ARTICLE |
Correspondence to: Kenji Saga, Dept. of Dermatology, Sapporo Medical U. School of Medicine, Minami 1 Nishi 16, Chyuo-ku, 060-8543 Sapporo, Japan. E-mail: ksaga@sapmed.ac.jp
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Summary |
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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 EGFEGFR 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 49:597601, 2001)
Key Words: confocal laser scanning, microscope, duct, activated epidermal growth, factor receptor, human sweat glands, immunohistochemistry, nucleus, tyrosine-phosphorylated, receptor
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Introduction |
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Previous studies have shown that human sweat contains epidermal growth factor (EGF) and that human sweat glands express EGF as well as the EGF receptor (EGFR).
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Materials and Methods |
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Antibodies
EGF was stained with anti-EGF MAb AM 26 from Ohtsuka (Tokushima, Japan) diluted to 45 µg/ml. This MAb was raised against recombinant human EGF (manufacturer's data sheet). Anti-activated EGFR MAb and anti-EGFR MAb were obtained from Transduction Laboratories (Lexington, KY). Anti-activated EGFR MAb (clone 74) was raised against tyrosine-phosphorylated EGFR isolated from EGF-challenged mouse L-cells expressing the human EGFR. This MAb reacts with 180-kD EGF-challenged EGFR. However, it does not react with non-stimulated EGFR (manufacturer's data sheet). Anti-EGFR MAb (clone 13) was raised against a synthetic peptide corresponding to residues 9961022 of human EGFR. This MAb reacts with both EGF-challenged and EGF-non-challenged mouse L-cells expressing the human EGFR (manufacturer's data sheet). These antibodies were used at the dilution of 10 µg/ml.
The positive site with anti-activated EGFR indicated the presence of activated EGFR. If anti-EGFR showed positive staining and anti-activated EGFR showed negative staining, non-activated EGFR should be present. However it was impossible to determine the presence of non-activated EGFR in the sites at which both anti-activated EGFR and anti-EGFR antibodies showed positive staining because the antibody specific to non-activated EGFR was not available.
Tissue Processing
Small pieces of human skin were obtained from the surgical margin of various non-inflammatory skin lesions. These tissues were mounted in OCT compound and quickly frozen by immersion in liquid nitrogen. Specimens were stored at -70C until use. Five-µm sections were cut with a cryostat and fixed in cold acetone for 10 min, then air-dried.
Immunohistochemistry
Nonspecific immunoglobulin binding sites were inhibited by preincubating the sections with ovalbumin in PBS for 10 min. After blotting the blocking solution, sections were incubated with the first antibody for 30 min at room temperature. After washing with three changes of PBS (5 min each), sections were then incubated with FITC-labeled anti-mouse IgG (Vector Laboratories; Burlingame, CA) for 30 min, followed by washing with PBS. Nuclei were counterstained by incubation with propidium iodide (50 µg/ml in PBS) for 30 min, then washed with PBS. Sections were mounted with Perma Fluor (Shandon; Pittsburgh, PA) aqueous mounting medium then sealed with a coverslip. Antibody binding sites were visualized by observing with a Olympus BX 50 microscope equipped with Olympus Fluoview confocal laser scanning and analysis unit (Olympus; Tokyo, Japan). Color green and color red indicate where FITC and propidium iodide are localized, respectively. Color yellow to orange indicates nuclei are stained with FITC.
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Results |
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Localization of EGF
Distribution of EGF in eccrine and apocrine sweat glands was studied by immunohistochemical staining with an MAb specific for EGF. The cytoplasm of secretory cells in eccrine and apocrine sweat glands showed positive staining with the anti-EGF MAb (data not shown), although nuclei of secretory cells and of ductal cells were not stained with the anti-EGF MAb as reported previously (
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Localization of EGFR in Eccrine Sweat Glands
Nuclei of secretory cells and those of ductal cells in eccrine sweat glands (Fig 2) were stained with the MAb that specifically binds to the activated form of EGFR. Luminal cell membranes and luminal cytoplasm of inner ductal cells in eccrine sweat glands were positively stained with anti-EGFR MAb (Fig 3), although they were not stained with the anti-activated EGFR MAb. Other areas of cytoplasm in ductal cells and cytoplasm of eccrine secretory cells were not stained with the anti-activated EGFR antibody. Myoepithelial cells were not stained with the anti-EGFR antibody or with the anti-activated EGFR MAb.
Localization of EGFR in Apocrine Sweat Glands
The anti-activated EGFR antibody stained cytoplasm and nuclei of the apocrine secretory cells (Fig 4) Some nuclei of secretory cells in apocrine sweat glands showed weak staining. The anti-EGFR antibody showed the same pattern of the staining as the antibody against activated EGFR. Myoepithelial cells were not stained with the anti-EGFR antibody nor with the anti-activated EGFR MAb.
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Discussion |
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Confocal laser scanning microscopic observation of the staining with the anti-EGF MAb was consistent with previous reports (
EGFR is a 180-kD tyrosine kinase that mediates signals of EGF and TGF- to the cells. On binding of ligands, the kinase is activated and phosphorylates the receptor's carboxyl terminus on tyrosine residues. Autophosphorylated tyrosine kinase phosphorylates substrates both bound to and not bound to the receptor. The conformational change induced to the substrates by tyrosine phosphorylation and/or binding to the receptor activates these substrates. These substrates then stimulate a series of downstream signaling pathways (
The anti-EGFR MAb showed positive reaction on the nuclei of sweat glands in our observations. Therefore, a different mode of activation from the one described in the previous paragraph must be working. A number of reports have indicated the presence of EGF (-thrombin caused a massive intranuclear increase of diacylglycerol and protein kinase C (
Because most of these studies were carried out using cultured cells, little is known about nuclear EGFR in vivo. An immunohistochemical study showed that EGFR was localized in the nuclei of ductal cells of eccrine sweat glands (
Nuclei of secretory cells in eccrine and apocrine sweat glands stained positively with anti-EGFR MAb specific for the activated form. Because the cytoplasm of eccrine and apocrine secretory cells contains EGF, it would activate EGFR in the nuclei of secretory cells in an intracrine manner (
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Acknowledgments |
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Supported in part by a Grant-in-Aid for Scientific Research (08670978) from The Ministry of Education, Science and Culture (KS).
Received for publication September 25, 2000; accepted January 3, 2001.
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