Nicholas Boulais

The epidermis: a sensory tissue

The skin is an efficient barrier which protects our bodies from the external environment but it is also an important site for the perception of various stimuli. Sensory neurones of the peripheral nervous system send many primary afferent fibres to the skin. They pass through the dermis and penetrate the basement membrane to innervate epidermal cells or remain as free endings. Nerve fibres are clearly involved in somatosensation. However, they are not always so numerous, for example in distal parts of the limbs, and some kinds of sensors can be at a distance of hundreds of micrometers from each other. The skin can detect patterns at a very fine and smaller scale, which suggests that nerve terminals are helped by epidermal sensors. All epidermal cells (keratinocytes, melanocytes, Langerhans cells and Merkel cells) express sensor proteins and neuropeptides regulating the neuro-immuno-cutaneous system. Hence, they must play a part in the epidermal sensory system. This review will consider the epidermal components of this forefront sensory system and the stimulations they perceive. The epidermis can be considered a true sensory tissue where sensor proteins and neurone-like properties enable epidermal cells to participate in the skin surface perception through interactions with nerve fibres.

Mechanisms of the sensory effects of tacrolimus on the skin

Background  Tacrolimus is an immunosuppressant drug currently used for the treatment of atopic dermatitis and pruritus. This topical therapy is effective and safe, but transient burning, stinging and itch are frequently reported.

Rat Merkel cells are mechanoreceptors and osmoreceptors.

Merkel cells (MCs) associated with nerve terminals constitute MC-neurite complexes, which are involved in slowly-adapting type I mechanoreception. Although MCs are known to express voltage-gated Ca2+ channels and hypotonic-induced membrane deformation is known to lead to Ca2+ transients, whether MCs initiate mechanotransduction is currently unknown. To answer to this question, rat MCs were transfected with a reporter vector, which enabled their identification. Their properties were investigated through electrophysiological studies. Voltage-gated K+, Ca2+ and Ca2+-activated K+ (KCa) channels were identified, as previously described. Here, we also report the activation of Ca2+ channels by histamine and their inhibition by acetylcholine. As a major finding, we demonstrated that direct mechanical stimulations induced strong inward Ca2+ currents in MCs. Depolarizations were dependent on the strength and the length of the stimulation. Moreover, touch-evoked currents were inhibited by the stretch channel antagonist gadolinium. These data confirm the mechanotransduction capabilities of MCs. Furthermore, we found that activation of the osmoreceptor TRPV4 in FM1-43-labeled MCs provoked neurosecretory granule exocytosis. Since FM1-43 blocks mechanosensory channels, this suggests that hypo-osmolarity activates MCs in the absence of mechanotransduction. Thus, mechanotransduction and osmoreception are likely distinct pathways.

Development of an in vitro coculture of primary sensitive pig neurons and keratinocytes for the study of cutaneous neurogenic inflammation.

Please cite this paper as: Development of an in vitro coculture of primary sensitive pig neurons and keratinocytes for the study of cutaneous neurogenic inflammation. Experimental Dermatology 2010; 19: 931–935.

Effects of the re‐innervation of organotypic skin explants on the epidermis

Abstract:  The nervous system takes part in skin homeostasis and interacts with skin cells. In in vitro organotypic skin models, these interactions are lost owing to the absence of nerve endings. We have developed an in vitro organotypic skin model based on a re‐innervated human skin explant using primary sensory neurons from the dorsal root ganglia of rats. After 10 days of co‐culture between skin explant and neurons, a dense network of nerve fibres was observed. The epidermis and dermis presented nerve fibres associated with cellular body from sensory neurons introduced in the co‐culture. Epidermal thickness, cell density and quality of re‐innervated skin explant were all higher when skin explants were re‐innervated by sensory neurons at 10 days of culture. Proliferation of epidermal cell was not modified, but the apoptosis was significantly diminished. Hence, this innovative model of co‐cultured skin explants and neurons allows better epidermal integrity and could be useful for studies concerning interactions between the skin and its peripheral nervous system.

Merkel Cells as Putative Regulatory Cells in Skin Disorders: An In Vitro Study

Merkel cells (MCs) are involved in mechanoreception, but several lines of evidence suggest that they may also participate in skin disorders through the release of neuropeptides and hormones. In addition, MC hyperplasias have been reported in inflammatory skin diseases. However, neither proliferation nor reactions to the epidermal environment have been demonstrated. We established a culture model enriched in swine MCs to analyze their proliferative capability and to discover MC survival factors and modulators of MC neuroendocrine properties. In culture, MCs reacted to bFGF by extending outgrowths. Conversely, neurotrophins failed to induce cell spreading, suggesting that they do not act as a growth factor for MCs. For the first time, we provide evidence of proliferation in culture through Ki-67 immunoreactivity. We also found that MCs reacted to histamine or activation of the proton gated/osmoreceptor TRPV4 by releasing vasoactive intestinal peptide (VIP). Since VIP is involved in many pathophysiological processes, its release suggests a putative regulatory role for MCs in skin disorders. Moreover, in contrast to mechanotransduction, neuropeptide exocytosis was Ca2+-independent, as inhibition of Ca2+ channels or culture in the absence of Ca2+ failed to decrease the amount of VIP released. We conclude that neuropeptide release and neurotransmitter exocytosis may be two distinct pathways that are differentially regulated.

Characterization of neurons from adult human skin-derived precursors in serum-free medium : a PCR array and immunocytological analysis.

Abstract:  Adult stem cells could be small sources of neurons or other cellular types for regenerative medicine and tissue engineering. Recently, pluripotent stem cells have been extracted from skin tissue, which opened a new accessible source for research. To routinely obtain a high yield of functional neurons from adult human skin stem cells with defined serum‐free medium, stem cells from abdominal skin were cultured in serum‐free medium. To differentiate them, we used a defined medium containing growth factors. Differentiated cells were identified using the following methods: (i) Oil‐red‐O staining for adipocytes, immunocytochemistry with antibodies recognising (ii) neurofilaments and PGP9.5 for neural differentiation, (iii) glial fibrillary acidic protein (GFAP) for glial differentiation, (iv) Ki‐67 for proliferative cells, (v) FM1‐43 staining to analyse vesicle trafficking in neuronal cells and (vi) a PCR array was used. Stem cells were floating in spheres and were maintained in culture for 4 months or more. They expressed nestin and Oct 4 and were proliferative. We induced specific differentiation into adipocytes, glial and neuronal cells. The yields of differentiated neurons were high and reproducible. They were maintained for long time (1 month) in the culture medium. Furthermore, these neurons incorporated FM1‐43 dye, which indicates a potent acquisition of synaptic features in neurons. Stem cells from adult human skin could be valuable and reproducible tools/source to obtain high numbers of functional specific cellular types, such as neurons, for tissue engineering. In this work, the possibility to obtain a high yield of differentiated neurons, with the ability of endocytosis and vesicle cell trafficking, was shown.

The whole epidermis as the forefront of the sensory system

Please cite this paper as: The whole epidermis as the forefront of the sensory system. Experimental Dermatology 2007; 16: 634–635. Skin is a major sensory organ. The awareness of the external environment goes through molecular receptors. We were highly interested in the paper from Denda (1), which proposes the concept of ‘epidermal keratinocytes as the forefront of the sensory system’. The authors suggest that keratinocytes are able to recognize environmental factors and then the information is processed and conveyed to the nervous system. Keratinocytes are equipped with sensing systems similar to those of neurons: (i) they express sensory receptors like the transient receptor potential (TRP) family or the ATP receptors; (ii) the stimulation of these receptors is followed by signal transduction and release of neurotransmitters by keratinocytes, whereas an electrochemical communication is possible due to the presence of voltage-gated channels; (iii) keratinocytes are able to process information from the nervous system through ionotropic and G-protein-coupled receptors for neurotransmitters. The close interactions between skin and nerve endings have lead to define integrated systems, which have been called skin-neuroendocrine system (2) or neuro-immunocutaneous system (3). These interactions are strongly enhanced by the existence of synapses between epidermal cells (keratinocytes, melanocytes, Langerhans cells and Merkel cells) and neurons (4). But all these neuron-like properties are not restricted to keratinocytes.

Effects of sangre de drago in an in vitro model of cutaneous neurogenic inflammation.

Please cite this paper as: Effects of sangre de drago in an in vitro model of cutaneous neurogenic inflammation. Experimental Dermatology 2010; 19: 796–799.

Presence of putative stem cells in Merkel cell carcinomas.

Editor Merkel cell carcinoma (MCC) has been recently described as probably secondary to clonal infection by a polyomavirus, which was named Merkel cell polyomavirus. Polyomaviruses are known to infect preferentially stem cells. Due to this argument, the possible co-localization of MCC with other skin tumours or atypical differentiations and the dermal location of MCC, it is probable that MCC cells are issued from Merkel cell (MC) progenitors rather than from mature MCs. In the skin, it has been largely demonstrated that melanoma contains a subpopulation of stem cells which can be considered as the source of the primary tumour mass and metastases. More recent data suggest that squamous cell carcinoma could originate from stem cells expressing cytokeratin 19 (CK19). Moreover, apocrine mixed tumours and dermatosarcoma protuberans appear to be tumours issued from nestinpositive cells. Many published findings are consistent with a neural-crest origin for the embryonic precursors of MCs and subsequent differentiation only once in the epidermis. In this hypothesis, MCs could arise from a skin precursor of neural-crest origin. Recently, new arguments strongly suggest that MCs could originate from epidermal stem cells expressing cytokeratin 14 by an Atoh1-dependent mechanism. In this study, we searched for the presence of putative stem cells expressing nestin, CK14 or CK19 in Merkel cell carcinomas, that could be putative cancer stem cells (CSCs). We examined 13 MCCs by immunohistochemistry by using monoclonal antibodies recognizing nestin (Santa Cruz, CA, USA), CK14 (Progen, Heidelberg, Germany) or CK19 (Progen). No double immunostaining was performed. Remarkably, we found immunoreactive cells for CK14 (Fig. 1), CK19 and nestin (Fig. 2) in MCCs. Amongst tumoural cells, an estimated positive rate was 1–3%, 5–15% and 20–30%, respectively. The staining intensity was strong in most samples for CK14 and nestin but weaker for CK19. Outside tumours, we noted immunoreactivity for CK14 on hair follicles and cells of the basal layer of the epidermis, for CK19 on hair follicles and rarely on basal epidermal cells and for nestin on hair follicles, sebaceous and sweat glands. The presence of numerous CK14 tumoural cells in MCCs is of a particular interest because convincing arguments have been recently given to show that MCs are descended from epidermal CK14 stem cells in embryos and adults and that they do not originate in neural-crest cells. We suggest that CK14-positive tumoural cells could be stem cells that have undergone a malignant transformation and could be CSCs in the MCC. Their presence in MCCs is a new argument in favour of an epidermal origin of MCs. The MCCs showing squamous metaplasia and mixed MCCs and squamous cell carcinomas as well as the increased expression of CK19 in squamous cell carcinomas suggest that MCCs could