LH7:2, a new monoclonal antibody for use in the diagnosis of epidermolysis bullosa

Monoclonal antibodies to keratin filament proteins can be used as markers of differentiation within the skin. Two major cell groups can be detected in the interfollicular skin—namely basal and suprabasal compartments. Within the hair follicle small subpopulations of cells stain with simple epithelial antibodies; in particular, the inner hair root sheath and a small zone of upper hair follicle cells. Previous biochemical studies suggested that basal cell carcinomas derive from the hair follicle. We have studied a series of basal cell carcinomas ( n = 2o) using (1) SDS gel electrophoresis and (2) immunocytochemistry, using a large panel of monoclonal antibodies including peptide-specific monoclonal antibodies (LE61 to keratin 18; LE41 to keratin 8 and LP2K to keratin 19). Biochemically, tumour cells failed to express high molecular weight (MW) keratins but variable expression of low MW keratins. Immunocytochemistry showed (a) failure to express suprabasal differentiation (LHP₁, LHP₂, LHP₃); (b) invariable expression of interfollicular basal antigens; and (c) occasional expression of simple epithelial antibodies only. There was no evidence to suggest that basal cell carcinoma cells originate from hair follicle cells.     

Adrenomedullin: expression and possible role in human skin and hair growth

BACKGROUND: Adrenomedullin (AM) is a regulatory peptide that is synthesized and secreted by a wide number of cells and tissues. AM is a potent vasodilator, but also exerts other functions, such as regulating cell growth and antimicrobial defence. Two receptors, L1 and calcitonin receptor-like receptor (CRLR), which are able to bind AM, have been cloned and characterized. OBJECTIVES: To investigate expression of AM protein and its receptors in human skin and during different stages of the human hair cycle and, moreover, because of the suggested antimicrobial function of AM in skin, to investigate AM immunoreactivity (IR) in inflammatory acne lesions compared with healthy pilosebaceous follicles. METHODS: We used immunohistochemistry to determine the distribution of AM and its receptors in human skin and during different stages of the human hair cycle. AM IR in inflammatory acne lesions was investigated to evaluate the antimicrobial function of the protein, and hair follicle cultures were performed to examine the role of AM in differentiation and proliferation of hair follicle keratinocytes. RESULTS: Strong IR for AM and its receptors was present in the suprabasal epidermis, in the melanocytes of the epidermis, and in sweat and sebaceous glands. In the hair follicle, AM protein was strongly expressed in the basal and suprabasal layers of the hair bulb and the proximal outer root sheath (ORS). In the distal ORS, AM expression was increasingly suprabasal, especially in proximity to the bulge region where the basal cell layer was free of IR. IR for the CRLR revealed a similar expression pattern to that seen for AM. In contrast, L1 IR showed a suprabasal pattern of IR throughout the ORS. Similar expression of AM and its receptors was observed in catagen and early anagen follicles. AM expression was not markedly upregulated in acne lesions, suggesting a minor role for this antimicrobial peptide in acne. Despite its well-documented mitogenic effects, particularly in oral and skin keratinocytes, AM had no significant effect on hair follicle growth in vitro. CONCLUSIONS: AM and its receptors are expressed in human hair follicles, and both AM and its receptors are colocalized in the same compartments and cell types of the skin. This finding is consistent with the proposed autocrine/paracrine mechanism in the physiology of AM.     

The calcium-binding protein calretinin is a marker of the companion cell layer of the human hair follicle

BACKGROUND: Ultrastructural studies of the hair follicle show that the outer root sheath (ORS) does not consist of a homogeneous cell population. The innermost cell layer of the ORS, also called the companion layer, is a single cell layer closely associated with the Henle layer of the inner root sheath. OBJECTIVES: To describe the immunohistochemical expression of calretinin, a calcium-binding protein, in the human hair follicle. METHODS: Immunohistochemical studies using two different antisera to calretinin were performed in paraffin-embedded and in frozen scalp specimens using standard techniques. RESULTS: Calretinin immunostaining was consistently and specifically found in the companion cell layer of hair follicles. CONCLUSIONS: These findings provide further evidence to support the notion that the companion layer is not only morphologically, but also immunohistochemically, different from the other cells of the ORS.     

Exosomes derived from human dermal papilla cells promote hair growth in cultured human hair follicles and augment the hair‐inductive capacity of cultured dermal papilla spheres

Dermal papillae (DP) play key roles in hair growth and regeneration by regulating follicular cell activity. Owing to the established roles of exosomes (Exos) in the regulation of cell functions, we investigated whether DP‐derived Exos, especially those from three‐dimensional (3D)‐cultured DP cells, affect hair growth, cycling and regeneration. Exos derived from 3D DP (3D DP‐Exos) promoted the proliferation of DP cells and outer root sheath (ORS) cells and increased the expression of growth factors (IGF‐1, KGF and HGF) in DP cells. 3D DP‐Exo treatment also increased hair shaft elongation in cultured human hair follicles. In addition, local injections of 3D DP‐Exos induced anagen from telogen and also prolonged anagen in mice. Moreover, Exo treatment in human DP spheres augmented hair follicle neogenesis when the DP spheres were implanted with mouse epidermal cells. Similar results were obtained using Exos derived from 2D‐cultured DP cells (2D DP‐Exo). Collectively, our data strongly suggest that Exos derived from DP cells promote hair growth and hair regeneration by regulating the activity of follicular dermal and epidermal cells; accordingly, these findings have implications for the development of therapeutic strategies for hair loss.     

The neuroepithelial stem cell protein nestin is a marker of the companion cell layer of the adult and developing human hair follicle

Background  The interface between the inner root sheath (IRS) and the outer root sheath (ORS) represents a slippage plane for the hair shaft to evolve from the pilar canal to the skin surface. Interposed between the IRS and ORS is a single cell layer which is believed to represent the angle point of that slippage plane, termed the companion cell layer (CCL). The CCL is cited in most of the literature as part of the ORS.
Objectives  To describe the expression pattern of nestin, a neuroepithelial stem cell protein, in the adult and developing human hair follicle.
Methods  Immunohistochemical evaluation with a monoclonal antibody against nestin was performed using standard techniques.
Results  Nestin is selectively expressed in the CCL of the adult anagen and late stage fetal hair follicles. Early stages of hair follicle development are negative for nestin expression.
Conclusions  The selective demarcation of the CCL by nestin highlights the unique feature of this follicular cell layer and raises the question of whether the CCL should not be better conceptualized as a part of the IRS rather than the ORS. The results of the present study, together with published ultrastructural data, also suggest that the slippage plane for the evolving hair shaft may be located at the interface between the CCL and the ORS.     

Transforming growth factor-beta receptor II is preferentially expressed in the companion layer of the human anagen hair follicle

BACKGROUND: Transforming growth factor (TGF)-beta is a multifunctional growth factor with multiple roles in skin including hair follicle development and cycling, where it regulates cell proliferation, differentiation and apoptosis, as well as in wound healing. While TGF-beta receptor I (TGF-beta RI) and receptor II (TGF-beta RII) expression helps define early human hair follicle morphogenesis, expression in the adult human hair follicle remains to be established. OBJECTIVES: To assess TGF-beta receptor expression in human scalp anagen hair follicles. METHODS: Immunohistochemical and double immunofluorescence analysis of TGF-beta RI and RII was conducted on frozen sections of haired human scalp obtained from 10 healthy individuals. RESULTS: TGF-beta RI expression was detected in the outer root sheath of anagen hair follicles while TGF-beta RII was expressed almost exclusively in the companion layer of inner root sheath and less so in premedulla keratinocytes. Both receptors were colocalized in the companion layer of the proximal and mid follicle. CONCLUSIONS: The well-described role of TGF-beta in keratinocyte apoptosis during catagen is likely to involve anagen-specific hair follicle components including the companion layer, as this layer provides the slippage plane supporting the inner root sheath and hair shaft as they ascend to the skin surface. Results of this study suggest that the colocalization of TGF-beta RI/RII complexes at the companion layer would facilitate TGF-beta signalling at this site to regulate apoptosis of the companion layer keratinocytes, facilitating shrinkage/contraction of this cell layer during hair follicle regression/catagen.     

Human hair follicle pigmentary unit as a direct target for modulators of melanogenesis, as studied by [14C]-2-Thiouracil incorporation

Abstract:  The purpose of this study was to evaluate human hair follicle melanogenic activity using the [¹⁴C]-2-thiouracil, which was known to incorporate into nascent melanins. Results obtained on pigmented, grey and non-pigmented hair follicles demonstrated that [¹⁴C]-2-TU incorporation was restricted to the melanogenic compartment with a strong accumulation located around dermal papilla and within the fibre of pigmented hair follicles. Quantitative analysis of [¹⁴C]-2-TU incorporation showed a significant increase in pigmented hair follicles upon stimulation with 1  μ m forskolin concomitant to an increase in tyrosinase levels. A strong significant decrease in [¹⁴C]-2-TU incorporation was noted, when hair follicles were incubated with the tyrosinase competitive inhibitor kojic acid (200  μ m ). Incubation with the MC1-R agonist α-MSH (0.2  μ m ) did not induce a significant stimulation of hair melanogenesis. The present model could thus represent a useful new tool to identify modulators of human hair pigmentation.     

Tenascin is overexpressed in vitiligo lesional skin and inhibits melanocyte adhesion

The aetiology of vitiligo remains obscure. In this study, the role of integrins in the observed inability of melanocytes to repopulate lesional skin was investigated. Antibodies directed to α₂, α₃, α₅, α_v, α₆, β₁ and β₃ integrin subunits were used. Immunohistology revealed no marked differences in the overall levels of expression of integrins between control, non-lesional, perilesional or lesional skin. Moreover, no differences were noted in the level of expression of integrins or the adhesive capacity between cultured control cells derived from three separate donors and vitiligo-derived melanocytes from two donors. Rather, it was clearly observed that towards the lesion, vitiligo skin contains increasing amounts of tenascin in the basal membrane and papillary dermis in five patients employing T2H5 antihuman tenascin antibody. The anti-adhesive effect observed in vitro for this extracellular matrix molecule using normal melanocytes may contribute to loss of pigment cells in vitiligo or to ineffective repopulation of the lesions.     

Red hair, fair skin and melanoma – melanocortin 1 receptor

POMC processing in human melanocytes has been widely documented, and the α-MSH/MC1R/cAMP cascade has been implicated in the control of pigmentation. Only very recently, a role of β-endorphin, one cleavage product of β-LPH, has been demonstrated to influence melanocyte growth, dendricity and melanin biosynthesis via the µ-opiate receptor. However, much earlier, it was shown that β-MSH, the other cleavage product of β-LPH, controls melanogenesis and melanin transfer in amphibians. To date, a specific receptor for β-MSH has not been identified. Earlier POMC processing has been found in melanosomes. Therefore, an MC1R-independent role of α-MSH was postulated and demonstrated in control of 6-tetrahydrobiopterin (6BH₄)-inhibited tyrosinase. Utilizing the depigmentation disorder vitiligo, we were now able to follow the fate of epidermal POMC processing in the presence of mM levels of hydrogen peroxide (H₂O₂). In vitiligo epidermal PC2 and 7B2 protein expression is increased, whereas α-MSH, β-MSH and β-endorphin are significantly decreased. Analysis of the peptide sequences revealed in all three cases H₂O₂ oxidation targets such as methionine and tryptophan yielding significant structural alterations. Moreover, we have identified a new function of β-MSH due to its capacity to bind the important cofactor 6BH₄ as well as its isomer 7BH₄. Hence, we propose for the first time that β-MSH can control both the supply of l -tyrosine from l -phenylalanine via phenylalanine hydroxylase and l -Dopa synthesis via tyrosinase hydroxylase in melanocytes and keratinocytes. Therefore, both melanogenesis and catecholamine synthesis could be regulated by this peptide.     

Prostaglandin metabolism in human hair follicle

Prostaglandins regulate a wide number of physiological functions. Recently PGF(2alpha) analogue such as latanoprost was shown to have a real impact on hair regrowth. The aim of this study was to investigate and describe the expression profile in human hair follicle of prostaglandin metabolism key enzymes, i.e. carbonyl reductase-1 (CBR1), microsomal prostaglandin E synthase-1 (mPGES-1) and microsomal prostaglandin E synthase-2 (mPGES-2), cytosolic prostaglandin E synthase (cPGES), the aldoketoreductase AKR1C1 and the prostaglandin F synthase AKR1C3. Quantitative RT-PCR on plucked hair follicles revealed some sex-related differences, mPGES-2 and AKR1C3 expression levels being higher in women. Cell and hair follicle compartment specificity was investigated using Western blot, PGE(2) and PGF(2alpha) ELISA assays and immunohistochemistry. Most of the hair cell types were endowed with prostaglandin metabolism machinery and were thus able to produce PGE(2) and/or PGF(2alpha). The epithelial part of the hair bulb was identified by immunohistology and EIA assays as the main source of prostaglandin synthesis and interconversion. All these observations support the concept that prostaglandins might be involved in hair growth and differentiation control.     

Gap junction development in the human fetal hair follicle and bulge region

BACKGROUND: Gap junctions, composed of connexin (Cx) subunits, are channels that allow intercellular communication between adjacent cells and are thought to play a key role in the regulation of cell proliferation and differentiation. The Cx expression pattern and formation of gap junctions in human fetal hair follicles has yet to be clarified, including the prominent follicular bulge region that is believed to be a site rich in stem cells. OBJECTIVES: To study the expression of two major Cxs, Cx26 and Cx43, in developing hair follicles in skin samples from a series of human fetuses of estimated gestational age (EGA) 88-163 days, and to determine quantitatively the presence of gap junctions. METHODS: We used immunofluorescence labelling to investigate the sequential expression pattern of Cx26 and Cx43 in developing human hair follicles. Gap junction formation was observed by electron microscopy and the numbers of gap junctions were analysed quantitatively. Results Both Cx26 and Cx43 expression were observed at 88 days' EGA in the inner part of the hair peg. At 135 days' EGA, Cx26 was expressed in the outer root sheath (ORS) and the inner root sheath (IRS), while Cx43 was expressed chiefly in the IRS, hair matrix and sebaceous glands. At 163 days' EGA, Cx26 expression was most intense in the outermost layer of the ORS, in contrast to Cx43 expression which was in the inner part of the ORS. In the bulge region, only Cx43 was expressed in a subset of cells in the bulge. Ultrastructurally, gap junctions were observed at 102 days' EGA in the hair peg, and the number of gap junctions increased as the hair follicle matured. Gap junctions were also observed between the bulge cells in considerable numbers. CONCLUSIONS: The changing expression patterns of Cx26 and Cx43 and the increasing gap junction numbers suggest a close association of Cx expression and gap junction formation with hair follicle morphogenesis. In addition, the present ultrastructural observations demonstrate that considerable numbers of the bulge cells, a putative site rich in hair follicle stem cells, form gap junctions during human hair follicle development.     

Syndecan-1 is strongly expressed in the anagen hair follicle outer root sheath and in the dermal papilla but expression diminishes with involution of the hair follicle

Syndecan-1 is the prototypic member of a family of heparan sulfate-bearing cell surface proteoglycans that function in adhesion, cell-extracellular matrix interactions, migration, and proliferation. During embryogenesis, syndecan-1 expression in the epithelium is downregulated when the epithelium gives rise to motile mesenchymal cells, whereas mesenchymal syndecan-1 expression is upregulated during organ formation. In aggressive basal cell carcinomas, syndecan-1 expression is evident in the stroma. Some neoplastic cells induce stroma to meet needs for growth, and it may be the mesenchymal cells that produce and shed syndecan-1 into the stroma. The physiologic mechanism by which the hair follicle undergoes its cyclic process of involution and formation of a new active hair follicle is not well understood. Sixty scalp biopsies and a large scalp resection were evaluated for syndecan-1 expression within hair follicles in the growing (anagen), involuting (catagen), and resting (telogen) phases. Strong syndecan-1 immunoreactivity was evident in the outer root sheath (ORS) of the anagen hair follicle, but this expression diminished in intensity with the involution and resting stages in the hair follicle cycle. The diminution of syndecan-1 immunoreactivity in the ORS of involuting and resting hair follicles may be a result of terminal keratinocyte differentiation. Syndecan-1 was also present in the dermal papilla of the anagen hair follicle, where it may promote growth factor-mediated cell signaling that induces and maintains growth of the hair shaft and the inner root sheath.     

Effects of 1α,25-dihydroxy-vitamin D3 and calcipotriol on organotypic cultures of outer root sheath cells: a potential model to evaluate antipsoriatic drugs

In the human hair follicle, outer root sheath (ORS) cells constitutively express the hyperproliferation-associated keratins 6, 16 and 17 instead of keratins 1 and 10 found in interfollicular epidermis. In organotypic cultures, ORS cells form a stratified epithelium which in many respects resembles psoriatic skin: it has a hyperplastic tissue architecture and a poorly developed granular layer, and expresses hyperproliferation-associated keratins. Therefore, we studied the effects of the antipsoriatic compounds 1,25-dihydroxy-vitamin D₃ (1,25-(OH)₂-D₃) and its synthetic derivative calcipotriol on cultured ORS cells. In monolayer cultures, 10^–6 M 1,25-(OH)₂-D₃ or calcipotriol completely blocked ORS cell proliferation. This inhibitory effect was substantially reduced at 10^–8 M. Incubation of organotypic ORS cultures with both vitamin D analogues resulted in a marked thinning of the living cell compartment concomitant with a thickening of the horny layer. A reduced expression of differentiation markers such as keratins 10,16 and 17, involucrin and filaggrin paralleled the thinning of the stratum Malpighi. As determined by quantification of BrdU-positive cells, ORS cell proliferation was apparently not affected by the vitamin D analogues, indicating that these compounds mainly operate by accelerating the differentiation pathway within the suprabasal living cell compartment. No alteration in the expression of the 6- and 1-integrin chains was found.     

Sox9 Increases the Proliferation and Colony-forming Activity of Outer Root Sheath Cells Cultured In Vitro

Background

β-catenin plays a pivotal role in hair follicle development and hair growth cycle.

Objective

The aim of this study was to identify β-catenin-regulated genes in cultured human hair outer root sheath (ORS) cells.

Methods

Primary cultured ORS cells were transduced with recombinant adenovirus expressing N-terminal truncated β-catenin (constitutive active form), and β-catenin-regulated genes were identified.

Results

Overexpression of the constitutively active form of β-catenin led to induction of Sox9 expression at both mRNA and protein levels. To investigate the potential role of Sox9, we made the recombinant adenovirus expressing green fluorescent protein-tagged Sox9, and then transduced into cultured ORS cells. Interestingly, Sox9 induced the expression of keratin 15, increased the proliferation of ORS cells in vitro, and enhanced colony-forming activity.

Conclusion

Our results suggest that Sox9 is a β-catenin-regulated gene in ORS cells, and has potential importance in the regulation of hair follicle homeostasis.     

Protein kinase C inhibits human hair follicle growth and hair fibre production in organ culture

Summary In this study we have used a human hair follicle whole-organ culture system to examine the effects of 12-O-tetradecanoyl-phorbol-l3-acetatc (TPA), a potent activator of protein kinase C (PKC), on hair follicle growth and hair fibre production. Anagen hair follicles were isolated from human facial skin by microdissection and placed in suspension culture in supplemented Williams E medium. Hair follicle and hair fibre lengths were measured daily using an inverted microscope and cumulative growth values were calculated. Treatment with TPA resulted in a potent, dose-dependent inhibition of total cumulative hair follicle growth (lC₅₀=1 nm). Hair follicles grew at a comparable rate for 4 days in the presence or absence of 10 n m TPA. after which growth of TPA-treated follicles ceased while control follicles grew by a further 0–8 mm over the subsequent 6 days. In contrast. 10 n m TPA treatment did not affect hair fibre elongation for a period of 8 days, after which TPA-treated fibre production ceased while control fibres grew by a further 0–79 mm over the subsequent 7 days. Incubation of hair follicles with TPA resulted in a 41% inhibition of hair fibre protein synthesis, as measured biochemically from the incorporation of ³H-leucine using a differential akali extraction method. The inhibitory effect of TPA on follicle growth was partially prevented by preincubation with the selective PKC inhibitor H-7, and almost completely prevented by preincubation with the more potent PKC inhibitor Ro 31-7549. Neither agent alone significantly affected follicle growth at concentrations that reversed the TPA response. These findings indicate that PKC is a negative regulator of hair follicle growth, and suggest that PKC may play a part in the transduction of follicular growth-inhibitory signals.