Ulysse Pereira

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.

Effect of human skin explants on the neurite growth of the PC12 cell line

The skin is a densely innervated organ. After a traumatic injury, such as an amputation, burn or skin graft, nerve growth and the recovery of sensitivity take a long time and are often incomplete. The roles played by growth factors and the process of neuronal growth are crucial. We developed an in vitro model of human skin explants co‐cultured with a rat pheochromocytoma cell line differentiated in neuron in presence of nerve growth factor (NGF). This model allowed the study of the influence of skin explants on nerve cells and nerve fibre growth, probably through mediators produced by the explant, in a simplified manner. The neurite length of differentiated PC12 cells co‐cultured with skin explants increased after 6 days. These observations demonstrated the influence of trophic factors produced by skin explants on PC12 cells.

Characterization of the first coculture between human primary keratinocytes and the dorsal root ganglion-derived neuronal cell line F-11.

The epidermis can be considered as a sensory organ. Sensory neurons of the peripheral nervous system send many primary afferent fibers to the skin, creating a dynamic communication with epidermal cells. However, little is known about the functional interactions between the sensory fibers and the keratinocytes. It is therefore difficult to reproduce these interactions in vitro. We have developed an in vitro model based on the coculture of primary human keratinocytes and the dorsal root ganglion cell line F-11. F-11 cells have been classically used to mimic authentic peptidergic nociceptive neurons. We first investigated the morphological and functional characteristics of F-11 cells cultured in basal keratinocyte medium and then analyzed the influence of keratinocytes on these properties. We found that F-11 cells survived and differentiated well in this medium. Therefore, the addition of neurotrophins did not enhance their survival or differentiation. These neurons expressed sensory neuron markers and were able to release neuropeptides after capsaicin activation. We noted that neuropeptides release were obtained even at a low calcium concentration and that axonal outgrowth was not influenced by external calcium (Ca(2+)) levels. These properties were reproduced when F-11 cells were cocultured with keratinocytes, but they had no significant influence on axonal development or neuropeptide release. In this study, we describe for the first time the culture of F-11 neurons with another cell type. This coculture model in which keratinocytes and neurons are maintained in low Ca(2+) concentrations may be a useful in vitro alternative for studying and characterizing the close communication between keratinocytes and sensory neurons.