Junichi Kumamoto

Oxytocin is expressed in epidermal keratinocytes and released upon stimulation with adenosine 5′-[γ-thio]triphosphate in vitro

Oxytocin is a neuropeptide produced primarily in the hypothalamus and is best known for its roles in parturition and lactation. It also influences behaviour, memory and mental state. Recent studies have suggested a variety of roles for oxytocin in peripheral tissues, including skin. Here we show that oxytocin is expressed in human skin. Immunohistochemical studies showed that oxytocin and its carrier protein, neurophysin I, are predominantly localized in epidermis. RT‐PCR confirmed the expression of oxytocin in both skin and cultured epidermal keratinocytes. We also show that oxytocin is released from keratinocytes after application of adenosine 5′‐[γ‐thio]triphosphate (ATPγS, a stable analogue of ATP) in a dose‐dependent manner. The ATPγS‐induced oxytocin release was inhibited by removal of extracellular calcium, or by the P2X receptor antagonist 2′,3′‐O‐(2,4,6‐trinitrophenyl)adenosine 5′‐triphosphate (TNP‐ATP). These results suggest that oxytocin is produced in human epidermal keratinocytes and is released in response to calcium influx via P2X receptors.

Low environmental humidity induces synthesis and release of cortisol in an epidermal organotypic culture system.

Dry environmental conditions induce a variety of skin pathologies and a recent report indicating that cortisol synthesis in epidermis was increased during wound healing led us to hypothesize that environmental dryness might induce increased cortisol secretion in epidermis. Therefore, we incubated a skin equivalent model under dry (relative humidity: less than 10%) and humid (relative humidity: approximately 100%) conditions for 48 hours and evaluated cortisol secretion and mRNA levels of cortisol‐synthesizing enzyme (steroid 11β‐hydroxylase, CYP11B1) and IL‐1β. Cortisol secretion was increased threefold, and CYP11B1 and IL‐1β mRNAs were increased 38‐fold and sixfold, respectively, in the dry condition versus the humid condition. Occlusion with a water‐impermeable plastic membrane partially blocked the increases in cortisol secretion and CYP11B1 and IL‐1β mRNA expression in the dry condition. Thus, environmental dryness might induce increased cortisol secretion in epidermis of diseased skin characterized by epidermal barrier dysfunction, potentially influencing mental state and systemic physiology.

Intracellular calcium response to high temperature is similar in undifferentiated and differentiated cultured human keratinocytes

Abstract:  A series of thermo‐sensitive proteins is expressed in epidermal keratinocytes, where they function as cation channels that are activated at temperatures in the range of 28–42°C. Here, we examined the influence of high temperature on intracellular calcium level in undifferentiated and differentiated cultured human keratinocytes, which are considered to be representative of deeper and upper epidermal layers, respectively. We found that the percentages of responsive keratinocytes among undifferentiated and differentiated cells exposed to temperatures in the warm–hot range (30–50°C) were essentially the same. In contrast, we previously demonstrated that undifferentiated keratinocytes were more sensitive than differentiated cells to low temperature (<22°C). Thus, the deeper layer of the epidermis is more sensitive to cold than the upper layer, but the response to warm–hot temperatures appears to be homogeneous throughout the epidermis.

Mathematical Modeling of Calcium Waves Induced by Mechanical Stimulation in Keratinocytes

Recent studies have shown that the behavior of calcium in the epidermis is closely related to the conditions of the skin, especially the differentiation of the epidermal keratinocytes and the permeability barrier function, and therefore a correct understanding of the calcium dynamics is important in explaining epidermal homeostasis. Here we report on experimental observations of in vitro calcium waves in keratinocytes induced by mechanical stimulation, and present a mathematical model that can describe the experimentally observed wave behavior that includes finite-range wave propagation and a ring-shaped pattern. A mechanism of the ring formation hypothesized by our model may be related to similar calcium propagation patterns observed during the wound healing process in the epidermis. We discuss a possible extension of our model that may serve as a tool for investigating the mechanisms of various skin diseases.

Distinct intracellular calcium responses of individual cultured human keratinocytes to air pressure changes

We previously showed that application of hydraulic pressure to cultured human keratinocytes induced elevation of intracellular calcium concentration ([Ca2+]i), but the absolute value of the pressure could not be determined.

Numerical and comparative three-dimensional structural analysis of peripheral nerve fibres in epidermis of patients with atopic dermatitis.

DEAR EDITOR, There is some evidence that the cause of pruritus in patients with atopic dermatitis (AD) is increased nerve fibre outgrowth in the epidermis. For example, itching requires the presence of epidermis, and although nerve fibres penetrate into the uppermost layer of the epidermis in healthy subjects, the number of nerve fibres in the skin of patients with AD is reported to be higher than in healthy controls. Also, levels of nerve growth factor (NGF) and its receptors in epidermis of patients with AD are increased, while production of semaphorin 3A, which retracts NGF-sensitive neurons, is decreased. Keratinocytes from patients with AD express an increased level of NGF mRNA and show enhanced outgrowth of calcitonin gene-related peptide-immunoreactive neurites in a coculture system of keratinocytes and neurons. However, nerve fibres in epidermis of healthy subjects and patients with AD have not been quantitatively compared. Two-photon microscopy is superior to conventional microscopy, including confocal microscopy, for precise imaging of thick tissue sections because excitation in the near-infrared region minimizes tissue absorption and background autofluorescence. Here we used two-photon microscopy to construct cross-sectional and three-dimensional images of nerve fibres in epidermis of healthy subjects and patients with AD, and quantified their structural features in order to examine the validity of the idea that outgrowth of nerve fibres occurs in epidermis of patients with AD. The details of the patients and the methods are given in Data S1 (see Supporting Information). Representative immunofluorescence staining images (Fig. 1a–c) and binary images (Fig. 1d– f) of sequential 10-lm cross-sectional images (Fig. S1; see Supporting Information) show that the epidermis is thickest in lichenified lesions, followed by unlichenified lesions then healthy skin. Although the number of nerve fibres in the epidermis was not significantly different among the three groups, the length of nerve fibres was greatest in lichenified lesions, followed by unlichenified lesions and healthy skin (Fig. 1d–f). These results are quantified in Figure 1g–n. For numerical analysis, we chose samples obtained only from back skin of male subjects to minimize possible influences of different skin regions and sex on nerve fibre distribution. Details of the numerical processing are given in Data S1 (see Supporting Information). The epidermis was thicker in lichenified lesions than in the other groups (Fig. 1g). The asperity of the basal membrane was also higher in lichenified lesions than in the other groups (Fig. 1h). The length of nerve fibres extending from the basal membrane in lichenified lesions was significantly greater than in the other groups (Fig. 1i). The percentages of nerve fibres within 0–15° of vertical with respect to the skin surface are shown in Figure 1j; the results indicate that nerve fibres in the epidermis of both unlichenified and lichenified lesions were more vertically oriented towards the skin surface than those in healthy skin. The length of nerve fibres in the epidermis per length of stratum corneum is shown in Figure 1k. The density of nerve fibres was highest in healthy skin (Fig. 1l). The number of nerve fibres per 1 mm basal membrane was also highest in healthy skin (Fig. 1m). Furthermore, the density of nerve fibres in the region to a depth of 50 lm from the stratum corneum was also highest in healthy skin (Fig. 1n). Healthy subjects (Fig. 2a–d) showed many nerve fibres in the epidermis. In the unlichenified skin of patients with AD, the epidermis was thickened, and thick nerve fibres were observed in the dermis, although the number of nerve fibres in the epidermis seemed to be lower than in healthy skin (Fig. 2e–h). These changes were more marked in lichenified skin (Fig. 2l). In particular, nerve fibres did not appear to penetrate far into the epidermis in lichenified lesions (Fig. 2l). Next, nerve fibres within 50 lm of the skin surface were examined. Figure 2m shows representative processed images in which nerve fibres are shown in black. Quantitative results are shown in Figure 2n. The numbers of black pixels were lower in unlichenified and lichenified lesions than in healthy skin, in agreement with the findings in Figure 1n. It has been suggested that an increase of nerve fibres in patients with AD might be the cause of pruritus. However, when we analysed the three-dimensional distribution by means of two-photon microscopy (Fig. S2; see Supporting Information), we found that the density of nerve fibres was higher in healthy subjects than in patients with AD (Fig. 1l– n). Although individual nerve fibres in lichenified and unlichenified lesions were longer (stretched) than in healthy skin, the total amount of nerve fibres was lower than in healthy skin, especially in the uppermost layer of the epidermis. These results are in contrast to previous findings in the antecubital fossa and abdomen of healthy subjects and patients with AD. Moreover, although stretched nerve fibres were observed in disease sites, they did not reach the upper layer of the epidermis (Fig. 2h,l). In addition, previous reports have

Frontiers in epidermal barrier homeostasis – an approach to mathematical modelling of epidermal calcium dynamics

Intact epidermal barrier function is crucial for survival and is associated with the presence of gradients of both calcium ion concentration and electric potential. Although many molecules, including ion channels and pumps, are known to contribute to maintenance of these gradients, the mechanisms involved in epidermal calcium ion dynamics have not been clarified. We have established that a variety of neurotransmitters and their receptors, originally found in the brain, are expressed in keratinocytes and are also associated with barrier homeostasis. Moreover, keratinocytes and neurons show some similarities of electrochemical behaviour. As mathematical modelling and computer simulation have been employed to understand electrochemical phenomena in brain science, we considered that a similar approach might be applicable to describe the dynamics of epidermal electrochemical phenomena associated with barrier homeostasis. Such methodology would also be potentially useful to address a number of difficult problems in clinical dermatology, such as ageing and itching. Although this work is at a very early stage, in this essay, we discuss the background to our approach and we present some preliminary results of simulation of barrier recovery.

External negative electric potential accelerates exocytosis of lamellar bodies in human skin ex vivo

Exocytosis of lamellar bodies at the uppermost nucleated layer of the epidermis is a crucial process for epidermal permeability barrier homoeostasis. We have previously suggested that skin surface electric potential might be associated with barrier homoeostasis. Thus, we hypothesized that the potential might drive exocytosis of lamellar bodies. In this study, we tested this idea by applying negative electric potential (−0.5 V) to human skin samples ex vivo for 2 h and observing the ultrastructure of the uppermost layer. The secretion of lamellar bodies was accelerated in the potential‐applied skin, compared to that in untreated control skin. Multiphoton observation indicated that extracellular lipid domains were more extensive in treated skin than in control skin. Moreover, the calcium ion gradient was greater at the uppermost layer of the epidermis of treated skin, compared to that in control skin. These results indicate that electric potential may regulate lamellar body secretion in healthy human skin.

In vitro formation of organized structure between keratinocytes and dorsal‐root‐ganglion cells

We recently found that the morphology in a co‐culture system of keratinocytes and dorsal‐root ganglion‐derived cells depended on the timing of seeding of the two cell types. In skin, epidermis is formed first, followed by construction of peripheral nerve structure. Therefore, we hypothesized that formation of peripheral nerve structure in the epidermis might be driven by interaction between keratinocytes and nerve cells. In the present study, we tested this idea by incubating keratinocytes and dorsal‐root ganglion cells in a spatially separated manner and observing the morphological changes in the co‐culture system. Extension of nerve fibre‐like structures from the ganglion cells was observed, and within 3 days after seeding, many nerve fibre‐like extensions penetrated into the keratinocyte cluster, subsequently forming a network that appeared to resemble the cutaneous peripheral nervous system. Our present model may be useful for studying the formation of peripheral nerve structure in the skin.

Abnormal Morphology of Blood Vessels in Erythematous Skin From Atopic Dermatitis Patients.

Abstract:Previous studies suggest that altered peripheral blood circulation might be associated with erythema or inflammation in atopic dermatitis (AD) patients. However, the overall structure of blood vessels and capillaries in AD skin is poorly understood because most studies have involved light-microscopic observation of thin skin sections. In the present study, we compared the 3-dimensional structures of peripheral blood vessels of healthy subjects and AD patients in detail by means of 2-photon microscopy. In skin from healthy subjects, superficial vascular plexus and capillaries originating from flexous blood vessels were observed. However, skin from AD patients contained thickened, flexuous blood vessels, which might be associated with increased blood flow, in both erythematous and nonlesional areas. However, patients with lichenification did not display these morphological changes. Bifurcation of vessels was not observed in either erythematous or lichenification lesions. These results might be helpful for developing new clinical strategies to treat erythema in AD patients.