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.     

Immunohistochemical studies of S-100 protein alpha and beta subunits in adenoid cystic carcinoma of salivary glands

Immunohistochemical studies were performed to explore the distribution of S-100 protein and its alpha, beta subunits in 76 adenoid cystic carcinomas (ACC) of the salivary glands. Histopathologically. ACC was divided into cribriform, tubular, basaloid and trabecular types which could be mixed in the same tumor. S-100 protein was usually positive in tumor cells forming cribriform structures; foci of strongly positive tumor cells were also distributed in the luminal layer of tubular structures, and in areas transitional between cribriform and tubular patterns. S-100 alpha staining was confined to some tumor cells in cribriform areas, to luminal tumor cells in tubular structures and to few tumor cells in basaloid structures. S-100 beta reaction was usually localized to luminal surfaces in a fine granular pattern in tubular and microtubular structures in a distribution somewhat similar to that in the normal salivary gland. Great heterogeneity in the immunohistochemical distribution of S-100, S-100 alpha and S-100 beta proteins was found in the various histologic types of ACC and the pattern was different from that seen in pleomorphic adenomas. It is possible that the ACC tumor cells positive for S-100 protein may be closely related to true or modified myoepithelial cells.     

Distribution of S-100 protein and its subunits in bovine exocrine glands

 The distribution of S-100 protein and its α- and β-subunits in bovine exocrine glands was studied by indirect immunohistochemistry. The entire spectrum of salivary glands, glands of the respiratory tract, intestinal glands, male and female genital glands, and skin glands was examined. S-100 and its β-subunit were identified in most serous secretory cells of mixed salivary glands, although secretory acini in some serous glands remained unreactive for these antigens. Mucous cells were constantly negative; mucoid cells were positive in the lacrimal and Harderian gland. The α-subunit of S-100 protein was identified in serous cells but the staining reaction was faint. Subunits of S-100 showed a characteristic distribution along the excretory duct systems of compound glands: S-100 and the β-subunit were present in intercalated duct epithelium, while striated duct epithelium stained for S100-α. Therefore, it is suggested that S100-α is related to resorption and secretion in striated ducts, while S100-β may govern acinar exocytosis and probably regulates proliferation and differentiation of glandular cells. Differing staining intensities for S-100 and its subunits in secretory cells of exocrine glands most probably indicate functional differences with regard to secretory activity and the cell cycle. Accepted: 11 February 1997     

Differential localization of "brain-specific" S-100 and its subunits in rat salivary glands

In the rat, the S-100 antigens in the submandibular gland were found to be immunochemically identical with those in the brain (glial cells) when compared using crossed immunoelectrophoresis. Specific antibodies against the S-100a non-beta and against the S-100 beta subunit were prepared from antibodies against crude S-100 protein and from S-100 components (S-100a and b) by affinity chromatography. In the rat salivary glands a differential distribution of subunit immunoreactivity was clearly evidenced using indirect immunofluorescence. Certain intercalated duct cells of the submandibular gland as well as Schwann cells contained the S-100 beta subunit immunoreactivity exclusively, while other duct cells in parotid, submandibular, and sublingual glands contained S-100a non-beta subunit immunoreactivity. Both subunits were present in astrocytes and ependymal cells. The immunocytochemical localization of alpha and beta subunits is a promising technique for the classification of various types of S-100-containing cells.     

Time course of differentiation of different cell types in 3D-reconstructed eccrine sweat glands

Epidermal basal cells invaginate into the dermis to form sweat ducts, which then grow downwards further to form secretory coils during the ontogenesis of eccrine sweat glands, but the time course of differentiation of different cell types in 3D-reconstructed eccrine sweat glands remain unclear. In this study, secretory cell-specific marker K7, clear secretory cell-specific marker CA II, dark secretory cell-specific marker GCDFP-15, myoepithelial cell-specific marker α-SMA, inner duct cell-specific marker S100P and outer duct cell-specific marker S100A2 were detected by immunofluorescence staining. The results showed that S100P and S100A2 were first detected at 2 weeks post implantation, K7 and α-SMA at 3 weeks, and GCDFP-15 and CA II at 4 weeks. The differentiation of ducts preceded secretory coils in 3D-reconstructed eccrine sweat glands. After 8 weeks post implantation, the distribution of these markers in 3D-reconstructed eccrine sweat glands was similar to that in native ones, and the percentage of the 3D-reconstructed glands expressing these markers maintained steady. We conclude that although the 3D-reconstructed and native eccrine sweat glands originated from different cells, the differentiation of different cell types in 3D-reconstructed eccrine sweat glands parallels the sequence observed during embryonic development.     

Expression of S100A2 and S100P in human eccrine sweat glands and their application in differentiating secretory coil-like from duct-like structures in the 3D reconstituted eccrine sweat spheroids

Secretory coils and ducts are two components of eccrine sweat glands with different structures and functions. In our previous study, we combined keratins and α-SMA to distinguish between secretory coils and ducts. However, the key deficiency of the method was that none of the antibodies used was specific for ducts. In this study, we first examined the co-localization of K5/K7, α-SMA/K14, K7/S100P and α-SMA/S100A2 by double-immunofluorescence staining to confirm the localization of S100P and S100A2 in native human eccrine sweat glands, and second we identified secretory coil-like and duct-like structures in the 3D reconstituted eccrine sweat gland spheroids by double-immunofluorescence staining for K7/S100P and α-SMA/S100A2. In native human eccrine sweat glands, S100A2 immunoreactivity was confined to the outer layer and S100P to the inner layer of the duct. In 12-week Matrigel plugs containing eccrine sweat gland cells, double-immunofluorescence staining for K7/S100P and α-SMA/S100A2 could easily distinguish duct-like structures from secretory coil-like structures. We conclude that S100A2 and S100P can be used as specific duct markers in eccrine sweat glands, and combined use of S100P or S100A2 with keratins enables easy to distinction between secretory coils and ducts.     

Immunohistochemical studies of S-100 protein α and β subunits in adenoid cystic carcinoma of salivary glands

Immunohistochemical studies were performed to explore the distribution of S-100 protein and its α,β subunits in 76 adenoid cystic carcinomas (ACC) of the salivary glands. Histopathologically, ACC was divided into cribriform, tubular, basaloid and trabecular types which could be mixed in the same tumor. S-100 protein was usually positive in tumor cells forming cribriform structures; foci of strongly positive tumor cells were also distributed in the luminal layer of tubular structures, and in areas transitional between cribriform and tubular patterns. S-100 α staining was confined to some tumor cells in cribriform areas, to luminal tumor cells in tubular structures and to few tumor cells in basaloid structures. S-100β reaction was usually localized to luminal surfaces in a fine granular pattern in tubular and microtubular structures in a distribution somewhat similar to that in the normal salivary gland. Great heterogeneity in the immunohistochemical distribution of S-100, S-100 a and S-100β proteins was found in the various histologic types of ACC and the pattern was different from that seen in pleomorphic adenomas. It is possible that the ACC tumor cells positive for S-100 protein may be closely related to true or modified myoepithelial cells.     

Distribution of hyaluronan and its CD44 receptor in the epithelia of human skin appendages.

Biotinylated hyaluronan (HA) binding complex (HABC) from bovine articular cartilage proteoglycan was used as a histological probe to study the localization of HA in human skin. The distribution of HA was compared with its presumptive cell surface receptor, CD44, using monoclonal antibodies. In epidermis both HA and CD44 were found in the basal and spinous cell layers, but neither was present in the stratum granulosum and stratum corneum. In the keratinizing parts of hair follicles, i.e. in the outer and inner epidermal root sheath, pilosebaceous duct and the actual hair, HA and CD44 were found between the vital but not the terminally differentiated cells. In the sebaceous glands a small amount of HA was found around all cells, whereas CD44 was restricted to the basal cell layer. The secretory acini of the sweat glands stained intensively with anti-CD44 antibodies but only weakly with HABC. In the sweat gland, CD44 was localized on the basal and lateral surfaces of the clear cells, whereas the dark cells and the myoepithelial cells were negative. Both the lower and upper layers of the sweat gland ducts showed a faint but constant staining for CD44 and only minor amounts of HA. While in the keratinizing skin epithelia both HA and its CD44 receptor showed an intense staining with a close co-distribution, in the sweat and sebaceous glands their distribution patterns were not similar. It is suggested that in epithelia with divergent differentiation programs the functions of CD44 and HA may be different.     

Localization of gamma-glutamyl transpeptidase in human sweat glands: an immunohistochemical study using a monoclonal antibody

We studied the distribution of gamma-glutamyl transpeptidase (gamma-GT) by use of a monoclonal antibody (MAb) against human kidney gamma-GT in human sweat glands. In the eccrine sweat gland, the enzyme was localized along the luminal membrane and small apocrine extrusions of the superficial cells of the secretory portion. The intercellular canaliculi between basal cells were occasionally immunoreactive. In the secretory portion of the apocrine gland, luminal membrane and apocrine extrusions of various sizes and stages at the apices of the secretory cells exhibited positive reactions. Immunoreaction was also seen in the Golgi area of the cuboidal secretory cells. No positive reaction was observed in the myoepithelial cells of either gland or in the excretory duct cells.     

Three-dimensional culture and identification of human eccrine sweat glands in matrigel basement membrane matrix

Interactions between the extracellular matrix (ECM) and epithelial cells are necessary for the proper organization and function of the epithelium. In the present study, we show that human eccrine sweat gland epithelial cells cultured in matrigel, a representation of ECM components, constitute a good model for studying three-dimensional reconstruction, wound repair and regeneration and differentiation of the human eccrine sweat gland. In matrigel, epithelial cells from the human eccrine sweat gland form tubular-like structures and then the tubular-like structures coil into sphere-like shapes that structurally resemble human eccrine sweat glands in vivo. One sphere-like shape can be linked to another sphere-like shape or to a cell monolayer via tubular-like structures. Hematoxylin and eosin staining has revealed that the tubular-like structures have a single layer or stratified epithelial cells located peripherally and a lumen at the center, similar to the secretory part or duct part, respectively, of the eccrine sweat gland in sections of skin tissue. Immunohistochemical analysis of the cultures has demonstrated that the cells express CK7, CK19, epithelial membrane antigen and actin. Thus, matrigel promotes the organization and differentiation of epithelial cells from the human eccrine sweat gland into eccrine sweat gland tissues.     

Cell-specific expression of epithelial sodium channel alpha, beta, and gamma subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry

A highly selective, amiloride-sensitive, epithelial sodium channel from rat colon (rENaC), composed of three homologous subunits termed alpha, beta, and gamma rENaC, has been cloned by functional expression and was proposed to mediate electrogenic sodium reabsorption in aldosterone- responsive epithelia. To determine whether rENaC could account for sodium absorption in vivo, we studied the cellular localization of the sodium channel messenger RNA subunits by in situ hybridization and their cellular and subcellular distribution by immunocytochemistry in the kidney, colon, salivary, and sweat glands of the rat. In the kidney, we show that the three subunit mRNAs are specifically co- expressed in the renal distal convoluted tubules (DCT), connecting tubules (CNT), cortical collecting ducts (CCD), and outer medullary collecting ducts (OMCD), but not in the inner medullary collecting ducts (IMCD). We demonstrate co-localization of alpha, beta, and gamma subunit proteins in the apical membrane of a majority of cells of CCD and OMCD. Our data indicate that alpha, beta, and gamma subunit mRNAs and proteins are co-expressed in the distal nephron (excepting IMCD), a localization that correlates with the previously described physiological expression of amiloride-sensitive electrogenic sodium transport. Our data, however, suggest that another sodium transport protein mediates electrogenic amiloride-sensitive sodium reabsorption in IMCD. We also localized rENaC to the surface epithelial cells of the distal colon and to the secretory ducts of the salivary gland and sweat gland, providing further evidence consistent with the hypothesis that the highly selective, amiloride-sensitive sodium channel is physiologically expressed in aldosterone-responsive cells.     

Structure of the plantar sweat glands of the Bank vole Clethrionomys glareolus

The plantar pads of the hind feet of 15 male and female, young and old Clethrionomys glareolus (Rodentia–family Muridae) were examined for the presence of tubular glands. Groups of eight or nine coiled glands were found in all of the 11 plantar pads. The secretory portion of the gland lies deep in the fatty hypodermis and communicates with the plantar surface by a coiled duct whose lining cells merge with those of the stratum basale of the undersurface of the foot. Groups of shallow depressions mark the site of the duct openings. They are not associated with any hairs.
Although light microscopy shows only one cell type, electron microscopy reveals two morphologically different cell types. Neither conform directly to the light and dark secretory cells of the human sweat glands although similarities exist. Myoepithelial cells surround the secretory cells, but do not appear to be present around the duct cells.     

Distribution of hyaluronan and its CD44 receptor in the epithelia of human skin appendages

Summary Biotinylated hyaluronan (HA) binding complex (HABC) from bovine articular cartilage proteoglycan was used as a histological probe to study the localization of HA in human skin. The distribution of HA was compared with its presumptive cell surface receptor, CD44, using monoclonal antibodies. In epidermis both HA and CD44 were found in the basal and spinous cell layers, but neither was present in the stratum granulosum and stratum corneum. In the keratinizing parts of hair follicles, i.e. in the outer and inner epidermal root sheath, pilosebaceous duct and the actual hair, HA and CD44 were found between the vital but not the terminally differentiated cells. In the sebaceous glands a small amount of HA was found around all cells, whereas CD44 was restricted to the basal cell layer. The secretory acini of the sweat glands stained intensively with anti-CD44 antibodies but only weakly with HABC. In the sweat gland, CD44 was localized on the basal and lateral surfaces of the clear cells, whereas the dark cells and the myoepithelial cells were negative. Both the lower and upper layers of the sweat gland ducts showed a faint but constant staining for CD44 and only minor amounts of HA. While in the keratinizing skin epithelia both HA and its CD44 receptor showed an intense staining with a close co-distribution, in the sweat and sebaceous glands their distribution patterns were not similar. It is suggested that in epithelia with divergent differentiation programs the functions of CD44 and HA may be different.     

Multiple expression of keratins,vimentin, and S-100 protein in pleomorphic salivary adenomas

Immunohistochemical staining for S-100 protein and the intermediate filaments keratin and vimentin, was made in 41 salivary adenomas. In pleomorphic adenomas, great heterogeneity in the staining, as well as multiple and co-expressions of these proteins were found in the outer tumor cells of tubulo-ductal structures and modified myoepithelial cells, but not in the luminal tumor cells. All the outer tumor cells stained for S-100 protein, 97% for K8.12 keratin and 85% for vimentin. Of these cells, 29% showed multiple expression of K8.12 keratin, vimentin, and S-100 protein, and 17% showed co-expression of K8.12 and S-100 protein. Modified and neoplastic myoepithelial cells showed similar expressions of these proteins to those of outer tumor cells; myoepithelioma cells displayed the most complicated pattern, being positive for KL1, PKK1, and K8.12 keratins, vimentin and S-100 protein. In luminal tumor cells there was a heterogeneous expression of KL1 and PKK1 in 82%, and of KL1, PKK1, and K8.12 in only 14.7%. Based on the immunohistochemical findings obtained with different monoclonal antibodies in pleomorphic salivary adenomas, outer tumor cells may be derived from ductal basal cells and luminal tumor cells from intercalated duct cells.     

Multiple expression of keratins, vimentin, and S-100 protein in pleomorphic salivary adenomas.

Immunohistochemical staining for S-100 protein and the intermediate filaments keratin and vimentin, was made in 41 salivary adenomas. In pleomorphic adenomas, great heterogeneity in the staining, as well as multiple and co-expressions of these proteins were found in the outer tumor cells of tubulo-ductal structures and modified myoepithelial cells, but not in the luminal tumor cells. All the outer tumor cells stained for S-100 protein, 97% for K8.12 keratin and 85% for vimentin. Of these cells, 29% showed multiple expression of K8.12 keratin, vimentin, and S-100 protein, and 17% showed co-expression of K8.12 and S-100 protein. Modified and neoplastic myoepithelial cells showed similar expressions of these proteins to those of outer tumor cells; myoepithelioma cells displayed the most complicated pattern, being positive for KL1, PKK1, and K8.12 keratins, vimentin and S-100 protein. In luminal tumor cells there was a heterogeneous expression of KL1 and PKK1 in 82%, and of KL1, PKK1, and K8.12 in only 14.7%. Based on the immunohistochemical findings obtained with different monoclonal antibodies in pleomorphic salivary adenomas, outer tumor cells may be derived from ductal basal cells and luminal tumor cells from intercalated duct cells.     

Immunohistochemical study on the distribution of alpha and beta subunits of S-100 protein in human neoplasm and normal tissues

The immunohistochemical distribution and localization of the alpha and beta subunits of S-100 protein in human neoplasms and normal tissues were studied by the PAP method using monospecific rabbit antibodies against each subunit. Beta subunit immunoreactivity was detected in all S-100-positive cells and tumors reported previously. In contrast alpha subunit immunoreactivity was absent from Schwann cells, schwannomas, neurofibromas, granular cell myoblastomas, pituicytes of the neurohypophysis, Langerhans cells, interdigitating reticulum cells, and histiocytosis X cells. Interestingly, only the alpha subunit was detected in neurons of both central and peripheral nervous system, and in lymph node macrophages. Human S-100-positive cells are divided into three groups; the first is composed of cells containing only the beta subunit (probably S-100b; beta beta), the second consists of cells containing both the alpha and beta subunits, and the third is composed of cells containing only the alpha subunit (probably S- 100ao ; alpha alpha). The ontogentic relationships between S-100-positive cells and tumors are discussed in the light of these findings.     

Gross Cystic Disease Fluid Protein 15 in Stratum Corneum Is a Potential Marker of Decreased Eccrine Sweating for Atopic Dermatitis

It is well known that eccrine sweating is attenuated in patients with atopic dermatitis (AD). We have reported by using proteome analysis that gross cystic disease fluid protein 15 (GCDFP15), a substance secreted from eccrine sweat glands, is decreased in tape-stripped stratum corneum (SC) samples from AD patients. The aim of this study was to evaluate GCDFP15 production by eccrine glands with SC samples and to assess sweating in AD. SC samples were obtained from 51 healthy control (HC) and 51 AD individuals. Sweat samples were from 18 HC and 12 AD subjects. GCDFP15 was quantified by ELISA. By immunohistochemistry, the expression of GCDFP15 in eccrine glands was examined in normal and AD skin specimens. To identify GCDFP15-producing cells, double immunofluorescence staining for GCDFP15 and S100 protein was performed in frozen sections. To address the mechanism underlying the decreased eccrine sweating in AD patients, we examined the expression of cholinergic receptor M3 (CHRM3), a receptor for acetylcholine-induced sweating, in eccrine sweat glands. The amounts of GCDFP15 in the SC extracts were significantly lower in AD than HC (P < 0.0001). The sweat samples from AD patients also had lower levels of GCDFP15 concentration (P < 0.05). Immunohistochemistry showed positive GCDFP15 staining in the eccrine gland secretory cells and the ductal and acrosyringial lumen in normal skin, but AD lacked clear staining. Immunofluorescence staining revealed that GCDFP15 was co-expressed with S100 protein, suggesting that the clear cell of eccrine glands produces GCDFP15. Finally, we found that the expression of CHRM3 was depressed in AD, suggesting contribution to the low sweating. The SC of AD patients contains a low amount of GCDFP15 due to both low sweating and low GCDFP15 concentration in the sweat. GCDFP15 in SC is a potential marker for dysregulated sweating in AD.     

S-100 alpha-like immunoreactivity in tubules of rat kidney. A clue to the function of a "brain-specific" protein

The localization of the alpha and beta subunits of S-100 protein was studied in normal tissue where the identification of three subclasses of S-100 containing cells was derived: i) cells that contain both alpha and beta subunits; ii) cells that contain only the alpha subunit; and iii) cells that contain only the beta subunit. In this study monospecific antibodies against the S-100 alpha and beta subunits were used to characterize the S-100-like immunoreactivity in the rat kidney: Certain cells in the distal nephron, i.e., the connecting piece, collecting ducts, and the thin limb of Henle's loop, contained S-100 alpha immunoreactivity. Proximal tubules, the thick ascending limb of Henle's loop, the distal tubules, and the juxtaglomerular apparatus were negative. No S-100 beta immunoreactivity was found in kidney tubules. However, Schwann cells of renal pelvic nerves contained S-100 beta immunoreactivity. The presence of S-100 alpha antigen in certain cells of the kidney gives further support to the assumption that the alpha subunit of S-100a is related to cells that are highly involved in pH, electrolyte, and water regulation.     

Induction of S-100b (beta beta) protein in human teratocarcinoma cells

Human teratocarcinoma NT2/D1 cells undergo differentiation into a variety of cell types, including neurons, treated with retinoic acid. In the present study, the concentrations of alpha S-100 and beta S-100 proteins (alpha and beta subunits of S-100 proteins), and three subunits (alpha, beta and gamma) of enolase in NT2/D1 cells were measured using the sensitive enzyme immunoassay method. The concentration of beta S-100 was markedly increased in the cells after treatment with retinoic acid, whereas the concentration of alpha S-100 was undetectably low, indicating that the S-100b (beta beta) protein was induced by retinoic acid. On the other hand, the concentrations of the three forms of enolase isozymes did not change in the same culture. The induction of S-100b protein was not observed in the NT2/D1 cells after treatment with forskolin, dibutyryl cyclic AMP or cholera toxin. The indirect double-labeled immunofluorescence, using antibodies specific to beta S-100 and monoclonal antibodies specific to neurofilaments, revealed that both the S-100b protein and the neurofilaments were induced in the same subpopulation of cells which underwent neuronal differentiation.     

AN ELECTRON MICROSCOPIC STUDY OF ECCRINE SWEAT GLANDS OF THE CAT FOOT AND TOE PADS—EVIDENCE FOR DUCTAL REABSORPTION IN THE HUMAN

The eccrine sweat glands of the cat foot and toe pads have been studied by light and electron microscopy before and after stimulation with mecholyl. The ultrastructure of these glands in the cat is found to be entirely comparable to that in the human (13). The ultrastructure and staining properties of the secretory segment of the two species are identical. The ductal part of the feline gland is shorter and the ductal cells have only scant mitochondria as compared with the human. Since Brusilow et al. (1) have observed that the secretion of the cat foot pad is isotonic as compared with human sweat, which is hypotonic, and since the secretory segments of the two species are structurally identical, the striking difference in the morphology of the duct is regarded as being responsible for the difference in the chemistry of the secretion of the two species. Thus the duct in the human is capable of reabsorbing sodium and chloride.