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Histochemical and Immunohistochemical Markers for Human Eccrine and Apocrine Sweat Glands: An Aid for Histopathologic Differentiation of Sweat Gland Tumors
Histochemical and Immunohistochemical Markers for Human Eccrine and Apocrine Sweat Glands: An Aid for Histopathologic Differentiation of Sweat Gland Tumors Kenji Saga
Department of Dermatology, Sapporo Medical University School of Medicine, Sapporo, Japan
from human milk fat globule membranes, and HMFG-1 (1.10.F3) monoclonal antibody produced by immunizing mice with defatted human milk fat globule membranes. Markers for eccrine sweat glands are as follows: dark cell granules that have chondroitinase ABC sensitive anionic sites detected by cationic gold at pH 2.0 after pretreatment with EGTA, and intercellular canaliculi with high activity of alkaline phosphatase. CEA and GCDFP-15 are expressed in both eccrine and apocrine sweat glands. Anti-EMA monoclonal antibody (E29) stains both eccrine and apocrine sweat glands. Key words: cationic gold/dark cell granule/epidermal growth factor/HMFG-1/ human milk fat globule membrane proteins/intercellular canaliculus. Journal of Investigative Dermatology Symposium Proceedings 6:49±53, 2001
Apocrine and eccrine sweat glands are distinct in function, although they are closely related to each other developmentally and morphologically. In certain sweat gland tumors, it is dif®cult to differentiate between eccrine or apocrine sweat glands. Therefore, this paper reviews histochemical and immunohistochemical markers to differentiate apocrine and eccrine sweat glands with the aim of better understanding the structural and functional characteristics of these sweat glands. Speci®c markers for apocrine sweat glands are as follows: neuraminidase sensitive anionic sites detected by cationic colloidal gold at pH 2.0, and mitochondrion-like secretory granules that have epidermal growth factor-like antigenicity. The following antibodies react with apocrine sweat glands but not with eccrine sweat glands; the antibodies raised against 70 kDa glycoprotein puri®ed
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of the eccrine sweat gland is the control of body temperature. The human eccrine sweat gland is composed of a single tubular structure with a blind end and another end opening to the surface of the skin. The size of the secretory coil is about 60±80 mm in diameter and 2± 5 mm in length (Sato et al, 1989). The diameter of the duct is slightly smaller and the length of the duct is about the same as the secretory portion. The secretory portion of the eccrine sweat gland consists of a pseudostrati®ed single layer of secretory cells and surrounding myoepithelial cells. The secretory portion of eccrine sweat glands consists of three types of cells: clear cells, dark cells, and myoepithelial cells. The dark cells border nearly all the luminal surface of the secretory tubule, whereas clear cells rest either directly on the basement membrane or on the myoepithelial cells. Where two or more clear cells abut, intercellular canaliculi are formed. Not infrequently, the lateral cell membrane of the dark cells forms part of the ori®ce of a canaliculus. Intercellular canaliculi are pouches extending from the luminal surface of secretory cells. Nearly isotonic primary sweat is produced in the secretory portion and the reabsorption of NaCl results in the secretion of hypotonic sweat to the surface of the skin (Sato et al, 1989). Myoepithelial cells in eccrine and apocrine sweat glands are spindle-shaped cells and they sit longitudinally or obliquely to the axis of the secretory tubule, between secretory cells and the basement membrane. Apocrine sweat glands, the larger sweat glands, are distributed mainly on the axillary and genital skin, but are also found in the external auditory meatus (ceruminous gland), the eyelid (Moll's gland), and within the areola (Robertshaw, 1991). They are an evolutionary remnant of an odorous organ of animals. A single layer
weat glands are usually classi®ed as either eccrine or apocrine, based on their respective mode of secretion. The secretion of eccrine sweat glands consists of the transfer of ¯uids across luminal cell membranes of secretory cells, whereas in apocrine sweat glands luminal portions of the cytoplasm of secretory cells are pinched off and released into the secretory lumen. These two types of sweat glands are different in size, structure, distribution, nervous control, and function. Nevertheless, it is sometimes dif®cult to assign the origin and the differentiation of sweat gland tumors to either eccrine or apocrine sweat glands because many of their characteristics are lost in such tumors. This paper reviews histochemical and immunohistochemical markers that can be used to differentiate eccrine and apocrine sweat glands (Table I). These markers should help the diagnosis of sweat gland tumors and help to understand the secretory function of these sweat glands in relation to the structural characteristics. ECCRINE, APOCRINE, AND APOECCRINE SWEAT GLANDS The human body has 2±3 million eccrine sweat glands distributed over almost the entire body surface. The most important function Manuscript received June 14, 2001; accepted for publication June 14, 2001. Reprint requests to: Dr. Kenji Saga, Department of Dermatology, Sapporo Medical University School of Medicine, Minami 1 Nishi 16, Chyuo-ku, Sapporo 060±8543, Japan. Email: [email protected] 1087-0024/01/$15.00
´ Copyright # 2001 by The Society for Investigative Dermatology, Inc. 49
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Table I. Histochemical and immunohistochemical markers of eccrine and apocrine sweat glands Eccrine sweat gland Dark cell granules with chondroitinase ABC-sensitive anionic sites after EGTA treatment Basal cell membranes with chondroitinase ABC-sensitive anionic sites Intercellular canaliculi with high activity of alkaline phosphatase Apocrine sweat gland Mitochondrion-like cytoplasmic granules containing EGF Luminal cell membranes with sialidase-sensitive anionic sites Antibodies that react with apocrine sweat glands but not with eccrine sweat glands HMFG-1 (1.10.F3) monoclonal antibody Antibody raised against puri®ed 70 kD glycoprotein in human milk fat globule membranes Common markers of eccrine and apocrine sweat glands GCDFP-15 Carcinoembryonic antigen (CEA) EMA detected by anti-EMA monoclonal antibody (E29)
of columnar secretory cells surrounded by myoepithelial cells form the tubular secretory portion of apocrine sweat glands. Light microscopic observation demonstrates that the luminal portion of secretory cells in apocrine sweat glands are pinched off and released into the secretory lumen. This mode of secretion is called decapitation secretion or apocrine secretion. The ultrastructure of secretory cells in apocrine sweat glands shows two types of cytoplasmic granules. Small granules are about 50 nm in diameter and have a round to elongated shape and mitochondrion-like internal structure. Large granules are round and variable in size and contain ®ne electron-dense particles. The luminal cell membranes have microvilli. The ducts of apocrine sweat glands open to the follicular infundibulum of the hair follicle close to the skin surface and super®cial to the sebaceous gland opening. A third type of sweat gland, the apoeccrine sweat gland, was discovered by Sato et al (1987). Apoeccrine sweat glands are larger than typical eccrine glands and smaller than typical apocrine glands. The apoeccrine gland has a long duct that opens directly onto the skin surface like the eccrine sweat duct, but is distinct from the apocrine duct, which is very short and opens into the upper portion of the hair follicle. The secretory tubule of the apoeccrine gland is irregularly dilated. The dilated segment of the secretory segment consists of an apocrine-like single layer of tall columnar epithelium, whereas the undilated segment consists of eccrine-like pseudostratifed epithelium. The apoeccrine glands appear to develop during puberty from the eccrine glands, or eccrine-like precursor glands. Apoeccrine glands secrete copious serous sweat in response to both methacholine and epinephrine (Sato and Sato, 1987). MARKERS TO BE USED FOR THE DIFFERENTIATION OF ECCRINE AND APOCRINE SWEAT GLANDS Distribution of anionic sites Cationic colloidal gold labels net negative charge on the histologic sections (Skutelsky and Roth, 1986; Saga, 1998). Therefore, an alteration of pH has a marked effect on the labeling with cationic gold. Cationic gold labels only sialic acid and sulfonic acid residues at low pH (= 2.0), which are known to be the only dissociated cellular constituents under such conditions (Skutelsky and Roth, 1986). Cationic gold labels carboxyl residues of carbohydrates in glycoproteins, glycolipids, and proteoglycans at neutral pH (Skutelsky and Roth, 1986; Vorbrodt, 1987; Goode et al, 1991). Cationic gold stains anionic sites on the histologic sections using the postembedding method. Pretreatment of the sections with enzymes eliminates anionic sites on the histologic sections. The advantage of cationic gold over traditional cationic dye is in postembedding staining. The problem of the penetration of cationic
Figure 1. Cationic gold at pH 2.0 labeled anionic sites on the dark cell granules after EGTA treatment in human eccrine sweat glands. Scale bar: 500 nm.
Figure 2. Cationic gold at pH 2.0 labeled anionic sites on the luminal cell membranes of apocrine secretory cells. Scale bar: 500 nm.
probe and enzymes that are used for the digestion of anionic sites can be eliminated by the post-embedding method. Another advantage is that cationic gold is clearly visible under an electron microscope, and silver enhancement enables light microscopic observation. Cationic gold labeled basolateral cell membranes of secretory cells in the eccrine sweat gland at pH 2.0 (Saga and Takahashi, 1993). These anionic sites were digested by preincubating the sections with chondroitinase ABC. Therefore chondroitin sulfate and/or dermatan sulfate constitute anionic sites on the secretory portion of eccrine sweat glands. Cationic gold at pH 2.0 labeled dark cell granules after pretreatment with EGTA (Fig 1). That implies calcium covers anionic sites on the dark cell granules. Chondroitinase ABC digested exposed anionic sites by pretreatment with EGTA. Therefore chondroitin sulfate and/or dermatan sulfate constitute anionic sites on dark cell granules. Ductal cells or myoepithelial cells of secretory cells were not labeled with cationic gold. In apocrine sweat glands, cationic gold at pH 2.0 labeled luminal cell membranes, microvilli, and submembranous vesicles
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Figure 3. Anti-EGF antibody stained mitochondrion-like cytoplasmic granules in apocrine secretory cells. Scale bar: 500 nm.
Figure 4. Intercellular canaliculi of eccrine sweat glands showed high activity of alkaline phosphatase. Scale bar: 500 nm.
of secretory cells (Fig 2). Neuraminidase digested these anionic sites, thus sialic acid is responsible for the anionic charge in the apocrine sweat gland (Saga and Takahashi, 1993); however, these anionic sites were not labeled with cationic gold at pH 7.4. Charge theory alone does not explain this paradoxical pHdependency observed in apocrine sweat glands. It is speculated that an effect of low pH may be to alter the structural con®guration of sialoprotein, thus rendering charge sites available for labeling with cationic colloidal gold (Saga and Takahashi, 1993; Saga, 1998). Post-embedding labeling with cationic gold and predigestion on the sections showed completely different distribution and responsible molecules between eccrine and apocrine sweat glands. Chondroitin sulfate and/or dermatan sulfate is a marker of secretory cells of the eccrine sweat gland. Dark cell granules that express chondroitin sulfate and/or dermatan sulfate are speci®c markers of dark secretory cells in the eccrine sweat gland. Sialic acid is a marker of secretory cells of the apocrine sweat gland. Some past reports using cationic dye are contradictory to the studies using cationic gold. According to studies with cationic dye such as alcian blue, both eccrine and apocrine sweat glands were decorated with sialic acid (Johnson and Helwig, 1963; Constantine and Mowry, 1966). The discrepancy may be due to the poor speci®city of the cationic dye to anionic sites and the lesser purity of enzymes used.
stained with anti-EGF antibodies. Therefore the mitochondrionlike granule containing EGF is a marker of secretory cells in apocrine sweat glands.
Epidermal growth factor (EGF) EGF, a 53 amino acid polypeptide, directly stimulates epidermal growth and differentiation. EGF is contained in various secretory ¯uids. The presence of EGF in sweat was ®rst reported by Pesonen et al (1987), who showed that the concentration of EGF (EGF/unit sweat volume) was much higher in armpit sweat than in breast sweat. That suggests apocrine sweat glands secrete more EGF than eccrine sweat glands. Sato et al (1989) con®rmed the presence of EGF in eccrine sweat, using precautions to minimize epidermal contamination during sweat collection. They also found that the concentration of EGF in eccrine sweat (EGF/unit sweat volume) increases in proportion to the increase in sweat rate. Thermally induced axillary sweat contains about 50 times more EGF than sweat obtained from other areas of the trunk. Immunohistochemical studies have shown that both eccrine and apocrine sweat glands express EGF in the cytoplasm of secretory cells. Immunoelectron microscopy showed that small secretory granules that have cristae of mitochondrion-like internal structure were heavily stained with anti-EGF antibody (Fig 3), whereas these granules were not found in the secretory cells of eccrine sweat glands (Saga and Takahashi, 1992). In eccrine sweat glands, EGF was not localized on any speci®c organella in the cytoplasm. Dark cell granules in eccrine sweat glands were not
70 kDa glycoprotein in human milk fat globule membrane Membranes from mammary epithelial cells can be found in the milk fat fraction. The cream fraction of milk consists of fat droplets surrounded by an external membrane. This membrane layer, known as the milk fat globule membrane, is mainly derived from the apical plasma membrane of lactating mammary secretory cells. Polyclonal and monoclonal antibodies raised against components of human milk fat globule membranes react not only with mammary glands but also with human sweat glands. Several glycoproteins were isolated and characterized from human milk fat globule membranes (Imam et al, 1981, 1982). The antibody raised against the 70 kDa glycoprotein puri®ed from a human milk fat globule membrane reacted with the mammary gland and the apocrine sweat gland, but not with the eccrine sweat gland (Imam et al, 1988). The antibody strongly stained the luminal membranes of secretory cells and that of ductal cells in apocrine sweat glands. In addition, the antibody weakly stained the luminal cytoplasm of apocrine secretory cells. On the other hand the antibody raised against 155 kDa glycoprotein puri®ed from human milk fat globule membrane reacted with the mammary gland, kidney, pancreas, salivary gland, and stomach, but not with the apocrine or eccrine sweat glands (Imam et al, 1986). Therefore, 70 kDa glycoprotein puri®ed from human milk fat globule membrane can be used for the differentiation of apocrine and eccrine sweat glands. HMFG-1 monoclonal antibody The monoclonal antibodies designated as HMFG-1 (1.10.F3) and HMFG-2 (3.14.A3) were produced by immunizing mice with delipidated preparations of the human milk fat globule (Taylor-Papadimitriou et al, 1981; Burchell et al, 1983). Western blot analyses showed that both antibodies recognize antigenic determinants found in high molecular weight components (> 400 kDa). HMFG-1 immunostains apocrine sweat glands; however, it does not react with eccrine sweat glands (Viragh et al, 1997). HMFG-1 monoclonal antibody stains luminal cells of the duct and the luminal cell membranes and the luminal cytoplasm of secretory cells in apocrine sweat glands. Comparative immunohistochemistry of HMFG-2 and antiepithelial membrane antigen monoclonal antibody (E29) showed a similar distribution of staining (Heyderman et al, 1985). Core proteins for several human mucins (MUC1-MUC7) have been identi®ed by molecular biology techniques. MUC1 is a membrane-associated glycoprotein consisting of three domains. HMFG-1 detects fully
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glycosylated MUC1 mucin, whereas HMFG-2 detects underglycosylated MUC-1 mucin (Taylor-Papadimitriou et al, 1981; Burchell et al, 1983; Yonezawa and Sato, 1997). Intercellular canaliculi with high activity of alkaline phosphatase Alkaline phosphatase (ALP) (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) is a group of enzymes that are membrane-associated glycoproteins. ALP catalyzes the hydrolysis of inorganic and organic monophosphate esters at alkaline pH. Although this enzyme is widely distributed in human tissues, we do not fully understand its physiologic functions. The high activity of ALP in the kidney, liver, and intestine has suggested that this enzyme might participate in membrane transport (Harris, 1989). Past reports have demonstrated the activity of ALP in the eccrine and apocrine sweat glands (Bunting et al, 1948; Shelley and Mescon, 1952). The presence of ALP was also reported in some sweat gland tumors such as eccrine hidrocystoma (Sperling and Sakas, 1982). An enzyme histochemical study showed that the activity of ALP was localized on the intercellular canaliculi of the eccrine sweat gland and the myoepithelial cell of apocrine sweat glands (Saga and Morimoto, 1995). The dark cells border nearly all the luminal surface of the secretory tubule, whereas clear cells rest either directly on the basement membrane or on the myoepithelial cells. In eccrine secretory cells, intercellular canaliculi showed positive reaction (Fig 4), whereas luminal cell membranes that were in continuity with intercellular canaliculi were negative for the enzyme activity. Intercellular canaliculi are pouches extending from the luminal surface of secretory cells. This result suggests that intercellular canaliculi are not simple extensions of luminal cell membranes but are differentiated structures that may play a key role in the production of primary sweat. COMMON MARKERS OF ECCRINE AND APOCRINE SWEAT GLANDS GCDFP-15 Gross cystic disease of the breast is a premenopausal disorder in which gross cysts are the predominant pathologic lesion. It is speculated that gross cysts are formed by the over-secretion of the mammary gland. Gross cystic disease ¯uid is a pathologic secretion from the breast composed of several glycoproteins, including GCDFP-70 (70 000 Da), human albumin, GCDFP-44, and GCDFP-15. GCDFP-15 is a unique monomer protein that has no normal plasma counterpart. Normal apocrine gland and various tumors derived from the apocrine gland stained with anti-GCDFP-15 antibody (Mazoujian et al, 1983). It was once believed that GCDFP-15 was a speci®c marker of the apocrine sweat gland and that it could differentiate eccrine and apocrine tumors; however, later studies showed that these gross cystic disease ¯uid proteins exist in both eccrine and apocrine sweat glands (Ordonez et al, 1987). Carcinoembryonic antigen (CEA) CEA was originally found in fetal tissues and in certain gastrointestinal tumors as an oncofetal antigen. Later it was found in various normal secretory epithelium of adults, including sweat glands (reviewed by HammarstroÈm, 1999). CEA is a glycoprotein containing approximately 50% carbohydrate with an approximate molecular weight of 200 kDa. CEA cross-reactive antigens were found in normal human tissue. These antigens include nonspeci®c crossreacting antigen (NCA) and biliary glycoprotein (BGP). Molecular cloning of cDNA for CEA, NCA, and BGP was carried out in the late 1980s. The CEA gene family belongs to the immunoglobulin superfamily. In total 29 genes/pseudogenes have been identi®ed in the human CEA gene family. Speci®c monoclonal antibodies are available for CEA, NCA, BGP, CEA gene family member 2 (CGM2), and for human pregnancyspeci®c 1 glycoproteins (PSG) as a group. Speci®c PCR primers and riboprobes have been constructed. Although the distribution of CEA is limited in certain tissues in normal adults, BGP and
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NCA show broader tissue distribution. The expression of CEA is observed in the gastrointestinal tract, in the prostate, in the cervix, and in sweat glands of human adults. CEA is present on the secretory lumen of secretory cells and the luminal surface of the duct in eccrine sweat glands (Penneys et al, 1981; Nap et al, 1988). The duct shows stronger labeling than the secretory portion. The luminal membranes of the apocrine secretory cells are also labeled with CEA. The biologic function of CEA is poorly understood. It is postulated that CEA and NCA play a role in the innate immune defense protecting the lumen of sweat glands from microbial insult. CEA and NCA probably bind and trap microorganisms, preventing them from reaching down to the microvilli of the epithelial cells and invading the epithelial cells (HammarstroÈm, 1999). Epithelial membrane antigen (EMA) Antisera raised against defatted human cream (defatted human milk fat globule membranes) have been shown to react with various normal epithelial tissues and carcinomas (Heyderman et al, 1979; Sloane and Ormerod, 1981). The glycoproteins with which these antisera react have been termed epithelial membrane antigen (EMA). The antigen has been partially puri®ed, and shown to consist of heterogeneous glycoproteins of high molecular weight (Ormerod et al, 1983). Ceriani et al (1977) reported that an antiEMA antibody reacted with cell membranes of normal breast and breast carcinoma cells; however, that antibody did not react with other carcinoma cells, lymphoma cells, melanoma cells, or mesenchymal cells. Carbohydrates form the major antigenic determinant, the principal sugars of which are galactose and Nacetyl-glucosamine (Ormerod et al, 1983). Later, a monoclonal antibody was produced by immunizing mice with delipidated human cream. This monoclonal antibody, designated as anti-EMA (E29), did not show a signi®cant difference of the distribution of immunostaining with polyclonal anti-EMA antibodies (Cordell et al, 1985; Heyderman et al, 1985). An immunoblot study showed that E29 antibody reacts with a wide range of molecular weights (265±400 kDa) (Cordell et al, 1985). E29 monoclonal antibody stains luminal membranes of both eccrine and apocrine sweat glands. Therefore it cannot be used for the differentiation of apocrine and eccrine sweat glands. CLINICAL APPLICATIONS Some of the histochemical and immunohistochemical markers for eccrine and apocrine sweat glands have been applied to the study of sweat gland tumors. Extramammary Paget's disease expresses anionic sites characteristic to the secretory portion of the apocrine sweat gland (Saga and Jimbow, 1999). Cationic gold at pH 2.0 labeled about 20% of Paget cells from genital areas. Neuraminidase digested these anionic sites indicating that sialic acid is responsible for the anionic charge. These results indicate extramammary Paget cells are differentiating to the secretory cells of the apocrine sweat gland. An antibody against a 70 kDa glycoprotein puri®ed from human milk fat globule membrane (MFGM-gp 70) reacted with the apocrine sweat gland but not with the eccrine sweat gland (Imam et al, 1988). This antibody stained luminal membranes of both secretory and ductal cells in normal apocrine sweat glands. The antibody reacted with Paget cells of extramammary Paget's disease (Imam et al, 1988). This result indicates that Paget cells of extramammary Paget's disease differentiate to the secretory cells or ductal cells of apocrine sweat glands. HMFG-1 monoclonal antibody stains secretory cells and ductal cells of apocrine sweat glands (de Viragh et al, 1997). Several studies have used this antibody to clarify the histogenesis of sweat gland tumors. A study by de Viragh showed that HMFG-1 antibody stained all apocrine cystadenomas and some eccrine hidrocystomas. They speculated that eccrine hidrocystomas might represent cystic tumors of the eccrine sweat duct, or they might represent cystic tumors of the apocrine sweat duct (de Viragh et al, 1997).
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Kamishima et al (1999) studied a case of pigmented hidrocystoma in the vulva using the HMFG-1 antibody. Negative staining with the HMFG-1 antibody and the results of other immunohistochemical staining showed that the tumor was not apocrine in nature but of eccrine derivation. HMFG-1 antibody stained less than 10% of tubular epithelial cells in spiradenoma and dermal cylindroma (Meybehm and Fischer, 1997). This result indicates positive tubular epithelial cells are differentiating to the secretory cells or ductal cells in apocrine sweat glands. REFERENCES Bunting H, Wislocki GB, Dempsey EW: The chemical histology of human eccrine and apocrine sweat gland. Anat Rec 100:61±77, 1948 Burchell J, Durbin H, Taylor-Papadimitriou J: Complexity of expression of antigenic determinants, recognized by monoclonal antibodies HMFG-1 and HMFG-2, in normal and malignant human mammary epithelial cells. J Immunol 131:508±513, 1983 Ceriani RL, Thompson K, Peterson JA, Abraham S: Surface differentiation antigens of human milk fat globule. Proc Natl Acad Sci 74:582±586, 1977 Constantine VS, Mowry RW: Histochemical demonstration of sialomucin in human eccrine sweat glands. J Invest Dermatol 46:536±541, 1966 Cordell J, Richardson TC, Pulford KAF, Ghosh AK, Gatter KC, Heyderman E, Mason DY: Production of monoclonal antibodies against human epithelial membrane antigen for use in diagnostic immunocytochemistry. Br J Cancer 52:347±354, 1985 Goode NP, Shires M, Crellin DM, Davison AM: Detection of glomerular anionic sites in post-embedded ultrathin sections using cationic colloidal gold. J Histochem Cytochem 39:965±972, 1991 HammarstroÈm S: The carcinoembryonic antigen (CEA) family. structures, suggested functions and expression in normal and malignant tissues. Semi Cancer Biol 9:67±81, 1999 Harris H: The human alkaline phosphatases. what we know and what we don't know. Clinica Chimica Acta 186:133±150, 1989 Heyderman E, Steele K, Ormerod MG: A new antigen on the epithelial membrane: its immunoperoxidase localisation in normal and neoplastic tissues. J Clin Pathol 32:35±39, 1979 Heyderman E, Strudley I, Powell G, Richardson TC, Cordell JL, Mason DY: A new monoclonal antibody to epithelial membrane. Br J Cancer 52:355±361, 1985 Imam A, Laurence JR, Neville AM: Isolation and characterization of a major glycoprotein from milk-fat-globule membrane of human breast milk. Biochem J 193:47±54, 1981 Imam A, Laurence DJR, Neville AM: Isolation and characterization of two individual glycoprotein components from human milk-fat-globule membranes. Biochem J 207:37±41, 1982 Imam A, Drushella MM, Taylor CR, Toekes ZA: Preferential expression of a Mr 155,000 milk-fat-globule membrane glycoprotein on luminal epithelium of lobules in human breast. Cancer Res 46:6374±6379, 1986 Imam A, Yoshida SO, Taylor CR: Distinguishing tumour cells of mammary from extramammary Paget's disease using antibodies to two different glycoproteins from human milk-fat-globule membrane. Br J Cancer 58:373±378, 1988 Johnson WC, Helwig EB: Histochemistry of the acid mucopolysaccharides of skin in normal and in certain pathologic conditions. Am J Clin Pathol 40:123±131, 1963 Kamishima T, Igarashi S, Takeuchi Y, Ito M, Fukuda T: Pigmented hidrocystoma of the eccrine secretory coil in the vulva: clinicopathologic, immunohistochemical and ultrastructural studies. J Cutan Pathol 26:145±149, 1999 Mazoujian G, Pinkus GS, Davis S Jr: DEH: Immunohistochemistry of a gross cystic disease ¯uid protein (GCDFP-15) of the breast. Am J Pathol 110:105±112, 1983 Meybehm M, Fischer HP: Spiradenoma and dermal cylindroma: comparative
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immunohistochemical analysis and histogenetic considerations. Am J Dermatopathol 19:154±161, 1997 Nap M, Mollgard K, Burtin P, Fleuren GJ: Immunohistochemistry of carcinoembryonic antigen in the embryo, fetus and adult. Tumor Biol 9:145±153, 1988 Ordonez NG, Awalt H, Mackay B: Mammary and extramammary Paget's disease: An immunocytochemical and ultrastructural study. Cancer 59:1173±1183, 1987 Ormerod MG, Steele K, Westwood JH, Mazzini MN: Epithelial membrane antigen. Partial puri®cation, assay and properties. Br J Cancer 48:533±541, 1983 Penneys NS, Nadji M, McKinney EC: Carcinoembryonic antigen present in human eccrine sweat. J Am Acad Dermatol 4:401±403, 1981 Pesonen K, Viinikka L, Koskimies A, Banks AR, Nicolson M, Perheentupa J: Size heterogeneity of epidermal growth factor in human body ¯uids. Life Sci 40:2489±2494, 1987 Robertshaw D: Apocrine sweat glands. In: Goldsmith LA, ed. Physiology, Biochemistry and Molecular Biology of the Skin. New York: Oxford University Press, 1991, pp 763±775 Saga K: Application of cationic colloidal gold staining for the demonstration of anionic sites. Acta Histochem Cytochem 31:1±8, 1998 Saga K, Jimbow K: Localization and characterization of anionic sites in extramammary Paget's disease with cationic colloidal gold. Acta Histochem Cytochem 32:415±421, 1999 Saga K, Morimoto Y: Ultrastructural localization of alkaline phosphatase activity in human eccrine and apocrine sweat glands. J Histochem Cytochem 43:927±932, 1995 Sato K, Sato F: Sweat secretion by human axillary apoeccrine sweat gland in vitro. Am J Physiol 251:R181±R187, 1987 Saga K, Takahashi M: Immunoelectron microscopic localization of epidermal growth factor in the eccrine and apocrine sweat glands. J Histochem Cytochem 40:241±249, 1992 Saga K, Takahashi M: Demonstration of anionic sites in human eccrine and apocrine sweat glands in post-embedded ultra-thin sections using cationic colloidal gold: the effect of enzyme digestion on these anionic sites. J Histochem Cytochem 41:1197±1207, 1993 Sato K, Leidal R, Sato F: Morphology and development of an apoeccrine sweat gland in the human axillae. Am J Physiol 252:R166±R180, 1987 Sato K, Kang WH, Saga K, Sato KT: Biology of sweat glands and its disorders. I. Normal sweat gland function. J Am Acad Dermatol 20:537±563, 1989 Shelley WB, Mescon H: Histochemical demonstration of secretory activity in human eccrine sweat glands. J Invest Dermatol 18:289±301, 1952 Skutelsky E, Roth J: Cationic colloidal gold-A new probe for the detection of anionic cell surface sites by electron microscopy. J Histochem Cytochem 34:693±696, 1986 Sloane JP, Ormerod MG: Distribution of epithelial membrane antigen in normal and neoplastic stussues and its value in diagnostic tumor pathology. Cancer 47:1786±1795, 1981 Sperling LC, Sakas EL: Eccrine hidrocystomas. J Am Acad Dermatol 7:763±770, 1982 Taylor-Papadimitriou J, Peterson JA, Arklie J, Burchell J, Ceriani RL: Monoclonal antibodies to epithelium-speci®c components of the human milk fat globule membrane: production and reaction with cells in culture. Int J Cancer 28:17±21, 1981 Viragh P, Ad Szeimies RM, Eckert F: Apocrine cystadenoma, apocrine hidrocystoma, and eccrine hidrocystoma: three distinct tumors de®ned by expression of keratins and human milk fat globulin 1. J Cutan Pathol 24:249±255, 1997 de Viragh PA, Szeimies RM, Eckert F: Apocrine cystadenoma, apocrine hidrocystoma, and eccrine hidrocystoma: three distinct tumors de®ned by expression of keratins and human milk fat globulin 1. J Cutan Pathol 24:249±255, 1997 Vorbrodt AW: Demonstration of anionic sites on the luminal and abluminal fronts of endothelial cells with poly-L-lysine-gold complex. J Histochem Cytochem 35:1261±1266, 1987 Yonezawa S, Sato E: Expression of mucin antigens in human cancers and its relationship with malignancy potential. Pathol Int 47:813±830, 1997
Department of Dermatology, Sapporo Medical University School of Medicine, Sapporo, Japan
from human milk fat globule membranes, and HMFG-1 (1.10.F3) monoclonal antibody produced by immunizing mice with defatted human milk fat globule membranes. Markers for eccrine sweat glands are as follows: dark cell granules that have chondroitinase ABC sensitive anionic sites detected by cationic gold at pH 2.0 after pretreatment with EGTA, and intercellular canaliculi with high activity of alkaline phosphatase. CEA and GCDFP-15 are expressed in both eccrine and apocrine sweat glands. Anti-EMA monoclonal antibody (E29) stains both eccrine and apocrine sweat glands. Key words: cationic gold/dark cell granule/epidermal growth factor/HMFG-1/ human milk fat globule membrane proteins/intercellular canaliculus. Journal of Investigative Dermatology Symposium Proceedings 6:49±53, 2001
Apocrine and eccrine sweat glands are distinct in function, although they are closely related to each other developmentally and morphologically. In certain sweat gland tumors, it is dif®cult to differentiate between eccrine or apocrine sweat glands. Therefore, this paper reviews histochemical and immunohistochemical markers to differentiate apocrine and eccrine sweat glands with the aim of better understanding the structural and functional characteristics of these sweat glands. Speci®c markers for apocrine sweat glands are as follows: neuraminidase sensitive anionic sites detected by cationic colloidal gold at pH 2.0, and mitochondrion-like secretory granules that have epidermal growth factor-like antigenicity. The following antibodies react with apocrine sweat glands but not with eccrine sweat glands; the antibodies raised against 70 kDa glycoprotein puri®ed
S
of the eccrine sweat gland is the control of body temperature. The human eccrine sweat gland is composed of a single tubular structure with a blind end and another end opening to the surface of the skin. The size of the secretory coil is about 60±80 mm in diameter and 2± 5 mm in length (Sato et al, 1989). The diameter of the duct is slightly smaller and the length of the duct is about the same as the secretory portion. The secretory portion of the eccrine sweat gland consists of a pseudostrati®ed single layer of secretory cells and surrounding myoepithelial cells. The secretory portion of eccrine sweat glands consists of three types of cells: clear cells, dark cells, and myoepithelial cells. The dark cells border nearly all the luminal surface of the secretory tubule, whereas clear cells rest either directly on the basement membrane or on the myoepithelial cells. Where two or more clear cells abut, intercellular canaliculi are formed. Not infrequently, the lateral cell membrane of the dark cells forms part of the ori®ce of a canaliculus. Intercellular canaliculi are pouches extending from the luminal surface of secretory cells. Nearly isotonic primary sweat is produced in the secretory portion and the reabsorption of NaCl results in the secretion of hypotonic sweat to the surface of the skin (Sato et al, 1989). Myoepithelial cells in eccrine and apocrine sweat glands are spindle-shaped cells and they sit longitudinally or obliquely to the axis of the secretory tubule, between secretory cells and the basement membrane. Apocrine sweat glands, the larger sweat glands, are distributed mainly on the axillary and genital skin, but are also found in the external auditory meatus (ceruminous gland), the eyelid (Moll's gland), and within the areola (Robertshaw, 1991). They are an evolutionary remnant of an odorous organ of animals. A single layer
weat glands are usually classi®ed as either eccrine or apocrine, based on their respective mode of secretion. The secretion of eccrine sweat glands consists of the transfer of ¯uids across luminal cell membranes of secretory cells, whereas in apocrine sweat glands luminal portions of the cytoplasm of secretory cells are pinched off and released into the secretory lumen. These two types of sweat glands are different in size, structure, distribution, nervous control, and function. Nevertheless, it is sometimes dif®cult to assign the origin and the differentiation of sweat gland tumors to either eccrine or apocrine sweat glands because many of their characteristics are lost in such tumors. This paper reviews histochemical and immunohistochemical markers that can be used to differentiate eccrine and apocrine sweat glands (Table I). These markers should help the diagnosis of sweat gland tumors and help to understand the secretory function of these sweat glands in relation to the structural characteristics. ECCRINE, APOCRINE, AND APOECCRINE SWEAT GLANDS The human body has 2±3 million eccrine sweat glands distributed over almost the entire body surface. The most important function Manuscript received June 14, 2001; accepted for publication June 14, 2001. Reprint requests to: Dr. Kenji Saga, Department of Dermatology, Sapporo Medical University School of Medicine, Minami 1 Nishi 16, Chyuo-ku, Sapporo 060±8543, Japan. Email: [email protected] 1087-0024/01/$15.00
´ Copyright # 2001 by The Society for Investigative Dermatology, Inc. 49
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Table I. Histochemical and immunohistochemical markers of eccrine and apocrine sweat glands Eccrine sweat gland Dark cell granules with chondroitinase ABC-sensitive anionic sites after EGTA treatment Basal cell membranes with chondroitinase ABC-sensitive anionic sites Intercellular canaliculi with high activity of alkaline phosphatase Apocrine sweat gland Mitochondrion-like cytoplasmic granules containing EGF Luminal cell membranes with sialidase-sensitive anionic sites Antibodies that react with apocrine sweat glands but not with eccrine sweat glands HMFG-1 (1.10.F3) monoclonal antibody Antibody raised against puri®ed 70 kD glycoprotein in human milk fat globule membranes Common markers of eccrine and apocrine sweat glands GCDFP-15 Carcinoembryonic antigen (CEA) EMA detected by anti-EMA monoclonal antibody (E29)
of columnar secretory cells surrounded by myoepithelial cells form the tubular secretory portion of apocrine sweat glands. Light microscopic observation demonstrates that the luminal portion of secretory cells in apocrine sweat glands are pinched off and released into the secretory lumen. This mode of secretion is called decapitation secretion or apocrine secretion. The ultrastructure of secretory cells in apocrine sweat glands shows two types of cytoplasmic granules. Small granules are about 50 nm in diameter and have a round to elongated shape and mitochondrion-like internal structure. Large granules are round and variable in size and contain ®ne electron-dense particles. The luminal cell membranes have microvilli. The ducts of apocrine sweat glands open to the follicular infundibulum of the hair follicle close to the skin surface and super®cial to the sebaceous gland opening. A third type of sweat gland, the apoeccrine sweat gland, was discovered by Sato et al (1987). Apoeccrine sweat glands are larger than typical eccrine glands and smaller than typical apocrine glands. The apoeccrine gland has a long duct that opens directly onto the skin surface like the eccrine sweat duct, but is distinct from the apocrine duct, which is very short and opens into the upper portion of the hair follicle. The secretory tubule of the apoeccrine gland is irregularly dilated. The dilated segment of the secretory segment consists of an apocrine-like single layer of tall columnar epithelium, whereas the undilated segment consists of eccrine-like pseudostratifed epithelium. The apoeccrine glands appear to develop during puberty from the eccrine glands, or eccrine-like precursor glands. Apoeccrine glands secrete copious serous sweat in response to both methacholine and epinephrine (Sato and Sato, 1987). MARKERS TO BE USED FOR THE DIFFERENTIATION OF ECCRINE AND APOCRINE SWEAT GLANDS Distribution of anionic sites Cationic colloidal gold labels net negative charge on the histologic sections (Skutelsky and Roth, 1986; Saga, 1998). Therefore, an alteration of pH has a marked effect on the labeling with cationic gold. Cationic gold labels only sialic acid and sulfonic acid residues at low pH (= 2.0), which are known to be the only dissociated cellular constituents under such conditions (Skutelsky and Roth, 1986). Cationic gold labels carboxyl residues of carbohydrates in glycoproteins, glycolipids, and proteoglycans at neutral pH (Skutelsky and Roth, 1986; Vorbrodt, 1987; Goode et al, 1991). Cationic gold stains anionic sites on the histologic sections using the postembedding method. Pretreatment of the sections with enzymes eliminates anionic sites on the histologic sections. The advantage of cationic gold over traditional cationic dye is in postembedding staining. The problem of the penetration of cationic
Figure 1. Cationic gold at pH 2.0 labeled anionic sites on the dark cell granules after EGTA treatment in human eccrine sweat glands. Scale bar: 500 nm.
Figure 2. Cationic gold at pH 2.0 labeled anionic sites on the luminal cell membranes of apocrine secretory cells. Scale bar: 500 nm.
probe and enzymes that are used for the digestion of anionic sites can be eliminated by the post-embedding method. Another advantage is that cationic gold is clearly visible under an electron microscope, and silver enhancement enables light microscopic observation. Cationic gold labeled basolateral cell membranes of secretory cells in the eccrine sweat gland at pH 2.0 (Saga and Takahashi, 1993). These anionic sites were digested by preincubating the sections with chondroitinase ABC. Therefore chondroitin sulfate and/or dermatan sulfate constitute anionic sites on the secretory portion of eccrine sweat glands. Cationic gold at pH 2.0 labeled dark cell granules after pretreatment with EGTA (Fig 1). That implies calcium covers anionic sites on the dark cell granules. Chondroitinase ABC digested exposed anionic sites by pretreatment with EGTA. Therefore chondroitin sulfate and/or dermatan sulfate constitute anionic sites on dark cell granules. Ductal cells or myoepithelial cells of secretory cells were not labeled with cationic gold. In apocrine sweat glands, cationic gold at pH 2.0 labeled luminal cell membranes, microvilli, and submembranous vesicles
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Figure 3. Anti-EGF antibody stained mitochondrion-like cytoplasmic granules in apocrine secretory cells. Scale bar: 500 nm.
Figure 4. Intercellular canaliculi of eccrine sweat glands showed high activity of alkaline phosphatase. Scale bar: 500 nm.
of secretory cells (Fig 2). Neuraminidase digested these anionic sites, thus sialic acid is responsible for the anionic charge in the apocrine sweat gland (Saga and Takahashi, 1993); however, these anionic sites were not labeled with cationic gold at pH 7.4. Charge theory alone does not explain this paradoxical pHdependency observed in apocrine sweat glands. It is speculated that an effect of low pH may be to alter the structural con®guration of sialoprotein, thus rendering charge sites available for labeling with cationic colloidal gold (Saga and Takahashi, 1993; Saga, 1998). Post-embedding labeling with cationic gold and predigestion on the sections showed completely different distribution and responsible molecules between eccrine and apocrine sweat glands. Chondroitin sulfate and/or dermatan sulfate is a marker of secretory cells of the eccrine sweat gland. Dark cell granules that express chondroitin sulfate and/or dermatan sulfate are speci®c markers of dark secretory cells in the eccrine sweat gland. Sialic acid is a marker of secretory cells of the apocrine sweat gland. Some past reports using cationic dye are contradictory to the studies using cationic gold. According to studies with cationic dye such as alcian blue, both eccrine and apocrine sweat glands were decorated with sialic acid (Johnson and Helwig, 1963; Constantine and Mowry, 1966). The discrepancy may be due to the poor speci®city of the cationic dye to anionic sites and the lesser purity of enzymes used.
stained with anti-EGF antibodies. Therefore the mitochondrionlike granule containing EGF is a marker of secretory cells in apocrine sweat glands.
Epidermal growth factor (EGF) EGF, a 53 amino acid polypeptide, directly stimulates epidermal growth and differentiation. EGF is contained in various secretory ¯uids. The presence of EGF in sweat was ®rst reported by Pesonen et al (1987), who showed that the concentration of EGF (EGF/unit sweat volume) was much higher in armpit sweat than in breast sweat. That suggests apocrine sweat glands secrete more EGF than eccrine sweat glands. Sato et al (1989) con®rmed the presence of EGF in eccrine sweat, using precautions to minimize epidermal contamination during sweat collection. They also found that the concentration of EGF in eccrine sweat (EGF/unit sweat volume) increases in proportion to the increase in sweat rate. Thermally induced axillary sweat contains about 50 times more EGF than sweat obtained from other areas of the trunk. Immunohistochemical studies have shown that both eccrine and apocrine sweat glands express EGF in the cytoplasm of secretory cells. Immunoelectron microscopy showed that small secretory granules that have cristae of mitochondrion-like internal structure were heavily stained with anti-EGF antibody (Fig 3), whereas these granules were not found in the secretory cells of eccrine sweat glands (Saga and Takahashi, 1992). In eccrine sweat glands, EGF was not localized on any speci®c organella in the cytoplasm. Dark cell granules in eccrine sweat glands were not
70 kDa glycoprotein in human milk fat globule membrane Membranes from mammary epithelial cells can be found in the milk fat fraction. The cream fraction of milk consists of fat droplets surrounded by an external membrane. This membrane layer, known as the milk fat globule membrane, is mainly derived from the apical plasma membrane of lactating mammary secretory cells. Polyclonal and monoclonal antibodies raised against components of human milk fat globule membranes react not only with mammary glands but also with human sweat glands. Several glycoproteins were isolated and characterized from human milk fat globule membranes (Imam et al, 1981, 1982). The antibody raised against the 70 kDa glycoprotein puri®ed from a human milk fat globule membrane reacted with the mammary gland and the apocrine sweat gland, but not with the eccrine sweat gland (Imam et al, 1988). The antibody strongly stained the luminal membranes of secretory cells and that of ductal cells in apocrine sweat glands. In addition, the antibody weakly stained the luminal cytoplasm of apocrine secretory cells. On the other hand the antibody raised against 155 kDa glycoprotein puri®ed from human milk fat globule membrane reacted with the mammary gland, kidney, pancreas, salivary gland, and stomach, but not with the apocrine or eccrine sweat glands (Imam et al, 1986). Therefore, 70 kDa glycoprotein puri®ed from human milk fat globule membrane can be used for the differentiation of apocrine and eccrine sweat glands. HMFG-1 monoclonal antibody The monoclonal antibodies designated as HMFG-1 (1.10.F3) and HMFG-2 (3.14.A3) were produced by immunizing mice with delipidated preparations of the human milk fat globule (Taylor-Papadimitriou et al, 1981; Burchell et al, 1983). Western blot analyses showed that both antibodies recognize antigenic determinants found in high molecular weight components (> 400 kDa). HMFG-1 immunostains apocrine sweat glands; however, it does not react with eccrine sweat glands (Viragh et al, 1997). HMFG-1 monoclonal antibody stains luminal cells of the duct and the luminal cell membranes and the luminal cytoplasm of secretory cells in apocrine sweat glands. Comparative immunohistochemistry of HMFG-2 and antiepithelial membrane antigen monoclonal antibody (E29) showed a similar distribution of staining (Heyderman et al, 1985). Core proteins for several human mucins (MUC1-MUC7) have been identi®ed by molecular biology techniques. MUC1 is a membrane-associated glycoprotein consisting of three domains. HMFG-1 detects fully
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glycosylated MUC1 mucin, whereas HMFG-2 detects underglycosylated MUC-1 mucin (Taylor-Papadimitriou et al, 1981; Burchell et al, 1983; Yonezawa and Sato, 1997). Intercellular canaliculi with high activity of alkaline phosphatase Alkaline phosphatase (ALP) (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) is a group of enzymes that are membrane-associated glycoproteins. ALP catalyzes the hydrolysis of inorganic and organic monophosphate esters at alkaline pH. Although this enzyme is widely distributed in human tissues, we do not fully understand its physiologic functions. The high activity of ALP in the kidney, liver, and intestine has suggested that this enzyme might participate in membrane transport (Harris, 1989). Past reports have demonstrated the activity of ALP in the eccrine and apocrine sweat glands (Bunting et al, 1948; Shelley and Mescon, 1952). The presence of ALP was also reported in some sweat gland tumors such as eccrine hidrocystoma (Sperling and Sakas, 1982). An enzyme histochemical study showed that the activity of ALP was localized on the intercellular canaliculi of the eccrine sweat gland and the myoepithelial cell of apocrine sweat glands (Saga and Morimoto, 1995). The dark cells border nearly all the luminal surface of the secretory tubule, whereas clear cells rest either directly on the basement membrane or on the myoepithelial cells. In eccrine secretory cells, intercellular canaliculi showed positive reaction (Fig 4), whereas luminal cell membranes that were in continuity with intercellular canaliculi were negative for the enzyme activity. Intercellular canaliculi are pouches extending from the luminal surface of secretory cells. This result suggests that intercellular canaliculi are not simple extensions of luminal cell membranes but are differentiated structures that may play a key role in the production of primary sweat. COMMON MARKERS OF ECCRINE AND APOCRINE SWEAT GLANDS GCDFP-15 Gross cystic disease of the breast is a premenopausal disorder in which gross cysts are the predominant pathologic lesion. It is speculated that gross cysts are formed by the over-secretion of the mammary gland. Gross cystic disease ¯uid is a pathologic secretion from the breast composed of several glycoproteins, including GCDFP-70 (70 000 Da), human albumin, GCDFP-44, and GCDFP-15. GCDFP-15 is a unique monomer protein that has no normal plasma counterpart. Normal apocrine gland and various tumors derived from the apocrine gland stained with anti-GCDFP-15 antibody (Mazoujian et al, 1983). It was once believed that GCDFP-15 was a speci®c marker of the apocrine sweat gland and that it could differentiate eccrine and apocrine tumors; however, later studies showed that these gross cystic disease ¯uid proteins exist in both eccrine and apocrine sweat glands (Ordonez et al, 1987). Carcinoembryonic antigen (CEA) CEA was originally found in fetal tissues and in certain gastrointestinal tumors as an oncofetal antigen. Later it was found in various normal secretory epithelium of adults, including sweat glands (reviewed by HammarstroÈm, 1999). CEA is a glycoprotein containing approximately 50% carbohydrate with an approximate molecular weight of 200 kDa. CEA cross-reactive antigens were found in normal human tissue. These antigens include nonspeci®c crossreacting antigen (NCA) and biliary glycoprotein (BGP). Molecular cloning of cDNA for CEA, NCA, and BGP was carried out in the late 1980s. The CEA gene family belongs to the immunoglobulin superfamily. In total 29 genes/pseudogenes have been identi®ed in the human CEA gene family. Speci®c monoclonal antibodies are available for CEA, NCA, BGP, CEA gene family member 2 (CGM2), and for human pregnancyspeci®c 1 glycoproteins (PSG) as a group. Speci®c PCR primers and riboprobes have been constructed. Although the distribution of CEA is limited in certain tissues in normal adults, BGP and
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NCA show broader tissue distribution. The expression of CEA is observed in the gastrointestinal tract, in the prostate, in the cervix, and in sweat glands of human adults. CEA is present on the secretory lumen of secretory cells and the luminal surface of the duct in eccrine sweat glands (Penneys et al, 1981; Nap et al, 1988). The duct shows stronger labeling than the secretory portion. The luminal membranes of the apocrine secretory cells are also labeled with CEA. The biologic function of CEA is poorly understood. It is postulated that CEA and NCA play a role in the innate immune defense protecting the lumen of sweat glands from microbial insult. CEA and NCA probably bind and trap microorganisms, preventing them from reaching down to the microvilli of the epithelial cells and invading the epithelial cells (HammarstroÈm, 1999). Epithelial membrane antigen (EMA) Antisera raised against defatted human cream (defatted human milk fat globule membranes) have been shown to react with various normal epithelial tissues and carcinomas (Heyderman et al, 1979; Sloane and Ormerod, 1981). The glycoproteins with which these antisera react have been termed epithelial membrane antigen (EMA). The antigen has been partially puri®ed, and shown to consist of heterogeneous glycoproteins of high molecular weight (Ormerod et al, 1983). Ceriani et al (1977) reported that an antiEMA antibody reacted with cell membranes of normal breast and breast carcinoma cells; however, that antibody did not react with other carcinoma cells, lymphoma cells, melanoma cells, or mesenchymal cells. Carbohydrates form the major antigenic determinant, the principal sugars of which are galactose and Nacetyl-glucosamine (Ormerod et al, 1983). Later, a monoclonal antibody was produced by immunizing mice with delipidated human cream. This monoclonal antibody, designated as anti-EMA (E29), did not show a signi®cant difference of the distribution of immunostaining with polyclonal anti-EMA antibodies (Cordell et al, 1985; Heyderman et al, 1985). An immunoblot study showed that E29 antibody reacts with a wide range of molecular weights (265±400 kDa) (Cordell et al, 1985). E29 monoclonal antibody stains luminal membranes of both eccrine and apocrine sweat glands. Therefore it cannot be used for the differentiation of apocrine and eccrine sweat glands. CLINICAL APPLICATIONS Some of the histochemical and immunohistochemical markers for eccrine and apocrine sweat glands have been applied to the study of sweat gland tumors. Extramammary Paget's disease expresses anionic sites characteristic to the secretory portion of the apocrine sweat gland (Saga and Jimbow, 1999). Cationic gold at pH 2.0 labeled about 20% of Paget cells from genital areas. Neuraminidase digested these anionic sites indicating that sialic acid is responsible for the anionic charge. These results indicate extramammary Paget cells are differentiating to the secretory cells of the apocrine sweat gland. An antibody against a 70 kDa glycoprotein puri®ed from human milk fat globule membrane (MFGM-gp 70) reacted with the apocrine sweat gland but not with the eccrine sweat gland (Imam et al, 1988). This antibody stained luminal membranes of both secretory and ductal cells in normal apocrine sweat glands. The antibody reacted with Paget cells of extramammary Paget's disease (Imam et al, 1988). This result indicates that Paget cells of extramammary Paget's disease differentiate to the secretory cells or ductal cells of apocrine sweat glands. HMFG-1 monoclonal antibody stains secretory cells and ductal cells of apocrine sweat glands (de Viragh et al, 1997). Several studies have used this antibody to clarify the histogenesis of sweat gland tumors. A study by de Viragh showed that HMFG-1 antibody stained all apocrine cystadenomas and some eccrine hidrocystomas. They speculated that eccrine hidrocystomas might represent cystic tumors of the eccrine sweat duct, or they might represent cystic tumors of the apocrine sweat duct (de Viragh et al, 1997).
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Kamishima et al (1999) studied a case of pigmented hidrocystoma in the vulva using the HMFG-1 antibody. Negative staining with the HMFG-1 antibody and the results of other immunohistochemical staining showed that the tumor was not apocrine in nature but of eccrine derivation. HMFG-1 antibody stained less than 10% of tubular epithelial cells in spiradenoma and dermal cylindroma (Meybehm and Fischer, 1997). This result indicates positive tubular epithelial cells are differentiating to the secretory cells or ductal cells in apocrine sweat glands. REFERENCES Bunting H, Wislocki GB, Dempsey EW: The chemical histology of human eccrine and apocrine sweat gland. Anat Rec 100:61±77, 1948 Burchell J, Durbin H, Taylor-Papadimitriou J: Complexity of expression of antigenic determinants, recognized by monoclonal antibodies HMFG-1 and HMFG-2, in normal and malignant human mammary epithelial cells. J Immunol 131:508±513, 1983 Ceriani RL, Thompson K, Peterson JA, Abraham S: Surface differentiation antigens of human milk fat globule. Proc Natl Acad Sci 74:582±586, 1977 Constantine VS, Mowry RW: Histochemical demonstration of sialomucin in human eccrine sweat glands. J Invest Dermatol 46:536±541, 1966 Cordell J, Richardson TC, Pulford KAF, Ghosh AK, Gatter KC, Heyderman E, Mason DY: Production of monoclonal antibodies against human epithelial membrane antigen for use in diagnostic immunocytochemistry. Br J Cancer 52:347±354, 1985 Goode NP, Shires M, Crellin DM, Davison AM: Detection of glomerular anionic sites in post-embedded ultrathin sections using cationic colloidal gold. J Histochem Cytochem 39:965±972, 1991 HammarstroÈm S: The carcinoembryonic antigen (CEA) family. structures, suggested functions and expression in normal and malignant tissues. Semi Cancer Biol 9:67±81, 1999 Harris H: The human alkaline phosphatases. what we know and what we don't know. Clinica Chimica Acta 186:133±150, 1989 Heyderman E, Steele K, Ormerod MG: A new antigen on the epithelial membrane: its immunoperoxidase localisation in normal and neoplastic tissues. J Clin Pathol 32:35±39, 1979 Heyderman E, Strudley I, Powell G, Richardson TC, Cordell JL, Mason DY: A new monoclonal antibody to epithelial membrane. Br J Cancer 52:355±361, 1985 Imam A, Laurence JR, Neville AM: Isolation and characterization of a major glycoprotein from milk-fat-globule membrane of human breast milk. Biochem J 193:47±54, 1981 Imam A, Laurence DJR, Neville AM: Isolation and characterization of two individual glycoprotein components from human milk-fat-globule membranes. Biochem J 207:37±41, 1982 Imam A, Drushella MM, Taylor CR, Toekes ZA: Preferential expression of a Mr 155,000 milk-fat-globule membrane glycoprotein on luminal epithelium of lobules in human breast. Cancer Res 46:6374±6379, 1986 Imam A, Yoshida SO, Taylor CR: Distinguishing tumour cells of mammary from extramammary Paget's disease using antibodies to two different glycoproteins from human milk-fat-globule membrane. Br J Cancer 58:373±378, 1988 Johnson WC, Helwig EB: Histochemistry of the acid mucopolysaccharides of skin in normal and in certain pathologic conditions. Am J Clin Pathol 40:123±131, 1963 Kamishima T, Igarashi S, Takeuchi Y, Ito M, Fukuda T: Pigmented hidrocystoma of the eccrine secretory coil in the vulva: clinicopathologic, immunohistochemical and ultrastructural studies. J Cutan Pathol 26:145±149, 1999 Mazoujian G, Pinkus GS, Davis S Jr: DEH: Immunohistochemistry of a gross cystic disease ¯uid protein (GCDFP-15) of the breast. Am J Pathol 110:105±112, 1983 Meybehm M, Fischer HP: Spiradenoma and dermal cylindroma: comparative
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