Inhibition of TNF-alpha induced-adhesion molecules by Avène Thermal Spring Water in human endothelial cells.

BACKGROUND: Cell adhesion molecules, such as E-selectin or intercellular adhesion molecule 1 (ICAM-1), play an important role in mediating leucocyte capture and rolling on the surface of blood vessels in atopic skin. The effectiveness of Avène hydrotherapy in patients suffering from atopic dermatitis has previously been demonstrated. Thus, we examined the effect of Avène Thermal Spring Water (TSW) on adhesion molecules to understand its mechanism of action. METHODS: Human endothelial cells EA.hy926 were treated with tumour necrosis factor-α (TNFα) in the presence or not of Avène TSW during 4 h. As nuclear factor-κB (NF-κB) is involved in the signalisation of inflammatory mediators such as the adhesion molecules, the translocation of NF-κB in endothelial cells was assessed by immunohistochemistry with anti-NF-κBp65. The protein and mRNA levels of TNFα-induced ICAM-1 and E-selectin were assessed by ELISA assay and RT-PCR. These adhesion molecules were also detected by immunohistochemistry. RESULTS: Tumour necrosis factor-α induced the activation of p65 NF-κB nuclear translocation. TNFα also induced E-selectin and ICAM-1 in a dose-dependant manner in EA.hy926 endothelial cells. In the presence of Avène TSW, a significant inhibition of the TNFα-induced E-selectin and ICAM-1 expression (-22% and -7%, respectively, P < 0.05) was observed. CONCLUSION: These data suggest that Avène TSW mediated inhibition of TNFα-induced E-selectin and ICAM-1 expression. The inhibition of such adhesion molecules is attributable to the suppression of NF-κB transcription factor pathway activation.

TLR2 sensing of F. nucleatum and S. sanguinis distinctly triggered gingival innate response.

Gingival tissue faces constant exposure to micro-organisms. It functions as part of the host response, an anti-microbial barrier that recognizes and discriminates between commensal and pathogenic bacteria. This study aimed to evaluate and compare the effects of cell wall extracts from different periodontal bacteria, commensals Streptococcus sanguinis and Fusobacterium nucleatum and the pathogen Porphyromonas gingivalis, on the innate immune response of gingival keratinocytes and the role of TLR2 in regulating this. We assayed mRNA levels to determine the expression of human beta-defensins (hbetaD2, hbetaD3), interleukin-1alpha, -1beta, 6 and 8 and matrix metalloproteinase-9. F. nucleatum extracts induced beta-defensin and inflammatory marker mRNA expression at higher levels than P. gingivalis. Extracts from the Gram-positive commensal S. sanguinis did not upregulate the host response. TLR2 extinction inhibited the upregulation of beta-defensin and cytokine transcripts by F. nucleatum extracts but, in contrast, led to a weak induction of hbetaD3 after challenge with S. sanguinis extracts. Although F. nucleatum strongly induces innate immune and inflammatory mediators, S. sanguinis limits their expression through TLR2. Together, our data demonstrate that gingival keratinocytes recognize and discriminate between Gram-positive and Gram-negative commensal extracts, in part through TLR2, to activate different signaling pathways of the innate immune host response.

Modulation of integrins and filaggrin expression by Propionibacterium acnes extracts on keratinocytes.

Propionibacterium acnes plays an important role in the pathogenesis of acne and it is established that this bacteria is involved in the induction and maintenance of the inflammatory phase of acne. The aim of our work was to determine if P. acnes extracts could modulate integrins and filaggrin in vitro expression by keratinocytes. Integrins and filaggrin expression was examined using immunohistochemistry technique both on Normal Human Epiderminal Keratinocytes (NHEK) and on deep-frozen sections of normal human skin explants incubated with three different P. acnes extracts. In addition, the expression of filaggrin was investigated on biopsies of acne lesions and by western-blot associated with its precursor profilaggrin. We demonstrated that P. acnes extracts induced beta1 integrin expression significantly on both proliferating keratinocytes and differentiated keratinocytes. In addition, P. acnes induced alpha3, alpha6s and alphaVbeta6 integrin expression and filaggrin expression on differentiated keratinocytes. Finally P. acnes extracts increased filaggrin expression by suprabasal layer of epidermis of explants. Western-blot confirmed that total amount of filaggrin was increased. These results indicate that P. acnes extracts are directly able to modulate the differentiation of keratinocytes suggesting that this bacteria play a role not only in the development of inflammatory acne lesions but also in the formation of the microcomedo.

Antimicrobial peptide modulation in a differentiated reconstructed gingival epithelium.

Gingival innate immunity has been studied by using biopsies and normal or transformed epithelial cell monolayers. To overcome individual biological variabilities and as a physiological alternative, we have proposed using a reconstructed tissue equivalent. In this study, we investigated the functionality and the stage of differentiation of a reconstructed human gingival epithelium. We also characterized this epithelium at the molecular level to investigate its differentiation stage compared with native human gingival epithelium. The expression levels and localization of markers related to proteins and lipids of well-differentiated stratified epithelium, such as cytokeratins, cornified envelope proteins and enzymes, or to factors in lipid synthesis and trafficking were examined. Immunohistochemistry revealed similar localization patterns in both types of epithelia and mRNA quantification showed a close resemblance of their expression profiles. We further revealed that, like native gingiva, reconstructed gingival epithelium was able to respond to pro-inflammatory or lipopolysaccharide stimuli by producing antimicrobial peptides hbetaD-2, hbetaD-3 or LL-37. Finally, we demonstrated that reconstructed human gingival epithelium, as a model, was good enough to be proposed as a functional equivalent for native human gingival epithelium in order to study the regulation of gingival innate immunity against periodontal infections.

Incorporation of phytosterols in human keratinocytes. Consequences on UVA-induced lipid peroxidation and calcium ionophore-induced prostaglandin release.

We have designed experimental conditions allowing the replacement of 50% of cholesterol of human keratinocytes (SVK14 line) with sitosterol or stigmasterol without affecting cellular viability. We have investigated the influence of incorporating phytosterol on the ultraviolet-A-induced formation of lipid-peroxidation products (thiobarbituric reactive substances (TBARS)) in these cells. Our results show that ultraviolet-A-induced lipid peroxidation depends on the nature of the phytosterol. Sitosterol induces a significant decrease (-30%) of TBARS relative to the control whereas stigmasterol markedly increases lipid peroxidation (+70%). We have also studied the effect of plant sterols on prostaglandin release by using the Ca(2+) ionophore A23187 as an in vitro model of the inflammation induced by UVA radiation. We show that in the presence of 50% of phytosterol (particularly stigmasterol), the release of prostaglandin (6-ketoPG(1alpha), PGE(2)) is increased compared to untreated cells. This pro-inflammatory effect of phytosterols is correlated with a loss of the regulation of the intracellular Ca(2+) concentration.

The peptidylarginine deiminases expressed in human epidermis differ in their substrate specificities and subcellular locations.

Deimination, a post-translational modification catalyzed by peptidylarginine deiminases (PADs), appears as a crucial Ca(2+)-dependent event in the last steps of epidermal differentiation. In normal human epidermis, where the deiminated proteins are filaggrin and keratins, PAD1, 2 and 3 are expressed but their relative role is unknown. The three PADs, produced as active recombinant forms, showed distinct synthetic-substrate specificities, various efficiencies to deiminate filaggrin and particular calcium and pH sensitivities. Immunoelectron microscopy demonstrated that PAD1 and PAD3 are co-located with filaggrin within the filamentous matrix of the deeper corneocytes where the protein is deiminated. This result strongly suggests that both isoforms are involved in the deimination of filaggrin, an essential step leading to free amino acid production necessary for epidermal barrier function. Moreover, PAD1 was shown to persist up to the upper corneocytes where it deiminates keratin K1, a modification supposed to be related to ultrastructural changes of the matrix.

Mitochondrial membrane potential (DeltaPsi) and Ca(2+)-induced differentiation in HaCaT keratinocytes.

We have used the human calcium- and temperature-dependent (HaCaT) keratinocyte cell line to elucidate mechanisms of switching from a proliferating to a differentiating state. When grown in low calcium medium (<0.1 mM) HaCaT cells proliferate. However, an increase in the calcium concentration of the culture medium, [Ca(2+)](0), induces growth arrest and the cells start to differentiate. Numerous studies have already shown that the increase in [Ca(2+)](0) results in acute and sustained increases in intracellular calcium concentration, [Ca(2+)](i). We find that the Ca(2+)-induced cell differentiation of HaCaT cells is also accompanied by a significant decrease in mitochondrial membrane potential, DeltaPsi. By combining patch-clamp electrophysiological recordings and microspectrofluorimetric measurements of DeltaPsi on single cells, we show that the increase in [Ca(2+)](i) led to DeltaPsi depolarization. In addition, we report that tetraethylammonium (TEA), a blocker of plasma membrane K(+) channels, which is known to inhibit cell proliferation, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a blocker of plasma membrane Cl(-) channels, also affect DeltaPsi. Both these agents stimulate HaCaT cell differentiation. These data therefore strongly suggest a direct causal relationship between depolarization of DeltaPsi and the inhibition of proliferation and induction of differentiation in HaCaT keratinocytes.

[Lipids and skin inflammation: role of phospholipases A2]. / Lipides et inflammation cutanée: place des phospholipases A2.

Phospholipases A2 (PLA2) are enzymes that catalyse the hydrolysis of glycerophospholipids at the sn-2 position, generating free fatty acids and lysophospholipids. At present, PLA2 family consists of 12 groups. PLA2 are involved in many pathophysiological processes such as barrier function, eicosanoid production, and inflammation. They are implicated in inflammatory diseases of the skin: psoriasis, eczema, atopy. The presence of PLA2 activity has been demonstrated several years ago, however the precise localization of all these PLA2 in the epidermis and its appendages has to be determined. Further studies have shown that these enzymes are expressed in various layers of epidermis. This differential localization suggests different roles for each PLA2 in skin physiology and during inflammation.

[Implication of VEGF, steroid hormones and neuropeptides in hair follicle cell responses]. / Rôles du VEGF, des hormones stéroïdes et des neuropeptides dans les réponses cellulaires des follicules pilaires.

Human hair follicles progress independently through the anagen, catagen, telogen and latency phases that correspond to growth arrest and hair shedding before initiation of a new anagen phase. Hair follicles are self-renewing and contain reservoirs of multi-potent stem cells. Identification of the messenger molecules and pathways operating in the growth and cycling of hair follicles, have provided substantial data. However, only a limited number of these signals is well understood. The specific response of hair follicle cells to these signals is correlated with the expression of their corresponding receptors. What regulates these responses? In this review, we will focus on the hair cycle and its control mechanisms. We will provide some elements in answer to these questions and present some of the markers of hair follicle cells, and hormonal and vascular growth factors, which may regulate respectively hair follicle cell metabolism and cycle, and the neuropeptide impact on hair follicle response and hair growth. The results of our study show the modifications in various expression patterns of receptors in dermal papilla cells, and demonstrate the cross-interaction between these different components. In conclusion, we present an accumulation of evidence suggesting that the regulation of hair growth requires a combination of hormonal, vascular and neuropeptide approaches that will provide further insight in defining new treatments for hair loss.

Environmental pollutants and skin cancer.

We are increasingly exposed to environmental pollution. Pollutants can be inhaled, ingested or come into contact with the skin depending on the form in which they occur. On metabolization, activation, or accumulation, pollutants can become extremely toxic for the vital organs and this is often related to a strong genotoxic effect. Since the skin acts as a barrier between the organism and the environment, it is frequently directly exposed to pollution. It is very often degraded by polluting agents and acts as an inlet toward other tissues. Numerous studies in man recognize and demonstrate the carcinogenic power of certain pollutants in the digestive and respiratory tracts. The "pollutants" that react most specifically with the skin are: ultraviolet radiation, polycyclic aromatic hydrocarbons (e.g., benzo[a]pyrene), volatile organic compounds (e.g., benzene), heavy metals, and ozone. Ultraviolet radiation, a "physical" pollutant, has been described as being the factor responsible for most skin cancers in man. The genotoxicity of UV light is well documented (type of lesion or mutation, etc.) and its carcinogenic effect is clearly demonstrated in vivo in man. A few epidemiological studies describe the carcinogenicity of certain pollutants such as arsenic or lead on the skin. However, most of the evidence for the role of pollutants in skin cancers comes from in vivo animal studies or from in vitro studies (e.g., PAHs). In this report, different studies are presented to illustrate the research strategies developed to investigate the mechanism of action of "chemical" pollutants and their potential role in human skin pathology. All the study models and the associated techniques of investigation are tools for a better understanding and thus more efficient prevention of the deleterious effects caused by the environment.

Immunomodulation induced by Avène spring water on Th1- and Th2-dependent cytokine production in healthy subjects and atopic dermatitis patients.

Avène spring water (ASW) is commonly used in France for treating atopic dermatitis and psoriasis. Previous works demonstrated modulation of cell membrane fluidity by ASW. The aims of the present study were (a) to investigate a possible in vitro effect of ASW on Th1- and Th2-dependent cytokine production using peripheral blood mononuclear cells from healthy individuals and (b) to investigate both the in vitro effect of ASW on AD patients' cells and the in vivo cellular and clinical modifications induced by a 3-week Avène Medical Spa water cure (AMSWC). The effect of ASW was tested on lymphocyte cultures, which were stimulated in vitro by various mitogens and a superantigen of staphylococcal origin. The lymphocyte proliferation and the production of the cytokines IL-2, IL-4 and IFN-gamma were tested. The results showed that ASW-containing medium enhanced the lymphoproliferative response to some mitogens. IL-2 and IFN-gamma production were also increased in stimulated culture supernatants. Conversely, ASW-containing medium induced a decrease in IL-4 production by normal peripheral blood lymphocytes. Furthermore, AMSWC was able to amend the clinical features as well as the immunological Th2 profile of atopic dermatitis.

Identification of two secreted phospholipases A2 in human epidermis.

Phospholipases A2 are enzymes that catalyze the release of fatty acids from the sn-2 position of phospholipids. Fatty acids have been suggested to play a key role in the barrier function of the epidermis. The aim of this study was to identify and characterize the type of secretory phospholipase A2 expressed in human epidermis. We report the molecular cloning of two secretory phospholipase A2 in the human epidermis. The first enzyme is identical to human pancreatic type IB phospholipase A2. Western blots revealed a 14 kDa protein localized in the soluble fraction. The second phospholipase A2 is identical to human synovial type IIA enzyme and is localized in the membrane fraction. By semiquantitative reverse transcription-polymerase chain reaction performed on horizontal sections of the epidermis, we found that the mRNAs of both phospholipases A2 were expressed mainly in the basal layers of the epidermis. Our data thus provide evidence for the expression of two secretory phospholipases A2 in human epidermis. The different localization of these two secretory proteins strongly suggests that each enzyme might have a specific role in skin physiology and probably in the barrier function. Taken together, these data validate the reverse transcription-polymerase chain reaction technique performed on thin sections as a first approach to detect gene expression in different layers of the epidermis.

Identification of pancreatic type I secreted phospholipase A2 in human epidermis and its determination by tape stripping.

Phospholipases A2 (PLA2) catalyse the release of fatty acids from the sn-2 position of phospholipids and have been suggested to play a key part in permeability barrier homeostasis. Using a sensitive and versatile fluorometric method, significant PLA2 activity has been detected in both human skin homogenates and tape strippings of stratum corneum. Based on various properties (resistance to heat and sulphuric acid treatment, neutral optimal pH, absolute requirement for millimolar calcium concentrations, inhibition by dithiothreitol and p-bromophenacyl bromide, and resistance to a trifluoromethyl ketone derivative of arachidonic acid, AACOCF3, a specific inhibitor of cytosolic PLA2), this enzyme was characterized as a secretory PLA2 (sPLA2). Immunohistochemistry revealed strong labelling of type I pancreatic sPLA2 at the stratum corneum-stratum granulosum junction, type II sPLA2 being undetectable. An increase in PLA2 activity in tape-stripped material from the deepest level of the stratum corneum was correlated with partial morphological disappearance of type I sPLA2 immunolabelling. Our data thus provide the first convincing evidence that pancreatic sPLA2 is significantly expressed in human epidermis, where it might participate in the accumulation of free fatty acids contributing to the permeability barrier. In addition, our method for determining PLA2 activity in easily available tape strippings should allow further clinical studies aimed to explore possible PLA2 abnormalities in various dermatoses.

Evaluation of cutaneous modifications in seventy-seven growth hormone-deficient children.

Cutaneous parameters such as dermal thickness, stiffness, elasticity, skin surface lipid and hydration were evaluated using noninvasive methods in 77 growth hormone-deficient (GHD) children before replacement therapy and in 70 non-GHD children. We showed that in GHD children, dermis was thinner (0.70 +/- 0.10 vs. 0.80 +/- 0.10 mm, p < 0.0001 for prepubertal children and 0.81 +/- 0.10 vs. 0.94 +/- 0.11 mm, p < 0.0001 for pubertal children), stiffer (178.5 +/- 57.3 vs. 113.09 +/- 37 kPa, p < 0.0001 for prepubertal children and 172.5 +/- 61.7 vs. 117.3 +/- 42.5 kPa for pubertal children, p < 0.001) and less elastic (0.44 +/- 0.09 vs. 0.39 +/- 0.06 (nonelasticity index), p < 0.01 for prepubertal children and 0.39 +/- 0.05 vs. 0.33 +/- 0.04, p < 0.001 for pubertal children) compared to controls. Fourteen GHD children were re-evaluated after 1 year of GH treatment: dermal thickness and skin stiffness were significantly improved (p < 0.001 and p < 0.05 respectively) while elasticity was not modified. During the same period, 11 controls did not show any significant cutaneous modification. IGF-1 values, but not IGFBP-3 values, correlated positively with dermal thickness in GHD children, before and after 1 year of GH treatment. To conclude, GHD children exhibited specific cutaneous modifications. In a subset of GHD children, we showed that these modifications were influenced by GH treatment. More extensive studies are needed to see if these changes correlated with other GH effects.

A case of dyskeratosis congenita associated with impaired DNA repair as shown by the comet assay.

An 18-year old boy with dyskeratosis congenita is presented. To examine the DNA metabolism of our patient, we applied the comet assay, a simple, quick and sensitive method that so far has not been used in this disease. After exposure to UVB, cells originating from the patient present abnormal DNA repair localized in the late step. We consider that such repair deficiency could be related to susceptibility to cancer. The comet assay seems to be a good procedure to investigate dyskeratosis congenita or other genodermatoses.

Optimisation of plant sterols incorporation in human keratinocyte plasma membrane and modulation of membrane fluidity.

The in vitro effects of plant sterols were investigated with regard to their uptake and membrane lipid fluidity in human keratinocytes. Among the different media tested to transport sterols (liposomes, micelles and organic solvents), the best results in terms of incorporation and viability were obtained by the use of the organic solvents dimethylsulfoxide and ethanol. After 48 h incubation exogenous sterol can account for about 30% of the total cell sterol content. The total sterol amount in plasma membranes increased 2-fold after incubation with cholesterol, whereas it was not altered when phytosterols were incorporated. The incorporation of cholesterol, sitosterol and stigmasterol led to an increase in the percent of unsaturated fatty acid C18:1 in the plasma membrane. The effect of this uptake on membrane fluidity was studied by means of fluorescence polarisation using DPH and TMA-DPH as fluorescent probes. Whereas cholesterol and sitosterol had no significant effect on the DPH fluorescence anisotropy (rs), the presence of stigmasterol induced a 12% decrease of rs reflecting an increase in membrane fluidity. We can conclude from this study that in the presence of sitosterol, the mean fluidity of the membrane is regulated whereas stigmasterol triggers a looseness of molecular packing of phospholipids acyl chains, in accordance with previous results obtained on purely lipid model membranes.

A mineral sunscreen affords genomic protection against ultraviolet (UV) B and UVA radiation: in vitro and in situ assays.

Ultraviolet (UV) radiation has been shown to be responsible for different biological effects on human skin, including the initiation of photocarcinogenesis. Both UVB and UVA have been described as mutagenic, but the processes by which they alter the DNA are different. Although cells can repair DNA damage, some deleterious mutations nevertheless appear and can promote cancer. The risk of photocarcinogenesis is acknowledged and the frequency of photogenodermatosis is increasing. In order to evaluate the protection efficacy of a high sun protection factor (SPF) mineral sunscreen against UVB- and UVA-induced genomic alterations, we have followed two approaches. First, we have tested the sunscreen for its ability to decrease the unscheduled DNA synthesis response in vitro in human fibroblasts, as an indirect measure of UVB-induced lesions (0.005 and 0.01 J/cm2), and second, we have verified its ability to reduce the in situ end-labelling intensity in human skin as a direct measure of UVA-induced single-strand breaks (10 J/cm2). Microscopic analysis clearly demonstrated the protective effect of the sunscreen against UVB and UVA. A dose-dependent effect of mineral sunscreens was observed. There was also a relationship between the SPF and genomic protection. By limiting the accumulation of UV-induced lesions on DNA, this mineral sunscreen could limit the mutation frequency.

Inhibitory effects of retinoids on vascular endothelial growth factor production by cultured human skin keratinocytes.

BACKGROUND: Vascular endothelial growth factor (VEGF), a potent angiogenic factor and vasodilator, is strongly expressed by epidermal keratinocytes in many angiogenesis-dependent skin disorders. Retinoids may modulate VEGF in skin and this may be related to an effect on rosacea. AIM: To investigate the effect of retinaldehyde on VEGF production by human keratinocytes. METHODS: The effects of different concentrations of retinoids (all-trans-retinal and all-trans-retinoic acid) on VEGF production by cultured human skin keratinocytes in both cell extracts and supernatants were determined. Expression of VEGF was analyzed by enzyme-linked immunosorbent assay (ELISA) and RT-PCR. RESULTS: The amount of cell-associated and secreted VEGF strongly decreased with retinoid concentration (e.g. 48, 69% inhibition at 0.1 microM all-trans-retinal and -retinoic acid, respectively, in the supernatants). In parallel, approximately 25% inhibition of VEGF mRNA expression was obtained in the presence of 0.01 microM all-trans-retinal. CONCLUSION: The decrease in VEGF expression by keratinocytes on contact with retinoids may prevent skin neoangiogenesis in certain skin diseases.

In vitro main pathways of steroid action in cultured hair follicle cells: vascular approach.

The known role of steroids on the hair follicle leads us to investigate their effects on hair follicle cell angiogenic responses in vitro. We verified, using the immunohistochemical technique, whether human occipital scalp follicle cells express steroid receptors in vitro. We showed that androgen and estrogen receptors were expressed by dermal papilla cells (DPC) and keratinocytes from the outer root sheath in vitro. With regard to steroidal enzymes (type I and II 5alpha-reductases and Cytochrome-p-450-aromatase), the type I 5alpha-reductase gene is much more expressed in DPC than in dermal fibroblasts; however, the type II 5a-reductase gene is transcribed more in dermal fibroblasts than in DPC. The transcription of the two 5alpha-reductase isoform genes in cultured DPC is regulated by a 5alpha-reductase inhibitor. We also demonstrated that DPC, dermal fibroblasts, and outer root shealth keratinocytes expressed cytochrome-p-450-aromatase. Using ELISA and reverse transcriptase-polymerase chain reaction, we investigated the role played by some steroids (estrogens, androgens, antiandrogens) in the modulation of vascular endothelial growth factor (VEGF) expression by DPC. The association of different treatments of DPC (5alpha-reductase inhibitor and androgen receptor antagonist) shows a great stimulation of VEGF and aromatase expression. Strong stimulation of VEGF protein and gene expression is observed in the presence of 17beta-estradiol. Also, the concentration-dependent inhibition of VEGF expression by DPC using the cytochrome-p-450-aromatase inhibitor, confirms the involvement of this estrogenic pathway in the regulation of VEGF expression in vitro.