Human salivary gland acinar cells spontaneously form three-dimensional structures and change the protein expression patterns

Applying tissue engineering principles to design an auto-secretory device is a potential solution for patients suffering loss of salivary gland function. However, the largest challenge in implementing this solution is the primary culture of human salivary gland cells, because the cells are highly differentiated and difficult to expand in vitro. This situation leads to the lack of reports on the in vitro cell biology and physiology of human salivary gland cells. This study used a low-calcium culture system to selectively cultivate human parotid gland acinar (PGAC) cells from tissues with high purity in cell composition. This condition enables PGAC cells to continuously proliferate and retain the phenotypes of epithelial acinar cells to express secreting products (α-amylase) and function-related proteins (aquaporin-3, aquaporin-5, and ZO-1). Notably, when the cells reached confluence, three-dimensional (3D) cell aggregates were observed in crowded regions. These self-formed cell spheres were termed post-confluence structures (PCSs). Unexpectedly, despite being cultured in the same media, cells in PCSs exhibited higher expression levels and different expression patterns of function-related proteins compared to the two-dimensional (2D) cells. Translocation of aquoporin-3 from cytosolic to alongside the cell boundaries, and of ZO-1 molecules to the boundary of the PCSs were also observed. These observations suggest that when PGAC cells cultured on the 2D substrate would form PCSs without the help of 3D scaffolds and retain certain differentiation and polarity. This phenomenon implies that it is possible to introduce 2D substrates instead of 3D scaffolds into artificial salivary gland tissue engineering.     

A novel chemokine ligand for CCR10 and CCR3 expressed by epithelial cells in mucosal tissues

Mucosae-associated epithelial chemokine (MEC) is a novel chemokine whose mRNA is most abundant in salivary gland, with strong expression in other mucosal sites, including colon, trachea, and mammary gland. MEC is constitutively expressed by epithelial cells; MEC mRNA is detected in cultured bronchial and mammary gland epithelial cell lines and in epithelia isolated from salivary gland and colon using laser capture microdissection, but not in the endothelial, hemolymphoid, or fibroblastic cell lines tested. Although MEC is poorly expressed in skin, its closest homologue is the keratinocyte-expressed cutaneous T cell-attracting chemokine (CTACK; CCL27), and MEC supports chemotaxis of transfected lymphoid cells expressing CCR10, a known CTACK receptor. In contrast to CTACK, however, MEC also supports migration through CCR3. Consistent with this, MEC attracts eosinophils in addition to memory lymphocyte subsets. These results suggest an important role for MEC in the physiology of extracutaneous epithelial tissues, including diverse mucosal organs.     

Generation of cells with phenotypes of both intercalated duct-type and myoepithelial cells in human parotid gland adenocarcinoma clonal cells grown in athymic nude mice

A neoplastic epithelial cell line was established from nude mice tumors grown after transplantation of surgical specimens from a human parotid gland adenocarcinoma. This cell line, which had ultrastructure similarities to salivary intercalated duct cells, was found by immunohistochemical techniques to contain amylase, but myosin was not detected. Ultrastructurally, cells of an intermediate type between intercalated ductal and myoepithelial cells were found in the transplanted tumors. Moreover, the expression of myosin in addition to the presence of amylase was detected in the tumors. These findings indicate that some transplanted tumor cells appear to be differentiating towards myoepithelial cells.     

Distinct immunohistochemical expression of osteopontin in the adult rat major salivary glands

Osteopontin is a multifunctional protein secreted by epithelial cells of various tissues. Its expression in the adult rat major salivary glands has not yet been studied. We examined osteopontin expression by immunohistochemistry using a well characterized monoclonal antibody. Submandibular glands of young adult male rats (70–100 days old) showed specific expression in secretion granules of granular duct cells but also in cells of the striated ducts and excretory duct. In the major sublingual as well as the parotid gland expression was found solely in the duct system. In addition, a few interstitial-like cells exhibiting very strong immunostaining for osteopontin could be found in either organ. Expression could neither be seen in acinar cells nor in cells of the intercalated ducts. Moreover, in submandibular glands of more aged rats (6- to 7-month old) which show well developed granular convoluted tubules, there was almost exclusive expression of osteopontin in granular duct cells as well as in some interstitial-like cells, but barely in the striated/excretory duct system. Western blot analysis of the submandibular gland showed a specific band migrating at approximately 74 kDa, detectable at both age stages. Osteopontin secreted fom granular duct cells may influence the compostion of the saliva, e.g. thereby modulating pathways affecting sialolithiasis. Its expression in striated duct cells may also hint to roles such as cell–cell attachment or cell differentiation. The cell-specific expression detected in the rat major salivary glands differs in part from that reported in mice, human and monkey.Nicholas Obermüller and Nikolaus Gassler contributed equally to this work.     

CD151 forms a functional complex with c-Met in human salivary gland cancer cells

In this study, we have attempted to elucidate the expression and function of CD151 in human salivary gland cancer cells. CD151 expression was detected in Acc2 and AccM cells, but not in normal tissues and primary cultured epithelial cells derived from human salivary gland. CD151 has been found to function as a molecular linker in the formation of complexes between c-Met/hepatocyte growth factor (HGF) receptor and integrin alpha3/alpha6. Knockdown of CD151 or integrin alpha3/alpha6 expression almost completely abrogated HGF-stimulated cell growth and migration. In contrast, forced expression of CD151 in Acc2 cells resulted in the increase of the HGF-dependent biological effects. These results suggest that CD151 forms a structural and functional complex with c-Met and integrin alpha3/alpha6, and exerts its oncogenic functions through excessive activation of the HGF/c-Met signalling pathway.     

Expression and localization of immunoreactive-sialomucin complex (Muc4) in salivary glands

Sialomucin Complex (SMC; Muc4) is a heterodimeric glycoprotein consisting of two subunits, the mucin component ASGP-1 and the transmembrane subunit ASGP-2. Northern blot and immunoblot analyses demonstrated the presence of SMC/Muc4 in submaxillary, sublingual and parotid salivary glands of the rat. Immunocytochemical staining of SMC using monoclonal antisera raised against ASGP-2 and glycosylated ASGP-1 on paraffin-embedded sections of parotid, submaxillary and sublingual tissues was performed to examine the localization of the mucin in the major rat salivary glands. Histological and immunocytochemical staining of cell markers showed that the salivary glands consisted of varying numbers of serous and mucous acini which are drained by ducts. Parotid glands were composed almost entirely of serous acini, sublingual glands were mainly mucous in composition and a mixture of serous and mucous acini were present in submaxillary glands. Since immunoreactive (ir)-SMC was specifically localized to the serous cells, staining was most abundant in parotid glands, intermediate levels in submaxillary glands and least in sublingual glands. Ir-SMC in sublingual glands was localized to caps of cells around mucous acini, known as serous demilunes, which are also present in submaxillary glands. Immunocytochemical staining of SMC in human parotid glands was localized to epithelial cells of serous acini and ducts. However, the staining pattern of epithelial cells was heterogeneous, with ir-SMC present in some acinar and ductal epithelial cells but not in others. This report provides a map of normal ir-SMC/Muc4 distribution in parotid, submaxillary and sublingual glands which can be used for the study of SMC/Muc4 expression in salivary gland tumors.     

Establishment of an abalone digestive gland cell line secreting various glycosidases in protein-free culture

A cell line designated as ADG was established from an abalone digestive gland using ERDF medium supplemented with 8% fetal bovine serum (FBS), 8% abalone hemolymph, and high concentrations of NaCl, KCl, MgCl₂, MgSO₄, and CaCl₂. ADG cells proliferated better in protein-free medium than in FBS-supplemented medium. Among 9 kinds of media examined, ERDF medium was shown to be optimal for cell growth. ADG cells secreted 13 different kinds of glycosidases in protein-free medium: α-L-fucosidase, β-L-fucosidase, α-D-galactosidase, β-D-galactosidase, N-acetyl-α-D-galactosaminidase, N-acetyl-β-D-galactosaminidase, α-D-glucosidase, β-D-glucosidase, N-acetyl-α-D-glucosaminidase, N-acetyl-β-D-glucosaminidase, α-D-mannosidase, β-D-mannosidase, β-D-xylosidase, and 1-3 xylanase. When ADG cells were cultured in Grace’s insect cell medium, the activity of some secreted glycosidases increased 25-fold to 65-fold per cell as compared with control cells cultured in ERDF medium. ADG - abalone digestive gland; ERDF - enriched RDF; FBS - fetal bovine serum; L-15 - Leibovitz’s L-15 media; DME - Dulbecco’s modified Eagle medium; F-12 - nutrient mixture (Ham); LDF - L-15; DME: F-12 = 10 : 7 : 3; MEM - minimum essential medium; RPMI - RPMI medium 1640; 199 - media 199; GIC - Grace’s insect cell medium; pNP -p -nitrophenol. This revised version was published online in July 2006 with corrections to the Cover Date.     

Immunohistochemical localization of carbonic anhydrase isoenzymes VI, II, and I in human parotid and submandibular glands

Human salivary carbonic anhydrase (HCA VI) was purified by inhibitor affinity chromatography and its location in the human parotid and submandibular glands identified, using a polyclonal antiserum raised against the purified enzyme in rabbits in conjunction with the peroxidase-antiperoxidase complex method. The antibodies raised against the purified enzyme in rabbits did not crossreact with the HCA II or I. However, they slightly recognized human IgA; the antiserum was therefore absorbed with human IgA before immunohistochemical use. HCA VI-specific staining was detected in the cytoplasm and particularly in the secretory granules of the serous acinar cells of both parotid and submandibular glands, the staining of the secretory granules being most distinct in paraformaldehyde-fixed tissues. Some epithelial cells and the luminal content of the striated ducts also gave a specific HCA VI staining. Staining specific for HCA II was also found in the granules of the serous acinar cells, particularly in the submandibular gland when Carnoy fluid fixation was used. Slight HCA II-specific staining was also detected in the striated ductal cells in the Carnoy fluid-fixed specimens. No staining specific for HCA I was detected. The results indicate that the serous acinar cells in human parotid and submandibular glands contain abundant HCA II and HCA VI. Interestingly, only HCA VI is secreted into the saliva, although both enzymes appear to be located in structures resembling the secretory granules in the acinar cells. The enzymes probably form a mutually complementary system regulating the salivary buffer capacity.     

Secretion of α-amylase in human parotid gland epithelial cell culture

The secretions of the salivary gland system are essential for the maintenance of oral health. The nature of cell-specific secretions of the various glands and their regulation is not completely understood. The objective of this study was to establish epithelial cell cultures from the human parotid gland that exhibit the tissue-specific function of α-amylase secretion. A specimen of normal human parotid gland was obtained at surgery and used to obtain primary cultures by the explant/outgrowth procedure. The cultures were maintained in keratinocyte basal medium, supplemented with insulin (5 μg/ml), EGF (10 ng/ml), hydrocortisone (0.5 μg/ml), bovine pituitary extract (25 μg/ml), and antibiotics. The cultures were passaged using 0.125% trypsin to dissociate the cells. Phase contrast and ultrastructural observations showed that the cells were polygonal and exhibited desmosomes. Their cytoplasm contained tonofilament bundles and abundant rough endoplasmic reticulum and Golgi complexes. Immunofluorescence studies showed that all cells were positive for cytokeratins. Immunoblot analysis revealed keratins with molecular weights of 58, 56, 52, 50, 48, 46, and 40 KD, which are characteristic of secretory epithelia. The cells have been passaged 35 times so far, undergoing a cumulative 120–140 population doublings. The serially passaged epithelial cell cultures produced and secreted α-amylase, a major component of parotid gland acinar cell secretion. The β-adrenergic agonist, isoproterenol (ISP), stimulated α-amylase secretion, which was accompanied by increased intracellular concentrations of cAMP. ISP-induced stimulation of amylase and cAMP was blocked by the β-adrenergic antagonist, propranolol. Further, dibutryl cAMP also enhanced the secretion of amylase. Thus we have established a long-term epithelial cell culture model of human parotid gland epithelial cells that exhibits differentiated function and retains the intact β-adrenergic receptor system. © 1993 Wiley-Liss, Inc.     

Integrin expression in developing human salivary glands

The development and complete differentiation of salivary glands is a complex process that involves a large number of co-ordinated events. Little is known about the molecular basis for salivary gland development. However, we have reported previously that integrins appear to play a role. Integrins are heterodimeric transmembrane receptors consisting of one α and one β subunit that play a pivotal role in the interaction of cells with the extracellular matrix. Such interactions regulate the organisation of cells of tissues and organs during development as well as cell proliferation and differentiation. Using immunohistochemistry and Western and Northern blot analysis, we mapped the localisation and expression of integrins β1, β3 and β4 in human salivary glands obtained from foetuses ranging from weeks 4–24 of gestation and compared it with adult salivary glands. Integrin β1 first appeared during the canalisation stage and during the differentiation stage. A message first appeared at week 6 of development. The expression of β4 integrin protein and message was observed only in the late stage of differentiation. Integrin β3 was not detected in the developing glands; however, integrins β1, β3 and β4 were all expressed in adult salivary gland tissues. The data suggest that integrins, particularly β1, have a role to play in salivary gland development and differentiation.     

Psophocarpus tetragonolobus agglutinin reveals N-acetyl galactosaminyl residues confined to endothelial cells and some epithelial cells in human tissues

We studied the binding of Psophocarpus tetragonolobus agglutinin (PTA) conjugates to human adult tissues. In all kidney specimens studied, PTA bound in a blood group-independent way to endothelia in glomerular and intertubular capillaries as well as in larger vessels. In addition, a heterogeneous binding to collecting duct cells was seen. In specimens of human smooth, cardiac, and skeletal muscle, cerebellum, lung, thyroid gland, liver, proliferative endometrium, and placenta, PTA bound only to endothelial of capillaries and larger vessels. In epidermis and gingiva, PTA conjugates additionally revealed reactivity with keratinocytes. Similarly, in salivary gland, urinary bladder, gastrointestinal tract, mammary gland, and renal pelvis, PTA reacted with some epithelial cell layers. The PTA conjugates gave an even cell surface membrane staining of cultured umbilical vein endothelial cells. Lectin-affinity binding of radioactively surface-labeled endothelial cells showed that PTA and Ulex europaeus I agglutinin (UEA-I) recognized related major cell surface glycoproteins. The results with PTA conjugates show that certain N-acetyl galactosaminyl residues are, in addition to some epithelial cells, confined to endothelial cells in human tissues.     

Detection of Epstein-Barr virus-associated antigens and DNA in salivary gland biopsies from patients with Sjogren's syndrome

Sjogren's syndrome (SS) is an autoimmune disorder characterized by lymphocytic infiltration of salivary and lacrimal glands. To determine whether Epstein-Barr virus (EBV) might play a role in the pathogenesis of this disorder, we used monoclonal antibodies and DNA probes to detect evidence of viral gene products and genomes in these patients' tissue biopsies and saliva. Cytoplasmic staining of epithelial cells (i.e., ductal and/or acinar cells) with monoclonal antibody against the EBV-encoded early antigen (EA-D) was noted in 8/14 salivary gland biopsies from SS patients. This antibody did not react with normal salivary glands or salivary gland tumors, nor with other tissues from SS patients. The reactive antigen in SS biopsies had a m.w. of 52,000 on the basis of immunoblotting experiments, similar to the EA-D antigen found in lymphoblastoid cells lytically infected with EBV. EBV DNA was detected in parotid biopsies from two SS patients in amounts ranging from 0.1 to 3 pg per 20 micrograms of cellular DNA. Southern blotting was used to demonstrate the reactivity with Bam V, Eco D, and Bam M probes. Parotid saliva samples from 8/20 SS patients contained EBV DNA detectable by slot blot hybridization. EBV DNA was not detected in saliva of age-matched controls, rheumatoid arthritis patients lacking sicca symptoms, or patients with benign parotid tumors. The presence of EBV in salivary gland and saliva samples was associated with clinically more severe SS, as manifested by extraglandular symptoms such as vasculitis, occurrence of "pseudolymphoma", and marked abnormalities in immunoglobulin levels. These results demonstrate an elevated content of EBV in salivary glands of SS patients, and suggest that this virus may play a role in pathogenesis.     

Redistribution of carbonic anhydrase VI expression from ducts to acini during development of ovine parotid and submandibular glands

 Carbonic anhydrase VI (CA VI) is a secreted enzyme produced predominantly by serous acinar cells of submandibular and parotid glands. We have investigated the developmental pattern of CA VI production by these glands in the sheep, from fetal life to adulthood, using immunohistochemistry. Also, a specific radioimmunoassay for CA VI was used to measure changes in enzyme expression in the parotid gland postnatally. CA VI is detectable by immunohistochemistry in parotid excretory ducts from 106 days gestation (term is 145 days), in striated ducts from 138 days and in acinar cells from 1 day postnatal. The duct cell content of CA VI declined as the acinar cell population increased, a feature also of CA VI immunoreactivity in the submandibular gland. Production of CA VI by submandibular duct cells was detectable initially at 125 days gestation, and acinar production was not seen before 29 days post-natal. Apart from the differing ontogeny of CA VI production in ducts and acini of parotid and submandibular glands, there was a parallel pattern of CA VI expression during the development of these major salivary glands.With the development of the acinar tissues in the postnatal lamb, there was a dramatic increase (about 600-fold) in the level of expression of CA VI in the parotid gland between days 7 and 59 as measured by radioimmunoassay. Accepted: 19 December 1996     

Epidermal growth factor (EGF) expression in human salivary glands. An immunohistochemical study.

Epidermal growth factor (EGF) is a biologically active peptide involved in differentiation, growth, regeneration and repair of human and animal tissues. Quantitative biochemical studies showed in man the highest concentration of EGF in the parotid gland. The aim of the present study was to define EGF immunolocalization in the individual segments of the human major salivary glands (salivon). The material consisted of sections obtained from the surgically removed salivary glands: parotid, submaxillary and sublingual. Immunohistochemical studies were performed by PAP method using monoclonal antibody against human epidermal growth factor. EGF expression was found almost exclusively in the efferent pathways of the salivary glands, mostly in the intercalated ducts and Pflüger salivary tubules. These segments of the salivon are most developed in the parotid gland in which the staining was stronger than in other salivary glands.     

Immunolocalization patterns of cytokeratins during salivary acinar cell development in mice

Embryonic development of the mouse salivary glands begins with epithelial thickening and continues with sequential changes from the pre-bud to terminal bud stages. After birth, morphogenesis proceeds, and the glands develop into a highly branched epithelial structure that terminates with saliva-producing acinar cells at the adult stage. Acinar cells derived from the epithelium are differentiated into serous, mucous, and seromucous types. During differentiation, cytokeratins, intermediate filaments found in most epithelial cells, play vital roles. Although the localization patterns and developmental roles of cytokeratins in different epithelial organs, including the mammary glands, circumvallate papilla, and sweat glands, have been well studied, their stage-specific localization and morphogenetic roles during salivary gland development have yet to be elucidated. Therefore, the aim of this study was to determine the stage and acinar cell type-specific localization pattern of cytokeratins 4, 5, 7, 8, 13, 14, 18, and 19 in the major salivary glands (submandibular, sublingual, and parotid glands) of the mouse at the E15.5, PN0, PN10, and adult stages. In addition, cell physiology, including cell proliferation, was examined during development via immunostaining for Ki67 to understand the cellular mechanisms that govern acinar cell differentiation during salivary gland morphogenesis. The distinct localization patterns of cytokeratins in conjunction with cell physiology will reveal the roles of epithelial cells in salivary gland formation during the differentiation of serous, mucous or seromucous salivary glands.     

Fine needle aspiration cytology of epithelial-myoepithelial carcinoma of salivary glands. A report of three cases.

BACKGROUND: Epithelial-myoepithelial carcinoma is a rare, low grade malignant tumor of the salivary glands. Histologically, it has a biphasic cellular composition and exhibits a high degree of differentiation. The fine needle aspiration cytology of this rare tumor is rarely described in the literature. CASES: We report the fine needle aspiration cytology of three epithelial-myoepithelial carcinomas, arising in the right parotid, left parotid and minor salivary gland of the hard palate. Cytology showed a biphasic population consisting of cells of ductal epithelial and myoepithelial origin arranged in small clusters and sheets. The myoepithelial cells had small, uniform nuclei; ample, clear cytoplasm and distinct cell borders, while the ductal epithelial cells had larger, mildly pleomorphic nuclei and scanty cytoplasm. These ductal cells tended to form tubules among background sheets of clear myoepithelial cells. This feature, if present, was an important diagnostic clue. Hyaline material surrounding cell clusters and focal adenoid cystic carcinoma-like areas with orangeophilic globules were also not uncommon. CONCLUSION: While the cytologic appearance of epithelial-myoepithelial carcinoma may closely mimic that of other salivary gland tumors, such as adenoid cystic carcinoma, pleomorphic adenoma and basal cell adenoma, certain peculiar cytologic features may allow a distinction to be made on fine needle aspiration biopsy.