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Gap junctions (GJ) are known to be involved in spontaneous wound healing in rodent skin. We analyzed the staining patterns of the GJ proteins Cx26, Cx30, and Cx43 in human cutaneous wound healing and compared ex vivo spontaneous wound healing to non-healing wounds (chronic leg ulcers) and to ex vivo accelerated wound healing after transplantation of cultured keratinocytes. We demonstrate a loss of Cx43 staining at the wound margins during initial wound healing and after transplantation of keratinocytes. In contrast, Cx43 remains present at the margins of most non-healing wounds. We show a subsequent induction of Cx26 and Cx30 near the wound margins in spontaneous wound healing and-even earlier-after the transplantation of keratinocytes. The cells at the wound margins remain negative until the commencement of epidermal regeneration. Cx26/30 are present at the wound margins of most non-healing wounds. Cx stainings are absent in the transplanted keratinocytes during early wound healing, but there is a subsequent induction. Our results suggest that the downregulation of Cx43 is an important event in human wound healing. We discuss the assumption that direct cell-cell communication via GJ contribute to the acceleration of wound healing after the transplantation of keratinocytes.

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The transmembrane protein claudin-1 is a major component of epidermal tight junctions (TJs), which create a dynamic paracellular barrier in the epidermis. Claudin-1 downregulation has been linked to atopic dermatitis (AD) pathogenesis but variable levels of claudin-1 have also been observed in healthy skin. To elucidate the impact of different levels of claudin-1 in healthy and diseased skin we determined claudin-1 levels in AD patients and controls and correlated them to TJ and skin barrier function. We observed a strikingly broad range of claudin-1 levels with stable TJ and overall skin barrier function in healthy and non-lesional skin. However, a significant decrease in TJ barrier function was detected in lesional AD skin where claudin-1 levels were further reduced. Investigations on reconstructed human epidermis expressing different levels of claudin-1 revealed that claudin-1 levels correlated with insideout and outside-in barrier function, with a higher coherence for smaller molecular tracers. Claudin-1 decrease induced keratinocyte-autonomous IL-1β expression and fostered inflammatory epidermal responses to non-pathogenic Staphylococci. In conclusion, claudin-1 decrease beyond a threshold level results in TJ and epidermal barrier function impairment and induces inflammation in human epidermis. Increasing claudin-1 levels might improve barrier function and decrease inflammation and therefore be a target for AD treatment. Tight junctions (TJs) are an important component of the complex epidermal barrier system. They are localized in the stratum granulosum (SG) of the epidermis and provide mechanical barrier function to ions and solutes of different molecular sizes 1-4. The transmembrane protein claudin-1 (Cldn-1) is a major component of TJs 5. It is also found outside of TJs in basal and suprabasal layers of the epidermis 2,5. Mice with a complete Cldn-1 knockout (KO) die at the first day of birth due to increased transepidermal water loss (TEWL) 5. They develop TJs leaky to a molecular tracer (Biotin-556) 5 , and a highly water permeable stratum corneum (SC) 6. Human subjects lacking Cldn-1 suffer from the Neonatal Ichthyosis-Sclerosing Cholangitis (NISCH) syndrome which includes an ichthyosiform skin phenotype 7. An archetypical disease of epidermal barrier dysfunction is atopic dermatitis (AD) 8. Cldn-1 single nucleotide polymorphisms were linked to AD in some cohorts 9-11 , but not in others 11,12. Using immunostaining-intensity measurements and western blot analyses, reduced Cldn-1 levels were found in lesional AD skin 13-16. For non-lesional skin, divergent observations were described. Some authors found decreased mRNA and immunointensity levels 10 , while others observed no alteration of Cldn-1 immunointensity and western-blot levels 14,16 .

Tight Junction (TJ) proteins have been shown to exert a barrier function within the skin. Here, we study the fate of TJ proteins during the challenge of the skin by bacterial colonization and infection. We investigated the influence of various exfoliative toxin-negative Staphylococcus strains on TJ, adherens junction (AJ), desmosomal proteins, and actin in a human keratinocyte infection culture and in a porcine skin infection model. We found that the pathogen Staphylococcus aureus downregulates TJ and subsequently AJ and desmosomal proteins, including atypical protein kinase C, an essential player in TJ formation, at the cell-cell borders of keratinocytes in a time and concentration dependent manner. Little changes in protein and RNA levels were seen, indicating redistribution of proteins. In cultured keratinocytes, a reduction of transepithelial resistance was observed. Staphylococcus epidermidis shows only minor effects. All strains induced enhanced expression of occludin and ZO-1 at the beginning of colonization/infection. Thus, we demonstrate that TJ are likely to be involved in skin infection of exfoliative toxin-negative S. aureus. As we did not find a change in actin, and as changes of TJ preceded alterations of AJs and desmosomes, we suggest that S. aureus targets TJ.

It has long been accepted that tight junctions (TJ) are crucial for the formation and maintenance of the paracellular barrier and for cell polarity in simple epithelia and endothelia. Moreover, it is long known that they play a role in barrier function of amphibian skin. However, only in recent years were TJ and TJ proteins identified in the epidermis of men and mice. Their involvement in the barrier function of mammalian skin has been shown. This review summarizes our current knowledge about TJ and TJ proteins in mammalian skin.

Few data are available on early regeneration of human epidermis in vivo. We have established a supravital skin organ culture model for epidermal wound healing by setting a central defect (3 mm diameter) in freshly excised skin specimens and culturing under air exposure. Re-epithelialization was followed for up to 7 d by histology and immunohistologic analysis of various markers for differentiation and proliferation. In 12 of 19 cases (63%; 5% fetal calf serum) or six of 21 cases (29%; 2% fetal calf serum), the wounds were re-epithelialized spontaneously after 7 d. After transplantation to the wounds of 1-2 x 10(6) dissociated allogenic cultured epidermal or about 1 x 10(6) autologous outer root sheath keratinocytes, 18 of 21 cases (86%; 5% fetal calf serum) or 17 of 21 cases (81%; 2% fetal calf serum) were healed within the same period. Histologically, early neoepithelium (3 d) was disordered after keratinocyte transplantation, whereas later (7 d) it had gained a more ordered stratification, exhibiting a thin discontinuous granular and a compact horny layer. At this stage, not only hyperproliferative (CK 6) but also, abundantly, maturation-associated cytokeratins (CK 1, CK 10) were detected immunohistochemically. Analyses of regenerated epidermis after transplantation of (i) keratinocytes labeled in vitro with BrdU and (ii) heterosexual keratinocytes by immunohistochemistry and fluorescence in situ hybridization for the Y chromosome, respectively, clearly showed that external keratinocytes are physically integrated into the regenerated epidermis and extendedly contribute to its formation. The data presented here demonstrate improvement and acceleration of epidermal re-epithelialization by transplantation of keratinocytes.