Reinhard Wanner

T-cell recognition of chemicals, protein allergens and drugs: towards the development of in vitro assays

Chemicals can elicit T-cell-mediated diseases such as allergic contact dermatitis and adverse drug reactions. Therefore, testing of chemicals, drugs and protein allergens for hazard identification and risk assessment is essential in regulatory toxicology. The seventh amendment of the EU Cosmetics Directive now prohibits the testing of cosmetic ingredients in mice, guinea pigs and other animal species to assess their sensitizing potential. In addition, the EU Chemicals Directive REACh requires the retesting of more than 30,000 chemicals for different toxicological endpoints, including sensitization, requiring vast numbers of animals. Therefore, alternative methods are urgently needed to eventually replace animal testing. Here, we summarize the outcome of an expert meeting in Rome on 7 November 2009 on the development of T-cell-based in vitro assays as tools in immunotoxicology to identify hazardous chemicals and drugs. In addition, we provide an overview of the development of the field over the last two decades.

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.

Human monocyte‐derived dendritic cells express TLR9 and react directly to the CpG‐A oligonucleotide D19

Oligodeoxynucleotides (ODNs) containing unmethylated CpG exhibit their immunostimulatory activities by binding to TLR. Here, we show that human monocyte‐derived dendritic cells (moDC) contain TLR9 protein, surprisingly, in amounts comparable with plasmacytoid DC (pDC). Immature moDC but not mature moDC nor monocytes captured CpG‐ODNs. moDC stimulation with the CpG‐A ODN D19 up‐regulated CD83, CD86, and HLA‐DR. Without CD40 ligand costimulation, full maturation was not achieved. D19‐stimulated moDC primed allogeneic CD4+‐T cells for proliferation and differentiation into IFN‐γ‐secreting Th1 cells. Neither IL‐12 nor IL‐6 or TNF‐α was involved. Microarray analysis pointed to a participation of Type I IFNs. In fact, D19‐stimulated moDC secreted considerable amounts of IFN‐α. This indicates that moDC themselves sense viral and bacterial DNA and do not need help from pDC.

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‐β1. Here, we compare maturation‐induced antigen expression and cytokine release of LCs with MoLCs. To achieve comparable cell populations, LCs andMoLCs 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, lipoplysaccharide (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.

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‐α 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‐α and IL‐6, and NF‐κB 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.

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.