title: A novel method to generate monocyte-derived dendritic cells during coculture with HaCaT facilitates detection of weak contact allergens in cosmetics.
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.
authors.affiliation: Institute of Clinical Pharmacology and Toxicology, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany.|Department of Dermatology and Allergy, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany.
authors.forename: Janna
authors.initials: J
authors.lastname: Frombach
authors.affiliation: Institute of Clinical Pharmacology and Toxicology, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Sonnenburg
authors.affiliation: Department of Plastic Surgery, St. Marien Hospital Berlin, Gallwitzallee 123, 12249, Berlin, Germany.
authors.forename: Björn-Dirk
authors.initials: BD
authors.lastname: Krapohl
authors.affiliation: Department of Dermatology and Allergy, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany.
authors.forename: Torsten
authors.initials: T
authors.lastname: Zuberbier
authors.affiliation: Institute of Clinical Pharmacology and Toxicology, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Stahlmann
authors.affiliation: Department of Internal Medicine (Abt. I), Bundeswehr Hospital, Bundeswehrkrankenhaus Berlin, Scharnhorststraße 13, 10115, Berlin, Germany. dr.max.schreiner@gmail.com.
authors.forename: Maximilian
authors.initials: M
authors.lastname: Schreiner
mesh_terms: D000485:Allergens / Q000032:analysis / Q000633:toxicity*; D051940:B7-2 Antigen / Q000819:agonists / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D002210:Caprylates / Q000009:adverse effects / Q000032:analysis; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D018920:Coculture Techniques; D003358:Cosmetics / Q000737:chemistry / Q000633:toxicity; D003713:Dendritic Cells / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D017449:Dermatitis, Allergic Contact / Q000209:etiology / Q000276:immunology / Q000378:metabolism / Q000473:pathology*; D005189:False Positive Reactions; D005230:Fatty Acids, Nonesterified / Q000009:adverse effects / Q000032:analysis; D057166:High-Throughput Screening Assays; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D007963:Leukocytes, Mononuclear / Q000166:cytology; D008956:Models, Chemical; D009000:Monocytes / Q000166:cytology; D010226:Parabens / Q000633:toxicity; D012118:Resorcinols / Q000633:toxicity; D015854:Up-Regulation
chemical_list: D000485:Allergens; D051940:B7-2 Antigen; D015415:Biomarkers; C497964:CD86 protein, human; D002210:Caprylates; D003358:Cosmetics; D005230:Fatty Acids, Nonesterified; D010226:Parabens; D012118:Resorcinols; C031492:octanoic acid; C031389:resorcinol; C006068:propylparaben
keywords: Coculture; Cosmetics; Dendritic cells; HaCaT; Keratinocytes; Sensitization assay