Electron Microscopic Immunogold Localization of Salivary Mucins MG1 and MG2 in Human Submandibular...

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ARTICLE

Volume 51(1): 69–79, 2003The Journal of Histochemistry & Cytochemistry

http://www.jhc.org

Electron Microscopic Immunogold Localization of Salivary Mucins MG1 and MG2 in Human Submandibular and Sublingual Glands

Marco Piludu, Sean A. Rayment, Bing Liu, Gwynneth D. Offner, Frank G. Oppenheim, Robert F. Troxler, and Arthur R. Hand

Departimento di Citomorfologia, Universita Degli Studi di Cagliari, Cagliari, Italy (MP); Department of Periodontology and Oral Biology, Goldman School of Dental Medicine (SAR,BL,FGO,RFT) and Departments of Biochemistry (GDO,FGO,RFT) and Medicine (GDO), Boston University, School of Medicine, Boston, Massachusetts; and Department of Pediatric Dentistry, School of Dental Medicine, University of Connecticut, Farmington, Connecticut (ARH)

SUMMARY

The human salivary mucins MG1 and MG2 are well characterized biochemi-cally and functionally. However, there is disagreement regarding their cellular and glandu-lar sources. The aim of this study was to define the localization and distribution of thesetwo mucins in human salivary glands using a postembedding immunogold labelingmethod. Normal salivary glands obtained at surgery were fixed in 3% paraformaldehyde–0.1% glutaraldehyde and embedded in Lowicryl K4M or LR Gold resin. Thin sections werelabeled with rabbit antibodies to MG1 or to an N-terminal synthetic peptide of MG2, fol-lowed by gold-labeled goat anti-rabbit IgG. The granules of all mucous cells of the sub-mandibular and sublingual glands were intensely reactive with anti-MG1. No reaction wasdetected in serous cells. With anti-MG2, the granules of both mucous and serous cellsshowed reactivity. The labeling was variable in both cell types, with mucous cells exhibitinga stronger reaction in some glands and serous cells in others. In serous granules, the elec-tron-lucent regions were more reactive than the dense cores. Intercalated duct cells nearthe acini displayed both MG1 and MG2 reactivity in their apical granules. In addition, thebasal and lateral membranes of intercalated duct cells were labeled with anti-MG2. These re-sults confirm those of earlier studies on MG1 localization in mucous cells and suggest thatMG2 is produced by both mucous and serous cells. They also indicate differences in proteinexpression patterns among salivary serous cells.

(J Histochem Cytochem 51:69–79, 2003)

alivary mucins

are large, highly glycosylated pro-teins that have multiple functions in the oral cavity(Tabak 1995). Mucins lubricate and protect the oraltissues, allowing them to slide over one another easily,and form a barrier to desiccation and to chemical andmechanical insults. They bind to oral microorganismsand viruses, aggregating them and facilitating theirclearance from the mouth by swallowing. Mucins alsobind to the exposed surfaces of the teeth, contributingto the acquired pellicle derived from salivary compo-

nents (Al-Hashimi and Levine 1989). In this location,the mucins may facilitate binding of microorganismsto the tooth surface. Finally, mucins in swallowed sa-liva may also aid in protection of the pharyngeal andesophageal mucosa (Rayment et al. 2000a).

Human saliva contains two major mucin compo-nents, MG1 and MG2. The high molecular weightMG1 consists mainly of the MUC5B gene product(Troxler et al. 1997; Nielsen et al. 1997: Thornton etal. 1999), whereas the low molecular weight MG2 is aproduct of the MUC7 gene (Bobek et al. 1993). Al-though there is general agreement that MG1 is pro-duced and localized in mucous cells of the submandib-ular, sublingual, and some minor salivary glands(Cohen et al. 1990; Audie et al. 1993; Nielsen et al.1996), this is not the case for MG2. Immunohis-

Correspondence to: Dr. Arthur R. Hand, Dept. of Pediatric Den-tistry, U. of Connecticut Health Center, 263 Farmington Avenue,Farmington, CT 06030-1610. E-mail: hand@nso1.uchc.edu

Received for publication December 10, 2001; accepted August23, 2002 (1A5700).

KEY WORDS

salivary glands

mucous cells

serous cells

intercalated ducts

immunohistochemistry/

postembedding

immunogold labeling

by guest on February 7, 2016jhc.sagepub.comDownloaded from

Piludu, Rayment, Liu, Offner, Oppenheim, Troxler, Hand

tochemical (IHC) and in situ hybridization (ISH) stud-ies by Cohen et al. (1991) and Khan et al. (1998) sug-gest that MG2 also is present in and produced bymucous cells in the submandibular glands. On theother hand, similar studies by Nielsen et al. (1996,1997) indicate that MG2 is present in a subset of theserous cells of these glands. Using the same antibodyand nucleotide probes, Sharma et al. (1998) describeda differential localization of MG1 and MG2 in mu-cous and serous cells, respectively, of human bron-chial submucosal glands. No reactivity for MG1 orMG2 was seen in the parotid gland or in salivarygland intercalated, striated, or excretory duct cells inany of these studies.

The purpose of the present study was to further in-vestigate the distribution of these two mucins in humansalivary gland tissue using immunoelectron microscopy.

Materials and Methods

Samples of normal submandibular and sublingual glandswere obtained from eight consenting male and female pa-tients, aged 44–69 years, undergoing surgery at the Otorhi-nolaryngology Clinic, University of Cagliari, Cagliari, Italy.All procedures were approved by the Human Experimenta-tion Committee, University of Cagliari. In addition, one sub-lingual gland was obtained from a 7-year-old boy undergo-ing sialadenectomy at the Hospital for Sick Children,Toronto, Canada. The procedure was approved by the Hos-pital Committee for Human Experimentation. The varioussamples studied are summarized in Table 1.

For light microscopic studies the glands were fixed over-night in 4% paraformaldehyde (Polysciences; Warrington,PA) in 0.1 M sodium cacodylate buffer, pH 7.2, then storedin 1% paraformaldehyde in cacodylate buffer at 4C. Thesamples were rinsed in 0.1 M cacodylate buffer, dehydratedin cold methanol, embedded in LR Gold resin (Polysciences),and polymerized under UV light (365 nm) at

20C. Onesample was dehydrated in ethanol, embedded in LR Whiteresin, and polymerized overnight at 50C. One-micrometer

sections were collected on glass slides and incubated for 90min at room temperature (RT) with rabbit polyclonal anti-bodies to MG1 (Troxler et al. 1995; Rayment et al. 2000b)diluted 1:1000–1:2000 in 1% bovine serum albumin (BSA)–5% normal goat serum (NGS) in PBS, or to an N-terminalsynthetic peptide of MG2 (Liu et al. 1999) diluted 1:500–1:1000 in a mixture of fish gelatin, ovalbumin, and Tween-20 in PBS. Sections incubated with medium devoid of pri-mary antibody or containing nonimmune rabbit serum wereused as controls. After incubation the sections were rinsedwith PBS and incubated for 60 min at RT with goat anti-rabbit IgG labeled with 5-nm gold particles (Amersham, Ar-lington Heights, IL) diluted 1:50 in 1% BSA–PBS. After rins-ing with PBS and distilled water, silver enhancement (BritishBioCell; Cardiff, UK) was performed to visualize the boundgold particles. The sections were lightly stained with 1%methylene blue–1% azure II and were observed and photo-graphed in a Leitz Orthoplan microscope.

Tissue processing for electron microscopy employed fixa-tion of small pieces (1 mm

) of the same samples in a mix-ture of 3% paraformaldehyde and 0.1% glutaraldehyde in0.1 M cacodylate buffer for 2 hr, after which the glandswere stored in 1% paraformaldehyde in cacodylate buffer.After rinsing in buffer, the tissues were dehydrated in coldmethanol, embedded in either LR Gold or Lowicryl K4Mresin (Polysciences), and polymerized as described above.One gland was fixed in 1% glutaraldehyde and embeddedin LR White resin. Ultrathin sections were collected onFormvar-coated nickel grids and treated with either 1%BSA–1% instant milk in PBS or fish gelatin, ovalbumin,and Tween-20 in PBS to block nonspecific binding. Thesections were incubated with anti-MG1 diluted 1:100–1:500 in 1% BSA–5% NGS in PBS for 60 min at RT orwith anti-MG2 diluted 1:50–1:200 in fish gelatin, ovalbu-min, and Tween-20 in PBS overnight at 4C. Omission ofthe primary antibody or incubation with nonimmune rab-bit serum was used as control. After rinsing with PBS, thegrids were incubated for 60 min at RT with goat anti-rab-bit IgG labeled with 10-nm gold particles (Amersham). Thegrids were washed with PBS and distilled water, stainedwith uranyl acetate and lead citrate, and observed and pho-tographed in a JEOL 100CX TEM.

Table 1

Summary of anti-MG1 and anti-MG2 labeling of human submandibular (SM) and sublingual (SL) glands

Sample no. GlandPatient age

(years) Sex

MG1 reactivity MG2 reactivity

Interc. duct Mucous Serous

Interc. ductMucous Serous

2093 SM 44 M �� n.d. �/��

2098 SM 50 M �� /� ��

2099 SM 54 M �� /�� /� n.d.2127 SM 49 M �� n.d. �/�� /��

2127 SL 49 M n.d. n.d. n.d. � �/��

2136 SM 49 F n.d. n.d. n.d. � �/��

2148 SM 69 M n.d. n.d. n.d. � � n.d.2174 SL 51 M �� /� � �/��

2176 SM 78 M n.d. n.d. n.d. � �/��

578 SL 7 M �� n.d. � �� /��

Labeling intensity:

��

, strong;

��

, moderate;

, weak;

, no reaction; n.d., not determined.

Immunogold Localization of Human Salivary Mucins

Figure 1 Human salivary glands: immunogold silver staining of 1-�m LR Gold sections with anti-MG1 and anti-MG2 antibodies. (A) Sublin-gual gland, anti-MG1; strong reactivity is seen in mucous cells (MC) but no labeling is present in demilune cells (DC). (B) Submandibulargland, anti-MG1; mucous cells (MC) are reactive but no labeling occurs in adjacent serous cells (SC). (C) Sublingual gland, anti-MG2; serouscell reactivity is strong, but little or no labeling of mucous cells (MC) is evident. (D,E) Submandibular gland, anti-MG2; labeling of small mu-cous cells (MC) is prominent, but less reactivity is seen in serous cells (SC). (F) Submandibular gland, anti-MG1; reactivity is observed in inter-calated duct cells (arrows) adjacent to the acini, whereas other duct cells (ID) and serous acinar cells are unlabeled. (G) Submandibulargland, anti-MG2; several intercalated duct cells (arrows) contain MG2 reactivity; lower levels of labeling are seen in serous cells (SC). Bars �20 �m.

Results

Specific reactivity for MG1 was detected at the lightmicroscopic level (Figures 1A and 1B) on the granulesof all mucous cells of the submandibular and sublin-gual glands examined at dilutions of the primary anti-body of 1:1000–1:2000. The granules of some interca-lated duct cells located adjacent to the acini alsoshowed labeling (Figure 1F). No reactivity was foundin serous acinar or demilune cells. Striated duct cellsalso were devoid of labeling.

In the same glands, MG2 labeling exhibited a dif-ferent distribution. Both mucous and serous compo-nents of the tissues showed reactivity at dilutions ofthe primary antibody of 1:500–1:1000. In most glandsexamined, the intensity of labeling of serous cells wasmoderate, whereas that of mucous cells was some-what weaker. The labeling of both cell types with theanti-MG2 antibody, however, exhibited significantvariability. In some glands, mucous cells, especiallythose of smaller size, labeled strongly (Figures 1D and1E). In the sublingual gland from the young individ-ual, the serous demilune cells were strongly reactive,whereas the mucous acinar cells exhibited relativelyweak labeling (Figure 1C). Intercalated duct cells lo-cated near acini exhibited labeling in their apical cyto-plasm (Figure 1G). These cells were more consistentlyreactive than the mucous and serous cells. No labelingwas seen in striated duct cells.

In the absence of the primary antibody, or whennonimmune IgG was substituted for the primary anti-body, no specific labeling was observed.

Electron microscopic immunogold labeling con-firmed and extended the light microscopic observa-tions. The granules of all mucous cells in the sub-mandibular and sublingual glands examined wereintensely labeled after incubation with anti-MG1 anti-body at dilutions of 1:100–1:500 (Figures 2A–2D).Nonspecific labeling of nuclei, mitochondria, and ex-tracellular spaces was minimal. No labeling was ob-served in serous cells, and no substantial differencesin the distribution and intensity of MG1 reactivitywere observed between submandibular and sublingualglands.

The granules of some intercalated duct cells, partic-ularly cells located near the acini, were labeled withthe anti-MG1 antibody (Figure 3). The gold particleswere present mainly over the electron-lucent portionof these granules. The labeling density of the interca-lated duct cell granules appeared to be less than that

of granules in the mucous acinar cells. In a few cases,gold particles were present along the luminal surfaceof the duct cells (Figure 3, inset), suggesting that thissurface may be coated with MG1.

Ultrathin sections of the submandibular and sublin-gual glands incubated with the anti-MG2 antibody(1:50–1:200) exhibited specific reactivity in both mu-cous and serous secretory granules (Figures 4 and 5).As observed by light microscopy, the distribution andintensity of labeling with the anti-MG2 antibody reac-tivity showed greater variability than that observedfor MG1. In the samples examined, the granules ofmost mucous cells had low levels of labeling, andmany serous cells had unreactive secretory granules(Figure 5B). Only a few serous acinar cells displayedlabeling in the lucent halo of their granules. In one ofthe submandibular glands and one of the sublingualglands (Figure 4) studied, a more intense and uniformlabeling was found in the electron-lucent halo of theserous granules, and mucous acinar cells in theseglands were either unreactive or only weakly labeled.The dense cores of the serous granules were almostunreactive. Occasionally they were labeled to a similarextent as in sections incubated with appropriate dilu-tions of normal rabbit serum, suggesting nonspecificbinding of the primary antibody to the electron-densecores of the serous granules. The endoplasmic reticu-lum and Golgi apparatus of the mucous and serouscells consistently were unlabeled.

Intercalated duct cells located near acini also dis-played MG2 reactivity in secretory granules and vesi-cles located in their apical cytoplasm (Figures 6A–6C).As noted for intercalated duct cells labeled with anti-MG1 antibody, gold particles representing MG2 reac-tivity were restricted mainly to the electron-lucentportion of the granules. The labeling density of the in-tercalated duct cell granules was similar to that seen ingranules of serous cells. In contrast, the labeling den-sity of the small vesicles appeared to be somewhatgreater. In addition, the Golgi apparatus of these cellswas labeled (Figure 6E) and reactivity was presentalong the basal and lateral plasma membranes, espe-cially in regions with prominent membrane folding(Figure 6D).

Discussion

MG1 was present in all mucous cells of the subman-dibular and sublingual glands. No reactivity for MG1

Figure 2

Human salivary glands: EM immunogold labeling with anti-MG1 antibody. (

) Submandibular gland; mucous granules (mg) inthe mucous cells are labeled but the secretory granules (sg) in the adjacent serous demilune cell are unlabeled. (

) Sublingual gland; la-beling is seen in the mucous granules (mg) of the mucous cells but no labeling is present in the granules (sg) of the serous demilune cell. N,nucleus. Bars

was observed in serous cells. This pattern of reactivityis similar to the distribution of MG1 described in aprevious IHC study (Nielsen et al. 1996). Cohen et al.(1990) also found that MG1 was restricted to mucouscells, but noted that some mucous cells were unreac-tive. A possible explanation for this difference is thatthe antibody used by Cohen et al. (1990) was to a car-bohydrate epitope, whereas the antibody used byNielsen et al. (1996) was a monoclonal antibody di-rected against an epitope on the apomucin. The

antibody used in the present study is a polyclonalantibody that recognizes both polypeptide and carbo-hydrate epitopes (R.F. Troxler and G.D. Offner,unpublished observations). Heterogeneity among mu-cous cells with regard to the carbohydrate structurespresent on mucins has been described in other studies(Laden et al. 1984). In addition, ISH with an MUC5B-specific probe revealed MG1 transcripts in all mucouscells of the submandibular and sublingual glands(Nielsen et al. 1997).

Figure 3 Human submandibular gland: EM immunogold labeling with anti-MG1 antibody. Secretory granules (sg) of intercalated duct cellsshow reactivity. (Inset) MG1 labeling is associated with the apical plasma membrane of some intercalated duct cells. L, lumen. Bars � 0.5 �m.

Although labeling of the mucous granules withanti-MG1 was intense, little or no labeling of othermucous cell compartments was present. There are sev-eral possible explanations for this observation. Theconcentration of MG1 in the lumina of the endoplas-mic reticulum and Golgi saccules may be below thelimit of detection allowed by the tissue preparationand labeling methods employed. The association ofthe apomucin with other molecules in these compart-ments, e.g., molecular chaperones in the endoplasmicreticulum or glycosyltransferases in the endoplasmicreticulum and Golgi apparatus, might interfere withantibody labeling. Possible differences in conforma-tion of the mucin polypeptide related to the extent ofglycosylation could alter the reactivity of variousepitopes with the antibody.

Labeling for MG2 was more variable than forMG1. In five of six submandibular glands and two of

three sublingual glands examined, mucous cells exhib-ited weak to moderate labeling. Labeling of serousgranules in the submandibular gland was highly vari-able, even within the same serous cell. Serous cells ofthe sublingual gland were more consistently labeledfor MG2. In one submandibular and one sublingualgland the mucous cells exhibited very little reactivity,whereas the serous cells exhibited the most prominentlabeling. Therefore, the previous discrepancies re-ported for MG2 localization may reflect variability inthe level of expression of MG2 in the mucous and se-rous cells of different individuals. It is also possiblethat the heterogeneous labeling for MG2 observed inthe present study may be due to restricted access of theantibody as a result of the presence of different carbo-hydrate moieties on the mucin molecules.

Recently, the presence of other mucins has been de-scribed in the salivary glands. MUC1 (Newland et al.

Figure 4 Human sublingual gland: EM immunogold labeling with anti-MG2 antibody. Serous demilune cells show more uniform reactivityin their secretory granules (sg) than that detected on submandibular serous cells. (Inset) Labeled mucous granules (mg) are present in a mu-cous cell. N, nucleus. Bars � 0.5 �m.

1997; Sengupta et al. 2001; Liu et al. 2002) andMUC4 (Liu et al. 2002), membrane-associated mu-cins, have been localized to the ducts and some serouscells of human major and minor salivary glands.Moreover, the transmembrane component of rMuc4is reported to be present in serous cells and duct cellsof the major rat salivary glands (Li et al. 2001). Thepresence of MG1 and MG2 in the intercalated ductcells, as described in this study, suggests that thesecells express several types of mucins. The granules ofthe duct cells were labeled for both MG1 and MG2.The presence of MG2 reactivity in apical vesiclescould indicate an alternative route for secretion orcould represent endocytosis of MG2 from the duct lu-men. Labeling for MG1 was occasionally presentalong the apical membrane of the duct cells, presum-ably due to binding of secreted mucin. Membrane-associated and adherent mucins at the luminal surfaceof the duct cells may function to maintain duct pa-tency by preventing adherence of the luminal mem-branes of opposing cells (Hilkens et al. 1992; Sen-gupta et al. 2001). Liu et al. (2002) have suggestedthat the cytoplasmic domain of membrane-associatedmucins may be involved in signal transduction. Inter-estingly, labeling for MG2 also was consistently ob-served on the basolateral membranes of these cells.The significance of this localization remains to be de-termined.

Secretory granules of serous cells of human salivaryglands typically exhibit a bi- or tripartite ultrastruc-ture, with an electron-dense core and/or electron-dense crescents in an electron-lucent matrix (Tandlerand Erlandson 1972; Riva et al. 1974). Granules witha similar substructure are present in intercalated ductcells located adjacent to acinar cells (Lantini et al.1988). It is generally believed that these substructuralpatterns reflect the differential distribution of variousgranule components. Previous studies of the distribu-tion of amylase and agglutinin (Takano et al. 1991)and the H and Lewis-b blood group antigens (Cossu etal. 1990,1994) in human salivary glands support thishypothesis. The localization of MG2 to the electron-lucent portion of the serous granules and MG1 andMG2 to the electron-lucent portion of intercalatedduct cell granules is consistent with these earlier obser-vations.

The presence of MG2 in submandibular and sublin-gual serous cells, as described in this study and byNielsen et al. (1996,1997), but not in parotid serouscells, indicates that protein expression patterns differ

among the serous cells of human salivary glands. Asimilar situation exists for the salivary glands of otherspecies (Hand et al. 1999). The expression of MG2and possibly other mucins by salivary gland serouscells contributes to a blurring of the classical histolog-ical distinction between serous and mucous cells. Italso bears on the debate, based largely on histochemi-cal staining for glycoconjugates, over the use of theterms “serous” or “seromucous” to describe thesecells (Pinkstaff 1993). Additional studies that furtherdefine the molecular phenotypes of the secretory cellsof human salivary glands may help to settle these is-sues.

Acknowledgments

Supported by a fellowship from the Universita DegliStudi di Cagliari (MP), the University of Connecticut HealthCenter, and by NIH grants DE11691, DE07652, andDK44619.

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Human submandibular gland: EM immunogold labeling with anti-MG2 antibody. (

) Secretory granules (sg) of a serous acinarcell show a highly variable distribution of MG2. (

) Mucous cells (MC) display reactivity but no labeling is seen in the granules (sg) of the ad-jacent serous demilune cells. (

) Labeled mucous secretory granules (mg) in a mucous cell. N, nucleus. Bars

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Figure 6

Human submandibular gland: EM immunogold labeling with anti-MG2 antibody. (

) Intercalated duct cells display reactivity insecretory granules (sg) and small vesicles (arrows in B) located in their apical cytoplasm. (

) Section through the proximal part of an interca-lated duct (ID) adjacent to the acinus. Labeling of small vesicles in the duct cell is greater than that of the granules of the adjacent acinarcells. (

) Labeling is present in Golgi saccules (arrows in

) and is associated with the basal plasma membrane (arrows in

) of the ductcells. L, lumen; AC, acinar cell. Bars

Electron Microscopic Immunogold Localization of Salivary Mucins MG1 and MG2 in Human Submandibular...

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