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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

Apocrine and eccrine sweat glands are distinct in from human milk fat globule membranes, and function, although they are closely related to each HMFG-1 (1.10.F3) monoclonal antibody produced other developmentally and morphologically. In cer- by immunizing mice with defatted human milk fat tain sweat gland tumors, it is dif®cult to differentiate globule membranes. Markers for eccrine sweat between eccrine or apocrine sweat glands. There- glands are as follows: dark cell granules that have fore, this paper reviews histochemical and immuno- chondroitinase ABC sensitive anionic sites detected histochemical markers to differentiate apocrine and by cationic gold at pH 2.0 after pretreatment with eccrine sweat glands with the aim of better under- EGTA, and intercellular canaliculi with high activity standing the structural and functional characteristics of alkaline phosphatase. CEA and GCDFP-15 are of these sweat glands. Speci®c markers for apocrine expressed in both eccrine and apocrine sweat glands. sweat glands are as follows: neuraminidase sensitive Anti-EMA monoclonal antibody (E29) stains both anionic sites detected by cationic colloidal gold at eccrine and apocrine sweat glands. Key words: cationic pH 2.0, and mitochondrion-like secretory granules gold/dark cell granule/epidermal growth factor/HMFG-1/ that have epidermal growth factor-like antigenicity. human milk fat globule membrane proteins/intercellular The following antibodies react with apocrine sweat canaliculus. Journal of Investigative Dermatology glands but not with eccrine sweat glands; the anti- Symposium Proceedings 6:49±53, 2001 bodies raised against 70 kDa glycoprotein puri®ed

weat glands are usually classi®ed as either eccrine or of the eccrine sweat gland is the control of body temperature. The apocrine, based on their respective mode of secretion. human eccrine sweat gland is composed of a single tubular structure The secretion of eccrine sweat glands consists of the with a blind end and another end opening to the surface of the skin. transfer of ¯uids across luminal cell membranes of The size of the secretory coil is about 60±80 mm in diameter and 2± Ssecretory cells, whereas in apocrine sweat glands luminal 5 mm in length (Sato et al, 1989). The diameter of the duct is portions of the cytoplasm of secretory cells are pinched off and slightly smaller and the length of the duct is about the same as the released into the secretory lumen. These two types of sweat glands secretory portion. The secretory portion of the eccrine sweat gland are different in size, structure, distribution, nervous control, and consists of a pseudostrati®ed single layer of secretory cells and function. Nevertheless, it is sometimes dif®cult to assign the origin surrounding myoepithelial cells. The secretory portion of eccrine and the differentiation of sweat gland tumors to either eccrine or sweat glands consists of three types of cells: clear cells, dark cells, apocrine sweat glands because many of their characteristics are lost and myoepithelial cells. The dark cells border nearly all the luminal in such tumors. This paper reviews histochemical and immuno- surface of the secretory tubule, whereas clear cells rest either histochemical markers that can be used to differentiate eccrine and directly on the basement membrane or on the myoepithelial cells. apocrine sweat glands (Table I). These markers should help the Where two or more clear cells abut, intercellular canaliculi are diagnosis of sweat gland tumors and help to understand the formed. Not infrequently, the lateral cell membrane of the dark secretory function of these sweat glands in relation to the structural cells forms part of the ori®ce of a canaliculus. Intercellular canaliculi characteristics. are pouches extending from the luminal surface of secretory cells. Nearly isotonic primary sweat is produced in the secretory portion ECCRINE, APOCRINE, AND APOECCRINE SWEAT and the reabsorption of NaCl results in the secretion of hypotonic GLANDS sweat to the surface of the skin (Sato et al, 1989). Myoepithelial cells in eccrine and apocrine sweat glands are spindle-shaped cells The human body has 2±3 million eccrine sweat glands distributed and they sit longitudinally or obliquely to the axis of the secretory over almost the entire body surface. The most important function tubule, between secretory cells and the basement membrane. Apocrine sweat glands, the larger sweat glands, are distributed Manuscript received June 14, 2001; accepted for publication June 14, mainly on the axillary and genital skin, but are also found in the 2001. external auditory meatus (ceruminous gland), the eyelid (Moll's Reprint requests to: Dr. Kenji Saga, Department of Dermatology, Sapporo Medical University School of Medicine, Minami 1 Nishi 16, gland), and within the areola (Robertshaw, 1991). They are an Chyuo-ku, Sapporo 060±8543, Japan. Email: [email protected] evolutionary remnant of an odorous organ of animals. A single layer

1087-0024/01/$15.00 ´ Copyright # 2001 by The Society for Investigative Dermatology, Inc. 49 50 SAGA JID SYMPOSIUM PROCEEDINGS

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 Figure 1. Cationic gold at pH 2.0 labeled anionic sites on the the tubular secretory portion of apocrine sweat glands. Light dark cell granules after EGTA treatment in human eccrine sweat microscopic observation demonstrates that the luminal portion of glands. Scale bar: 500 nm. 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, Figure 2. Cationic gold at pH 2.0 labeled anionic sites on the whereas the undilated segment consists of eccrine-like pseudos- luminal cell membranes of apocrine secretory cells. Scale bar: tratifed epithelium. The apoeccrine glands appear to develop 500 nm. 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). probe and enzymes that are used for the digestion of anionic sites can be eliminated by the post-embedding method. Another MARKERS TO BE USED FOR THE DIFFERENTIATION advantage is that cationic gold is clearly visible under an electron OF ECCRINE AND APOCRINE SWEAT GLANDS microscope, and silver enhancement enables light microscopic observation. Distribution of anionic sites Cationic colloidal gold labels Cationic gold labeled basolateral cell membranes of secretory net negative charge on the histologic sections (Skutelsky and cells in the eccrine sweat gland at pH 2.0 (Saga and Takahashi, Roth, 1986; Saga, 1998). Therefore, an alteration of pH has a 1993). These anionic sites were digested by preincubating the marked effect on the labeling with cationic gold. Cationic gold sections with chondroitinase ABC. Therefore chondroitin sulfate labels only sialic acid and sulfonic acid residues at low pH and/or dermatan sulfate constitute anionic sites on the secretory (= 2.0), which are known to be the only dissociated cellular portion of eccrine sweat glands. Cationic gold at pH 2.0 labeled constituents under such conditions (Skutelsky and Roth, 1986). dark cell granules after pretreatment with EGTA (Fig 1). That Cationic gold labels carboxyl residues of carbohydrates in implies calcium covers anionic sites on the dark cell granules. glycoproteins, glycolipids, and proteoglycans at neutral pH Chondroitinase ABC digested exposed anionic sites by pretreat- (Skutelsky and Roth, 1986; Vorbrodt, 1987; Goode et al, 1991). ment with EGTA. Therefore chondroitin sulfate and/or derma- Cationic gold stains anionic sites on the histologic sections using tan sulfate constitute anionic sites on dark cell granules. Ductal the postembedding method. Pretreatment of the sections with cells or myoepithelial cells of secretory cells were not labeled enzymes eliminates anionic sites on the histologic sections. The with cationic gold. advantage of cationic gold over traditional cationic dye is in post- In apocrine sweat glands, cationic gold at pH 2.0 labeled embedding staining. The problem of the penetration of cationic luminal cell membranes, microvilli, and submembranous vesicles VOL. 6, NO. 1 NOVEMBER 2001 HISTOCHEMICAL MARKERS FOR HUMAN SWEAT GLANDS 51

Figure 3. Anti-EGF antibody stained mitochondrion-like Figure 4. Intercellular canaliculi of eccrine sweat glands showed cytoplasmic granules in apocrine secretory cells. Scale bar: 500 nm. high activity of alkaline phosphatase. Scale bar: 500 nm. of secretory cells (Fig 2). Neuraminidase digested these anionic stained with anti-EGF antibodies. Therefore the mitochondrion- sites, thus sialic acid is responsible for the anionic charge in the like granule containing EGF is a marker of secretory cells in apocrine sweat gland (Saga and Takahashi, 1993); however, these apocrine sweat glands. anionic sites were not labeled with cationic gold at pH 7.4. 70 kDa glycoprotein in human milk fat globule Charge theory alone does not explain this paradoxical pH- membrane Membranes from mammary epithelial cells can be dependency observed in apocrine sweat glands. It is speculated found in the milk fat fraction. The cream fraction of milk that an effect of low pH may be to alter the structural consists of fat droplets surrounded by an external membrane. con®guration of sialoprotein, thus rendering charge sites available This membrane layer, known as the milk fat globule membrane, for labeling with cationic colloidal gold (Saga and Takahashi, is mainly derived from the apical plasma membrane of lactating 1993; Saga, 1998). Post-embedding labeling with cationic gold and predigestion mammary secretory cells. Polyclonal and monoclonal antibodies on the sections showed completely different distribution and raised against components of human milk fat globule membranes responsible molecules between eccrine and apocrine sweat glands. react not only with mammary glands but also with human sweat Chondroitin sulfate and/or dermatan sulfate is a marker of glands. secretory cells of the eccrine sweat gland. Dark cell granules that Several glycoproteins were isolated and characterized from express chondroitin sulfate and/or dermatan sulfate are speci®c human milk fat globule membranes (Imam et al, 1981, 1982). markers of dark secretory cells in the eccrine sweat gland. Sialic The antibody raised against the 70 kDa glycoprotein puri®ed acid is a marker of secretory cells of the apocrine sweat gland. from a human milk fat globule membrane reacted with the Some past reports using cationic dye are contradictory to the mammary gland and the apocrine sweat gland, but not with the studies using cationic gold. According to studies with cationic eccrine sweat gland (Imam et al, 1988). The antibody strongly dye such as alcian blue, both eccrine and apocrine sweat glands stained the luminal membranes of secretory cells and that of were decorated with sialic acid (Johnson and Helwig, 1963; ductal cells in apocrine sweat glands. In addition, the antibody Constantine and Mowry, 1966). The discrepancy may be due to weakly stained the luminal cytoplasm of apocrine secretory cells. the poor speci®city of the cationic dye to anionic sites and the On the other hand the antibody raised against 155 kDa lesser purity of enzymes used. glycoprotein puri®ed from human milk fat globule membrane reacted with the mammary gland, kidney, pancreas, salivary Epidermal growth factor (EGF) EGF, a 53 amino acid gland, and stomach, but not with the apocrine or eccrine sweat polypeptide, directly stimulates epidermal growth and glands (Imam et al, 1986). Therefore, 70 kDa glycoprotein puri- differentiation. EGF is contained in various secretory ¯uids. The ®ed from human milk fat globule membrane can be used for the presence of EGF in sweat was ®rst reported by Pesonen et al differentiation of apocrine and eccrine sweat glands. (1987), who showed that the concentration of EGF (EGF/unit sweat volume) was much higher in armpit sweat than in breast HMFG-1 monoclonal antibody The monoclonal antibodies sweat. That suggests apocrine sweat glands secrete more EGF designated as HMFG-1 (1.10.F3) and HMFG-2 (3.14.A3) were than eccrine sweat glands. Sato et al (1989) con®rmed the produced by immunizing mice with delipidated preparations of presence of EGF in eccrine sweat, using precautions to minimize the human milk fat globule (Taylor-Papadimitriou et al, 1981; epidermal contamination during sweat collection. They also Burchell et al, 1983). Western blot analyses showed that both found that the concentration of EGF in eccrine sweat (EGF/unit antibodies recognize antigenic determinants found in high sweat volume) increases in proportion to the increase in sweat molecular weight components (> 400 kDa). HMFG-1 rate. Thermally induced axillary sweat contains about 50 times immunostains apocrine sweat glands; however, it does not react more EGF than sweat obtained from other areas of the trunk. with eccrine sweat glands (Viragh et al, 1997). HMFG-1 Immunohistochemical studies have shown that both eccrine monoclonal antibody stains luminal cells of the duct and the and apocrine sweat glands express EGF in the cytoplasm of luminal cell membranes and the luminal cytoplasm of secretory secretory cells. Immunoelectron microscopy showed that small cells in apocrine sweat glands. Comparative immunohisto- secretory granules that have cristae of mitochondrion-like internal chemistry of HMFG-2 and antiepithelial membrane antigen structure were heavily stained with anti-EGF antibody (Fig 3), monoclonal antibody (E29) showed a similar distribution of whereas these granules were not found in the secretory cells of staining (Heyderman et al, 1985). Core proteins for several eccrine sweat glands (Saga and Takahashi, 1992). In eccrine sweat human mucins (MUC1-MUC7) have been identi®ed by glands, EGF was not localized on any speci®c organella in the molecular biology techniques. MUC1 is a membrane-associated cytoplasm. Dark cell granules in eccrine sweat glands were not glycoprotein consisting of three domains. HMFG-1 detects fully 52 SAGA JID SYMPOSIUM PROCEEDINGS glycosylated MUC1 mucin, whereas HMFG-2 detects NCA show broader tissue distribution. The expression of CEA is underglycosylated MUC-1 mucin (Taylor-Papadimitriou et al, observed in the gastrointestinal tract, in the prostate, in the 1981; Burchell et al, 1983; Yonezawa and Sato, 1997). cervix, and in sweat glands of human adults. CEA is present on the secretory lumen of secretory cells and Intercellular canaliculi with high activity of alkaline the luminal surface of the duct in eccrine sweat glands (Penneys phosphatase Alkaline phosphatase (ALP) (orthophosphoric et al, 1981; Nap et al, 1988). The duct shows stronger labeling monoester phosphohydrolase, EC 3.1.3.1) is a group of enzymes than the secretory portion. The luminal membranes of the that are membrane-associated glycoproteins. ALP catalyzes the apocrine secretory cells are also labeled with CEA. The biologic hydrolysis of inorganic and organic monophosphate esters at function of CEA is poorly understood. It is postulated that CEA alkaline pH. Although this enzyme is widely distributed in and NCA play a role in the innate immune defense protecting human tissues, we do not fully understand its physiologic the lumen of sweat glands from microbial insult. CEA and NCA functions. The high activity of ALP in the kidney, liver, and probably bind and trap microorganisms, preventing them from intestine has suggested that this enzyme might participate in reaching down to the microvilli of the epithelial cells and membrane transport (Harris, 1989). invading the epithelial cells (HammarstroÈm, 1999). Past reports have demonstrated the activity of ALP in the eccrine and apocrine sweat glands (Bunting et al, 1948; Shelley Epithelial membrane antigen (EMA) Antisera raised against and Mescon, 1952). The presence of ALP was also reported in defatted human cream (defatted human milk fat globule some sweat gland tumors such as eccrine hidrocystoma (Sperling membranes) have been shown to react with various normal and Sakas, 1982). An enzyme histochemical study showed that epithelial tissues and carcinomas (Heyderman et al, 1979; Sloane the activity of ALP was localized on the intercellular canaliculi of and Ormerod, 1981). The glycoproteins with which these the eccrine sweat gland and the myoepithelial cell of apocrine antisera react have been termed epithelial membrane antigen sweat glands (Saga and Morimoto, 1995). The dark cells border (EMA). The antigen has been partially puri®ed, and shown to nearly all the luminal surface of the secretory tubule, whereas consist of heterogeneous glycoproteins of high molecular weight clear cells rest either directly on the basement membrane or on (Ormerod et al, 1983). Ceriani et al (1977) reported that an anti- the myoepithelial cells. In eccrine secretory cells, intercellular EMA antibody reacted with cell membranes of normal breast and canaliculi showed positive reaction (Fig 4), whereas luminal cell breast carcinoma cells; however, that antibody did not react with membranes that were in continuity with intercellular canaliculi other carcinoma cells, lymphoma cells, melanoma cells, or were negative for the enzyme activity. Intercellular canaliculi are mesenchymal cells. Carbohydrates form the major antigenic pouches extending from the luminal surface of secretory cells. determinant, the principal sugars of which are galactose and N- This result suggests that intercellular canaliculi are not simple acetyl-glucosamine (Ormerod et al, 1983). extensions of luminal cell membranes but are differentiated Later, a monoclonal antibody was produced by immunizing structures that may play a key role in the production of primary mice with delipidated human cream. This monoclonal antibody, sweat. designated as anti-EMA (E29), did not show a signi®cant difference of the distribution of immunostaining with polyclonal COMMON MARKERS OF ECCRINE AND APOCRINE anti-EMA antibodies (Cordell et al, 1985; Heyderman et al, SWEAT GLANDS 1985). An immunoblot study showed that E29 antibody reacts with a wide range of molecular weights (265±400 kDa) (Cordell GCDFP-15 Gross cystic disease of the breast is a et al, 1985). E29 monoclonal antibody stains luminal membranes premenopausal disorder in which gross cysts are the predominant of both eccrine and apocrine sweat glands. Therefore it cannot pathologic lesion. It is speculated that gross cysts are formed by be used for the differentiation of apocrine and eccrine sweat the over-secretion of the mammary gland. Gross cystic disease glands. ¯uid is a pathologic secretion from the breast composed of several glycoproteins, including GCDFP-70 (70 000 Da), human CLINICAL APPLICATIONS albumin, GCDFP-44, and GCDFP-15. GCDFP-15 is a unique Some of the histochemical and immunohistochemical markers for monomer protein that has no normal plasma counterpart. eccrine and apocrine sweat glands have been applied to the study of Normal apocrine gland and various tumors derived from the sweat gland tumors. Extramammary Paget's disease expresses apocrine gland stained with anti-GCDFP-15 antibody anionic sites characteristic to the secretory portion of the apocrine (Mazoujian et al, 1983). It was once believed that GCDFP-15 sweat gland (Saga and Jimbow, 1999). Cationic gold at pH 2.0 was a speci®c marker of the apocrine sweat gland and that it labeled about 20% of Paget cells from genital areas. Neuraminidase could differentiate eccrine and apocrine tumors; however, later digested these anionic sites indicating that sialic acid is responsible studies showed that these gross cystic disease ¯uid proteins exist for the anionic charge. These results indicate extramammary Paget in both eccrine and apocrine sweat glands (Ordonez et al, 1987). cells are differentiating to the secretory cells of the apocrine sweat Carcinoembryonic antigen (CEA) CEA was originally gland. found in fetal tissues and in certain gastrointestinal tumors as an An antibody against a 70 kDa glycoprotein puri®ed from human oncofetal antigen. Later it was found in various normal secretory milk fat globule membrane (MFGM-gp 70) reacted with the epithelium of adults, including sweat glands (reviewed by apocrine sweat gland but not with the eccrine sweat gland (Imam HammarstroÈm, 1999). CEA is a glycoprotein containing et al, 1988). This antibody stained luminal membranes of both approximately 50% carbohydrate with an approximate molecular secretory and ductal cells in normal apocrine sweat glands. The weight of 200 kDa. CEA cross-reactive antigens were found in antibody reacted with Paget cells of extramammary Paget's disease normal human tissue. These antigens include nonspeci®c cross- (Imam et al, 1988). This result indicates that Paget cells of reacting antigen (NCA) and biliary glycoprotein (BGP). extramammary Paget's disease differentiate to the secretory cells Molecular cloning of cDNA for CEA, NCA, and BGP was or ductal cells of apocrine sweat glands. carried out in the late 1980s. The CEA gene family belongs to HMFG-1 monoclonal antibody stains secretory cells and ductal the immunoglobulin superfamily. In total 29 genes/pseudogenes cells of apocrine sweat glands (de Viragh et al, 1997). Several studies have been identi®ed in the human CEA gene family. Speci®c have used this antibody to clarify the histogenesis of sweat gland monoclonal antibodies are available for CEA, NCA, BGP, CEA tumors. A study by de Viragh showed that HMFG-1 antibody gene family member 2 (CGM2), and for human pregnancy- stained all apocrine cystadenomas and some eccrine hidrocystomas. speci®c 1 glycoproteins (PSG) as a group. Speci®c PCR primers They speculated that eccrine hidrocystomas might represent cystic and riboprobes have been constructed. Although the distribution tumors of the eccrine sweat duct, or they might represent cystic of CEA is limited in certain tissues in normal adults, BGP and tumors of the apocrine sweat duct (de Viragh et al, 1997). VOL. 6, NO. 1 NOVEMBER 2001 HISTOCHEMICAL MARKERS FOR HUMAN SWEAT GLANDS 53

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