ARTICLE |
Correspondence to: Arthur R. Hand, Dept. of Pediatric Dentistry, U. of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-1610. E-mail: hand@nso1.uchc.edu
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The human salivary mucins MG1 and MG2 are well characterized biochemically and functionally. However, there is disagreement regarding their cellular and glandular sources. The aim of this study was to define the localization and distribution of these two mucins in human salivary glands using a postembedding immunogold labeling method. Normal salivary glands obtained at surgery were fixed in 3% paraformaldehyde0.1% glutaraldehyde and embedded in Lowicryl K4M or LR Gold resin. Thin sections were labeled with rabbit antibodies to MG1 or to an N-terminal synthetic peptide of MG2, followed by gold-labeled goat anti-rabbit IgG. The granules of all mucous cells of the submandibular and sublingual glands were intensely reactive with anti-MG1. No reaction was detected in serous cells. With anti-MG2, the granules of both mucous and serous cells showed reactivity. The labeling was variable in both cell types, with mucous cells exhibiting a stronger reaction in some glands and serous cells in others. In serous granules, the electron-lucent regions were more reactive than the dense cores. Intercalated duct cells near the acini displayed both MG1 and MG2 reactivity in their apical granules. In addition, the basal and lateral membranes of intercalated duct cells were labeled with anti-MG2. These results confirm those of earlier studies on MG1 localization in mucous cells and suggest that MG2 is produced by both mucous and serous cells. They also indicate differences in protein expression patterns among salivary serous cells. (J Histochem Cytochem 51:6979, 2003)
Key Words: salivary glands, mucous cells, serous cells, intercalated ducts, immunohistochemistry/ postembedding, immunogold labeling
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SALIVARY MUCINS are large, highly glycosylated proteins that have multiple functions in the oral cavity (
Human saliva contains two major mucin components, MG1 and MG2. The high molecular weight MG1 consists mainly of the MUC5B gene product (
The purpose of the present study was to further investigate the distribution of these two mucins in human salivary gland tissue using immunoelectron microscopy.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Samples of normal submandibular and sublingual glands were obtained from eight consenting male and female patients, aged 4469 years, undergoing surgery at the Otorhinolaryngology Clinic, University of Cagliari, Cagliari, Italy. All procedures were approved by the Human Experimentation Committee, University of Cagliari. In addition, one sublingual gland was obtained from a 7-year-old boy undergoing sialadenectomy at the Hospital for Sick Children, Toronto, Canada. The procedure was approved by the Hospital Committee for Human Experimentation. The various samples studied are summarized in Table 1.
|
For light microscopic studies the glands were fixed overnight in 4% paraformaldehyde (Polysciences; Warrington, PA) in 0.1 M sodium cacodylate buffer, pH 7.2, then stored in 1% paraformaldehyde in cacodylate buffer at 4C. The samples were rinsed in 0.1 M cacodylate buffer, dehydrated in cold methanol, embedded in LR Gold resin (Polysciences), and polymerized under UV light (365 nm) at -20C. One sample was dehydrated in ethanol, embedded in LR White resin, and polymerized overnight at 50C. One-micrometer sections were collected on glass slides and incubated for 90 min at room temperature (RT) with rabbit polyclonal antibodies to MG1 (
Tissue processing for electron microscopy employed fixation of small pieces (1 mm3) of the same samples in a mixture of 3% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer for 2 hr, after which the glands were stored in 1% paraformaldehyde in cacodylate buffer. After rinsing in buffer, the tissues were dehydrated in cold methanol, embedded in either LR Gold or Lowicryl K4M resin (Polysciences), and polymerized as described above. One gland was fixed in 1% glutaraldehyde and embedded in LR White resin. Ultrathin sections were collected on Formvar-coated nickel grids and treated with either 1% BSA1% instant milk in PBS or fish gelatin, ovalbumin, and Tween-20 in PBS to block nonspecific binding. The sections were incubated with anti-MG1 diluted 1:1001:500 in 1% BSA5% NGS in PBS for 60 min at RT or with anti-MG2 diluted 1:501:200 in fish gelatin, ovalbumin, and Tween-20 in PBS overnight at 4C. Omission of the primary antibody or incubation with nonimmune rabbit serum was used as control. After rinsing with PBS, the grids were incubated for 60 min at RT with goat anti-rabbit IgG labeled with 10-nm gold particles (Amersham). The grids were washed with PBS and distilled water, stained with uranyl acetate and lead citrate, and observed and photographed in a JEOL 100CX TEM.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Specific reactivity for MG1 was detected at the light microscopic level (Fig 1A and Fig 1B) on the granules of all mucous cells of the submandibular and sublingual glands examined at dilutions of the primary antibody of 1:10001:2000. The granules of some intercalated duct cells located adjacent to the acini also showed labeling (Fig 1F). No reactivity was found in serous acinar or demilune cells. Striated duct cells also were devoid of labeling.
|
In the same glands, MG2 labeling exhibited a different distribution. Both mucous and serous components of the tissues showed reactivity at dilutions of the primary antibody of 1:5001:1000. In most glands examined, the intensity of labeling of serous cells was moderate, whereas that of mucous cells was somewhat weaker. The labeling of both cell types with the anti-MG2 antibody, however, exhibited significant variability. In some glands, mucous cells, especially those of smaller size, labeled strongly (Fig 1D and Fig 1E). In the sublingual gland from the young individual, the serous demilune cells were strongly reactive, whereas the mucous acinar cells exhibited relatively weak labeling (Fig 1C). Intercalated duct cells located near acini exhibited labeling in their apical cytoplasm (Fig 1G). These cells were more consistently reactive than the mucous and serous cells. No labeling was seen in striated duct cells.
In the absence of the primary antibody, or when nonimmune IgG was substituted for the primary antibody, no specific labeling was observed.
Electron microscopic immunogold labeling confirmed and extended the light microscopic observations. The granules of all mucous cells in the submandibular and sublingual glands examined were intensely labeled after incubation with anti-MG1 antibody at dilutions of 1:1001:500 (Fig 2A2D). Nonspecific labeling of nuclei, mitochondria, and extracellular spaces was minimal. No labeling was observed in serous cells, and no substantial differences in the distribution and intensity of MG1 reactivity were observed between submandibular and sublingual glands.
|
The granules of some intercalated duct cells, particularly cells located near the acini, were labeled with the anti-MG1 antibody (Fig 3). The gold particles were present mainly over the electron-lucent portion of these granules. The labeling density of the intercalated 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 surface of the duct cells (Fig 3, inset), suggesting that this surface may be coated with MG1.
|
Ultrathin sections of the submandibular and sublingual glands incubated with the anti-MG2 antibody (1:501:200) exhibited specific reactivity in both mucous and serous secretory granules (Fig 4 and Fig 5). As observed by light microscopy, the distribution and intensity of labeling with the anti-MG2 antibody reactivity showed greater variability than that observed for MG1. In the samples examined, the granules of most mucous cells had low levels of labeling, and many serous cells had unreactive secretory granules (Fig 5B). Only a few serous acinar cells displayed labeling in the lucent halo of their granules. In one of the submandibular glands and one of the sublingual glands (Fig 4) studied, a more intense and uniform labeling was found in the electron-lucent halo of the serous granules, and mucous acinar cells in these glands were either unreactive or only weakly labeled. The dense cores of the serous granules were almost unreactive. Occasionally they were labeled to a similar extent as in sections incubated with appropriate dilutions of normal rabbit serum, suggesting nonspecific binding of the primary antibody to the electron-dense cores of the serous granules. The endoplasmic reticulum and Golgi apparatus of the mucous and serous cells consistently were unlabeled.
|
|
Intercalated duct cells located near acini also displayed MG2 reactivity in secretory granules and vesicles located in their apical cytoplasm (Fig 6A6C). As noted for intercalated duct cells labeled with anti-MG1 antibody, gold particles representing MG2 reactivity were restricted mainly to the electron-lucent portion of the granules. The labeling density of the intercalated duct cell granules was similar to that seen in granules of serous cells. In contrast, the labeling density of the small vesicles appeared to be somewhat greater. In addition, the Golgi apparatus of these cells was labeled (Fig 6E) and reactivity was present along the basal and lateral plasma membranes, especially in regions with prominent membrane folding (Fig 6D).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
MG1 was present in all mucous cells of the submandibular and sublingual glands. No reactivity for MG1 was observed in serous cells. This pattern of reactivity is similar to the distribution of MG1 described in a previous IHC study (
Although labeling of the mucous granules with anti-MG1 was intense, little or no labeling of other mucous cell compartments was present. There are several possible explanations for this observation. The concentration of MG1 in the lumina of the endoplasmic reticulum and Golgi saccules may be below the limit of detection allowed by the tissue preparation and labeling methods employed. The association of the apomucin with other molecules in these compartments, e.g., molecular chaperones in the endoplasmic reticulum or glycosyltransferases in the endoplasmic reticulum and Golgi apparatus, might interfere with antibody labeling. Possible differences in conformation of the mucin polypeptide related to the extent of glycosylation could alter the reactivity of various epitopes with the antibody.
Labeling for MG2 was more variable than for MG1. In five of six submandibular glands and two of three sublingual glands examined, mucous cells exhibited weak to moderate labeling. Labeling of serous granules in the submandibular gland was highly variable, even within the same serous cell. Serous cells of the sublingual gland were more consistently labeled for MG2. In one submandibular and one sublingual gland the mucous cells exhibited very little reactivity, whereas the serous cells exhibited the most prominent labeling. Therefore, the previous discrepancies reported for MG2 localization may reflect variability in the level of expression of MG2 in the mucous and serous cells of different individuals. It is also possible that the heterogeneous labeling for MG2 observed in the present study may be due to restricted access of the antibody as a result of the presence of different carbohydrate moieties on the mucin molecules.
Recently, the presence of other mucins has been described in the salivary glands. MUC1 (
Secretory granules of serous cells of human salivary glands typically exhibit a bi- or tripartite ultrastructure, with an electron-dense core and/or electron-dense crescents in an electron-lucent matrix (
The presence of MG2 in submandibular and sublingual serous cells, as described in this study and by
![]() |
Acknowledgments |
---|
Supported by a fellowship from the Universita Degli Studi di Cagliari (MP), the University of Connecticut Health Center, and by NIH grants DE11691, DE07652, and DK44619.
Received for publication December 10, 2001; accepted August 23, 2002.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Al-Hashimi I, Levine MJ (1989) Characterization of in vivo salivary-derived enamel pellicle. Arch Oral Biol 34:289-295[Medline]
Audie JP, Janin A, Porchet N, Copin MC, Gosselin B, Aubert JP (1993) Expression of human mucin genes in respiratory, digestive, and reproductive tracts ascertained by in situ hybridization. J Histochem Cytochem 41:1479-1485
Bobek LA, Tsai G, Biesbrock AR, Levine MJ (1993) Molecular cloning, sequence and specificity of expression of the gene encoding the low molecular weight human salivary mucin (MUC7). J Biol Chem 268:20563-20569
Cohen RE, Aguirre A, Neiders ME, Levine MJ, Jones PC, Reddy MS, Haar JG (1990) Immunochemistry of high molecular weight human salivary mucin. Arch Oral Biol 35:127-136[Medline]
Cohen RE, Aguirre A, Neiders ME, Levine MJ, Jones PC, Reddy MS, Haar JG (1991) Immunochemistry and immunogenicity of low molecular weight human salivary mucin. Arch Oral Biol 36:347-356[Medline]
Cossu M, Lantini MS, Puxeddu R (1994) Immunocytochemical localization of Lewis blood group antigens in human salivary glands. J Histochem Cytochem 42:1135-1142
Cossu M, Riva A, Lantini MS (1990) Subcellular localization of blood group substances ABH in human salivary glands. J Histochem Cytochem 38:1165-1172[Abstract]
Hand AR, Pathmanathan D, Field RB (1999) Morphological features of the minor salivary glands. Arch Oral Biol 44:S3-10[Medline]
Hilkens J, Ligtenberg MJL, Vos HL, Litvinov SV (1992) Cell membrane-associated mucins and their adhesion-modulating property. Trends Biochem Sci 17:359-363[Medline]
Khan SH, Aguirre A, Bobek LA (1998) In-situ hybridization localized MUC7 mucin gene expression to the mucous acinar cells of human and MUC7-transgenic mouse salivary glands. Glycoconjugate J 15:1125-1132[Medline]
Laden SA, Schulte BA, Spicer SS (1984) Histochemical evaluation of secretory glycoproteins in human salivary glands with lectin-horseradish peroxidase conjugates. J Histochem Cytochem 32:965-972[Abstract]
Lantini MS, Valentino L, Riva A (1988) A granular cell in the proximal intercalated duct of human parotid and submandibular glands. J Submicrosc Cytol Pathol 20:147-152[Medline]
Li P, Arango ME, Perez RE, Reis CA, Bonfante EL, Weed D, Carraway KL (2001) Expression and localization of immunoreactive-sialomucin complex (Muc4) in salivary glands. Tissue Cell 33:111-118[Medline]
Liu B, Lague JR, Nunes DP, Toselli P, Oppenheim FG, Soares RV, Troxler RF, Offner GD (2002) Expression of membrane-associated mucins MUC1 and MUC4 in major human salivary glands. J Histochem Cytochem 50:811-820
Liu B, Rayment S, Oppenheim FG, Troxler RF (1999) Isolation of human salivary mucin MG2 by a novel method and characterization of its interactions with oral bacteria. Arch Biochem Biophys 364:286-293[Medline]
Newland JR, Jacob R, McDaniel RK, Durban EM (1997) MUC-1 mucin immunodetection in human salivary glands. J Dent Res 76:342
Nielsen PA, Bennett EP, Wandall HH, Therkildsen MH, Hannibal J, Clausen H (1997) Identification of a major human high molecular weight salivary mucin (MG1) as tracheobronchial mucin MUC5B. Glycobiology 7:413-419[Abstract]
Nielsen PA, Mandel U, Therkildsen MH, Clausen H (1996) Differential expression of human high-molecular-weight salivary mucin (MG1) and low-molecular-weight salivary mucin (MG2). J Dent Res 75:1820-1826[Abstract]
Pinkstaff CA (1993) Serous, seromucous, and special serous cells in salivary glands. Microsc Res Tech 26:21-31[Medline]
Rayment SA, Liu B, Offner GD, Oppenheim FG, Troxler RF (2000a) Salivary mucin: a factor in the lower prevalence of gastroesophageal reflux disease in African-Americans? Am J Gastroenterol 95:3064-3070[Medline]
Rayment SA, Liu B, Offner GD, Oppenheim FG, Troxler RF (2000b) Immunoquantification of human salivary mucins MG1 and MG2 in stimulated whole saliva: factors influencing mucin levels. J Dent Res 79:1765-1772[Abstract]
Riva A, Motta G, RivaTesta F (1974) Ultrastructural diversity in secretory granules of human major salivary glands. Am J Anat 139:293-298[Medline]
Sengupta A, Valdramidou D, Huntley S, Hicks SJ, Carrington SD, Corfield AP (2001) Distribution of MUC1 in the normal human oral cavity is localized to the ducts of minor salivary glands. Arch Oral Biol 46:529-538[Medline]
Sharma P, Dudus L, Nielsen PA, Clausen H, Yankaskas JR, Hollingsworth MA, Engelhardt JF (1998) MUC5B and MUC7 are differentially expressed in mucous and serous cells of submucosal glands in human bronchial airways. Am J Respir Cell Mol Biol 19:30-37
Tabak LA (1995) In defense of the oral cavity: structure, biosynthesis, and function of salivary mucins. Annu Rev Physiol 57:547-564[Medline]
Takano K, Bogert M, Malamud D, Lally E, Hand AR (1991) Differential distribution of salivary agglutinin and amylase in the Golgi apparatus and secretory granules of human salivary gland acinar cells. Anat Rec 230:307-318[Medline]
Tandler B, Erlandson RA (1972) Ultrastructure of the human submaxillary gland. IV. Serous granules. Am J Anat 135:419-433[Medline]
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
Troxler RF, Iontcheva I, Oppenheim FG, Nunes DP, Offner GD (1997) Molecular characterization of a major high molecular weight mucin from human sublingual gland. Glycobiology 7:965-973[Abstract]
Troxler RF, Offner GD, Zhang F, Iontcheva I, Oppenheim FG (1995) Molecular cloning of a novel high molecular weight mucin from human sublingual gland. Biochem Biophys Res Commun 217:1112-1119[Medline]