PCS/GENEX-Experimental Toxicology, Novartis Pharma AG, 4002 Basel, Switzerland
Keywords: human; dendritic cells; skin; allergy; sensitization; cell cultures: organ-specific; reduction; replacement; toxicity testing: contact allergens; toxicity testing: sensitising potential
Duration: 2 years Project Completion: 2003
Background and Aim
The only tests for contact allergic potential that are currently accepted by regulatory agencies are in vivo tests. The introduction of new methods (for example, the Murine Local Lymph Node Assay) has contributed to a reduction in both the number of animals used and in the stress experienced by the animals. The development and validation of in vitro methods using human material could lead to the replacement of animal tests entirely in the evaluation of the contact allergic potential of chemicals.
Human dendritic cells form an excellent basis for in vitro test systems since they play a pivotal role in the induction of contact allergy. In this project the reactions induced by contact allergens in human monocyte-derived dendritic cells (M-DC) will be investigated with the goal of developing a routine testing protocol.
Method and Results
The technology to culture dendritic cells from human blood monocytes has recently become available. Preliminary results from our laboratory indicate that human monocyte-derived dendritic cell technology has the potential to become a standard tool in contact allergy screening (Coutant et al., Toxicological Sciences, 1999). However, considerable work is still necessary to clarify the cellular, subcellular and biochemical events in M-DC following treatment with contact allergens. Within the ongoing project we refined the protocol for the generation of DC from human blood monocytes. CD86 up-regulation was used as a marker for M-DC activation. The M-DC were exposed to a dilution series of contact allergens or irritants in order to find the ranges necessary to produce a a concentration dependent up-regualtion of CD86. With our method we were able to distinguish between contact allergens and irritants in their mode of M-DC activation: contact allergens were able to activate the M-DC in the sub-toxic range, whereas irritants only activated M-DC at cytotoxic concentrations. For the screening method, both concentration ranges will be tested, and the measurement of activation markers and markers of cytotoxicity will be carried out in parallel. In addition, using genomic methods, we will search for a yet more sensitive marker to discriminate between contact allergens and irritants.
Conclusions and Relevance for 3R
Understanding the different effects of contact allergens and irritants on M-DC at the sub-cellular and molecular level is essential to provide the scientific basis for a standard in vitro testing protocol with human monocyte-derived dendritic cells. This project will deliver this background information. New markers that can discriminate even more sensitively between the mechanisms of M-DC activation by irritants and contact allergens may be found by new genomic methods. These will have to be validated using a large set of control substances.
1. Coutant Karine D., Fraissinette Anne, Cordier André and Ulrich Peter (1999) Modulation of the activity of human monocyte-derived dendritic cells by chemical haptens, a metal allergen and a staphylococcal superantigen, Toxicological Sciences 52, 189-198.
2. Straube Frank, Grenet Olivier, Bruegger Peter and Peter Ulrich (2005) Contact Allergens and Irritants show discrete differences in the activation of Human Monocyte derived Dendritic Cells: Consequences for in vitro Detecion of Contact Allergens, Archives of Toxicology, 79 (1), 37-46.