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Key Event Title
|Level of Biological Organization|
Key Event Components
|cytokine production involved in inflammatory response||increased|
Key Event Overview
AOPs Including This Key Event
Key Event Description
Keratinocytes are the major cell type of the epidermis of the skin. They are known to be the primary site of skin metabolism and play an important role in epithelial Dendritic Cells (DC) activation. Uptake of the hapten-protein complex formed during covalent binding by keratinocytes activates multiple events, including the release of pro-inflammatory cytokines (i.e. IL-18) and the induction of cyto-protective cellular pathways. Hapten-protein complexes can activate the inflammasome (;) and in so doing induce IL-18 production. Activation of the pro-inflammatory cytokine IL-18 results from cleavage of inactive IL-18 precursor protein by inflammasome-associated caspase-1. Intracellular Nod-like receptors (NLR) contain sensors for a number of cellular insults. Upon activation, NLRs oligomerise form molecular complexes (i.e. inflammasomes) that are involved in the activation of inflammatory-associated caspases, including caspase-1. Induction of intracellular levels of IL-18 exhibit responses upon exposure to hapten-protein complexes which can be used to establish potency. Keratinocyte exposure to allergens also results in induction of antioxidant/electrophile response element ARE/EpRE-dependent pathways. Briefly, reactive chemicals bind to Keap1 (Kelch-like ECH-associates protein 1) that normally inhibit the nuclear erythroid 2-related factor 2 (Nrf2). Released Nrf2 interacts with other nuclear proteins and binds to and activates ARE/EpREdependent pathways, including the cytoprotective genes NADPH-quinone oxidoreductase 1 (NQO1) and glutathione S-transferase (GSHST), among others (;).
How It Is Measured or Detected
Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible?
Investigations have focused on the DNA antioxidant-response elements (ARE), also known as electrophile response element. OECD TG 442D is the validated test guideline for measuring the activation of the antioxidant/electrophile response element (ARE) - dependant pathway). Currently, the only in vitro ARE-Nrf2 luciferase test method covered by this Test Guideline is the KeratinoSensTM. This assay uses a luciferase reporter gene under control of a single copy of the ARE element of the human AKR1C2 gene stably inserted into immortalized human keratinocytes (HaCaT cells). The KeratinoSensTM protocol can be found in the EURL ECVAM Database Service on Alternative Methods to animal experimentation (DB-ALM): Protocol No155 for KeratinoSensTM. The Keap1/Nrf2/ARE/EpRE cell signalling assay is also the mechanistic basis for the work on skin sensitisation chemicals at CeeTox Inc.. This work includes quantitative realtime polymerase chain reaction measurements of the relative abundance of mRNA for eleven selected genes whose expression is controlled by one of the three following pathways: Keap1/Nrf 2/ARE/EpRE, ARNT/AhR/XRE, and Nrf1/MTF/MRE. Interestingly, both Emter et al. and McKim et al. combine their cell signalling results with chemical reactivity data in algorithms, which can be viewed as a first step in using the AOP in quantitative assessment.
In vitro assays based on IL-18 induction in human keratinocytes (cell line NCTC 2544) or IL-8 induction in THP-1 cells have also been developed to identify allergens. Other studies have described chemokines (e.g. CCL2, CCL4) and receptor (e.g. CCR7) (see).
Domain of Applicability
A dose-dependent release of IL-18 has been shown following exposure of the murine keratinocyte cell line HEL-30 to sensitisers. Moreover, a concentration-dependant increase in intracellular IL-18 at non-cytotoxic concentrations of chemicals was observed in the human keratinocyte cell line NCTC2455 following 24-h treatment.
- Sutterwala FS, Ogura Y, Szczepanik M, Lara-Tejero M, Lichtenberger GS, Grant EP, Bertin J, Coyle AJ, Galán JE, Askenase PW, Flavell RA. 2006. Critical role for NALP3/CIAS1/Cryopyrin in innate and adaptive immunity through its regulation of caspase-1. Immunity 24: 317-327.
- Watanabe H, Gaide O, Pétrilli V, Martinon F, Contassot E, Roques S, Kummer JA, Tschopp J, French LE. 2007. Activation of the IL-1beta-processing inflammasome is involved in contact hypersensitivity. J. Invest. Dermatol. 127: 1956-1963.
- Martinon F, Mayor A and Tschopp J. 2009. The inflammasomes: guardians of the body. Ann. Rev. Immunol. 27: 229-265.
- Van Och FMM, Van Loveren H, Van Wolfswinkel JC, Machielsen AJC, Vandebriel RJ. 2005. Assessment of potency of allergenic activity of low molecular weight compounds based on IL-1α and IL-18 production by a murine and human keratinocyte cell line. Toxicology 210: 95-109.
- Natsch A and Emter R. 2008. Skin sensitizers induce antioxidant response element dependent genes: Application to the in vitro testing of the sensitisation potential of chemicals. Toxicol. Sci. 102:110-119.
- Ade N, Leon F, Pallardy M, Pfeiffer JL, Kerdine-Romer S, Tissier MH, Bonnet PA, Fabre I Ourlin JC. 2009. HMOX1 and NQO1 genes are upregulated in response to contact sensitizers in dendritic cells and THP-1 cell line: role of the Keap1/Nrf2 pathway. Toxicol. Sci. 107: 451-460.
- OECD 2015. Test No442D: In vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method. OECD Guidelines for the Testing of Chemicals, Section 4: Health effects. OECD Publishing. Doi: 10.1787/9789264229822-en.
- Emter R, Ellis G, Natsch A. 2010. Performance of a novel keratinocyte-based reporter cell line in screen skin sensitizers in vitro. Toxicol. Appl. Pharmacol. 245: 281-290.
- EURL ECVAM DB-ALM. Protocol No155: KeratinoSensTM. Available on: http://ecvam-dbalm.jrc.ec.europa.eu/.
- McKim JM Jr, Keller DJ III, Gorski JR. 2010. A new in vitro method for identifying chemical sensitizers combining peptide binding with ARE/EpRE-mediated gene expression in human skin cells. Cutan. Ocul. Toxicol. 29: 171-192.
- Corsini E, Mitjans M, Galbiati V, Lucchi L, Galli CL, Marinovich M. 2009. Use of IL-18 production in a human keratinocyte cell line to discriminate contact sensitizers from irritants and low molecular weight respiratory allergens. Toxicol. In Vitro 23: 769-796.
- Mitjans M, Galbiati V, Lucchi L, Viviani B, Marinovich M, Galli CL, Corsini E. 2010. Use of IL-8 release and p38 MAPK activation in THP-1 cells to identify allergens and to assess their potency in vitro. Toxicol. In Vitro 24: 1803-1809.
- dos Santos GG, Reinders J, Ouwhand K, Rustemeyer T, Scheper RJ, Gibbs S. 2009. Progress on the development of human in vitro dendritic cell based assays for assessment of skin sensitizing potential of compounds. Toxicol. Appl. Pharmacol. 236: 372-382.