Acta Medica et Biologica Vol. 38, No.2, 51-67, 1990

The Morphology and Cell Biology of the Apparatus : Recent Advances

Masaaki ITO

Department of Dermatology, Niigata University School of Medicine, Niigata 951, Japan

Received March 20, 1990

Summary. Recent advances in knowledge of the mor­ basal cell layer of the epidermis (periderm),1-3) simi­ phology and biology of hair apparatuses are introduced. lar to other skin appendages such as sebaceous The hair apparatus morphologically shows a cyclic glands, apocrine and eccrine sweat glands. Amongst change "hair cycle" from anagen through catagen to these appendages, the hair apparatus producing hair telogen. In anagen, the hair apparatus is composed of shows the most complicated structure, being com­ eight epithelial cell layers, one of which has been very recently discovered: the innermost cell layer of the posed of several cell layers and displaying the most outer root sheath. When the ultrastructures of these dynamic cell kinetics indicated by the cyclic mor­ cell layers are compared with each other, the cells of phological changes from anagen phase through each layer reveal unique ultrastructural characteristics catagen and telogen phases, and back to anagen 4 in differentiation and keratinization. By bromodeoxyur­ phase again. ) The changes in the hair apparatus are idine stain, the germinative cells in anagen hair and called "hair cycle"; grow in anagen phase, stop hair follicles can be divided into four groups. The basal their growth in catagen phase, fall out at the end of lamina in the hair follicle gradually becomes thickened telogen phase, and then regenerate in the next anagen during anagen. In catagen, as the inner hair structures phase. atrophy and retract, the hyaline membrane including In the present article, recent investigations of the the thickened basal lamina becomes corrugated. Im­ munoblot analyses and immunohistochemical studies hair apparatus are introduced and summarized for a using several anti-hair monoclonal antibodies clearer understanding of the biology of the hair. have shown that the cells of the hair shaft and inner root sheath express hard keratin and that the inner­ I. Structures of Anagen Hair and Hair Follicles most cell layer of the outer root sheath is an indepen­ dent cell layer. The various anti-keratin monoclonal antibodies that are available are useful for either Levels of anagen hair apparatus differentiation of normal skin appendages or diagnosis The anagen hair apparatus is an epithelial tissue of skin epithelial tumors. Remaining for further biolog­ which has a continuity with epidermis and extends ical and biochemical investigation are the roles of in dermis into subcutaneous fat tissue. The deepest cytoplasmic interfilamentous substances in filament formation in hair cells and the nature of the cell portion of the hair epithelium, into which a small membranes of the hair cells. Proper cell culture mass of connective tissue invaginates from its bot­ methods for the cells deriving from hair apparatuses tom side, is swollen and called "hair bulb". The are required for further advances in studies of hair invaginated connective tissue is called "hair dermal biology. papilla" and contains unique mesenchymal cells which interconnect through desmosome-like struc­ tures, some fibrils, abundant mucin deposi­ tions and a blood capillary.5) Above the hair bulb, the INTRODUCTION hair epithelium forms a columnar structure, in the center of which a hair is organized and extends In fetal life, the hair apparatus generates from the through a hair canal to the outside of the skin. The

51 52 M. ITO: wall of the hair canal is called "hair follicle", which means "sheath of hair", Near the epidermis, a seba­ ceous gland adheres to the follicular epithelium and its duct opens into the hair canal. Just beneath the sebaceous duct opening, the hair follicle is slightly reduced in width; this portion is called "follicular isthmus", The portion above the opening is "follicular infundibulum", which is formed by a circularly arran­ ged epithelium surrounding a pilar pore and showing a cell differentiation similar to that of the epidermis (Figs. 1, 2). -- -e- HC

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ORS

IMC

Me Co KZ liS CH

CI

Hu I kS Ill'

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Leve I s- Fig. 1. Histological schema of the anagen hair appara­ tus. The painted areas indicate keratinizing or keratin­ ized portions. CH, cuticle of hair; CI, cuticle of inner root Fig. 2. Light micrograph of the human anagen appara­ sheath; Co, cortex; CTS, connective tissue sheath; HC, tus. CH, cuticle of hair; CI, cuticle of inner root sheath; hair canal, HDP, hair dermal papilla; He, Henle's layer; Co, cortex; CTS, connective tissue sheath; h, hyaline HM, hair matrix; HS, hair shaft; Hu, Huxley's layer; membrane; HDP, hair dermal papilla; He, Henle's layer; IMC, innermost cell layer of the outer root sheath; IRS, HM, hair matrix; Hu, Huxley's layer; IMC, innermost cell inner root sheath; KZ, keratogenous zone; Me, medulla; layer of outer root sheath; KIRS, keratinized inner root ORS, outer root sheath; SD, sebaceous duct; TK, tri­ sheath; Me, medulla; ORS, outer root sheath. chilemmal keratinization. Hematoxylin-eosin stain. The Morphology and Cell Biology of the Hair Apparatus 53

Fig. 3. Electron micrograph of the suprabulbar portion of the human anagen hair apparatus. Mitotic cells (asterisks) are seen in the outer root sheath (ORS). The Huxley's cells (Hu) produce many trichohyaline granules (t), whereas the Henle's cells (He) are already keratinized. b, basal lamina; CH, cuticle of hair; Cl, cuticle of inner root sheath; Co, cortex; CTS, connective tissue sheath; IMC, innermost cell layer of outer root sheath. Uranyl acetate-lead citrate stain. X 1,500

hair bulb facing the hair dermal papilla and move Production and development 0/ cell layers upwards in development, showing individual cell The anagen hair apparatus is composed of surround­ characteristics (Fig. 3). By an autoradiographic study ing connective tissue (connective tissue sheath, CTS) using 3H-thymidine, many S-phase cells were shown 10 and three groups of cell layers: hair shaft, inner root to be localized in the hair matrix area. ) Further­ sheath (IRS) and outer root sheath (ORS). Further­ more, a recent cell kinetic study using bromodeox­ more, the first "hair shaft" group is composed of yuridine (BrdU), which is an analog of thymidine and medulla, cortex and hair cuticle layers, the second similarly incorporated into the nuclei in S-phase, and "IRS" group of IRS cuticle, Huxley's and Henle's anti-BrdU monoclonal antibody, has shown more layers, and the last "ORS" group of the innermost cell precise localizations of such S-phase cells in human 1 (IMC) layer of the ORS6-9) and the layer of other ORS and mouse hair apparatuses. 1,12) The S-phase cells cells. These eight cell layers are laminatedly and are present in several cell layer in the hair matrix cylindrically arranged in the order cited above from area around the hair dermal papilla and divided into the center of the hair to the outer part of the hair three groups in the hair bulb (Fig. 4). The cells in the follicle (Figs. 1, 2). The IRS, ORS and CTS form the medulla and cortex may originate from the inner and hair follicle. All the cells in the hair shaft, IRS and central group of the germinative cells (group A), the IMC layer generate in the hair matrix area in the those in the hair cuticle, IRS cuticle and Huxley's 54 M. ITO:

layer from the middle group (group B), and those in the Henle's layer, IMC layer of the ORS and bulbar ORS from the outer group (group C)l2) (Figs. 4, 5). A recent DNA-flow cytometric study of human anagen hair bulbs has revealed that 6.7% and 5.3% of the cells in a hair bulb cell suspension are in S-phase and Gz M phase, respectively.13) These cell numbers are '-21 so o

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Fig. 5. Schema of the location of germinative cells and cell movement in the anagen hair apparatus. The ger­ Fig. 4. Bromodeoxyuridine (BrdU) stain of the human minative cells (circles) are divided into the four groups A, anagen hair apparatus. Positive cells, which are indicated B, C and D. Cells produced in area A compose the medulla by a dark (originally brown) nuclear stain (arrowheads) and cortex, those in area B compose the cuticle of hair, and considered to be in S phase, in the areas A, Band C cuticle of inner root sheath, Huxley's layer and Henle's in the hair bulb and in the area D in the outer root sheath layer, those in area C compose the innermost cell layer (ORS). CH, cuticle of hair; CI, cuticle of inner root sheath; (IMC) of the outer root sheath and bulbar outer root Co, cortex; CTS, connective tissue sheath; h, hyaline sheath, and those in area D compose most of the outer membrane; HDP, hair dermal papilla; He, Henle's layer; root sheath (ORS). Arrows indicate the directions of the Hu, Huxley's layer; IMC, innermost cell layer of the outer movement of the cells produced. HDP, hair dermal papil­ root sheath. la; SD, sebaceous duct. The Morphology and Cell Biology of the Hair Apparatus 55 significantly more than those in a cell suspension of normal epidermis, where 2.7% epidermal cells are in 14 S-phase and 1.3% are in Gz +M phase. ) The data indicate that the anagen hair bulb is one of the rapidly renewing tissues. The ORS shows more complicated cell kinetics than that in the other cell layers described above. In the hair bulb, the ORS is composed of two cell layers of flattened cells; out of the two, the inner cell layer is the immature IMC layer of the ORSS). At the suprabulbar level, the outer cells in the ORS abruptly increase in number, resulting in a thickening of the ORS (Figs. 2, 3). In this area, figures of mitotic cells are often observed not only in the outermost (CTS­ side) cell layer but also in the inner (IMC-side) cell layers of the ORS, although the IMC layer of the ORS shows no mitotic figure but an already differentiated 7 S cell feature. • ) Corresponding to this morphological finding, many S-phase cells detected by BrdU­ immunohistochemical staining are present in this 1Z area ) (Fig. 4). In the upper part of the ORS up to the isthmus levels, the BrdU-positive cells gradually decrease in number and are localized more in the 1Z outer (CTS-side) layer of the ORS ) (Fig. 5). These findings suggest that the ORS cells derive mainly from the lateral part at the suprabulbar level and partly from the basal (CTS-side) cell layer at every level of the hair follicle, forming a cell stream moving from the deep lateral portion in an upper­ inner direction. They finally fall into the hair canal at the isthmus level (Fig. 5). Fig. 6. Electron micrograph of a hair matrix of the human anagen hair apparatus. The hair matrix cells (HM) are immature and contain many free ribosomes, but Ultrastructures of the immature, differentiating and no tonofilaments, in the cytoplasm. b, basal lamina; HDP, keratinizing cells in each cell layer hair dermal papilla; M, mesenchymal cell. Uranyl acetate-lead citrate stain. X 6,000 1) Hair matrix The hair matrix cells, which are germinative cells of hair and hair follicle as described above, surround the between a few layers of the matrix cells. hair dermal papilla in the hair bulb (Figs. 1, 2). These cells ultrastructurally show an undifferentiated cell 2) Medulla feature; their cytoplasm is extremely rich in The medulla layer is located in the center of hair ribosomes but lacking in tonofilaments (keratin shaft (Figs. 1, 2). The developing medulla cells pro­ filaments). They have a few undeveloped desmosomes duce medulla granules in the cytoplasm; the granules at their cell peripheries, but do not form hemides­ are round electron dense depositions of similar size mosome structures facing the hair dermal papilla, without a limiting membrane but surrounded by although they have a basal lamina on the papilla-side free ribosomes. They form a small number of cell surface, including the whole papilla tissue (Fig. tonofilaments and have melanosomes, which have 6). Several melanocytes are intermingled with the been transferred from melanocytes, in the cytoplasm. suprapapillary matrix cells adjacent to the papilla; Interestingly, the medulla granules are distributed the distribution of the melanocytes is limited to the evenly over the whole cytoplasm, whereas the area A as shown in Fig. 5. They produce many melanosomes are found only in its upper half, form­ melanosomes in the cytoplasm and extend well devel­ ing a so-called epinuclear cap of melanosomes (Fig. 7) oped dendritic processes into the intercellular spaces as seen in the basal cells in the epidermis. When the 56 M. ITO:

Fig. 7. Electron micrograph of the suprabulbar portion of mouse anagen hair apparatus. In the medulla cells (Me), melanosomes (m) are distributed in the epinuclear area, whereas medullary granules (g) cover the entire area of the cytoplasm. CH, cuticle of hair; CI, cuticle of inner root sheath; Co, cortex; CTS, connective tissue sheath; He, Henle's layer; Hu, Huxley's layer; IMC, innermost cell layer of outer root sheath; DRS, outer root sheath; t, trichohyaline granule. Uranyl acetate-lead citrate stain. x 5,000 hair keratinizes, the medulla cells become degenerat­ ultrastructural studyJ6) has revealed that the develop­ ed, leaving an empty medulla space. The medulla ing cortical cells in human hairs produce small (OJ -0.3 layer is well developed and occupies a large part of ,urn) round electron dense granules in the cytoplasm the hair volume in mice (Fig. 7) and guinea pigs. In at a limited level of about 150 ,urn length of the humans, the medulla layer is often lost or poorly keratogenous zone. These granules have no limiting developed in thin hairs as those of Caucasians, while membrane and are surrounded by ribosomes and fuse it is more frequently found in thick hairs as those of to tonofilaments (Fig. 8); therefore, the granules Mongoloids and Negroids, although there seem to be should be a kind of trichohyaline granule and termed variations in degree of the medulla-formation among "cortical trichohyaline granule".16) After the fusion of individuals. the granules to the filaments, the filaments become more electron dense. Similar granules are produced 3) Cortex by cortical cells of anagen hairs in guinea pigs but not The cortex layer, a main structure, consists of a hair in mice. 16 ) The developing cortical cells lose shaft located between the medulla layer and the hair desmosome-structures, a nucleus and other cell cuticle layer (Figs. 1, 2). The cortical cells contain organellae and finally keratinize at the level of 700­ many melanosomes transferred from melanocytes. 800 ,urn distance from the hair matrix area; this level The developing cortical cells gradually become spin­ is the top (end) of the keratogenous zone. At this dle-shaped and fairly elongated in a direction parallel level, the Huxley's layer is also keratinizing (Fig. 1). to the hair long axis at the suprabulbar level. As they The disappearance of the cell organellae may be their move upwards in the keratogenous zone, the cyto­ digestion by lysosomal enzymes, which the cortical plasmic tonofilaments gradually increase in number cells themselves produce. In the case of melanosomes (Fig. 8) and finally fill their cytoplasm; all of the incorporated into the cortical cells, eumelanosomes filaments run along the hair axis direction. as seen in human or mouse black hairs are very Textbooks5,15) have taught that no trichohyaline sub­ difficult to be digested by such lysosomes, whereas stance is produced by cortical cells; however, an pheomelanosomes as seen in reduced-colored hairs of The Morphology and Cell Biology of the Hair Apparatus 57

Fig. 8. Trichohyaline granules in the keratogenous zone Fig. 9. High power view of a cross-section of keratin­ of the cortex in the human anagen hair apparatus. Round ized cortex cells in a human hair. The cytoplasm of the homogenous trichohyaline granules (t) surrounded by free cortical cells is entirely filled with a large number of ribosomes are seen to fuse to the (f) in the cross-sectioned electron lucent keratin filaments and a cytoplasm. Uranyl acetate-lead citrate stain. x 50,000 highly electron dense interfilamentous substance. The cortical cells show a close cell-to-cell contact (arrow­ heads). Uranyl acetate-lead citrate stain. x 82,000 some mice are easily digested during the cortical cell band structure,19,21) and that the keratinized hair I7 development ). cortex does not show such a membranous fluores­ The cytoplasm of keratinized cortical cells is filled cence but rather a diffuse and homogenous with compactly packed keratin filaments; these fluorescence.22 - 24 ) The SS-staining pattern of the filaments are less electron dense and embedded in a cortex seems to be also related to the presence of highly electron dense matrix (interfilamentous) sub­ SS-rich in the cytoplasm of the cortical cells, stance (Fig. 9), which may partly derive from cortical especially proteins of cortical keratin filaments and trichohyaline granules. Cytoplasmic filaments are matrix substances.25 ) Although no marginal band is present in the cell periphery just inside the cell formed in the cortical cells, "involucrin", which is one membrane; this is one characteristic ultrastructure of of the precursor proteins of the marginal band,26) can keratinized cortical cells differing from those of be immunohistochemically detected in the ker­ epidermal horny cells. More specifically, the latter atogenous zone of the cortex (Fig. 10) (data not yet cells have a highly electron dense zone of 150 ,urn published). Furthermore, transglutaminase, which is thicknesss located on the inner surface of the cell considered to playa role in the formation of involu­ membrane, which is called "marginal band",18) while crin polymers,27l has also been detected in the same the cortical cells do not form a marginal band (Fig. part of the cortex by a fluorescence-histochemical 9). Histochemically, by DACM (N -(7-dimethylamino­ method using monodancylcadeverine (Fig. lOb) (data 4-methyl-3-coumarinyl)maleimide) stainI9,20) for SS not yet published). There are some discrepancies linkages of proteins present in tissues, it has been between the morphological and the histochemical shown that the epidermal horny cells reveal a mem­ results; the roles of involucrin and transglutaminase branous fluorescence, which is considered to be due to in the cortex remain to be further studied. The SS linkages abundantly present in their marginal cortical cells closely adhere to each other (Fig. 9); 58 M. ITO: biochemical natures of the cell-to-cell contact are unknown. Membrane-coating granules (cement­ 28l somes ) are not produced by the cortical cells.

4) Hair cuticle The hair cuticle layer is a single cell layer surround­ ing the cortex (Figs. 1, 2) to form the surface struc­ ture of hair. The hair cuticle cells first acquire a cuboid cell shape in the hair bulb and gradually become flattened and oblong during their differentiation (Figs. 3, 7). During this change, each cuticle cell is covered by the next (lower) one, result­ ing in a bamboo sheath-like lamination of the cuticle cells forming imbricated scales on the hair surface (Fig. 3). The developing hair cuticle cells produce some tonofilaments and many small (up to 0.1 ,urn in diameter) round trichohyaline granules, both of which are mainly localized in the outer half of the cytoplasm. This half of the cytoplasm forms a well organized keratin pattern and a marginal band along the inner surface of the cell membrane and is called "exocuticle", whereas the inner half (endocuticle) does not form such structures and is filled with elec­ 5 tron dense substances. ) Several cell layers of the hair cuticle can be observed when a hair is cross­ sectioned; the inner hair cuticle cells are the upper ones. These cells show a close cell-to-cell contact similar to that of cortical cells and form some small invaginations of the cell membranes with each Fig. 10. a. Immunohistochemical stain of human anagen other.5l hair apparatus by anti-involucrin antibody (by Drs T. Maruyama and M. Ito, Niigata University School of 5) IRS Medicine). A positive reaction is indicated by the slightly The IRS cuticle layer is a single cell layer located at dark (originally brown) stain and is present in the cyto­ the inner side of the IRS, facing the hair cuticle layer plasm of the cortex (Co), cuticle of hair, inner root sheath (Fig. 1). Its cells also change their cell shape from (IRS) and outer root sheath (ORS). The hair matrix (HM) cuboid to flattened and form a laminated cell layer to and the outermost cells of the outer root sheath (ORS) are fit its inner border with the outer border of the hair negative. b, Fluorescence-histochemical transglutaminase cuticle layer (Fig. 3). During development, they pro­ stain of the human anagen hair apparatus using monodan­ duce some relatively large trichohyaline granules and cy1cadeverine (by Drs T. Maruyama and M. Ito, Niigata University School of Medicine). Almost the same positive a few tonofilaments evenly in the cytoplasm. When areas as shown in Fig. lOa can be recognized. the hair cuticle layer is keratinized, the IRS cuticle cells also keratinize and lose contact with the hair cuticle cells. The keratinized IRS cells have a homogeneous The Huxley's and Henle's layers are respectively cytoplasm (Figs. 3, 7, lla, llb), in which randomly located in the middle and outer parts in the IRS and running electron dense keratin filaments and less display similar cell ultrastructures, although the for­ electron dense interfilamentous substance are mer keratinizes at the top (end) of the keratogenous contained; no keratin pattern is observed (Fig. llb). zone and the latter keratinizes at the suprabulbar They form a marginal band on the inner surface of level prior to keratinization of any other cell layers the cell membrane (Fig. llb) and connect with each in the hair and hair follicle. The cells in both layers other by desmosomes. The keratinized IRS is preser­ produce many huge trichohyaline granules and a ved up to the lower isthmus level and then becomes small number of tonofilaments in the cytoplasm (Figs. degenerated. The patterns of the IRS degeneration 3, 7). are morphologically different between human hair The Morphology and Cell Biology of the Hair Apparatus 59

Fig. 11. Electron micrographs of the innermost cell layer of the outer root sheath in the suprabulbar portion of the human anagen hair apparatus. a, The innermost cell (lMC) accumulates tonofilaments (t) in the Henle's side of the cytoplasm. b, enlargement of tonofilaments in an innermost cell. All the tonofilaments (f) show cross-sections, indicating that they are running transversely against the hair long axis and surrounding inner hair structures. The keratinized Henle's cell (He) is filled with sparsely distributed electron dense keratin filaments and a less electron dense interfilamentous substance. d, desmosome. Uranyl acetate-lead citrate stain. a x 6,000, b x 60,000 follicles and animal ones. In humans, the keratinized tonofilaments in the inner (Henle's) side of the cyto­ cells suddenly lose their electron density and are plasm; these tonofilaments always run transversely destroyed; the destruction occurs first in Huxley's against the long axis of the hair surrounding the IRS cells, then in IRS cuticle cells, and lastly in Henle's structure (Figs. lla, lIb). Such a unique arrangement cells.7,8) On the other hand, in mice8,29) and guinea of tonofilaments has been ultrastructurally observed pigs,29) the keratinized IRS cells gradually lose their in a previous report,30) although the significance and cell thickness and become thin, resulting in the pres­ roles were unknown until recent studies. 1,7-9,29) When ence of a thin layer of laminated cell membranes on a part of a Huxley's cell protrudes to the ORS the hair canal-side surface of the ORS. These through a Henle's pore, the adjacent IMC shows a degenerated IRS cells vanish before ORS cells start massive accumulation of tonofilaments facing the to keratinize at the middle level of the isthmus.7,8,29) protrusion. The IMC layer may playa role in suppor­ It is clear that, morphologically, the IRS plays a ting and covering the inner hair structures tightly role in guiding the developing cell layers to form a like hoops of a barrel.9) The IMCs gradually increase hair shaft, itself forming an inner part of follicular the number of the tonofilaments lying entirely in their wall surrounding the hair canal. When the role is cytoplasm; their cell shape changes from flat through completed, the ORS keratinize towards the hair canal cuboid and lozenge, finally to extremely flattened. and the IRS vanishes into the hair canaL The lamellar cell is almost parallel to the direction of the long axis of the hair and is attached to keratin­ 6) ORS ized Henle's cells at their inner-upper portion, at the As described above, the IMC layer of the ORS has lower isthmus level,7,9) At the level where the keratin­ been found to be an independent single cell layer ized IRS becomes degenerated and the IMCs gradu­ located just outside the Henle's layer (Figs. 1, 3). In ally become lamellar in cell shape, the IMCs some­ the hair bulb, the IMCs are flat and immature, times produce a few small keratohyaline granules in containing free ribosomes in the cytoplasm. At the the cytoplasm and then keratinize prior to the suprabulbar level, the IMCs gradually form keratinization of the other ORS cells. Keratinized 60 M. ITO:

IMCs are filled with electron dense keratin filaments, anagen ORS cells occasionally produce some kerato­ showing no keratin pattern, and form a marginal hyaline granules, and do not seem to be mor­ band at the cell periphery.7) The intercellular spaces phologically similar to such epithelial cells (data not between the keratinized IMCs and some remaining yet published). As a characteristic cell feature, these keratinized Henle's cells are often dilated, containing ORS cells produce a large number of membrane amorphous substances.9) These morphological coating granules in the cytoplasm and excrete them findings of the IMC layer are common to human/,9) into the intercellular spaces before keratinization.7.33l mouse8 ,29) and guinea pig29 ) hair follicles. The cells in other layers including the medulla, cor­ The ORS layer except for the IMCs shows a unique tex, hair cuticle, IRS, and the IMC layer of the ORS, cell production as described above. The ORS cells produce no membrane coating granules. Neverthe­ produced at the suprabulbar level ultrastructurally less, the ultrastructures of the keratinized cells, such show irregular cell shapes (Fig. 3), contain a small as the marginal band and keratin filaments, show no number of tonofilaments in the cytoplasm, and gradu­ differences between the IMCs (see above) and the ally become rich in glycogen granules. The other ORS cells. tonofilaments form thin bundles running in various The morphological characteristics of the directions in the cytoplasm. During their upward differentiating and keratinizing cells in all the layers movement, they produce more tonofilaments in the of anagen hair and hair follicles as described above cytoplasm and gradually become elongated, then are summarized in Table I. They are compared with finally lamellar, at the lower isthmus level. At the those of epidermal keratinocytes (Table I). middle to upper levels of the isthmus, these ORS cells move in an inner direction, keratinize and fall down II. Morphological Changes of the Hair Apparatus into the hair canal. It has been found that this ker­ during the Hair Cycle atinization is not accompanied by the production of keratohyaline granules and is the same as those of 1) Anagen 3 the epithelium of a sebaceous cyst !) and of the ORS In the early anagen phase, the CTS is ultrastructural­ surrounding the club root in telogen.32l This is so­ ly composed of a basal lamina, is adjacent to the called "trichilemmal keratinization". However, the outer border of the ORS, and forms a collagen tissue.

Table I. Ultrastructural comparison of cell differentiation and keratinization between hair cell layers and epidermis Hair and hair follicles ORS Structures Cortex Hair Epidermis Medulla cuticle IRS Outer (Human) (Mouse) IMC layer# Differentiating cells: Tonofilaments ± 3+ 3+ +* + 2+* 3+ 3+ Membrane coating granules x x x x x x 0 0 Keratohyaline or trichohyaline © 0 x 0 © /::; /::; © granules MG TG TG TG KG KG KG Keratinized cells: Keratin kattern x 0 o 0* x x x 0 Marginal band x x x 0* 0 0 0 © Remnants of desmosomes x x x x /::; /::; /::; 0 Cell-to-cell contact L C C C CL C--L C--L IMC innermost cell layer; IRS inner root sheath; ORS outer root sheath KG keratohyaline granules; MG medullary granules; TG trichohyaline granules # not including infundibular epithelium ± very small number; + small number; 2+ large number; 3+ very large number x not produced (formed); /::; occasionally or incompletely produced (formed) o produced (formed); © always or richly produced (formed) * localized to one side; C close; L loose; C--L changing from close to loose The Morphology and Cell Biology of the Hair Apparatus 61

Fig. 12. Electron micrographs of a connective tissue sheath in a human anagen hair follicle. a, middle anagen. The inner layer (In) of the connective tissue sheath (CTS) is composed of collagen fibers running longitudinally parallel to the long axis of the hair whereas the collagen fibers in the middle layer (Mi) are always running transversely against the long axis and surrounding the inner hair layers. The basal lamina (b) is thin, but shows a partial thickening by lamination (asterisks). b, late anagen. The basal lamina (b) is entirely thickened. F, fibroblast; ORS, outer root sheath. Uranyl acetate-lead citrate stain. a and b x 6,350

The collagen tissue gradually grows and thickens 2) Catagen during the development of the hair follicle. 34 ) In the In the early catagen phase, when the hair matrix mature anagen hair apparatus, the collagen tissue is stops cell production and is no longer labeled by separated into three layers: The inner collagen layer, BrdU staining,II,12) it becomes degenerated prior to just outside the basal lamina, is thin and composed of any other structural changes of the hair and hair collagen fibers running longitudinally parallel to the follicle. Then, since the cells of the inner layers hair long axis (Figs. 12a, 12b). The middle layer is including the hair shaft, the IRS and the IMC of the very thick and its collagen fibers run transversely ORS are no longer produced, the cell layers lose their against the hair axis, surrounding the inner hair cylindrical structures and become atrophic, leaving tissue. In this layer many fibroblasts are present an epithelial cord composed of ORS cells. The basal among the collagen fibers, whereas the inner layer lamina and the inner collagen layer become corru­ contains almost no cells (Figs. 12a, 12b). The outer gated, resulting in a thickening of the hyaline collagen layer is made of collagen fibers running membrane.4,34) The epithelial cord gradually becomes longitudinally or transversely as well as cellular shorter and shorter, and the bottom of the hair structures such fibroblasts, blood vessels and some apparatus finally goes up to the isthmus level, form­ fat cells (Fig. 12a). In the late anagen phase, the basal ing a telogen hair apparatus. The reduction of the lamina becomes very thick and is composed of fine epithelial cord is achieved by cell degeneration and filamentous or granular structures (Fig. 12b). The phagocytosis of the ORS cells. Namely, the cells complex of the thickened basal lamina and the inner become degenerated here and there in the cord and collagen layer is light microscopically recognized as are phagocytized by still living neighboring cells; this a hyaline membrane around the hair follicle. 34,35) phenomenon may occur repeatedlY,4,34) resulting in reductions of the volume and length of the cord. The remaining connective tissue, which derives from the 62 M. ITO:

CTS in the anagen hair follicle, in the dermis through subcutaneous fat tissue shows partly degeneration and partly production of collagen tissue.34)

3) Telogen In telogen, the club root is directly surrounded by an ORS epithelium, which shows keratinization without any formation of keratohyaline granules. The mesen­ chymal cells in the anagen hair dermal papilla are known to remain in the connective tissue just beneath 4 1 - 2 - 3 - 4 - 5 the epithelium. ) At the bottom of the ORS, a secon­ Fig. 13 Histological schema of structural changes of the dary hair germ generates, develops and forms the hair apparatus during the hair cycle. 1, anagen; 2, early next anagen hair apparatus. The newly produced hair catagen; 3, middle catagen; 4, telogen; 5, early anagen; h, pushes the old hair out of the hair follicle. hyaline membrane. The histological changes of hair and hair follicles during the hair cycle are schematically shown in Fig. 13. the electrophoretic bands of either hair or epidermal keratin, whereas HKN-5, HKN-6 and HKN-7 show III. Keratin Biology of Hair and Hair Follicle positive reactions only with hair keratin bands.6,37) By immunohistochemical surveys using normal human Keratin is one of the cytoplasmic intermediate tissues, the former MoAb decorate not only the hair filament proteins, and is specific for epithelial cells. apparatus including ORS but also the epidermis and When compared with other kinds of intermediate other epidermal appendages, whereas the latter filament proteins such as , , glial MoAb positively stain only the hair apparatus except fibrillary acidic and protein for the major part of the ORS.6,37) These data indicate (specific for mesenchymal cells, muscular cells, glial that hair hard keratin molecules have either indepen­ cells and neurons, respectively), keratin is known to dent specific antigenic sites or ones common to soft be most variable protein. It has been divided into four keratin molecules. Such specific antigenic sites do not types; a stratified epithelium type, a simple epith­ seem to be localized in a small part of the hair elium type, a complex type of both, and hard keratin molecule, but may exist in various parts of keratin.36 ) The first three types are called "soft ker­ the molecule. An immunoblot analysis after 2D SDS­ atin" as opposed to the last of "hard keratin". In the PAGE44 ) has shown that HKN-4 and HKN-5 react skin, the epidermis, sebaceous glands, ducts and positively with a group of basic polypeptides of hair myoepithelial cells of sweat glands, and ORS37) have keratin, whereas HKN-2, HKN-6 and HKN-7 react a stratified epithelium type of keratin, while the positively with both acidic and basic groups (Fig. 14) secretory cells of sweat glands38 ) and Merkel cells39,40) (data not yet published). Interestingly, HKN-5 im­ express a simple epithelium type of keratin. On the munohistochemically stains the IMC layer of the other hand, the medulla, cortex, hair cuticle and IRS ORS, but not other ORS cells6) (Fig. 15a). This and of the hair apparatus and some parts of the are other morphological findings of the IMC layer de­ known to possess hard . Biochemically, the scribed above indicate that this layer is an indepen­ single-dimensional (lD)41) and two-dimensional dent layer in the anagen hair follicle. Recently, some (2D)42,43) electrophoretic patterns of hair keratin are investigators have extracted hair keratins from much different from those of epidermal keratin. plucked anagen hair roots, which may contain living Furthermore, there are distinct immunological and developing hair shaft cells, and produced poly­ differences in antigenicity between these keratins. clonal antibodies (PoAb) against the extracts. By Monoclonal antibodies (MoAb) have been produced such immunohistochemical studies,45) the PoAb have against the hair keratin extracted from human hair been found to react only with developing cells in the and characterized by immunoblot analyses and im­ hair shaft layers and IRS in anagen hair tissue and munohistochemical surveys.6,37) By immunoblot ana­ never decorate the IMC layer of the ORS, epidermis lyses after 1D sodium dodecyl sulfate (SDS)-poly­ nor any other epithelial tissues. The discrepancy in acrylamide gel electrophoresis (PAGE), HKN-2 and the immunohistochemical findings between the PoAb HKN-4 out of the anti-hair keratin MoAb react with and the MoAb seems to be due to the difference in The Morphology and Cell Biology of the Hair Apparatus 63

b

a

HKN2 HKN4

a a

HKN5 HKN6 HKN7 Fig. 14. Immunoblot analyses of anti-hair keratin monoclonal antibodies against electrophor­ etic polypeptide spots of human hair fibrous proteins (HFP) by two-dimensional SDS­ polyacrylamide gel electrophoresis (by Drs K. Katsuumi, T, Tazawa and M. Ito, Niigata University School of Medicine). Upper left, Coomassie brilliant blue stain of the gel. The proteins are separated mainly into two groups (a and b) in IP ph 5.1-6.8. The molecular weight of the polypeptides in group b is 59kD and those of group a are 43kD and 41.5kD. Others, immunoblot analyses. HKN-2, HKN-6 and HKN-7 react with the polypeptides in both groups a and b, whereas HKN-4 and HKN-5 react only with those in group b. Asterisk, Bio-Lyte; NEPHG, nonequilibrium pH gradient gel electrophoresis; SDS, sodium dodecyl sulfate­ polyacrylamide gel electrophoresis. sensitivities of both antibodies, since the PoAb may hair cells, has become possible. Their 2D electro­ be too low in sensitivety to detect a minor antigenic phoretic patterns are varied, probably depending on 44 sites in hair keratin molecules, which can be detected inherent factors. ) Although the biology of keratin by some MoAb. Although PoAb are considered to be filaments has rapidly advanced in scope, little infor­ important in a rough examination of the outline of mation about such interfilamentous substances is immunological characteristics of a substance, MoAb presently available. In order to obtain more knowl­ should be used to determine the precise localization edge on the filament formation in hair cells, further of an antigenic site. studies of the satellite substances around the filaments seem to be important.

IV. Further Investigations of the Hair Apparatus 2) Cell membranes of hair cells Ultrastructurally, the keratinized cells in the cortex 1) Interfilamentous substances and hair cuticle show a very close cell-to-cell contact Recently, 2D SDS-PAGE of high sulfur proteins, with each other. However, membrane coating gran­ which are considered to be a matrix or inter­ ules (cementsomes28») are not produced in these cells filamentous proteins in the cytoplasm of keratinized and desmosomes are no longer present between them, 64 M. ITO:

Fig. 15. Immunohistochemical stain by anti-hair keratin monoclonal anti­ body HKN -5. a, suprabulbar portion of human anagen hair apparatus. A positive reaction is seen in the medulla (Me), cortex (Co), cuticle of hair (CH) and inner root sheath (IRS). In the outer root sheath (ORS), only the inner­ most cell layer (IMC) shows a positive reaction. b, basal cell epithelioma. Positive cells are seen mainly in the centers of the tumor nests. as described above. In the epidermis, steroid sulfates tochemical surveys of the differentiation of various are known to playa role in the cell-to-cell contact skin tumors and given us abundant new dermatohis­ among the epidermal horny cells46,47) and the natures topathological information on skin epithelial of the cell membranes of the epidermal keratinocytes tumors.38,40,56-61) Briefly, basal cell epitheliomas (Fig. are also known to change gradually during 15b),57) squamous cell carcinomas55 ) and trichilemmal differentiation and keratinization.48 ) However, no tumors54,55) express keratin similar to that of the ORS biochemical data on the cell membranes of the hair in the anagen hair follicle; all the cells of the tumors cells nor on the intercellular substances between are positive for HKN-2 and HKN-4 and some of the them are available and further studies are required. tumor cells are always positive for HKN-5, whereas the tumors never show a positive reaction with 3) Cell culture of the cells of the hair and hair HKN-6 or HKN-7. Pilomatricomas,57) an intraepider­ follicle mal pilar epithelioma,56) and a trichomatricoma62 ) Methods for culturing epidermal keratinocytes have show positive reactions with all the MoAb; this been markedly developed and used for various pur­ reaction pattern is the same as that of the anagen poses. Moreover, some cell lines have been estab­ hair cortex. On the other hand, the tumor cells lished from trichilemmomas.49.50 ) On the other hand, of seborrheic keratosis,57) Bowen's disease and no established culturing methods for the cells of squamous cell carcinomas arising from Bowen's normal hair apparatus are known at present, disease indicate positivities for HKN-2 and HKN-4, although some investigators have tried to culture but negativities for HKN-5, HKN-6 and HKN-760 ; ORS cells50 and others have succeeded in culturing this is the same as the reaction pattern of normal the cells of the whole murine hair apparatus, includ­ epidermal keratinocytes. Furthermore, Merkel cell ing the hair matrix, medulla, cortex, IRS and ORS, carcinomas,40) Paget's disease38) and some kinds of though only for about 10 days.52) Since mesenchymal sweat gland tumors61 ) immunohistochemically cells in the hair dermal papilla have been able to be express a simple epithelia-type of keratin. They are separated from anagen hair roots and cultured,53) the not stained by HKN-5, HKN-6 or HKN-7, although collagen synthesis54 ) and hormone sensitivities55) of the so-called mixed tumors of the skin show a posi­ these cells have been investigated. It seems very tive reaction with HKN_5. 53 ) important for further advances in hair biology to In spite of these significant results, there remain establish adequate culturing methods for those cells some problems which are under further investigation. deriving from the hair and hair follicle. One can not immunohistochemically distinguish the cortex from IRS nor the epidermis from ORS except 4) Keratin expression of skin epithelial tumors for the IMC layer of the ORS in keratin expressions Anti-hair keratin MoAb as well as other kinds of using any anti-keratin MoAb. It is unknown whether anti-keratin MoAb have been utilized for immunohis- or not the proteins of the tumor cells detected by the The Morphology and Cell Biology of the Hair Apparatus 65 anti-hair keratin MoAb are biochemically the same sheath in anagen hair follicle: light and electron as those in the normal hair apparatus detected by the microscopic study. Arch Dermatol Res 279: 112-119, same MoAb. Moreover, the morphological and cyto­ 1986. logical characteristics of the tumor cells positively 8) Ito M: Electron microscopic study on cell differentiation in anagen hair follicles in mice. stained by the anti-hair keratin MoAb are still un­ I Invest Dermatol 90: 65-72, 1988. known. 9) Ito M: Biologic roles of the innermost cell layer of the outer root sheath in human anagen hair follicle: 5) Keratin expressions in the hair cycle further electron microscopic study. 281: 254-259, Keratin expressions of the anagen hair apparatus and 1989. morphological changes in hair tissues during hair 10) Epstein WL, Maibach HI: Cell proliferation and generation and hair cycle have been well studied as movement in human hair bulbs. In: Montagna W, described above. However, changes in the keratin Dobson RL (eds) Advances in biology of skin, vol. expressions of hair apparatuses during morpho­ IX, hair growth. Pergamon Press, Oxford, 1967, 83­ logical changes are unknown. This might possibly be 97. 11) Tezuka M: Cell kinetic study in generating hair examined using murine or rat hair apparatuses, tissues using anti-bromodeoxyuridine monoclonal because they show a synchronous hair cycle and antibody. lap I Dermatol (Tokyo) 100: 153-161, immunohistochemically react with the anti-hair ker­ 1990. (in Japanese with English abstracts) 62 atin MoAb. ) 12) Tezuka M, Ito M, Ito K, Sato Y: Cell kinetic study of human and mouse hair tissues using anti­ 6) Controls of hair growth and hair cycles bromodeoxyuridine monoclonal antibody. I Der­ Hair growth and hair cycles have been known to be matol Sci (in press) controlled or influenced by hormones,63) physical or 13) Deinlein E, Schell H, Fenner M, Katsuoka K: Cell psychological stress64 ) and seasonal changes65J as well kinetics of human anagen hair bulbs as determined 66 68 by DNA flow cytometry. 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