Eccrine Sweat Gland Cells Research Articles

PAR-2 receptor-induced effects on human eccrine sweat gland cells

Serine proteases can induce cell signaling by stimulating G-protein-coupled receptors, called proteinase-activated receptors (PAR's) on a variety of epithelial cells. While PAR-2, one such receptor, activates cell signaling in a secretory cell line derived from human sweat glands, there was no information on their presence and effects on intact sweat glands. PAR-2 presence and activation of eccrine sweat glands isolated from human skin samples was investigated using Western blot analysis, immunohistochemistry, electron microscopy (EM) and Ca(2+) imaging. Anti-human PAR-2 antibody demonstrated the presence of these receptors in eccrine sweat glands. EM showed that PAR-2 activation resulted in degranulation of secretory cells. Ca(2+) imaging using PAR-2 activators demonstrated a two phase increase in [Ca(2+)](i) which was dependent on extracellular Ca(2+) for the second phase, and that the response could be blocked by prior incubation with xestospongin, the IP(3) receptor blocker. The results demonstrated that PAR-2 receptors are present in human sweat gland secretory cells and that these receptors are functionally active and can induce changes associated with secretory events in eccrine glands.

Estradiol rapidly induces the translocation and activation of the intermediate conductance calcium activated potassium channel in human eccrine sweat gland cells

Background and aims Steroid hormones target K + channels as a means of regulating electrolyte and fluid transport. In this study, ion transporter targets of Estradiol (E2) were investigated in the human eccrine sweat gland cell line NCL-SG3. Results Whole cell patch-clamp studies revealed E2 (10 nM) rapidly activates a whole cell K + conductance, which is abolished by clotrimazole (30 μM), an inhibitor of the intermediate conductance calcium activated K + channel (IK Ca). The estrogen receptor (ER) antagonist ICI 182, 780 had no effect on this E2 activated K + conductance, suggesting an estrogen receptor independent mechanism of activation. Confocal microscopy studies revealed under basal conditions that the IK Ca channel is located within the cell cytoplasm and in the presence of E2, rapidly translocates to both the apical and basolateral membrane. In the presence of E2, tyrosine phosphorylation of calmodulin, which is known to regulate trafficking of the IK Ca channel, is increased, and treatment of cells with the calmodulin inhibitor trifluoperazine (TFP) prevents the E2-induced translocation. Conclusions Estradiol rapidly regulates a K + conductance through the IK Ca channel in an estrogen receptor independent manner. E2 stimulates the translocation of IK Ca to the cell membrane in a calmodulin dependent manner, representing a novel paradigm of estrogen action in sweat gland epithelial cells.

PAR‐2 receptor‐induced intracellular signalling in isolated intact human eccrine sweat gland cells and primary tissue culture cells

PAR‐2 receptors can evoke intracellular signalling and anion transport in a cell line derived from human sweat glands, however, it is unknown whether PAR‐2 receptors are present and play a role in intact human sweat gland secretory cells. PAR‐2 receptors were investigated in isolated sweat glands and tissue culture cells derived from primary explants. Skin samples were obtained by informed consent and ethics approval from volunteers for immunohistochemistry and sweat gland isolation for; Western blot analysis; calcium imaging or explanted for tissue culture. Immunohistochemistry and Western blot using an anti human PAR‐2 antibody demonstrated the presence of these receptors in sweat gland secretory cells. Calcium imaging experiments using trypsin and the agonist peptide SLIGKV showed that both could increase intracellular calcium levels in intact glands and in the primary sweat gland cultures. Results also demonstrated that the effects of the agonist peptide were reversible upon washout, but not trypsin, and that the reverse peptide VKGILS failed to initiate changes in intracellular calcium. The results of this study demonstrate that PAR‐2 receptors are present in isolated human sweat gland secretory cells and in primary cultured cells and that these receptors are functionally active and can induce increases in intracellular calcium.The study was supported by Glasgow Caledonian University.

Activation of chloride secretion via proteinase‐activated receptor 2 in a human eccrine sweat gland cell line – NCL‐SG3

Proteinase-activated receptor 2 (PAR-2) has been shown to elicit secretion in a variety of secretory epithelial cells by the transepithelial movement of chloride ions across the apical membrane. However, it is not known whether these receptors are present and/or functional in the secretory epithelial cells of the human eccrine sweat gland. To investigate this possibility mRNA analysis, Ca2+ microspectrofluorimetry and the short circuit current (Isc) technique were used to quantify electrolyte transport in a cell line (NCL-SG3) derived from human eccrine sweat gland secretory epithelia. The results provided molecular and functional evidence of the presence of PAR-2 receptors in the NCL-SG3 cells and show that these receptors can activate transepithelial Cl- secretion possibly via Ca2+-activated Cl- channels.

A male infant case of lipofibromatosis in the submental region exhibited the expression of the connective tissue growth factor

We encountered a case of lipofibromatosis in a 21-month-old male patient and examined its histopathologic properties. We examined morphological aspects of the tumor and immunohistochemical patterns. Tumor proliferation was infiltrative, which did not show apparent encapsulation. Positive immunoreactivity was found for CD-34, CD-99, Ki-67, and connective tissue growth factor/CCN2 in the fibrous region, S-100 in the adipose region, and Notch1 stain was observed in the eccrine sweat gland cells juxtaposed to the tumor adipose tissue, but no reactivity for Bcl-2, alphaSMA, Notch 2-4, CCN1, and CCN3. The clinical process and immunohistochemical pattern of this case was consistent with the criteria of reported lipofibromatosis. Specific expression of CCN2 might be significant for the development of the tumor.

神崎病の発見・確立とその後の展開―１９８６〜２００６―

A 48-year-old Japanese lady was examined in 1989. She was apparently healthy but showed numerous numbers of small petechiae-like angiokeratoma on her lower torso to upper thighs, axillae and beneath the breasts. Electron microscopic examination of the skin revealed largely dilated electron lucent lysosomes with fuzzy filamentous materials in vascular endothelial cells, fibroblasts, eccrine sweat gland cells and others. Urinary examination revealed unusual glycopeptides with GalNAc-Ser/Thr moieties. This disease was reported as a novel lysosomal storage disease with angiokeratoma corporis diffusum, crowned Kanzaki disease (MIM#104170). Soon, this disease was found to be caused by a deficit of α-N-acetylgalactosaminidase (α-NAGA, 4. 3. 2. 49) activity and a point mutation was found in the gene (R329W) encoding the enzyme. Another patient, 47-year-old Japanese woman, was found, and she also was apparently healthy, but had less angiokeratoma as compared to the first one. The gene mutation was found and the resultant mutant enzyme was R329Q. She excreted less amount of GalNAc-Ser in urine as compared to the first patient. These phenotypical differences between case 1 and 2 were estimated to be caused by the differences in the three-dimensional structures in mutated α-NAGAs (R329W v. s. R329Q). Schindler disease also shows α-NAGA deficiency but shows very severe central nervous symptoms before the age of one. Electron microscopically electron-dence material deposited in lysosomes in Schindler disease. Electron-dense material means that the substance are probably lipid- or lipoprotein-containg materials. This quiet contrasts to the findings in Kanzaki disease. These evidences suggest that Kanzaki disease is caused by a pure α-NAGA deficiency but Schindler disease is probably caused by together α-NAGA deficiency with some other factors.

Characterization of a Novel Human Type II Epithelial Keratin K1b, Specifically Expressed in Eccrine Sweat Glands

In this study, we show that a novel human type II epithelial keratin, K1b, is exclusively expressed in luminal duct cells of eccrine sweat glands. Taking this luminal K1b expression as a reference, we have used antibodies against a plethora of epithelial keratins to systematically investigate their expression in the secretory globule and the two-layered sweat duct, which was divided into the intraglandular, intradermal, and intraepidermal (acrosyringium) segments, the latter being further subdivided into the sweat duct ridge and upper intraepidermal duct. We show that (i) each of the eccrine sweat gland tissue compartments expresses their own keratin patterns, (ii) the peripheral and luminal duct layers exhibit a sequential keratin expression, with both representing self-renewing cell layers, (iii) the intradermal duct and the sweat duct ridge display hitherto unknown length variations, and (iv) out of all cell layers, the luminal cell layer is the most robust layer and expresses the highest number of keratins, these being concentrated at the apical side of the cells to form the cuticle. We provide evidence that the cellular and intercellular properties of the peripheral and the luminal layers reflect adaptations to different functions.

Distribution of cannabinoid receptor 1 (CB1) and 2 (CB2) on sensory nerve fibers and adnexal structures in human skin

Cannabinoid receptors mediate the psychopharmacological action of marijuana and have been localized in the central and peripheral nervous system as well as on cells of the immune system. Up to now, two cannabinoid receptors (CB1 and CB2) have been cloned and recent studies on animal tissue gave evidence for the presence of cannabinoid receptors in the skin. In the present immunohistochemical investigation we determined the precise localization of CB1 and CB2 in sections of human skin and in one case of mastocytosis. CB1 and CB2 immunoreactivity was observed in cutaneous nerve fiber bundles, mast cells, macrophages, epidermal keratinocytes, and the epithelial cells of hair follicles, sebocytes and eccrine sweat glands. In epidermal keratinocytes, hair follicle and sebaceous glands, CB1 and CB2 were distributed in a complementary fashion. Double-immunostaining with an anti-CGRP antibody suggested the presence of cannabinoid receptors on small afferent peptidergic nerves. The abundant distribution of cannabinoid receptors on skin nerve fibers and mast cells provides implications for an anti-inflammatory, anti-nociceptive action of cannabinoid receptor agonists and suggests their putatively broad therapeutic potential.

Novel aspects in cutaneous biology of acetylcholine synthesis and acetylcholine receptors

Extraneuronal acetylcholine (ACh) has been demonstrated to influence a plethora of cutaneous cell functions in an autocrine, paracrine and endocrine fashion. Through the differentiation-specific expression of its different nicotinic (nACh-R) and muscarinic (mACh-R) receptors, ACh acts upon keratinocyte proliferation and migration, terminal differentiation and barrier formation, sweat and sebum secretion as well as microcirculation and angiogenesis. Only very recently it has been recognized that acetylcholinesterase, but not cholineacetyltransferase, activity is regulated by hydrogen peroxide. Considering that the outer layer of the human skin can be a target for UV-generated H2O2 in the millimolar range, this mechanism needs to be taken into account for the regulation of ACh homeostasis in skin biology. Consequently, ACh can accumulate, as shown, for example, in the depigmentation process in vitiligo. There is a highly regulated distribution of ACh-R in human epidermis and adnexal structures, supporting previously observed effects of cholinergic compounds on keratinocyte biology. Most significantly, the regulated expression of ACh-R in sebaceous glands advocates a role for ACh in sebum production and as a promoter of sebocyte differentiation, thus offering an explanation for skin diseases associated with altered sebum production after chronic nicotine exposure. So far, ACh-induced sweat production has been thought to be under the exclusive control of mACh-R. However, recently, the presence of both different nACh-R and mACh-R in myoepithelial and acinar cells of eccrine sweat glands has been documented, indicating a more complex regulation of sweat production and expulsion.

Hepatitis C virus replicates in sweat glands and is released into sweat in patients with chronic hepatitis C.

Hepatitis C virus (HCV) replicates in salivary glands of chronic hepatitis C patients and is released into the saliva, suggesting that HCV may replicate in other exocrine glands. The presence of positive and negative HCV RNA strands was demonstrated by in situ hybridization, and of HCV core protein by immunohistochemistry, in sweat glands and keratinocytes in healthy skin biopsies from 15 patients with chronic hepatitis C and 10 anti-HCV negative patients with chronic liver disease. Positive and negative HCV RNA strands were detected in 9.6 +/- 5.2% and 4.2 +/- 3.8%, respectively, of the epithelial cells of eccrine sweat glands. Core protein was detected in 6.0 +/- 3.93% of these cells. HCV RNA resistant to RNase digestion (encapsidated HCV RNA) was detected in 10/10 sweat samples from HCV-infected patients. Positive and negative HCV RNA strands were detected in 6.7 +/- 2.97% and 3.0 +/- 3.08% of the keratinocytes, respectively. HCV core protein was found in 4.5 +/- 2.76% of these cells. No HCV RNA or HCV core protein was detected in the skin biopsies from the 10 anti-HCV negative patients. In conclusion, HCV replicates in eccrine sweat glands cells and keratinocytes in healthy skin and is released into the sweat.

Immunoelectron microscopic analysis of lysosomal deposits in α- N-acetylgalactosaminidase deficiency with angiokeratoma corporis diffusum

α- N-Acetylgalactosaminidase (α-NAGA) deficiency with angiokeratoma corporis diffusum (AKCD) is one of the lysosomal storage diseases. GalNAcα1- O-Ser/Thr (Tn) is theoretically deposited in lysosomes, but substances with attached galactose and neuraminic (sialic) acid (T and sialosyl Tn, respectively) are excreted in patients’ urine. In this study, in two Japanese cases we analyzed the material accumulated in lysosomes using immunoelectron microscopy with mouse antibodies to Tn, sialosyl Tn and T (Thomsen–Friedenreich) antigens in order to find out what substance(s) is really deposited in lysosomes. We found that only GalNAcα1- O-Ser/Thr (Tn) was actually accumulated in vacuolated lysosomes of vascular endothelial cells, eccrine sweat gland cells, fibroblasts and pericytes. Galactosylation and sialylation of Tn appears to occur in cells other than those in the skin. The results suggest that this disease is caused by a single enzyme deficiency.

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)

Epiplakin, a Novel Member of the Plakin Family Originally Identified as a 450-kDa Human Epidermal Autoantigen: STRUCTURE AND TISSUE LOCALIZATION

A 450-kDa human epidermal autoantigen was originally identified as a protein that reacted with the serum from an individual with a subepidermal blistering disease. Molecular cloning of this protein has now shown that it contains 5065 amino acids and has a molecular mass of 552 kDa. As reported previously this protein, which we call epiplakin, belongs to the plakin family, but it has some very unusual features. Epiplakin has 13 domains that are homologous to the B domain in the COOH-terminal region of desmoplakin. The last five of these B domains, together with their associated linker regions, are particularly strongly conserved. However, epiplakin lacks a coiled-coil rod domain and an amino-terminal domain, both of which are found in all other known members of the plakin family. Furthermore, no dimerization motif was found in the sequence. Thus, it is likely that epiplakin exists in vivo as a single-chain structure. Epitope mapping experiments showed that the original patient's serum recognized a sequence unique to epiplakin, which was not found in plectin. Immunofluorescence staining revealed the presence of epiplakin in whole sheets of epidermis and esophagus, in glandular cells of eccrine sweat and parotid glands and in mucous epithelial cells in the stomach and colon.

Nucleotide-evoked ion transport and [Ca 2+] i changes in normal and hyperhidrotic human sweat gland cells

Apical and basolateral application of ATP and UTP evoked [Ca 2+] i and short circuit current (Isc) increases in normal and hyperhidrotic human eccrine sweat gland cells grown into functionally polarised epithelia on permeable supports. Basolateral application to hyperhidrotic cells exhibited a markedly greater increase in Isc than in normal cells. Hyperhidrotic cells also demonstrated differences from the normal in [Ca 2+] i and Isc responses to ATP when pre-treated with thapsigargin. The data demonstrate the presence of apical and basolateral receptors that allow nucleotides to increase [Ca 2+] i and Isc. The results suggest that changes from the normal in transepithelial ion transport contribute to the characteristic excessive fluid production of hyperhidrotic sweat glands.

Bikunin, a Serine Protease Inhibitor, is Present on the Cell Boundary of Epidermis

Bikunin, which is an inhibitor of serine proteases, is widely distributed in human tissues, including liver, kidney, and mucous membranes of the stomach and colon. The aim of this study was to clarify whether bikunin is expressed in human epidermis and its appendages. Immunoblot analysis using a specific polyclonal antibody to bikunin revealed that a single 43 kDa protein is present in the cell lysate from the human keratinocyte cell line HaCaT. Immunohistochemically, dotted reaction products stained with anti-bikunin antibody were localized on the cell boundary in both basal and spinous cell layers, except on the cell boundary of the basal cells facing the basal membrane. There were no reaction products in the granular-horny cell layers. Reaction products stained with anti-bikunin antibody were also observed on the hair bulb cells and eccrine sweat gland cells, but not on apocrine sweat glands. Also, reaction products were observed on the luminal surface of the renal proximal tubules and in the cytoplasm of these cells. In immunoelectron microscopy, gold particles were observed on the cell membranes close to the desmosomal structures. Reverse transcription-polymerase chain reaction and northern blot analyses showed that mRNA specific for bikunin was expressed in HaCaT cells and human epidermal keratinocytes obtained from suction blisters, and was contained in a commercially available human keratinocyte cDNA preparation. These findings indicate that bikunin is expressed in keratinocytes and may play an important part in regulating keratinocytes in either mitosis or inflammation.

Expression of CD44 splice variants in human skin and epidermal tumours

Splice variants of the adhesion molecule CD44 (CD44v) are important in the lymphatic spread of rat carcinoma cells. In several human tumours expression of CD44v correlates with tumour progression. However, little is known about the physiological functions of distinct variant exons. Here we report on the immunohistological evaluation of CD44 expression in normal human skin and epidermal tumours which do not metastasise, or do so vary rarely. Frozen tissues were stained with a panel of monoclonal antibodies, recognizing epitopes of the CD44 standard isoform, as well as of variant exons v5, v6, v7, v7-v8 and v10. Stratum basale and spinosum as well as the root shaft of hairs reacted strongly with the whole panel of anti-CD44 antibodies. Stratum corneum, acinar cells of sebaceous and eccrine sweat glands stained with anti-CD44v5, anti-CD44v6 and anti-CD44v7, but not with anti-CD44v10, the latter recognizing the "epithelial isoform" (CD44v8-v10) of CD44. Ductal cells of glands and apocrine glands did not express CD44v. Compared with its expression in normal human skin, CD44v expression was reduced in basal cell carcinoma and squamous cell carcinoma of the skin. This was particularly true of CD44v10. The expression of CD44v in normal skin and dermal appendages indicates that not all combinations of variant exons are involved in tumour progression. Since the epithelial isoform is particularly downregulated in basal cell carcinoma and squamous cell carcinoma of the skin, it is unlikely that exons v8-v10 play a role in tumour progression. Rather, they may be of functional importance in maintenance of the epidermal structure.

Immunoreactive analogues of erythrocyte ankyrin in human epidermal keratinocytes.

Using immunoblot and immunofluorescence analysis, we demonstrated the presence and localization of an immunoreactive form of erythrocyte ankyrin in human epidermal keratinocytes. Immunoblot analysis revealed that both human epidermis and cultured epidermal keratinocytes contained ankyrin-like proteins of molecular mass 210 kDa that crossreacted with antihuman erythrocyte ankyrin antibodies. Immunofluorescence microscopy revealed that the plasma membrane of epidermal keratinocytes was stained. Eccrine sweat gland cells and ductal cells were also stained. These results indicate that in human epidermal keratinocytes, eccrine sweat gland cells and ductal cells, an ankyrin-like protein is present as one of the membrane proteins. The present findings and our recent previous studies showing the presence of a spectrin-like protein (fodrin) and 4.1-like proteins in these cells enable us to suggest that a membrane skeletal protein lattice may exist in these cells.

Proliferating cells in human eccrine and apocrine sweat glands.

Morphological observations of sweat glands showed degenerated debris of secretory cells in the secretory lumen in both apocrine and eccrine sweat glands. This suggested that dead secretory cells of human eccrine and apocrine sweat glands were released into the lumen and replaced by other cells. However, we did not know which type of cells replaced lost secretory cells. Therefore, we studied the proliferating cells in human eccrine and apocrine sweat glands by labeling S-phase cells in vitro with 5-bromo-2'-deoxyuridine (BrdUrd) and by immunostaining proliferation-associated proliferating cell nuclear antigen (PCNA) with anti-PCNA monoclonal antibody. BrdUrd and anti-PCNA antibody labeled a few secretory cells in eccrine and apocrine sweat glands, but neither method labeled myoepithelial cells. Luminal and peripheral cells of the eccrine and apocrine coiled duct were labeled with both BrdUrd and PCNA. However, we could not find any highly proliferative germinative cells in coiled ducts. Our results suggest that lost secretory cells could be replaced by proliferation of secretory cells themselves rather than by proliferation of myoepithelial cells or duct cells.

CD44 expression in normal human skin and skin tumors.

CD44 is thought to be a principal cell surface receptor for hyaluronic acid. Although the distribution of hyalulonic acid has been studied, little is known about the distribution of the CD44 molecule in the human skin and skin tumors. This study was undertaken to investigate the distribution of the CD44 molecule in normal human skin as well as in benign and malignant skin tumors. In normal skin, CD44 was expressed on 1) keratinocyte cell surfaces throughout the epidermis except for the granular and horny layers, 2) hair follicular cells, 3) eccrine sweat gland cells, and 4) cell surfaces of dendritic cells in the dermis. In skin tumors, although CD44 was expressed on the tumor cell surface of seborreic keratosis, Bowen's disease, and squamous cell carcinoma as in normal skin, we could not detect any CD44 expression on the cell surface of the tumor cells of basal cell carcinoma. However, CD44 positive dendritic cells were observed in the tumor islands of basal cell carcinoma. Phenotypic analysis suggested that these CD44 positive cells were melanocytes.

Histopathologic and Ultrastructural Studies of Angiokeratoma Corporis Diffusum in Kanzaki Disease

A novel metabolic disease, angiokeratoma corporis diffusum (Kanzaki), was the subject of an extensive histopathologic and ultrastructural study. Findings included dilated lymph and blood vessels in the upper dermis with an orthokeratortic, thickened, horny layer in well developed angiokeratoma. In the early papules, a few sporadic dyskeratotic keratinocytes were present in the epidermis with or without a thickened horny layer. Vesicular clear vacuolation was clearly observed in the cytoplasm of the secretory portion of the eccrine sweat glands, but none was observed in the vascular endothelial cells with hematoxylin-eosin staining. Using electron microscopy, lysosomal vacuolation was observed in many cell types, including eccrine sweat gland cells, vascular endothelial cells, dermal fibroblasts, dermal neural cells, lymphocytes of peripheral blood in the skin, and glomerular endothelial cells, but none was noted in the epithelial cells of the kidney. Widely dilated vacuoles were found to contain only a small amount of fuzzy filamentous material in the vascular endothelial cells, filamentous or electron-dense granular substances in fibroblasts, and electron-dense, lamellated or homogeneous structures in eccrine sweat gland cells and in neural cells. Ultracytochemical examination revealed glycoconjugates in dilated lysosomes. Characteristics of Kanzaki Disease were shown to differ from those of Fabry disease or any other lysosomal storage disease.