Localization of calcium in murine epidermis following disruption and repair of the permeability barrier

Summary Perturbation of the cutaneous permeability barrier results in rapid secretion of epidermal lamellar bodies, and synthesis and secretion of new lamellar bodies leading to barrier repair. Since external Ca²⁺ significantly impedes the repair response, we applied ion capture cytochemistry to localize Ca²⁺ in murine epidermis following barrier disruption. In controls, the numbers of Ca²⁺ precipitates in the basal layer were small, increasing suprabasally and reaching the highest density in the stratum granulosum. Barrier disruption with acetone produced an immediate, marked decrease in Ca²⁺ in the stratum granulosum, accompanied by secretion of lamellar bodies. Loss of this pattern of Ca²⁺ distribution was associated with the appearance of large Ca²⁺ aggregates within the intercellular spaces of the stratum corneum. The Ca²⁺-containing precipitates progressively reappeared in parallel with barrier recovery over 24 h. Disruption of the barrier with tape stripping also resulted in loss of Ca²⁺ from the nucleated layers of the epidermis, but small foci persisted where the stratum corneum was not removed; in these sites the Ca²⁺ distribution did not change and accelerated secretion of lamellar bodies was not observed. Following acetone-induced barrier disruption and immersion in isoosmolar sucrose, the epidermal Ca²⁺ gradient did not return, and both lamellar body secretion and barrier recovery occurred. However, with immersion in isoosmolar sucrose plus Ca²⁺, the epidermal Ca²⁺ reservoir was replenished, and both secretion of lamellar bodies and barrier recovery were impeded. These results demonstrate that barrier disruption results in loss of the epidermal Ca²⁺ reservoir, which may be the signal that initiates lamellar body secretion leading to barrier repair.     

Frequent blood-brain barrier disruption in the human cerebral cortex

1. The blood–brain barrier (BBB) protects the brain from circulating xenobiotic agents. The pathophysiology, time span, spatial pattern, and pathophysiological consequences of BBB disruptions are not known.2. Here, we report the quantification of BBB disruption by measuring enhancement levels in computerized tomography brain images.3. Pathological diffuse enhancement associated with elevated albumin levels in the cerebrospinal fluid (CSF) was observed in the cerebral cortex of 28 out of 43 patients, but not in controls. Four patients displayed weeks-long focal BBB impairment. In 19 other patients, BBB disruption was significantly associated with elevated blood pressure, body temperature, serum cortisol, and stress-associated CSF readthrough acetylcholinesterase. Multielectrode electroencephalography revealed enhanced slow-wave activities in areas of focal BBB disruption. Thus, quantification of BBB disruption using minimally invasive procedures, demonstrated correlations with molecular, clinical, and physiological stress-associated indices.4. These sequelae accompany a wide range of neurological disorders, suggesting that persistent, detrimental BBB disruption is considerably more frequent than previously assumed.     

Myosin light chain kinase mediates intestinal barrier disruption following burn injury

Background

Severe burn injury results in the loss of intestinal barrier function, however, the underlying mechanism remains unclear. Myosin light chain (MLC) phosphorylation mediated by MLC kinase (MLCK) is critical to the pathophysiological regulation of intestinal barrier function. We hypothesized that the MLCK-dependent MLC phosphorylation mediates the regulation of intestinal barrier function following burn injury, and that MLCK inhibition attenuates the burn-induced intestinal barrier disfunction.

Methodology/Principal Findings

Male balb/c mice were assigned randomly to either sham burn (control) or 30% total body surface area (TBSA) full thickness burn without or with intraperitoneal injection of ML-9 (2 mg/kg), an MLCK inhibitor. In vivo intestinal permeability to fluorescein isothiocyanate (FITC)-dextran was measured. Intestinal mucosa injury was assessed histologically. Tight junction proteins ZO-1, occludin and claudin-1 was analyzed by immunofluorescent assay. Expression of MLCK and phosphorylated MLC in ileal mucosa was assessed by Western blot. Intestinal permeability was increased significantly after burn injury, which was accompanied by mucosa injury, tight junction protein alterations, and increase of both MLCK and MLC phosphorylation. Treatment with ML-9 attenuated the burn-caused increase of intestinal permeability, mucosa injury, tight junction protein alterations, and decreased MLC phosphorylation, but not MLCK expression.

Conclusions/Significance

The MLCK-dependent MLC phosphorylation mediates intestinal epithelial barrier dysfunction after severe burn injury. It is suggested that MLCK-dependent MLC phosphorylation may be a critical target for the therapeutic treatment of intestinal epithelial barrier disruption after severe burn injury.     

The permeability barrier in mammalian epidermis

The structural basis of the permeability barrier in mammalian epidermis was examined by tracer and freeze-fracture techniques. Water-soluble tracers (horesradish peroxidase, lanthanum, ferritin) were injected into neonatal mice or into isolated upper epidermal sheets obtained with staphylococcal exfoliatin. Tracers percolated through the intercellular spaces to the upper stratum granulosum, where further egress was impeded by extruded contents of lamellar bodies. The lamellar contents initially remain segregated in pockets, then fuse to form broad sheets which fill intercellular regions of the stratum corneum, obscuring the outer leaflet of the plasma membrane. These striated intercellular regions are interrupted by periodic bulbous dilatations. When adequately preserved, the interstices of the stratum corneum are wider, by a factor of 5-10 times that previously appreciated. Freeze-fracture replicas of granular cell membranes revealed desmosomes, sparse plasma membrane particles, and accumulating intercellular lamellae, but no tight junctions. Fractured stratum corneum displayed large, smooth, multilaminated fracture faces. By freeze-substitution, proof was obtained that the fracture plane had diverted from the usual intramembranous route in the stratum granulosum to the intercellular space in the stratum corneum. We conclude that: (a) the primary barrier to water loss is formed in the stratum granulosum and is subserved by intercellular deposition of lamellar bodies, rather than occluding zonules; (b) a novel, intercellular freeze-fracture plane occurs within the stratum corneum; (c) intercellular regions of the stratum corneum comprise an expanded, structurally complex, presumably lipid-rich region which may play an important role in percutaneous transport.     

The dynamics of axolemmal disruption in guinea pig spinal cord following compression

Membrane damage has been postulated as a critical factor in mediating axonal degeneration in brain and spinal cord trauma. Despite compelling evidence of membrane disruption as a result of physical insults in both in vivo and in vitro studies, the dynamics of such damage over the time post injury in in vivo studies has not been well documented. Using a well-characterized in vivo guinea pig spinal cord compression model and horseradish peroxidase exclusion assay, we have documented significant membrane disruption at 1 hr, 3 days, and 7 days following injury. Furthermore, the membrane damage was found to spread laterally 10 mm beyond the center of original compression site in both rostral and caudal directions. A second-degree polynomial fit of the measured data predicts a bilateral spread of approximately 20–21 mm of membrane disruption from the epicenter of injury over a period of about 20 days. Thus, this study shows that membrane damage exists days, and possibly weeks, after spinal cord trauma in live guinea pigs. This provides the evidence necessary to investigate the role of membrane damage in triggering axonal deterioration in the future. Furthermore, this study has also revealed a long therapeutical window for membrane repair and functional enhancement following traumatic injury in the central nervous system.     

Viral disruption of the blood-brain barrier

The blood-brain barrier (BBB) provides significant protection against microbial invasion of the brain. However, the BBB is not impenetrable, and mechanisms by which viruses breach it are becoming clearer. In vivo and in vitro model systems are enabling identification of host and viral factors contributing to breakdown of the unique BBB tight junctions. Key mechanisms of tight junction damage from inside and outside cells are disruption of the actin cytoskeleton and matrix metalloproteinase activity, respectively. Viral proteins acting in BBB disruption are described for HIV-1, currently the most studied encephalitic virus; other viruses are also discussed.     

Characterization of label-retaining cells in the epidermis of a human skin equivalent

Abstract. The human skin equivalent (HSE) is an in vitro reconstructed model that resembles skin morphologically and biochemically. The HSE is formed by overlaying a fibroblast-populated collagen matrix with a suspension of epidermal cells. Basal keratinocytes attach to the dermal equivalent via a newly formed basement membrane and multiply to form a stratified, differentiated epidermis. The aim of the studies described here was to characterize the basal cells of the HSE in terms of their cell cycling potential. The experiments utilized long-term labelling of the cells with tritiated thymidine ([³H]dT), followed by irradiation with ultraviolet light. [³H]dT incorporation was analysed via routine autoradiography. Irradiation with 100 J/m² UV light increased the number of labelled basal cells by 58% over the control, the maximal stimulation observed. Decreased numbers of labelled basal cells were observed at doses of UV light greater than 100 J/m². The maximal number of labelled basal cells was observed on day 14 and decreased over time; the number of labelled suprabasal cells increased concomitantly. Label-retaining cells (12%) persisted in the stratum basale of control HSEs after 32 days in culture. Labelled cells were observed in the apical layers of the stratum granulosum of control HSEs after 22 days in culture. These data suggest that the stratum basale of the HSE contains a population of slow-cycling cells whose characteristics resemble a subpopulation of slowly cycling cells found in normal human skin.     

Dynamic changes of cell-surface glycoconjugates in human palmar epidermis following friction-blisters

Summary Damage and repair of cell-surface glycoconjugates were examined in human palmar skin following friction-blister injury, using biotinylated lectins and the avidinbiotin complex method. In normal skin, concanavalin A, Ricinus communis, and Triticum vulgaris bound to the surface of cells from the basal layer to the granular layer. After injury, binding of concanavalin A was absent in the plasma membrane, but appeared in the cytoplasm at perinuclear sites. The surface reaction was recovered in basal and spinous cells, but not in granular cells, when cell maturation began at 5 days after injury. In contrast, binding of Ricinus communis and Triticum vulgaris was, in general, much more resistant to tissue damage. Even in some cells, where the surface staining became obscure at an early period, a normal staining pattern reappeared by 6 h after injury. Staining of Ulex europeus I and Glycine max, detected on the surface of upper spinous and granular cells in normal skin, disappeared immediately after the injury, but recovered quickly on the surfaces of the differentiated cells. These findings suggest that at least 2 oligosaccharide sequences, one binding with concanavalin A, and the other with Ricinus communis and Triticum vulgaris, may exist on epidermal cells. Addition of terminal carbohydrates, detectable with binding of Ulex europeus I and Glycine max, appears to occur on the Ricinus communis I and Triticum vulgaris-bound oligosaccharide chain.     

Compromising human skin in vivo and ex vivo to study skin barrier repair

Ex vivo regenerated stratum corneum (SC) after tape-stripping can be used as a model to study the barrier function of compromised skin. Yet, details about how close the regenerated SC model mimics the lipid properties (e.g. lipid composition and lipid ordering) of the in vivo situation are not known. Here, we examined using a comprehensive ceramide analysis whether human ex vivo regenerated SC showed similar lipid properties as human in vivo regenerated SC. Both in vivo and ex vivo regenerated SC had an altered ceramide subclass composition, with increased percentages of sphingosine-based subclass and decreased percentages of phytosphingosine-based subclass ceramides, a reduced mean ceramide chain length, and a higher percentage of unsaturated ceramides. Overall, regenerated SC ex vivo showed more pronounced but similar changes compared to the in vivo response. One of the purposes of these models is to use them to mimic compromised skin of inflammatory skin diseases. The altered lipid properties in regenerated SC were comparable to those observed in several inflammatory skin diseases, which makes them a valuable model for the barrier properties in inflammatory skin diseases.     

The use of PIXE in experimental studies of the physiology of human skin epidermis

In order to understand the normal and pathological physiologies of the epidermal cells, the simultaneous determination of several elements in the different cellular strata is of crucial importance. In recent years the electron microprobe (EMP) has become an established technique in this field. Its high spatial resolution, in principle, allows measurements of various cell organelles. However, the limited (intrinsic) sensitivity of the EMP represents a serious drawback to the technique. The introduction of the proton microprobe (PMP) has significantly improved the sensitivity, although the ultimate spatial resolution of the PMP is much less than that of the EMP. When studying the elemental profiles in skin epidermis, it is possible to use skin sections with a thickness of the order of 10 μm, then the spatial resolution of the PMP is equal to or better than that of the EMP since the electrons are scattered to a significant degree in the sample. The characteristics of the two methods have been compared by analysis of parallel duplicate freeze-dried sections of normal human skin. The distributions of the elements P, S, Cl, and K, obtained with the two techniques, were in good agreement. In addition, the PMP provided distributions of the important elements Ca, Fe, and Zn. In a recently started study, the useful features of the PMP will be used for studying how efficient a barrier the skin is to nickel and chromate ions. A preliminary experiment has been performed by exposing cadaverous skin, not older than 24-h postmortem, to solutions of the two ions. After an 18-h exposure, samples were prepared by shock-freezing and sectioning. The first results from PMP analysis of these samples demonstrate the presence of a nickel and chromium gradient in the outer strata in the epidermis (mainly stratum corneum). A third experiment deals with the physiology of psoriatic skin. Calcium is an important element in the differentiation. Hence, the higher sensitivity of the PMP has been used in analysis of sections from psoriatic skin epidermis. Preliminary results are presented.     

Development and homeostasis of the skin epidermis

The skin epidermis is a stratified epithelium that forms a barrier that protects animals from dehydration, mechanical stress, and infections. The epidermis encompasses different appendages, such as the hair follicle (HF), the sebaceous gland (SG), the sweat gland, and the touch dome, that are essential for thermoregulation, sensing the environment, and influencing social behavior. The epidermis undergoes a constant turnover and distinct stem cells (SCs) are responsible for the homeostasis of the different epidermal compartments. Deregulation of the signaling pathways controlling the balance between renewal and differentiation often leads to cancer formation.     

Changes in the microstructure of the human epidermis following local vacuum exfoliation]

Structural rearrangements of the human epidermis have been studied after its local vacuum exfoliation. Blisters have been formed in 48 men-volunteers by means of negative pressure up to 0.7 kg/cm2 and during the following 72 h structure of the exfoliated epidermis has been investigated. Immediately after the blister formation the epidermal basal layer is traumatized, a part of its cells die in some time after the lesion. In the center of most of the cells of the spinous layer there is a large vacuole which presses back the nucleus. However, the whole epidermis is not ruined, and during 24 h actively regenerates. The remaining viable cells into the blister lumen. By the end of the first 24 h span they practically cover from below the whole surface of the exfoliated epidermis. In the cells of the spinous layer amount and size of vacuoles decrease, the nuclei return to the central position. In 48 h in the spinous layer keratohyalin granules are revealed, moreover, in the cells, arranging on the border with the basal layer. By 72 h within the epidermis of the number of necrotic areas sharply increases. All the arrangements in the epidermal structure occur at the absence of mitotic division of cells.     

Reconstructed human epidermis for skin absorption testing: results of the German prevalidation study

Exposure to chemicals absorbed by the skin can threaten human health. In order to standardise the predictive testing of percutaneous absorption for regulatory purposes, the OECD adopted guideline 428, which describes methods for assessing absorption by using human and animal skin. In this study, a protocol based on the OECD principles was developed and prevalidated by using reconstructed human epidermis (RHE). The permeation of the OECD standard compounds, caffeine and testosterone, through commercially available RHE models was compared to that of human epidermis and animal skin. In comparison to human epidermis, the permeation of the chemicals was overestimated when using RHE. The following ranking of the permeation coefficients for testosterone was obtained: SkinEthic > EpiDerm, EPISKIN > human epidermis, bovine udder skin, pig skin. The ranking for caffeine was: SkinEthic, EPISKIN > bovine udder skin, EpiDerm, pig skin, human epidermis. The inter-laboratory and intra-laboratory reproducibility was good. Long and variable lag times, which are a matter of concern when using human and pig skin, did not occur with RHE. Due to the successful transfer of the protocol, it is now in the validation process.     

Kallikrein-related peptidase-8 (KLK8) is an active serine protease in human epidermis and sweat and is involved in a skin barrier proteolytic cascade

Kallikrein-related peptidase-8 (KLK8) is a relatively uncharacterized epidermal protease. Although proposed to regulate skin-barrier desquamation and recovery, the catalytic activity of KLK8 was never demonstrated in human epidermis, and its regulators and targets remain unknown. Herein, we elucidated for the first time KLK8 activity in human non-palmoplantar stratum corneum and sweat ex vivo. The majority of stratum corneum and sweat KLK8 was catalytically active, displaying optimal activity at pH 8.5 and considerable activity at pH 5. We also showed that KLK8 is a keratinocyte-specific protease, not secreted by human melanocytes or dermal fibroblasts. KLK8 secretion increased significantly upon calcium induction of terminal keratinocyte differentiation, suggesting an active role for this protease in upper epidermis. Potential activators, regulators, and targets of KLK8 activity were identified by in vitro kinetic assays using pro-KLK8 and mature KLK8 recombinant proteins produced in Pichia pastoris. Mature KLK8 activity was enhanced by calcium and magnesium ions and attenuated by zinc ions and by autocleavage after Arg(164). Upon screening KLK8 cleavage of a library of FRET-quenched peptides, trypsin-like specificity was observed with the highest preference for (R/K)(S/T)(A/V) at P1-P1'-P2'. We also demonstrated that KLK5 and lysyl endopeptidase activate latent pro-KLK8, whereas active KLK8 targets pro-KLK11, pro-KLK1, and LL-37 antimicrobial peptide activation in vitro. Together, our data identify KLK8 as a new active serine protease in human stratum corneum and sweat, and we propose regulators and targets that augment its involvement in a skin barrier proteolytic cascade. The implications of KLK8 elevation and hyperactivity in desquamatory and inflammatory skin disease conditions remain to be studied.     

CD44 standard and variant isoform expression in normal human skin appendages and epidermis

 CD44 isoforms have been implicated in tumor progression and metastasis formation. This study presents a thorough immunohistochemical analysis of CD44 standard and isoform expression in normal human skin appendages and epidermis applying monoclonal antibodies against CD44s, CD44v3, -v4, -v5, -v6, and -v9. An improved immunohistochemical protocol with microwave-based antigen retrieval in paraffin sections and heavy metal amplification of the diaminobenzidine reaction product provided enhanced resolution and sensitivity as compared to studies on frozen sections. The hair follicle, the seborrheic and eccrine sweat glands were strongly positive for all CD44 isoforms studied. In the latter, the clear cells but not the dark (intercalated) cells were positive. The sudoriferous ducts adjacent to the glands were weakly positive for all CD44 isoforms and strongly positive near the skin surface. In the apocrine glands, the basal cells showed only a moderate positivity. The myoepithelial cells expressed only CD44s. In the epidermis, all CD44 isoforms were detectable, with strongest CD44 immunostaining in the lower third of the stratum spinosum and weaker staining in the stratum basale and the upper two-thirds of the stratum granulosum. The stratum granulosum and corneum were unreactive. Thus, a regional and cell type-specific CD44 expression was revealed. Accepted: 10 May 1996     

Ingestion of coffee polyphenols suppresses deterioration of skin barrier function after barrier disruption,concomitant with the modulation of autonomic nervous system activity in healthy subjects

The aim of this study was to evaluate the effect of consumption of coffee polyphenols (CPPs) on the autonomic nervous system activity and decreased skin barrier function caused by sodium dodecyl sulfate (SDS) treatment. In this single-blind, placebo-controlled study, ten healthy male subjects consumed either a beverage containing CPPs or a placebo beverage for four weeks. CPPs significantly suppressed the deterioration in skin barrier function and skin moisture content induced by SDS treatment after the third week. Furthermore, in the heart rate variability analysis, CPPs significantly produced an increase in parasympathetic nervous activity, and a decrease in sympathetic nervous activity after the four weeks of beverage consumption. These results suggest that CPPs might influence the regulation of the autonomic nervous system and contribute to the suppressive effect on deterioration of skin barrier function.