Induction of eosinophil‐ and Th2‐attracting epidermal chemokines and cutaneous late‐phase reaction in tape‐stripped skin

Abstract: Skin barrier damage induces various harmful or even protective reactions in the skin, as represented by enhancement of keratinocyte cytokine production. To investigate whether acute removal of stratum corneum modulates the production of chemokines by epidermal cells, we treated ears of BALB/c and C57BL/6 mice by tape‐stripping, or acetone‐rubbing as a control of acute barrier disruption procedure. There was no difference between the tape‐stripped and acetone‐rubbed skin sites in the increased and recovered levels of transepidermal water loss. The mRNA expression levels of all the chemokines tested, including Th1 chemokines (CXCL10, CXCL9 and CXCL11), Th2 chemokines (CCL17 and CCL22) and eosinophil chemoattractant (CCL5), were higher in the epidermal cells from BALB/c than in those of C57BL/6 mice. In particular, CCL17, CCL22 and CCL5 were remarkably elevated in BALB/c mice and augmented by tape‐stripping more markedly than acetone‐rubbing, whereas Th1 chemokines were enhanced by acetone‐rubbing more remarkably. Tape‐stripping induced dermal infiltration of eosinophils in BALB/c but not C57BL/6 mice. In a contact hypersensitivity model, where BALB/c mice were sensitized on the abdomen and challenged on the ears with fluorescein isothiocyanate, mice exhibited higher ear swelling responses at the late‐phase as well as delayed‐type reactions, when challenged via the tape‐stripped skin. The challenge via tape‐stripped skin augmented the expression of IL‐4 and CCR4 in the skin homogenated samples, indicating infiltration of Th2 cells. These findings suggest that acute barrier removal induces the expression of Th2 and eosinophil chemokines by epidermal cells and easily evokes the late phase reaction upon challenge with antigen.     

Topical tacalcitol (1,24-(R)-dihydroxyvitamin D3) induces a transient increase in thymidine incorporation and calmodulin content in pig epidermis following tape stripping in vivo

Tape stripping induces transient increase in keratinocyte proliferation in vivo. The effects of tacalcitol (l,24-(R)-dihydroxyvitamin D₃) ointment on the cell kinetics of pig epidermis after the tape stripping were investigated. The tacalcitol ointment (2 μg/g) was applied once to the back of pigs immediately after the tape stripping. The pig epidermal cell kinetics were analyzed at various times following the treatment. Tape stripping transiently increased thymidine incorporation of keratinocytes; the maximal effect was observed at 24 h. Tape stripping-induced increase in thymidine incorporation was markedly augmented by tacalcitol treatment. At 24 h following the tape stripping DNA-flow cytometry revealed an accelerated transition from G0/1 to S phase of cell cycle in tacalcitol treated epidermis. There was no significant difference, however, in mitotic counts and G2/M phase fractions between tape stripping-treated and tape stripping plus tacalcitol ointment-treated epidermis. We also measured calmodulin content of pig epidermis following the treatments. Although tape stripping slightly increased calmodulin content of pig epidermis, this was statistically not significant. Tape stripping plus tacalcitol ointment treatment resulted in a significant increase in calmodulin content at 24 h following the treatment. There was no significant difference in calmodulin content between tape stripping treated- and tape stripping plus tacalcitol-treated epidermis.     

Superoxide dismutase in epidermis: its relation to keratinocyte proliferation

Decreased superoxide dismutase (SOD) activity has been reported in various hyperproliferative tissues. In order to elucidate the significance of SOD activity in keratinocyte proliferation, we measured the SOD activity in several pathologic epidermal conditions. The SOD activity was significantly decreased in psoriatic hyperproliferative epidermis as well as in basal cell epithelioma, squamous cell carcinoma and seborrheic keratosis. Following tape stripping in vivo, pig epidermis revealed a marked increase in thymidine uptake and mitotic counts. The peak of the thymidine uptake was observed at 24 h, which was followed by the increased mitotic counts (peak at 48 h). The pig skin revealed a decreased epidermal SOD activity following the tape stripping. A decline of SOD activity was observed within 7 days after the tape stripping and about a 50% decrease was observed at 48 h. Epidermis is known to contain both Cu, Zn-SOD and Mn-SOD activities; the decreased SOD activity after tape stripping was mainly due to decreased Cu, Zn-SOD activity without any significant alteration of Mn-SOD activity. Following UVB irradiation (2MED) in vivo, pig epidermis revealed decreased thymidine incorporation as well as decreased mitotic counts. Epidermal SOD activity, however, was not altered by the UVBirradiation. Pig epidermis is also known to indicate decreased cell proliferative activity in the presence of various chemicals in vitro. These modalities, which include retinoid (Ro 10-1670), colchicine and protein synthesis inhibitors (puromycin, actinomycin D, cycloheximide), had no effect on the epidermal SOD activity. Our results indicate that, although the decreased SOD activity appears to be comcomitant with keratinocyte hyperproliferation, no alteration of SOD activity is observed in hypoproliferative epidermis. The significance of the altered SOD activity in the regulatory mechanism of keratinocyte proliferation remains unknown at present.     

Calcium and potassium inhibit barrier recovery after disruption, independent of the type of insult in hairless mice

Abstract Disruption of the cutaneous permeability barrier induces metabolic responses in the epidermis which result in barrier recovery. Barrier disruption by either solvent treatment or tape stripping results in the loss of the epidermal calcium gradient. Previous studies in acetone treated hairless mice have shown that maintaining this calcium gradient inhibits barrier repair, suggesting that alterations in the epidermal calcium concentration may be an important signal for barrier homeostasis. In the present study, we show that in hairless mice disruption of the barrier by treatment with the detergent. SDS, also results in the loss of the calcium gradient, as demonstrated both semi-quantitatively with ultrastructural cytochemical localization and quantitatively using proton induced X-ray emission (PIXE). Additionally, immersion in calcium containing solutions delays barrier repair after either detergent (SDS treatment) or mechanical (tape stripping) disruption of the barrier, as reported previously for acetone treated skin. These results indicate that barrier disruption, regardless of the insult, induces changes in the epidermal calcium gradient which may play an important role in signaling the metabolic changes required for barrier homeostasis.     

Large scale study of epidermal recovery after stratum corneum removal: dynamics of genomic response

Please cite this paper as: Large scale study of epidermal recovery after stratum corneum removal: dynamics of genomic response. Experimental Dermatology 2010; 19: 259–268. Abstract: The stratum corneum (SC) is a superficial skin compartment that protects the body from the outside environment. Any disturbance of this function induces cascading steps of molecular and cellular repair in the whole epidermis. The aim of this study was to investigate epidermal gene expression following SC removal by tape stripping. Twenty‐nine healthy male volunteers were included (27 ± 4 years old). Tape stripping was processed on one inner forearm, the other unstripped forearm served as a control. Epidermis samples were collected at 2, 6, 19, 30 and 72 h after tape stripping. Trans‐epidermal water loss measurements were performed at each step to monitor barrier restoration. Total RNA was extracted from collected epidermis samples and analysed by using DermArray® cDNA microarrays. Among 4000 genes under investigation, we found that the expression of 370 genes varied significantly at least once during the time following stripping. Using an original clustering method, the modulated genes were gathered into eight groups. A functional characterization of the clusters enabled us to get a dynamic and global view of the main molecular processes taking place during epidermal recovery.     

The morphologic changes in lamellar bodies and intercorneocyte lipids after tape stripping and occlusion with a water vapor-impermeable membrane

It has been reported that artificial restoration of barrier function by a water vapor-impermeable membrane after tape stripping induces barrier abrogation in hairless mice, impeding rather than enhancing barrier recovery. To address this issue, we examined the morphologic changes in the epidermis after tape stripping and occlusion with a water vapor-impermeable membrane in murine skin. Male hairless mice were used for all studies of barrier perturbation and occlusion. Barrier disruption was achieved by repeated application of cellophane tape. Immediately after tape stripping the animals were wrapped in a tightly fitting water vapor-impermeable membrane. Transepidermal water loss (TEWL) was measured 20 min after tape stripping and 14, 24, 36, 48 and 60 h after occlusion. For electron microscopy the samples were treated with osmium tetroxide (OsO ₄ ) or ruthenium tetroxide (RuO ₄ ). When tape-stripped animals were wrapped in a water vapor-impermeable membrane, thereby preventing water flux, barrier function did not recover normally. These results demonstrate that an artificial block to TEWL with an impermeable membrane did not enhance barrier recovery. By electron microscopy many transitional cells and lacunae of various sizes were seen within the intercellular spaces of the stratum corneum after occlusion following tape stripping. Occlusion also caused alterations in both lipid lamellar membrane structures in the stratum corneum interstices and the lamellar bodies in the cytosol of granulocytes and transitional cells. Secreted lamellar body contents also appeared to be abnormal in the stratum corneum-stratum granulosum junction. Received: 17 December 1996 / Accepted: 14 November 1997     

Association of cyclic adenosine monophosphate with permeability barrier homeostasis of murine skin

Activation of Gs protein increases the intracellular cyclic adenosine monophosphate (cAMP) level, and the Gs protein-linked receptor has been implicated in the skin barrier homeostasis. In this study, we investigated the role of cAMP in epidermal barrier function. The barrier was disrupted by tape stripping or treatment with acetone. Immediately after barrier disruption, reagents affecting the cAMP level were topically applied. Topical application of forskolin, which activates cAMP synthesis delayed barrier recovery, whereas application of the antagonist of cAMP, cAMP-Rp, accelerated barrier recovery. Moreover, application of 9-cyclopentyladenine, an inhibitor of cAMP synthesis also accelerated barrier recovery. Tape stripping was found to increase the cAMP in the epidermis. Light and electron microscopic observations showed the delay of lamellar body secretion by forskolin and acceleration of the lamellar body secretion by cAMP-Rp. Application of an inhibitor of protein kinase A did not affect the barrier recovery rate. The delay of barrier recovery induced by forskolin was blocked by the voltage-gated calcium channel blockers, nifedipine and verapamil. In cultured keratinocytes, forskolin increased the intracellular calcium concentration and both nifedipine and verapamil blocked the increase. These results suggest that intracellular cAMP in the epidermis is involved in skin barrier homeostasis.     

Multiparameter flow cytometric characterization of epidermal cell suspensions prepared from normal and hyperproliferative human skin using an optimized thermolysin-trypsin protocol

Reliable flow cytometric analysis of normal and diseased skin requires pure epidermal single-cell suspensions. Several methods to separate the dermis from the epidermis are available. The proteolytic enzyme thermolysin separates the epidermis from the dermis at the lamina lucida and therefore permits reliable dermoepidermal separation. In the present study an optimized cell isolation procedure using thermolysin and trypsin is described, which is particularly suitable for punch biopsies. A 16–20-h (overnight) incubation of biopsies taken from normal and hyperproliferative skin with thermolysin (0.5 mg/ml) at 4°C produced a selective separation of the dermis and epidermis. After a 30-min trypsin incubation (0.25 mg/ml) at 37°C a cell suspension was produced which was characterized by minimal cell damage (cellular debris and clumps), a high recovery of basal cells and high quality DNA histograms. Furthermore, dermal contamination was very low. The thermolysin-trypsin separation methodology followed by triple-labelling flow cytometry provided a precise quantification of the percentage of keratin 10-positive cells, vimentin-positive cells and cells in S and G₂M phases. Proliferative activity was selectively measured in the basal, the suprabasal and the non-keratinocyte compartment at various time intervals during epidermal regeneration after adhesive tape stripping. In contrast to the non-keratinocytes, the percentage of cells in S and G₂M phases in the basal keratinocytes and in the suprabasal compartment increased 44–48 h after stripping. The increased proliferation following tape stripping was paralleled by an increased invasion of vimentin-positive cells into the epidermis and preceded by a decreased number of keratin 10-positive cells. Thermolysin-trypsin separation followed by three-colour flow cytometry permits a highly selective characterization of normal and hyperproliferative epidermis.     

Topical stratum corneum lipids accelerate barrier repair after tape stripping, solvent treatment and some but not all types of detergent treatment

Summary Topical act-tone treatment extracts lipids from the stratum corneum. and disrupts the permeability barrier, resulting in a homeostatic response in the viable epidermis that ultimately repairs the barrier. Recently, we have developed an optimal lipid mixture (cholesterol, ceramide. palmitate and linoleate 4–3:2.3:1:1.08) that, when applied topically, accelerates barrier repair following extensive disruption of the barrier by acetone. The present study determined if topical treatment with this optimal lipid mixture would have beneticial effects following disruption of the barrier by petroleum ether, tape stripping, or by detergent treatment. Also, we determined if barrier repair was accelerated after moderate disturbances of barrier function. Following moderate or extensive disruption of the barrier by acetone or petroleum ether (solvents), or tape stripping (mechanical), application of the optimal lipid mixture accelerated barrier repair. Additionally, following barrier disruption with V-laurosarcosine free acid or dodecylbenzensulphuric acid (detergents), the optimal lipid mixture similarly accelerated barrier repair. However, following disruption of the barrier with different detergents, sodium dodecyl sulphate and ammonium lauryl sulphosuccinate. the optimal lipid mixture did not improve barrier recovery. Thus, the optimal lipid mixture is capable of accelerating barrier repair following disruption of the barrier by solvent treatment or tape stripping (mechanical), and by certain detergents such as Sarkosyl and dodecylbenzensulphuric acid. The ability of the opiimal lipid mixture to accelerate barrier repair after both moderate and extensive degrees of barrier disruption suggests a potential clinical use for this approach.     

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins of newborn rat skin. I. Cell strata and nuclear proteins.

The proteins obtained from separated cells of neonatal rat dermis, four cell populations of epidermis, and an epidermal nuclear preparation were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Comparison of the results of the insoluble proteins of the dermis and epidermis show no similarity of the major protein bands, indicating the effective separation of the dermis and epidermis and absence of cross-contamination. The gels of the soluble proteins of the basal, spinous, and granular layers of the epidermid are very similar. Only the pattern of bands of the cornified cells differs in that some of these bands are absent and at least three new bands are present. The insoluble proteins have specific differences in the protein content related to the cell structure. An example is the nuclear protein bands which correspond with the most prominent bands in the gels of basal and spinous layer proteins, but are absent, with the possible exception of one band, from gels of cornified cell proteins.     

Stimulated growth of human and pig epidermal nerve fibers by tape stripping

Nerve growth studies in adults usually rely upon nerve regeneration that follows axon disruption. In this study elongation of the epidermal nerve fibers occurred in human and pig epidermis stimulated to hypertrophy by removing the stratum corneum with repetitive applications of tape (tape stripping). Epidermal thickening was accompanied by elongation of the epidermal nerve fibers. This study demonstrates that changing the cellular and chemical environment of nerves by tape stripping is a feasible method to study nerve fiber growth in a physiological manner.     

Tape stripping on a human nail: quantification of removal

Background/purpose: Tape stripping is commonly used to investigate the stratum corneum (SC). This study assesses if protein quantitative tape stripping method was suitable for human nails. Method: We used a colorimetric method to quantify proteins removed by the tape. Water barrier functions as a result of tape stripping were also observed by changes in transonychial water loss (TOWL) from the baseline. Results: Using tape stripping, we observed no difference between nails in the protein quantity removed by tape stripping (P=0.39). The mean TOWL before and after tape stripping were 6.9 and 9.3 g/m²/h, respectively; this was significantly increased in tape stripped nails (P<0.0001). Conclusion: Tape stripping seems to be an effective method to extract proteins from human nail plate and may aid the study of nail structure and function. Further studies are needed to extend our results in terms of age, gender, ethnicity and disease.     

The effects of anti-prostaglandin agents on epidermal proliferation induced by dermal inflammation

Experimentally induced dermal inflammatory lesions cause epidermal hyperplasia and increased thymidine autoradiographic indices in the overlying epidermis. In the experiments described here anti-prostaglandin agents (indomethacin, aspirin, prednisolone and polyphloretin phosphate) were given to guinea-pigs with experimentally induced dermal granulomata in an effort to learn more about the dermal control of epidermal proliferation. Indomethacin caused a significant reduction in the epidermal hyperplasia and labelling index in the epidermis overlying carageenan granulomata. Similar but less marked reductions in epidermal proliferation were seen with the other agents used. Some reduction, which proved not to be statistically significant, was also seen in the epidermal proliferation due to kaolin granulomata. Slight reduction in epidermal hyperplasia, but no effect on thymidine incorporation, was seen in proliferation induced by tape stripping.     

Effect of endothelial nitric oxide synthase on epidermal permeability barrier recovery after disruption

Background We previously demonstrated that neuronal nitric oxide synthase (nNOS) in epidermal keratinocytes is associated with epidermal permeability barrier homeostasis. Objectives In the present study, we examined the contributions of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) to epidermal permeability barrier homeostasis. Methods We measured the barrier recovery rate after tape stripping of the epidermis of iNOS and eNOS knockout mice, and carried out electron‐microscopic observation of the epidermis after acetone treatment. Results The barrier recovery rate of eNOS knockout mice was significantly faster than that of the wild‐type control, while no significant difference was observed between iNOS knockout mice and wild‐type mice. Electron‐microscopic observation at 1 h after acetone treatment indicated that barrier recovery of both nNOS and eNOS mice was faster than that of wild‐type mice, and lamellar body secretion was accelerated in both types of knockout mice. Conclusions These results suggested that both nNOS and eNOS play roles in epidermal barrier homeostasis and lamellar body secretion.     

Auftrennung wasserlöslicher,epidermaler Proteine mittles Disc-Elektrophorese

Zusammenfassung Die Auftrennung wäßriger Extrakte von normaler und psoriatischer Epidermis sowie von Psoriasisschuppen mittels Disc-Elektrophorese ergab 4–5 Proteinbanden mit größerer elektrophoretischer Beweglichkeit als Albumin. Diese Banden konnten als Nucleoproteine identifiziert werden. In normaler Hornschicht und Callus wurde nur eine Proteinbande vor dem Albumin beobachtet, die der am schnellsten wandernden Bande der Epidermisextrakte entspricht.In den Extrakten von psoriatischer Epidermis und von Psoriasisschuppen wurden mehrere Nucleoproteine in der Globulinfraktion des Proteinspektrums nachgewiesen, die in normaler Epidermis und Hornschicht nicht angetroffen werden; besonders ausgeprägt sind zwei benachbarte Proteinbanden.

---

Separation of water soluble epidermal proteins by means of disc-electrophoresis

Summary Separation of proteins of water soluble extracts from normal and from psoriatic epidermis, as well as from psoriatic scales by means of disc electrophoresis yielded 4–5 protein bands showing greater electrophoretic mobility than does albumin. These proteins could be identified as nucleoproteins. In normal stratum corneum and callus only one protein band was observed in front of albumin. This protein seems to be identical with the fastest moving band obtained with the epidermal extracts.In extracts of psoriatic scales and psoriatic epidermis the globulin fraction of the proteinspectrum showed several nucleoproteins not found in normal epidermis and stratum corneum. Most characteristic were two adjacent bands.

---

---

Herrn Prof. Dr. med. Franz Herrmann zum 70. Geburtstag gewidmet.

---

Wir danken der Deutschen Forschungsgemeinschaft für die Unterstützung der Arbeit.     

Permeability barrier disruption increases the level of serine palmitoyltransferase in human epidermis

Sphingolipids play an important role in the homeostasis and barrier function of human stratum corneum. A disturbance of sphingolipid formation is supposed to be a crucial factor for the increased transepidermal water loss in common skin diseases like atopic eczema or psoriasis. The key enzyme for de novo sphingolipid synthesis is serine palmitoyltransferase, which consists of two different subunits, named LCB1 and LCB2 proteins. In order to investigate the induction of LCB2 synthesis in human epidermis, skin barrier disruption was performed by tape stripping on the forearm of healthy volunteers enough to obtain a 3-4-fold increase in transepidermal water loss. Skin punch biopsies were taken before and 0.5, 2, 4, and 8 h after tape stripping by each volunteer to measure LCB2 at the mRNA level. Additional biopsies taken before and 12 h after tape stripping were used to evaluate LCB2 at the protein level. Our results show that 0.5 and 2 h after tape stripping the LCB2 mRNA expression was decreased compared to control in all cases. A significant increase in LCB2 mRNA expression was detectable 4 h after barrier disruption, with individual variations; no further increase was detectable 8 h after tape stripping. Immunohistochemical analysis 12 h after barrier disruption showed increased LCB2 immunolocalization in the inner epidermis, whereas in the outer epidermis it was similar to control. LCB2 mRNA expression preceded the expression of the corresponding protein by 4-8 h. Our findings support the concept that an increase in transepidermal water loss is an obligatory trigger for the upregulation of serine palmitoyltransferase mRNA expression in humans.     

The changes of epidermal calcium gradient and transitional cells after prolonged occlusion following tape stripping in the murine epidermis.

Disruption of the epidermal permeability barrier causes an immediate loss of the calcium gradient, and barrier recovery is parallel with the restoration of the calcium gradient in the epidermis. Artificial restoration of the barrier function by occlusion with a water vapor-impermeable membrane abrogate the expected increase in lipid synthesis and retard the barrier recovery, as well as block the normalization of the epidermal calcium gradient. To clarify the long-term effects of occlusion after acute barrier perturbation, we studied the calcium distribution and epidermal keratinocytes response after occlusion with a water vapor-impermeable membrane immediately following tape stripping in the murine epidermis. Acute barrier disruption caused an immediate depletion of most calcium ions in the upper epidermis, obliterating the normal calcium gradient. When the skin barrier function was artificially corrected by occlusion, the return of calcium ions to the epidermis was blocked. After 2 h of air exposure or occlusion, the density of epidermal calcium precipitates remained negligible. The transitional cell layers appeared with occlusion, but not or negligibly with air exposure. By 6 h though, calcium precipitates could be seen, the density of the calcium precipitates with occlusion was more sparse than with air exposure. With the air exposure, the thickness of the stratum corneum had normalized and the calcium gradient nearly recovered to normal after 24 h. The longer the occlusion period, the greater was the increase of transitional cells. By 60 h of occlusion, the thickness of the stratum corneum had increased and the transitional cell layers had disappeared, in parallel with the calcium gradient which was almost normalized. These results show that prolonged occlusion of tape-stripped epidermis induced transitional cells and delayed the restoration of the epidermal calcium gradient, the stratum corneum was then restored, transitional cells having disappeared, in parallel with normalization of the epidermal calcium gradient.