title: Development of a co-culture of keratinocytes and immune cells for in vitro investigation of cutaneous sulfur mustard toxicity.
abstract: Sulfur mustard (SM) is a chemical warfare agent causing skin blistering, ulceration and delayed wound healing. Inflammation and extrinsic apoptosis are known to have an important role in SM-induced cytotoxicity. As immune cells are involved in those processes, they may significantly modulate SM toxicity, but the extent of those effects is unknown. We adapted a co-culture model of immortalized keratinocytes (HaCaT) and immune cells (THP-1) and exposed this model to SM. Changes in necrosis, apoptosis and inflammation, depending on SM challenge, absence or presence and number of THP-1 cells were investigated. THP-1 were co-cultured for 24h prior to SM exposure in order to model SM effects on immune cells continuously present in the skin. Our results indicate that the presence of THP-1 strongly increased necrosis, apoptosis and inflammation. This effect was already significant when the ratio of THP-1 and HaCaT cells was similar to the ratio of Langerhans immune cells and keratinocytes in vivo. Any further increases in the number of THP-1 had only slight additional effects on SM-induced cytotoxicity. In order to assess the effects of immune cells migrating into skin areas damaged by SM, we added non-exposed THP-1 to SM-exposed HaCaT. Those THP-1 had only slight effects on SM-induced cytotoxicity. Notably, in HaCaT exposed to 300μM SM, necrosis and inflammation were slightly reduced by adding intact THP-1. This effect was dependent on the number of immune cells, steadily increasing with the number of unexposed THP-1 added. In summary, we have demonstrated that (a) the presented co-culture is a robust model to assess SM toxicity and can be used to test the efficacy of potential antidotes in vitro; (b) immune cells, damaged by SM strongly amplified cytotoxicity, (c) in contrast, unexposed THP-1 (simulating migration of immune cells into affected areas after exposure in vivo) had no pronounced adverse, but exhibited some protective effects. Thus, protecting immune cells from SM toxicity may help to reduce overall injury.
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstraße 11, 80937 Munich, Germany. Electronic address: frankbalszuweit@bundeswehr.org.
authors.forename: Frank
authors.initials: F
authors.lastname: Balszuweit
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstraße 11, 80937 Munich, Germany.
authors.forename: Georg
authors.initials: G
authors.lastname: Menacher
authors.affiliation: Department of Environmental Toxicology, University of Trier, Universitätsring 15, 54296 Trier, Germany.
authors.forename: Brunhilde
authors.initials: B
authors.lastname: Bloemeke
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstraße 11, 80937 Munich, Germany; Department of Molecular and Cellular Sports Medicine, German Sports University, Am Sportpark Müngersdorf 6, 50933 Cologne, Germany.
authors.forename: Annette
authors.initials: A
authors.lastname: Schmidt
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstraße 11, 80937 Munich, Germany.
authors.forename: Franz
authors.initials: F
authors.lastname: Worek
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstraße 11, 80937 Munich, Germany.
authors.forename: Horst
authors.initials: H
authors.lastname: Thiermann
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstraße 11, 80937 Munich, Germany; Walther Straub Institute of Pharmacology and Toxicology, University of Munich, Goethestr. 33, 80336 Munich, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Steinritz
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line; D002619:Chemical Warfare Agents / Q000633:toxicity*; D018920:Coculture Techniques; D006801:Humans; D007249:Inflammation / Q000139:chemically induced / Q000276:immunology / Q000473:pathology; D015850:Interleukin-6 / Q000096:biosynthesis; D015603:Keratinocytes / Q000187:drug effects* / Q000276:immunology* / Q000473:pathology; D007801:Langerhans Cells / Q000187:drug effects / Q000276:immunology / Q000473:pathology; D009000:Monocytes / Q000187:drug effects* / Q000276:immunology* / Q000473:pathology; D009151:Mustard Gas / Q000633:toxicity*; D009336:Necrosis
chemical_list: D002619:Chemical Warfare Agents; C508600:IL6 protein, human; D015850:Interleukin-6; D009151:Mustard Gas
keywords: Cell death; Inflammation; Keratinocytes; Sulfur mustard; THP-1 immune cells