cAMP-dependent Cl(-) channel protein (CFTR) and its mRNA are expressed in the secretory portion of human eccrine sweat gland.

Immunoreactive cystic fibrosis transport regulator (CFTR) proteins in human sweat ducts has been documented but CFTR expression in the secretory coil has remained uncertain. Using monoclonal antibodies (MAbs) against epitopes in the R-domain and C-terminus, we observed the following: Formalin fixation masks the CFTR epitopes but the epitopes are exposed by treatment with urea and heat (antigen retrieval). Pen-Fix fixation preserves CFTR epitopes. The secretory coil also expresses CFTR epitopes for the R-domain and C-terminus. An MAb against C-terminus amino acids 1466-1480 coupled to keyhole limpet hemocyanin (MAb WC) stained dark cells predominantly. Staining by MAbs against the C-terminus was completely blocked by a C-terminus peptide. mRNA for CFTR was amplified by RT-PCR in both the duct and the secretory coil. In situ hybridization for CFTR mRNA after 3SR amplification indicates that mRNA is localized in the dark cells and perhaps also in the clear cell cytoplasm near the secretory coil. mRNA is present in both the luminal and basal duct cells. We conclude that CFTR is expressed equally well in both the duct and the secretory coil, suggesting that cAMP-dependent Cl(-) channels are involved in regulation of sweat secretion and duct absorption.     

Localisation of the Vacuolar Proton Pump (V-H+-ATPase) and Carbonic Anhydrase II in the Human Eccrine Sweat Gland

The localisation of the vacuolar proton pump (V-H+ -ATPase) and the enzyme carbonic anhydrase II (CAII) was investigated in the human eccrine sweat gland employing standard immunohistochemical techniques after antigen retrieval using microwave heat treatment and high pressure. The high-pressure antigen retrieval unmasked the presence of V-H+ -ATPase in the clear cells of the secretory coil, with a distribution similar to that previously observed for CAII. However, the dark cells were unreactive to both antibodies. In addition, heat and high-pressure antigen retrieval demonstrated the presence of CAII in the apical zone of luminal cells of the reabsorptive duct, a location not previously reported. The localisation of V-H+ -ATPase and CAII in the secretory coil clear cells suggests that the formation of HCO3- and H+ by carbonic anhydrase II and the transport of H+ by V-H+ -ATPase may play an role in sweat fluid secretion. Their presence at the apex of the duct cells indicates involvement in ductal ion reabsorption.     

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.     

Immunohistochemical localization of activated EGF receptor in human eccrine and apocrine sweat glands.

Epidermal growth factor (EGF) is secreted into sweat from secretory cells of human sweat glands. The function of EGF in sweat is poorly understood. The biological function of EGF is exerted by the binding of EGF to the receptor (EGFR) and its activation. Therefore, we immunohistochemically localized the activated form of EGFR in human eccrine and apocrine sweat glands to assess the functional importance of the EGF-EGFR system in human sweat glands. Frozen sections of human skin were stained with a monoclonal antibody (MAb) specific for tyrosine-phosphorylated (activated) EGFR and with an MAb that stains both activated and non-activated EGFR. In the secretory portion of eccrine sweat glands, nuclei of the secretory cells were stained with the anti-activated EGFR MAb. In coiled and straight portions of eccrine sweat ducts, nuclei of luminal and peripheral cells were stained with the antibody specific for activated EGFR. Luminal cell membranes and luminal cytoplasm of inner ductal cells possessed non-activated EGFR. In the secretory portion of apocrine sweat glands, activated EGFRs were present in cytoplasm and nuclei of secretory cells. These data suggest that EGF, already known to be present in the cytoplasm of secretory cells in eccrine and apocrine sweat glands, activates EGFR in the nuclei of secretory cells themselves in an intracrine manner. Because ductal cells do not express EGF, EGF in the sweat secreted from the secretory cells should activate EGFR in the ductal cells in a paracrine manner. (J Histochem Cytochem 49:597-601, 2001)     

Ultrastructural localization of ouabain-sensitive, K-dependent p-nitrophenyl phosphatase activity in monkey eccrine sweat gland

We studied the electron microscopic localization of ouabain-sensitive, potassium-dependent p-nitrophenyl phosphatase (K-pNPPase) activity of the Na K-ATPase complex in Rhesus monkey eccrine sweat gland by use of the one-step lead citrate method of Mayahara et al. (Histochemistry 1980; 67:125). Reaction product was observed predominantly in the cytoplasmic side of the basolateral membranes of clear (secretory) cells, especially in the interdigitating membrane folds in the basal labryinth, and were completely abolished by 10 mM ouabain or by removal of K+. Little or no enzyme activity was noted in membrane processes in the intercellular canaliculi and in the secretory coil lumen. Basolateral membranes of the dark cells also showed moderate enzyme activity. The myoepithelial cell membrane was devoid of reaction product, except in a few membrane processes arising from the inner aspect of myoepithelial cells. In the coiled duct, K-pNPPase activity was present predominantly in the entire cell membrane of the peripheral ductal cells. The predominantly basolateral distribution of Na-K-ATPase in the eccrine sweat secretory cells is consistent with the concept that a Na-K-Cl co-transport model may be involved in the mechanism of eccrine sweat secretion.     

Vacuolar-type H+ -ATPase distribution in unstimulated and acetylcholine-activated isolated human eccrine sweat glands

The presence and cellular distribution of subunits of the V₁ sector of the vacuolar-type H⁺-ATPase (V-ATPase) was investigated in isolated human eccrine sweat glands. In every instance, V-ATPase was located in the cytoplasm and apical membranes of the luminal cells of the reabsorptive duct segment. In the secretory coil, both diffuse and perinuclear staining was demonstrated in the secretory cells, with additional expression at the apical and basolateral membranes and on the intercellular canaliculi. There was no detectable difference in V-ATPase expression as a result of prior application of 100µM acetylcholine.     

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.     

Immunohistochemical localization by monoclonal antibodies of S-100 alpha and beta proteins in mixed tumours and adenomas of the skin

A study using monoclonal antibodies was made to evaluate the immunohistochemical localization of S-100 protein subunits alpha and beta in a total of 41 mixed tumours and adenomas of sweat gland origin. Normal eccrine glands showed positive staining for S-100 alpha in the secretory portion and in epithelial cells located in the transitional area from the coiled duct to the intraepidermal duct, as well as granular deposition of S-100 beta at the luminal surface of the secretory coil and duct. The myoepithelial cells were negative for S-100 alpha and beta. In mixed tumours, the tumour cells were round or oval in shape and displayed markedly positive staining for S-100 alpha and slightly positive or negative staining for S-100 beta. S-100 alpha staining in clear cell tumours was typically more intense than in any other sweat gland tumour. It is possible that clear cell tumours may arise from the transitional area of sweat glands. Spindle cell tumours displayed on abundance of S-100 alpha subunits but little S-100 beta. Occasional spindle cells located in the outer layer of tubular structures within tumours gave positive S-100 alpha staining. This result was different from that seen in pleomorphic salivary adenomas. Cells having undergone chondroidal changes revealed a positive S-100 reaction.     

Immunolocalization of band 3 protein in normal and cystic fibrosis skin

Current evidence indicates that the defect in cystic fibrosis (CF) involves chloride transport in various epithelial cells. The sweat gland, one site of altered chloride transport in CF, was examined immunocytochemically for localization of a chloride-channel membrane protein, designated band 3 protein. Immunoreactivity was observed in sweat duct cell membranes of both normal and CF samples, whereas secretory coil regions were entirely unreactive. No difference was observed in the pattern or intensity of immunoreactivity between the two groups at the light microscopic (LM) level of resolution.     

NKCC1 and NHE1 are abundantly expressed in the basolateral plasma membrane of secretory coil cells in rat, mouse, and human sweat glands

In isolated sweat glands, bumetanide inhibits sweat secretion. The mRNA encoding bumetanide-sensitive Na⁺-K⁺-Cl^– cotransporter (NKCC) isoform 1 (NKCC1) has been detected in sweat glands; however, the cellular and subcellular protein localization is unknown. Na⁺/H⁺ exchanger (NHE) isoform 1 (NHE1) protein has been localized to both the duct and secretory coil of human sweat duct; however, the NHE1 abundance in the duct was not compared with that in the secretory coil. The aim of this study was to test whether mRNA encoding NKCC1, NKCC2, and Na⁺-coupled acid-base transporters and the corresponding proteins are expressed in rodent sweat glands and, if expressed, to determine the cellular and subcellular localization in rat, mouse, and human eccrine sweat glands. NKCC1 mRNA was demonstrated in rat palmar tissue, including sweat glands, using RT-PCR, whereas NKCC2 mRNA was absent. Also, NHE1 mRNA was demonstrated in rat palmar tissue, whereas NHE2, NHE3, NHE4, electrogenic Na⁺-HCO₃^– cotransporter 1 NBCe1, NBCe2, electroneutral Na⁺-HCO₃^– cotransporter NBCn1, and Na⁺-dependent Cl^–/HCO₃^– exchanger NCBE mRNA were not detected. The expression of NKCC1 and NHE1 proteins was confirmed in rat palmar skin by immunoblotting, whereas NKCC2, NHE2, and NHE3 proteins were not detected. Immunohistochemistry was performed using sections from rat, mouse, and human palmar tissue. Immunoperoxidase labeling revealed abundant expression of NKCC1 and NHE1 in the basolateral domain of secretory coils of rat, mouse, and human sweat glands and low expression was found in the coiled part of the ducts. In contrast, NKCC1 and NHE1 labeling was absent from rat, mouse, and human epidermis. Immunoelectron microscopy demonstrated abundant NKCC1 and NHE1 labeling of the basolateral plasma membrane of mouse sweat glands, with no labeling of the apical plasma membranes or intracellular structures. The basolateral NKCC1 of the secretory coils of sweat glands would most likely account for the observed bumetanide-sensitive NaCl secretion in the secretory coils, and the basolateral NHE1 is likely to be involved in Na⁺-coupled acid-base transport. bumetanide; eccrine glands; immunohistochemistry; immunoblotting     

Immunoelectron microscopic localization of epidermal growth factor in the eccrine and apocrine sweat glands.

We studied the localization of the epidermal growth factor (EGF) in eccrine and apocrine sweat glands with light microscopic and electron microscopic immunohistochemistry. Anti-human EGF (anti-hEGF) polyclonal antiserum and anti-hEGF monoclonal antibody (MAb) were used for the study. Light microscopic immunohistochemistry with monoclonal and polyclonal antibodies showed that hEGF-like immunoreactivity was strongly positive in the myoepithelial cells and weakly positive in the secretory cells of eccrine sweat glands. In apocrine sweat glands, it was strongly positive in the secretory cells as well as in the myoepithelial cells. Immunoelectron microscopy with polyclonal antibody showed that hEGF-like immunoreactivity was present in secretory granules of apocrine secretory cells. These granules had mitochondrion-like internal structure. No reactivity was observed on the eccrine secretory cells by immunoelectron microscopy. Neither dark cell granules nor mitochondria in eccrine secretory cells were labeled with anti-hEGF antibody. In both eccrine and apocrine sweat glands, hEGF-like immunoreactivity was diffusely present in the cytoplasm of myoepithelial cells. However, nuclei and mitochondria of myoepithelial cells were devoid of immunoreactivity for hEGF. Our observations indicate that apocrine sweat glands may secrete more hEGF in the sweat than eccrine sweat glands.     

High-level expression of biologically active human prolactin from recombinant baculovirus in insect cells

We examined the feasibility of high-level production of recombinant human prolactin, a multifunctional protein hormone, in insect cells using a baculovirus expression system. The human prolactin cDNA with and without the secretory signal sequence was cloned into pFastBac1 baculovirus vector under the control of polyhedrin promoter. Prolactin was produced upon infection of either Sf9 or High-Five cells with the recombinant baculovirus containing the human prolactin cDNA. The production of recombinant prolactin varied from 20 to 40 mg/L of monolayer culture, depending on the cell types. The prolactin polypeptide with its own secretory signal was secreted into the medium. N-terminal amino acid sequence analysis of the recombinant polypeptide purified from the culture medium indicated that the protein was processed similar to human pituitary prolactin. Carbohydrate analysis of the purified protein indicated that a fraction of the recombinant prolactin made in insect cells appeared to be glycosylated. Also, both secreted and nonsecreted forms of the recombinant prolactin in insect cells were biologically equivalent to the native human prolactin (pituitary derived) in the Nb2 lymphoma cell proliferation assay.     

Functional and morphological changes in the eccrine sweat gland with heat acclimation

Three adult male patas monkeys (11-15 kg) were heat acclimated by continuous exposure to an ambient temperature of 33 +/- 1 degree C at 13% relative humidity for 9 mo. During the last month, they were also exposed to 45 degrees C at 10% relative humidity for 4 h/day and 5 days/wk. Before and after 3 wk of acclimation, the animals were given a heat-tolerance test in which rectal (Tre) and mean skin (Tsk) temperatures, heart rate, and sweat rate (msw) were monitored during a 90-min exposure to 45 degrees C heat with 24% relative humidity under lenperone (1.0-1.4 mg/kg im) tranquilization. Maximal in vivo msw was also determined in response to subcutaneous injections (1 and 10% solutions) of methacholine (MCh). Before and after 9 wk and 9 mo of acclimation, sweat glands were dissected from biopsy specimens of the lateral calf, cannulated, and stimulated in vitro with MCh. Morphological measurements of isolated tubules were compared with maximal secretory rates produced by MCh stimulation. Three weeks of acclimation 1) reduced Tre and Tsk and increased msw during the heat tolerance test and 2) significantly increased maximal msw in response to MCh stimulation. Acclimation also increased (P less than 0.05) sweat gland size, as measured by tubular length and tubular volume. Maximal in vitro msw produced by MCh stimulation and msw per unit length of secretory coil also increased significantly. We conclude that heat acclimation increases the size of eccrine sweat glands and that these larger glands produce more sweat. They are also more efficient because they produce more sweat per unit length of secretory coil.     

Ion transport in primary cultures from human sweat gland coils studied with X-ray microanalysis

Chloride secretion in primary cultures of cells originating from the secretory coil of human sweat glands was investigated by electron probe X-ray microanalysis. The total intracellular Cl concentration was lowered by muscarinic agonists (carbachol and acetylcholine), as well as by the calcium ionophore A23187. The muscarinic agonists also lowered the cellular K concentration. Cl^- secretion induced by these agonists could be inhibited by the chloride channel blocker NPPB. After cAMP stimulation, the frequency distribution of the Cl concentration changed from Gaussian to bimodal, indicating that cAMP induces Cl^- secretion only from a subpopulation of the cells. Also ATP stimulated Cl^- secretion, indicating the presence of purinergic receptors. The results suggest that some of the cells in addition to Ca²⁺ -regulated Cl^- channels also possess cAMP-activated Cl^- channels. Hence, the primary cultures still possess the Cl^- transport mechanisms known to be present in intact glands. It can, however, not be excluded that some coil cells have acquired ductal characteristics during culture.     

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.     

Low abundance of sweat duct Cl- channel CFTR in both healthy and cystic fibrosis athletes with exceptionally salty sweat during exercise

To understand potential mechanisms explaining interindividual variability observed in human sweat sodium concentration ([Na(+)]), we investigated the relationship among [Na(+)] of thermoregulatory sweat, plasma membrane expression of Na(+) and Cl(-) transport proteins in biopsied human eccrine sweat ducts, and basal levels of vasopressin (AVP) and aldosterone. Lower ductal luminal membrane expression of the Cl(-) channel cystic fibrosis transmembrane conductance regulator (CFTR) was observed in immunofluorescent staining of sweat glands from healthy young adults identified as exceptionally "salty sweaters" (SS) (n = 6, P < 0.05) and from patients with cystic fibrosis (CF) (n = 6, P < 0.005) compared with ducts from healthy young adults with "typical" sweat [Na(+)] (control, n = 6). Genetic testing of healthy subjects did not reveal any heterozygotes ("carriers") for any of the 39 most common disease-causing CFTR mutations in the United States. SS had higher baseline plasma [AVP] compared with control (P = 0.029). Immunostaining to investigate a potential relationship between higher plasma [AVP] (and sweat [Na(+)]) and ductal membrane aquaporin-5 revealed for all groups a relatively sparse and location-dependent ductal expression of the water channel with localization primarily to the secretory coil. Availability of CFTR for NaCl transport across the ductal membrane appears related to the significant physiological variability observed in sweat salt concentration in apparently healthy humans. At present, a heritable link between healthy salty sweaters and the most prevalent disease-causing CFTR mutations cannot be established.     

THE ULTRASTRUCTURE AND HISTOPHYSIOLOGY OF HUMAN ECCRINE SWEAT GLANDS

The electron microscopy of human eccrine sweat glands has been studied before and after stimulation by pilocarpine iontophoresis. The identity of the dark and clear cells in the secretory segment as defined by Montagna et al. (23) was determined by studying serial sections, thin for electron microscopy and thick for light microscopy. Cells with numerous apical secretory vacuoles are termed mucoid (dark) cells, since these vacuoles stain positively for acid mucopolysaccharide. Clear cells are intimately associated with intercellular canaliculi. The "cuticular border" of surface cells of the duct is a condensation of tonofilaments and granules. Numerous mitochondria are concentrated in basal cells of the duct. The presence of mucoid cells in the secretory segment may bear on the interpretation of the pathologic findings in the disease cystic fibrosis of the pancreas, and suggests that this disease may be due to a basic disorder of mucopolysaccharide production. The possible roles of the various cellular components in the elaboration of sweat are discussed.     

Origin of periodic acid-Schiff-reactive glycoprotein in human eccrine sweat

To evaluate the possible involvement of ductal blockade with periodic acid-Schiff (PAS)-positive materials in the mechanism of hidromeiosis in humans, skin slices were incubated with methacholine for 2 h and PAS-positive materials localized histologically in the ductal lumen. In 20% of the glands complete ductal blockade with PAS-positive materials was noted. The characteristics and origin of such PAS-positive glycoproteins in human sweat were then studied using various electrophoretic techniques. One-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis (1-D SDS-PAGE) demonstrated considerable individual variation in the electrophoretic pattern; however, four major bands at 45, 28, 20, and 18K shared by different individuals, were PAS positive. Further studies using two-dimensional SDS-PAGE, immunodiffusion and immunoaffinity chromatography demonstrated that the PAS-positive glycoproteins are not derived directly from serum because they are electrophoretically and antigenically distinct from serum proteins, including alpha 1-glycoprotein, alpha 2-HS-glycoprotein, and alpha 1-antitrypsin. Since only dark cell granules are densely stained in the histochemical PAS staining, and because antiserum produced against the PAS-positive band selectively stained cells facing the secretory coil lumen (which are most likely dark cells), it is suggested that PAS-positive sweat glycoproteins are derived predominantly from the dark cells.     

A melanocyte–melanoma precursor niche in sweat glands of volar skin

Determination of the niche for early‐stage cancer remains a challenging issue. Melanoma is an aggressive cancer of the melanocyte lineage. Early melanoma cells are often found in the epidermis around sweat ducts of human volar skin, and the skin pigmentation pattern is an early diagnostic sign of acral melanoma. However, the niche for melanoma precursors has not been determined yet. Here, we report that the secretory portion (SP) of eccrine sweat glands provide an anatomical niche for melanocyte–melanoma precursor cells. Using lineage‐tagged H2B‐GFP reporter mice, we found that melanoblasts that colonize sweat glands during development are maintained in an immature, slow‐cycling state but renew themselves in response to genomic stress and provide their differentiating progeny to the epidermis. FISH analysis of human acral melanoma expanding in the epidermis revealed that unpigmented melanoblasts with significant cyclin D1 gene amplification reside deep in the SP of particular sweat gland(s). These findings indicate that sweat glands maintain melanocyte–melanoma precursors in an immature state in the niche and explain the preferential distribution of early melanoma cells around sweat glands in human volar skin.     

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.