Histochem Cell Biol (2007) 127:433–438

DOI 10.1007/s00418-006-0246-6

ORIGINAL PAPER

MUC16 in the lacrimal apparatus

Kristin Jäger · Guangxi Wu · Saadettin Sel · Fabian Garreis · Lars Bräuer · Friedrich P. Paulsen

Accepted: 6 October 2006 / Published online: 9 January 2007© Springer-Verlag 2007

Abstract The aim of the present study was to deter-mine the possible expression of the mucin MUC16 inthe lacrimal apparatus. Expression and distribution ofMUC16 in lacrimal gland, accessory lacrimal glands,and nasolacrimal ducts was monitored by RT-PCR andimmunohistochemistry. MUC16 was expressed anddetected in all tissues investigated. Comparable to con-junctiva and cornea it was membrane-anchored inaccessory lacrimal glands whereas in lacrimal gland aci-nar cells and columnar cells of the nasolacrimal ducts itwas stored in intracytoplasmic vesicles without mem-brane-association. Subepithelial serous glands of thenasolacrimal ducts revealed staining of the secretionproduct. Intracelluar production of MUC16 is presentin lacrimal gland and epithelial cells of the nasolacri-mal ducts but it is not clear whether this MUC16 issecreted. MUC16 seems to be shedded or secretedfrom the epithelial surface of subepithelial serousglands of the nasolacrimal ducts. Our results show thatMUC16 is present in the whole lacrimal apparatus. Itsdistribution pattern suggests diVerent physiological

functions with regard to tear Wlm physiology and tearoutXow. Moreover, the results demonstrate the exis-tence of so far not recognized qualitative diVerences inthe secretion product of main lacrimal gland and acces-sory lacrimal glands (glands of Krause).

Keywords Mucin · Lacrimal gland · Accessory lacrimal gland · Tear Wlm · Nasolacrimal ducts

Introduction

MUC16 is a high-molecular-weight glycoprotein (Yinand Lloyd 2001) of the mucin family initially known asthe CA125 antigen, that was Wrst detected in ovariancarcinoma cell lines of epithelial origin and in the lumi-nal surface of tumor tissues taken from patients withovarian cancer (Bast et al. 1981). Today, MUC16domains provide novel opportunities to develop newassays and reWne current tools to improve the sensitiv-ity and speciWcity of CA125 for population-basedscreening guidelines (McLemore and Aouizerat 2005).

In addition to its function as a tumor marker, studieshave also revealed the presence of CA125 antigen in anumber of normal tissues mainly ocular surface epithe-lia like cornea and conjunctiva but also nose, uvula andlarynx (Paulsen 2006 and unpublished results). MUC16has therefore become the latest addition to the list ofmembrane-spanning ocular surface mucins (MUC1and MUC4) (Argüeso et al. 2003). MUC16 is locatedat the tips of epithelial processes of cornea and con-junctiva “protruding” into the preocular Xuid (Argüesoet al. 2003) and augmented by exogenous retinol (Horiet al. 2004, 2005). Its function is not deWnitively deter-mined; some authors suggest that MUC16 is particu-

K. Jäger · G. Wu · F. Garreis · L. Bräuer · F. P. Paulsen (&)Department of Anatomy and Cell Biology, Martin-Luther-University Halle-Wittenberg, Grosse Steinstr. 52, 06097 Halle, Saale, Germanye-mail: friedrich.paulsen@medizin.uni-halle.de

G. WuSchool of Life Science, Fudan University, Shanghai, People’s Republic of China

S. SelDepartment of Ophthalmology, Martin-Luther-University Halle-Wittenberg, Halle, Saale, Germany

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larly important as hydrophilic molecule involved inmaintenance of a healthy ocular surface (Hori et al.2005; Argüeso et al. 2006a, b), others connect it todiVerences in Pseudomonas aeruginosa adhesion(McNamara et al. 2005) to open and closed-eye tears.In dry eyes there is an increase in ocular surface reac-tivity with an antibody recognizing an oligosaccharide(Danjo et al. 1998) now known to be decoratingMUC16. Moreover, there is some evidence that theextracellular domain of MUC16 is shed into the tearWlm (Hori et al. 2006).

The hypothetically high impact of MUC16 on theintegrity of ocular surface epithelia, on the rheologicand probably also antimicrobial properties of the tearWlm and its possible changes in dry eye led us to adetailed analysis of the MUC16 expression and distri-bution in the lacrimal apparatus.

Materials and methods

Tissues

The study was conducted in compliance with Institu-tional Review Board regulations, informed consentregulations, and the provisions of the declaration ofHelsinki. Lacrimal glands, upper eye lids, conjunctivas,corneas, and nasolacrimal systems (consisting of lacri-mal sac and nasolacrimal duct) were obtained fromcadavers (six males, eight females, aged 53–86 years)donated to the Department of Anatomy and Cell Biol-ogy, Martin-Luther-University Halle-Wittenberg, Ger-many. The donors were free of recent trauma, eye andnasal infections or diseases involving or aVecting lacri-mal function. Biopsy samples for molecular biologicalanalysis were taken from the bulbar conjunctiva. Tis-sues from the right eye each were prepared for paraYnembedding and Wxed in 4% paraformaldehyde; tissuesfrom the left eye each were used for molecular biologi-cal investigations and were immediately frozen at80°C.

RNA preparation and complementary DNA synthesis (cDNA)

For conventional reverse transcriptase polymerasechain reaction (RT-PCR), tissue biopsies from 14 lacri-mal glands, upper eye lids, conjunctivas, corneas andnasolacrimal systems were crushed in an agate mortarunder liquid nitrogen, then homogenized in 5 ml peq-gold RNA pure solution (peqLab Biotechnologie,Erlangen, Germany) with a Polytron homogenizer.Insoluble material was removed by centrifugation

(12,000 g, 5 min, 4°C). Total RNA was isolated usingRNeasy-Kit (Qiagen, Hilden, Germany). Crude RNAwas puriWed with isopropanol and repeated ethanolprecipitation and contaminating DNA was destroyedby digestion with RNase-free DNase I (27.27 Kunitzunits; 20 min 25°C; Boehringer, Mannheim, Germany).This enzyme was heat-inactivated for 15 min at 65°C.500 ng RNA were used for each reaction: cDNA wasgenerated with 50 ng/�l (20 pmol) oligo (dT)15 primer(Amersham Pharmacia Biotech, Uppsala, Sweden)and 0.8 �l superscript RNase H¡ reverse transcriptase(100 U; Gibco, Paisley, UK) for 60 min at 37°C. Integ-rity of RNA was controlled by RT-PCR of GAPDHwith speciWc primers. No PCR products larger than360 bp were detected. These would indicate contami-nating DNA.

RT-PCR

For conventional RT-PCR, 4 �l cDNA (from eachsample) were incubated with 30.5 �l water, 4 �l 25 mMMgCl2, 1 �l dNTP, 5 �l 10£ PCR buVer, 0.5 �l (2.5 U)platinum taq DNA polymerase (Gibco) and 2.5 �l(10 pmol) of the following primer pairs: MUC16forward 5�-GCCTCTACCTTAACGGTTACAATGAA-3�, reverse 5�-GGTACCCCATGGCTGTTGTG-3�

(114 bp, annealing temperature 60°C). 0.1 �M of glyc-eraldehyde-3-phosphate dehydrogenase (GADPH)intron-spanning primer pair (Varoga et al. 2004)served as the internal control for cDNA. The primerswere synthesized by MWG-Biotech AG, Ebersberg,Germany. Human cornea and conjunctiva samplesknown to express MUC16 were included in each exper-iment. The cDNA was replaced with water for a nega-tive control reaction.

Immunohistochemistry

For analysis by immunohistochemistry, 14 lacrimalglands, upper eye lids, conjunctivas, corneas, and naso-lacrimal systems were Wxed in 4% formalin, embeddedin paraYn, sectioned, and dewaxed.

Immunohistochemical staining was performed withan antibody to the mucin peptide core epitope ofMUC16 (monoclonal mouse anti-human MUC16 anti-body, OC125, DAKO, Glostrup, Denmark, 1:50 dilu-tion). Sections were microwaved for 10 min andnonspeciWc binding inhibited by incubation with rabbitnormal serum 1:5 in Tris buVered saline [TBS]. Theprimary antibody was applied overnight at room tem-perature. The secondary antibody rabbit anti-mouse(1:200) (DAKO) was incubated at room temperaturefor at least 4 h. Visualization was achieved with peroxi-

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dase-labeled streptavidin-biotin and diaminobenzidine(DAB) for at least 5 min. After counterstaining withhemalum, the sections were mounted in Aquatex(Boehringer, Mannheim, Germany). Two negativecontrol sections were used in each case: one was incu-bated with the secondary antibody only, the other withthe primary antibody only. The sections of human con-junctiva and cornea were used for positive control. Theslides were examined with a Zeiss axiophot micro-scope.

Results

Expression of MUC16 in tissue

MUC16-speciWc ampliWcation products (114 bp) weredetected in all lacrimal glands (Fig. 1a), accessory lacri-mal glands (Krause’s gland; Fig. 1b), and nasolacrimalducts (both from lacrimal sac and nasolacrimal duct;Fig. 1b). Human cornea (Fig. 1a) and bulbar conjunc-tiva (Fig. 1b) also yielded 114 bp ampliWcation prod-ucts.

Distribution of MUC16 in cadaveric tissue

ParaYn-embedded 7 �m sections from 14 lacrimalglands, accessory lacrimal glands (Krause’s glands), con-junctiva, cornea, and nasolacrimal ducts were anayzed.

Lacrimal gland

MUC16-positive acinar cells are rare, but present in all14 lacrimal glands (Fig. 2a), with numbers varyingamong specimens. MUC16 is detected in intracytoplas-mic organelles localized mainly above the nucleus(Fig. 2b). No membrane-anchored MUC16 is visible.

Accessory lacrimal gland (Krause)

All the accessory lacrimal glands in sections from 14diVerent donors were positive for MUC16 (Fig. 2c). Incontrast to the major lacrimal gland, the majority ofaccessory lacrimal glands acini displayed cell surface-related MUC16 (Fig. 2c).

Conjunctiva and Cornea

Membrane-anchored MUC16 was present in the super-Wcial conjunctival and corneal epithelium from alldonors (Fig. 2d, e).

Nasolacrimal ducts

MUC16 was detected in all 14 eVerent tear duct systemsinvestigated (Fig. 2f–h). Strong MUC16 reactivity wasvisible perinuclearly in the cells of the double-layeredepithelium of the lacrimal sac and nasolacrimal duct, andvery weak in the apical part of high columnar epithelialcells (Fig. 2g). Reactivity was absent in goblet cells andintraepithelial mucous glands (Fig. 2f), but was presentin the serous acini and secretion products of subepithe-lial serous glands of the nasolacrimal passage (Fig. 2h).No diVerences in MUC16 distribution were seenbetween the lacrimal sac and the nasolacrimal duct.

Discussion

Our study conWrms the presence of membrane-anchored MUC16 at the surface of corneal and con-junctival epithelial cells (Argüeso et al. 2003) and indi-cates production of MUC16 mRNA and protein in thelacrimal gland, accessory lacrimal glands (glands ofKrause), and eVerent tear ducts. In contrast to MUC1,which is membrane-anchored in the lacrimal gland andat the ocular surface, and to MUC4, which shows bothmembrane-anchored and cytoplasmic occurrence inacinar cells of the lacrimal gland and ocular surface(Paulsen et al. 2004; for review see Paulsen 2006; Paul-sen and Berry 2006) MUC16 is immunohistochemicallydetectable in intracytoplasmic organelles of lacrimalgland acinar cells mainly above the nucleus suggesting

Fig. 1 Expression of MUC16: RT-PCR. Samples from two diVer-ent donors are shown for each tissue: a lacrimal glands (LG),cornea Cr; b conjunctiva (Cj), nasolacrimal ducts (ND), andaccessory lacrimal gland (aLG)

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that only a soluble form of MUC16 is produced by aci-nar cells or that the mucin detected is immature mucin.Then not seeing it on apical membranes might eithermean that the turnover is great, or that the Wxing hasdestroyed the mucin or epitope. However, the latter isunlikely as otherwise also membrane anchoredMUC16 of conjunctiva and cornea should not havebeen visible in the tissue specimens that were takenfrom the same individuals.

Moreover, it should be mentioned that MUC16-pos-itive acinar cells are very rare in the lacrimal gland,with numbers varying among individual specimens. In

contrast, the MUC16 distribution pattern of accessorylacrimal glands (Krause’s glands) is diVerent from themajor lacrimal gland. In accessory lacrimal glands,MUC16 occurs membrane bound throughout all glan-dular acinar cells and no supranuclear staining hasbeen detected. Of further interest is the Wnding thatreactivity for MUC16 is present in the serous acini andof subepithelial serous glands of the nasolacrimalducts, suggesting that this mucin might be secreted inthe nasolacrimal passage.

Together with MUC1 and MUC4, MUC16 isthought to anchor the tear Wlm onto the surface of the

Fig. 2 Distribution of MUC16 in tissues of the lacri-mal apparatus. a MUC16 reactivity (arrows) is only visi-ble in a few acini of the lacri-mal gland (female, 53 years). b MagniWcation of lacrimal gland acinus positive for MUC16 (box in a). Reactivity against MUC16 is restricted to supranuclear vesicles. c Reac-tivity in an accessory lacrimal gland (Krause’s gland). The gland is located subepthelially beneath the tarsal conjunctiva which also reacts positive with the antibody. The staining pattern demonstrates mem-brane-anchored MUC16. d Section through the tarsal conjunctiva revealing mem-brane-anchored reactivity against MUC16. e SuperWcial corneal epithelial cells reveal membrane-anchored reactiv-ity (arrows). f High prismatic epithelial cells of human naso-lacrimal duct stain positive with the antibody. Reactivity is absent in goblet cells and in-traepithelial mucous glands. For (small box see g). g High-er magniWcation (small box in f) reveals strong MUC16 reac-tivity perinuclearly in the cells of the double-layered epithe-lium and very weak in the api-cal surface of high columnar epithelial cells (arrows). h Reactivity is also visible in se-rous acini and secretion prod-ucts of subepithelial serous glands (arrows below) and within the lumen of the naso-lacrimal passage (arrows on the right side). Counterstains: a–h hemalum. Scale bars a, h 82,5 �m; b 16,5 �m; c, d, f 56 �m; e, g 36 �m

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eye by intertwining with the secreted mucins in the pre-ocular Xuid. Moreover, all three membrane-boundmucins can occur in both membrane-anchored as wellas soluble form (Carraway et al. 2003; Ligtenberg et al.1992). Most likely, the soluble form is a result of alter-native splicing in which the membrane-anchoreddomain is split oV post-transcriptionally (Moniauxet al. 2001; Gender 2001; Higuchi et al. 2004). How-ever, cleavage of the exodomain (alpha domain) of themucin from the cellular surface has also been shown(Rossi et al. 1996). In this process, MUCs 1, 4, and 16all appear to be shed from the epithelial surface (Lig-tenberg et al. 1992; Sheng et al. 1990; Auersperg et al.1999) although the mechanism and its regulation isunclear. There is evidence that not all membrane-anchored mucins are controlled by the same mecha-nisms: a selective augmentation of MUC4 and MUC16,but not MUC1 was observed after retinoic acid orserum was added to a conjunctival cell line (Hori et al.2004), while the eicosanoid 15-(S)-hydroxy-5,8,11,13-eicosatetraenoic acid (15(S)-HETE) is a selectivesecretogogue for MUC1 (Jumblatt et al. 2002). Laurieet al. (2006) demonstrated just recently in HCE cellsthat lacritin, a mulitipurpose growth and secretory fac-tor produced by the lacrimal gland promotes mRNAexpression and protein production of MUC16 and pos-sibly could be one mechanism by which lacritin is pro-tective against tumor necrosis factor alpha (TNF�).

It is well documented that some membrane-span-ning mucins aid cell detachment and desquamationbeing potent anti-adhesives and repressors of apoptosis(Lomako et al. 2005, 2006) of epithelia at the ocularsurface. These studies suggest that proteosomal andlysosomal degradation play a role in both quality andquantity control of membrane-anchored mucin synthe-sis and represent Wrst evidence for proteosome andlysosome involvement in mucin distribution among celllayers. Such features might also play a role for MUC16at the ocular surface but also for renewal of cells in thelacrimal structures where MUC16 is absent in the cellmembrane. However, nothing is known so far aboutMUC16 with regard to such features.

The regional diVerences in the distribution patternof MUC16 between lacrimal gland, accessory lacrimalglands, ocular surface epithelia and the epithelium ofthe lacrimal sac and nasolacrimal duct might inXuencethe rheology of the tear Wlm and Xow of tears besideother factors through the eVerent tear passages. Thereis a speculation that at the ocular surface, mucin com-position, distribution, and function are inXuenced byshear forces generated during blinking (for review seeAuersperg et al. 1999). Recent evidence indicates thatunder dynamic Xow conditions mucin O-glycans of

membrane-associated mucins (especially of MUC16)prevent cell–cell adhesion in human corneal epithelialcells and suggest that glycosylated membrane-anchored mucins may contribute to the disadhesiveproperties of the apical epithelial surfaces on the eye(Argüeso et al. 2006a, b). In the nasolacrimal ducts,shear forces, and disadhesive properties are absent ornot necessary to ease the Xow of tears. This might walkalong with diVerences in the MUC16 distribution in thenasolacrimal passage. Here, MUC16 seems to besecreted from subepithelial serous glands. However,and comparable to the lacrimal gland the Wnding ofsupranuclear staining of MUC16 in epithelial cells ofthe lacrimal sac and nasolacrimal duct without mem-brane-anchored staining remains unclear and thewhole discussion about rheological properties ofMUC16 is hypothetically as the role of MUC16 in Xuidmovement has not been studied so far.

In conclusion, the present results show that besideconjunctiva and cornea MUC16 occurs also membraneanchored in accessory lacrimal glands. Moreover,MUC16 is detected intracellularly in some areas of lac-rimal gland and epithelial cells of the nasolacrimalducts as well as in secretory form in subepithelialglands of the nasolacrimal ducts. Finally, this investiga-tion suggests for the Wrst time that qualitative diVer-ences exist in the secretion product of main andaccessory lacrimal glands (Krause’s glands) at leastwith regard to MUC16.

Acknowledgments The authors thank Ute Beyer and MichaelaRisch for expert technical assistance as well as Monica Berry,Academic Unit of Ophthalmology, University of Bristol, UK forher critical suggestions. Supported in part by the Deutsche Fors-chungs-gemeinschaft (DFG)—program grants PA 738/9-1 andPA 738/1-5, the BMBF—Wilhelm Roux Program, Halle, Ger-many—program grants FKZ 9/18, 12/08, and 13/08, as well as Sic-ca Forschungsförderung of Association of GermanOphthalmologists.

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