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

  • Immunotoxicology
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An Erratum to this article was published on 25 June 2016

Abstract

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.

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Acknowledgments

Studies were supported by a grant from the German Federal Ministry of Education and Research (grant no. 31P5924). We are also grateful for a grant of the Sonnenfeld Stiftung, Berlin, providing financial support for the FACS Calibur.

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Authors and Affiliations

  1. Institute of Clinical Pharmacology and Toxicology, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany

    Janna Frombach, Anna Sonnenburg & Ralf Stahlmann

  2. Department of Dermatology and Allergy, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany

    Janna Frombach & Torsten Zuberbier

  3. Department of Plastic Surgery, St. Marien Hospital Berlin, Gallwitzallee 123, 12249, Berlin, Germany

    Björn-Dirk Krapohl

  4. Department of Internal Medicine (Abt. I), Bundeswehr Hospital, Bundeswehrkrankenhaus Berlin, Scharnhorststraße 13, 10115, Berlin, Germany

    Maximilian Schreiner

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  1. Janna Frombach
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  2. Anna Sonnenburg
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  3. Björn-Dirk Krapohl
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  4. Torsten Zuberbier
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  6. Maximilian Schreiner
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Correspondence to Maximilian Schreiner.

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Conflict of interest

The LCSA is patented in Germany with the application number DE102007006736B4 (patent holder: Mrs. Dagmar Briechle). The authors have no financial relationship with the patent holder and declare no conflict of interest.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. This article does not contain any studies with animals performed by any of the authors.

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An erratum to this article is available at http://dx.doi.org/10.1007/s00204-016-1748-1.

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Frombach, J., Sonnenburg, A., Krapohl, BD. et al. A novel method to generate monocyte-derived dendritic cells during coculture with HaCaT facilitates detection of weak contact allergens in cosmetics. Arch Toxicol 91, 339–350 (2017). https://doi.org/10.1007/s00204-016-1722-y

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