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Notes: The hair follicle offers an exquisite model for the experimental exploration of key issues of cutaneous neuroimmunology, for example, how local, intracutaneous and systemic stress–response systems are integrated with the skin immune system and with epithelial–mesenchymal interactions (as they occur during hair follicle growth and cycling). Previously, we had shown that skin mast cells, which operate as central switchboards of inflammation and tissue remodelling, also are important regulators of hair growth in mice and that endogenous, immunomodulatory mast cell secretagogues are potent hair growth modulators. This is true both for secretagogues that are generated by the hair follicle epithelium itself (e.g. ACTH) and for mast cell-activating neuropeptides synthesized by the sensory hair follicle innervation (e.g. SP). Also, we had shown that the prototypic stress-associated neuropeptide, SP, plays a crucial role in mediating the hair growth-inhibitory, mast cell-activating, inflammation- and catagen-promoting properties of chronic psychoemotional stress on murine hair follicles. Now, we show that the immunomodulatory and mast cell-activating neurotrophin, NGF, is also crucially involved in mediating the inhibitory effects of stress on murine hair growth. Furthermore, the central, stress-related neurohormone CRH, a recognized mast cell secretagogue which is expressed by the hair follicle epithelium, also is a hair growth inhibitor and activates a fully functional peripheral equivalent of the hypothalamic-pituitary-adrenal axis within organ-cultured human scalp hair follicles, including the synthesis and secretion of cortisol as well as the induction of classical feedback loops. We also demonstrate that one of the melanocortins whose intrafollicular synthesis is stimulated by CRH (α-MSH) is a potent suppressor of MHC class I expression in situ and is thus capable of restoring the collapsed immune privilege of human anagen hair bulbs, while SP upregulates the ectopic expression of MHC class I, thus endangering the hair follicle immune privilege. Finally, we show that vanilloids long exploited as experimental tools for neuroimmunological research in the skin (capsaicin) can, in fact, directly modulate human hair growth via the stimulation of vanilloid receptors (VR1) expressed by the follicle epithelium, in addition to stimulating vanilloid expressed by skin mast cells. Therefore, the hair follicle offers an ideal, highly instructive and clinically most relevant research model for dissecting how nervous system, central and peripheral (neuro-) endocrine signalling loops and the immune system interact in order to adapt skin functions to changing environmental conditions (e.g. in response to external stressors, by alterating, e.g. keratinocyte proliferation/apoptosis, skin immune status, as well as defined cutaneous metabolic and endocrine activities).

Notes: Background  Human hair growth can currently be studied in vitro by the use of organ-cultured scalp hair follicles (HFs). However, simplified organotypic systems are needed for dissecting the underlying epithelial–mesenchymal interactions and as screening tools for candidate hair growth-modulatory agents.Objectives  To optimize the design and culture conditions of previously published organotypic systems that imitate epithelial–mesenchymal interactions in the human HF as closely as possible.Materials and methods  Continuous submerged organotypic ‘sandwich’ cultures were established. These consist of a pseudodermis (collagen I mixed with and contracted by human interfollicular dermal fibroblasts) on which one of two upper layers is placed: either a mixture of MatrigelTM basement membrane matrix (BD Biosciences, Bedford, MA, U.S.A.) and follicular dermal papilla fibroblasts (DPC), with outer root sheath keratinocytes (ORSK) layered on the top (‘layered’ system), or a mixture of MatrigelTM, DPC and ORSK (‘mixed’ system). Morphological and functional characteristics of these ‘folliculoid sandwiches’ were then assessed by routine histology, histomorphometry and immunohistochemistry.Results  In both ‘layered’ and ‘mixed’ systems, the ORSK formed spheroid epithelial cell aggregates, which retained their characteristic keratin expression pattern (i.e. cytokeratin 6). In the ‘mixed’ sandwich model the size of the epithelial cell aggregates was smaller, but the numbers of ORSK were significantly higher than in the ‘layered’ model at day 14 in the culture. ORSK proliferated better in the ‘mixed’ than in the ‘layered’ sandwich system, regardless of the calcium or serum content of the media, whereas apoptosis of ORSK was lowest in the ‘mixed’ system in serum-free, low calcium medium. The kinetics of proliferation and apoptosis of DPC, which retained their characteristic expression of versican, were similar in both systems. However, proliferation and apoptosis of DPC were higher in the presence of serum and/or under high calcium conditions.Conclusions  Our results underscore the importance of structural design and medium composition for epithelial–mesenchymal interactions as they occur in the human HF. Specifically, we report a new organotypic submerged ‘folliculoid sandwich’ system with serum-free, low calcium medium and a mixture of interacting human DPC and ORSK, which offers several advantages over previously available assays. This system allows the standardized assessment of the effects of a test agent on the proliferation, apoptosis and key marker expression of human ORSK and DPC under substantially simplified in vitro conditions which approximate the in vivo situation.

Notes: We have studied the expressions of various protein kinase C (PKC) isoenzymes in T cells and monocytes from patients with systemic lupus erythematosus (SLE), in comparison to those of healthy controls and patients with other immunological disorders. As measured by Western blotting, the levels of PKCβ, δ, η, ε, θ and ζ (but not of PKCα) significantly decreased in T cells of SLE patients. In monocytes, however, we observed marked suppressions only in the expressions of PKCδ, ε and ζ but not in the expressions of other PKC isoforms. In vivo corticosteroid application, as well as in vitro steroid treatment of monocytes, elevated the expressions of most isoforms close to normal values; however, the decreased levels of PKCθ and ζ were not affected by steroid application. These alterations were characteristic to SLE because we could not detect any changes in the PKC levels in mononuclear cells of primary Sjögren's syndrome and mixed connective tissue disease patients. These results suggest that impaired PKC isoenzyme pattern may exist in the T cells and monocytes of SLE patients. Furthermore, the clinically efficient glucocorticoid application in SLE can increase the expression of some members of PKC system.

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.