0022-1554/70/$3.

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The J o d of Histochemistry and Cytochemistry Copyright Q 1790 by The Histochemical Society, Inc.

Vol. 38. No. 12, pp. 1815-1821, 1790

Printed in U.S.A.

Original Article

I
Ultrastructural Immunocytochemical Studies of Blood Group Substances in Human Eccrine Glands'
SHINICHI YOTSUMOTO, SHINICHIRO TSUYAMA, MASAAKI TASHIRO, and FUSAYOSHI MURATA
Departments of Dermatology (SYMg and Anatomy (STFM), Faculty of Medicine, ffigosbima University, ffigosbima 890, Japan.
Received for publication April 12, 1990 and in revised form July 10. 1990; accepted July 14, 1990 (OA1961).

We investigated the ultrastructure of blood group antigens A, B, and H in human eccrine glands by means of the immunogold labeling technique. Blood group antigens A, B, and H were found in the Golgi apparatus, secretory granules, and over the apical and basolated cell membranes of dark cells of eccrine glands depending on the blood group phenotype of the donors. Both A and B antigens were found in the dark cells of AB donors. The labeling pattern of the Golgi stacks seemed to have a polarity whereby the ana-blood group A antibody labeled all the stacks, whereas anti-blood

groups B and H bound to the traas side of the Golgi complex. These observations suggest that the blood group substances are secreted into the lumen after being processed through the Golgi apparatus and the immature and mature granules in the dark cells of human eccrine glands. ( J
Nistochem Cgtochem 38:1815-1821, 1990)
KEY WORDS:Blood group substances; Monoclonal antibodies; Im-

munogold labeling; Human eccrine gland; Golgi apparatus; Secretory granules.

irrespective of secretor status. However, little work has been done at the electron microscopic level. Blood group antigens are a group of carbohydrate determinants Glpolipids and glycoproteins with blood group specificitieshave typically found in erythrocytes. Their specificitiesare also expressed been regarded as differentiation antigens, defining particular cell in many epithelial tissues, and they act as major alloantigenic sysor tissue types representing developmental pathways and specific tems in the human (33,34). functions. To investigate the blood group antigen expression in norBlood group A, B, and H antigens are either glycolipids or glymal eccrine gland and tumors derived from eccrine gland cells is coproteins and carry their specific determinants at the ends of their very important because changes of these antigenic systems followcarbohydrate chains. The A and B antigens and their immediate ing tumorigenesis occur in various organs, including sweat glands precursor, H antigen, are carried by branched or unbranched type (4,5,7,8,15,19,25,39). Fundamentally, it is very meaningful to clar1 ((Galpl-3Gl~NAc)~~1-3Gal-R)2 ((Galpl-4Gl~NAc)~P1or type ify the intracellular blood group antigenic sites in normal human 3Gal-R) chains, where Gal is D-galactose and GlcNAc is N-acetyleccrine gland cell. D-glucosamine, depending on the type of tissue (9,16). In this work, we studied the ultrastructural localization of blood There haw been many studies concerning the expression of blood group substances in human eccrine glands using monoclonal antigroup substances in normal tissues and concerning proliferative leblood group A, B, and H antibodies by means of the immunogold sions, including the malignant transformation of various organs labeling technique. (4,5,8,15,19,25,39), and a few reports on the blood group isoantigenicity of human skin appendages (7,17). The first study of the systemic histochemical distribution of human blood group subMaterials and Methods stances using human sera was published by Szulman (33,34). These Spechem. Elcven specimens were investigated in this study. Four were two reports noted the presence of A, B, and H antigens in human blood group A (two secretors and two non-secretors), two group B (both eccrine glands and that the sweat glands produced the antigens secretors), three group 0 (twosecretors and one unknown), and two group
~~~ ~~

Introduction

Supported by Grant-in-Aid No. 62570012 from the Ministry of Education. Science and Culture, Japan. Correspondence to: Shinichi Yotsumoto, MD,Department of Dermatology, Faculty of Medicine, Kagoshima University, 1208-1 Usukicho, Kagoshima 890.Japan.

'

AB (one secretor and one non-secretor). The name, age, sex, blood group phenotype, and secretor status of each individual are listed in Table 1.

Tissue Processing. Human eccrine glands were obtained from skin biopsy or surgery. Normal parts of the skin were cut into small pieces and fmed in a solution containing 2.5% glutaraldehyde and 2% paraformaldehyde in a 0.1 M phosphate buffer (pH 7.4) for 2 hr at 4'C. The tissue blocks
1815

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YOTSUMOTO. TSUYAMA, USHIRO, MuRATA

Table 1. Blood p u p a n d secretor status of 11

examined individuals

acetate solution for 45 sec. The sections were then examined with a JEOL 1OOB electron microscope. With the controls, non-immune mouse serum was substituted for the primary antibody and staining was done with gold-conjugated anti-mouse IgM alone.
Le ( a - b + ) Le (a- b + ) Lc ( a + b - ) Le(a+b-) Le(a-b+) Le(a-b+) Lc(a-b+) Le ( a - b + ) Lc (a- b - ) Lc(a-b+) Le ( a + b - )

1
2

HS I1

3 4 5 6 7
8

MU MY MY
TH

EM
MT IS KK KS

9 10 11

79 39 37 45 75 72 75 16 49 45 27

M
F M F F F F F M

Results
Cytoarchitecture of the Secretory Segment of Human Eccrine Glands
Fixation with 2.5% glutaraldehyde combined with 2 % paraformaldehyde in 0.1 M PB, pH 7.4, and embedding in Lowicryl K4M resin resulted in satisfactory preservation of the ultrastructure and enabled us to ascertain cytoplasmic organelles such as endoplasmic reticula, Golgi complexes, mitochondria, secretory granules, and nuclei. Two different cell types were observed in the secretory segment of human eccrine glands; these cells are called dark or mucoid cells and clear cells. At the electron microscopic level, cells containing abundant ribosomes and many apical secretory granules appeared to correspond to the dark cells. Their shape was pyramidal, with the apical end being broader than the base (Figure 1). The clear cells were associated with the intercellularcanaliculi. They had relatively few ribosomes but a considerable amount of glycogen. The endoplasmic reticulum was poorly developed. No secretory precursors were identified within their cytoplasm. This cell was characterized by prominent interdigitations with neighboring cells.

were then dehydrated and infiltrated in the following steps: 30% ethanol at O'C for 30 min; 50% ethanol at -2O'C for 60 min; 70% ethanol at - 35'C for 60 min; 95% ethanol at - 35'C for 60 min; 100% ethanol at - 35'C for 60 min; twice, 100% ethano1:Lowicryl K4M (1:l) at -35'C for 60 min; 100% ethanol:K4M (1:2) at -35'C for 60 min; K4M at -35'C for 60 min. twice; overnight infiltration in K4M at - 35%. Specimens were embedded in Lowicryl K4M at -35'C (3.22) and polymerized for 24 hr under w irradiation using low-temperature polymerization apparatus 'ID 010 (Balzen; Liechtenstein) as reported previously. Ultra-thin sections were cut and mounted on gold grids with formw-supported film. Blood group typing of the tissue donors was performed by applying a routine hemag glutination test to peripheral blood using human antiserum against A, B, and AB antigens and goat antiserum against Lewis a and b antigens. The secretor status of the tissue donor was deduced from the Lewis blood type because the saliva ABH secretor types are closely related to the eryth. rocyte Lewis types (37). Lewis (a- b + ) patients will be referred to as secretors and h i s (a+ b-) patients as non-secretors. Reagents. Mouse monoclonal anti-blood group A, B, and H antibodies were purchased from Dako (Santa Barbara, CA). The antibodies A, B, (1-3) and H are specifically directed against the a-D-N-acetylgdactosaminc (a-Lfucox (1-2)) f3-~-galactox chain, a-D-gdactox (1-3) (a-&cox (1-2)) f3-D-gdactose chain and a-Lfucose (1-2)-f3-D-~actose (1-4)-&~-Nacetylglucosamine chain, respectively (2.9.16). Ten-nm gold-conjugated goat anti-moux IgM (p) antibody was obtained from Jansscn ( k n e , Belgium). T h e Lowicryl K4M kit was obtained from Polyxience (Warrington, PA).

Localization o Blood Group Antigen A f

Examination of sections from blood group A donors incubated in the monoclonal antibody against blood group antigen A revealed labeling of dark cells (Figure 1). However, no labeling was observed in the clear cells (Figure 1). No labeling was observed in the B or 0 types. In the A group donors, both the immature and mature secretory granules, apical microvilli, and basolateral membranes were labeled, although the adjacent clear cells were not labeled (Figure 2). The Golgi apparatus and following immature and mature granules were also labeled (Figure 3). In the AB group donors, the secretory granules and apical microvilli were labeled similarly to the A donors (Figure 6). The Golgi apparatus was also labeled (not shown). Endothelial cells and red blood cells of the dermal vcssels in the group A donor were labeled by the monoclonal antiblood group A antibody (Figures 1la and llb).

knmunocgtoch~al Spining Rocedurc. Ultra-thin sections mounted on gold grids were placed consecutively on top of a drop of the following reagents in a moist chamber. Ultra-thin sections were rinsed with 0.01 M PBS, pH 7.0, for 5 min, pre-incubated with 1% bovine serum albumin (BSA) in 0.01 M PBS, pH 7.0, for 10 min, rinsed twice with 0.01 M PBS, pH 7.0, at room temperature, 5 min each time, and incubated with mouse monoclonal antibodies (IgM class) against blood-group antigens A, B, and H diluted 1:100 in 0.01 M PBS, pH 7.0. overnight at 4'C. After the application of the monoclonal antibodies, the sections were rinsed five times with 0.01 M PBS, pH 7.0, for I min each time, and floated twice on 0.1% BSA in 0.02 M liis-buffcred saline (TBS), pH 8.2. for 5 min. Finally, 10-nm gold-conjugated goat a n t i - m o w IgM (p) antibody solution diluted 1:10 in 0.02 M TBS. pH 8.2, was applied and incubated for 20 min at room temperature. After washing with 0.02 M TBS, p H 8.2, five times, each for 5 min, and then with distilled water, each section was counterstained with a saturated aqueous uranyl acetate solution for 3 min and then with a lead

Localization of Blood Group Antigen B

Application of the monoclonal antibody against B antigen in bloodgroup B donors resulted in the labeling of secretory granules, apical and lateral membrane (Figure 4), and the Golgi apparatus (Fig ure 5 ) . Gold labeling was usually seen in the trans side of the Golgi stacks (Figure 5 ) . Labeling also appeared in the heterochromatin of the nucleus (Figure 5 ) . In the AB donors, gold labeling against B antigen was observed in the secretory granules and apical cell membrane (Figure 7). It was also seen in the Golgi complex (not shown).

BLOOD GROUP ANTIGENS IN HUMAN ECCRINE GLANDS

1817

Figures 1-11 show electron photomicrographsof human eccrine glands (Figures 1-10) and dermal vessels (Figure l l a ) and erythrocytes (Figure l l b ) embedded in Lowicryl K4M and stained with monoclonal antibodies to bloodgroup substances. Abbreviations: DC, dark cell; CC, clear cell; L. lumen; N. nucleus; G. Golgi apparatus; m. mitochondria; g, secretory granules; mv, microvilli; EC, endothelial cell; RBC, red blood cell. Figure 1. Dark and clear cells of a bloodgroup A donor eccrine gland stained by monoclonal anti-blood group A antibody. Golgi apparatus, secretory granules, and apical microvilliand lateral membranes are labeled in the dark cell, whereas clear cells are devoid of labeling. Original magnification x 14,300. Bar = 1 pm. Figure 2. Apical microvilli, immature light granules and mature dense granules and lateral cell membranes of the dark cell in a bloodgroup A donor are labeled by monoclonal anti-blood group A antibody. Original magnification x 17,800. Bar = 1 pm. Figure3. Golgi apparatus, immatureand mature secretory granules of a dark cell in a bloodgroup A donor are labeled by monoclonal anti-blood group A antibody. Original magnification x 23.700.Bar 1 pm.
I

Localization o Blood Group Antigen H f

After treatment of sections obtained from group 0 donors with a monoclonal antibody against H antigen, labeling was seen in some secretory granules, apical membrane microvilli, and Golgi com-

plm (Figures 8 and 9). Peripheral labeling was more often seen in granules. The trans side of the Golgi stacks also appeared to be labeled (Figure 9). Sections in which non-immune mouse serum had been substituted for the primary antibody had negative staining (Figure 10).

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YOTSUMOTO. TSUYAMA, TASHIRO. MURATA

Figure 4. Apical and lateral cell membranes, secretory granules of the three dark cells in a blood-group B donor are stained by monoclonal anti-bloodgroup B antibody. Original magnification x 20,800. Bar = 1 ym. Figure 5. Golgi region of the dark cell in a bloodgroup B donor incubated with monoclonal anti-blood group B antibody. T i n s lamellaeof the Golgi apparatus are stained. Weak labeling is also seen in the nucleus. Original magnification x 44,400.Bar = 0.5 ym.

I
I

'.

Figure 6. A dark cell in a blood-group AB donor incubated with monoclonal anti-blood group A antibody. Microvilli and secretory granules are stained. Original magnification x 25,500. Bar = 0.5 pm. Figure 7. A dark cell in a bloodgroup AB donor incubated with monoclonal anti-blood group B antibody. In this photograph. apical membrane and secretory granules are also labeled. Original magnification x 21,700. Bar = 0.5 pm. Figure 8. A dark cell in a blood-group 0 donor incubated with monoclonal anti-blood group H antibody. Apical membrane and secretory granules are stained. Peripheral labeling was often observed in granules. Original magnification x 24.300. Bar = 0.5 pm. Figure 9. The Golgi region of the dark cell in a bloodgroup 0 donor incubated with monoclonal anti-H antibody. Labeling of the lfans side of the Golgi stacks is discernible in this photograph. Original magnification x 28.400.Bar = 0.5 pm. Figure 10. Control section of the dark cell in a bloodgroup B donor incubated with non-ir"ne mouse serum substituted for the first monoclonal antibody. No labeling is seen in the apical and lateral membranes, secretory granules, and Golgi complex. Original magnification x 14,000. Bar = 1 prn. Figure 11. (a) Endothelial cell of a bloodgroup A donor stained by monoclonal anti-blood group A antibody. The cell surface of this cell is labeled with this antibody. (b) Erythrocytes of a bloodgroup A donor stained by monoclonal anti-blood group A antibody. The cell surface is labeled with this antibody (arrowhead). Original magnifications: a x 16,000; b x 17,000. Bars = 0.5 pm.

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YOTSUMOTO, TSUYAMA, TASHIRO, MURATA

Discussion
We applied immunogold labeling using monoclonal antibodies against human blood-group antigens in the secretory segments of human eccrine glands. We were able to verfi that the intracytochcmical localization in the dark cells was the Golgi apparatus, cytoplasmic granules, and cell membranes. The distribution of blood-group antigens in normal human skin at the light microscopic level has been reported before (17). Although Hinglais et al. (10) have reported the ultrastructural localization of blood group A antigen in normal human kidney, their antibody belongs to the polyclonal IgG type. To the best of our knowledge, the present report is the first to show the intracellular localization of blood group antigens A, B, and H in human eccrine glands at the electron microscopic level using monoclonal antibodies. In our previous experiment, we tried to find intracellular antigenic sites using a two- three-step pre-embedding method. Monoor clonal antibody, HRP-labeled goat IgG F(ab)2 anti-mouse IgM antibody sequences (or biotinylated goat anti-mouse IgM), and HRP-labeled streptavidin sequences were applied to 8-pm thick sucrose-infused cryostat sections. As we anticipated, this preembedding method stained only the surface of the dark cell. Intracellular antigenic sites were not visualized owing to failure of penetration of the IgM type monoclonal antibody. Instead of this pre-embedding method, in the present experiment we used postembedding staining and succeeded in finding the intracellular reactive sites. Theoretically, post-embedding staining using monoclonal antibody, biotinylated goat anti-mouse IgM antibody, and colloidal gold-conjugated streptavidin is also suitable for this purpose. The authors spent much time using this sequence, but more background staining was observed than with the present two-step method. The monoclonal anti-blood group antigen A, B, and H antibodies labeled the Golgi complex, immature or mature granules, and cell membranes in the dark cells of eccrine glands, depending on the blood group phenotype of the donors. It is consistent with this that the blood group substances are processed through the Golgi apparatus following the secretory granules, and then secreted to the lumen. The reaction of monoclonal anti-blood group A antibody, which specifically binds to a-D-GalNAc (1-3) (a-L-Fuc (1-2)) P-D-Gal chain, revealed the whole lamellae of the Golgi complex, whereas the monoclonal anti-blood group B and H antibody, which binds to a-D-Gal (1-3) (a-L-Fuc (1-2)) P-D-Gal and a-L-Fuc (1-2) p-DGal (1-4) P-D-GIcNAcchain, seemed to label the trans side of the Golgi apparatus. These findings may suggest a functional polarity of the Golgi stacks in the dark cells of human eccrine glands. There have been many reports on sugar chain processing in the Golgi complex using various lectins (26.36.38) and monoclonal or affinitypurified antibodies against enzymes (6,27,28,30,32). According to these reports, the terminal GalNAc residue seems to localize at the cis and/or trans sides of the Golgi stacks; on the other hand, terminal Gal and Fuc residues are located at the trans side of the Golgi stacks. This may also support the findings in the Golgi stacks in the dark cells of human eccrine glands. Peripheral labeling of morphologically homogeneous secretory granules was sometimes found in anti-blood group H antibody staining. The significance of this labeling pattern is unclear at present.

Weak labeling of monoclonal anti-blood group B antibody was seen in the heterochromatin of nuclei in the blood-group B phenotype donor (Figure 5 ) . This suggests that there are blood-group B substances in the intranuclear heterochromatin. Recently, the presence of glycoconjugates in the nucleus has come to be reported progressively. These reports are based on biochemical analyses of purified nuclei (11,12) and various histochemical methods (1,18, 20,21,26,31). The precise roles of nuclear glycoconjugatcs are not yet known. However, there is a line of evidence for an involvement of glycoprotein in transcriptional processes (13,14). In relation to the present immunocytochemical study, Nakapma et al. (24) studied goblet cell mucin in 39 human colons stained with three lectins (DBA, GSA-1, PNA) and immunostained for blood group isoantigens A, B, H, and T. These authors showed that some lectin reactions to goblet cells might be related to blood group antigens but that others were due to the different specificity between lectins and monoclonal anti-blood group antibodies. Lectin histochemistry is often applied to normal and abnormal sweat glands. Terui et al. (35) applied three lectins (PNA, SBA, and DBA) to a patient diagnosed with idiopathic acquired generalized anhidrosis and showed a deficient lectin binding pattern of the glandular cells. Saida et al. (29) showed that the PHA lectin was a useful histochemical marker of acrosyringium and the distal portion of intradermal sweat duct. Immunogold labeling of blood group antigens in human salivary glands using monoclonal antibodies has been reported by Nakajima et al. (23). These authors detected no labeling in the Golgi complex, and labeling was detected only in mature secretory granules. In contrast, we were able to detect labeling with blood group antibodies in the Golgi apparatus and in immature and mature secretory granules of the dark cells of human eccrine glands. Hinglais et al. (10) found blood group A antigen in the Golgi complex at the ultrastructural level by use of a two-step pre-embedding method using purified rabbit anti-A antibody and peroxidaselabeled sheep anti-rabbit IgG F(ab)2 antibody in several components of human kidney. From these results, they suggested in situ synthesis of blood-group substances in these organs. Because bloodgroup substances are either glycoproteins or glycolipids, it is reasonable to assume that the Golgi apparatus plays an important role in the synthesis of these substances. In conclusion, the post-embedding immunogold method using monoclonal antibodies clearly revealed intracellular localization of blood group antigens A, B. and H in human eccrine glands. Further examination with this method of malignant tumors of the skin will provide valuable information concerning the cytochemical features of blood group substances.

Acknowledgments We thank Prof Z Suganuma, Department of Anatomy, Miyazaki Medical

College andMs K.Ihi& fortheir helpfuldiscusrions, Mr M. Goreforreviewing the English, and Mr S. Nonaka for technical assistance.

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