The innermost cell layer of the outer root sheath is positive with Ki-67

The expression of a cell proliferation-associated human nuclear antigen was immunohistochemically studied in human anagen hair and hair follicles using the monoclonal antibody Ki-67. The reaction of Ki-67 in mature anagen hair follicles was observed in the hair matrix cells and outer root sheath (ORS) cells. Nuclear staining was seen in a small number of matrix cells and in some ORS cells; this finding corresponded to the thymidine or bromodeoxyuridine labeling studies previously reported. In addition, there were two different patterns of cytoplasmic staining in the ORS: strong staining of the innermost cells (IMC) and weaker staining of the other ORS cells in the isthmus. Ki-67 reactivity of the IMC layer was observed at each stage of anagen and was regularly seen from the upper bulb to the isthmus. Ki-67 is a commercially available antibody that detects cycling cells. However, the IMCs in anagen hair follicles showed cytoplasmic labeling by Ki-67 from the matrix cells in the upper bulb to the distal portion of the isthmus.     

The immunohistochemical expression of CD34 in human hair follicles: a comparative study with the bulge marker CK15

BACKGROUND: Anti-CD34 antibodies label the bulge region of mouse hair follicles. However, in human hair follicles, CD34 immunoreactivity is found in the outer root sheath below the bulge zone. The immunohistochemical staining of CD34 in catagen and telogen follicles has not been evaluated. AIMS: To characterize the expression of CD34 immunoreactivity at different stages of the hair cycle in human terminal hair follicles, and to compare the immunostaining pattern of CD34 with that of CK15, used here as a marker of the bulge region. METHOD: Serial vertical sections of human hair follicles in anagen, catagen and telogen phases were immunostained with anti-CD34 (QBEnd 10) and anti-CK15 (LHK15 and C8/144B) antibodies. Double-labelling immunofluorescence was also performed. RESULTS: The catagen and telogen follicles studied did not show CD34 immunoreactivity in the outer root sheath. The location of CD34 and CK15 immunoreactivity in anagen follicles reveals a different staining pattern: CD34-positive cells are located in the outer root sheath below the attachment zone of the arrector pili muscle, whereas CK15-positive cells are located in the outer root sheath above the attachment zone of the arrector pili muscle. CONCLUSIONS: Only anagen human hair follicles show CD34 immunoreactivity. CD34 and CK15 recognize different types of cells or cells at different stages of differentiation.     

Expression of basement membrane proteins and interstitial collagens in dermal papillae of human hair follicles

The expression of basement membrane molecules and interstitial collagens in human hair follicle mesenchyme was studied by immunohistochemical staining of tissue sections and of cells cultured from dermal papillae. Type I and type III collagens were found in the dermal sheath and in the dermal papilla throughout the hair cycle. Laminin and type IV collagen were expressed at the outer root sheath basement membrane and in the extracellular matrix of the dermal papilla of anagen and catagen follicles. In telogen follicles, where the volume of the dermal papilla extracellular matrix is much reduced, outline staining of dermal papilla cells for laminin and type IV collagen was still apparent. Staining for bullous pemphigoid antigen was also seen at the outer root sheath basement membrane extending to the lower tip of the hair bulb. In anagen follicles, there was no staining for bullous pemphigoid antigen at the interface between hair bulb epithelium and the dermal papilla and no staining within the dermal papilla. However, linear staining for bullous pemphigoid antigen became continuous around hair follicle epithelium during catagen and telogen. Cells cultured from human dermal papillae also stained for interstitial collagens, type IV collagen and laminin. However, similar results were obtained when cultured dermal fibroblasts were stained with the same antibodies. The expression of basement membrane proteins in human dermal papillae resembles that seen in follicles from other mammalian species and suggests that this is relevant to dermal papilla function. Cultured dermal papilla cells express a similar pattern of interstitial collagens and basement membrane proteins to those seen in tissue sections but this finding is not specific to dermal papilla cells.     

Immunohistochemical study of angiotensin receptors in human anagen hair follicles and basal cell carcinoma

BACKGROUND: Angiotensin receptors are the specific receptors of angiotensin II of the renin-angiotensin system. However, expression of the receptors in hair follicles has not been determined. OBJECTS: To clarify the expression and localization of angiotensin receptors in human anagen hair follicles and basal cell carcinomas. METHODS: We studied immunohistochemically the expression of angiotensin type 1(AT1) and type 2 (AT2) receptors in human anagen hair follicles and in 16 cases of basal cell carcinoma (BCC) (nine of solid BCC of the circumscribed type, two of adenoid BCC, five of BCC with follicular differentiation). RESULTS: Our experiments demonstrated the localization of AT1 in the inner root sheath and the inner layers of the outer root sheath. In BCC, positive staining with AT1 was revealed in the tumour cells of basal cell carcinoma with follicular differentiation. CONCLUSIONS: AT1 may have a role in association with follicular keratinization. Studying AT1 distribution may be useful in understanding the pathophysiology of human hair follicles and the hair follicle-associated tumours.     

Accordion-like Structure of Terminal Hair Follicles of the Human Scalp

Terminal hair follicles extracted after ethylenediaminetetraacetic acid treatment preserved the basal cell surface of the outer root sheath well enough to examine the outer surface in detail. Light microscopic observations of whole mount specimens revealed several sawtooth-like structures of follicular epithelium in the lower portion of terminal hair follicle. Vertical sections of the human scalp also showed several invaginations of the outer root sheath of terminal hair follicles in the lower portion, not in association with the arrector pili muscle. Scanning electron microscopy of the same specimens demonstrated accordion-like structures below the sebaceous gland in the lower half of anagen terminal hair follicles and surrounding the entire circumference of the follicles. Sawtooth-like structures were unilaterally observed at the level of the middle or lower portion below the sebaceous gland. With transmission electron microscopy, these structures were seen as undulations of basal cells which preserved hemidesmosomes, although the basal lamina had been peeled off during the extraction procedure. Thus, this accordion-like structure is a genuine structural variation of some human anagen hair follicles and not an artifact.     

Immunohistochemical localization of cytokeratins and involucrin in calcifying epithelioma: comparative studies with normal skin

The expression of cytokeratins and involucrin varies greatly in different epithelia, and this raises the possibility that detailed analysis of these epidermal proteins might provide a means of identifying various skin tumours. The present study was conducted to determine the immunohistochemical distribution of cytokeratins and involucrin in calcifying epithelioma of Malherbe, in order to elucidate the nature and differentiation of this tumour. To correlate the immunohistochemical profile with the most frequest histological patterns, we categorized the basophilic, transitional, shadow, and squamoid cells, and the shreds of keratin. Comparative studies with normal skin showed that the shadow and transitional cells corresponded to hair cortex cells, the squamoid cells to the outer root sheath, the basophilic cells adjacent to the stroma to the outermost cell layer of the outer root sheath between the lower permanent portion and upper transient portion of the follicles, and the basophilic cells adjacent to the transitional cells to the hair matrix. The expression of cytokeratins in most shreds of keratin was similar to that in squamoid cells. Calcifying epithelioma was, therefore, shown to be composed of tumour cells differentiating into both the hair cortex and outer root sheath. These tumour cells were differentiated from basophilic cells, which showed the same staining patterns as the outermost cell layer of the outer root sheath between the lower permanent portion and upper transient portion of the hair follicles, supporting the hypothesis that the keratinocytes in the outermost cell layer can differentiate into the transitional portion of the follicle and anagen hair.     

CD10 and CD34 in fetal and adult human hair follicles: dynamic changes in their immunohistochemical expression during embryogenesis and hair cycling

Background  CD10 and CD34 have been detected in both epithelial and mesenchymal components of anagen human hair follicles.
Objectives  To analyse the expression of CD10 and CD34 in human hair follicle development as well as in different phases of the hair cycle.
Methods  Fetal and adult hair follicles at different stages of the hair cycle were examined by immunohistochemistry for CD10 and CD34.
Results  In fetal follicles, CD10 is expressed by the cells of the placodes, and CD34 by the mesenchymal cells of the dermal condensate. As the follicle matures, CD10 can be seen in the matrix cells, inner root sheath and dermal sheath. In adult follicles, the expression of CD10 in the follicular epithelium is present in anagen follicles, but tends to disappear in catagen, and is not detected in telogen. The CD10 positivity of the dermal sheath is more intense in catagen than in anagen follicles. CD34 immunostaining of the external root sheath was seen in adult anagen follicles but not in fetal follicles. This staining of the anagen outer sheath tends to disappear in catagen and is not detected in telogen.
Conclusions  CD10 and CD34 are not proteins constantly present in a specific cell type of the hair follicle, but are proteins that can be expressed by both epithelial and mesenchymal cells depending on the stage of development and hair cycle. The distribution of the immunoreactivity to CD10 in the placode and CD34 in the dermal condensate suggests a role of these proteins in initial stages of hair formation.     

The innermost cells of the outer root sheath in human anagen hair follicles undergo specialized keratinization mediated by apoptosis

The innermost cell layer of the outer root sheath (IORS) is a special single cell layer located just outside Henle's layer. In situ endlabeling immunohistochemistry for apoptosis showed that labeled cells were most consistently located in the IORS from the suprabulbar portion to the infundibulum of anagen terminal hair follicles of the human scalp. Labeled cells were also sparsely scattered in the middle portion, including the bulge area of the outer root sheath of anagen hair, the regressing lower portion of catagen hair and the bulb of telogen hair. Ultrastructurally, the cells of the innermost layer underwent cellular degeneration through cytoplasmic vacuolization and nuclear pyknosis without keratohyalin production. These were compatible with the morphology of apoptotic cells. These findings confirmed that the innermost cell layer is different from other layers of the outer root sheath, not only by previously demonstrated criteria such as Ki67 immunostainability and characteristic ultrastructure but also by the mode of cell death.     

Cell kinetic study of human and mouse hair tissues using anti-bromodeoxyuridine monoclonal antibody

To determine sites of cell proliferation in hair tissues, in vitro and in vivo labeling with bromodeoxyuridine (BrdU) and immunohistochemical demonstration of BrdU incorporation sites by anti-BrdU monoclonal antibody were performed on human and mouse hairs and hair follicles. The germinative area of the hair bulb of human anagen hair was divided into three portions: (A) the upper and inner portion, (B) the middle portion and (C) the lowest outer portion. A-cells intermingled with melanocytes, were regarded as germinative cells of the hair cortex. B-cells appeared to develop into Huxley's layer, cuticle of inner root sheath (IRS), and hair cuticle. C-cells seemed to develop into bulbar outer root sheath (ORS), the innermost cell (IMC) layer of the ORS and Henle's layer. The suprabulbar portion, where the ORS abruptly increased in thickness, was found to be the fourth main germinative portion (D). The ORS cells, except for the IMCs, seemed to originate mostly from the D-cells. In the late anagen phase, first, C-cells became BrdU negative, then, A- and B-cells gradually turned negative, and finally, D-cells lost their germinative activity. In catagen and telogen hair tissues, BrdU-positive cells were found in the two outer cell layers in the ORS. The structure of anagen hair tissues seems to be maintained by the coordinated mitotic activities of characteristically distributed germinative cells of various hair cell layers. The sequential cessation of mitotic activity of these cells is associated with the morphological changes from anagen through catagen to telogen. These findings were common to both human and mouse hair tissues.     

Expression of HLA-DR by anagen hair follicles in alopecia areata

The expression of HLA-DR within hair follicles in alopecia areata was studied using an immunoperoxidase method. Scalp biopsies were taken from 12 patients with alopecia areata and from 6 normal control subjects. Frozen sections were stained with a panel of 4 anti-HLA-DR monoclonal antibodies, Leu 2, Leu 3, Leu 4, and T6 antibodies. The expression of DR in normal hair follicles and in most anagen follicles from nonlesional alopecia skin was confined to dendritic cells which were sparse below the level of the arrector pilorum insertion. Of the 37 anagen follicles examined in lesional skin, 25 displayed staining for DR on epithelial cells in the precortical matrix and presumptive cortex. Six follicles showed DR staining in other epithelial compartments, the lower bulb matrix, inner root sheath, and outer root sheath. Infiltration of the hair bulb matrix by T cells was seen in the majority of follicles where epithelial cells were DR+. The aberrant expression of DR antigens by hair follicle epithelium provides direct evidence that immune mechanisms are operating in the pathogenesis of alopecia areata. In a previous study of alopecia areata we found evidence of cell injury confined to the precortical matrix and presumptive cortex in lesional anagen follicles. The relative restriction of epithelial DR expression to the same site suggests that this region of the follicle is of fundamental importance in the disease process.     

Ber-EP4 antigen is a marker for a cell population related to the secondary hair germ

Ber-EP4 is an antibody to a cell membrane glycoprotein of unknown function. In the skin, Ber-EP4 immunoreactivity has been reported to be localized in structures composed of basaloid epithelial cells, i.e. fetal epithelial germ cells, basal cell carcinoma, and trichoepithelioma as well as eccrine or apocrine ducts. In this study, we further characterized the follicular expression of Ber-EP4 immunoreactivity at different stages of the hair cycle of human terminal hair follicles. In addition, to clarify the location of Ber-EP4(+) cells, we compared the Ber-EP4 immunoreactivity with the expression of keratin 15 and keratin 19. Positive staining by Ber-EP4 was found in the lower part of the epithelial strand of late catagen hair follicles, in the secondary hair germ of telogen hair follicles, and in the matrix of early anagen hair follicles but not in any parts of mature anagen hair follicles. In contrast, the follicular expression of keratin 15 detected by using LHK15 antibody was restricted to two distinct parts of anagen hair follicles, i.e. the outer root sheath above the hair bulb and that of the isthmus including the bulge area, and to the outer root sheath of late catagen and telogen hair follicles. The follicular expressions of keratin 19 and that of keratin 15 were apparently superimposed, whereas keratin 15 expression was more extended. The immunoreactivity of LHK15 antibody and antikeratin 19 antibody against the secondary hair germ of telogen follicles was negative or dim. Our results suggest that Ber-EP4 reacts with the secondary hair germ and possibly a cell population related to the secondary hair germ but not with the presumptive stem cell population as revealed immunohistochemically either by the keratin 15 or keratin 19 expression.     

Preliminary findings on lectin binding in human follicular epithelium

Glycoconjugates in the cell membranes of the outer and the inner root sheath of human anagen hair follicles were histochemically characterized by means of the following lectins: UEA-I, DBA, PNA, WGA, SBA, RCA, and Con A (avidin-biotin technique, counter-staining with methyl green). We observed intense labeling of the outer root sheath with UEA-I, DBA, Con A, and PNA, whereas the other markers were only weakly positive. VEA-I, DBA and SBA labeling was confiried to the upper two thirds of the outer root sheath. As to the inner root sheath, we found intense labeling with UEA-I, WGA, RCA, and SBA; all other lectins were only weakly represented. Our results suggest that the cell membranes of the outer and inner root sheaths of human hair follicles are characterized by the prevalence of different glycoconjugates; moreover, labeling with UEA-I, SBA, and DBA is obviously related to the process of differentiation within the follicle.     

Heparanase 1: a key participant of inner root sheath differentiation program and hair follicle homeostasis

Heparanase is a heparan sulphate endo-glycosidase which was previously detected in the outer root sheath of murine hair follicles. Heparanase overexpression was reported to improve mouse hair (re)growth. In this study, we investigated its involvement in human hair biology. Immunofluorescence detection was used to explore heparanase distribution in both anagen and catagen hair follicles. Heparanase functionality was assessed in in vitro cultured hair follicles, in the presence of a heparanase activity inhibitor. Our results showed that heparanase expression was (i) primarily located in the inner root sheath (IRS) of human hair follicle, and there (ii) restricted to anagen phase. Furthermore, inhibition of heparanase in in vitro cultured hair follicles induced a catagen-like process. Hair shaft retreat upward was accompanied by a decrease in Ki67-positive cells, the formation of an epithelial strand as evidenced by K14 keratin expression, and the loss of IRS as assessed by transglutaminase 1 and desmoglein labelling. IRS distribution of heparanase and the induction of catagen-like involution of hair follicles when a potent heparanase inhibitor is added suggest that heparanase is a key actor of IRS differentiation and hair homeostasis.     

Abnormal expression of Ki-67 antigen in hair follicle of alopecia areata.

The monoclonal antibody Ki-67 was used to determine the numbers of cycling cells in hair follicles both in alopecia areata and in normal scalp skin. Pronounced nuclear staining was limited to the area below the critical line of Auber and the exterior part of the outer root sheath. In alopecia areata there is reduced nuclear Ki-67 binding in the bulb of anagen hair follicles. These findings indicate that inhibition of keratinocyte proliferation might be a pathogenetic mechanism in alopecia areata.     

Differential expression of the 4F2 activation antigen on human follicular epithelium in hair cycle

The expression of the 4F2 activation molecule has been studied on keratinocytes of human skin and hair follicle using immunoperoxidase staining with three different anti-4F2 monoclonal antibodies. Membranes of basal layer keratinocytes of the skin uniformly expressed this antigen, whereas a differential expression of this antigen was located in specific areas of the hair follicle. Follicles in the complete anagen phase displayed a strong 4F2 positive staining at the matrix and the outer root sheath cells. This positive staining gradually decreased along the proliferation zone, and became negative at the migration zone. Positivity was recovered in follicular cells at the duct of the sebaceous gland and was maintained in the upper outer root sheath where those cells fuse with the keratinocyte basal monolayer. Changes were also detected on different phases of the hair cycle. Follicles in the catagen-telogen phase expressed a very low number of positive cells in the matrix. Positive labeling progressively increased when the follicle was at the initial anagen stage, reaching a complete staining pattern in hair at the anagen phase. These results suggest that the expression of this activation antigen on hair keratinocytes may be related to the proliferation, active metabolism, and/or activation states of these cell types.     

Scanning electron microscopic observations of extracted terminal hair follicles of the adult human scalp and eyebrow with special references to the bulge area

Scanning electron microscopic studies of human terminal hair follicles of the scalp and eyebrow have previously been limited to the hair shaft. In this study we investigated EDTA-treated extracted whole hair follicles in which most of the basal cell surface of the outer root sheath was well preserved. In the bulge area of scalp follicles there were many knob-like or villous projections. These were located in some specimens on one side of the follicle, while in others they were located around the entire circumference of the follicle. These projections were thought to represent the anchoring points of the branched follicular end of the arrector muscles. Ring-like elevations with groove-like depressions above and below were also observed surrounding the entire follicle. These were thought to represent the track of circumfollicular arrector muscles which depressed the outer root sheath when they repeatedly contracted. Most anagen eyebrow follicles showed morphological variations in the bulge area such as lattice-window-like structures and undulation patterns. In telogen follicles, the bulge became indistinguishable from the clubbed end. The lower end of these telogen follicles showed irregularly shaped bulge areas, but did not show lattice-window-like structures or undulation patterns as observed in anagen follicles. Interestingly, a hole was found in some bulge areas of both anagen and telogen follicles. Serial vertical sections of follicles revealed invaginated areas, which seemed to correspond to the openings seen in whole mounts. In vertical sections of eyebrow follicles some keratinocytes of the outer root sheaths of the bulge area were seen to be melanized to various extents. This phenomen was independent of hair cycle phase.     

Cell differentiation in human anagen hair and hair follicles studied with anti-hair keratin monoclonal antibodies

By a hybridoma technique using BALB/c mice and Sp2/0-Ag14 mouse myeloma cells, monoclonal antibodies against hair fibrous proteins (HFP) were produced. Two monoclonal antibodies, designated as HKN-5 and HKN-7, were chosen. Either HFP or epidermal fibrous proteins (EFP) were electrophoretically separated on polyacrylamide gels with sodium dodecyl sulfate. By immunoblot analyses, HKN-5 and HKN-7 decorated the electrophoretic bands of HFP but not those of EFP. Immunohistochemically, these monoclonal antibodies stained the medulla, cortex, cuticle, and inner root sheath in the keratogenous zone of anagen hairs, but not hair matrix cells. HKN-5 further reacted with the innermost cells (IMC) of the outer root sheath; these cells formed a single cell layer located outside of the Henle's layer. HKN-7 did not react with the outer root sheath including the IMC. Neither monoclonal antibody reacted with any other skin components or any tissues of other organs examined. Ultrastructurally, the IMC of the outer root sheath showed a unique cell differentiation forming an independent cell layer. It is suggested that the cells in the medulla, cortex, cuticle, and inner root sheath of anagen hair and hair follicles possess a similar keratin expression and that the IMC of the outer root sheath display a unique keratin expression and their own cell differentiation, resulting in 2 types of keratinization of the outer root sheath; keratinization of IMC and trichilemmal keratinization.     

Expression and location of phospho-Artemis (Serine516) in hair follicles during induced growth of mouse hair

Artemis has been implicated in having a role in NHEJ, and it is also a multifunctional protein. Previous studies have found Omenn syndrome-like phenotype due to Artemis mutations and associated with alopecia. As Artemis phosphorylation in its c-terminus including Serine516 is prerequisite for the Artemis endonuclease reaction, we postulate that Artemis (Serine516) may be expressed in hair follicle and relate to hair cycling. In this study, hair growth in C57BL/6 mice was induced by plucking the telogen hair on the back. Expression of Artemis (Serine516) in hair follicles during the hair growth cycle was evaluated by immunofluorescence using cryosections and a specific polyclonal anti-Artemis (Serine516) immunoglobulin G (IgG) antibody. It was detected in germ cells, cap, and club hair adjoining the epidermis in telogen. In anagen II, intense staining for Artemis (Serine516) was found in the whole interfollicular epidermis, and in strand keratinocytes. In anagen IV, intense staining for Artemis (Serine516) was detected in basal cells and upper of outer root sheath (ORS) and inner root sheath (IRS). But only upper ORS and lower medulla were stained positive in anagen VI. Upper ORS and lower cortex were positively stained with Artemis (Serine516) in catagen. Based on the phenomenon that the expression of Artemis (Serine516) in mid-anagen and mature anagen was stronger than that in telogen and catagen, we suggest it may take roles in induced growth of mouse hair.