THE ANATOMICAL RECORD 228:1-6 (1990)

Glycoconjugate Expression of Cells of Human Anagen Hair Follicles During Keratinization J U N OHNO, KIMIE FUKUYAMA, AND WILLIAM L. EPSTEIN Department of Oral Pathology, School of Dentistry, University of Meikai, Sakado, Saitama, Japan (J.O.); Department of Dermatology, University of California, Sun Francisco, California (K.F., W.L.E.)

ABSTRACT Changes in the expression of glycoconjugates in cells of the inner root sheath (IRS) and outer root sheath (ORS) of human anagen hair follicles were investigated by lectin histochemistry. Concanavalin A (Con A) and Ricinus comrnunis (RCA-I) stained hair follicle cells regardless of their differentiation stages. In IRS, Ulex europeaus-I (UEA-I) bound to the surface of the cells as soon as they were morphologically defined, and Glycine rnax (SBA) stained a s their differentiation progressed. Innermost (IM) cells of ORS layers were reactive with UEA-I at the stage where Henle’s cells were keratinized, while the reactivity of UEA-I was lost at the site of the completion of IRS keratinization where SBA reaction was detected. Staining of both UEA-I and SBA was prominent in other ORS cells at the levels where SBA binding in IM cells became strong. The staining intensity increased up to the position of the follicular isthmus. In addition, a sugar residue recognized by Dolichos biflorus (DBA) was detected in differentiated cells of ORS. In contrast, the DBA reaction was not found a t all in cells of IRS, infundibulum, and epidermis. These findings identified a complexity of carbohydrate metabolism in the cells of different layers at various stages of keratinization. IM cells differentiate independently from other ORS cells but seem responsive to the degree of IRS keratinization. All ORS cells possess a unique sugar moiety not found in other keratinocytes either in the hair or epidermis. Hair follicles develop from the epidermis in the third fetal month (Hashimoto, 1970; Montagna, 1976). While epidermal cells maintain a singular compartment, hair follicle cells are separated into several compartments, and each unit undergoes a n independent process of differentiation totally different from that seen in the epidermis. The hair follicle cells are merged with epidermal cells at the infundibulum (Fig. 1). The area below the entry of the sebaceous duct is called the follicular isthmus, where the inner root sheath (IRS) already has disintegrated and the outer root sheath (ORS) demonstrates so-called “trichilemmal”keratinization without the formation of keratohyalin granules (Pinkus, 1969). Two cell types, innermost (IM) and outer layer (OL) cells, are identified in ORS before keratinization (Ito, 1986). IRS comprises Henle’s and Huxley’s cells and its cuticle. The keratinization process begins with the formation of trichohyalin granules, first in the Henle’s cells and then in the cuticle and Huxley’s cells. In contrast to the precise description of morphological changes for differentiation of hair follicle cells (Hashimoto and Shibazaki, 19761, relatively little is known about the associated biochemical changes, except for detection of disulfide bond formation (Taneda et al., 1980) and various keratin types (Lynch et al., 1986; Ito et al., 1986; Heid et al., 1986; Stark et al., 1987). The relationship between keratinocyte maturation and cell surface glycoconjugates has been established in the epidermis (Holt et al., 1979; Brabec e t al., 1980; Reano et al., 1982; Zieske and Bernstein, 1982; 0 1990 WILEY-LISS, INC

Nemanic et al., 1983; Ookusa et al., 1983; Schaumburg-Lever et al., 1984; Virtanen et al., 1986, Brown et al., 1987). Binding of Ulex europeaus-I (UEA-I) and Glycine max (SBA) occurs before terminal differentiation indicating that expression of a-fucose (Fuc) and a-N-acetylgalactosamine (aGalNAc), respectively, is the specific biochemical event in epidermal keratinocytes. In this study we investigated changes in the expression of glycoconjugates in hair follicles during the keratinization process by lectin histochemistry. In spite of the morphological differences in hair follicle keratinocytes of either IRS or ORS, binding of UEA-I and SBA also was detected prior to keratinization. In addition, differentiated cells of ORS were found to possess a surface disaccharides detectable with Dolichos biflorus (DBA) (Baker et al., 1983), not found in the epidermis. DBA binding disappears from cells in the infundibulum, indicating that expression of this sugar moiety is a unique phenomenon for hair ORS keratinocytes. MATERIALS A N D M E T H O D S Fifteen paraffin blocks of normal human scalp, which had been processed and stored at the Dermatol-

Received July 5, 1989; accepted December 5, 1989. Address reprint requests to Kimie Fukuyama, M.D., Ph.D., Department of Dermatology, Box 0536, University of California, San Francisco, CA 94143-0536.

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ogy Research Laboratories of the University of California, San Francisco, were used in this study. These specimens were fixed in buffered-neutral 10% (v/v) formalin, dehydrated through graded alcohols, and embedded in paraffin by routine procedures. Serial sections, either longitudinal or transverse, were cut at 4 pm and placed on gelatin-coated glass slides. They were stained with hematoxylin and eosin to survey the histology (Fig. 1). Detection of glycoconjugates in anagen hair follicles was performed by the method of Hsu et al. (1981). UEA-I was used for identification of a-fucose (Fuc), SBA was used for the terminal disaccharide aN-acetylgalactosamine (aGalNAc) 1+3 D-galactose, and DBA was used for aGalNAac 1-3 GalNAc. Concanavalin A (Con A) and Ricinus communis (RCA-I) were used for staining a-D-mannose and D-galactose, respectively. These reagents were purchased from Vector Laboratories, Inc., Burlingame, CA. Scalp sections were first deparaffinized and immersed in 1% (vlv) H,O, in methanol for 20 min to block endogenous peroxidase activity. They were washed three times in 0.1 M phosphate-buffered saline (PBS), pH 7.4, containing 0.1 mM MgCl,, CaCl,, and MnC1, for 5 min each, and incubated with 1% ( w h ) bovine serum albumin in PBS to reduce background staining. The sections were reacted with the biotinylated lectins (10 pgl ml) in PBS for 30 min followed by reaction with avidinbiotin complex (ABC) solution for 30 min at 22°C. At each step they were washed three times in PBS for 5 min each. Sites of lectin binding were visualized by immersion in 0.05% (w/v) diaminobenzidine tetrahydrochloride (Sigma Chemical Co., St. Louis) in 0.05 M Tris-HC1 buffer, pH 7.4, together with 0.015% ( v h ) H,O, for 5 min. The sections were counterstained with methyl green, rinsed in water, dehydrated, cleared, and mounted in Permount. Controls for the specificity of lectin staining included substitution of unconjugated lectins for the lectin-biotin conjugates, replacement of the biotinylated lectins by PBS followed by the ABC solution, and reaction with biotinylated lectins mixed with saccharides that specifically bind to each lectin. RESULTS Binding patterns of UEA-I and SBA

creased andlor disappeared in IM cells facing the keratinized Huxley’s cells, whereas SBA binding appeared on the cell surface and the inner side of the cytoplasm (Fig. 4b). A limited number of OL cells showed the UEA-I reaction. At the site of the completion of IRS keratinization the SBA reactivity increased in IM cells, especially at the contact portion with the IRS layer (Fig. 5a,b). The number of OL cells stained with both UEA-I and SBA increased a s they differentiated. While UEA-I stained the cell outline, SBA stained both the cytoplasm and cell surface at this stage (Fig. 6a,b). In the follicular isthmus (Fig. 1A) and infundibulum, binding of both UEA-I and SBA was detected on the surface of ORS cells (Fig. 7a,b), and staining patterns were similar to those seen in the epidermis. Binding Patterns of DBA

All cells in the epidermis, infundibulum, and IRS, irrespective of the degree of differentiation, failed to stain (Figs. 2c, 3c). In the ORS, no reaction was seen up to the sites of keratinization of Henle’s cells (Fig. 3c). At level C in Figure 1 (Fig. 4c), the inner side of the cytoplasm of IM cells facing the keratinized IRS showed weak staining. At the site where keratinization of IRS cells was completed (Fig. lB), distinct cytoplasmic staining of IM cells became apparent (Fig. 5c). The DBA staining extended into OL cells as the follicles approached the surface (Fig. 6c). The reaction in OL cells appeared both in the cytoplasm and on the cell surface at the same level that cytoplasmic SBA staining was noted. Strong reactivity also was seen in the cytoplasm of IM cells at this level. In the follicular isthmus (Fig. lA), both the surface and cytoplasm of OL cells were intensely stained (Fig. 7c). Binding Patterns of Con A and RCA-I

Con A and RCA-I bound to the surface of almost all cells in the epidermis and infundibulum, except for the cornified cells. The two lectins reacted on cells of both the IRS and ORS layers at all stages of differentiation. DISCUSSION During fetal development, epidermal cells give rise to hair follicle cells, and they maintain gene expression of the same cell lineage, namely, keratinocytes that possess keratin protein-forming property. The present study demonstrated a n additional biochemical property, which is mutually retained on the cell surface of the two embryologically cross-related cells. We detected binding of UEA-I and SBA in cells of the epidermis, IRS, and ORS prior to terminal differentiation. As

In the epidermis UEA-I binding appeared in suprabasal cells, and the reaction became more intense in upper spinous and granular cells. SBA binding was limited to the upper spinous and granular cells. At the bulb to suprabulbar portion of scalp hair (Fig. lE,F), UEA-I reactivity became detectable at the cell surface of both Huxley’s and Henle’s layers. The surface of IM cells also stained weakly (Fig. 2a). SBA binding was seen on Henle’s cells and cuticle, but not on Huxley’s cells, and none of the ORS cells showed the reactivity (Fig. 2b). At level D in Figure 1, staining of UEA-I and IRS SBA became obscure in keratinized Henle’s cells (Fig. ORS 3a,b). However, Huxley’s cells were still reactive with IM UEA-I, and additional SBA reaction appeared (Fig. OL Con A 3b). The staining intensity of UEA-I increased on the RCA-I surface of IM cells, but no SBA staining was found. UEA-I Coexistence of non-keratinized and keratinized Hux- SBA ley’s cells was observed at level C, shown in Figure 1. DBA Fuc Binding of both UEA-I and SBA disappeared from aGalNAc keratinized cells (Fig. 4a,b). Staining of UEA-I de- PBS

A bbreuiations

inner root sheath outer root sheath innermost layer cells outer layer cells concanavalin A Ricrnus communis Ulex europeaus-I Glycine max Dolichos biflorus fucose a-N-acetylgalactosamine phosphate-buffered saline

LECTIN BINDING OF HUMAN HAIR FOLLICLE CELLS

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Sebaceous duct

Trichilemmal keratinization

Hair (a) and follicular cell layers: Inner root sheath (IRS) Cuticle (b) Huxley’s cells (c) Henle’s cells (d) Outer root sheath (ORS) Innermost cells (e) Outer layer cells (9 Basal cells (9)

F

Fig. 1 . Diagrammatic and photographic representation of morphological changes of cells in human anagen hair follicles. The composite scheme is based on both longitudinal and transverse sections stained with hematoxylin and eosin. A The follicular isthmus where the sebaceous duct (SD) enters the hair follicles. Hair (a) is surrounded by ORS cells composed of OL cells (f) and basal cells (g), which undergo trichilemmal keratinization (4).B: The sites where IRS keratiniza-

tion is complete. IM cells (e) are seen as a single layer of cuboid cells. C: Huxley’s cells (c) are partially keratinized (*I. Tricohyalin granules are seen in non-keratinized (*) cells. D: Keratinization of Henle’s cells. Huxley’s cells are filled with trichohyalin granules. IM cells (e) and OL cells (f). E: The suprabulbar portion. Three layers of IRS are established. F: The hair bulb. Cuticle (b), Huxley’s (c) and Henle’s (d) cells. Magnification A,D-F, x 760; B,C, X 640. Bars = 15 km.

seen in epidermal cells (Brabec et al., 1980; Zieske and Bernstein, 1982; Nemanic et al., 1983; Virtanen et al., 1986; Brown et al., 1987), UEA-I binding was a n earlier event than SBA binding in hair follicle cells, indicating that genes that control cell surface expression of

terminal Fuc and subsequent aGalNAc are conserved in hair follicle cells. Con A and RCA-I, which have a broad specificity, also reacted with those cells regardless of their differentiation stages. However, DBA staining disclosed that the surface

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Fig. 2. a d : Lectin reaction on serial sections from F in Figure 1. Binding of UEA-I (a) is seen on the surface of both Huxley’s and Henle’s cells. Some IM cells (arrow) are also stained. SBA (b) stained Henle’s cells and cuticle. No reaction with DBA (c). Magnification ~ 8 4 0Bar . = 15 pm. Fig. 3. a-c: Sections of hair follicles from D in Figure 1stained with UEA-I (a), SBA (b), and DBA (c). Huxley’s cells ( a ) react with

both UEA-I and SBA, whereas only UEA-I stains the surface of IM cells (arrow). There is no DBA staining. Magnification x 640. Bar = 15 pm.

Fig. 4. a-c: Lectin staining of hair follicles a t C in Figure 1. UEA-I (a) and SBA (b) stain nonkeratinized IRS cells ( a ) and IM cells (arrow) facing the keratinized IRS cells. DBA (c) binds to IM cells adjacent to keratinized cells. Magnification x 840. Bar = 15 pm.

LECTIN BINDING OF HUMAN HAIR FOLLICLE CELLS

Fig. 5. a-c: Binding of UEA-I (a), SBA (b), DBA (c) in hair follicles at B in Figure 1. The reactivity with UEA-I increases on the surface of OL cells, but reduces in IM cells (*). At this level, IRS cells are strongly stained with methyl green, which caused the black-toned staining of the IRS in these black and white photographs. SBA reacts intensely with the cytoplasm of IM cells and the cell surfaces of centrally located OL cells. Reaction of DBA in IM cells increases. Magnification ~ 8 4 0 Bar . = 15 pm.

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Fig. 6. a-c: At the level just above that shown in Figure 5. UEA-I staining (a) extends to most ORS cells devoid of IM and basal cells. The surface and cytoplasm of both IM and OL cells are stained with SBA (b) and DBA (c). Magnification x 840. Bar = 15 pm. Fig. 7. a-c: Lectin staining a t the follicular isthmus (Fig. 1A).UEAI (a) and SBA (b) bind preferentially to the surface of OL cells, whereas DBA binding (c) is observed both on the surface and in the cytoplasm of OL cells. Sebaceous duct (SD). Magnification x 630. Bar = 15 pm.

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property of ORS cells may be distinct from that of IRS and epidermal cells. DBA is a lectin that binds to the terminal disaccharide, aGalNAc 1-3 GalNAc (Baker et al., 1983) and has been shown to react with some specific cell types among the heterogenic cell populations present in kidney (Brown et al., 1985), gastric mucosa (Kessimian et al., 1986), and salivary glands (Laden e t al., 1984).There seems to be no specific common function identified for the cells bearing this glycoconjugate, though most of the cells that have been described act to secrete intrinsic substances rather than to absorb external substances. It is difficult to relate the functional and physiological roles of ORS cells to a secretion cell type with the information currently available. Deposition of glycogen occurs in ORS cells (Montagna et al., 1951; Uno e t al., 1968), and carbohydrate metabolism of the cells appears to differ from normal IRS and epidermal cells. The DBAbinding disaccharide may be a product of ORS cells as a part of this unique metabolism. Lectin binding to cell surface glycoconjugates in hair follicle cells elucidated the vertical relationship of the cell layers preceding keratinization in much greater detail than morphological observation based on hematoxylin and eosin staining. The findings emphasize the events in IM cells that occur independently from OL cells, even though both IM and OL cells have been classified as ORS cells. In IM cells UEA-I binding begins with keratinization of Henle’s cells and addition of SBA reaction to Huxley’s cells. When Huxley’s cells keratinize, SBA becomes reactive on IM cells, while those lectins bind to OL cells at more superficial levels of hair follicles. On the other hand, where DBA binding is concerned, IM and OL cells present a similar metabolic nature not related to IRS cells. Ito (1986)reported the differences between IM and OL cells using light and electron microscopy. Subsequently, Ito et al. (1986) found two monoclonal antibodies against hair keratin proteins, using immunohistochemical methods. They found that both antibodies reacted with IRS cells and one of them was positive in IM cells, whereas both were negative in OL cells. The present results support the conclusion reached by Ito’s prpers concerning the relationship of IM cells to IRS and ORS. Clinical biopsy specimens for histopathological diagnosis of scalp are usually cut in transverse section. Headington (1984) reported quantitative morphometric analyses of the follicles and hair from transverse sections of scalp specimens. We believe that lectin histochemistry can be useful in the clinical analysis of disorders of hair growth. ACKNOWLEDGMENTS

This study was supported in part by grant number AR12433 from the National Institutes of Health. LITERATURE CITED Baker, D.A., S. Sugii, E.A. Kabat, R.M. Ratcliffe, and P. Hermentin 1983 Immunochemical studies on the combining sites of Forssman hapten reactive hemagglutinins from Dolzchos biflorus, Helix pomatia, and Wistaria floribunda. Biochemistry, 22: 2741-2750. Brabec, R.K., B.P. Peters, LA. Bernstein, R.H. Gray, and I.J. Goldstein 1980 Differential lectin binding to cellular membranes in the epidermis of the newborn rat. Pro. Natl. Acad. Sci. U.S.A., 77t477-479.

Brown, D., J. Roth, and L. Orci 1985 Lectin-gold cytochemistry reveals intercalated cell heterogeneity along rat kidney collecting ducts. Am. J. Physiol., 248rC348-C356. Brown, R., W.W. Ku, and I.A. Bernstein 1987 Changes in lectin binding by differentiating cutaneous keratinocytes from the newborn rat. J. Invest. Dermatol., 88:719-726. Hashimoto, K. 1970 The ultrastructure of the skin of human embryos. V. The hair germ and perifollicular mesenchymal cells. Hair germ-mesenchyme interaction. Br. J. Dermatol., 83t167-176. Hashimoto, K., and S. Shibazaki 1976 Ultrastructural study on differentiation and function of hair. In: Biology and Disease of the Hair. T. Kobori and W. Montagna, eds. University ofTokyo Press, Tokvo. DD. 23-57. Headington, J.T. 1984 Transverse microscopic anatomy of the human scalp. Arch. Dermatol., 120:449-456. Heid, H.W., E. Werner, and W.W. Franke 1986 The comolement of native a-keratin polypeptides of hair-forming cells: A subset of eight polypeptides that differ from epithelial cytokeratins. Differentiation, 32r101-119. Holt, P.J.A., J. Hill Anglin, Jr., and R.E. Nordquist 1979 Localization of specific carbohydrate configurations in human skin using fluorescein-labelled lectins. Br. J . Dermatol., 100:237-245. Hsu, S.M., L. Raine, and H. Fanger 1981 Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem., 29r577-580. Ito, M. 1986 The innermost cell layer of the outer root sheath in anagen hair follicle: Light and electron microscopic study. Arch. Dermatol. Res., 279.112-1 19. Ito, M., T. Tazawa, N. Shimizu, K. Ito, K. Katsuumi, Y. Sato, and K. Hashimoto 1986 Cell differentiation in human anagen hair and hair follicles studied with anti-hair keratin monoclonal antibodies. J . Invest. Dermatol., 86t563-569. Kessimian, N., B.J. Langner, P.N. McMillan, and H.O. Jauregui 1986 Lectin binding to parietal cells of human gastric mucosa. J . Histochem. Cytochem., 34.237-243. Laden, S.A., B.A. Schulte, and S.S. Spicer 1984 Histochemical evaluation of secretory glycoproteins in human salivary glands with lectin-horseradish peroxidase conjugates. J. Histochem. Cytochem., 32t965 -972. Lynch, M.H., W.M. OGuin, C. Hardy, L. Mak, and T.T. Sun 1986 Acidic and basic hairinail ( “ h a r d ) keratins: Their colocalization in upper cortical and cuticle cells of the human hair follicle and their relationship to “soft” keratins. J. Cell Biol., 103:2593-2606. Montagna, W. 1976 General review of the anatomy, growth, and development of hair in man. In: Biology and Disease of the Hair. T. Kobori and W. Montagna, eds. University of Tokyo Press, Tokyo, pp. xxi-xxxi. Montagna, W., H.B. Chase, and J.B. Hamilton 1951 The distribution of glycogen and lipids in human skin. J. Invest. Dermatol., 17: 147-157. Nemanic, M.K., J.S. Whitehead, and P.M. Elias 1983 Alterations in membrane sugars during epidermal differentiation: Visualization with lectins and role of glycosidases. J. Histochem. Cytochem., 31t887-897. Ookusa, Y., K. Takata, M. Nagashima, and H. Hirano 1983 Distribution of glycoconjugates in normal human skin using biotinyl lectins and avidin-horseradish peroxidase. Histochemistry, 79: 1-7. Reano, A,, M. Faure, Y. Jacques, U. Reichert, H. Schaefer, and J. Thivolet 1982 Lectins as markers of human epidermal cell differentiation. Differentiation, 22t205-210. Pinkus, H. 1969 ‘Sebaceous cysts’ are trichilemmal cysts. Arch. Dermatol., 99t544-555. Schaumburg-Lever, G., J. Alroy, A. Ucci, and W.F. Lever 1984 Distribution of carbohydrate residues in normal skin. Arch. Dermatol. Res., 276:216-223. Stark, H.J., D. Breitkreutz, A. Limat, P. Bowden, and N.E. Fusenig 1987 Keratins of the human hair follicle: “Hyperproliferative” keratins consistently expressed in outer root sheath cells in vivo and in vitro. Differentiation, 35,236-248. Taneda, A., H. Ogawa, and K. Hashimoto 1980 The histochemical demonstration of protein-bound sulfhydryl groups and disulfide bonds in human hair by a new staining method (DACM staining). J. Invest. Dermatol., 75r365-369. Uno, H., K. Adachi, and W. Montagna 1968 Glycogen contents of primate hair follicles. J. Invest. Dermatol., 51r197-199. Virtanen, I., A.L. Kariniemi, H. Holthofer, and V.P. Lehto 1986 Fluorochrome-coupled lectins reveal distinct cellular domains in human epidermis. J. Histochem. Cytochem., 34t307-315. Zieske, J.D., and LA. Bernstein 1982 Modification of cell surface glycoprotein: Addition of fucosyl residues during epidermal differentiation. J. Cell Biol., 95t626-631. ~