Effect of dihydrocaffeic acid on UV irradiation of human keratinocyte HaCaT cells

Dihydrocaffeic acid, a dietary constituent and a microbial metabolite of flavonoids, is an antioxidant, but few biological effects have been examined. After its production by microflora in the colon, dihydrocaffeic acid is absorbed and found in plasma as a combination of free and metabolized forms. Excess solar UV radiation provokes damage and initiates immune response and inflammation in skin, sometimes leading to cancer. Dihydrocaffeic acid reduced the cytotoxicity and pro-inflammatory cytokine production (interleukin-6 and -8) in HaCaT cells, a keratinocyte model, following UV radiation. The effect of dihydrocaffeic acid may result from a combination of direct radical scavenging of the reactive oxygen species formed or reinforcement of the antioxidant potential of the keratinocytes, as well as a direct interference with the pathway involved in cytokine stimulation. The minimum structure required for such an effect appears to consist of a propionate side chain attached to a catechol moiety, as indicated by the efficacy of caffeic acid, but not of the methyl and glucuronide conjugates of dihydrocaffeic acid. The data obtained suggest that dihydrocaffeic acid is a potential candidate for photo-protection by interfering with the events initiated after UV exposure in keratinocytes.     

Cdc42 is crucial for the maturation of primordial cell junctions in keratinocytes independent of Rac1

Cell-cell contacts are crucial for the integrity of all tissues. Contrasting reports have been published about the role of Cdc42 in epithelial cell-cell contacts in vitro. In keratinocytes, it was suggested that Rac1 and not Cdc42 is crucial for the formation of mature epithelial junctions, based on dominant negative inhibition experiments. Deletion of the Cdc42 gene in keratinocytes in vivo slowly impaired the maintenance of cell-cell contacts by an increased degradation of β-catenin. Whether Cdc42 is required for the formation of mature junctions was not tested.We show now that Cdc42-deficient immortalized and primary keratinocytes form only punctate primordial cell contacts in vitro, which cannot mature into belt-like junctions. This defect was independent of enhanced degradation of β-catenin, but correlated to an impaired activation and localization of aPKCζ in the Cdc42-null keratinocytes. Inhibition of aPKCζ by the inhibitor Gö6983 reproduced the phenotype, suggesting that decreased activation of aPKCζ was sufficient to explain the defective junctional maturation. In the absence of Cdc42, Rac1 activation was strongly decreased, indicating that Cdc42 is upstream of Rac1 activation. These data reveal that Cdc42 is crucial for the formation of mature epithelial cell junctions between keratinocytes by regulating activation of aPKCζ.     

An alginate hydrogel matrix for the localised delivery of a fibroblast/keratinocyte co-culture

There is significant interest in the development of tissue-engineered skin analogues, which replace both the dermal and the epidermal layer, without the use of animal or human derived products such as collagen or de-epidermalised dermis. In this study, we proposed that alginate hydrogel could be used to encapsulate fibroblasts and that keratinocytes could be cultured on the surface to form a bilayered structure, which could be used to deliver the co-culture to a wound bed, initially providing wound closure and eventually expediting the healing process. Encapsulation of fibroblasts in 2 and 5% w/v alginate hydrogel effectively inhibited their proliferation, whilst maintaining cell viability allowing keratinocytes to grow uninhibited by fibroblast overgrowth to produce a stratified epidermal layer. It was shown that the alginate degradation process was not influenced by the presence of fibroblasts within the hydrogel and that lowering the alginate concentration from 5 to 2% w/v increased the rate of degradation. Fibroblasts released from the scaffold were able to secrete extracellular matrix (ECM) and thus should replace the degrading scaffold with normal ECM following application to the wound site. These findings demonstrate that alginate hydrogel may be an effective delivery vehicle and scaffold for the healing of full-thickness skin wounds.     

Streptococcus pyogenes M49 plasminogen/plasmin binding facilitates keratinocyte invasion via integrin-integrin-linked kinase (ILK) pathways and protects from macrophage killing

The entry into epithelial cells and the prevention of primary immune responses are a prerequisite for a successful colonization and subsequent infection of the human host by Streptococcus pyogenes (group A streptococci, GAS). Here, we demonstrate that interaction of GAS with plasminogen promotes an integrin-mediated internalization of the bacteria into keratinocytes, which is independent from the serine protease activity of potentially generated plasmin. α(1)β(1)- and α(5)β(1)-integrins were identified as the major keratinocyte receptors involved in this process. Inhibition of integrin-linked kinase (ILK) expression by siRNA silencing or blocking of PI3K and Akt with specific inhibitors, reduced the GAS M49-plasminogen/plasmin-mediated invasion of keratinocytes. In addition, blocking of actin polymerization significantly reduced GAS internalization into keratinocytes. Altogether, these results provide a first model of plasminogen-mediated GAS invasion into keratinocytes. Furthermore, we demonstrate that plasminogen binding protects the bacteria against macrophage killing.     

Cytokines as therapeutic targets in skin inflammation

This review focuses on treatment targets for the most common inflammatory skin diseases, eczema and psoriasis with an emphasis on cytokines expressed in the uppermost layer of the skin which is easily accessible for diagnostic and therapeutic approaches. Recently, a significant body of research has highlighted the influence of the skin barrier and the patients’ microbiome on skin inflammatory responses and we will comment on their impact on mediator regulation. Itch is a prominent dermatology symptom which is influenced by cytokines and can via itch–scratch cycle impact on the skin barrier and mediator expression associated with damage. Taking the contribution of pruritus and superficial skin damage into account, we address cytokines as targets for stratified treatment approaches in subgroups of eczema and psoriasis.     

Ex Vivo Reconstruction of the Epidermis With Melanocytes and the Influence of UVB

To study pigmentation, we have reconstructed an epidermis ex vivo with keratinocytes and melanocytes. Keratinocytes and melanocytes were grown first in primary cocultures and separately in secondary cultures, then seeded on a dead deepidermized dermis (Pruniéras type) at a 1:20 melanocyte/keratinocyte ratio. Reconstructed epidermis were grown in a special medium enriched with calcium and fetal bovine serum lifted for 15 days at the air-liquid interface. Using histology, immunohistochemistry and electron microscopy we have shown an excellent level of differentiation of the reconstructed epidermis and a physiologic distribution of dendritic melanocytes in the basal layer capable of melanosome transfer to keratinocytes. UVB irradiation 0.15 J/cm²× 5 consecutive days increased melanocyte numbers and stimulated pigmentation as evidenced macroscopically and microscopically and at the biochemical level. Following UVB irradiation melanosome transfer was markedly increased and isolated or clumps of melanosomes were seen in the basal layers as well as in the stratum corneum. This model allows the study of the physiology of pigmentation ex vivo.     

MICA, a new polymorphic HLA-related antigen, is expressed mainly by keratinocytes, endothelial cells, and monocytes

 MICA is a new polymorphic gene in the HLA region expressed in epithelial cell lines and gastrointestinal epithelium. Little is yet known about the MICA protein, and the pattern of its expression by freshly isolated cells has not been established. In the present experiments, we used antibodies raised in rabbits against α1 and α2 domain-peptides to study the expression of MICA. By western blot and immunoprecipitation, we detected a band of 62 000 M _r in various cell lines (THP-1, U937, HeLa, A431, Raji, MOLT-4, and HUV-EC-C) and in freshly isolated keratinocytes, endothelial cells, and monocytes but not in CD4⁺ and CD8⁺ T cells, and CD19⁺ cells (B lymphocytes). It was not possible to up-regulate the expression of MICA in different cells by stimulation with γ-interferon, but the expression of MICA was induced in phytohemagglutinin-stimulated T cells. We confirmed that MICA is expressed at the cell surface by flow cytometry. Results of immunoprecipitation studies of β₂-microglobulin (β₂m)- or MICA-depleted, metabolically labeled HeLa cells indicated that MICA was not associated with β₂m. Although the function of MICA is still unknown, its restricted pattern of tissue expression, the fact that it is expressed on the cell surface, and its polymorphic nature suggest that this new molecule, encoded close to HLA class I, may play a role in the interaction between epithelial cells and cells of the immune system. Received: 21 May 1997 / Revised: 15 July 1997     

Skin Innervation and Its Effects on the Epidermis

Sensory innervation of the skin subserves protective sensations for the body to prevent thermal and noxious injuries. Neurophysiologically, they belong to the categories of A and C fibers, usually with caliber less than one µm in diameter. Morphological demonstration of the terminals of these nerves in the epidermis has been recognized recently by sensitive immunocytochemistry and an axonal marker, the protein gene product 9.5 (PGP). PGP is a ubiquitin C-terminal hydrolase, which is abundantly present in the nervous system, and particularly enriched in the unmyelinated nerves. Sensory nerves positive for PGP arise from the dorsal root ganglion, pass through the dermis, parallel the epidermis-dermis border, penetrate the basement membrane, move vertically and upwards in the epidermis with tortuous course and knobby appearance, and finally terminate at the granular layers of the epidermis. In rodents, denervation of the skin results in degeneration of epidermal nerves within 48 h of nerve transection, and thinning of the epidermis. In humans, application of this technique to evaluate disorders of the peripheral nervous system makes study of the degeneration of sensory nerve terminals possible. Patients with sensory neuropathy had fewer epidermal nerves than normal subjects, consistent with the notion of distal axonopathy. This approach has the potential to evaluate human sensory neuropathy in temporal and spatial domains. In addition, the influences of epidermal denervation open a new field to explore the interactions between sensory nerves and keratinocytes.