Specific demonstration of myoepithelial cells by anti-alpha smooth muscle actin antibody

The myoepithelial cells of the sweat, mammary, tracheobronchial, and salivary glands are specifically identified by monoclonal antibody alpha-SM-1, which recognizes alpha smooth muscle actin and not the other actin isoforms. Basal or "reserve" cells in the stratified epithelia and excretory ducts of the salivary glands are negative, as well as all other epithelial cells in various organs. The reaction can be performed in routinely fixed and embedded tissues and is of practical interest in diagnostic histopathology. In immunoelectron cytochemistry, alpha-SM-1 antibody binds to the microfilament bundles in myoepithelial cells of the breast, but does not stain luminal cells and occasional basally located epithelial cells. These basal cells are morphologically and immunocytochemically distinct from the myoepithelial cells, and their nature and significance remain to be clarified.     

On the myoepithelium of human salivary glands. An immunocytochemical study

This study investigated the immunocytochemical characteristics of normal myoepithelial cells (MECs) of human major and minor salivary glands using the LSAB method. Other human exocrine glands were used as controls. Immunoreactivity of MECs was observed exclusively with fully differentiated smooth muscle antibodies (a-SMA; SMMS-1; CALP; hCD) and with epithelial markers (cytokeratins) Ck14 and Ck17. This epithelial-muscular immunophenotype was similarly expressed in the MECs of other human exocrine glands used as control. In the salivary MECs, we did not observe evidence for neuroectodermic phenotype (S-100 protein, GFAP, NSE). On the contrary, positivity was observed for S-100 protein in Mecs of control glands (mammary, bronchial and sweat glands). Immunoreaction for extracellular matrix markers (fibronectin, laminin and collagen IV) and vimentin always were negative. Our data show that in normal "non transformed" MECs of the salivary glands the smooth muscle phenotype is in a state of complete differentiation, while the epithelial phenotype expresses only Ck14 and Ck17. This double and simultaneous immunoreactivity represents a differential marker of MECs from the epithelial basal cell (EBCs).     

P63 is expressed in basal and myoepithelial cells of human normal and tumor salivary gland tissues.

p63 is essential for epithelial cell survival and may function as an oncogene. We examined by immunohistochemistry p63 expression in human normal and tumor salivary gland tissues. In normal salivary glands, p63 was expressed in the nuclei of myoepithelial and basal duct cells. Among 68 representative salivary gland tumors, 63 displayed p63 reactivity. In all tumor types differentiated towards luminal and myoepithelial lineages (pleomorphic adenomas, basal cell adenomas, adenoid cystic carcinomas, and epithelial-myoepithelial carcinomas), p63 was expressed in myoepithelial cells, whereas luminal cells were always negative. Similarly, in mucoepidermoid carcinomas, basal, intermediate, and squamous cells expressed p63, in contrast to luminal mucous cells. p63 reactivity was also restricted to basal cells in Warthin tumors and oncocytomas. Myoepitheliomas and myoepithelial carcinomas all expressed p63. The only five negative tumors were three of four acinar cell carcinomas and two of three adenocarcinomas. In conclusion, p63 is expressed in the nuclei of normal human salivary gland myoepithelial and basal duct cells. p63 expression is retained in the modified myoepithelial and basal cells of human salivary gland tumors, which suggests a role for p63 in oncogenesis of these complex tumors.     

Expression of CD44 isoforms in normal salivary gland tissue: an immunohistochemical and ultrastructural study

We studied the expression of CD44 isoforms immunoreactivity in normal human salivary gland tissue, aiming at its full characterisation in normal epithelial and myoepithelial cell types. Optical immunohistochemistry techniques using monoclonal antibodies anti-CD44v3, CD44v4/5 and, for CD44v6, together with immunoelectron microscopy, were performed in serous, seromucinous and mucinous glands. Normal human breast and a case of lactating breast adenoma were used for comparative purposes and as controls. CD44v3 was positive in acinar and myoepithelial cells and was absent in mucin-producing cells from the different gland types. CD44v4/5 was consistently negative in all types of salivary tissue. CD44v6 was constantly positive in serous acinar cells, focally positive in basal cells of ducts, and myoepithelial cells consistently expressed it. At the ultrastructural level, CD44v6 was localised to the interdigitating processes of acinar cells, whenever they were not covered by basal lamina and to the cell membrane facing myoepithelial cells. In myoepithelial cells, immunolabelling was found at the membranes facing the acinar cells and in caveolae present at this interface. No labelling was found at cell membranes of both acinar and myoepithelial cells in contact with basal lamina or at the luminal aspect of the former. The finding of CD44v3 and v6 in myoepithelium of normal salivary glands may argue in favour of the role of these molecules in the regulation of growth and renewal of normal tissues and, potentially, on the morphogenesis of salivary gland neoplasms.     

The ultrastructural aspects of neoplastic myoepithelial cell in pleomorphic adenomas of salivary glands

The purpose of this study has been to establish the major ultrastructural aspects of the myoepithelial cell and the myoepithelial-like cells proliferated in the pleomorphic adenomas of salivary glands. Thus, twelve benign pleomorphic adenomas of salivary glands have been studied by electron-microscopy transmission techniques. Our analysis has proved the proliferation of two major cellular populations, one of ductal type and one of myoepithelial type, which tried to reproduce the tubulo-acinar cytoarchitecture from the normal salivary glands. We have also noticed the key role of the so-called 'modified' myoepithelial cells from the periphery of the proliferating epithelial units in the genesis of the myxoid and chondromyxoid tumoral stromal areas. All these ultrastructural aspects have explained the great histological diversity of these salivary gland neoplasms as well as the key role of the myoepithelial cell in its histogenesis.     

Par-1b is required for morphogenesis and differentiation of myoepithelial cells during salivary gland development

The salivary epithelium initiates as a solid mass of epithelial cells that are organized into a primary bud that undergoes morphogenesis and differentiation to yield bilayered acini consisting of interior secretory acinar cells that are surrounded by contractile myoepithelial cells in mature salivary glands. How the primary bud transitions into acini has not been previously documented. We document here that the outer epithelial cells subsequently undergo a vertical compression as they express smooth muscle α-actin and differentiate into myoepithelial cells. The outermost layer of polarized epithelial cells assemble and organize the basal deposition of basement membrane, which requires basal positioning of the polarity protein, Par-1b. Whether Par-1b is required for the vertical compression and differentiation of the myoepithelial cells is unknown. Following manipulation of Par-1b in salivary gland organ explants, Par-1b-inhibited explants showed both a reduced vertical compression of differentiating myoepithelial cells and reduced levels of smooth muscle α-actin. Rac1 knockdown and inhibition of Rac GTPase function also inhibited branching morphogenesis. Since Rac regulates cellular morphology, we investigated a contribution for Rac in myoepithelial cell differentiation. Inhibition of Rac GTPase activity showed a similar reduction in vertical compression and smooth muscle α-actin levels while decreasing the levels of Par-1b protein and altering its basal localization in the outer cells. Inhibition of ROCK, which is required for basal positioning of Par-1b, resulted in mislocalization of Par-1b and loss of vertical cellular compression, but did not significantly alter levels of smooth muscle α-actin in these cells. Overexpression of Par-1b in the presence of Rac inhibition restored basement membrane protein levels and localization. Our results indicate that the basal localization of Par-1b in the outer epithelial cells is required for myoepithelial cell compression, and Par-1b is required for myoepithelial differentiation, regardless of its localization.     

A high resolution sem study of human minor salivary glands

By SEM we have investigated the human minor salivary glands using the NaOH method for the visualization of endpieces and myoepithelial cells, and the osmium maceration technique that reveals membranous intracellular structures. With the former method all minor glands, including the posterior deep (Ebner's) lingual glands, consist of tubules sometimes dilated into alveoli, while true acini of the kind observed in human major salivary glands, are absent. Tubules of the posterior deep lingual gland exhibit stellate myoepitelial cells that leave a substantial part of the secretory cells uncovered. The latter cells, at variance with serous cells of major glands, do not show basal folds. In contrast, tubules of the other minor glands, like the mucous ones of major glands, are covered almost completely by band-like myoepithelial cells. The osmium maceration method clearly demonstrates that posterior deep lingual glands are serous in character and that all the other minor glands, together with the predominant mucous cells, possess a variable number of seromucous cells that, despite variations among individuals, increase in order from palatine and posterior superficial lingual (Weber's), to minor sublingual, labial, anterior lingual (Blandin and Nuhn's), and buccal glands.     

Intermediate-sized filaments of the prekeratin type in myoepithelial cells

Myoepithelial cells from mammary glands, the modified sweat glands of bovine muzzle, and salivary glands have been studied by electron microscopy and by immunofluorescence microscopy in frozen sections in an attempt to further characterize the type of intermediate-sized filaments present in these cells. Electron microscopy has shown that all myoepithelial cells contain extensive meshworks of intermediate-sized (7--11-nm) filaments, many of which are anchored at typical desmosomes or hemidesmosomes. The intermediate-sized filaments are also intimately associated with masses of contractile elements, identified as bundles of typical 5--6-nm microfilaments and with characteristically spaced dense bodies. This organization resembles that described for various smooth muscle cells. In immunofluorescence microscopy, using antibodies specific for the various classes of intermediate-sized filaments, the myoepithelial cells are strongly decorated by antibodies to prekeratin. They are not specifically stained by antibodies to vimentin, which stain mesenchymal cells, nor by antibodies to chick gizzard desmin, which decorate fibrils in smooth muscle Z bands and intercalated disks in skeletal and cardiac muscle of mammals. Myoepithelial cells are also strongly stained by antibodies to actin. The observations show (a) that the epithelial character, as indicated by the presence of intermediate-sized filaments of the prekeratin type, is maintained in the differentiated contractile myoepithelial cell, and (b) that desmin and desmin-containing filaments are not generally associated with musclelike cell specialization for contraction but are specific to myogenic differentiation. The data also suggest that in myoepithelial cells prekeratin filaments are arranged--and might function--in a manner similar to the desmin filaments in smooth muscle cells.     

Ultrastructure of human labial salivary glands. 3. Myoepithelium and ducts

Myoepithelial cells were present between the basal lamina and the acinar secretory cells of human labial salivary glands. In form and disposition, they resembled myoepithelial cells in the major salivary glands. Many of these cells possessed single cilia on their upper surfaces. Such cilia occasionally extended into invaginations of the overlying secretory cell. The intercalated ducts were variable in occurrence. Their epithelium ranged from columnar to squamous, and showed few signs of secretory activity. Few intralobular ducts possessed basal striations. While mitochondria were abundant in non-striated cells, they were randomly disposed in both basal and apical cytoplasm, and the basal plasmalemma showed only occasional infoldings. The paucity of true striated ducts in labial salivary glands may be responsible for the high concentration of sodium and chloride in unstimulated labial gland salivary secretions.     

Immunolocalization of PTHrP in the submandibular glands of three rodent species.

The present study deals with immunohistochemical localization of PTHrP in bank vole, pine vole and white mouse submandibular glands. PTHrP immunoreactivity was observed in epithelial cells of all ductal segments (intercalated, striated, interlobular and main excretory ducts) of the salivary glands in all the three animal species tested. We also found PTHrP expression in myoepithelial cells surrounding the mucous alveoli of submandibular glands in those animals. The reaction was less intense than that found in the epithelial cells of excretory ducts. We occasionally observed a very slight positive reaction for PTHrP in smooth muscle cells of small blood vessels. We also found PTHrP expression in the neurons of ganglion in the submandibular gland.     

The interest of actin immunocytochemistry in diagnostic histopathology

The immunohistochemical detection of different isoforms of actin provides useful data in the histopathological diagnosis of human tumours. The marked resistance of this protein to fixation and embedding procedures makes it a practical alternative to the ultrastructural search of microfilaments in routine tissue sections. Staining with monoclonal antibodies against alpha smooth muscle actin is helpful in the diagnosis of benign and malignant smooth muscle tumours and allows the differentiation of various types of spindle cell sarcomas. These antibodies are also useful in tracing the myoepithelial cells in normal and various pathological conditions of the breast and salivary glands. Skeletal muscle actin is a reliable marker in the diagnosis of rhabdomyosarcomas. Its use should be combined and complemented with other markers (i.e. desmin; foetal, slow and fast myosins; myoglobin) to monitor the degree of rhabdomyoblastic differentiation of the neoplastic cells in single cases, a parameter of potential prognostic value.     

Immunohistochemical demonstration of lysozyme and lactoferrin in salivary pleomorphic adenomas

Immunohistochemical identification of lysozyme and lactoferrin was made in salivary pleomorphic adenomas (147 cases) and the staining patterns were evaluated with respect to the histological features and histogenesis. In normal salivary glands, the intercalated duct cells gave positive staining for lysozyme in major glands, and serous acinar cells, demilune cells, and interlobular duct cells were positive in minor glands. Lactoferrin staining was irregularly positive in serous cells and ductal epithelium. In pleomorphic adenomas, the reaction for lysozyme was positive in 14% (21/147) of the cases, and was confined to luminal cells of tubulo-ductal structures. Lactoferrin in pleomorphic adenomas was distributed in luminal tumor cells (51%; 75/147), in outer tumor cells (3%; 4/147), and in both luminal and outer tumor cells (5%; 7/147) in tubulo-ductal structures; it was also detected in plasmacytoid myoepithelial cells (5%, 8/147). However, modified myoepithelial cells and other types of neoplastic myoepithelial participants were negative for lactoferrin staining. The occurrence of both lysozyme and lactoferrin in salivary pleomorphic adenomas suggests their participation in the local defense mechanism in the tumor.     

Microcinematographic demonstration of synchronous and asynchronous myoepithelial contractions in mouse submandibular gland rudiments in organotypic culture

Summary Time-lapse phase-contrast cinematography revealed contractile activity within mouse submandibular salivary gland rudiments in organotypic culture. Three types of contraction were distinguishable. In type I (voiding contractions), all portions of the gland contracted synchronously, and the active state ranged from 30 min to 2 hr. In type II (priming contractions), all portions of the gland contracted synchronously, but the active state was shorter, ranging from 4 to 10 min. In type III (churning contractions), isolated foci in lobules or secretory units throughout the gland contracted asynchronously and had very short active states of about 1 min. By electron microscopy, myoepithelial cells could first be demonstrated in submandibular glands developing either in vitro or in vivo, at 21 days postconception. Contractions in the cultured rudiments began as early as 18 days postconception. Since neither smooth nor striated muscle could be identified in these glands by electron microscopy, the contractions are believed to result from myoepithelial activity that apparently may begin before ultrastructural evidence of myoepithelial differentiation is contractile function and indirect evidence has lent ample support to this presumption, the present study represents the first direct cinematographic demonstration and characterization of myoepithelial contractions, under conditions in vitro.     

Myoepithelial cell contraction and milk ejection are impaired in mammary glands of mice lacking smooth muscle alpha-actin

Mammary myoepithelial cells are specialized smooth musclelike epithelial cells that express the smooth muscle actin isoform: smooth muscle alpha-actin (ACTA2). These cells contract in response to oxytocin to generate the contractile force required for milk ejection during lactation. It is believed that ACTA2 contributes to myoepithelial contractile force generation; however, this hypothesis has not been directly tested. To evaluate the contribution of ACTA2 to mammary myoepithelial cell contraction, Acta2 null mice were utilized and milk ejection and myoepithelial cell contractile force generation were evaluated. Pups suckling on Acta2 null dams had a significant reduction in weight gain starting immediately postbirth. Cross-fostering demonstrated the lactation defect is with the Acta2 null dams. Carmine alum whole mounts and conventional histology revealed no underlying structural defects in Acta2 null mammary glands that could account for the lactation defect. In addition, myoepithelial cell formation and organization appeared normal in Acta2 null lactating mammary glands as evaluated using an Acta2 promoter-GFP transgene or phalloidin staining to visualize myoepithelial cells. However, mammary myoepithelial cell contraction in response to oxytocin was significantly reduced in isolated Acta2 null lactating mammary glands and in in vivo studies using Acta2 null lactating dams. These results demonstrate that lack of ACTA2 expression impairs mammary myoepithelial cell contraction and milk ejection and suggests that ACTA2 expression in mammary myoepithelial cells has the functional consequence of enhancing contractile force generation required for milk ejection.     

Cytochemical demonstration of actin filaments in myoepithelial cells of the human parotid gland

Ultrastructurally, myoepithelial cells were shown to contain numerous fine filaments in their cytoplasm and resembled smooth muscle cells. The myoepithelial cell of the salivary gland has been considered to play an important role in the secretion of saliva. The present study showed that all the thin filaments (actin filaments) in the myoepithelial cell of the human parotid gland bound heavy meromyosin (HMM) and formed characteristic arrowhead structures. These filaments ran in two opposite directions with the poles at different ends. On the other hand, there was no binding of HMM with thicker filaments (10-nm filaments), plasma membrane, nuclear membrane, collagen fibrils, basement membrane or other cytoplasmic organelles. The present results strongly suggest that myoepithelial cells possess a contractile function parallel to the long axis of the cell for supporting the secretion of saliva in the parotid gland.     

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.     

Immunohistochemical localization of the α and β subunits of S-100 protein in pleomorphic adenoma of the salivary glands

The immunohistochemical expression of the alpha and beta subunits of S-100 protein in reactive, modified and transformed of myoepithelial cells, salivary pleomorphic was investigated using monoclonal antibodies. With S-100 alpha, normal salivary glands showed strong staining in serous acinar cells and moderate to slight staining in ductal segments, and with S-100 beta staining was slight or negative in acinar cells, but strong in nerve fibres. In pleomorphic salivary adenomas, the immunohistochemical distribution of S-100 alpha and beta proteins indicated great variation in the tumour cells. Some neoplastic cells gave similar staining for both S-100 alpha and beta, others were strongly positive for S-100 alpha and stained only slightly for S-100 beta, or vice versa. Yet other cells were positive for S-100 alpha and negative for S-100 beta, or vice versa. Pleomorphic salivary adenomas were classified both by histopathological criteria and by their staining pattern for S-100 alpha and beta proteins. Great heterogeneity in S-100 alpha and beta protein expression was found in individual tumour cells of both ductal and myoepithelial origin, and no regular pattern was identified. The cellular origin of salivary pleomorphic adenomas is discussed in terms of S-100 alpha and beta protein immunohistochemistry. Pleomorphic adenoma cells may be transformed from reserve cells into tumour cells displaying biologic properties of myoepithelial cells, ductal cells, or a mixture of both.     

The expression of water channel proteins during human salivary gland development: a topographic study of aquaporins 1, 3 and 5

Some members of aquaporin family (AQP) plays crucial functions in salivary synthesis and secretion. These proteins expression has already been reported during salivary gland formation, however no previous studies in human developing glands have been performed. We evaluated AQP1, 3 and 5 expression through the stages of human salivary gland morphogenesis and discuss the possible role of AQP for glandular maturation. Human salivary glands derived from foetuses aged between 14 and 25 weeks were submitted to immunohistochemistry. At the bud stage, membrane expression of AQP1, 3 and 5 were observed within the epithelial bud cells presenting a similar apicolateral pattern, also found at the pseudoglandular stage, present within the terminal portions of future acini, while AQP5 was also particularly strong at the apical membrane of pre-acinar and pre-ductal cells. AQP5 was co-localised with Cytokeratin 7. Similar AQP1, 3 and 5 expression were observed at the following canalicular stage, where distinct and strongly luminal and acinar AQP5 expression is present. During the final terminal bud stage, AQP1 was only identified in serous acini, myoepithelial and endothelial cells, while differentiated mucous acinar cells and ducts were negative. AQP3 was detected at apicolateral membranes of both mucous and serous acini. AQP5 also showed a diffuse expression in mucous and serous acini, in addition to strong apical membrane expression within lumen of intercalated ductal cells. This topographic analysis of AQP1, 3 and 5 revealed differences in the expression pattern throughout salivary gland developmental stages, suggesting different roles for each protein in human glandular maturation.