somatostatin.

Excretory lacrimal gland ducts are lined by cuboidal cells in one layer (beginningof ducts) or two layers (greater part of ducts). In a manner comparable to the acinarcells, the superficial lining cells of the excretory duct system contain luminally tight

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junctions, resulting in polarized cells, whereas the superficial and basal cells of theducts are connected by gap junctions, creating a syncytium. In the lining cells as well,the nucleus is in the basal portion of the cell, with a more apical location of theendoplasmic reticulum and Golgi apparatus. The cells have been shown to secretemainly water and electrolytes, but a limited protein secretion potential has also beendemonstrated. Basal epithelial cells of the lacrimal gland ducts secrete, for example,bactericidal permeability-increasing protein (BPI), which occurs in a relatively highconcentration in tears and plays a substantial antibacterial role in human tears(Peuravuori et al., 2005). The main excretory ducts often contain single goblet cellsor groups of goblet cells forming intraepithelial glands near their transition into theconjunctival fornix.

Acinar and ductal cells are basally surrounded by myoepithelial cells. In a mannercomparable to the salivary glands, the myoepithelial cells are thought to contributeto transport of secretions. They express receptors for several neurotransmittersknown to stimulate acinar cell protein secretion (Lemullois et al., 1996; Hodgeset al., 1997). It has also been hypothesized that myoepithelial cells may play astructural role in helping the gland maintain its shape.

The lacrimal gland is thought to be an integral part of the ocular mucosal immunesystem. It contains cell populations of lymphocytes, plasma cells (expressing allimmunoglobulins [A, G, E, M, D]), mast cells, macrophages and dendritic cells. Ofthe mononuclear cells, over 50% are plasma cells, and most secrete IgA – a criticalcomponent of the ocular mucosal immune system (for review, see Stern andPflugfelder, 2004; Pflugfelder and Stern, 2005; Knop et al., 2005).

2.2. Eyelid and ocular surface

2.2.1. Eyelid

By the sixth week of life, small folds of ectoderm with a mesenchymal core appearjust cranial and caudal of the developing cornea. These upper and lower eyelidprimordia rapidly grow towards each other, meeting and fusing by the eighth week.The space between the fused eyelids and the cornea, lined with ectoderm-derivedepithelium, is known as the conjunctival sac. The eyelids then separate between thefifth and seventh months.

The normal lid margin, measured from its posterior border to the lash line, growscontinuously over the first 20 years of life and is about 1.5mm wide in children and2.0mm in adults (Bron et al., 1991). In young adults it presents a smooth, flat,mucocutaneous surface, the posterior conjunctival margin of which roughly forms aright angle with the tarsal conjunctiva, conforming to the contour of the globe.

The ‘‘skeleton’’ of the eyelid is a collagen plate called the tarsus (Fig. 4). Itcontains a row of branched alveolar sebaceous glands unrelated to the eyelashes(Fig. 5). These tarsal, or Meibomian, glands have punctate openings along the freeedge of the eyelid close to its posterior margin. They produce a lipid substance,synthesis of which depends on neuronal, hormonal, and vascular factors. Althoughrecent investigations have more fully defined the composition of the Meibomiangland lipids, complete characterization of meibum is an ongoing process (for a

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Fig. 4. Anatomy of the lid. The figure shows a histological section through a upper eyelid in a

sagittal plane: (1) superior tarsal muscle (smooth muscle), (2) accessory lacrimal gland (Krause

gland), (3) fornical part of conjunctiva, (4) tarsus, (5) palpebral part of conjunctiva, (6)

meibomian gland, (7) ciliary (Moll’s) gland, (8) eyelash, (9) orbicularis oculi muscle, (10) outer

surface (stratified keratinized squamous epithelium).


review, see McCulley and Shine, 2003). Meibum is fluid that spreads easily, is asurfactant, provides a smooth optical surface for the cornea at the air interface,reduces evaporation from the tear film, enhances the stability of the tear film,enhances spreading of the tear film, prevents spill-over of tear from the lid margin,prevents contamination of the tear film by sebum, must remain functional after ablink and seals the apposed lid margins during sleep (Foulks and Bron, 2003). TheMeibomian lipids have a specific composition to satisfy these requirements. Thesecretion might be modified by lipases produced by ocular bacteria after delivery,

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Fig. 5. Histology of different glands as well as fornical and palpebral conjunctiva of the eyelid:

(a) tarsal (Meibomian) gland, a specialized sebaceous gland; (b) ciliary (Moll’s) gland, an

apocrine gland related to the eyelash; (c) accessory lacrimal gland (Krause) in the fornix;

(d) fornical conjunctiva with high columnar epithelium and large goblet cells (arrows);

(e) palpebral conjunctiva with stratified nonkeratinized squamous epithelium and small goblet

cells (arrow) partly arranged in small groups forming small intraepithelial glands (not visible).

a–e, Azan staining; bars a–c, e 55 mm, d, 27.5mm.


and modifications in the lipid components can lead to unique disease states (for areview, see McCulley and Shine, 2003). Sexual hormones, and androgens inparticular, appear to play a decisive role in Meibomian physiology (Sullivan et al.,2000).

The cross-striated orbicularis oculi muscle, the fibre bundles of whose palpebralpart overlap one another like tiles on a roof, is located in the middle of the lid(Fig. 4). The tendon of the cross-striated levator palpebral muscle inserts into thetarsus, with the smooth tarsalis muscle beneath it. The tone of the latter isdetermined by autonomic nervous impulses and is presumably responsible foradjusting the width of the palpebral opening. Two or three rows of stiff hairs – theeyelashes – are located close to the anterior margin of the eyelids. The apocrine orciliary glands (Moll’s glands) open near the eyelashes (Fig. 5). These glands areactive from birth on in production of antimicrobial agents in the eyelid shaft and onthe ocular surface, i.e. lysozyme, beta-defensin-2, adrenomedullin, lactoferrin, andIgA (Stockelhuber et al., 2003). The eyelid also contains small accessory lacrimalglands in the conjunctival fornix (Krause’s glands, Wolfring’s glands) (Fig. 5).Although much smaller, these glands are histologically comparable to the main

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lacrimal gland. However, less is known about the secretions of these small glandsand their contribution to tear film physiology. The anterior surface of the lid iscovered by thin stratified, keratinized squamous epithelium, its posterior surface bystratified, nonkeratinized squamous epithelium with small goblet cells forming partlygroups (intraepithelial glands) and (towards the fornix) columnar epithelium withintegrated goblet cells. The posterior epithelium is the palpebral conjunctiva (Fig. 5).

The mucocutaneous junction is an important watershed zone that segregates thetear-wettable conjunctival mucous membrane from the oil-wettable marginal skin. Inyouth it runs as a smooth, continuous line, parallel to the posterior lid margin, but inthe elderly it runs a more irregular course (Norn, 1985).

2.2.2. Ocular surface

The apical surface of the ocular surface epithelia, both corneal and conjunctival(with bulbar, fornical and palpebral segments), features a specialized interfacebetween tear fluid and epithelium that stabilizes the fluid layer. This interfaceincludes the undulating membrane ridges on the cells’ apical membrane, termedmicroplicae, as well as a polysaccharide matrix that is produced and anchored at thecell membrane termed the glycocalyx. This matrix covers the cell surface includingthe microplicae. Conjunctival and corneal epithelial cells react to pathogen invasionsby producing (inducible) antimicrobial peptides (McDermott, 2004; Paulsen et al.,2005). Besides these innate immune defence potentials, the conjunctiva is also anintegral part of the adaptive immune system of the ocular surface. In most casesthere is a diffuse infiltrate of variable intensity within the lamina propria, consistingpredominantly of T lymphocytes with scattered CD20 and CD45RA-positive B cellsas well as many plasma cells, most of them IgA positive. Aggregated follicles arepresent in nearly one-third of the specimens analyzed, fulfilling the criteria fordesignation as conjunctiva-associated lymphoid tissue (CALT) (Osterlind, 1944;Hingorani et al., 1997; Chodosh et al., 1998; Wotherspoon et al., 1994; Knop andKnop, 2000; Knop et al. 2005).

2.3. The nasolacrimal ducts

The ectoderm at the center of each nasal placode invaginates to form an oval nasalpit in the sixth week, thus dividing the raised rim of the placode into lateral andmedial nasal processes. The groove between the lateral nasal process and theadjacent maxillary swelling is the nasolacrimal groove. During the seventh week ofdevelopment, the ectoderm at the floor of this groove invaginates into the underlyingmesenchyme to form a tube called the nasolacrimal duct. This duct is invested bybone during the maxillary ossification process. The upper part enlarges and formsthe lacrimal sac. The exact development of the upper and lower canaliculi, theconnection between the conjunctival sac and the lacrimal sac, is still unknown.Upper and lower canaliculus, lacrimal sac and nasolacrimal duct are subsumedunder the terms ‘‘nasolacrimal ducts,’’ ‘‘efferent tear ducts,’’ or ‘‘lacrimal passages.’’After birth, the function of the nasolacrimal ducts is to drain tear fluid into theinferior meatus of the nose (Fig. 6). The nasolacrimal ducts consist of a bony passage

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Fig. 6. Macroscopy of the nasolacrimal ducts: (a) view of a prepared nasolacrimal system in

situ. Parts of the lateral nasal wall have been removed, i.e. the heads of the middle (mt) and

inferior (it) turbinate. The lacrimal bone surroundig the nasolacrimal duct (nt) has been

removed with a diamond trephine; (b) macroscopic view of the lower part of a prepared

nasolacrimal duct removed from its bony channel. nd – nasolacrimal duct; mmn – middle

meatus of the nose; imn – inferior meatus of the nose; ss – sphenoid sinus; hv – Hasner’s valve

(opening of the nasolacrimal duct into inferior meatus of the nose); nm – nasal mucosa.


and a membranous lacrimal passage (Duke-Elder, 1961). The bony passage isformed at the anterior end by the frontal maxillary process and a the posterior endby the lacrimal bone. The membranous lacrimal passages include the lacrimalcanaliculi, the lacrimal sac and the nasolacrimal duct (for reviews, see Paulsen, 2003;Paulsen et al., 2003a).

The upper and lower canaliculi are lined by a pseudostratified/stratified columnarepithelium and are surrounded by a dense ring of connective tissue as well as byfibres of Horner’s muscle (the lacrimal portion of the orbicularis oculi muscle;Halben, 1904). The lacrimal sac and the nasolacrimal duct are lined with a double-layered epithelium comprising a superficial columnar layer and a deep, flattenedlayer of basal cells (Tsuda, 1952; Duke-Elder, 1961) (Fig. 7). Both layers sometimesappear as a pseudo-stratified epithelium. Superficial epithelial cells containnumerous secretory vesicles in their cytoplasm (Paulsen et al., 1998) and have beenshown to secrete many different antimicrobial proteins and peptides (Paulsen et al.,2001a, 2002a). In addition to epithelial cells, goblet cells are integrated in theepithelium as single cells or, more frequently, in a characteristic arrangementforming intraepithelial mucous glands (Werncke, 1905; Paulsen et al., 1998) (Fig. 7).Moreover, kinociliae (motile ciliae) have been described as commonly occurring insome epithelial cells (Radnot and Bolcs, 1971; Radnot, 1977). Most epithelial cellsare, however, lined by microvilli (for a review, see Paulsen, 2003a). Rivas et al. (1991)

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Fig. 7. Histological cross section through the lacrimal sac. The epithelium (small arrows)

surrounding the lumen (L) consists of a basal cell layer and a superficial columnar layer.

Goblet cells are integrated as solitary cells or in a characteristic arrangement forming

intraepithelial mucous glands (large arrows). Resorcin-fuchsine-thiacine picric-acid staining,

bar 55 mm.


described small serous glands in the lamina propria, especially in the fundus of thelacrimal sac.

The walls of the lacrimal sac and the nasolacrimal duct are made up of a helicalsystem of different connective tissue fibres. Embedded in this helical system, whichfunctions as a cavernous body, are wide luminal plexus with specialized blood vessels(Paulsen et al., 2000a; Ayub et al., 2003). Caudally, the vascular system is connectedto the cavernous body of the inferior turbinate. Upon lid closure, the system distendsand may be ‘‘wrung out’’ due to its medial attachment and helically arranged fibrillarstructures, whereby tear fluid is drained distally (Thale et al., 1998). The embeddedblood vessels and epithelial layer are under vegetative control. This was emphasizedby a high density of nerve fibres as well as the presence of various neuropeptides(Paulsen et al., 2000b). Via the innervation, the specialized blood vessels permitregulation of blood flow by opening and subsidence of the cavernous body, at thesame time regulating tear outflow. Relevant related functions include a role in theoccurrence of epiphora related to emotional responses.

The nasolacrimal ducts are also an integral part of the ocular mucosal immunesystem. In most cases there is a diffuse infiltrate of variable intensity within thelamina propria, consisting predominantly of T lymphocytes with scattered CD20and CD45RA-positive B cells as well as many plasma cells, most of them IgApositive. Aggregated follicles are present in nearly one-third of nasolacrimal ducts,fulfilling the criteria for designation as mucosa-associated lymphoid tissue (MALT)or tear duct-associated lymphoid tissue (TALT) (for a review, see Paulsen, 2003;Paulsen et al., 2000c, 2002b, 2003a).

As a draining and secretory system, the nasolacrimal ducts play a role in teartransport and non-specific immune defences. Moreover, components of tear fluid are

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absorbed in the nasolacrimal passage and transported into the surrounding vascularsystem (Paulsen et al., 2002d).

It has been suggested that, under normal conditions, tear fluid components areconstantly absorbed into the blood vessels of the surrounding cavernous body. Thesevessels are connected to the blood vessels of the outer eye and may act as a feedbacksignal for tear fluid production, which is therefore stopped if these tear componentsare not absorbed (Paulsen 2003; Paulsen et al., 2003a).

3. The preocular tear film

Today it is generally accepted that the preocular fluid is a complex secretion withstructure on all scales: lipids, an aqueous component dissolving a great variety ofchemical entities, and mucins contribute to a stable and continuous layer coveringthe external ocular epithelia and anchored onto their apical surfaces (Fig. 8). Thelipid component is secreted by the Meibomian glands in the eyelid. The aqueouscomponent is secreted by the main lacrimal gland and the accessory lacrimal glands(glands of Krause; glands of Wolfring) of the lids. The mucus component is theproduct of conjunctival goblet and epithelial cells, corneal epithelial cells, acinar cellsand excretory duct cells of the lacrimal gland, and probably also of acinar cells of theaccessory lacrimal glands (for review, see Paulsen et al., 2003a). Tear film thickness isstill a matter of debate. Recent studies have used refraction analysis, freezesubstitution electron microscopy, measurement of tear film electrical profile,examination of reflectance spectra or optical coherence tomography and suggest

Fig. 8. Source and components of the tear film.

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that human tear film is approximately 3 mm thick (King-Smith et al., 2000; Wanget al., 2003) whereas the corresponding figure in mice averages 7 mm (Tran et al.,2003) and the range in rats is 2–6 mm (Chen et al., 1997; for a review, see Johnson andMurphy, 2004). Using slightly different interferometry methods the thickness of thehuman tear film was evaluated as 34–45 mm, thinned by N-acetyl cysteine to 11 mmand recovering after some 40min (Prydal et al., 1992, 1993; Prydal and Campbell,1992).

These differing results for rodents and humans underline just how difficult it is toapply results from animal models to humans. Most of the species used to determinetear film structure have markedly greater tear stability and a much lower blink rate(Duke-Elder and Gloster, 1968) than humans. It is well known, for example, thatrabbits have a very low eye-blink rate, with an average of one blink every 20–30min(Gormezano et al., 1962), and can also manage for long periods without drinkingwater (Tarjan et al., 1984; Denton et al., 1985), suggesting special enablingmechanisms. Also, many animals commonly used in laboratory research have a largegland filling the posterior part of the orbit and surrounding the optic nerve, called theHarderian gland, which humans do not have and the function of which is still poorlyunderstood, although it certainly contributes to tear film thickness. Therefore, studiesby Chen et al. (1997) and Tran et al. (2003) who observed a homogenous, finenetwork-like structure throughout the tear film in rodents, warrant caution.

The whole epithelium covering the ocular surface (comprising the cornea as well asthe bulbar, fornical and palpebral conjunctiva) produces mucins. As will be reviewedlater, lacrimal gland acinar and ductal cells also produce mucins that contribute tomaintenance of the tear film. However, variations are noted in the pattern ofexpression and type of mucin production in the different structures of the ocularsurface, so that different functions are attributed to each zone.

4. Properties of mucins and TFF peptides

4.1. Mucins

An evolutionarily ancient protective molecular type, the defining and unifyingfeature of mucins is the presence of a large number of oligosaccharide chains inwhich N-acetylgalactosamine (GalNAc) is O-linked to a hydroxylated amino-acid(Ser or Thr) in the peptide core. Oligosaccharides are not evenly distributed alongthe linear molecule, but concentrated in discrete regions, encoded by repeatsequences in the mucin gene (tandem repeats). These features, preserved in all thewet epithelia mucins protect, impart on large polymeric mucins an appearanceapproaching a feathered wiggly worm: a central core with tufts of sugar chains.

The peptide core, which is the stiffest part of these long polymers, is most likelycoiled in solution, while the oligosaccharide chains that richly and unevenly decorateit occupy most of and determine the size of the spheroid molecular volume.However, the majority of these large polymers are part of mucosal gels, and thus in a

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variety of linkages to neighboring polymers. In the case of small mucins, e.g.membrane-spanning members of the class, a more extended configuration isexpected: the O glycosidic link forces the peptide core into a linear conformation(Carlstedt et al., 1983). Mucin domains are not exclusively found in mucin molecules,they are also part of adhesion O-glycoproteins (Porchet and Aubert, 2004).

It is believed that mucins evolved in tandem with bacteria: there are indeed mutualinteraction between barrier forming mucins and bacteria, which result in theformation and maintenance of a local flora, which in turn affects gene expression inmucosal cells. Examples here range from the effect of Pseudomonas aeruginosa andother bacteria on mucin secretion in the airways (Li et al., 1997; Basbaum et al.,1999; Lemjabbar and Basbaum, 2002; Wang et al., 2002), to the gut where in theabsence of commensals sets of genes are either not switched on or switched off,resulting in a poor gel and an impaired absorption (Enss et al., 2000; Vimal et al.,2000; Corfield et al., 2001; Einerhand et al., 2002).

It is becoming apparent that mucins also are capable of altering bacterial geneexpression: in bacterial vaginosis bacterial species secrete enzymes not encounteredwhen they are in different habitats (Roberton et al., 2005).

Interactions between mucosal epithelia and cells of the immune system are also, atleast partly, achieved by mucin molecules or mucin domains (Leiper et al., 2001;Mitoma et al., 2003; Wahrenbrock et al., 2003; Aknin et al., 2004). Thus, thepermeability of epithelia and both secretion and synthesis of mucins are complexresponses to environmental and physiological factors.

The similarity in general architectures of mucin genes leads to the hypothesis thatthey arose by duplication of an common ancestral gene (Desseyn et al., 2000), withperhaps further local doubling and mobilization to a protective and interactivefunction. This is however accompanied by a large degree of variability, both geneticand epigenetic. The former manifests itself in the variability in the number of repeatsthat encode the heavily glycosylated domains – the relevance of this allelic variationis not yet fully understood: small population studies suggest that there might be arelation between mucin gene alleles and susceptibility to disease. Epigeneticpolydispersity arises as a result of the competition between the different enzymesthat add sugars to the lengthening oligosaccharide chains combined with the typeand availability of donor sugars and response to the present bacteria (Lamblin et al.,2001; Schulz et al., 2005). When a mature secretion is analyzed some of thepolydispersity may also follow from cleavages of peptide and sugars that occurfollowing the action of other components of the fluid.

Mucins are complexly expressed at the cell and tissue level: the combinatorialnature of sugar sequences and of substitutions at the periphery (sialylation,sulphation, etc.), are likely not to merely represent the rich possibilities of glyco-chemistry, but a signalling language that would be fascinating to understand(Lamblin et al., 2001).

4.1.1. Classification and general properties

Encoded by a family of genes preserved through evolution, mucins come in anumber of flavours, depending on the classification criteria used and the interest or

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formation of the researcher. Questions related to the function of the mucin in oroutside the cell, range of expression, expression in tumor cells, association to cellulardifferentiation or tumor progression, all these criteria and more could result indifferent classifications (Porchet and Aubert, 2004). The complex functionality ofmucins at the ocular surface (which is taken to include the tears) is not yet fullyunderstood. Hence, we will restrict ourselves to distinguish secreted andtransmembrane or membrane-spanning mucins. The former are secreted from cellsas mostly (very) long polymers and are, even at relatively low concentrations, themain constituents of mucosal gels. A few small secreted mucins are an exception tothis rule: one is MUC7, the small mucin of saliva and also secreted by the lacrimalglands, that is considered non-gel forming. This mucin is also an exception in that itpolymerizes not as an N–N and C–C linear polymer but around a central structure,somewhat like spokes of a wheel (Mehrotra et al., 1998). Membrane-anchoredmucins have hydrophobic core peptide domains that integrate the mucin in the cellmembrane; some have glycosylphosphatidylinositol anchors and many have at leasta potential link with the cytoskeleton. These mucins are shed rather than secreted.The distinction is however somewhat blurred by the fact that some splice alternativescan be either secreted or membrane-spanning (Moniaux et al., 2000), as is the casefor MUC4 in the eye. This classification, as related to ocular mucins, is presentedmore fully in Table 1. Proposed classifications criteria rely on sequence similaritiesand expression areas (Porchet and Aubert, 2004).

4.1.1.1. Nomenclature and classification. Mucin gene nomenclature is under theaegis of the Human Gene Nomenclature Committee file: localhost (http://www.gene.ucl.ac.uk.nomenclature). A gene is defined as ‘‘a DNA segment thatcontributes to phenotype/function. In the absence of demonstrated function a genemay be characterized by sequence, transcription or homology’’. The numberingreflects the order of gene description. Their products, the mucin molecules areregularly referred to by the same names, MUC1, MUC2, etc., for humans, Muc1,Muc2, etc. for their animal orthologues. Simultaneous discovery brought, forexample, MUC5A, MUC5B and MUC5C. However, two of the proposed genesturned out to be one, hence MUC5AC. Occasionally what was believed to be onegene was later shown as two distinct sequences or functions, as in the case of MUC3,whose approved symbols now are MUC3A and MUC3B, both located at 7q22. Thegenetic determinant of the mucin, the peptide core, does also dictate the maximalnumber of oligosaccharide chains, and influences the efficiency of glycosyl-transferases, the enzymes that add sugars to the chains, like beads on a string.

Common regulatory mechanisms might account for the capability of mucous-secreting cells to express several mucin genes simultaneously (Van Seuningen et al.,2001). 11p15 mucin genes are regulated at the transcriptional level by pro-inflammatory cytokines (interleukins IL-1b, IL-6, tumor necrosis factor-a),pleiotropic cytokines (IL-4, IL-13, IL-9), bacterial lipopolysaccharide, growthfactors (epidermal growth factor – EGF, tumor growth factor – TGF-a), lipidmediators (platelet aggregating factor), retinoids and hormones (Van Seuningenet al., 2001), as well as extracellular Ca2+ (Verdugo et al., 1987a), injury induced

http://www.gene.ucl.ac.uk.nomenclature

http://www.gene.ucl.ac.uk.nomenclature

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Table 1. Epithelial secreted and membrane-spanning mucins at the ocular surface

Gene

symbol

Gene name Location Repeat

size

mRNA

(kb)

At the ocular

surface

MUC1 Mucin 1,

transmembrane

1q21–24 20 4-6, 8 K, J, LG, T

MUC2 Mucin 2, sercreted

intestinal/tracheal

11p15 23 14–16 J, T

MUC4 Mucin 4,

transmembrane,

tracheobronchial

3q29 16 16 K, J, LG, T

MUC5AC Mucin 5, subtypes A

and C, secreted,

tracheobronchial/

gastric

11p15.5 8 17–18 J, LG, T

MUC5B Mucin 5, subtype B,

secreted (MG1)

11p15.5 29 17.5 J, LG

MUC7 Mucin 7, salivary 4q13–q21 23 2.7 J, LG

MUC16 Mucin 16 (CA125,

FLJ14303)

19p13.2 165 ? K, J, LG*, T

Notes: Most authors do not class MUC7 together with the typical secreted mucins. MUC16 is not yet

officially classified. Ocular surface epithelia express it as membrane-spanning (Argueso et al. 2003).

*Paulsen et al., unpublished results.

Abbreviations: K, Cornea; J, Conjunctiva; LG, Lacrimal gland; T, Tears.


pathways (Basbaum et al., 1999), and cells of the immune system. These pathwayshave not yet been extensively studied for tears and the ocular surface, though theinterplay between inflammatory and healing mechanisms is understood to beessential for vision.

4.1.1.2. Membrane-bound mucins. Also known as membrane-spanning mucins orcell surface mucins, these are dimeric or multimeric molecules with extracellulartransmembrane and cytoplasmic domains. These domains are encoded in the geneand undergo cleavage early in synthesis (perhaps simultaneous with translation), andfurther cleavage when shed from the membrane. The second cleavage occurs in theSEA domain (sea urchin sperm protein, enterokinase and agrin). This domain is alsoinvolved in the, yet incompletely understood, bond between the subunits. At theocular surface there at least 3 mucins are expressed: MUC1, MUC4 and MUC16.The structure of MUC1 and MUC4 is presented in Fig. 9. Note that MUC4 is not atypical membrane-spanner, since it does not possess an SEA domain. It however hasan EGF-like domain involved in signalling (see below). These mucins can beidentified in tissue, in impression cytology, in tears and among highly purified mucinsfrom these sources (Berry et al., 2000a, 2002b, 2004b; Argueso et al., 2003).

Cleavage – involvement of neutrophil enzymes: An important role has beenattributed to membrane spanning mucins: they are thought to anchor the tear film

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Fig. 9. Gene structure of membrane-spanning mucins MUC1 and MUC4. Note the different

sizes of the two genes and the absence of EGF-like domains in MUC1. The two domains are

cleaved during synthesis and re-assembled in the membrane.


onto the surface of the eye by intertwining with the secreted mucins in the preocularfluid. MUC4 length varies between 1.2 and 2 mm, MUC1 and MUC16 some 50 nm,while the thickness of the glycocalyx is estimated at 10 nm. Membrane spanners arethus extending beyond the glycocalyx, creating a continuous barrier apical to theepithelia, and into the mucosal gels. We calculated the persistence lengths of m-longmucins (Round et al., 2002) at around 30nm, suggesting very flexible polymers,capable of reptating through gel pores. Furthermore, smaller mucins penetrated amodel gel more – a larger proportion of the molecular length diffused into anagarose gel – than in the case of long polymers (Berry and McMaster, unpublishedresults).

There is evidence that not all membrane-bound mucins are controlled by the samemechanism: a selective augmentation of MUC4 and MUC16, but not MUC1 wasobserved after retinoic acid or serum was added to a conjunctival cell line (Horiet al., 2004), while the eicosanoid 15-(S)-hydroxy-5,8,11,13-eicosatetraenoic acid(15(S)-HETE) is a selective secretogogue for MUC1 (Jumblatt et al., 2002).

4.1.1.3. Secreted mucins. Genes encoding secreted mucins have, as discussedabove, the common features of the mucin class. These genes are very large, and yieldvery large mRNAs (Table 1, Fig. 10).

Mucin synthesis is not a fast process: pulse-chase studies indicated that it takes2–4 h (Sheehan et al., 2004) to detect the first labelled mucins in the cells. Mucinpolypeptide is synthesized and undergoes dimerization, then becomes substitutedwith GalNAc residues, and then glycosylation proceeds to produce a mucin dimer.The last step in the process is multimerization. These oligomeric mucins follow asimilar assembly to the von Willebrand factor glycoprotein to yield long lineardisulfide-linked chains (Sheehan et al., 2004).

Secreted mucins are stored in secretion granules from which water is excluded,often in specialized cells – goblet cells of the conjunctiva, or acinar cells of thelacrimal gland (Paulsen et al., 2004a) and goblet cells of the nasolacrimal ducts(Paulsen et al., 2003a), or in sub-apical granules as described for corneal epithelium(Gipson et al., 1995).

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Fig. 10. General structure of genes encoding secreted mucins. The D domains are similar to

domains in the von Willbrand factor (vWf), and might be involved in mucin assembly. Trefoil

family factor peptides interact with vWf domains in mucins. Note the Cys-rich domains that

might participate in intra- and inter-molecular bonds. In ocular mucins the non- or poorly

glycosylated domains of the peptide core can be tens of nanometers long, while tandem repeat

domains that are most glycosylated bear short sugar chains.


Ca2+, which is also important in mucin packaging (Paz et al., 2003), is massivelyreleased before mucin secretion and hydration (Verdugo et al., 1987b). Ca2+ mightbe then exchanged for extracellular Na+, causing a change in the polymer matrixphase, i.e. hydration. The process is then driven by Donnan equilibrium, and, as aresult the formation of small mucin gels that anneal to other existing gels (Verdugo,1991). Donnan equilibrium can be expressed as an imbalance between the salt in thegel and that in the bathing solution or an osmotic potential excess within the gel(Hodson, 1997). If however the gels are also linked with disulfide bonds, thisannealing would be more difficult. We have shown that disulfide bond reducingagents have a disastrous effect on ocular mucin gels (Berry et al., 2004a): these resultsare not necessarily contradictory, since the existence of mucin polymers depends ondisulfide bonds.

Use of disulphide bond breaking compounds, such as N-acetyl cysteine in dry eyeclinics or dithiothreitol in mucin research, dissolves mucin aggregates and renders‘‘insoluble’’ mucins amenable to biochemical analysis (Carlstedt et al., 1995).Insoluble mucins are physiological in several mucosal systems (Davies et al., 1996;Herrmann et al., 1999), including the eye (Berry et al., 2004b). The physiologicalrelevance remains however unclear.

Mucins also undergo proteolysis, at or near secretion, which shortens them tomerely long polymers. This phenomenon is not totally understood: it might be amore general storage device – pituitary hormones are also stored in a long chain thenpartitioned upon release. The cleavage site is encoded in the mucin gene (Herrmannet al., 1999; Wickstrom and Carlstedt, 2001). It is likely that further cleavages occurduring the physiological life of the molecule in gel or solution, some as a result ofbacterial enzymes (Berry et al., 2002a). More than a single mucolytic activity isnecessary to degrade the mucin gel and penetrate into the epithelium; in someinfections this is achieved by consortia of microbes rather than a single species(Roberton et al., 2005).

The glycan repertoire of ocular mucins is yet little known. The oligosaccharidesare short – often referred to as truncated as found in tumours of other mucosae, andrich in sialic acids (Berry et al., 1996; Argueso et al., 1998; Ellingham et al., 1999). Anumber of functions have been ascribed to these highly charged epitopes: they might

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determine the spacing of neighboring polymer or vicinal oligosaccharide chains andthus the gel pore size, they might interact with cations in the solution and stabilizethe gel (cations would bridge between one negatively charged moiety and another),or they may serve as ligands to immune cells or bacteria. In human ocular mucins wehave found that the majority of sialic acids are di- or mono-acetylated – and that thefood derived N-glycolyl form can also be found (Corfield et al., unpublished results).The addition of an acetyl group prevents bacterial cleavage. In dogs the N-acetylneuraminic acids are common at the normal ocular surface, and though sialic acidgroups increase in kertoconjunctivitis sicca, there is a specific loss of the mono-O-acetylated form (Corfield et al., 2005).

Sampling the conjunctiva is widely performed by impression cytology (for a reviewsee Calonge et al., 2004). If cellulose acetate is used and little if any pressure themucin layer is lifted with few if any surface cells. Use of other membranes allows anumber of surface layers to be obtained, with very little discomfort and no adverseeffects to the patient. Using the first method, we took sequential impressions at thesame site, fixed using a membrane mask. Only very few groups of cells couldbe identified as repeatedly sampled (Ellingham RB, Ph.D. Thesis, University ofBristol, 1999). We have however observed a similarity in the distribution ofgoblet cell impression sizes in a number of individuals, with some regional variation(Fig. 11a). The distribution of impression sizes in sequential impressions was varied(Fig. 11b), although the first, most superficial impression, yielded the smallestimpression sizes.

4.1.1.3.1. Gel-forming mucins. Many functions of ocular surface mucins and ofother components of tears are carried out in a gel phase. As secreted mucins are notextended in solution they can form gels just by entanglement. However, the diffusion

Fig. 11. Impression cytology – sampling ocular surface mucins. Examples are shown of three

healthy individuals, woman, 40 (F40, light gray with blue surrounding), and men 36 (M36,

black) and 20 (M20, dark gray) whose conjunctivae were sequentially impressed at the same

place. The general distribution of impression sizes is quite similar (a), though the areas

(quantities) obtained vary with depth (b).

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of molecules in mucin gels indicates that other linkages exist, both covalent and non-covalent. Bridging between negative charges on mucin molecules can be achieved bydivalent cations – addition of Ca2+ tightens the gel (Raynal et al., 2003); its chelationcauses gel expansion accompanied by release of material into solution (Berry et al.,unpublished results).

At the ocular surface the main gel former is MUC5AC, that is secreted by thegoblet cells of the conjunctiva, and also by epithelial cells of the lacrimal glands andnasolacrimal ducts. MUC2 is also found, secreted by epithelial cells of the ocularsurface, as well as by the lacrimal apparatus (Berry et al., 2000a; McKenzie et al.,2000; Corrales et al., 2003a; Paulsen et al., 2004a; Schafer et al., 2005). MUC4 issecreted in the lacrimal system (Arango et al., 2001; Paulsen et al., 2004a,b), butmembrane bound in corneal (Tei et al., 1999; Carraway et al., 2000; Hu et al., 2000;Lomako et al., 2005) and conjunctival (Berry et al., 2000a) apical epithelia, and insome petrygia. MUC4 is part of the preocular gel (Berry et al., 2004b) and dissolvedin tears (Pflugfelder et al., 2000). This mucin can also be recovered from deposits oncontact lenses (Berry et al., 2003).

Secreted mucins protect the ocular surface from infection and physical trauma. Itis likely, though the evidence is scant, that secreted mucins form small gels oraggregates around foreign bodies or dead cells and eliminate them from the ocularsurface. In this way there is little friction between the mucin-wrapped entity and thelids or the ocular surface (Berry et al., 2001).

Next to nothing is known about the factors which determine the duration ofmucin residence in tears. Degradation by tear enzymes and commensal biota is likelyto play a major role, but mechanisms and time scales are yet to be understood.Neutrophil enzymes are able to cleave mucins from apical cell membranes (Kimet al., 1987; Leiper et al., 2001). And neutrophils are dominating the tear film underclose eyelids (Sakata et al., 1997; Sack et al., 2000, 2003).

4.1.1.3.2. Small soluble mucins. These are mucins that are secreted, butmonomeric, and do not form linear multimers. MUC 7 is the representative ofthis class of mucins at the ocular surface (Corrales et al., 2003a, b; Jumblatt et al.,2003; Berry et al., 2004b). In saliva this mucin is a low-specificity but avid bacterialbinder, and thus fulfils an antibacterial role. It is expected that MUC7 has the samefunction in the tears.

4.2. TFF peptides

TFF peptides (TFFs) are a family of short peptides, rich in disulfide bonds thatform intramolecular loops (Thim and May, 2005). TFFs are intimately associatedwith mucins: in amphibians TFFs are encoded by mucin genes (Hoffmann and Joba,1995). In humans the separate genes encoding members of the trefoil peptide familyare clustered on chromosome 21q22.3 (Chinery et al., 1996).

4.2.1. Classification and general properties

4.2.1.1. Nomenclature and classification. Three human TFF peptides have beendescribed: TFF1, 2 and 3. The peptides contain trefoil domains, conserved sequences

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of 42 or 43 aminoacids in which 6 conserved residues are found in specific disulfidebridges: 1–5, 2–4, 3–6. These residues form a stable core with protruding, clover-like,loops. The structure, functions and interactions of TFF peptides, have recently beenthe subject of authoritative reviews (Baus-Loncar and Giraud, 2005; Baus-Loncaret al., 2005; Hoffmann, 2005; Otto and Thim, 2005; Thim and May, 2005; Tomasettoand Rio, 2005). TFFs have distinct intracellular and mucosal actions. The latter arelargely protective and perhaps stabilizing of secreted mucins or mucus gels: theyinclude restitution, wound healing, apoptosis, cell motility, and adhesion, amongstothers (Otto and Thim, 2005). Increase in gel viscosity is spectacular on additionTFF2 (tenfold), and with TFF3 dimers. Monomers do not have this property (Thimet al., 2002).

It is thought that TFFs act as ligands to a receptor that has yet to be identified.Potential binders and their interaction have been recently reviewed (Otto and Thim,2005).

Synthesis and release of TFFs is regulated by a number of environmental andlocal agents, amongst which we can cite: their own and other TFF release,estrogens, pro-inflammatory and anti-inflammatory cytokines (for a reviewsee Baus-Loncar and Giraud, 2005). This complex cytoprotective response tomucosal damage or chemical alteration in the immediate environment underlinesthat the complex functionality of this peptide family is yet to be completelyunderstood.

TFF dimers are resistant to trypsin, chymotrypsin and carboxypeptidases, whichsupports a protective function in the gastrointestinal tract, but not cocktails ofbacterial enzymes (Thim and May, 2005), which might suggest that interactions withmucins enhance this role.

TFF1, also known as pS2, is a 60 amino-acids long mature peptide and contains asingle trefoil domain. The cysteine (Cys), three residues from the acid C-terminus,permits homo- and hetero-dimerization. The TFF1 dimer is the predominant formthat co-elutes with MUC5AC on gradient centrifugation and is precipitated by ananti-MUC5AC antibody (Ruchaud-Sparagano et al., 2004).

Mature TFF2 (spasmolytic polypeptide) contains 106 aminoacids, withtwo trefoil domains and two Cys residues outside the trefoil domains.Human TFF2 is the only member of the family to have one N-linked gly-cosylation site: all gastric tissue TFF2 is glycosylated, whereas in normal gastricjuice both forms are present. The two loops of TFF2 are not only linked by

disulfide bonds but also by a peptide sequence, resulting in a very compactmolecule.

TFF3 was also called intestinal trefoil factor (ITF). The mature 59 peptideaminoacids, is secreted as a monomer or dimer. TFF3 has one TFF domain and afree Cys residue in the C terminal. TFF3 promotes migration of epithelial cells invitro and enhance mucosal healing and epithelial restitution in vivo in thegastrointestinal mucosa, where it co-localizes with MUC2.

Along the human gastrointestinal tract TFF peptides and 11p5.5 mucins aresecreted in a site specific fashion. TFFs accumulate at the luminal aspect of themucosa (Longman et al., 2000).

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5. Mucins and TFF peptides of the tear film

5.1. Mucins of the tear film

The ocular surface and tears are rich in diverse mucins adapted to maintaining atransparent and well hydrated gel. The properties of the gel are derived from thecharacteristics of the backbone polymers, their glycosylation and interactions withother chemical entities in the preocular fluid.

5.1.1. Membrane bound mucins

5.1.1.1. MUC1. It is a ubiquitous epithelial membrane spanning mucin. MUC1has a trans-membrane domain, a GPI anchor and a cytoplasmic tail, and is the onlymucin in its class without an EGF domain.

The protein-enterokinase-agrin (SEA) module, associated with proteins receivingextensive O-glycosylation (Engelmann et al., 2005), is part of the extracellulardomain and contains a putative proteolytic cleavage site. When cleavage occurs, asignal may be given to the cell. The remaining trans-membrane unit might then act asa ligand to the soluble mucin subunit, which, on re-association might also signalthrough membranal changes (Wreschner et al., 2002).

Clusters of sequence-variant repeats are interspersed in the repeat domain ofMUC1 at high frequency, and contribute to the structural and immunologicalfeatures of the mucin. The normal IgG responses to MUC1 are directed to thesesequence variants (von Mensdorff-Pouilly et al., 2005). Such repeats have justrecently been described in human ocular surface tissues (Imbert et al., 2006).

This mucin is sensitive to hormonal control: activation of the androgen receptoraugmentsMUC1 expression in prostatic cell lines (Mitchell et al., 2002). This phenomenonmight also occur in the lacrimal gland and the external ocular epithelia, contributing to thefragility of the tear film in age-related dry eyes. However, the charge profile of the mucinseems to be conserved, since normal profiles of MUC1 (Fig. 12a) are similar in preocularfluid from patients with meibomian gland disease (MGD), aqueous tear deficiency (ATD)and Sjogren’s syndrome (SJS) (Khan-Lim and Berry, unpublished results).

Further control of MUC1 is exerted by inflammatory mediators, released in response tosignals from dendritic cells present in the inflamed conjunctiva (Baudouin, 2001), possiblythrough IL6 (Tsubota et al., 1999).

Vitamin A deficiency in the diet did not affect the expression of rMuc1 in corneal orconjunctival epithelia (Tei et al., 2000).

5.1.1.2. MUC4. The two dissimilar subunits of this high molecular mass mucin areencoded by the MUC4 gene. They are proteolytically cleaved before substantialglycosylation and reassembled in the membrane, probably through a non-covalentbond. The dynamics of both membrane-spanning and alternatively spliced, soluble,Muc4 are similar (Komatsu et al., 2002). This mucin has two EGF-like domains thatare essential for secretion and interact with the receptor tyrosine kinase ErbB2,which, in turn, can trans-activate the gene promoter (Perez et al., 2003). In cells

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Fig. 12. MUC1 and MUC4 in preocular fluid. Mucins were isolated on CsCl gradients:

layering at different densities reflects molecular charge and therefore how densely glycosylated

the molecules are. Classical mature mucins peak in the range 1.3–1.5 g/ml: (a) membrane

spanning mucins: MUC1 (solid symbols) is mainly contained in this classical elution range,

while MUC4 (open symbols) is mainly less rich in oligosaccharide chains; (b) secreted mucins:

MUC5AC (solid symbols) and MUC2 (open symbols) are mainly encountered as with mature

glycosylation.


transiently transfected with SMC/Muc4, ErbB2 is translocated to the apicalmembrane – where the mucin is expressed in polarised epithelia – suggesting amucin role in ErbB signalling (Ramsauer et al., 2003).

Muc4/SMC, the animal orthologue of MUC4 is highly correlated with celldisadhesion and migration: at the ocular surface loose and desquamated cells arerichly decorated with this mucin (Lomako et al., 2005).

Mature fully glycosylated MUC4 and less charged species, are present in surfacemucus (Fig. 12b). Molecules less charged than classical mucins might reflect somedegradation in the preocular fluid.

5.1.1.3. MUC16. This latest addition to the list of membrane-spanning ocularsurface mucins (Argueso et al., 2003) is located at the tips of epithelial processes intothe preocular fluid (Argueso et al., 2003), and augmented by exogenous retinol (Horiet al., 2004). Its function is not definitively determined; some authors connect thismucin to differences in P. aeruginosa adhesion (McNamara et al., 2005) to open- orclosed-eye tears. In dry eyes there is an increase in ocular surface reactivity with anantibody recognizing an oligosaccharide (Danjo et al., 1998) now known to bedecorating MUC16. The cause of this increase reactivity is yet to be elucidated.

5.1.2. Secreted mucins

5.1.2.1. MUC5AC. Secretion of MUC5AC is concurrent with specialization ofgoblet cells. Its message is richly detected in the conjunctiva (McKenzie et al., 2000),while the mature mucin is found in the preocular gel , dissolved in tears and coatingcontact lenses. Long-term wear of contact lenses did not appear to diminish thenumber of MUC5AC-positive cells, though the reactivity with the anti-MUC5ACantibody was reduced (Pisella et al., 2001). It is not known whether this reduction isdue to differences in MUC5AC glycosylation, which might affect anti-M1 reactivity

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with the mucin, or whether mucin production is diminished by asymptomatic lenswear. The antibody anti-M1 is directed to the peptide core of the MUC5AC mucin.In a group of patients with symptoms of dry eyes we have observed differences inreactivity with two antibodies that recognize epitopes of the core peptides outside therichly glycosylated tandem repeat domains of MUC5AC (Berry et al., 2004b). Wehave interpreted these differences, not observed in normal eyes, as pathology-associated changes in mucin processing.

In dry eyes, the elution patterns of MUC5AC collected from the ocular surface areshifted towards lower charges compared with normal mucins (Khan-Lim and Berry,unpublished results). A notable exception here is MGD where MUC5AC wascomparable in size with that found in normal preocular fluid. The lower charge andsmaller hydrodynamic volumes may lead to gels with properties different fromnormal. This important aspect of ocular surface physiology has not beensystematically studied so far.

5.1.2.2. MUC2. This secreted mucin is found in a minority of goblet cells(Ellingham and Berry, unpublished results), and can be recovered from all surfaceepithelia, the preocular fluid and lens deposits. In MGD, we were unable to detectMUC2, though it was found in soluble and insoluble forms in other dry eyessubcategories (Khan-Lim and Berry, unpublished results). In mild dry eyes MUC2reactivity peaks in the classic range of mature mucins (1.35–1.45 g/ml); the morerichly glycosylated MUC2 population is polydisperse in hydrodynamic volume, as innormals. In floppy eyelid syndrome (FES) there is an increase in MUC2, andespecially in the insoluble form of the mucin (the mucin requiring disulfide bondbreaking to be solubilised – Ellingham and Berry, unpublished results, Fig. 13).

5.1.2.3. MUC5B and MUC7. MUC5B is a mucin with a high potential for selfaggregation. This tendency may explain the antibacterial function and the gelqualities of saliva. It is a highly polymorphic mucin, with a number of cysteinedomains in its peptide core, and with a large and distinct repertoire ofoligosaccharides in saliva. We have detected MUC5B in normal conjunctivalepithelial cells, in organ-cultured corneas and in stratified immortalized humancorneal epithelial cell lines (Skintethic) (Fig. 14).

MUC7 is secreted by conjunctival epithelial cells (Jumblatt et al., 2003). Its role atthe ocular surface is likely to be similar to that in saliva: this mucin is a generalistbacterial ligand.

6. Mucins and TFF peptides of the lacrimal gland

6.1. Lacrimal gland mucins

Supported by older studies (Jensen et al., 1969; Ito and Shibasaki, 1964; Kuhnel,1968; Allen et al., 1972; Millar et al., 1996) and the finding that rat lacrimal glands

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Fig. 13. MUC5AC and MUC2 in floppy eyelid syndrome. This syndrome usually affects

obese middle-age men, who may present thickened conjunctival epithelium with vascular

papillae and pseudoglands and chronic inflammatory cell infiltrate in the substantia propria.

Mucins extracted in Guanidinium chloride are termed soluble. A subsequent extraction, in the

presence of the reducing agent dithiothreitol yields the insoluble mucins. Characteristics of

MUC5AC (solid symbols) and MUC2 (open symbols) are compared in normal and floppy eye

syndrome (FES) tissue. The FES soluble MUC5AC (solid squares) are more glycosylated than

their insoluble counterparts (solid diamonds). Insoluble MUC2 (open diamonds) are

substantially elevated in FES. Normal MUC5AC (filled circles) and MUC2 (open circles)

are presented to substantiate the disease-induced changes in mucin distribution.

Fig. 14. MUC5B in cultured epithelium. Stratified immortalized corneal epithelium,

(SkintethicTM, Nice, France) synthesize and secrete mucins, among which MUC5B: (a)

Distribution of MUC5B in cells (solid symbols) and secreted in the medium (open symbols).

Note that the majority of mature mucin is secreted. (b) Secreted MUC5B are polymorphic in

polymer size: from very large (Vo) to less large but eluting in order of size from the column

(Vi), to the smallest that are not resolved by the chromatography used (Vt). These variation in

polymer length is characteristic to all ocular surface mucins.


synthesize rMuc4 (Arango et al., 2001), recent studies have demonstrated mucinexpression and protein synthesis of a spectrum of mucins (Jumblatt et al., 2003;Paulsen et al., 2004a; Table 2).

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Table 2. Mucins in the lacrimal glanda

Mucinb Expression in

lacrimal gland

Localization in lacrimal gland Changes in

dry eye

MUC1 Positive Membrane bound at the luminal

lining of acinar epithelial cells

No

MUC2 Negative Not present No

MUC4 Sporadic If present, membrane bound

and cytoplasmic in acinar cells

Yes

MUC5AC Positive Goblet cells of larger excretory

ducts

Yes

MUC5B Positive Cytoplasmic in many acinar

cells

Yes

MUC6 Sporadic Not present No

MUC7 Positive Cytoplasmic in many acinar

cells and columnar epithelial

cells of excretory ducts

Yes

MUC8 NDc Not present NDc

MUC16 Positive Supranuclear in the Golgi in

single groups of acinar cells

NDc

aCompare Fig. 15.bNo data exist about MUCs 3A, 3B, 9, 11, 12, 13, 15, 17, 19, and 20.cND—not determined.


6.1.1. Membrane-anchored mucins

6.1.1.1. MUC1. MUC1 mRNA and protein have been demonstrated in specimensof human lacrimal gland tissue. MUC1 shows intense membrane-spanning reactivityat the luminal lining of acinar epithelial cells (Paulsen et al., 2004a). It has beensuggested that its specific localization on the apical surface of epithelial cells has asignalling function and may be important as a sensor mechanism in response toinvasion or damage of epithelia (Ren et al., 2002).

6.1.1.2. MUC4. There are marked differences in the expression and production ofmembrane-spanning mucins, MUC1 and MUC4, in the lacrimal gland. Both havebeen shown to provide steric protection of epithelial surfaces. Muc4 has beendetected as a main mucin of the rat lacrimal gland (Arango et al., 2001). Humanlacrimal gland message for MUC4 is present in some lacrimal glands only (Paulsenet al., 2004a). MUC4 protein has been detected, but only in individuals aged 87 yearsor older, and not in younger donors, irrespective of the presence of MUC4 mRNA(Paulsen et al., 2004a). When present, MUC4 is not only membrane-associated butalso cytoplasmic in acinar cells, with single acinar cells revealing intensivecytoplasmic staining.

The low detection rate of MUC4 might be due to the fact that localization ofMUC4, and indeed of all mucins in tissue sections, has been fraught with problemsof antibody specificity. As a result of the rich O-glycosylation of mucins, access of

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antibodies to the protein core is restricted. This is especially relevant to MUC4,whose extracellular subunit is the mucin subunit. Most specific MUC4 antibodies aredirected to the ASGP2 or b-subunit, the membrane-associated/cytoplasmic domain,since this lacks the heavily O-glycosylated regions found in the mucin ectodomain(Pflugfelder et al., 2000). Our own experience supports this, revealing that MUC4 ismore readily detected in paraffin-embedded tissue sections if an antibody directed tothe membrane-spanning domain is used.

Studies of rMuc4 (Carraway et al., 2002) have revealed that the EGF-like domainsplay a role in the regulation of epithelial growth in the lacrimal gland. rMuc4 cantrigger a specific phosphorylation of the receptor tyrosine kinase ErbB2 (Jepsonet al., 2002) and potentiates neuregulin activation of the ErbB3 receptor. Thesereceptor interactions suggest that rMuc4 might be involved in regulation of epithelialcell growth. Moreover, experimental evidence indicates that ErbB2 can activate Tlymphocytes (Fisk et al., 1997; Kuerer et al., 2002). Activation of T lymphocytes is acommon feature in certain lacrimal gland and ocular surface pathologies (i.e. in dry-eye syndromes), leading to abnormal apoptosis in terminally differentiated acinarepithelial cells of the lacrimal gland (Gao et al., 1998). Arango et al. (2001) andCarraway et al. (2002) showed that rMuc4 and ErbB2 interact in the healthy lacrimalgland, while Liu et al. (2000a) found increased expression of ErbB2 and ErbB3 in theconjunctival epithelium of patients with non-Sjogren keratoconjunctivitis sicca.These results point to the possibility that lacrimal gland MUC4 contributes to T cellactivation.

6.1.1.3. MUC16. MUC16 is a high-molecular-weight glycoprotein (Yin andLloyd, 2001), initially known as the CA125 antigen, that was initially detected inovarian carcinoma cell lines of epithelial origin and in the luminal surface of tumortissues taken from patients with ovarian cancer (Bast et al., 1981). Today, MUC16domains are providing new opportunities to develop new assays and refine currenttools to improve the sensitivity and specificity of CA125 for population-basedscreening guidelines (McLemore and Aouizerat, 2005). In addition to its function astumor marker, studies have also revealed the presence of CA125 antigen in a numberof normal tissues such as endocervical epithelium, endometrium, pleura, pericardiumand peritoneum (Kabawat et al., 1983), in seminal plasma (Halila, 1985), milksecretions (Hanisch et al., 1985), in cervical mucus (Kabawat et al., 1983; Halila,1985; Hanisch et al., 1985), and just recently in human ocular surface epithelia aswell (Argueso et al. 2003). To date, neither MUC16 expression nor protein have beendetected in the human lacrimal gland. However, our own unpublished observationsindicate production of MUC16 mRNA and protein in the lacrimal gland. In contrastto MUC1, which is membrane-anchored in the lacrimal gland, and to MUC4, whichshows both membrane-associated and cytoplasmic occurrence in acinar cells (seeabove), MUC16 is immunohistochemically visible in intracytoplasmic organelleslocalized mainly above the nucleus. Our unpublished immunohistochemical resultsindicate that only a soluble form of MUC16 is produced by acinar cells, but nomembrane-anchored MUC16. MUC16-positive acinar cells are very rare in thelacrimal gland, with numbers varying among individual specimens. Immunodot blot

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analysis of tears from healthy volunteers indicates that they are positive for MUC16(Paulsen, unpublished observations) that might derive from either the lacrimal glandor the surface epithelia.


6.1.2.1. MUC5B. Our results indicate that message for MUC5B is nearly alwayspresent in the lacrimal gland, as is its mucin product (Paulsen et al., 2004a),expanding on the observations of Jumblatt et al. (Jumblatt et al., 2003). MUC5B isproduced by many acinar cells of lacrimal gland lobules. Other areas in the lacrimalgland are completely devoid of MUC5B (Paulsen et al., 2004a). MUC5B has beenshown to participate in bacterial adhesion (Baughan et al., 2000; Thomsson et al.,2002). MUC5B secretion is also familiar from serous cells of salivary glands (Nielsenet al., 1997). The secretory activities of the lacrimal gland are not unlike those of theserous cells of salivary glands.

6.1.2.2. MUC7. Recent studies by Jumblatt et al. (2003) have demonstratedMUC7 mRNA and protein in four of six samples of human lacrimal tissue RNA.Using in situ hybridization, they demonstrated the presence of message in some ofthe acinar cells of the gland. MUC7 has also been detected in cellular extracts oflacrimal gland by immunoblot, but was not detectable in tear samples assayed withthe same technique (Jumblatt et al., 2003). Our investigations confirm message forMUC7, while immunohistochemistry reveals cytoplasmic MUC7 in some, but notall, acinar cells, as well as in the columnar epithelial cells of excretory ducts of thelacrimal gland. Failure to detect MUC7 in tears, as reported by Jumblatt et al.(2003), is likely to be due to its low concentration. Together with MUC5B, MUC7has been shown to participate in bacterial adhesion (Liu et al, 2000b; Situ andBobek, 2000) and to possess antifungal activity (Wei and Bobek, 2004, 2005).Moreover, MUC7 has a lubricative function in saliva (Gururaja et al., 1998, 1999)and may play a similar role at the ocular surface following secretion from thelacrimal gland.

6.1.2.3. MUC5AC and others. MUC5AC is demonstrably present in the excretoryduct system of the human lacrimal gland (Paulsen et al., 2004a), where it isassociated with single goblet cells of larger excretory ducts. It appears that theMUC5AC positive goblet cells are those which have migrated from the fornicalconjunctiva and therefore are not components of the lacrimal gland. However,acinar cells of the lacrimal gland also seem to be able to produce MUC5AC. Weobserved MUC5AC production in lacrimal glands of elderly women who receivedtreatment for dry eyes (Paulsen et al., 2004a).

Message and protein for mucins MUC2 and MUC8 are absent in the humanlacrimal gland (Paulsen et al., 2004a). MUC6 mucin, on the other hand, was notdetected even though message for this mucin was present in half of the samplesanalyzed (Paulsen et al., 2004a).

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6.2. Accessory lacrimal gland mucins

Accessory lacrimal glands were first described by Carl Friedrich Krause fromHannover in the conjunctival fornix and at the edge of the upper tarsus (Krause,1842). These glands were again described by Wilhelm Johann Krause fromGottingen (different Krause, 1876) as well as by Emil von Wolfring from Warsaw(von Wolfring, 1872), who seems to have been unaware of the original publicationby C.F. Krause. Ever since, the glands in the fornix have been referred to as theglands of Krause and the glands of Wolfring. Although the accessory lacrimalglands contribute to the aqueous phase of tear secretion and are believed to playan important role in pathogenesis of dry eye, there are only a small number ofpublications on the structure of the glandular elements, their blood vessels andtheir neural supply (Seifert et al., 1993, 1997; Seifert and Spitznas, 1994a).Histologically, this appears to be a mixed population of cells, very similar to themain lacrimal gland, with both serous and mucus secretory vesicles and anappearance much like that of other submucosal glands (Seifert et al., 1994b).Presumably, these glands also produce mucins, but they have not been tested formucin production to date.

6.3. Relationship between lacrimal gland mucins and tear film

Current models of tear film suggest structure on all scales: a mucus gel hydratedand subtended by the aqueous tear fluid, and a lipid-rich layer at the air interface.The lids move over the eyes with a maximum acceleration of 5879.47978.3mm/s2,and reach a maximal speed evaluated at 206.5717.4mm/s using a 60Hz film camera(Somia et al., 2000). After a blink, as the lids accelerate upwards, the tear film movesrapidly upwards before stabilizing, within 1.0570.30 s in normal humans (Owensand Phillips, 2001). Blinking, part of ocular surface physiology, is considered thekinematic support to trap and remove debris and cellular components from thesurface of the epithelium. The preocular gel is likely to display non-Newtonianshear-thinning, as expected from mucin and gel structure (Carlstedt et al., 1985;Corfield et al., 1997), and cushion eyelid movements, in an adhesion-less gliding asobserved between ocular mucin macromolecular assemblies (Berry et al., 2001). Allmucin species extracted from conjunctival tissue were also recovered from thepreocular surface after N-acetyl cysteine washings, from mucins dissolved in thetears – extracted from the distal part of the Schirmer strips that were not in contactwith the lids (Khan-Lim et al., unpublished results) and from deposits on contactlenses, indicating that secreted and cleaved mucins are part of the preocular fluid(Berry et al., 2002b, 2003, 2004a, b). It is not clearly understood yet how mucinsmove across the ocular surface and within the gel: long polymers diffuse more readilythrough a gel than within a viscous solution (Celli et al., 2005). Mucin concentrationsin the preocular gel in liquid tears are yet to be assessed, and therefore there is no apriori answer to mucin mobility on the ocular surface (Mucin quantification isfraught with difficulties that are outwith the scope of this review).

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6.4. Function of lacrimal gland mucins in antimicrobial defence

Mucin production is an evolutionary defence strategy. Beside lubrication of themucosa and waterproofing to regulate epithelial cell hydration, mucus protectsmucosal surfaces against potentially harmful substances such as particles, aggressivechemical agents, enzymes, food lectins, toxins and bacteria or other infectious agents(Faillard and Schauer, 1972; Hutch, 1970; Walker, 1976; Forstner, 1978; Schauer,1992; Reuter et al., 1992). Intestinal mucus has been observed to carry away bacteria(Florey, 1933). Moreover, it has been shown that mucus possesses structuresmimicking the receptor sites for microorganisms on epithelial cells that facilitatetrapping and subsequent disposal of bacteria (Abraham and Beachey, 1985) andviruses (Reuter et al., 1988). A variety of oral and intestinal bacteria produceneuraminidase (sialidase), an enzyme that can degrade mucins by removal of sialicacid (Corfield, 1992). In addition, oral, intestinal and female reproductive tractbacteria synthesize an array of other glycosidases, proteases, and sulfatases (so calledmucinases), enzymes that can degrade mucins (Schauer, 1997; Wiggins et al., 2001;Roberton et al. 2005).

At the ocular surface mucus binds potential harmful tear contaminants and acts asa debris-removal system. The expectation is that mucin secretion, together with thesecretion of other antibacterial proteins, diminishes bacterial, viral and fungalcolonization. Antibacterial substances are secreted by the ocular surface epithelia,and to a great extent by the lacrimal gland. The antibacterials, presumably togetherwith tear film mucins, maintain bacteria at low levels. Some 40% of ocular surfacesare culture-negative (Willcox and Stapleton, 1996; Berry et al., 2002a), andculturable bacteria are not correlated to ocular surface symptoms. Mucin decreasesbacterial adherence to the cornea (Fleiszig et al., 1994). Steady renewal of tear filmmucins is a necessity: commensal bacteria possess mucinolytic activity, targeted todiscrete sites in the mucin molecule. Inhibition of bacterial growth by ocular mucinscan be seen as an element in the system of mucosal control of microbiota (Berryet al., 2002a).

The innate immune system as manifested at the local ocular environment, with itsantimicrobial substances, including mucins, exchanges signals with the adaptiveimmune system (Willcox et al., 1997; Thakur and Willcox, 1998; McNamara, 2003).Although leukocytes are rarely seen in normal open-eye tear fluid (Norn,1989; Willcox et al., 1997; Thakur and Willcox, 1998; McNamara, 2003), theyare numerous in the tear film under closed lids (Sack et al., 1992, 2000; Sakataet al., 1997). Interactions between mucins and neutrophils have been demon-strated in a number of mucosal tissues. For example, MUC7 from salivary glandshas been described as associated with neutrophils from the oral cavity (Prakobpholet al., 1999). Neutrophil products such as elastase and cathepsin G can, in turn,release mucin secretion (Kim et al., 1987, 2003; Dwyer and Farley, 2000; Shimet al., 2001; Fischer and Voynow, 2002; Kohri et al., 2002; Pettersen and Adler,2002; Fischer et al., 2003). Neutrophil defensins in particular show an ability toenhance MUC5AC and MUC5B gene expression in cultured epithelial cell lines(Aarbiou et al., 2004). On the other hand, we have shown that normal mucin

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glycosylation is important for neutrophil activation at mucosal surfaces (Akninet al., 2004).

Interactions between mucins and neutrophils in the lacrimal gland and in tear filmstill require elucidation. Any interactions between lacrimal gland mucins and otherdefence cells such as macrophages, mast cells or lymphocytes also remain to beclarified.

6.5. Lacrimal gland TFF peptides

Whether TFF peptides are produced by the lacrimal gland is unclear at present.Message for TFF1 was detected in only approximately 30% of samples (Paulsenet al., unpublished results), whereas TFF3 message was present in all (Paulsen et al.,2004b). TFF2 was not detected. Immunohistological localization reveals weakcytoplasmic staining of acinar cells and strong staining of some single cells betweenlacrimal acini that are not lymphocytes or CD68 positive macrophages (Paulsen,unpublished observations). The detected peptide has barely half the size of normalTFF3 (Paulsen et al., 2004b), which might reflect protein degradation due to use ofpost-mortem material. Whether TFF3 is secreted by acinar cells and whether it has afunction in acinar cell physiology must still be determined.

7. Age-related changes in the composition of lacrimal gland and tear

film mucins

7.1. Age-related dry eye

The term ‘‘dry eye’’ or dry eye syndrome refers to a common but highlyheterogeneous group of ocular surface disorders (Dana and Hamrah, 2002;Schaumberg et al., 2002). Dry eye accounts for the largest group of patients seekingophthalmic treatment. Most of the cases result from changes associated with aging ofthe glands and ocular surface epithelia, although the condition can occur at any age.An estimated 20% of people over the age of 45 will experience dry eyes.

In view of the high prevalence of dry eyes there is an urgent need forinternationally agreed criteria for differential diagnosis of the various conditionsthat are covered by this term. The Dry Eye Workshop, that involves leadingacademics and ophthalmologists, is reviewing the accumulated data on symptoms,signs, aetiology, pathology and treatment of these conditions. The harmonizedclassification and recommendations resulting from these deliberations will advancepatient treatment and focus research in ocular surface physiology.

7.2. Age-related changes in lacrimal gland mucins

Recently, we analyzed aging of the lacrimal gland with regard to mucinproduction (Paulsen et al., 2004a; Table 2). Membrane-spanning mucins, MUC1

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and MUC4, showed marked differences in expression and production. As statedabove, MUC1 was detectable in all of the lacrimal glands analyzed at the luminallining of acinar epithelial cells. No change in MUC1 related to age or to cases treatedwith artificial tears has been observed, suggesting that MUC1 is not involved in theprocesses occurring in this entity of dry eye.

MUC4 protein has been detected in lacrimal gland samples of women treated withartificial tears and in specimens aged 87 years or older but was not detectable insamples aged o87 years, regardless of the presence of MUC4 mRNA (Paulsen et al.,2004a). When present, MUC4 is not only membrane-associated but also cytoplasmicin acinar cells, with single acinar cells revealing intense cytoplasmic staining.

Zhao et al. (2001) quantified levels of MUC5AC in tears of healthy subjects and indry eye patients. No correlations between the amount of MUC5AC and age orgender in the healthy population were found. However, decreased levels of the mucinwere observed in the tears of patients with dry eye. A similar finding was reported byArgueso et al. (2002), who found decreased levels of MUC5AC in tears of patientswith SJS. Based on these results, a deficiency of MUC5AC mucin in tears wasproposed as one of the mechanisms responsible for tear film instability in SJS.However, mucin quantification by antibody reactivity is not a straightforwardexercise; these conclusions need to be interpreted with caution.

In the lacrimal gland, a strong increase in immunodot blot staining intensity ofMUC 4, 5AC and 5B was observed in lacrimal glands of elderly women who receivedtreatment for dry eyes (Paulsen et al., 2004a). In such cases, MUC5AC is producednot only by the goblet cells of excretory ducts, but also by some acinar cells of thelacrimal gland.

Reactivity with antimucin antibodies strongly increased in the lacrimal glands ofelderly women treated with artificial tears (Paulsen et al., 2004a). It is not clearwhether this increase reflects an increase in mucin production or a change inglycosylation, or both, associated with symptoms of dry eyes. It also remains toestablish whether the changes are brought about by hormonal, inflammatory ormicrobial factors.

7.3. Age-related changes in tear film mucins

Despite a number of epidemiological studies on the prevalence of dry eyes in theelderly population, no systematic study of age-related changes in ocular surface mucinshas been published to date. We have assessed the overall charge/mass characteristics ofmucins purified from the conjunctivae of several individuals aged 40–90 years, andnoted the conservation of the range of mobilities in agarose gels. We are awaiting theconclusion of the Dry Eye Workshop which will establish the criteria for normal anddry eyes. Then a study of changes with age will be more readily undertaken.

7.4. Neuronal regulation of lacrimal gland and tear film mucins

Demonstrably, acinar cells of the lacrimal gland as well as cornea and conjunctivaare densely supplied by nervous tissue and peptidergic nerve fibers (Seifert et al.,

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1996; for review see Hodges and Dartt, 2003; Belmonte et al., 2004). It has beensuggested that this innervation pattern is part of the ‘‘functional ocular surface-central nervous system-lacrimal gland sensory-autonomic neural network’’ thatmaintains ocular surface health and homeostasis (Song et al., 2003). This ‘‘lacrimalfunctional unit’’ comprises the ocular surface tissues (cornea and conjunctiva,including goblet cells and Meibomian glands), the lacrimal glands (main andaccessory [Krause and Wolfring]) and their interconnecting sensory (CN V) andautonomic (CN VII) innervation (Stern et al., 1998; Stern and Pflugfelder, 2004).

Activation of parasympathetic and sympathetic nerves surrounding lacrimal glandacini brings about the release of their classic and peptide neurotransmitters, whichthen bind to their specific receptors on myoepithelial and acinar cells and lead toregulated protein secretion. Of the three types of nerves, parasympathetic,sympathetic and sensory, that innervate the lacrimal gland, only parasympatheticand sympathetic nerves are major stimulators of lacrimal gland protein secretion.The parasympathetic neurotransmitters acetylcholine and vasoactive intestinalpeptide and the sympathetic neurotransmitter norepinephrine are potent stimuli oflacrimal gland secretion, whereas others have either minor stimulatory effects ornone at all (for review see Hodges and Dartt, 2003). Acetylcholine released fromparasympathetic nerves activates the M3 glandular subtype receptor, the only one ofthe five muscarinic receptor subtypes present in human lacrimal gland. M3 is locatedon the basolateral membranes of acinar and the plasma membranes of myoepithelialcells (Hodges and Dartt, 2003) and has obtained attention as a marker of SJS(Bacman et al., 2001; Gao et al., 2004).

The sensory innervation of the cornea and the conjunctiva includes differentfunctional classes of sensory receptor fibres identified as mechano-, polymodalnociceptor, and cold receptor (Belmonte et al., 1991, 2004; Gallar et al., 1993). Inaddition, a small number of low-threshold mechanoreceptor nerve endings are foundin the limbus and perilimbal bulbar conjunctiva (Gallar et al., 1993). Reflex tearsecretion caused by corneal stimulation seems to be chiefly due to activation ofcorneal polymodal nociceptors, whereas selective excitation of corneal mechano-, orcold receptors, appears to be less effective in evoking augmented lacrimal secretion.Conjunctival receptors stimulated at equivalent levels do not evoke an increased tearsecretion (Acosta et al., 2004).

Data from rat conjuctiva have shown that conjunctival goblet cell mucoussecretion can be neurally mediated and serve as an immediate response directed atprotection of the ocular surface (Kessler et al., 1995; Kovacs et al., 2005). Thepresence of nerves and their receptors in conjunctival goblet cells has been observedin humans as well (Diebold et al., 2001) and recent work by Suuronen et al. (2004)has shown with a corneal tissue engineering model that corneal innervation isnecessary for production of a mucus layer. However, other cell lines produce mucinson stratification in the absence of innervation (e.g. primary conjunctival epithelium,SkinethicTM human corneal epithelium constructs, Berry, unpublished results). Nodata exist to date on lacrimal gland innervation and mucin secretion. However, it islikely that lacrimal gland mucin production depends on stimuli from the nervesinnervating the lacrimal gland. In a murine model of SJS, it has been shown that

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activation of nerves of lacrimal glands infiltrated with lymphocytes does not increasethe release of neurotransmitters, resulting in impaired secretion from these glands(Zoukhri and Kublin, 2001). In another model, it was demonstrated that mice with adeficiency of neurturin – a neurotrophic factor for parasympathetic neurons –develop defective parasympathetic innervation of their lacrimal glands, leading todry eye symptoms (Song et al., 2003). Nguyen et al. (2004) showed that, followingloss of parasympathetic control in the rat lacrimal gland, tear secretion wassignificantly reduced and corneal ulcers developed in all experimental eyes. Lightmicroscopy revealed a breakdown of the acinar structure of the lacrimal gland, andDNA microarray analysis revealed downregulation of genes associated with theendoplasmic reticulum and Golgi bodies, including genes involved in protein foldingand processing. Conversely, transcripts for cytoskeleton and extracellular matrixcomponents, inflammation, and apoptosis were upregulated (Nguyen et al., 2004).Hence, it might be expected that mucin production by the lacrimal gland is alsoaltered with loss of neuronal control, a connection that requires further research.

7.5. Hormonal regulation of lacrimal gland and tear film mucins

Hormonal regulation of mucin is likely to be organ-specific. In the mouse, ocularsurface mucin is not regulated by either androgens or progesterone (Lange et al.,2003). Regulation of MUC5B seems to be dependent on hormonal regulation, asshown for the endocervical cycle (Gipson et al., 2001). The expression of thesialomucin complex (SMC; rMUC4; the rat homologue of MUC4) in the rat uterusis down-regulated by progesterone (McNeer et al., 1998), as is MUC5B in the humanendocervix (Gipson et al., 2001). Therefore, postmenopausal down-regulation ofprogesterone could account for changes in MUC4 and MUC5B production in age-related dry eye. Moreover, androgens regulate both lacrimal and Meibomian glandfunction, and androgen deficiency correlates with the occurrence of dry eye (Sullivanet al., 1999). However, with the exception of MUC1 (Mitchell et al., 2002), no dataexist regarding androgen-dependent regulation of mucins. Recent results obtained byour group (Paulsen et al., 2004a) indicate that MUC1 production by the lacrimalgland is unaffected in cases of age-related dry eye.

8. Interaction of mucins with TFF peptides

The localization of TFF1 and TFF3 to the human conjunctival goblet cellsmatches that of the secretory mucin MUC5AC. Conjunctival TFF1 and TFF3 musttherefore be considered typical mucin-associated peptides. It is postulated that TFFpeptides function as link peptides interacting noncovalently with mucins andinfluencing the rheological properties of mucous gels (Hauser et al., 1993). Thishypothesis has been confirmed by in vitro studies demonstrating that TFF peptidesincrease the viscosity of mucin preparations (Thim et al., 2002) and that trefoilsinteract with the von Willebrand Factor-like domains of mucins (Tomasetto et al.,

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2000). The dimeric structure of TFF1 and TFF3 is ideally suited to form anentangled network (Hoffmann and Jagla, 2001) with MUC5AC in the ocular mucus,also the structure of the two trefoil factors may give rise to different ways ofinteracting with their ligands (Muskett et al., 2003). The precise nature of theinteraction between TFF peptides and mucins is not yet known. We have shown thatbinding of the glycosylated form of TFF2 to ocular mucins resulted in a change inmucin elasticity, while the non-glycosylated form did not affect the persistence lengthof the polymer (Brayshaw et al., unpublished results). TFF2 is not synthesized at theocular surface but is present in systemic circulation. We have recently shown that thecornea is also able to produce TFF3, but only under certain disease states (Stevenet al., 2004), e.g. following corneal injury (Jansen et al., 2004). Secreted TFF3 fromthe corneal wound could stimulate goblet cell mucus secretion by way of a yetunknown mechanism (Paulsen et al., unpublished results). On the other hand,intracellular TFF peptides might be involved, on their secretory pathway, in thecomplex oligomerization and packaging of secretory mucins.

Mucin deficiency disorders (Tseng et al., 1984) such as ocular cicatricialpemphigoid, Steven–Johnson syndrome or xerophthalmia may lower levels ofTFF peptides parallel to the decrease in goblet cell density. By contrast, a highernumber of goblet cells (Connor et al., 1994) might enhance TFF peptide secretionsimilarly to daily wearing of contact lenses. TFF peptide secretion might also beinfluenced by alterations in glycosylation of goblet cell mucins as they occur inpatients with dry eye symptoms (Danjo et al., 1998).

Using impression cytology we observed an alteration of the distribution of trefoilpeptides on the conjunctival surface of dry eye patients. In evaporative dry eye(MGD), TFF-positive areas were numerous and mainly in between goblet cellimpressions. ATD (SJS and Graft versus Host Disease) was characterised by diffuse‘‘clouds’’ of TFFs (Khan-Lim at al., unpublished results).

9. Mucins and TFF peptides of the nasolacrimal ducts

9.1. Efferent tear duct mucins

The physiology of lacrimal drainage has been under study for more than acentury, but the forces that cause tear flow are not completely understood. Variousmechanisms have been proposed to explain tear drainage. These include an activelacrimal pump mechanism that functions by contraction of the orbicularis eyemuscle (Jones, 1958; Amrith et al., 2005; Pavlidis et al., 2005; Kakizaki et al., 2005); a‘‘wringing-out mechanism’’ involving a system of helically arranged fibrillarstructures (Thale et al., 1998); and the action of a cavernous body surroundingthe lacrimal sac and the nasolacrimal duct (Paulsen et al., 2000a; Ayub et al., 2003).Physical factors such as capillarity, gravity and the respiration, evaporation andreabsorption of tear fluid through the lining epithelium of the efferent tear ductshave also been invoked (for a review see Paulsen et al., 2002d, 2003a; Paulsen, 2003).

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Both the upper and lower canaliculus are lined with a thick, non-keratinized,stratified epithelium (Paulsen et al., 1998). To date, no data are available on mucinsin the canaliculi, although such information would be very interesting with regard totear flow through the canaliculi. At the transition from the canaliculi into thelacrimal sac, the stratified epithelium produces a double-layered epithelial area thatis rich in goblet cells and intraepithelial mucous glands (Paulsen et al., 1998). Theepithelial lining of the lacrimal sac and the nasolacrimal duct consists of pseudo-stratified, columnar epithelia that is rich in goblet cells. The principal secretoryproducts of the epithelial cells are mucins, plus TFF peptides (Paulsen et al.,2002d, e, 2003a, b). Small serous glands are situated subepithelially in some lacrimalsystems in addition to epithelial and goblet cells (Paulsen et al., 1998).

9.1.1. Membrane-spanning mucins

The membrane-anchored mucins MUC1 and MUC4 have not been detected in thelacrimal sac and nasolacrimal duct, although of message was present in allnasolacrimal ducts, and some lacrimal sacs (Paulsen et al., 2003b). Comparableresults were obtained by Lin et al. (2001), who were also unable to detect membrane-spanning mucins in the mucosa of the middle ear, even though the mucosa of theauditory tube was positive for MUCs 1 and 4. Use of an antibody (1G8) to themembrane-binding domain of MUC4 revealed membrane-spanning as well ascytoplasmic MUC4 in high columnar epithelial cells of the lacrimal sac andnasolacrimal duct (Paulsen, unpublished observations; Fig. 15a, Table 3). As statedabove, MUC1, if present, and MUC4 may function in signalling and may beimportant as sensor mechanisms in response to invasion or damage of epithelia, aswell as in T cell activation.

Our unpublished observations indicate the presence of mRNA and MUC16protein in nasolacrimal ducts (Table 3). Immunohistochemistry reveals perinuclearreactivity in the cells of the double-layered epithelium of the lacrimal sac andnasolacrimal duct. Perinuclear reactivity correlates with immature mucins still in theGolgi apparatus. Reactivity for MUC16 is absent in goblet cells and intraepithelialmucous glands, but is present in the serous acini and secretion products ofsubepithelial serous glands of the nasolacrimal ducts, suggesting that MUC16 mightbe secreted in the nasolacrimal passage.


The epithelium of the lacrimal sac and nasolacrimal duct produce MUC5B andMUC7 and, to a lesser extent, MUC5AC and MUC2. The gel forming MUC2,MUC5AC and MUC5B are produced by single goblet cells and goblet cells formingintraepithelial mucous glands, but not by columnar epithelial cells (Paulsen et al.,2003b; Table 3). The low-molecular weight, non-gel-forming mucin MUC7 isproduced by columnar epithelial cells of the lacrimal sac and nasolacrimal duct,similar to its distribution in the submandibular and lacrimal glands, in which MUC7is the secretory product of salivary and lacrimal serous cells (Tabak, 1995). MUC6could not be detected with available antibodies, although message for this mucin wasobserved in half of the samples (Paulsen et al., 2003b).

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Fig. 15. MUC4 and MUC8 distribution in human nasolacrimal ducts: (a) MUC4 (red) in and

on (membrane bound and cytoplasmic) the surface of high prismatic epithelial cells of the

nasolacrimal duct. Goblet cells appear intraepithelially as empty spaces. (b) MUC8 (red) in

high columnar epithelial cells of the nasolacrimal duct. Counterstain: hemalum; bars a, b,

27.5mm.

Table 3. Mucins in the nasolacrimal ductsa

Mucinb Expression in

nasolacrimal ducts

Localisation in nasolacrimal

ducts

Changes in

PANDOc

MUC1 Positive Not detected No

MUC2 Positive Single goblet cells and

intraepithelial mucous glands

Yes

MUC4 Positive Cytoplasmic in acinar cells No

MUC5AC Positive Single goblet cells and


Yes

MUC5B Positive Single goblet cells and


Yes

MUC6 Sporadic Not present No

MUC7 Positive Cytoplasmic in all high columnar

epithelial cells

No

MUC8 Positive Cytoplasmic in all high columnar

epithelial cells

NDd

MUC16 Positive Supranuclear in the Golgi in all

high columnar epithelial cells and

single basal cells as well as serous

cells of subepithelial glands

NDd

aThe mucin distribution is based on investigations about the epithelia of the lacrimal sac and

nasolacrimal duct. No data are available regarding mucins of the canaliculi.bNo data exist about MUCs 3A, 3B, 9, 11, 12, 13, 15, 17, 19, and 20.cPANDO—primary aquired nasolacrimal duct obstruction.dND—not determined.


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Unpublished results reveal that the columnar epithelial cells of the lacrimal sacand the nasolacrimal duct also produce MUC8 and secrete it into the lacrimalpassage (Fig. 15b). MUC8 has been demonstrated in particular as a ciliated cellmarker in human nasal epithelium and the epithelium of the paranasal sinuses (Yoonet al., 1999; Jung et al., 2000; Kim et al., 2005) and is upregulated byproinflammatory cytokines and under chronic inflammatory conditions (Yoonet al., 1999; Jung et al., 2000; Kim et al., 2005).

9.2. TFF peptides of the efferent tear ducts

The human efferent tear ducts synthesize and store the secretory peptides TFF1and TFF3, but not TFF2 (Paulsen et al., 2002e). TFF3 is produced by the columnarepithelial cells of the efferent tear ducts as well as the serous cells of seromucousglands, but not – or in small amounts only – by goblet cells. The clear contrast of thedistribution pattern to most other epithelia – for example that of the gastrointestinaland respiratory tracts (Hoffmann et al., 2001; Hoffmann and Jagla 2001) as well asthat of the ocular surface (see below), where TFF3 is normally found in goblet cells,is unexpected. TFF1 and TFF3 in the efferent tear ducts are generally expected tohave protective effects, as in the gastrointestinal tract (for review, see Hoffmannet al., 2001; Hoffmann and Jagla 2001). It is interesting in this regard that thecolumnar epithelial cells of the efferent tear ducts also produce a broad spectrum ofantimicrobial peptides including lysozyme, lactoferrin, secretory phospholipase A2,human b-defensin 1, and human-inducible b-defensin 2 (Paulsen et al., 2001a, 2002a,2005). An interaction of TFF3 with the antimicrobial peptides might be possible asboth peptides are products of the same cells. Remarkably, a combined secretion ofTFF peptides and lysozyme has also been observed in a specific gland-like structuretermed the ulcer-associated cell line (UACL) (Wright, 1998). The antiapoptoticeffect of TFF3 (Taupin et al., 2000; Chen et al., 2000) should also be considered inthe context with bacterial or viral infection.

9.3. Comparison of efferent tear duct mucins and TFF peptides with mucins and

TFF peptides in the tear film

9.3.1. Mucins

The lacrimal sac and nasolacrimal duct contain the same mucins as the ocularsurface. Conjunctiva mainly express MUC5AC, and, at much lower levels, MUC2(McKenzie et al., 2000; Table 1). In addition to these mucins, MUC7, as well asprobably also MUC5B and MUC16, are present in the tear film originating from thelacrimal gland and probably also from the ocular surface epithelia (Jumblatt et al.,2003; Paulsen et al., 2004a; Argueso et al., 2003; Hori et al., 2004; Paulsen et al.unpublished observations; Table 1). The epithelia of the lacrimal sac andnasolacrimal duct produce mainly MUC5B and MUC7 as well as MUC5AC andMUC2 (Paulsen et al., 2003a; Table 3). MUC16 and MUC8 are also secreted (ownunpublished results). Whether the ocular surface epithelia also produce MUC8 hasnot been determined as yet. The lacrimal gland, at any rate, does not. MUC8 in the

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nasolacrimal ducts could be interpreted as a mucin in the transitional stage fromocular surface mucin production to the nasal mucosa that produces MUC8 as amain mucin. Besides MUC8, nasal mucosa has been shown to produce MUCs 4,5AC, 5B and 7. MUC2 and MUC6 are not present (Yoon et al., 1999; Jung et al.,2000; Table 3). However, quantitative comparisons of mucins in the different regions– ocular surface, nasolacrimal ducts and nasal mucosa – are lacking, as is acomparative localization study.

9.3.2. TFF peptides

In the nasolacrimal ducts, TFF3 is produced by the columnar epithelial cells aswell as the serous cells of seromucous glands, but not – or in small amounts only – bygoblet cells (Table 4; see above, Paulsen et al., 2002e). This result is in agreementwith a observation concerning TFF3 in the major salivary glands (Jagla et al., 1999).This distribution pattern contrasts to that observed in human and porcineconjunctiva (Langer et al., 1999), in which the peptide is localized in goblet cells.As in most other mucous epithelia, the distribution of TFF3 in the efferent tear ductsdoes not overlap that of TFF1. The latter is localized in goblet cells of the efferenttear ducts. The presence of trace amounts of TFF1 in human efferent tear ducts(Table 4; Paulsen et al., 2002e) is similar to the salivary glands (Jagla et al., 1999) andagrees with earlier work (Rio et al., 1988; Pigott et al., 1991). However, quantitativecomparisons in the different regions – ocular surface, nasolacrimal ducts and nasalmucosa – have yet not been performed for TFF peptides, nor has a comparativelocalization study been carried out.

9.4. Relevance to tear outflow

The regional distribution of secretory mucins in the lacrimal sac and nasolacrimalduct might influence the rheology and flow of tears through the efferent tearpassages. There is speculation that at the ocular surface, mucin composition,distribution, and function are influenced by shear forces generated during blinking(for review see Corfield et al., 1997). In the nasolacrimal ducts, such forces are

Table 4. TFF peptides in the nasolacrimal ductsa

TFF peptide Expression in

nasolacrimal

ducts

Localization in nasolacrimal

ducts

Changes in

dacryoliths

TFF1 Positive Associated with single goblet

cells and intraepithelial mucous

glands

Yes

TFF2 Negative Not present Yes

TFF3 Positive High columnar epithelial cells Yes

aThe TFF peptide distribution is based on investigations about the epithelia of the lacrimal sac and

nasolacrimal duct. No data are available regarding TFF peptides of the canaliculi.

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absent, and other mechanisms are necessary to ease the flow of tears. From the dataavailable so far it can only be said that mucins and TFF peptides are likely tointeract and thus maybe affect tear outflow. However, any hard data addressing thisfeature are lacking.

9.5. Mucin changes in diseases of the nasolacrimal ducts

As is the case with all mucosae, the surfaces of lacrimal sac and the nasolacrimalduct are in constant interaction with environmental microorganisms and aretherefore vulnerable to infection. Previous studies have shown that the efferent tearduct mucosa has developed a variety of strategies to prevent infective colonization bymicroorganisms. These strategies are needed to thwart attacks by microorganismsthat cause dacryocystitis, the most frequent disease of the nasolacrimal ducts (Perraet al., 1995; Paulsen et al., 1998, 2000c, 2001a, 2002a, 2003a; Sirigu et al., 2000; Knopand Knop, 2001). MUC5B and MUC7 support these antimicrobial mechanisms inthat they participate in bacterial agglutination (Baughan et al., 2000; Liu et al.,2000b; Situ and Bobek, 2000).

Idiopathic or primary acquired dacryostenosis, synonymous with primaryacquired nasolacrimal duct obstruction (PANDO), is a syndrome of unknownetiology that accounts for most of the non-traumatic cases observed in adults.Pathological investigations indicate that it results from fibrous obstructionsecondary to chronic inflammation (Busse and Muller, 1977; Linberg andMcCormick, 1986; Mauriello et al., 1992). However, the pathophysiology offunctional dacryostenosis (i.e. patients with epiphora even though their lacrimalpassages are patent on syringing) is yet to be understood. The attendant epithelialpathology is characterized by squamous metaplasia with a loss of goblet cells(Paulsen et al., 2001b). Consistent with this condition, a marked reduction ofmessage for goblet cell-associated mucins MUC2, MUC5AC and MUC5B has beenobserved in PANDO specimens (Paulsen et al., 2003b; Table 3). Despite metaplasiaof the columnar cells, no change has been detected for MUC7 expression.Unchanged production of the antimicrobial mucin MUC7 may contribute to anexplanation of why dacryocystitis never develops in some patients with epiphora dueto postsaccal stenosis (Paulsen et al., 2003b; Table 3). Reduced levels of mRNA ofsecretory mucins in functional dacryostenosis support the assumption that mucinsfacilitate tear flow through the efferent tearducts.

9.6. TFF peptide changes in diseases of the nasolacrimal ducts

Functional dacryostenosis (PANDO) – presence of epiphora in spite of efferenttear duct passages that are patent when syringed – may result not only from anonfunctional segment in the efferent tear duct passage (Paulsen et al., 2001b) butalso from the downregulation of TFF peptides in this segment. Dacryoliths (lacrimalstones, calculi) are commonly observed by lacrimal surgeons during dacryocystorhi-nostomy (DCR) procedures. They are one of the causes of PANDO. Despite thelengthy history of dacryolith studies, our understanding of the pathophysiology

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involved is still in the Dark Ages according to Linberg (2001). It was recentlydemonstrated that dacryoliths consist partly of secreted mucins, comparable to themucin spectrum of the epithelium of healthy nasolacrimal ducts (Paulsen et al.,2006). Moreover, TFF1 and TFF3 appear to be augmented in dacryoliths andTFF2, which is not present under healthy conditions, is additionally induced andsecreted in dacryolithiasis (Table 4). It has been hypothesized that the rheologicalproperties of TFF peptides may play a functional role in dacryolith formation(Paulsen et al., 2006). However, it is not clear from these findings whether or notTFF peptides influence dacryolith formation per se and if their secretion is merely asecondary phenomenon.

10. Concluding remarks

The human lacrimal gland, the epithelia of the ocular surface (conjunctiva andcornea) and the epithelial structures of the nasolacrimal ducts each synthesize aspecific spectrum of mucins and TFF peptides. The diversity of mucins and TFFpeptides can be linked to cell signalling, tear film rheology, antimicrobial defencesand anti-apoptosis; the production of mucins and TFF peptides in the healthylacrimal sac and nasolacrimal duct is associated with enhanced tear transport andantimicrobial activity. Some lacrimal gland mucins may also correlate with age. Bothsecreted and membrane-associated mucins show alterations in dry eye disease at theocular surface. Increased TFF3 production occurs in association with ocular surfaceinjury. The presence of epiphora-associated reduced levels of mucin mRNA in non-functional, but patent, segments of the lacrimal passage suggests that mucins easetear flow through the efferent tear ducts. Increased levels of TFF peptide mRNA indacryolithiasis suggest that TFF peptides play a role in tear outflow.

A fuller understanding of the molecular function of mucins and TFF peptides ofthe lacrimal gland and ocular surface, as well as at the mucosal surface of the efferenttear duct passage, will provide further insight into the pathophysiology of dry eye,ocular surface infections and dacryocystitis. The latter can lead to residual functionalimpairment of the lacrimal system and result in ocular discomfort or other problemsaround the medial canthus. The factors controlling the production of mucins andTFF peptides associated with the lacrimal apparatus and ocular surface are largelyunknown at present, but it seems that old age, changes in hormonal status(postmenopausal women), and immunodeficiency are associated with them. It hasbeen suggested that the normally constant flow of tears ensures mucin and TFFpeptide production by means of a feedback control mechanism and that thisproduction comes to a halt if tears are not drained into the nose (Paulsen et al.,2002c, d, 2003a; Paulsen, 2003).

There has been tremendous progress in the cloning of mucins and TFF peptidesand characterization of their structures and physiological and pathologicalfunctions. However, whether each of the TFF peptides or of the secreted ormembrane spanning mucins have a specific function at the ocular surface is not yet

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known. Mucin and TFF peptide regulation, in particular as related to goblet celldifferentiation and disease, the interactions with other tear constituents are still to beinvestigated.

Obtaining deeper insights into these features and functions of the lacrimalapparatus and ocular surface will be particularly challenging in view of theconsiderable difficulties involved in realization of cell culture models and cell lines oflacrimal gland or epithelia of the efferent tear ducts. Results obtained with animalmodels, as discussed above, are difficult to extrapolate to humans, especiallyregarding tear film. Such investigations will be of great relevance, since the numberof patients with dry eye is still increasing, as is the number of contact lens wearersand surgical ocular surface manipulations, all of which affect normal mucinfunction. Progress in the analysis of the mucus secretory apparatus will suggest noveltherapies beneficial in the fight against diseases of the lacrimal apparatus and ocularsurface.

Acknowledgments

We acknowledge support by the Deutsche Forschungsgemeinschaft (DFG,German Research Foundation), Bonn, Germany (Program Grants No.: PA738/1-2, PA 738/1-3, PA 738/1-4, PA 738/1-5), the National Eye Research Center (Grant2000/013), Bristol, UK, the Wilhelm Roux program, Halle, Germany (FKZ 9/18 and12/08), and the Sicca Forschungsforderung (Dry Eye Research Foundation ofAssociation of German Ophthalmologists), Berlin, Germany.

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Mucins and TFF peptides of the tear film and lacrimal apparatus

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