Chemicals with weak skin sensitizing properties can be identified using low-density microarrays on immature dendritic cells.

A critical step in the induction of allergic contact dermatitis is the interaction of haptens with immature dendritic cells (iDC) leading to their activation. Therefore iDC appear as suitable targets for the evaluation of the sensitizing properties of haptens with the aim of developing in vitro toxicologic methods. Here, using a low-density cDNA-array, we analyzed the expression of 165 genes related to dendritic cell biology in human iDC following a 24h incubation with four haptens representative of strong (DNBS), moderate (isoeugenol) and weak (eugenol, hydroxycitronellal) contact sensitizers and with one irritant sodium dodecyl sulphate (SDS). Results show that 21/165 iDC genes were significantly modulated by hapten treatment. Some genes were preferentially modulated by a given chemical. Thus, DNBS, isoeugenol, eugenol and hydroxycitronellal consistently modulated CCR5, CCL27, CCL2 and CCR7, respectively, whereas the CXCL10 gene was regulated by SDS. When subjected to principal component analysis, the 21 target genes fell into four groups associated with a particular type of chemical endowed with distinct sensitizing or irritant properties. Thus, gene profiling of iDC using low-density microarray allows, for screening of chemicals, the indentification of weak haptens with potential skin sensitizing properties. These results suggest that gene profiling of iDC using low-density microarrays may be useful to identify chemicals with weak skin sensitizing properties.

DNA repair capacities of cutaneous fibroblasts: effect of sun exposure, age and smoking on response to an acute oxidative stress.

BACKGROUND: Sun irradiation causes skin ageing and cancer through the accumulation of damage to cell components. Intrinsic ageing is also associated with accumulation of oxidized macromolecules. OBJECTIVES: In this study we investigated the effects of sun exposure on response to an acute in vitro oxidative stress (H(2)O(2)) using normal human fibroblasts prepared from biopsies from 10 volunteers taken from sun-protected and sun-exposed sites. METHODS: Time-course experiments measuring repair of DNA strand-breaks and formamidopyrimidine DNA N-glycosylase-sensitive sites were conducted using the single-cell gel electrophoresis (comet) assay. RESULTS: Our results demonstrated that repair of strand-breaks was slower in sun-exposed compared with sun-protected cells. Interestingly, ageing was also associated with decreased DNA repair capacities for single-strand breaks in both sun-exposed and sun-protected cells whereas for formamidopyrimidine glycosylase (Fpg)-sensitive sites, this feature was in evidence only in sun-protected cells. Smoking, associated with age, was shown to have a markedly negative impact on DNA repair. CONCLUSIONS: Taken together our data suggest that stresses like ageing, sun exposure and smoking might have an additive effect contributing to the overall heterogeneity and decrease of DNA repair capacities in human cells and so increase the danger of sun exposure for health. They also emphasize the importance of the quality of the biological samples when repair studies on skin cells are to be conducted.

Study of housekeeping gene expression in human keratinocytes using OLISA, a long-oligonucleotide microarray and q RT-PCR.

In recent years, applications of microarray platforms have been extended to different areas of research including cosmetic and pharmaceutical. Although microarray technology is still improving its sensitivity and flexibility, researchers often turn toward quantitative RT-PCR for data validation. Assessment of messenger RNA quantity by these methods is based on comparison with internal standard genes, mainly housekeeping genes, so called because their synthesis occurs normally at a constant level. However, numerous studies showed that expression of these genes could vary in given situations. Here, we report results on four housekeeping genes (GAPDH, beta-2 microglobulin, S40 and S26 ribosomal sub-units) with constant expression levels established on OLISA microarray using different keratinocyte cultures. Moreover, qRT-PCR validation demonstrates that S26 ribosomal is a good housekeeping gene on keratinocytes and skin studies. Our data indicate that S26 gene can be routinely used to standardize results to investigate differentially expressed genes in a healthy human skin.

Expression and function of neurotrophins and their receptors in human melanocytes.

Melanocytes and cells of the nervous system are of common ectodermal origin and neurotrophins (NT) have been shown to be released by human keratinocytes. We investigated the expression and function of NT [nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), NT-3, NT-4/-5] and their receptors in human melanocytes. Human melanocytes produce all NT in different amounts, whereas they only release NT-4. NT-4 release is downregulated, whereas NT-3 is upregulated by ultraviolet (UVB) irradiation. Melanocytes treated with phorbol 12-myristate 13-acetate (PMA) express TrkA and TrkB, but not TrkC. NT fail to stimulate melanocyte proliferation, whereas they stimulate the synthesis of tyrosinase and tyrosinase-related protein-1 (TRP-1). Finally, NT-3, NT-4 and NGF increase melanin production. Taken together, these results demonstrate an intriguing interaction between melanocytes and the nervous system. We speculate that NT could be considered the target of therapy for disorders of skin pigmentation.

Photoageing shows histological features of chronic skin inflammation without clinical and molecular abnormalities.

BACKGROUND: Photodamage is characterized by degradation of collagen and accumulation of abnormal elastin in the superficial dermis. Mast cells and macrophages, which are found in higher numbers in photoaged skin, have been implicated in this process. OBJECTIVES: To analyse the phenotype of haematopoietic-derived infiltrating cells in photodamaged skin. METHODS: Chronically sun-exposed (preauricular) and control sun-protected (postauricular) skin was recovered from eight healthy subjects undergoing cosmetic surgery (facial lifting). RESULTS: Histological analysis showed that sun-exposed skin harboured more infiltrating mononuclear cells than sun-protected skin. Cellular infiltrates were found at the periphery of areas of elastolysis around hair follicles in sun-exposed sites, whereas they were found in the interfollicular dermis around blood vessels and around hair follicles in sun-protected samples. Immunohistochemical analysis revealed an increased number of mast cells, macrophages and CD4+ CD45RO+ T cells in sun-exposed dermis as well as a higher number of CD1a+ dendritic cells in sun-exposed epidermis, compared with the sun-protected samples. Thus photoageing displays histological features of chronic skin inflammation. However, no molecular sign of inflammation was observed and we even found a decreased expression of interleukin-1beta mRNA in sun-exposed compared with sun-protected sites. Furthermore, the patients' skin looked normal and did not display any clinical inflammation. CONCLUSIONS: Collectively, these data show that chronic ultraviolet irradiation induces alterations of innate immune cells which are recruited in sun-exposed skin without being activated.

Decreased expression of keratinocyte beta1 integrins in chronically sun-exposed skin in vivo.

BACKGROUND: Chronic exposure to ultraviolet (UV) radiation induces changes in the skin structure which are mostly found in the superficial dermis and at the dermal-epidermal junction. Keratinocytes and fibroblasts contribute both to the synthesis and to the degradation of the molecules important for the integrity of this skin site. While several studies have reported on alterations of dermal components and of the functions of fibroblasts in vivo and in vitro after UV exposure, recent data suggested that keratinocytes could be the main skin cell type involved in the photoageing process. OBJECTIVES: In this study, we analysed the expression of two keratinocyte molecules namely, beta1 integrin (a proliferation marker) and involucrin (a differentiation marker) in sun-exposed and sun-protected facial skin of 16 healthy patients undergoing facial lifting. METHODS: Methods included histology, immunohistochemistry and quantitative reverse transcriptase-polymerase chain reaction analysis. RESULTS: Sun-exposed skin displayed the characteristic morphological and molecular features of dermal photoageing, compared with sun-protected skin, including dermal elastosis, diminished fibrillin and type VII collagen expression. Analysis of the epidermis in sun-exposed vs. sun-protected skin showed no histological differences, but dramatic changes in the expression of beta1 integrin and involucrin. In sun-exposed skin, expression of beta1 integrin protein by epidermal basal cells was reduced, paralleling a downregulation of beta1 integrin mRNA, whereas involucrin protein expression was greatly enhanced in the superficial epidermal cell layers. Interestingly, the ratio between involucrin and beta1 integrin protein expression was consistently increased in sun-exposed skin sites. CONCLUSIONS: Collectively these results demonstrate that epidermal homeostasis is impaired by chronic UV exposure, and define beta1 integrin expression as a molecular marker of the epidermal photoageing process.