In vitro detection of contact allergens: Development of an optimized protocol using human peripheral blood monocyte-derived dendritic cells

Abstract

Allergic contact dermatitis is a delayed T-cell mediated allergic response associated with relevant social and economic impacts. Animal experiments (e.g. the local lymph node assay) are still supplying most of the data used to assess the sensitization potential of new chemicals. However, the 7th amendment to the EU Cosmetic Directive will introduce a testing ban for cosmetic ingredients after 2013. In vitro alternative methods are thus being actively developed. Although promising results have been obtained with cell lines, their reduced functionality and inherent genomic instability led us to reinvestigate the use of peripheral blood monocyte-derived dendritic cells (PBMDCs) for the establishment of a reliable in vitro sensitization test. To solve the issues associated with the use of primary cells, the culture and exposure conditions (cytokine concentrations, incubation time, readout, pooled vs. single donors and cytotoxicity) were re-assessed and optimized. Here we propose a stable and reproducible protocol based on PBMDCs. This should allow a wider acceptance of PBMDCs as a reliable test system for the detection of human skin sensitizers and the inclusion of this protocol in an integrated testing strategy.

Introduction

Allergic contact dermatitis (ACD) is a type-IV allergy of the skin associated with significant social and economic impacts. It has a high frequency in the general population and thus represents a significant public health challenge (Schafer et al., 2001). It is the results of a delayed T-cell-mediated allergic reaction to skin proteins modified by exposure to low molecular weight reactive chemicals (haptens). The source of haptens, also referred to as sensitizers, can be very diverse, e.g. antibiotics, antimycotics, metal ions or cosmetic ingredients. Therefore a comprehensive risk assessment of new chemicals is crucial to identify potential ACD inducers and avoid unacceptable exposure level of involved workers or end users. Currently, animal experiments (e.g. the local lymph node assay (LLNA) or the guinea pig maximization test) were supplying most of the data needed for risk assessment and decision-making (Basketter and Scholes, 1992, Kimber et al., 1994, Dearman et al., 1999, Gerberick et al., 2000, Basketter et al., 2002, Kimber et al., 2002). However, the 7th Amendment to the European Cosmetics Directive (Directive 76/768/EEC) will severely restrict the use of animal tests. Skin sensitization tests are considered as repeated dose toxicity assays, and the deadline for the corresponding animal test ban has been set to 2013. After this date, the cosmetic industry will face a marketing ban for new ingredients examined in animal experiments. The development of in vitro alternative test methods for skin sensitisation is thus required by the cosmetic industry and is also raising much interest in other industry sectors. To develop these alternative in vitro methods, an array of approaches is being investigated. These range from biophysical/in silico approaches to cell based systems. For cellular assays, a variety of cells known to be involved in the biology of ACD is being evaluated, i.e. keratinocytes, dendritic cells (DCs) and T-cells. DCs are known to play a central role during the induction of skin sensitization through their ability to uptake, process and present haptenated self-proteins to T-cells, and represent the most abundant antigen presenting cell population in the skin (Novak et al., 1999, Toebak et al., 2009). In vitro assay development has thus rapidly focused on available sources of DC-like cells including primary cells (cord or peripheral blood) or cell lines (e.g. U937, THP-1, Mutz-3, KP-1, HL-60, K562, reviewed in dos Santos et al. (2009)). Assays based on primary cells require specific cell sources that might represent a limiting factor, especially in the case of cord blood based assays. Donor to donor variability was also noted as a critical point (Aiba et al., 1997, Basketter and Maxwell, 2007) and many research groups directed their efforts towards the development of in vitro test systems using cell line. Cell line based assays were expected to overcome the main limitations of the primary cells. Being widely available through cell banks and considered as easy to cultivate, cell lines represented an obvious alternative. Most cell lines evaluated were of myeloid origin, mainly from hematopoietic neoplasms. However, the genome of these tumorigenic and transformed cells is severely altered and due to their intrinsic genomic instability, there may accumulate further genomic abnormalities over prolonged culture period. As a consequence some lost their typical homeostasis and have only limited functional properties (e.g. U937 decreased response to lipopolysaccharide (LPS)). Moreover it is known that an adequate usage of these lines is limited by cell passages due to their progressive loss of response to chemical stress (Sakaguchi et al., 2010). Nevertheless, in case of the U937 and THP-1 cell lines, promising assays could be developed by using carefully controlled culture conditions ensuring a relatively stable cell phenotype (Ade et al., 2006, Ashikaga et al., 2006, Sakaguchi et al., 2006, Python et al., 2007). The h-CLAT and MUSST test protocols based on THP-1 or U937 cells respectively are now under consideration for pre-validation by the European Centre for the Validation of Alternative Methods (ECVAM). Another cell line, Mutz-3, is being intensively investigated; it is relatively immature and can be forced to differentiate into a Langerhans-like phenotype (Azam et al., 2006, Kim et al., 2006, Larsson et al., 2006, Nelissen et al., 2009, Python et al., 2009, Ouwehand et al., 2010). However, Mutz-3 propagation requires complex culture procedures (e.g. co-culture), thereby reducing their perceived advantages and inducing unexpected intra- or inter-laboratories variability.

The difficulties associated with cell lines led us to reinvestigate the use of peripheral blood monocyte derived DCs as a reliable in vitro test system for detecting skin sensitizers. As cell culture and exposure conditions described in the literature were not always consistent a fundamental protocol review and optimization phase was initiated (Degwert et al., 1997, Reutter et al., 1997, Aeby et al., 2004, Staquet et al., 2004). This included the determination of the optimal cell isolation procedure (use of positive or negative depletion protocols), the use of monocytes obtained from single donors instead of pooled cells and the optimization of the culture (cytokine concentrations) and exposure conditions. The result is an improved and stable protocol with reduced donor to donor variability, reproducible results and a significant correlation with the LLNA results. This should allow a wider acceptance of monocyte derived DCs as cells of choice for detecting human skin sensitizers.