MICROSCOPY RESEARCH AND TECHNIQUE 00:00–00 (2014)
Immunocytological Detection of Salivary Mucins (MUC5B) on the Mucosal Pellicle Lining Human Epithelial Buccal Cells ENE MARTINE MORZEL,1,2,3* SIYING TAI,1,2,3 HEL BRIGNOT,1,2,3 AND JEANNINE LHERMINIER4 1
CNRS, UMR6265 Centre des Sciences du Go^ ut et de l’Alimentation, F-21000 Dijon, France INRA, UMR1324 Centre des Sciences du Go^ ut et de l’Alimentation, F-21000 Dijon, France Universite de Bourgogne, UMR Centre des Sciences du Go^ ut et de l’Alimentation, F-21000 Dijon, France 4 INRA, UMR1347 Agroecologie, ERL CNRS 6300, Plateforme DImaCell, Centre de Microscopie INRA/Universite de Bourgogne, F-21000 Dijon, France 2 3
KEY WORDS
salivary film; oral epithelium; high-pressure freezing; transmission immunoelectron microscopy
ABSTRACT The mucosal pellicle is defined as the protein film adsorbed onto oral mucosa. This study aimed at characterizing the ultrastructure of human epithelial buccal cells and localizing salivary mucins MUC5B, a major constituent of the mucosal pellicle. Cells were sampled from the buccal surface and prepared for Transmission Electron Microscopy using high-pressure freezing/cryosubstitution followed by immunogold labelling of MUC5B. Morphologically, cells were visualized as typical cells of the superficial layer of a squamous nonkeratinized epithelium with a partly degraded plasma membrane. The outer surface of the plasma membrane was lined with a biological material of medium electron density. MUC5B were detected in the extracellular space, and particularly in the vicinity of the plasma membrane, sometimes onto fibrils protruding from the membrane. This area was, therefore, considered as constituting the mucosal pellicle, which appeared as a mixed film of both salivary and epithelial components. The distribution of gold particles suggested that the surface of the pellicle was not uniform, and that the film thickness could reach up to 100 nm. This work showed the feasibility of visualizing and characterizing the mucosal pellicle directly on human epithelial buccal cells sampled in a noninvasive manner. Microsc. Res. Tech. 00:000–000, 2014. V 2014 Wiley Periodicals, Inc. C
INTRODUCTION Surfaces of the oral cavity are exposed to intense friction during mastication, elocution, or deglutition. To protect the oral soft tissues, lubrication is provided by saliva and by a biological film bound to epithelial cells termed the mucosal pellicle (Bradway et al., 1989). The mucosal pellicle also plays a significant role in bacterial colonization of the oral surfaces (Bradway et al., 1992), and it was recently proposed that the structure and/or thickness of this film on the tongue may modulate taste perception by modifying accessibility of tastants to the taste receptors (Dsamou et al., 2012). It has been reported that the mucosal pellicle contains salivary proteins such as amylase and cystatins (Bradway et al., 1992) or carbonic anhydrase VI, IgA, and Secretory Component (Gibbins et al., in press). However, the most prominent constituents of pellicles are mucins (Gibbins et al., in press, Tabak et al., 1982), which can form hydrated gels lubricating the oral surfaces (Amerongen et al., 1995). Two types of mucins are secreted by salivary glands, the low molecular weight and high molecular weight species (Tabak, 1990) encoded, respectively, by the MUC7 and MUC5B genes. MUC5B mucin possesses lubricating properties exceeding that of MUC7 (Aguirre et al., 1989), which is due mainly to the carbohydrate portion of the molecule (Tabak, 1990). MUC5B is expressed as many glycoforms (Thornton et al., 1999), the carbohydrate moiety C V
2014 WILEY PERIODICALS, INC.
of the protein being constituted by a large variety of oligosaccharides (Thomsson et al., 2002). The current structural view of the pellicles on soft tissues, based on in vitro studies, is that of a heterogeneous film consisting of an inner anchoring dense layer and a lubricious outer looser layer containing mainly mucins (Macakova et al., 2011). This structure is somehow comparable to the enamel acquired pellicle formed in situ on buccal sites in 2 h, although this initial structure is modified by so-called “maturation.” Thus, after 24 h, the outer layer becomes dense, homogenous, and granular (Hannig and Joiner, 2006). Specifically for the mucosal pellicle, adsorption of salivary proteins to epithelial cells is thought to occur onto the partially degraded plasma membrane and the underlying cytoskeleton (Bradway et al., 1989). One should note, however, that the direct microscopical visualization of this film onto epithelial cells is not available. The objective of this study was to describe the morphology of human epithelial buccal cells and to precise the localization of MUC5B on such cells. We opted for cryo-fixation of samples, which is less susceptible to *Correspondence to: Martine Morzel, INRA-CSGA, 17 rue Sully, 21000 Dijon, France. E-mail: [email protected] Received 10 January 2014; accepted in revised form 25 March 2014 REVIEW EDITOR: Prof. George Perry DOI 10.1002/jemt.22366 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com).
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affect the cell morphology and modify the structure of the film in comparison to conventional chemical fixation. MATERIALS AND METHODS Cell Sampling Sampling was performed on two female donors with no overt sign of oral pathology, 2 h after teeth brushing. Immediately after saliva deglutition, cells were collected by scraping the buccal surface gently with a plastic spatula. The harvested cells were directly placed in the specimen carrier adapted to the highpressure equipment. Preparation of Samples for TEM Samples were immediately frozen, without the addition of cryoprotectant, in a Leica EM HPM 100 high-pressure freezer. Samples were stored into liquid nitrogen and freeze substitution was then performed using an automatic FS instrument (Leica EM AFS1) precooled to 290 C. The samples were substituted in a solution of anhydrous acetone/ 0.25% uranyl acetate at 290 C for 86 h. The temperature was gradually increased (slope 5 C/h) to 280 C for 8 h, 250 C for 24 h and maintained at this temperature. The samples were then rinsed with pure acetone for 24 h. They were then gradually infiltrated with mixtures of acetone/Lowicryl HM20 (increasing the concentrations of resin) and finally embedded in Lowicryl HM20. Polymerization was carried out at 250 C for 48 h, followed by 24 h at 235 C, and finally 24 h at 0 C. Embedded samples were stored at room temperature. Ultrathin sections were cut on a Reichert Ultracut E ultramicrotome (Leica, Rueil-Malmaison, France) and were collected onto carbon-collodion-coated nickel grids. Immunogold Labelling of MUC5B on Thin Sections of Buccal Cells Grids were treated with 10 mM TBS pH 7.2, supplemented with 0.1% glycine for 15 min to inactivate free aldehyde groups. They were then treated in TBS containing 0.5% acetylated Bovine Serum Albumin (BSAc) and 10% normal goat serum for 30 min. After rinsing with 0.5% BSA-c in TBS, sections were incubated with the mouse monoclonal antibody anti MUC5B F2 (gift of Professor Veerman, Amsterdam VrijeUniversiteit) at a 1/100 dilution in TBS 1 0.1% BSA-c for 1 h at room temperature. The F2 antibody recognizes the SO323Galb1–3GlcNAc moiety of sulfo-Lewisa epitope (Veerman et al., 1997). Sections incubated with medium devoid of primary antibody were used as negative controls. Antigen-antibody reaction was detected with ultra-small 1 nm gold-labeled goat anti-mouse IgG (Aurion) diluted 1/25 in TBS 1 0.1% BSA-c for 1 h at room temperature. After a chemical stabilization of the complex with TBS containing 2% glutaraldehyde, a silver enhancement procedure was performed to enlarge the 1 nm gold particles (Aurion, R-GENT SEEM). Grids were then examined with a Hitachi H7500 transmission electron microscope (Hitachi Scientific Instruments Co., Tokyo, Japan) operating at 80 kV and equipped with an AMT camera driven by AMT software (AMT, Danvers, USA).
RESULTS Morphological Observations The sampled cells exhibited a rather flattened shape and a remarkable scarcity of intracellular organelles (Fig. 1a). Remains of nuclei were visible on some cells (as pointed at in Fig. 1b). The cytoplasm was quite densely packed with tonofilaments and the plasma membrane presented deep ridges. The extracellular space showed filamentous material (see arrows on Fig. 1c), probably corresponding to saliva. At a higher magnification, it was observed that the outer surface of the plasma membrane was lined with a biological material with a medium electron density (Fig. 1d). The plasma membrane was partly discontinuous (see arrows on Fig. 1d), indicating a partial loss of integrity. Immunolocalization of MUC5B Figure 2a shows a typical picture of a negative immunological control, where virtually no gold particles were detected. When incubating with the F2 antibody, a heterogeneous distribution of gold particles was observed. For example in some cases, MUC5B was detected on extracellular filamentous material, most likely to be mobile saliva sampled with the cells, and not on the epithelial cells (Fig. 2b). However, in most cases, MUC5B was also detected close to the external leaflet of the plasma membrane, especially in the zone of medium electron density area observed at the surface of these cells (Figs. 2c and 2d). In such cases, gold particles were not distributed evenly along the plasma membrane. They often clustered as small groups of 2– 4 particles. In addition, MUC5B appeared occasionally attached to fibrils (arrows on Fig. 2d) protruding from the plasma membrane. Estimation of the Mucosal Pellicle Thickness Measurement of the distance between the gold particles and the cell membrane was attempted as an estimation of the mucosal pellicle thickness. Gold particles detected on the extracellular salivary filaments were not taken into account. When the plasma membrane appeared partly degraded, as in Figure 2d for example, its position was extrapolated. Focusing on gold particles detected in the zone of medium electron intensity near the plasma membrane, i.e., MUC5B adsorbed onto the plasma membrane as circled on Figure 2c for example, distance varied from 0 to 120 nm approximately. DISCUSSION From a morphological point of view, the sampled buccal cells had the typical appearance of cells from the superficial layer of a squamous nonkeratinized epithelium described for example by Squier (1991). In particular, the absence or paucity of organelles and the digitated plasma membrane were previously observed (Zelickson and Hartmann, 1962), while the density of tonofilaments present as a diffuse network was also noted in primary cultures of buccal epithelial cells (Selvaratnam et al., 2001). Membrane-coating granules, also considered as typical of epithelial cells, were not clearly distinguishable. However, in theory, such structures should be very rare in the top cell layer of the epithelium since they fuse with the plasma Microscopy Research and Technique
MICROSCOPICAL OBSERVATION OF HUMAN ORAL MUCOSAL PELLICLE
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Fig. 1. Morphological observations of cryo-fixed epithelial buccal cells. a: General appearance of buccal cells. b: Remains of a nucleus (indicated by an arrow). c: Buccal cell and extracellular filamentous material (indicated by arrows). d: Detail of the plasma membrane, showing some partial loss of integrity (indicated by arrows). The double arrows mark the thickness of the mucosal pellicle.
membrane and extrude their contents into the intercellular space in the intermediate epithelium cell layer (Squier and Kremer, 2001). This confirms that gentle scraping of the buccal surface resulted in sampling mainly superficial cells, i.e., those onto which the mucosal salivary film is adsorbed. This study also provided information on the morphology of the plasma membrane, which appeared as partly degraded. This observation is consistent with the suggestion that salivary proteins may adsorb onto partially denuded epithelial cell envelopes or the cytoskeleton associated to the epithelial plasma membranes (Bradway et al., 1989). The primary objective of this study was to visualize the mucosal pellicle on epithelial cells sampled on buccal surfaces of healthy subjects, and MUC5B was used as a constitutive marker of this pellicle. This methodological approach presents two main limitations. First, sampling and sample treatment does not allow to separate completely the mucosal pellicle from the mobile Microscopy Research and Technique
salivary film (or residual fluid) retained on mucosa after saliva deglutition, described and characterized for example by Collins and Dawes (1987). Indeed, sampling of cells immediately after deglutition limits the amount of whole flowing saliva in the sample, but the residual fluid is expected to be sampled together with the epithelial cells lined with mucosal pellicle. Decision on what constituted the mucosal pellicle, therefore, relied on its morphological appearance, and we considered very likely that the material of medium electron density lining the plasma membrane represents, at least in part, the mucosal pellicle. Analogy can be made with the enamel pellicle formed on buccal sites within 6 h, which consists of a thin inner electron-dense layer and on outer looser layer with both granular and globular formed areas (Hannig, 1997). Here, the inner dense layer was not clearly distinguishable from the plasma membrane. The outer layer appeared looser but no globular structures were observed.
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Fig. 2. Immunocytological detection of MUC5B on cryo-fixed epithelial buccal cells. a: Negative immunological control. b: Gold particles observed on extracellular filamentous material (saliva). c: Gold particles detected predominantly in the vicinity of the cell membrane. d: Gold particles attached to fine fibrils (indicated by arrows) protruding from the cell membrane.
The second limitation of the methodological approach is that MUC5B is not specific to the mucosal pellicle because it is present in whole saliva and in the mucosal residual fluid (Pramanik et al., 2010). Assignment of MUC5B to the mucosal pellicle, therefore, relied on the close vicinity of the detected epitopes to the plasma membrane and their presence in the material of medium electron density described above. Proteomic analysis of the enamel pellicle revealed that it is composed of secreted salivary proteins, proteins of epithelial cells and proteins of plasmatic origin (Siqueira et al., 2007). Here, the plasma membranes were partly discontinuous, suggesting that some intracellular content extruding from the plasma membrane could be incorporated into the mucosal pellicle. The present morphological observation supports the hypothesis that, comparably to the enamel pellicle, the mucosal pellicle comprises among other components constituents of epithelial origin and, therefore, that the mucosal pellicle is most probably a mixed film of epithelial and salivary proteins.
The immunolocalization of MUC5B onto buccal cells brought three main types of information. First, it showed that MUC5B was occasionally attached to microfibrils. Additional research would be necessary to determine the nature of these fibrils. It may for example be proposed that they are bundles of cytoskeletal proteins, which would be consistent with the suggestion of Bradway et al. (1989). Second, the distribution of gold particles suggested that mucin molecules or glycosylated parts of the molecules formed clusters and therefore that the surface of the mucosal pellicle was probably irregular rather than uniform. An alternative explanation may be that the epitope was not accessible on the particular planes of the observed sections. However, MUC5B is an oligomeric mucin (Wickstrom et al., 1998) which forms spontaneously aggregates on hydrophobic surfaces (Cardenas et al., 2007) and can form a reticular porous network when freeze-dried (Teubl et al., 2013). Our observations are, therefore, in line and support the overall vision of an irregular surface of the mucosal pellicle. Finally, the Microscopy Research and Technique
MICROSCOPICAL OBSERVATION OF HUMAN ORAL MUCOSAL PELLICLE
third type of information brought by this study relates to the dimension of the pellicle. Again, distance from gold particles to the plasma membrane (or to the extrapolated position of the plasma membrane when it was degraded) varied, in relation to the above described uneven topography of the film, and/or the unstructured nature of mucins which implies that they can adopt very different spatial conformations. However, the maximum distance measured was 120 nm. Taking into account the length of the primary and secondary antibodies’ assembly (20 nm), this suggests that the film can reach up to 100 nm in thickness. The mucosal pellicle on buccal cells are, therefore, thinner than enamel pellicles found on the buccal sides of the 1st molar or the 1st premolar, which can reach up to 500 nm and 300 nm, respectively (Hannig et al., 2009). The observed value is, however, above the calculated thickness of salivary film adsorbed onto a hydrophobic synthetic surface, which was reported to be around 20 nm at physiological ionic strength (Macakova et al., 2010). Another study reported a quite comparable average thickness when whole saliva adsorbs onto hydrophobized silica, but noted that larger aggregates of up to 50 nm in height were present (Cardenas et al., 2007). In this study, the maximum thickness may suggest that even larger mucin aggregates are included in the mucosal pellicle. Such results should be taken into account in the design of a realistic cellular model of the human buccal mucosa, comprising epithelial cells, the mucosal pellicle and also the mobile salivary film (or residual fluid) retained on mucosa after saliva deglutition. To conclude, this study showed the feasibility of visualizing and characterizing the mucosal pellicle on human epithelial buccal cells which opens new perspectives in oral sciences. In particular, it would be of interest to characterize between-subject variability in mucosal pellicle structure and/or thickness, which may be related to various oral health (e.g., xerostomia) or comfort (e.g., during speech or eating) indicators, or even to sensory perception experienced during food consumption. ACKNOWLEDGMENTS The authors thank Jo€el Michel (INRA, Plateforme DImaCell, Centre de Microscopie INRA/Universite de Bourgogne) for technical assistance in sample preparation. Dr Francis Canon is gratefully acknowledged for skilful preparation of the figures. REFERENCES Aguirre A, Mendoza B, Levine MJ, Hatton MN, Douglas WH. 1989. In vitro characterization of human salivary lubrication. Arch Oral Biol 34:675–677. Amerongen AVN, Bolscher JGM, Veerman ECI. 1995. Salivary mucins: Protective functions in relation to their diversity. Glycobiology 5:733–740. Bradway SD, Bergey EJ, Jones PC, Levine MJ. 1989. Oral mucosal pellicle—Adsorption and transpeptidation of salivary components to buccal epithelial cells. Biochem J 261:887–896.
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Bradway SD, Bergey EJ, Scannapieco FA, Ramasubbu N, Zawacki S, Levine MJ. 1992. Formation of salivary mucosal pellicle—The role of transglutaminase. Biochem J 284:557–564. Cardenas M, Elofsson U, Lindh L. 2007. Salivary mucin MUC5B could be an important component of in vitro pellicles of human saliva: An in situ ellipsometry and atomic force microscopy study. Biomacromolecules 8:1149–1156. Collins LMC, Dawes C. 1987. The surface area of the adult human mouth and thickness of the salivary film covering the teeth and oral mucosa. J Dent Res 66:1300–1302. Dsamou M, Palicki O, Septier C, Chabanet C, Lucchi G, Ducoroy P, Chagnon MC, Morzel M. 2012. Salivary protein profiles and sensitivity to the bitter taste of caffeine. Chem Senses 37:87–95. Gibbins HL, Proctor GB, Yakubov GE, Wilson S, Carpenter GH. Concentration of salivary protective proteins within the bound oral mucosal pellicle. Oral Dis, in press; DOI 10.1111/odi.12194. Hannig M. 1997. Transmision electron microscopic study of in vivo pellicle formation on dental restorative materials. Eur J Oral Sci 105:422–433. Hannig M, Joiner A. 2006. The structure, function and properties of the acquired pellicle. Monogr Oral Sci 19:29–64. Hannig C, Berndt D, Hoth-Hannig W, Hannig M. 2009. The effect of acidic beverages on the ultrastructure of the acquired pellicle-An in situ study. Arch Oral Biol 54:518–526. Macakova L, Yakubov GE, Plunkett MA, Stokes JR. 2010. Influence of ionic strength changes on the structure of pre-adsorbed salivary films. A response of a natural multi-component layer. Colloid Surface B 77:31–39. Macakova L, Yakubov GE, Plunkett MA, Stokes JR. 2011. Influence of ionic strength on the tribological properties of pre-adsorbed salivary films. Tribol Int 44:956–962. Pramanik R, Osailan SM, Challacombe SJ, Urquhart D, Proctor GB. 2010. Protein and mucin retention on oral mucosal surafces in dry mouth patients. Eur J Oral Sci 118:245–253. Selvaratnam L, Cruchley AT, Navsaria H, Wertz PW, Hagi-Pavli EP, Leigh IM, Squier CA, Williams DM. 2001. Permeability barrier properties of oral keratinocyte cultures: A model of intact human oral mucosa. Oral Dis 7:252–258. Siqueira WL, Zhang W, Helmerhorst EJ, Gygi SP, Oppenheim FG. 2007. Identification of protein components in in vivo human acquired enamel pellicle using LC-ESI-MS/MS. J Proteome Res 6: 2152–2160. Squier CA. 1991. The permeability of oral mucosa. Crit Rev Oral Biol M 2:13–32. Squier CA, Kremer M. 2001. Biology of oral mucosa and esophagus. JNCI Monogr 29:7–15. Tabak LA. 1990. Structure and function of human salivary mucins. Crit Rev Oral Biol M 1:229–234. Tabak LA, Levine MJ, Mandel ID, Ellison SA. 1982. Role of salivary mucins in the protection of the oral cavity. J Oral Pathol Med 11:1–17. Teubl BJ, Absenger M, Frohlich E, Leitinger G, Zimmer A, Roblegg E. 2013. The oral cavity as a biological barrier system: Design of an advanced buccal in vitro permeability model. Eur J Pharm Biopharm 84:386–393. Thomsson KA, Prakobphol A, Leffler H, Reddy MS, Levine MJ, Fisher SJ, Hansson GC. 2002. The salivary mucin MG1 (MUC5B) carries a repertoire of unique oligosaccharides that is large and diverse. Glycobiology 12:1–14. Thornton DJ, Khan N, Mehrotra R, Howard M, Veerman E, Packer NH, Sheehan JK. 1999. Salivary mucin MG1 is comprised almost entirely of different glycosylated forms of the MUC5B gene product. Glycobiology 9:293–302. Veerman ECI, Bolscher JGM, Appelmelk BJ, Bloemena E, vandenBerg TK, Amerongen A VN. 1997. A monoclonal antibody directed against high M(r) salivary mucins recognizes the SO33Gal beta 1-3GlcNAc moiety of sulfo-Lewis(a): A histochemical survey of human and rat tissue. Glycobiology 7:37–43. Wickstrom C, Davies JR, Eriksen GV, Veerman ECI, Carlstedt I. 1998. MUC5B is a major gel-forming, oligomeric mucin from human salivary gland, respiratory tract and endocervix: identification of glycoforms and C-terminal cleavage. Biochem J 334:685–693. Zelickson A, Hartmann J. 1962. An electron microscope study of normal human non-keratinizing oral mucosa. J Invest Dermatol 38: 99–109.
Immunocytological Detection of Salivary Mucins (MUC5B) on the Mucosal Pellicle Lining Human Epithelial Buccal Cells ENE MARTINE MORZEL,1,2,3* SIYING TAI,1,2,3 HEL BRIGNOT,1,2,3 AND JEANNINE LHERMINIER4 1
CNRS, UMR6265 Centre des Sciences du Go^ ut et de l’Alimentation, F-21000 Dijon, France INRA, UMR1324 Centre des Sciences du Go^ ut et de l’Alimentation, F-21000 Dijon, France Universite de Bourgogne, UMR Centre des Sciences du Go^ ut et de l’Alimentation, F-21000 Dijon, France 4 INRA, UMR1347 Agroecologie, ERL CNRS 6300, Plateforme DImaCell, Centre de Microscopie INRA/Universite de Bourgogne, F-21000 Dijon, France 2 3
KEY WORDS
salivary film; oral epithelium; high-pressure freezing; transmission immunoelectron microscopy
ABSTRACT The mucosal pellicle is defined as the protein film adsorbed onto oral mucosa. This study aimed at characterizing the ultrastructure of human epithelial buccal cells and localizing salivary mucins MUC5B, a major constituent of the mucosal pellicle. Cells were sampled from the buccal surface and prepared for Transmission Electron Microscopy using high-pressure freezing/cryosubstitution followed by immunogold labelling of MUC5B. Morphologically, cells were visualized as typical cells of the superficial layer of a squamous nonkeratinized epithelium with a partly degraded plasma membrane. The outer surface of the plasma membrane was lined with a biological material of medium electron density. MUC5B were detected in the extracellular space, and particularly in the vicinity of the plasma membrane, sometimes onto fibrils protruding from the membrane. This area was, therefore, considered as constituting the mucosal pellicle, which appeared as a mixed film of both salivary and epithelial components. The distribution of gold particles suggested that the surface of the pellicle was not uniform, and that the film thickness could reach up to 100 nm. This work showed the feasibility of visualizing and characterizing the mucosal pellicle directly on human epithelial buccal cells sampled in a noninvasive manner. Microsc. Res. Tech. 00:000–000, 2014. V 2014 Wiley Periodicals, Inc. C
INTRODUCTION Surfaces of the oral cavity are exposed to intense friction during mastication, elocution, or deglutition. To protect the oral soft tissues, lubrication is provided by saliva and by a biological film bound to epithelial cells termed the mucosal pellicle (Bradway et al., 1989). The mucosal pellicle also plays a significant role in bacterial colonization of the oral surfaces (Bradway et al., 1992), and it was recently proposed that the structure and/or thickness of this film on the tongue may modulate taste perception by modifying accessibility of tastants to the taste receptors (Dsamou et al., 2012). It has been reported that the mucosal pellicle contains salivary proteins such as amylase and cystatins (Bradway et al., 1992) or carbonic anhydrase VI, IgA, and Secretory Component (Gibbins et al., in press). However, the most prominent constituents of pellicles are mucins (Gibbins et al., in press, Tabak et al., 1982), which can form hydrated gels lubricating the oral surfaces (Amerongen et al., 1995). Two types of mucins are secreted by salivary glands, the low molecular weight and high molecular weight species (Tabak, 1990) encoded, respectively, by the MUC7 and MUC5B genes. MUC5B mucin possesses lubricating properties exceeding that of MUC7 (Aguirre et al., 1989), which is due mainly to the carbohydrate portion of the molecule (Tabak, 1990). MUC5B is expressed as many glycoforms (Thornton et al., 1999), the carbohydrate moiety C V
2014 WILEY PERIODICALS, INC.
of the protein being constituted by a large variety of oligosaccharides (Thomsson et al., 2002). The current structural view of the pellicles on soft tissues, based on in vitro studies, is that of a heterogeneous film consisting of an inner anchoring dense layer and a lubricious outer looser layer containing mainly mucins (Macakova et al., 2011). This structure is somehow comparable to the enamel acquired pellicle formed in situ on buccal sites in 2 h, although this initial structure is modified by so-called “maturation.” Thus, after 24 h, the outer layer becomes dense, homogenous, and granular (Hannig and Joiner, 2006). Specifically for the mucosal pellicle, adsorption of salivary proteins to epithelial cells is thought to occur onto the partially degraded plasma membrane and the underlying cytoskeleton (Bradway et al., 1989). One should note, however, that the direct microscopical visualization of this film onto epithelial cells is not available. The objective of this study was to describe the morphology of human epithelial buccal cells and to precise the localization of MUC5B on such cells. We opted for cryo-fixation of samples, which is less susceptible to *Correspondence to: Martine Morzel, INRA-CSGA, 17 rue Sully, 21000 Dijon, France. E-mail: [email protected] Received 10 January 2014; accepted in revised form 25 March 2014 REVIEW EDITOR: Prof. George Perry DOI 10.1002/jemt.22366 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com).
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affect the cell morphology and modify the structure of the film in comparison to conventional chemical fixation. MATERIALS AND METHODS Cell Sampling Sampling was performed on two female donors with no overt sign of oral pathology, 2 h after teeth brushing. Immediately after saliva deglutition, cells were collected by scraping the buccal surface gently with a plastic spatula. The harvested cells were directly placed in the specimen carrier adapted to the highpressure equipment. Preparation of Samples for TEM Samples were immediately frozen, without the addition of cryoprotectant, in a Leica EM HPM 100 high-pressure freezer. Samples were stored into liquid nitrogen and freeze substitution was then performed using an automatic FS instrument (Leica EM AFS1) precooled to 290 C. The samples were substituted in a solution of anhydrous acetone/ 0.25% uranyl acetate at 290 C for 86 h. The temperature was gradually increased (slope 5 C/h) to 280 C for 8 h, 250 C for 24 h and maintained at this temperature. The samples were then rinsed with pure acetone for 24 h. They were then gradually infiltrated with mixtures of acetone/Lowicryl HM20 (increasing the concentrations of resin) and finally embedded in Lowicryl HM20. Polymerization was carried out at 250 C for 48 h, followed by 24 h at 235 C, and finally 24 h at 0 C. Embedded samples were stored at room temperature. Ultrathin sections were cut on a Reichert Ultracut E ultramicrotome (Leica, Rueil-Malmaison, France) and were collected onto carbon-collodion-coated nickel grids. Immunogold Labelling of MUC5B on Thin Sections of Buccal Cells Grids were treated with 10 mM TBS pH 7.2, supplemented with 0.1% glycine for 15 min to inactivate free aldehyde groups. They were then treated in TBS containing 0.5% acetylated Bovine Serum Albumin (BSAc) and 10% normal goat serum for 30 min. After rinsing with 0.5% BSA-c in TBS, sections were incubated with the mouse monoclonal antibody anti MUC5B F2 (gift of Professor Veerman, Amsterdam VrijeUniversiteit) at a 1/100 dilution in TBS 1 0.1% BSA-c for 1 h at room temperature. The F2 antibody recognizes the SO323Galb1–3GlcNAc moiety of sulfo-Lewisa epitope (Veerman et al., 1997). Sections incubated with medium devoid of primary antibody were used as negative controls. Antigen-antibody reaction was detected with ultra-small 1 nm gold-labeled goat anti-mouse IgG (Aurion) diluted 1/25 in TBS 1 0.1% BSA-c for 1 h at room temperature. After a chemical stabilization of the complex with TBS containing 2% glutaraldehyde, a silver enhancement procedure was performed to enlarge the 1 nm gold particles (Aurion, R-GENT SEEM). Grids were then examined with a Hitachi H7500 transmission electron microscope (Hitachi Scientific Instruments Co., Tokyo, Japan) operating at 80 kV and equipped with an AMT camera driven by AMT software (AMT, Danvers, USA).
RESULTS Morphological Observations The sampled cells exhibited a rather flattened shape and a remarkable scarcity of intracellular organelles (Fig. 1a). Remains of nuclei were visible on some cells (as pointed at in Fig. 1b). The cytoplasm was quite densely packed with tonofilaments and the plasma membrane presented deep ridges. The extracellular space showed filamentous material (see arrows on Fig. 1c), probably corresponding to saliva. At a higher magnification, it was observed that the outer surface of the plasma membrane was lined with a biological material with a medium electron density (Fig. 1d). The plasma membrane was partly discontinuous (see arrows on Fig. 1d), indicating a partial loss of integrity. Immunolocalization of MUC5B Figure 2a shows a typical picture of a negative immunological control, where virtually no gold particles were detected. When incubating with the F2 antibody, a heterogeneous distribution of gold particles was observed. For example in some cases, MUC5B was detected on extracellular filamentous material, most likely to be mobile saliva sampled with the cells, and not on the epithelial cells (Fig. 2b). However, in most cases, MUC5B was also detected close to the external leaflet of the plasma membrane, especially in the zone of medium electron density area observed at the surface of these cells (Figs. 2c and 2d). In such cases, gold particles were not distributed evenly along the plasma membrane. They often clustered as small groups of 2– 4 particles. In addition, MUC5B appeared occasionally attached to fibrils (arrows on Fig. 2d) protruding from the plasma membrane. Estimation of the Mucosal Pellicle Thickness Measurement of the distance between the gold particles and the cell membrane was attempted as an estimation of the mucosal pellicle thickness. Gold particles detected on the extracellular salivary filaments were not taken into account. When the plasma membrane appeared partly degraded, as in Figure 2d for example, its position was extrapolated. Focusing on gold particles detected in the zone of medium electron intensity near the plasma membrane, i.e., MUC5B adsorbed onto the plasma membrane as circled on Figure 2c for example, distance varied from 0 to 120 nm approximately. DISCUSSION From a morphological point of view, the sampled buccal cells had the typical appearance of cells from the superficial layer of a squamous nonkeratinized epithelium described for example by Squier (1991). In particular, the absence or paucity of organelles and the digitated plasma membrane were previously observed (Zelickson and Hartmann, 1962), while the density of tonofilaments present as a diffuse network was also noted in primary cultures of buccal epithelial cells (Selvaratnam et al., 2001). Membrane-coating granules, also considered as typical of epithelial cells, were not clearly distinguishable. However, in theory, such structures should be very rare in the top cell layer of the epithelium since they fuse with the plasma Microscopy Research and Technique
MICROSCOPICAL OBSERVATION OF HUMAN ORAL MUCOSAL PELLICLE
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Fig. 1. Morphological observations of cryo-fixed epithelial buccal cells. a: General appearance of buccal cells. b: Remains of a nucleus (indicated by an arrow). c: Buccal cell and extracellular filamentous material (indicated by arrows). d: Detail of the plasma membrane, showing some partial loss of integrity (indicated by arrows). The double arrows mark the thickness of the mucosal pellicle.
membrane and extrude their contents into the intercellular space in the intermediate epithelium cell layer (Squier and Kremer, 2001). This confirms that gentle scraping of the buccal surface resulted in sampling mainly superficial cells, i.e., those onto which the mucosal salivary film is adsorbed. This study also provided information on the morphology of the plasma membrane, which appeared as partly degraded. This observation is consistent with the suggestion that salivary proteins may adsorb onto partially denuded epithelial cell envelopes or the cytoskeleton associated to the epithelial plasma membranes (Bradway et al., 1989). The primary objective of this study was to visualize the mucosal pellicle on epithelial cells sampled on buccal surfaces of healthy subjects, and MUC5B was used as a constitutive marker of this pellicle. This methodological approach presents two main limitations. First, sampling and sample treatment does not allow to separate completely the mucosal pellicle from the mobile Microscopy Research and Technique
salivary film (or residual fluid) retained on mucosa after saliva deglutition, described and characterized for example by Collins and Dawes (1987). Indeed, sampling of cells immediately after deglutition limits the amount of whole flowing saliva in the sample, but the residual fluid is expected to be sampled together with the epithelial cells lined with mucosal pellicle. Decision on what constituted the mucosal pellicle, therefore, relied on its morphological appearance, and we considered very likely that the material of medium electron density lining the plasma membrane represents, at least in part, the mucosal pellicle. Analogy can be made with the enamel pellicle formed on buccal sites within 6 h, which consists of a thin inner electron-dense layer and on outer looser layer with both granular and globular formed areas (Hannig, 1997). Here, the inner dense layer was not clearly distinguishable from the plasma membrane. The outer layer appeared looser but no globular structures were observed.
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Fig. 2. Immunocytological detection of MUC5B on cryo-fixed epithelial buccal cells. a: Negative immunological control. b: Gold particles observed on extracellular filamentous material (saliva). c: Gold particles detected predominantly in the vicinity of the cell membrane. d: Gold particles attached to fine fibrils (indicated by arrows) protruding from the cell membrane.
The second limitation of the methodological approach is that MUC5B is not specific to the mucosal pellicle because it is present in whole saliva and in the mucosal residual fluid (Pramanik et al., 2010). Assignment of MUC5B to the mucosal pellicle, therefore, relied on the close vicinity of the detected epitopes to the plasma membrane and their presence in the material of medium electron density described above. Proteomic analysis of the enamel pellicle revealed that it is composed of secreted salivary proteins, proteins of epithelial cells and proteins of plasmatic origin (Siqueira et al., 2007). Here, the plasma membranes were partly discontinuous, suggesting that some intracellular content extruding from the plasma membrane could be incorporated into the mucosal pellicle. The present morphological observation supports the hypothesis that, comparably to the enamel pellicle, the mucosal pellicle comprises among other components constituents of epithelial origin and, therefore, that the mucosal pellicle is most probably a mixed film of epithelial and salivary proteins.
The immunolocalization of MUC5B onto buccal cells brought three main types of information. First, it showed that MUC5B was occasionally attached to microfibrils. Additional research would be necessary to determine the nature of these fibrils. It may for example be proposed that they are bundles of cytoskeletal proteins, which would be consistent with the suggestion of Bradway et al. (1989). Second, the distribution of gold particles suggested that mucin molecules or glycosylated parts of the molecules formed clusters and therefore that the surface of the mucosal pellicle was probably irregular rather than uniform. An alternative explanation may be that the epitope was not accessible on the particular planes of the observed sections. However, MUC5B is an oligomeric mucin (Wickstrom et al., 1998) which forms spontaneously aggregates on hydrophobic surfaces (Cardenas et al., 2007) and can form a reticular porous network when freeze-dried (Teubl et al., 2013). Our observations are, therefore, in line and support the overall vision of an irregular surface of the mucosal pellicle. Finally, the Microscopy Research and Technique
MICROSCOPICAL OBSERVATION OF HUMAN ORAL MUCOSAL PELLICLE
third type of information brought by this study relates to the dimension of the pellicle. Again, distance from gold particles to the plasma membrane (or to the extrapolated position of the plasma membrane when it was degraded) varied, in relation to the above described uneven topography of the film, and/or the unstructured nature of mucins which implies that they can adopt very different spatial conformations. However, the maximum distance measured was 120 nm. Taking into account the length of the primary and secondary antibodies’ assembly (20 nm), this suggests that the film can reach up to 100 nm in thickness. The mucosal pellicle on buccal cells are, therefore, thinner than enamel pellicles found on the buccal sides of the 1st molar or the 1st premolar, which can reach up to 500 nm and 300 nm, respectively (Hannig et al., 2009). The observed value is, however, above the calculated thickness of salivary film adsorbed onto a hydrophobic synthetic surface, which was reported to be around 20 nm at physiological ionic strength (Macakova et al., 2010). Another study reported a quite comparable average thickness when whole saliva adsorbs onto hydrophobized silica, but noted that larger aggregates of up to 50 nm in height were present (Cardenas et al., 2007). In this study, the maximum thickness may suggest that even larger mucin aggregates are included in the mucosal pellicle. Such results should be taken into account in the design of a realistic cellular model of the human buccal mucosa, comprising epithelial cells, the mucosal pellicle and also the mobile salivary film (or residual fluid) retained on mucosa after saliva deglutition. To conclude, this study showed the feasibility of visualizing and characterizing the mucosal pellicle on human epithelial buccal cells which opens new perspectives in oral sciences. In particular, it would be of interest to characterize between-subject variability in mucosal pellicle structure and/or thickness, which may be related to various oral health (e.g., xerostomia) or comfort (e.g., during speech or eating) indicators, or even to sensory perception experienced during food consumption. ACKNOWLEDGMENTS The authors thank Jo€el Michel (INRA, Plateforme DImaCell, Centre de Microscopie INRA/Universite de Bourgogne) for technical assistance in sample preparation. Dr Francis Canon is gratefully acknowledged for skilful preparation of the figures. REFERENCES Aguirre A, Mendoza B, Levine MJ, Hatton MN, Douglas WH. 1989. In vitro characterization of human salivary lubrication. Arch Oral Biol 34:675–677. Amerongen AVN, Bolscher JGM, Veerman ECI. 1995. Salivary mucins: Protective functions in relation to their diversity. Glycobiology 5:733–740. Bradway SD, Bergey EJ, Jones PC, Levine MJ. 1989. Oral mucosal pellicle—Adsorption and transpeptidation of salivary components to buccal epithelial cells. Biochem J 261:887–896.
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