Lymphocyte surface markers and cytokines are suitable for detection and potency assessment of skin-sensitizing chemicals in an in vitro model of allergic contact dermatitis: the LCSA-ly.

Allergic contact dermatitis is a widespread health disorder and occupational skin disease. Hence, screening for contact-sensitizing chemicals is highly relevant to toxicology, dermatology, and occupational medicine. The use of animal tests for this purpose is constrained by ethical considerations, need for high-throughput screening, and legislation (e.g., for cosmetics in the European Union). T cell activation is the final and most specific key event of the "adverse outcome pathway" for skin sensitization and therefore a promising target for the development of in vitro sensitization assays. We present a novel in vitro sensitization assay with a lymphocyte endpoint as an add-on to the loose-fit coculture-based sensitization assay (LCSA): the LCSA-ly. While the LCSA measures dendritic cell activation, the LCSA-ly offers the option for an additional lymphocyte endpoint which can be measured concurrently. We incorporated lymphocytes in our previously established coculture of primary human keratinocytes and monocyte-derived dendritic cells and tested nine substances: five sensitizers [2,4-dinitrochlorobenzene (DNCB) 1.25-15 µmol/l, p-phenylenediamine (PPD) 15.6-125 µmol/l, 2-mercaptobenzothiazole (MBT) 50-1000 µmol/l, coumarin, and resorcinol (both: 250-1500 µmol/l)] and four non-sensitizers (monochlorobenzene, caprylic acid, glycerol, and salicylic acid (all: 125-1000 µmol/l)]. DNCB and MBT increased a subset of IL-23 receptor+/IFN-γ receptor 1 (CD119)+ lymphocytes. DNCB, PPD, and MBT enhanced a subunit of the IL-4 receptor (CD124) and a memory marker (CD44) on lymphocytes. Remarkably, DNCB, PPD, and MBT raised IL-4 concentrations in coculture supernatants while IFN-γ levels decreased, which might point to Th2 activation in vitro. Coumarin, resorcinol, and non-sensitizers did not alter any of the tested surface markers or cytokines. IL-17 was not affected by any of the substances. Relative strength of sensitizers according to lymphocyte markers was DNCB > PPD > MBT, which corresponds to earlier results from the LCSA without lymphocyte endpoint, the murine local lymph node assay, and human data. This study is the first to prove the suitability of lymphocyte surface markers for sensitization testing and potency assessment.

Multiparameter toxicity assessment of novel DOPO-derived organophosphorus flame retardants.

Halogen-free organophosphorus flame retardants are considered as replacements for the phased-out class of polybrominated diphenyl ethers (PBDEs). However, toxicological information on new flame retardants is still limited. Based on their excellent flame retardation potential, we have selected three novel 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide (DOPO) derivatives and assessed their toxicological profile using a battery of in vitro test systems in order to provide toxicological information before their large-scale production and use. PBDE-99, applied as a reference compound, exhibited distinct neuro-selective cytotoxicity at concentrations ≥10 µM. 6-(2-((6-oxido-6H-dibenzo[c,e][1,2]oxaphosphinin-6-yl)amino)ethoxy)-6H-dibenzo[c,e][1,2]oxaphosphinine 6-oxide (ETA-DOPO) and 6,6'-(ethane-1,2-diylbis(oxy))bis(6H-dibenzo[c,e][1,2]oxaphosphinine-6-oxide) (EG-DOPO) displayed adverse effects at concentrations >10 µM in test systems reflecting the properties of human central and peripheral nervous system neurons, as well as in a set of non-neuronal cell types. DOPO and its derivative 6,6'-(ethane-1,2-diylbis(azanediyl))bis(6H-dibenzo[c,e][1,2]oxaphosphinine-6-oxide) (EDA-DOPO) were neither neurotoxic, nor did they exhibit an influence on neural crest cell migration, or on the integrity of human skin equivalents. The two compounds furthermore displayed no inflammatory activation potential, nor did they affect algae growth or daphnia viability at concentrations ≤400 µM. Based on the superior flame retardation properties, biophysical features suited for use in polyurethane foams, and low cytotoxicity of EDA-DOPO, our results suggest that it is a candidate for the replacement of currently applied flame retardants.

Assessment of Anti-TNF-α Activities in Keratinocytes Expressing Inducible TNF- α: A Novel Tool for Anti-TNF-α Drug Screening.

Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine important in normal and pathological biological processes. Newly synthesized pro-TNF-α is expressed on the plasma membrane and cleaved to release soluble TNF-α protein: both are biologically active. Secreted TNF-α signals through TNF receptors and the membrane-bound TNF-α acts by cell contact-dependent signaling. Anti-TNF-α antibodies have been used effectively for treatment of chronic inflammation, however with adverse side effects. Thus, there is a need for new anti-TNF-α small molecule compounds. Anti-TNF-α activity assays involve treatment of keratinocytes with exogenous TNF-α before or after anti-TNF-α incubation. However, this model fails to address the dual signaling of TNF-α. Here we describe a Doxycycline (Dox)-inducible TNF-α (HaCaT-TNF-α) expression system in keratinocytes. Using this in-vitro model, we show cell inhibition and induced expression of pro-inflammatory cytokines and markers, including IL-1ß, IL-6, IL-8, NF-κB1, and KRT-16, similar to cells treated with exogenous TNF-α. Sufficient secreted TNF-α produced also activated IL-1ß and IL-8 expression in wt HaCaT cells. Importantly, stimulated expression of IL-1ß and IL-8 in HaCaT-TNF-α were blocked by Quercetin, a flavanol shown to possess anti-TNF-α activities. This novel in vitro cell model provides an efficient tool to investigate the dual signaling of TNF-α. Importantly, this model provides an effective, fast, and simple screening for compounds with anti-TNF-α activities for chronic inflammatory disease therapies.

Neuromodulatory and anti-inflammatory ingredient for sensitive skin: in vitro assessment.

The manifestation of sensitive skin occurs as a consequence of increased permeability of the Stratum corneum, besides the involvement of neuro-immune-endocrine system. In this study, we evaluated the effects of an active ingredient SensC on the production of neuropeptides substance P (SP), enkephalin and ß-endorphin; eicosanoids prostaglandin E2 (PGE2) and leukotriene B4 (LTB4); histamine, transient receptor potential vanilloid subfamily member 1 (TRPV1), and envelope proteins filaggrin and involucrin, using an in vitro model of human cell culture. Our results demonstrated that treatment of keratinocyte cultures with SensC prevented the increase of all evaluated inflammatory mediators induced by interleukin-1 alpha (IL-1α). As the same way, SensC provides decrease in the synthesis of TRPV1. Regarding the synthesis of envelope proteins, SensC promoted increases for filaggrin and involucrin levels, when compared to control group. Considering the absence of appropriate treatment, the availability of ingredients, such as SensC, with antiinflammatory and protective barrier properties can be a significant tool for preventing neurosensorial symptoms associated with sensitive skin.

Histamine induces proliferation in keratinocytes from patients with atopic dermatitis through the histamine 4 receptor.

BACKGROUND: Epidermal hyperproliferation resulting in acanthosis is an important clinical observation in patients with atopic dermatitis, and its underlying mechanisms are not completely understood. OBJECTIVE: Because increased levels of histamine are present in lesional skin, we investigated the effect of histamine, especially with regard to histamine 4 receptor (H4R) activation, on the proliferation of human and murine keratinocytes. METHODS: The expression of H4R on human and murine keratinocytes was detected by using real-time PCR. Keratinocyte proliferation was evaluated by using different in vitro cell proliferation assays, scratch assays, and measurement of the epidermal thickness of murine skin. RESULTS: We detected H4R mRNA on foreskin keratinocytes and on outer root sheath keratinocytes; H4R mRNA was more abundant in keratinocytes from patients with atopic dermatitis compared with those from nonatopic donors. Stimulation of foreskin keratinocytes, atopic dermatitis outer root sheath keratinocytes, and H4R-transfected HaCaT cells with histamine and H4R agonist resulted in an increase in proliferation, which was blocked with the H4R-specific antagonist JNJ7777120. Abdominal epidermis of H4R-deficient mice was significantly thinner, and the in vitro proliferation of keratinocytes derived from H4R-deficient mice was lower compared with that seen in control mice. Interestingly, we only detected H4R expression on murine keratinocytes after stimulation with LPS and peptidoglycan. CONCLUSION: H4R is highly expressed on keratinocytes from patients with atopic dermatitis, and its stimulation induces keratinocyte proliferation. This might represent a mechanism that contributes to the epidermal hyperplasia observed in patients with atopic dermatitis.

Identifying targets for topical RNAi therapeutics in psoriasis: assessment of a new in vitro psoriasis model.

Diseases of the skin are amenable to RNAi-based therapies and targeting key components in the pathophysiology of psoriasis using RNAi may represent a successful new therapeutic strategy. We aimed to develop a straightforward and highly reproducible in vitro psoriasis model useful to study the effects of gene knockdown by RNAi and to identify new targets for topical RNAi therapeutics. We evaluated the use of keratinocytes derived from psoriatic plaques and normal human keratinocytes (NHKs). To induce a psoriatic phenotype in NHKs, combinations of pro-inflammatory cytokines (IL-1α, IL-17A, IL-6 and TNF-α) were tested. The model based on NHK met our needs of a reliable and predictive preclinical model, and this model was further selected for gene expression analyses, comprising a panel of 55 psoriasis-associated genes and five micro-RNAs (miRNAs). Gene silencing studies were conducted by using small interfering RNAs (siRNAs) and miRNA inhibitors directed against potential target genes such as CAMP and DEFB4 and miRNAs such as miR-203. We describe a robust and highly reproducible in vitro psoriasis model that recapitulates expression of a large panel of genes and miRNAs relevant to the pathogenesis of psoriasis. Furthermore, we show that our model is a powerful first step model system for testing and screening RNAi-based therapeutics.

Assessment of potency of allergenic activity of low molecular weight compounds based on IL-1alpha and IL-18 production by a murine and human keratinocyte cell line.

Assessment of allergenic potency of low molecular weight compounds is generally performed using animal models, such as the guinea pig maximisation test and the murine local lymph node assay (LLNA). Progress in unravelling the mechanisms of skin sensitisation, including effects on the production of cytokines by the different cell types of the skin, provides us with the opportunity to develop in vitro tests as an alternative to in vivo sensitisation testing. The aim of the present study was to establish an in vitro method to assess the potency of allergens, on the basis of their induction of cytokine production by murine and human keratinocytes. In the present study we used test systems comprised of the murine epidermal keratinocyte cell line HEL-30 and the human keratinocyte cell line HaCaT. We exposed these cell lines to the allergens ethyl-p-aminobenzoate (benzocaine), diethylamine (DEA), 2,4-dinitrochlorobenzene (DNCB), and phthalic anhydride (PA). IL-1alpha and IL-18 dose-response data were evaluated by non-linear regression analysis and at a stimulation index of 3 of cytokine production of treatment versus control, the corresponding allergen concentration was calculated. For HEL-30, for both cytokines DNCB showed the strongest potency followed in this order by PA, benzocaine, and DEA. This classification was similar to our previous findings obtained in the LLNA. For HaCaT, unfortunately, such ranking proved to be much less feasible. In conclusion, to assess the potency of allergens the murine keratinocyte cell line HEL-30 may be a useful in vitro test system, alternative to in vivo models, although this requires further testing using a much wider range of compounds.

[New advances in contact allergology]. / Nouvelles avancées en allergologie de contact.

In October 1996, dermatologists commemorated the centenary of the Patch test that Jadassohn had developed in 1896 at Breslau. A great amount of scientific information came to light in the following years due to progress in immunology applied to dermatology. To consider the most important recent advances in the field of contact allergic dermatitis, it is necessary to recognise the role of adhesion molecules specific for antigen presenting dendritic cells, also keratinocytes as antigen presenting cells, the importance of CD8 cells, IL10, IL12 and IL1 and their role in modulation of the contact allergy reaction. The chemistry of haptens should lead to a decision as to whether a new molecule is allergic or not, as well as the different possibilities of bonding to form sensitizing complexes. Epidemiology and the present development of epicutaneous tests are at the centre of studies that are mentioned in this general review.

Properties of the carcinoma-associated antigen MH 99/KS 1/4 in normal and transformed human keratinocytes: regulation of synthesis, molecular cross-linking and ultrastructural localization.

A 38-kDa cell-surface glycoprotein defined by monoclonal antibody MH 99 is markedly increased in many epithelial tumours. In normal human skin, it is a characteristic marker for germ-cell phenotypic tissues. Although the gene encoding the MH 99 antigen has recently been cloned, and several histological and biochemical studies have been performed, the biological function of this interesting antigen still remains unknown. In the present study, we examined the synthesis of MH 99 in keratinocyte populations showing different in vitro differentiation capacity. Normal keratinocytes, spontaneously immortalized keratinocytes (cell line HaCaT), three SV-40-transformed keratinocyte lines (130, 425, and HaSV), and two squamous cell carcinoma lines (SCL-1 and SCL-2), were compared. Radioimmunoprecipitation revealed the highest levels of synthesis in cell populations with the least differentiation. This was paralleled by an increase of MH 99 synthesis in normal keratinocytes cultured in low concentrations of Ca2+ and by an increase of MH 99 synthesis during subculture of normal keratinocytes. Both phenomena were paralleled by an opposite behaviour of a differentiation marker. Molecular cross-linking and subsequent immunoprecipitation led to a decrease of the MH 99 signal, but an increase of a high molecular weight protein signal was seen. After cleavage of the crosslinker, the MH 99 signal reappeared, whereas the signal of the large protein remained unchanged. Thus, the MH 99 antigen may be associated with a high molecular weight protein on the cell surface, supporting the suggestion of a receptor-like function. Phosphorylation of the molecule could not be detected. Immunoelectron microscopy revealed homogeneous distribution on the cell surface, but cells of the same culture exhibited clear differences in their MH 99 expression. A concept for MH 99 regulation in normal and transformed human keratinocyte populations in vitro is proposed, showing that the synthesis of MH 99 is inversely correlated with cell differentiation. The association with a high molecular weight protein supports the suggestion that the MH 99 antigen interacts with other molecules.

The human hair follicle: glycoprotein-related antigenic profile of distinct keratinocyte populations in vivo and their alterations in vitro.

The cell surface expression of three glycoprotein antigens, as defined by the monoclonal antibodies BT 15, T 43, and MH 99, was investigated in follicular keratinocyte populations in vivo. In addition, the regulation of glycoprotein synthesis was studied in follicular and interfollicular keratinocytes cultured in vitro. The BT 15 antigen was strongly expressed in the inner root sheath and the area above Auber's line of the hair bulb, whereas the T43 antigen was mainly seen in the outer root sheath. Selectively high expression of the MH 99 antigen was found only in outgrowing germ buds of early anagen follicles. Radioimmunoprecipitation revealed strong signals with BT 15 in freshly prepared follicular keratinocytes, two to three times stronger than those in interfollicular keratinocytes, but the signals clearly decreased by 80% under continuing culture conditions. The T 43 antigen was found by FACS analysis and radioimmunoprecipitation in initially low amounts in both populations, but the signals increased dramatically (up to 50 times) in long-term cultures and in subcultures. The MH 99 antigen was also initially present only in low amounts, in interfollicular rather than in follicular keratinocytes, but its expression increased up to 15-fold with continuing culture and any differences between the two populations disappeared. Our investigation revealed that at least three populations of hair follicle keratinocytes are characterized by different surface glycoprotein antigens, clearly related to their state of differentiation and proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)