Allergic contact dermatitis is a widespread health disorder and occupational skin disease. Hence, screening for contact-sensitizing chemicals is highly relevant to toxicology, dermatology, and occupational medicine. The use of animal tests for this purpose is constrained by ethical considerations, need for high-throughput screening, and legislation (e.g., for cosmetics in the European Union). T cell activation is the final and most specific key event of the “adverse outcome pathway” for skin sensitization and therefore a promising target for the development of in vitro sensitization assays. We present a novel in vitro sensitization assay with a lymphocyte endpoint as an add-on to the loose-fit coculture-based sensitization assay (LCSA): the LCSA-ly. While the LCSA measures dendritic cell activation, the LCSA-ly offers the option for an additional lymphocyte endpoint which can be measured concurrently. We incorporated lymphocytes in our previously established coculture of primary human keratinocytes and monocyte-derived dendritic cells and tested nine substances: five sensitizers [2,4-dinitrochlorobenzene (DNCB) 1.25–15 µmol/l, p-phenylenediamine (PPD) 15.6–125 µmol/l, 2-mercaptobenzothiazole (MBT) 50–1000 µmol/l, coumarin, and resorcinol (both: 250–1500 µmol/l)] and four non-sensitizers (monochlorobenzene, caprylic acid, glycerol, and salicylic acid (all: 125–1000 µmol/l)]. DNCB and MBT increased a subset of IL-23 receptor+/IFN-γ receptor 1 (CD119)+ lymphocytes. DNCB, PPD, and MBT enhanced a subunit of the IL-4 receptor (CD124) and a memory marker (CD44) on lymphocytes. Remarkably, DNCB, PPD, and MBT raised IL-4 concentrations in coculture supernatants while IFN-γ levels decreased, which might point to Th2 activation in vitro. Coumarin, resorcinol, and non-sensitizers did not alter any of the tested surface markers or cytokines. IL-17 was not affected by any of the substances. Relative strength of sensitizers according to lymphocyte markers was DNCB > PPD > MBT, which corresponds to earlier results from the LCSA without lymphocyte endpoint, the murine local lymph node assay, and human data. This study is the first to prove the suitability of lymphocyte surface markers for sensitization testing and potency assessment.
In vitro sensitization assay Lymphocytes Coculture Allergic contact dermatitis
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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 from the Sonnenfeld Stiftung, Berlin, providing financial support for the FACS Calibur. Some experiments in this study were part of Janna Frombach’s master thesis.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
Ashby J, Basketter DA, Paton D, Kimber I (1995) Structure activity relationships in skin sensitization using the murine local lymph node assay. Toxicology 103(3):177–194CrossRefPubMedGoogle Scholar
Ashikaga T, Yoshida Y, Hirota M et al (2006) Development of an in vitro skin sensitization test using human cell lines: the human cell line activation test (h-CLAT). I. Optimization of the h-CLAT protocol. Toxicol In vitro Int J Publ Assoc BIBRA 20(5):767–773. https://doi.org/10.1016/j.tiv.2005.10.012CrossRefGoogle Scholar
Bellinghausen I, Brand U, Enk AH, Knop J, Saloga J (1999) Signals involved in the early TH1/TH2 polarization of an immune response depending on the type of antigen. J Allergy Clin Immunol 103(2 Pt 1):298–306CrossRefPubMedGoogle Scholar
Dearman RJ, Moussavi A, Kemeny DM, Kimber I (1996) Contribution of CD4+ and CD8+ T lymphocyte subsets to the cytokine secretion patterns induced in mice during sensitization to contact and respiratory chemical allergens. Immunology 89(4):502–510CrossRefPubMedPubMedCentralGoogle Scholar
Novelli F, D’Elios MM, Bernabei P et al (1997) Expression and role in apoptosis of the alpha- and beta-chains of the IFN-gamma receptor on human Th1 and Th2 clones. J Immunol 159(1):206–213PubMedGoogle Scholar
OECD (2014) The adverse outcome pathway for skin sensitisation initiated by covalent binding to proteins. OECD, ParisCrossRefGoogle Scholar
OECD (2016) Annex I: guidance document on the reporting of defined approaches and individual information sources to be used within integrated approaches to testing and assessment (IATA) for skin sensitisation. OECD, ParisGoogle Scholar
Parham C, Chirica M, Timans J et al (2002) A receptor for the heterodimeric cytokine IL-23 is composed of IL-12Rbeta1 and a novel cytokine receptor subunit, IL-23R. J Immunol 168(11):5699–5708CrossRefPubMedGoogle Scholar
Rougier N, Redziniak G, Mougin D, Schmitt D, Vincent C (2000) In vitro evaluation of the sensitization potential of weak contact allergens using langerhans-like dendritic cells and autologous T cells. Toxicology 145(1):73–82CrossRefPubMedGoogle Scholar