Maximilian Schreiner

TLR2‐activated human langerhans cells promote Th17 polarization via IL‐1β, TGF‐β and IL‐23

The cytokines IL‐6, IL‐1β, TGF‐β, and IL‐23 are considered to promote Th17 commitment. Langerhans cells (LC) represent DC in the outer skin layers of the epidermis, an environment extensively exposed to pathogenic attack. The question whether organ‐resident DC like LC can evoke Th17 immune response is still open. Our results show that upon stimulation by bacterial agonists, epidermal LC and LC‐like cells TLR2‐dependently acquire the capacity to polarize Th17 cells. In Th17 cells, expression of retinoid orphan receptor γβ was detected. To clarify if IL‐17+cells could arise per se by stimulated LC we did not repress Th1/Th2 driving pathways by antibodies inhibiting differentiation. In CD1c+/langerin+ monocyte‐derived LC‐like cells (MoLC), macrophage‐activating lipopeptide 2, and peptidoglycan (PGN) induced the release of the cytokines IL‐6, IL‐1β, and IL‐23. TGF‐β, a cytokine required for LC differentiation and survival, was found to be secreted constitutively. Anti‐TLR2 inhibited secretion of IL‐6, IL‐1β, and IL‐23 by MoLC, while TGF‐β was unaffected. The amount of IL‐17 and the ratio of IL‐17 to IFN‐γ expression was higher in MoLC‐ than in monocyte‐derived DC‐cocultured Th cells. Anti‐IL‐1β, ‐TGF‐β and ‐IL‐23 decreased the induction of Th17 cells. Interestingly, blockage of TLR2 on PGN‐stimulated MoLC prevented polarization of Th cells into Th17 cells. Thus, our findings indicate a role of TLR2 in eliciting Th17 immune responses in inflamed skin.

A loose‐fit coculture of activated keratinocytes and dendritic cell‐related cells for prediction of sensitizing potential

Protection against contact allergy begins with the collection of reliable data about the sensitizing potential of chemicals. Today, the local lymph node assay (LLNA) in mice is widely used to identify sensitizing substances. For several reasons, an in vitro assay could be preferable to animal experiments. We propose an in vitro test for the detection of a sensitizing potential of a chemical composed of a single layer of human nondifferentiating keratinocytes and of allogenic floating monocytes which are cocultured in serum‐free medium in the presence of a cytokine cocktail. Within days, the coculture develops to an allergen‐ sensitive system consisting of activated keratinocytes and of mobile dendritic cell‐related cells (DC‐related cell). The sensitizing potential can be determined by analyzing the expression of the dendritic cell maturation marker CD86. For the model contact allergens tested so far [trinitrobenzenesulfonic acid (TNBS), phenylendiamine, and 4‐aminoacetanilide], the strength of the reaction was in concordance with results from the LLNA. Sensitivity of the assay allowed testing at concentrations without general cytotoxicity. Thus, a differentiation between allergens and irritants was possible. Regarding cytokine secretion, the assay distinguished between the allergen TNBS and the Toll‐like receptor ligand lipopolysaccharide. The coculture can be set up from cryopreserved cells. The assay is easy to perform and reproducible. Donor‐variance is negligible. This in vitro assay based on a loose‐fit coculture is a reasonable approach to screen for the sensitizing potential of xenobiotics and might partially replace the LLNA and other animal tests.

A new dendritic cell type suitable as sentinel of contact allergens

Establishing of alternatives to animal tests is ethically desirable and gains in importance in context of new European Union regulations such as REACH. We have refined our new in vitro assay for prediction of the sensitizing potency of xenobiotics. Monocytes cocultured with primary human keratinocytes develop to a novel class of in vitro generated dendritic cells after treatment with transforming growth factor beta and Interleukin-4 in serum-free medium. These dendritic cell-related cells (DCrc) are the key players in the loose-fit coculture-based sensitization assay (LCSA). Assay duration and cytokine consumption could be cut down without impairing the assays functionality. DCrc showed a dose-dependent upregulation of CD86 after treatment with the contact allergens 2,4,6-trinitrobenzenesulfonic acid, the prohapten isoeugenol, and alpha-hexyl cinnamic aldehyde. The metal allergens nickel and cobalt could be detected by measuring Interleukin-6 and macrophage inflammatory protein 1-beta (MIP-1beta, CCL-4) in coculture supernatants. The irritant zinc elicited no reaction. Lipopolysaccharide produced upregulation of CD86, IL-6 and MIP-1beta. Determination of tolerable concentrations of an allergen in consumer products requires a widely accepted sharp quantitative assay. Animal-based assays do not meet this requirement. The LCSA provides dose-response information, thereby allowing prediction of the relative ability of a substance to induce sensitization.

Assessment of the sensitizing potential of textile disperse dyes and some of their metabolites by the loose-fit coculture-based sensitization assay (LCSA)

Certain textile disperse dyes are known to cause allergic reactions of the human skin. Here, we examined 8 disperse dyes and 7 products of azo-cleavage of these dyes in an in vitro assay. We used the loose-fit coculture-based sensitization assay (LCSA) of primary human keratinocytes and of allogenic dendritic cell-related cells for combined testing of the sensitizing and irritative properties of these substances. The obtained data were compared to data generated in a modified version of the local lymph node assay by our working group. Disperse Blue 1 (DB1), p-nitroaniline (pNA) and p-aminoacetanilide (AAA) showed no sensitizing potential under our experimental conditions. Disperse Blue 124 (DB124), Disperse Yellow 3 (DY3), Disperse Orange 37/76 (DO37), Disperse Blue 106 (DB106), Disperse Red 1 (DR1), 2-amino-p-cresol (ApC), Disperse Orange 3 (DO3) and 2,6-dichloro-4-nitroaniline (DCh) were categorized as extreme sensitizers. Para-phenylenediamine (pPD) was categorized as strong sensitizer, and 2-amino-5-nitrothiazole (ANT) and 2-(N-ethylanilino)-ethanol (EAE) as weak sensitizers. All dyes, except for DB1, and ApC turned out to be strong irritants. DB1, ANT and DCh showed only weak irritative potential. PPD, pNA, EAE and AAA did not show any irritative effect at the concentration range tested. These results correlate with data derived from the modified version of LLNA and human data. Therefore, the LCSA represents a suitable test system to simultaneously analyse two crucial properties of substances relevant for allergy induction.

A novel method to generate monocyte-derived dendritic cells during coculture with HaCaT facilitates detection of weak contact allergens in cosmetics

The in vitro sensitization assay LCSA (Loose-fit Coculture-based Sensitization Assay) has proved reliable for the detection of contact sensitizers in the past. However, the coculture of human monocyte-derived dendritic cells (DCs) with primary human keratinocytes (KCs) in serum-free medium is relatively complex compared to other sensitization assays which use continuous cell lines. To facilitate high-throughput screening of chemicals, we replaced KCs with the HaCaT cell line under various culture conditions. Coculture of HaCaT with peripheral blood mononuclear cells in serum-supplemented medium leads to generation of CD1a+/CD1c+ DCs after addition of GM-CSF, IL-4, and TGF-β1 (as opposed to CD1a−/CD1c− DCs which arise in the “classic” LCSA coculture). These cells resemble monocyte-derived DCs generated in monoculture, but, unlike those, they show a marked upregulation CD86 after treatment with contact allergens. All of the nine sensitizers in this study were correctly identified by CD1a+/CD1c+ DCs in coculture with HaCaT. Among the substances were weak contact allergens such as propylparaben (which is false negative in the local lymph node assay in mice) and resorcinol (which was not detected by CD1a−/CD1c− DCs in the “classic” LCSA). The level of CD86 upregulation on CD1a+/CD1c+ DCs was higher for most allergens compared to CD1a−/CD1c− DCs, thus improving the assay’s discriminatory power. Three out of four non-sensitizers were also correctly assessed by the coculture assay. A false-positive reaction to caprylic (octanoic) acid confirms earlier results that some fatty acids are able to induce CD86 on DC in vitro. In conclusion, change of the LCSA protocol led to reduction of time and cost while even increasing the assay’s sensitivity and discriminatory power.

Unsaturated compounds induce up-regulation of CD86 on dendritic cells in the in vitro sensitization assay LCSA.

Unsaturated compounds are known to cause false-positive reactions in the local lymph node assay (LLNA) but not in the guinea pig maximization test. We have tested a panel of substances (succinic acid, undecylenic acid, 1-octyn-3-ol, fumaric acid, maleic acid, linoleic acid, oleic acid, alpha-linolenic acid, squalene, and arachidonic acid) in the loose-fit coculture-based sensitization assay (LCSA) to evaluate whether unspecific activation of dendritic cells is a confounder for sensitization testing in vitro. Eight out of 10 tested substances caused significant up-regulation of CD86 on dendritic cells cocultured with keratinocytes and would have been classified as sensitizers; only succinic acid was tested negative, and squalene had to be excluded from data analysis due to poor solubility in cell culture medium. Based on human data, only undecylenic acid can be considered a true sensitizer. The true sensitizing potential of 1-octyn-3-ol is uncertain. Fumaric acid and its isomer maleic acid are not known as sensitizers, but their esters are contact allergens. A group of 18- to 20-carbon chain unsaturated fatty acids (linoleic acid, oleic acid, alpha-linolenic acid, and arachidonic acid) elicited the strongest reaction in vitro. This is possibly due to the formation of pro-inflammatory lipid mediators in the cell culture causing nonspecific activation of dendritic cells. In conclusion, both the LLNA and the LCSA seem to provide false-positive results for unsaturated fatty acids. The inclusion of T cells in dendritic cell-based in vitro sensitization assays may help to eliminate false-positive results due to nonspecific dendritic cell activation. This would lead to more accurate prediction of sensitizers, which is paramount for consumer health protection and occupational safety.

Erratum to: A novel method to generate monocyte-derived dendritic cells during coculture with HaCaT facilitates detection of weak contact allergens in cosmetics

Authors would like to correct the errors in the published article. The first sentence of the second paragraph of the materials and methods section needs to be changed to: “Dimethylsulfoxide (DMSO; Sigma-Aldrich) was used as a vehicle for DNCB, MBT, caprylic acid, and parabens.” The two EC50sens values for caprylic acid in LCSA setup B and C were accidentally interchanged. The fourth sentence of the second paragraph of the results section needs to be changed to: “Thus, EC50sens was >252 μmol/l (setup A), >219 μmol/l (setup B), and >669 μmol/l (setup C).” In the last row of Table 1, EC50sens should be “>219” in column B and “>669” in column C.