The ‘follicular trochanter’: an epithelial compartment of the human hair follicle bulge region in need of further characterization
British Journal of Dermatology
Volume 157 Issue 5 Page 1013-1016, November 2007
Recent articles on hair follicle stem cells have summarized the current state of knowledge of what has been termed the hair follicle ‘bulge’. During the course of immunohistological studies aimed at characterizing the expression of selected extracellular matrix proteins in the – as yet insufficiently characterized – niche of human bulge hair follicle stem cells, we have recently come across a largely forgotten, peculiar epithelial protrusion of the outer root sheath, which was visible in only a minority of all examined hair follicles. The morphology and immunoreactivity patterns of this structure, the ‘follicular trochanter’, are described herein.
The ‘bulge’ region of the outer root sheath (ORS) of both mouse and human hair follicles, i.e. the area where the arrector pili muscle (APM) is inserted into the distal ORS, is now firmly established as at least one major site of epithelial hair follicle stem cells (Fig. 1a).1 During the embryonal development of human scalp hair follicles (hHFs), two separated ORS protrusions (lower and upper) begin to grow at the posterior side of the follicle. At first, the lower one, the ‘bulge’, is the larger one of the two, but later on it becomes relatively smaller and is the future attachment site of the developing APM.2–8 Normally, the APM attaches to this central ORS compartment without any major ORS protrusion detectable. The upper protrusion develops into the sebaceous gland and its duct (in apocrine pilosebaceous units, an additional protrusion forms into the apocrine gland and its duct).
Even though this special ORS region had already been described in hHFs by 19th century authorities and is prominent during fetal hHF development, it is difficult to identify in adult hHFs. Therefore, the insertion of the APM is generally employed as a lead structure to point one to the bulge region, whose identity is then further supported by immunohistology, e.g. using cytokeratin 15 and CD200 immunostaining.8,9
However, among 144 examined normal scalp hHFs from 12 different individuals of both sexes above 40 years of age (mean 52·5 years), we noted a stringently localized ORS protuberance at the site of APM insertion in 8% of the randomly sectioned hHFs. We have provisionally termed this protuberance a ‘follicular trochanter’ (FT). The rate of occurrence possibly increases in serially sectioned hHFs, but we have been unable to find a sufficient number of properly sectioned telogen and catagen follicles in our scalp skin samples that would allow us to make reliable comments on the presence, absence or frequency of this FT. The protuberance occurred in 42% of all the different human scalp skin samples we examined, suggesting that the occurrence of this structure is not that rare and that it is more than a biologically irrelevant structural peculiarity displayed only by the odd individual.
As scalp skin samples arose from facelift surgery performed in older individuals, it is well advised to investigate whether there are age-dependent differences in the frequency with which an FT can be identified. Interestingly, very similar structures were first described more than a decade ago in vellus hair follicles of vertical sections of human facial skin, showing bilateral knob-like protrusions and villous projections at the level of the sebaceous gland.10 As this publication has been widely ignored in the hair research literature, we would hope that the current, extended re-examination of this phenomenon in terminal scalp hHFs will encourage further systematic characterization of the FT.10
This beak-, hook- or papilloma-like protrusion of the ORS (Fig. 1b–m) of terminal hHFs may reflect ‘snapshots’ of distinct developmental stages in a dynamic process (Fig. 1n). It will be interesting to clarify, in future studies, whether these different developmental stages are linked to distinct stages of hHF cycling. Based on morphology alone, the FT may form an integral part of the bulge of adult terminal hHFs, but may demarcate functionally distinct subdomains within this hHF compartment.
This ORS protrusion was seen in the area of the APM insertion of anagen VI hHFs. In longitudinal cryosections immunostained with various markers, this structure forms a prominent protuberance of the ORS into the connective tissue sheath (CTS) of the hHF and its surrounding mesenchyme. It was found exclusively in the area of the APM insertion belonging to the proximal isthmus region (Fig. 1c, h, j). Morphologically, this structure does not represent a disintegrated inner root sheath and is not caused by trichilemmal keratin which lines the upper isthmus, extending to the entry of the sebaceous duct at the base of the infundibulum.11
In honour of its ossary ‘look-alike’ in the femur, the term ‘follicular trochanter’ may be appropriate for this intriguing ORS protrusion at the point of APM insertion (Fig. 1c, h, j). The FT structure is surrounded by the basement membrane (BM) which separates the ORS from the CTS as shown by the staining of nidogen, which is a major component of the BM (Fig. 1f). The expression of α6 integrin is present throughout the whole length of the hHF and shows no changes at the basal pole of ORS cells adherent to the BM in the FT structure (Fig. 1b). The interior part of the FT demonstrates more densely clutched keratinocytes, as visualized by haematoxylin staining (blue) (Fig. 1c, e, i, j, l). The attachment of the APM was speculated by Narisawa et al.10 in 1994; however, we underline this hypothesis by immunostaining and immunofluorescence (Fig. 1c, h, j). Indeed, this suggests that APM fibres exploit this FT-like structure as a point of insertion and fixation to the hHF epithelium, but this will have to be confirmed ultrastructurally.
We found upregulated extracellular matrix (ECM) proteins such as tenascin-C, nidogen and fibrillin-2 in the proximal and distal CTS and basal ORS layer surrounding the FT structure. Tenascin-C shows a high expression throughout the postulated bulge region of the hHF (Fig. 1g, h), as fibrillin-2 does (Fig. 1c). However, tenascin-C immunoreactivity is most intense in the FT periphery, next to the CTS and the APM insertion. The current data, showing tenascin-C in the stroma, suggest that the cells (e.g. hHF stem cells) in the FT are secreting it to create a ‘niche’, as it is the case for murine follicles.12 Interestingly, also nidogen, involved in mechanical signal transduction mechanisms, which is discussed among the other investigated proteins as an important stem cell niche regulator, shows strong immunoreactivity inside the FT, where a dense epithelial cell population is embedded (Fig. 1f).13
CD200, used as a marker for stem cells or immature progenitor cells in the hHF epithelium, is prominently expressed in the proximal FT (Fig. 1i, j).9 The CD200 immunoreactivity is slightly stronger here than in the rest of the bulge (Fig. 1j). Another marker, whose expression is restricted to the outermost ORS and appears to identify epithelial hHF progenitor cells, including bulge epithelial stem cells, is cytokeratin 15.8,9,14 Strong cytokeratin 15 immunoreactivity is seen around and inside that part of the ORS which forms the FT. Interestingly, cytokeratin 15 immunoreactivity increases towards the point of the APM anchorage on the central ORS (Fig. 1k, l).
Taken together, the above markers may distinguish a distinct region, separate from the CTS, in which the FT is embedded in a specialized stroma related to the hHF stem cell niche.
Interestingly, Narisawa et al. described an apoptotic pocket-like structure in the ORS of the bulge area at the anagen stage of hHFs, which is clearly distinct from the FT characterized here and the previously described ‘knob-like protrusions and villous projections’.10,15 In fact, Ki-67/TUNEL staining reveals no apoptotic cells inside the FT (Fig. 1m). This demonstrates that this apoptotic and vacuolated structure tended to be present on the side where the major part of the APMs anchored, and showed no simultaneous appearance nor any structural similarity with the ORS protuberance presented here.
In addition, vertical sections of the human scalp skin also revealed the presence of ORS invaginations in the lower portion of terminal hHFs, unassociated with the APM.16 These invaginated structures also clearly differ from the outgrowing FT in localization and morphology.
In 1996, Narisawa and Kohda17 additionally described an ORS protuberance of the early anagen terminal hHF in the region of the new secondary hair germ, associated with a lateral protuberance of basaloid cells which might represent the bulge of the new anagen hHF. Interestingly, the APM bundle was divided into two branches, one inserted into the original, clubbed end and the other into this protuberance of the secondary hair germ. In the light of our current findings, these observations deserve careful and systematic follow-up.10
To clarify whether the FT originates in hHF ORS-derived cells or if the FT undergoes differentiation pathways (e.g. for sebocytes), we have stained the FT with the hHF-specific ORS cell marker cytokeratin 6 and additionally with cytokeratin 19, which is upregulated in differentiating sebocytes.18 Cytokeratin 6 shows a clear staining of cells originating in the ORS ingrowing towards the FT (Fig. 1d). The upregulation of the in vitro sebocyte differentiation marker cytokeratin 19 in our hands is unclear, because there is a prominent intra-FT staining (Fig. 1e). Nevertheless, we found no cytokeratin 19 immunoreactivity in or next to the sebaceous gland. For that reason we like to conclude that the upregulation of cytokeratin 19 in the FT structure demonstrates no sebocyte differentiation but more likely an epithelialization process.
The FT identified here bears some resemblance to Pinkus’‘Mantelhaare’, which show apron-like buds that can display basaloid cells, and whose frequency is greatly increased in patients with acquired hypertrichosis lanuginosa.19,20 Our understanding of ‘Mantelhaare’, which remain to be systematically investigated, is that they represent unusually large, ‘activated’ vellus hair follicles that are in the process of transformation into terminal or lanugo hairs. As we have exclusively investigated regular terminal scalp hHFs in anagen VI, it is highly unlikely that the FT described here is identical with the apron-like buds of Pinkus’ Mantelhaare. However, it deserves further investigation whether these apron-like buds of basaloid cells, which more than 100 years after their first description remain uncharacterized and enigmatic, are functionally related to the FT described here. Specifically, it deserves further scrutiny whether both structures reflect morphologically distinct protrusions of the ORS that are enriched in epithelial hHF stem cells.
Taking these factors together, it is intriguing to note that the APM has been speculated to contribute to the niche for the epithelial stem and/or progenitor cells in the bulge region.21 In this context, the expression pattern of putative ‘stem cell markers’ and ECM proteins that we identify in this compartment raises the question whether the FT serves as more than a point of muscle fibre insertion: does the FT reflect the morphological structure of a dynamically remodelled ORS niche for hHF epithelial stem cells?8,12,22
Irrespective of whatever function the FT will turn out to have, this ORS protrusion already can serve as a morphological leading structure for the bulge. It helps to localize the elusive bulge region of adult hHFs, thus facilitating, for example, microdissection approaches in the continuing quest for defining the biology of this crucial compartment of the hHF.