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Notes: Background Hair loss following skin inflammation may in part be mediated by keratinocyte (KC) apoptosis. While the effects of different cytokines or other apoptosis stimulating agents such as interferon (IFN)-γ or tumour necrosis factor (TNF)-α on KC apoptosis in vitro have been addressed in several studies, little is known about the effects of proinflammatory cytokines on KC apoptosis in vivo. Objectives To study the effects of intradermally injected TNF-α, interleukin (IL)-1β and IFN-γ on KC apoptosis in the back skin of C57BL/6 mice. Methods Apoptosis in epidermal and hair bulb KCs was analysed by immunohistology using TUNEL staining. Results Injection of TNF-α induced a significantly higher number of apoptotic cells within the epidermis than vehicle; all three proinflammatory cytokines together further increased their number. Intrafollicular hair bulb KCs were much more susceptible to apoptosis induction by TNF-α or IL-1β; their injection significantly upregulated apoptosis after 6 h, which was further increased after 24 h. The combination of all cytokines together accelerated intrafollicular apoptosis after 6 h by doubling the number of apoptotic cells per hair bulb, compared with the effects of TNF-α or IL-1β alone. Conclusions These data suggest that programmed cell death of proliferating KCs in vivo can be induced by proinflammatory cytokines.

Notes: The immunology of the hair follicle, its relationship with the ‘skin immune system’ and its role in hair diseases remain biologically intriguing and clinically important. In this study, we analysed the immunoreactivity patterns of 15 immunodermatological markers to determine the cellular composition and immune privilege of the human hair follicle immune system in anagen VI (growth phase). The most prominent cells located in or around the hair follicle were Langerhans cells, CD4+ or CD8+ T cells, macrophages and mast cells, whereas B cells, natural killer cells and γδ T cells were found very rarely. Langerhans cells (CD1a+, major histocompatibility complex, MHC class II+), and T cells (CD4+ or CD8+) were predominantly distributed in the distal hair follicle epithelium, whereas macrophages (CD68+, MHC class II+) and mast cells (Giemsa+) were located in the perifollicular connective tissue sheath. Transmission electron microscopy confirmed low numbers of immune cells in the proximal hair follicle epithelium, and very few macrophages and Langerhans cells were seen in the dermal papilla. Melanophages were observed in the connective tissue sheath and dermal papilla. MHC class I (HLA-A, -B, -C) and β2-microglobulin immunoreactivity was found on most skin cells, but was substantially reduced on isthmus keratinocytes and virtually absent in the proximal hair follicle epithelium. Apart from the absence of Fas ligand immunoreactivity, the sharply reduced numbers of T cells and Langerhans cells, and the virtual absence of MHC class I expression all suggest that the anagen proximal hair follicle constitutes an area of immune privilege within the hair follicle immune system, whose collapse may be crucial for the pathogenesis of alopecia areata.

Notes: Despite more than a hundred years of professinal hair research, and substantial recent progress in unravelling the molecular controls of hair follicle morphogenesis, the chronobiological control system that cyclically drives the hair follicle through dramatic remodelling processes between phases of growth (anagen), regression (catagen), and relative resting (telogen) have remained disappointingly obscure. In view of the vast literature that has become available over the past decades on numerous genetic, biochemical, cellular and pharmacological aspects of hair growth control under physiological and pathological conditions, it is astounding how comparatievely few researchers in the field have published theoretical concepts that explore how hair follicle cycling might be controlled. Since this question is at the very heart of basic and clinically applied hair biology, it deserves a much more systematic and serious public exploration, which the following contributions are designed to stimulate.

Notes: The role of adhesion molecules in the control of hair follicle (HF) morphogenesis, regression and cycling is still rather enigmatic. Since the adhesion molecules E- and P-cadherin (Ecad and Pcad) are functionally important, e.g. during embryonic pattern formation, we have studied their expression patterns during neonatal HF morphogenesis and cycling in C57/BL6 mice by immunohistology and semi-quantitative RT-PCR. The expression of both cadherins was strikingly hair cycle-dependent and restricted to distinct anatomical HF compartments. During HF morphogenesis, hair bud keratinocytes displayed strong Ecad and Pcad immunoreactivity (IR). While neonatal epidermis showed Ecad IR in all epidermal layers, Pcad IR was restricted to the basal layer. During later stages of HF morphogenesis and during anagen IV-VI of the adolescent murine hair cycle, the outer root sheath showed strong E- and Pcad IR. Instead, the outermost portion of the hair matrix and the inner root sheath displayed isolated Ecad IR, while the innermost portion of the hair matrix exhibited isolated Pcad IR. During telogen, all epidermal and follicular keratinocytes showed strong Ecad IR. This is in contrast to Pcad, whose IR was stringently restricted to matrix and secondary hair germ keratinocytes which are in closest proximity to the dermal papilla. These findings suggest that isolated or combined E- and/or Pcad expression is involved in follicular pattern formation by segregating HF keratinocytes into functionally distinct subpopulations; most notably, isolated Pcad expression may segregate those hair matrix keratinocytes into one functional epithelial tissue unit, which is particularly susceptible to growth control by dermal papilla-derived morphogens. The next challenge is to define which secreted agents implicated in hair growth control modulate these follicular cadherin expression patterns, and to define how these basic parameters of HF topobiology are altered during common hair growth disorders.

Notes: Abstract: Since we have recently shown that the β2-adrenoreceptor (β2-AR) expression of selected regions of the hair follicle (HF) epithelium as well as the number of adrenergic nerve fibers in murine skin change in a hair cycle-dependent manner, this has raised the possibility that adrenergic nerves may exert “trophic” functions during HF cycling. To further explore this concept, we have investigated the effect of neuro-pharmacological manipulations on hair growth (anagen) induction in quiescent telogen mouse skin in vivo. Here, we demonstrate that subcutaneous injections of the noradrenaline (NA)-depleting agent guanethidine, or of the neurotoxin 6-hydroxydopaine, but not of the β2-AR agonist isoproterenol induce a premature onset of anagen in the lower back skin of C57BL/6 mice. On day 20 after the start of treatment, more than 80% of the guanethidine-treated mice and ca. 65% of the 6-hydroxydopamine-treated (6-OHDA) mice exhibited premature skin darkening and hair growth at the site of drug application, whereas less than one-third of all control animals showed macroscopic signs of anagen developent. This was confirmed by histology, demonstrating mature anagen VI HFs only at the immediate site of treatment with guanethidine of 6-OHDA as opposed to resting telogen HFs in the neighboring untreated skin area. This observation further supports the concept that sympathetic nerves are intimately involved in hair growth control and invites one to explore the neuro-pharmacological manipulation of piloneural interactions as a novel therapeutic strategy for the management of hair growth disorders.

Notes: In 1989, mice bearing mutations at the hr (hairless) locus were first proposed as a model for the human hair growth disorder papular atrichia, since in both these mice and in corresponding patients, a complete hair loss develops due to disintegration of the normal follicle structure into dermal cysts and so-called utriculi. Recently, the human hairless gene was characterized, and pathogenetic mutations were found to be associated with a recessively inherited form atrichia with papular lesions; however, the functions of hr gene remain unclear. Allelic mutations in the murine hairless gene represent a potentially powerful tool to elucidate the role of the hairless gene protein product in hair follicle physiology. In 1980, several naked animals were discovered in a breeding colony of B10.R109/Y mice maintained in the Laboratory of Experimental Biological Models (L.E.B.M., Yurlovo, Moscow District, Russia). By cross breeding with hairles HRS/J hr/hr mice, this mutation was shown to be allelic with hairless. Here, we describe the molecular basis of the hrrhY mutation in mice, which consists of a 13 bp insertion in exon 16 of the hr gene. Histological evaluation of Yurlovo mouse skin revealed some differences as compared to the hairless and rhino mutations, with the formation of dermal megacysts being the most specific peculiarity of the Yurlovo mutation. These results, together with previous studies of hrrhY/hrrhY mutant mice, suggest that the rhino Yurlovo (hrrhY) mutation represents a third and potentially more severe variation of the hairless phenotype.

Notes: After the initial discovery that, in vivo, mammalian skin both transcribes and translates the proopiomelanocortin (POMC) gene, and processes its product into melanocortins (Slominski et al., Experientia 1992), it has become increasingly appreciated that the hair follicle – including the human one – is a prime source and target not only of POMC-derived “pituitary” hormones, e.g. alpha-MSH, ACTH and ß-endorphin, but also expresses the most proximal control element of the hypothalamic-pituitary-adrenal (HPA) axis, corticotropin-releasing hormone (CRH) and its receptor (e.g. Roloff et al. FASEB J 1998, Ito et al. J Invest Dermatol 2004). However, while all proximal elements of the HPA are expressed in both murine and human hair follicles (CRH, CHR-R, POMC, ACTH and ACTH-R), it has neither been shown that these are functionally linked (i.e., is CRH actually capable of modulating intrafollicular POMC gene expression and ACTH production?), nor has it been known whether the most distal HPA component – cortisol synthesis – is also present in the hair follicle. Therefore, we have investigated whether the stimulation of microdissected, organ-cultured human hair follicles with CRH or ACTH elicits responses inside this peripheral miniorgan that imitate a functional HPA – in the absence of any systemic or neural connections and under serum-free culture conditions. Here, we show that CRH stimulation of organ-cultured human scalp hair follicles in the anagen VI stage of the hair cycle indeed results in significant upregulation of POMC transcription, and of alpha-MSH, ACTH, MC1, MC2 and glucocorticoid receptor (GR) immunoreactivity in situ (immunofluorescence). ACTH stimulation, in turn, significantly up-regulates the – already constitutively present!– cortisol-immunoreactivity as well as cortisol secretion into the culture medium. This represents the first available evidence that normal human skin (more precisely: the hair follicle) can actually synthesize the “adrenal” steroid hormone cortisol in situ, and that this acticity is regulated by the same “hypothalamic” and “pituitary” hormones that operate as key controls of adrenal cortisol synthesis. Moreover, we show that cortisol stimulation exerts classical feedback responses inside the human anagen hair follicle recognized for the central HPA: cortisol up-regulates GR, while it down-regulates CRH expression. Given that the HPA operates as the major system for coordinating stress-responses of the mammalian organism and for integrating them into changing metabolic demands and neuro-endocrine-immune signaling circuits, it has fascinating implications (e.g. for general skin physiology and dermatological therapy), and raises most intriguing questions, that human hair follicles are utilizing a fully functional peripheral equivalent of the central HPA.

Notes: The functional role of VR1, which we and others have recently identified on several epithelial and mesenchymal human skin cell populations, was investigated in the human hair follicle (HF), as a prototypic epithelial–mesenchymal interaction system. VR1 immunoreactivity was confined to distinct epithelial compartments of HFs in anagen and catagen, while dermal papilla fibroblasts and HF melanocytes were VR1 negative. In organ culture, VR1 activation by capsaicin resulted in a dose-dependent and VR1-specific inhibition of hair shaft elongation, suppression of proliferation, promotion of apoptosis, and induction of catagen transformation, possibly due to upregulation of a potent hair growth inhibitor TGFβ2. Cultured outer root sheath (ORS), as well as HaCaT, keratinocytes also expressed functional VR1, whose stimulation inhibited proliferation, induced apoptosis, and elevated intracellular calcium concentration. Finally, VR1 stimulation of cultured ORS keratinocytes upregulated the expression of recognized endogenous hair growth inhibitors (IL-1β and TGFβ2) and downregulated the expression of stimulators (HGF, IGF-1, and SCF), while key differentiation markers (CK17, CK14, filaggrin, and involucrin) remained unaffected. In conclusion, VR1 is a significant novel player in human hair growth control underscoring that its physiological functions in human skin far extend beyond sensory neuron-coupled nociception.

Notes: Recently, we introduced a mouse model launching experimental evidence for stress-induced hair growth inhibition (HGI), pointing to the existence of a brain-hair follicle axis (BFA). We suggested that nerve growth factor (NGF), besides neuropeptide substance P (SP), is a candidate mediator along the BFA. Published data further indicate that stress-related neuropeptides, e.g. calcitonin gene-related peptide (CGRP) and SP may be involved in HGI. SP and CGRP are synthesized in dorsal root ganglia (DRG) and released after axonal transport in the skin. Thus, aim of the present study was to investigate the effect of stress or subcutaneous injection of NGF, which mimics stress and regulates neuropeptide genes in sensory neurons, on the expression of SP and CGRP in DRG. Anagen was induced in C57BL/6 mice by depilation and retrograde tracing was employed on day 9 post-depilation (PD). On day 14 PD, mice were either exposed to sound stress (n = 4) injected subcutaneously with NGF (n = 4) or served as control (n = 4). On day 16 PD, DRG (mean of 30/mouse) were harvested and SP and CGRP in skin-specific sensory neurons, as identified by the tracer dye, were labelled by immunohistochemistry and counted. Stress exposure as well as NGF injection leads to a significant induction of SP and CGRP in retrograde-labelled neurons. This allows us to conclude that sensitive dermal nerve fibres are likely to originate from the presently identified neuropeptide-positive neurons. Peripheral activation of SP-expressing afferent nerve fibres via NGF-dependent pathways may cause neurogenic inflammation, eventually resulting in HGI.