Normal Human Scalp Research Articles

Human Female Hair Follicles Are a Direct, Nonclassical Target for Thyroid-Stimulating Hormone

Pituitary thyroid-stimulating hormone (TSH) regulates thyroid hormone synthesis via receptors (TSH-R) expressed on thyroid epithelial cells. As the hair follicle (HF) is uniquely hormone-sensitive and, hypothyroidism with its associated, increased TSH serum levels clinically can lead to hair loss, we asked whether human HFs are a direct target for TSH. Here, we report that normal human scalp skin and microdissected human HFs express TSH-R mRNA. TSH-R-like immunoreactivity is limited to the mesenchymal skin compartments in situ. TSH may alter HF mesenchymal functions, as it upregulates alpha-smooth muscle actin expression in HF fibroblasts. TSH-R stimulation by its natural ligand in organ culture changes the expression of several genes of human scalp HFs (for example keratin K5), upregulates the transcription of classical TSH target genes and enhances cAMP production. Although the functional role of TSH in human HF biology awaits further dissection, these findings document that intracutaneous TSH-Rs are fully functional in situ and that HFs of female individuals are direct targets for nonclassical, extrathyroidal TSH bioregulation. This suggests that organ-cultured scalp HFs provide an instructive and physiologically relevant human model for exploring nonclassical functions of TSH, in and beyond the skin.

Human hair follicle epithelium has an antimicrobial defence system that includes the inducible antimicrobial peptide psoriasin (S100A7) and RNase 7

Hair follicle (HF) ostia represent a potential port of microbial entry into the skin. However, they rarely show clinical signs of infection. This suggests the presence of local, efficient, antimicrobial defence systems, which may include antimicrobial peptides (AMPs). We determined the presence and distribution of the major AMPs, RNase 7 and psoriasin (S100A7), in human scalp HFs. We investigated whether HF production of these AMPs was induced by prototypic microbial products and proinflammatory cytokines, i.e. interferon (IFN)-gamma. Finally, we examined whether the classical pathways for AMP induction, such as toll-like receptor (TLR)4 and TLR5 expression, are present in human HFs and up-regulated after stimulation with bacterium-associated ligands. Cryosections from fresh or organ-cultured full-thickness normal human scalp skin treated with lipopolysaccharide (LPS), flagellin, protein A, lipoteichoic acid (LTA) or IFN-gamma were stained for psoriasin and RNase 7 immunoreactivity (IR) as well as for TLR4 and TLR5. In addition, outer root sheath cell culture and semiquantitative analysis of mRNA expression levels of RNase 7 and psoriasin were performed. Specific RNase 7 IR was present throughout the entire HF outer root sheath in situ and in cell culture, whereas psoriasin IR was present only in the most distal compartment and not detectable in cultured ORS cells. Upon treatment with Gram-positive (LTA, protein A) or Gram-negative bacterial (LPS, flagellin) cell wall components, or with the cytokine IFN-gamma, the IR of both psoriasin and RNase 7 was modified. TLR4 and TLR5 IR was detected in the normal HF epithelium and were upregulated after treatment with their respective ligand. The mRNA analysis confirmed the immunohistochemistry results. This pilot study suggests that normal human scalp HF epithelium possesses a functional antimicrobial defence system, which includes the AMPs RNase 7 and psoriasin, and TLRs, and that these are induced by classical microbial products.

Evidence that the bulge region is a site of relative immune privilege in human hair follicles

Recent gene profiling data suggest that, besides the anagen hair bulb, the epithelial stem cell region in the outer root sheath of hair follicles (HFs), termed the bulge, may also represent an area of relative immune privilege (IP). To investigate whether the human HF bulge is a site of relative IP within anagen VI HFs. Anagen VI HFs from normal human scalp skin were analysed using immunohistological staining techniques, quantitative histomorphometry and statistical analysis. For functional evidence we performed full-thickness human scalp skin organ cultures to investigate whether interferon (IFN)-gamma, a key inducer of IP collapse in hair bulbs, has a similar effect on the putative bulge IP. Major histocompatibility complex (MHC) class Ia, beta(2)-microglobulin and MHC class II immunoreactivity are downregulated in the human bulge. The immunosuppressants alpha-melanocyte stimulating hormone, transforming growth factor-beta2, macrophage migration inhibitory factor and indoleamine-2,3-dioxygenase (IDO) are upregulated in the CD200+, stem cell-rich bulge region. These CD200+ cells also co-express HLA-E. Furthermore, IFN-gamma induces significant ectopic MHC class Ia expression in bulge cells of organ-cultured human scalp skin. These data suggest that the bulge of human anagen HFs represents a hitherto unrecognized site of relative IP in human skin. Simultaneously, we present the first evidence of IDO and HLA-E protein expression in normal human HFs. Bulge IP presumably protects the HF epithelial stem cell reservoir from autoaggressive immune attack whereas a loss of bulge IP may play a central role in the pathogenesis of cicatricial alopecias.

Thyroid Hormones Directly Alter Human Hair Follicle Functions: Anagen Prolongation and Stimulation of Both Hair Matrix Keratinocyte Proliferation and Hair Pigmentation

Both insufficient and excess levels of thyroid hormones (T3 and T4) can result in altered hair/skin structure and function (e.g. effluvium). However, it is still unclear whether T3 and T4 exert any direct effects on human hair follicles (HFs), and if so, how exactly human HFs respond to T3/T4 stimulation. Our objective was to asses the impact of T3/T4 on human HF in vitro. Human anagen HFs were isolated from skin obtained from females undergoing facelift surgery. HFs from euthyroid females between 40 and 69 yr (average, 56 yr) were cultured and treated with T3/T4. Studying microdissected, organ-cultured normal human scalp HFs, we show here that T4 up-regulates the proliferation of hair matrix keratinocytes, whereas their apoptosis is down-regulated by T3 and T4. T4 also prolongs the duration of the hair growth phase (anagen) in vitro, possibly due to the down-regulation of TGF-beta2, the key anagen-inhibitory growth factor. Because we show here that human HFs transcribe deiodinase genes (D2 and D3), they may be capable of converting T4 to T3. Intrafollicular immunoreactivity for the recognized thyroid hormone-responsive keratins cytokeratin (CK) 6 and CK14 is significantly modulated by T3 and T4 (CK6 is enhanced, CK14 down-regulated). Both T3 and T4 also significantly stimulate intrafollicular melanin synthesis. Thus, we present the first evidence that human HFs are direct targets of thyroid hormones and demonstrate that T3 and/or T4 modulate multiple hair biology parameters, ranging from HF cycling to pigmentation.

Ultrastructure of Amelanotic Melanocytes from Human Hair Follicles

Objective To investigate the ultra structure of amelanotic melanocytes (AMMC). Methods The hair follicles obtained from normal human scalp by 0.50% collagenase type V treatment were washed with 0.1 mol/L phosphate buffer salt (PBS). Hair-follicle cell suspensions were prepared by trypsin treatment and cultured in melanocyte medium. Remaining keratinocytes were removed by differential trypsinization. 100μg/ml geneticin was used to eliminate the contaminating fibroblasts. At third passage, the cells were trypsinized, and then washed in phosphate-buffered saline and processed for transmission electron microscopy. Results Under transmission electron microscope, the cultured cells showed round or oval shape, with single large nuclear and the karyotheca were double deck. There were obvious euchromosome within the nucleus, and sparse heterochromosome. There were various organelles in the cytoplasm, including plentiful melanosomes with nearly similar size, mitochondria, rough endoplasmic reticule (RER) and ribosome. The electron density granules in most of the melanosomes disposed along concentric circularities. Golgi apparatus in the cells was inconspicuous. Conclusion The ultra structure of AMMC from human hair follicles is different from that of epidermal melanocytes, and these characteristics determine the functional immature of AMMC.

Towards the development of a pragmatic technique for isolating and differentiating nestin‐positive cells from human scalp skin into neuronal and glial cell populations: generating neurons from human skin?

Nestin+ hair follicle-associated cells of murine skin can be isolated and differentiated in vitro into neuronal and glial cells. Therefore, we have asked whether human skin also contains nestin+ cells, and whether these can be differentiated in vitro into neuronal and/or glial cell populations. In this methodological pilot study, we show that both are indeed the case - employing purposely only very simple techniques for isolating, propagating, and differentiating nestin+ cells from normal human scalp skin and its appendages that do not require selective microdissection and tissue compartment isolation prior to cell culture. We show that, it is in principle, possible to maintain and propagate human skin nestin+ cells for extended passage numbers and to differentiate them into both neuronal (i.e. neurofilament+ and/or PGP9.5+) and glial (i.e. GFAP+, MBP+ and/or O4+) cell populations. Therefore, human scalp skin can serve as a highly accessible, abundant, and convenient source for autologous adult stem cell-like cells that offer themselves to be exploited for neuroregenerative medicine purposes.

Analysis of the expression pattern of glial cell line‐derived neurotrophic factor, neurturin, their cognate receptors GFRα‐1 and GFRα‐2, and a common signal transduction element c‐Ret in the human scalp skin

Glial cell line-derived neurotrophic factor (GDNF) and a related family member, neurturin (NTN), as well as their cognate receptors (GDNF receptors, GFRalpha-1 and GFRalpha-2, respectively) are involved in nervous system development and murine hair cycle control. To date, their expression in human scalp skin is still unknown. The expression pattern of these proteins was examined in human scalp skin by immunofluorescence and immunoalkaline phosphatase staining methods as well as RT-PCR (GDNF). A total of 50 normal human scalp skin biopsy specimens were examined (healthy females, 53-57 years). The expression of GDNF protein was strong in the epidermis and sebaceous and sweat glands. In the epidermis, GDNF protein expression was seen in all layers except the stratum corneum. It was strong in the basal layer and decreased gradually towards the granular layer. The results of RT-PCR analysis revealed that GDNF protein is synthesised in the epidermis. The expression of NTN, GFRalpha-1, and GFRalpha-2 proteins was strong in the papillary dermis and sebaceous and sweat glands. In the epidermis, NTN protein expression was absent. The expression of GFRalpha-1 and GFRalpha-2 proteins was moderate in the epidermis. The expression of c-Ret protein was consistently strong in the epidermis and sebaceous and sweat glands. These proteins were strongly expressed in both epithelial and mesenchymal compartments of human anagen VI scalp hair follicles. Our investigation reports, for the first time, the expression patterns of GDNF, NTN, GFRalpha-1, GFRalpha-2, and c-Ret proteins in human scalp skin. The expression of these proteins in the skin suggests their possible roles in skin homeostasis. The clinical ramifications of these observations mandate further investigations. Adly MA, Assaf HA, Hussein MR, Paus R. Analysis of the expression pattern of glial cell line-derived neurotrophic factor, neurturin, their cognate receptors GFRalpha-1 and GFRalpha-2, and a common signal transduction element c-Ret in the human scalp skin.

New frontiers in human hair follicle (neuro‐)endocrinology

Recently, we have shown that normal human scalp hair follicles in anagen VI (a) synthesize CRH, ACTH, alpha‐MSH and cortisol and display a fully functional equivalent of the hypothalamus–pituitary–adrenal axis (Ito et al. FASEB J 2005) (b) synthesize melatonin, up‐regulate melatonin synthesis and secretion upon stimulation with noradrenaline (just like in the pineal gland), and express melatonin receptors in a hair cycle‐dependent manner (Kobayashi et al. FASEB J 2005) and (c) synthesize prolactin, express functional prolactin receptors, and respond to their stimulation by premature catagen induction, which is mediated in part via up‐regulation of TGFβ2 (Foitzik et al. Am J Pathol, in press). Besides these, by now established, aspects of hair follicle (neuro‐) endocrinology, we present preliminary evidence that points to exciting new frontiers in this field: for example, human anagen VI scalp hair follicles also express erythropoietin and its receptor (EPO and EPO‐R) on the gene and protein level and up‐regulate EPO‐R expression under conditions of hypoxia, and that these hair follicles may have established yet another hypothalamus–pituitary axis equivalent, the TRH‐TSH system. Studying the effects of calcitonin gene‐related peptide (CGRP), a key skin neuropeptide associated, e.g., with stress responses and neurogenic inflammation, on organ‐cultured human hair follicles, we also noted that CGRP inhibits both hair matrix keratinocyte proliferation in situ and hair follicle pigmentation. After defining the most immediately pressing open questions that remain to be addressed in these areas, our own data are discussed in the context of how the organ‐culture of human scalp hair follicles can be exploited for addressing questions of general importance in human dermatoendocrinology.

Expression of nerve growth factor and its high‐affinity receptor, tyrosine kinase A proteins, in the human scalp skin

Nerve growth factor (NGF) and its high-affinity receptor, tyrosine kinase A (TrkA), are members of the neurotrophin family. NGF-TrkA are involved in murine hair morphogenesis and cycling. To date, their expression in human hair follicle (HF) is unknown. In this investigation, we hypothesize that NGF-TrkA proteins are expressed in the human scalp skin. Moreover, NGF-TrkA expression in HF changes with the transitions from anagen-->>catagen-->>telogen stages. To test our hypothesis and to fill this existing gap in literature, the immunostaining values (semiquantitative evaluation of protein expression: SI, staining intensity; PP, percentage of positive cells; and IR score, immunoreactivity score) of NGF and TrkA proteins were examined in human scalp skin by immunofluorescent and immunoperoxidase staining methods. Fifty normal human scalp skin biopsy specimens were examined (healthy females, 53-57 years). In each case, 50 HFs were analyzed (35, 10, and five follicles in anagen, catagen, and telogen, respectively). The IR scores were statistically significantly higher (p < 0.001) in anagen as compared with either catagen or telogen HF (9.61 +/- 0.12 vs. 1.4 +/- 0.10 vs. 0.6 +/- 0.10 for NGF and 3.31 +/- 0.02 vs. 0.5 +/- 0.10 vs. 0.2 +/- 0.10 for TrkA). In the anagen HF, high expression values were seen in the distal region, followed by upper distal, lower distal, and bulb regions for both NGF (10.6 +/- 0.21 vs. 10.3 +/- 0.21 vs. 9.2 +/- 0.40 vs. 8.1 +/- 0.30) and TrkA (3.54 +/- 0.07 vs. 3.45 +/- 0.07 vs. 3.31 +/- 0.06 vs. 3.13 +/- 0.04). Both NGF and TrkA proteins showed prominent expression in the melanocytes (7.6 +/- 0.15 vs. 2.50 +/- 0.07), keratinocytes (10.2 +/- 0.40 vs. 2.71 +/- 0.06), sebaceous glands (10.2 +/- 0.40 vs. 2.72 +/- 0.06), and sweat glands (10.4 +/- 0.40 vs. 2.84 +/- 0.05). Our findings report, for the first time, the expression pattern of NGF and TrkA proteins in human scalp skin and HF. The differential expression of these proteins during HF cycling suggests their possible roles in human HF biology. The clinical ramifications of these observations mandate further investigations.

Modulation of the human hair follicle pigmentary unit by corticotropin‐releasing hormone and urocortin peptides

Human skin is a local source of corticotropin-releasing hormone (CRH) and expresses CRH and CRH receptors (CRH-R) at mRNA and protein levels. Epidermal melanocytes respond to CRH by induction of cAMP with up-regulation of pro-opiomelanocortin gene expression and subsequent production of adrenocorticotropin hormone. However, the role of CRH/CRH-R in melanocyte biology is complicated by the significant heterogeneity of cutaneous melanocyte subpopulations, from continuously active and UV-responsive melanocytes in epidermis to UV nonresponsive, hair growth cycle-coupled melanogenesis in hair follicles. In the present study we report that normal human scalp hair follicle melanocytes express CRH at the mRNA level. Furthermore, CRH, urocortin and CRH-R 1 and 2 were differentially expressed in follicular melanocytes, fibroblasts, and keratinocytes depending on anatomic location and differentiation status in situ and in vitro. Stimulation of follicular melanocytes with CRH and CRH peptides, modified for selectivity for CRH-R1 and/or CRH-R2, variably induced cell melanogenesis, dendricity, and proliferation. CRH-peptides also stimulated the expression and activity of Tyrosinase, and expression of Tyrosinase-related protein-1 and-2. However, a modified urocortin peptide highly selective for CRH-R2 down-regulated melanocyte differentiation phenotype. This study indicates that CRH peptides can differentially influence hair follicle melanocyte behavior not only via CRH-R1 signaling but also by complex cross-talk between CRH-R1 and CRH-R2.

Expression of the heat shock protein-27 in the adult human scalp skin and hair follicle: Hair cycle–dependent changes

Heat shock protein (HSP) is a molecular chaperone involved in protein folding, assembly, and transport and in the regulation of cell growth and differentiation. HSP27 protein is expressed in murine hair follicle (HF) and in human skin during fetal development. In this investigation we hypothesized that HSP27 protein is expressed in the human scalp skin and its expression in HF changes with the transitions form anagen --> catagen --> telogen stages. To test this hypothesis, the immunoreactivity of HSP27 protein was examined in human scalp skin by immunofluorescent method. A total of 50 normal human scalp skin biopsy specimens were examined (healthy women, age 53-57 years). In each case, 50 HF were analyzed (35, 10, and 5 follicles in anagen, catagen, and telogen, respectively). HSP27 protein expression was prominent in human scalp anagen, and weak in both catagen and telogen HFs. Within HF, HSP27 protein immunoreactivity was prominent in the outer root sheath, inner root sheath, precorteocytes, and corteocytes of the hair shaft. In addition, HSP27 protein expression was prominent in the epidermis, sebaceous glands, sweat glands, and arrector pili muscles. Only some types of heat shock proteins are known to date. Also, our knowledge about the exact molecular mechanisms involved in the interactions among these protein and other molecular chaperones is still incomplete. Our investigation reports, for the first time, the expression patterns of HSP27 in human scalp skin and HF. The differential expression of HSP27 during HF cycling suggests its possible roles in human HF biology.

A modified method for purifying amelanotic melanocytes from human hair follicles

We describe a modified method for establishing long-term pure cultures of amelanotic melanocytes (AMMC) derived from human hair follicles. Normal human corpse scalp (just after death, 1 h) was transected 1 mm below the epidermis, and hair follicles in the remaining dermis were isolated by a two-step enzyme treatment. Hair follicle cell suspensions were prepared by 0.50% trypsin treatment for 30 min and cultured in an optimized melanoblast proliferation nature mitogen medium. Cells attached to the substratum were mostly amelanotic melanocytic in character with small, bipolar shapes in the early stage; only a few keratinocytes and rare fibroblasts were observed. Keratinocytes were easily removed by differential trypsinization. After the third passage, the proliferating cells were all amelanotic melanocytes as confirmed by immunostaining with polyclonal antibodies to alphaPEP7h, which recognized the tyrosinase protein located on melanosomes and NKI/beteb, which is a pre-melanosomal antigen against synthetic peptides corresponding to the carboxyl termini of human melanosomal protein GP100. Cultured AMMC were highly positive to L-dopa reactivity after the addition of IBMX to the culture medium for 7 days. Many stage I and II melanosomes and occasional stage III melanosomes without stage IV melanosomes were found in the cytoplasm by transmission electron microscope. This modified technique is potentially more suitable for cultivating amelanotic melanocytes. The availability of pure cultures of hair-follicle amelanotic melanocytes will facilitate investigations of the roles of those cells in migration and differentiation during treatment of vitiligo.

Human Scalp Hair Follicles Are Both a Target and a Source of Prolactin, which Serves as an Autocrine and/or Paracrine Promoter of Apoptosis-Driven Hair Follicle Regression

The prototypic pituitary hormone prolactin (PRL) exerts a wide variety of bioregulatory effects in mammals and is also found in extrapituitary sites, including murine skin. Here, we show by reverse transcriptase-polymerase chain reaction and immunohistology that, contrary to a previous report, human skin and normal human scalp hair follicles (HFs), in particular, express both PRL and PRL receptors (PRL-R) at the mRNA and protein level. PRL and PRL-R immunoreactivity can be detected in the epithelium of human anagen VI HFs, while the HF mesenchyme is negative. During the HF transformation from growth (anagen) to apoptosis-driven regression (catagen), PRL and PRL-R immunoreactivity appear up-regulated. Treatment of organ-cultured human scalp HFs with high-dose PRL (400 ng/ml) results in a significant inhibition of hair shaft elongation and premature catagen development, along with reduced proliferation and increased apoptosis of hair bulb keratinocytes (Ki-67/terminal dUTP nick-end labeling immunohistomorphometry). This shows that PRL receptors, expressed in HFs, are functional and that human skin and human scalp HFs are both direct targets and sources of PRL. Our data suggest that PRL acts as an autocrine hair growth modulator with catagen-promoting functions and that the hair growth-inhibitory effects of PRL demonstrated here may underlie the as yet ill-understood hair loss in patients with hyper-prolactinemia.

Expression of CD1d in human scalp skin and hair follicles: hair cycle related alterations

Background: CD1d belongs to a family of antigen presenting molecules that are structurally and distantly related to the classic major histocompatibility complex class I (MHC I) proteins. However, unlike MHC...

Human scalp skin and hair follicles express neurotrophin-3 and its high-affinity receptor tyrosine kinase C, and show hair cycle-dependent alterations in expression

Neurotrophin (NT)-3 and its high-affinity receptor tyrosine kinase C (Trk C) are essential for nervous system development. These members of the NT family are also involved in murine hair morphogenesis and cycling. However, their role in human hair follicle (HF) biology remains to be elucidated. To explore the role of NTs in human skin and HF biology. The immunoreactivity (IR) of NT-3 and Trk C was studied in human scalp skin and HFs by immunofluorescent and light microscopic immunohistology. Skin biopsies were obtained from normal human scalp containing mainly anagen VI HFs from women (age 53-57 years) undergoing elective plastic surgery. Both NT-3 and Trk C showed prominent, yet distinct, IR patterns in human scalp anagen HFs (anagen VI), whereas they were weakly expressed in catagen and increased again in telogen HFs. Within HF compartments, NT-3 IR was prominent in the outer root sheath, inner root sheath, dermal papilla and connective tissue sheath. Trk C IR was prominent in all HF epithelial and mesenchymal compartments. Outside the HF, both NT-3 and Trk C showed prominent IR in the epidermis, sebaceous glands and sweat glands. These observations provide the first indication that NT-3 and Trk C are expressed in human scalp skin and HFs, and suggest that Trk C-mediated signalling is involved not only in murine but also in human HF biology. They may be useful in determining therapeutic strategies for the treatment of hair cycle and skin-related disorders.

Differential expression of nitric oxide synthases in human scalp epidermal and hair follicle pigmentary units: implications for regulation of melanogenesis

Nitric oxide (NO) is a ubiquitous gaseous lipophilic molecule generated from the conversion of L-arginine to L-citrulline by the NO synthases (NOSs). Ultraviolet radiation (UVR)-induced NO production appears to stimulate epidermal melanogenesis. However, given their relative protection from UVR, it is unclear whether NO plays a similar role in hair bulb melanocytes. We aimed to identify the expression profiles of the NOS isoforms endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS) and of phosphorylated eNOS and nitrotyrosine within the epidermal and follicular melanin units of normal human haired scalp during the hair growth cycle. This study employed single and double immunohistochemical and immunofluorescence staining techniques using haired scalp from 10 healthy individuals (six women and four men). Melanocytes in the basal layer of the epidermis expressed eNOS, nNOS and nitrotyrosine. By contrast, melanogenically active melanocytes of the anagen hair bulb were wholly negative for these markers. However, other follicular melanocytes not actively involved in pigment production, including undifferentiated melanocytes located in the outer root sheath and melanocytes surviving the apoptosis-driven hair follicle (HF) regression during catagen/telogen, expressed eNOS, nNOS and nitrotyrosine. While iNOS was only weakly expressed in the basal layer of the human epidermis, it was highly expressed in keratinocytes of the inner root sheath (IRS), where it colocalized with trichohyalin, a differentiation-associated protein of the IRS that requires enzyme-catalysed conversion of arginine to citrulline. The NOS isoforms and nitrotyrosine are differentially expressed in different cutaneous melanocyte subpopulations. Results of this study suggest a possible role for eNOS, nNOS, iNOS and nitrotyrosine in melanocyte biology, particularly with respect to melanogenesis and melanocyte survival during HF regression. Another example of possible NO involvement in HF biology is the postsynthetic modification of trichohyalin in differentiating keratinocytes of the IRS. These results suggest that NO may influence several aspects of HF biology.

Human hair follicles display a functional equivalent of the hypothalamic‐pituitary‐adrenal (HPA) axis and synthesize cortisol

The skin and its major appendages are prominent target organs and potent sources of key players along the classical hypothalamic-pituitary axis, such as corticotropin releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and alpha melanocyte stimulating hormone (alpha-MSH), and even express key steroidogenic enzymes. Therefore, it may have established local stress response systems that resemble the hypothalamic-pituitary-adrenal (HPA) axis. However, functional evidence that this is indeed the case in normal human skin in situ has still been missing. We show that microdissected, organ-cultured human scalp hair follicles respond to CRH stimulation by up-regulating proopiomelanocortin (POMC) transcription and immunoreactivity (IR) for ACTH and alpha-MSH, which must have been processed from POMC. CRH, alpha-MSH, and ACTH also modulate expression of their cognate receptors (CRH-R1, MC1-R, MC2-R). In addition, the strongest stimulus for adrenal cortisol production, ACTH, also up-regulates cortisol-IR in the hair follicles. Isolated human hair follicles secrete substantial levels of cortisol into the culture medium, and this activity is further up-regulated by CRH. CRH also modulates important functional hair growth parameters in vitro (hair shaft elongation, catagen induction, hair keratinocyte proliferation, melanin production). Finally, human hair follicles display HPA axis-like regulatory feedback systems, since the glucocorticoid receptor agonist hydrocortisone down-regulates follicular CRH expression. Thus, even in the absence of endocrine, neural, or vascular systemic connections, normal human scalp hair follicles directly respond to CRH stimulation in a strikingly similar manner to what is seen in the classical HPA axis, including synthesis and secretion of cortisol and activation of prototypic neuroendocrine feedback loops.

Interferon-gamma is a potent inducer of catagen-like changes in cultured human anagen hair follicles

Interferon (IFN)-gamma appears to be an important hair cycle modulator in mice. It is unclear whether it has similar hair growth modulatory functions in human hair follicles. To study whether IFN-gamma can be exploited to modulate the growth, pigmentation and/or cycling of organ-cultured human anagen scalp hair follicles, as an in vitro indicator system for how IFN-gamma affects human hair growth in vivo. This was correlated with the hair follicle expression patterns of IFN-gamma receptors alpha and beta. In addition, we wanted to establish a new, simple tool for the rapid experimental induction of catagen in vitro. Normal human scalp hair follicles in the anagen VI stage of the hair cycle were cultured according to the method of Philpott et al., with or without IFN-gamma (50-1000 IU mL(-1)). Hair shaft elongation and pigmentation changes were measured, complemented by quantitative histomorphometry to assess changes in hair follicle cycling (hair cycle score), proliferation (Ki-67), melanogenesis (Masson-Fontana) and apoptosis (TUNEL). IFN-gamma receptors were also localized by immunofluorescence and EnVision technique. As transforming growth factor (TGF)-beta2 is a recognized key inducer of catagen in human hair follicles, TGF-beta2 expression was investigated by tyramide signal amplification and reverse transcription-polymerase chain reaction in anagen hair follicles treated with vehicle (phosphate-buffered saline) or IFN-gamma. IFN-gamma rapidly inhibited hair elongation in cultured human anagen hair follicles and induced morphological signs of catagen transformation after only 4 days of culture, i.e. faster than with other reported catagen-inducers (e.g. TGF-beta2). Proliferation was inhibited, apoptosis was increased and follicular melanogenesis was switched off in hair bulb keratinocytes treated in situ with IFN-gamma. Anagen hair follicles displayed strong IFN-gamma receptor alpha-like immunoreactivity, while the immunoreactivity for IFN-gamma receptor beta in the hair matrix was only weak. TGF-beta2 immunoreactivity and mRNA transcript levels were enhanced in hair follicles treated with IFN-gamma. These data suggest that IFN-gamma is a potent catagen inducer in normal human scalp hair follicles, which express cognate receptors, and show that IFN-gamma administration offers an excellent tool for experimental catagen induction in organ-cultured human hair follicles. This catagen induction probably occurs at least in part via upregulation of the recognized catagen-stimulatory growth factor TGF-beta2.

Isolation and Culture of Amelanotic Melanocytes from Human Hair Follicles

We report a method to establish amelanotic melanocytes (AMMC) in culture and we investigate the effects of various components in the culture medium. Normal human scalp from cadaver donors was transected 1 mm below the epidermis, and hair follicles in the remaining dermis were isolated by a two-step enzyme treatment. The individual hair follicles were washed exhaustively and suspensions of hair follicle cells were prepared and cultured in Eagle's minimum essential medium supplemented with 12-o-tetradecanoyl-phorbol-13-acetate (TPA), cholera toxin and keratinocyte serum-free medium (K-SFM). Geneticin was used to eliminate contaminating fibroblasts. Proliferation of AMMC was observed after addition of TPA and K-SFM including bovine pituitary extract (BPE) into the culture medium. Cell type was determined by staining with monoclonal antibodies, NKI/beteb and HMB-45, which recognize premelanosomal and melanosomal antigens, respectively. The AMMC were also examined using transmission electron microscopy. Treatment with geneticin eliminates the majority of fibroblasts and does not impair the growth of keratinocytes or AMMC. After contaminating fibroblasts and keratinocytes were removed, two distinct cell morphologies remained: (1) large, dendritic and deeply pigmented cells, which did not proliferate and which disappeared by the third passage, and (2) small bipolar cells, which initially were unpigmented and proliferated very rapidly. We observed that TPA at various concentrations stimulated the proliferation of the cells, and at high concentrations could induce the formation of multiple dendrites. K-SFM including BPE accelerated the proliferation of the cells in a dose-dependent manner. After passage 3, almost all cells expressed premelanosomal and melanosomal antigens, recognized by NKI/beteb and HMB-45, respectively. Active mitochondria, abundant rough endoplasmic reticulum, Golgi complexes, ribosomes and melannosomes (predominantly in stages I, II or III with some at stage IV in some AMMC) were observed ultrastructurally in the cytoplasm of the cultured cells.

The contribution of the arrector pili muscle and sebaceous glands to the follicular unit structure

BackgroundThe evidence of hairs grouped into well-defined follicular units has given a new vision of hair anatomy and pathology. The sebaceous glands and the arrector pili muscle, as part of the pilosebaceous unit, should be viewed as important parts of this organized follicular unit structure. ObjectiveThis study was performed to define the morphology and the relationships between the sebaceous glands, muscles, and follicles within the context of the follicular unit structure. MethodsThis study analyzes horizontal, microscopic serial sections of large areas of normal human scalp skin stained with hematoxylin and eosin, Masson's trichrome, and desmin. The course of the arrector pili muscles from their superficial origins to their follicular attachments was followed in each section, which enabled us to match each muscle with its corresponding follicular unit. ResultsSerial, horizontal sections show that, at the upper isthmus, the arrector pili muscle is arranged as a muscular unit structure at the periphery of each follicular unit. Then, at a lower level, the muscle divides and encircles the sebaceous gland, and penetrates between the sebaceous lobules towards the follicular attachment zone. ConclusionMicrophotographic evaluation of large areas of scalp is important for assessing the anatomical relationships between the sebaceous glands, arrector pili muscles, and hair follicles as components of the follicular unit. We introduce the anatomical concept of a follicular unit served by a muscular unit, which can be identified in horizontal sections made at the upper level of the isthmus. This muscular unit results from the merging of the arrector pili muscles that originate from the hair follicles contained in that particular follicular unit. This anatomical disposition suggests that the arrector pili muscles could play an important role in the integrity of the follicular unit as well as in the secretion of the sebum contents.