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 assay's 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.

Human Langerhans cells selectively activated via Toll-like receptor 2 agonists acquire migratory and CD4+T cell stimulatory capacity.

In epidermal Langerhans cells (LCs), the expression pattern and the functions of TLRs have been poorly characterized. By using mAb, we show that LCs from human skin express TLR1, -2, -5, -6, and -9, the cognate receptors for detection of specific bacteria-derived molecules. As compared with other TLR agonists, LCs acquired a more matured phenotype when activated by specific bacterial or synthetic TLR2 agonists. In addition, monocyte-derived Langerin(+)/CD1c(+)LCs (CD1c(+)MoLCs) secreted higher amounts of IL-6 and TNF-alpha by stimulation via TLR2 than by stimulation via TLR3, -4, -5, -8, and -9. In contrast to MoLCs, dendritic cells, generated from the same donor monocytes, were activated by agonists of TLRs other than TLR2 as well. Lipopeptides triggering TLR2 induced IL-1R-associated kinase-1 phosphorylation and migration toward the chemokines CCL19 and CCL21 in epidermal LCs and CD1c(+)MoLCs. Up-regulation of CD86, CD83, and CCR7, TNF-alpha and IL-6, and NF-kappaB activation and proliferation of CD4(+)T cells could be inhibited TLR2-specific blockage using antibodies prior to TLR2 activation. Application of anti-TLR1, anti-TLR6, and anti-TLR2 indicated an exclusive role of TLR2 in IL-6 induction in human LCs. Collectively, our results show that TLR2 expressed by LCs mediates inflammatory responses to lipopeptides, which implicates a central role in sensing pathogens in human skin.

Human epidermal Langerhans cells differ from monocyte-derived Langerhans cells in CD80 expression and in secretion of IL-12 after CD40 cross-linking.

Langerhans cells (LCs) represent an immature population of myeloid dendritic cells (DCs). As a result of their unique Birbeck granules (BGs), langerin expression, and heterogeneous maturation process, they differ from other immature DCs. Monocyte-derived LCs (MoLCs) mimic epidermal LCs. MoLCs with characteristic BGs are generated by culturing blood-derived monocytes with granulocyte macrophage-colony stimulating factor, interleukin (IL)-4, and transforming growth factor-beta1. Here, we compare maturation-induced antigen expression and cytokine release of LCs with MoLCs. To achieve comparable cell populations, LCs and MoLCs were isolated by CD1c cell sorting, resulting in high purity. In unstimulated cells, CD40 was expressed at equal levels. After stimulation with CD40 ligand (CD40L), LCs and MoLCs acquired CD83 and increased CD86. High CD80 expression was exclusively detected in CD1c-sorted MoLCs. Human leukocyte antigen-DR and CD54 expression was found in all cell populations, however, at different intensities. CD40 triggering increased the potency of LCs and MoLCs to stimulate CD4+ T cell proliferation. Activated MoLCs released IL-12p70 and simultaneously, anti-inflammatory IL-10. The application of the Toll-like receptor ligands peptidoglycan, flagellin, and in particular, lipopolysaccharide (LPS) increased the corelease of these cytokines. LCs secreted IL-10 at a comparable level with MoLCs but failed to produce high amounts of IL-12p70 after application of danger signals. These data indicate that MoLCs as well as LCs display no maturation arrest concerning CD83 and CD86 expression. In difference to MoLCs, LCs resisted activation by CD40L and LPS in terms of IL-12 production. This shows that natural and generated LCs share similar features but differ in relevant functions.

Keratinocytes rapidly readjust ceramide-sphingomyelin homeostasis and contain a phosphatidylcholine-sphingomyelin transacylase.

Ceramide as central second messenger of the apoptosis-related sphingomyelin signaling pathway is a potential target for the control of cancer. A complex metabolizing network defines cell type and stage-specific final ceramide concentrations. Successful therapeutic control of ceramide levels requires a knowledge of multiple related turnover rates. The metabolism of ceramide and sphingomyelin was studied in keratinocytes under the condition of an unstimulated sphingomyelin signaling pathway. Preparations enriched in plasma membranes contain a neutral Mg(2+)-dependent sphingomyelinase and a Mg(2+)-independent sphingomyelin synthase that vigorously preserve balanced ceramide and sphingomyelin levels. Ceramide regulates neutral sphingomyelinase. Inhibition of sphingomyelin synthase by D609 treatment results in temporary loss of intercelluar contacts and in cellular shrinking. It is ineffective for sustained elevation of ceramide levels. Ceramide phosphorylating and deacylating activities are insignificant. Recently, fatty-acid remodeling in sphingomyelin was reported as likely to counteract the membrane-rigidifying effects of cholesterol. Keratinocytes transfer fluorescence labeled acyl-chains between phosphatidylcholine and sphingomyelin. A transferase of that kind would allow rapid adjustment of local lipid composition in response to acutely changed conditions. In addition, this transferase might have a function in the formation of the epidermal permeability barrier.