Event: 397

Key Event Title


Response, Keratinocytes

Short name


Response, Keratinocytes

Biological Context


Level of Biological Organization

Cell term


Organ term


Key Event Components


Process Object Action

Key Event Overview

AOPs Including This Key Event




Taxonomic Applicability


Life Stages


Sex Applicability


Key Event Description


Haptens can also react with cell surface proteins and activate response pathways in keratinocytes (see [1]). Uptake of the hapten by keratinocytes activates multiple events, including the release of pro-inflammatory cytokines and the induction of cyto-protective cellular pathways. Activation of the pro-inflammatory cytokine IL-18 results from cleavage of inactive IL-18 precursor protein by inflammasome-associated caspase-1[2]. Sensitizers can activate the inflammasome ([3];[4]) and in so doing induce IL-18 production. Intracellular Nodlike receptors (NLR) contain sensors for a number of cellular insults. Upon activation (by a currently unknown mechanism), NLRs oligomerise form molecular complexes (i.e. inflammasomes) that are involved in the activation of inflammatory-associated caspases, including caspase-1. Inductions of intracellular levels of IL-18 exhibit responses upon exposure to sensitizers which can be used to establish potency[5].

Keratinocyte exposure to sensitizers also results in induction of antioxidant/electrophile response element ARE/EpRE-dependent pathways[6]. 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 (NQ01) and glutathione S-transferase (GSHST), among others ([6];[7]). An in vitro reporter assay based on activation via the ARE/EpRE response element has been shown to be responsive to known sensitizers in HaCaT keratinocytes[8]. Expression of ARE/EpRE-dependent genes and other cytoprotective genes (including CYP1A1, MT1 and MT2) in HaCaT cells are part of a proprietary in vitro battery approach to determining sensitisation potency[9]. Both the Natsch and McKim groups have shown that this signalling pathway responds in a quantitative fashion, which is related to LLNA potency (e.g. strong, moderate, and weak).

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?

Biochemical Pathways Related to Skin Sensitisation

Biochemical or intracellular pathways affected by the action of reactive chemicals are in general thought to be related to skin sensitisation; however, current knowledge is incomplete. The c-Jun Nterminal kinases and the p38 kinases have been shown to be activated upon exposure to protein-binding chemicals [10]. However, these studies are based on only a few compounds, in particular, 1-chloro-2,4-dinitrobenzene. A few investigations have examined this possibility by measuring changes in kinase expression in different signal transduction pathways (e.g. p38 MAPK, ERK, PGK, and NFκB) (see [11]). Miyazawa and co-workers ([12] and [13]) examined P38 MAPK (mitogen-activated protein kinase) and ERK, (extracellular signal-regulated kinase) in THP-1 cells and showed that 1-chloro-2,4-dinitrobenzene, an extreme sensitizer, activates both cellular pathways and stimulates TNF-α release and subsequent phenotypic changes in the cells. As noted, the Keap1/Nrf2/ARE/EpRE cell signalling assay is a potential cellular marker for sensitisation because Keap1 is a thiol-rich sensor protein which has been shown to be covalently modified by electrophiles that leads to activation of ARE-dependent genes[14].

Events in Keratinocyte

In recent years, investigations have focused on the DNA antioxidant-response element (ARE), also known as electrophile response element (see [15]). The KeratinoSens system of Natsch’s group 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)[8]. The experimental design is robust with chemicals routinely tested at twelve concentrations in triplicate before evaluating for significant induction of gene activity [16]. One advantage of this assay is that it appears to address the issue of metabolism[8]. While several variants of the luciferase-based ARE assay have been developed based on data derived from the assay described by Natsch and Emter[6], Natsch et al.[17] evaluated the predictive performances of the ARE assay evaluated against LLNA data for more than 100 chemicals. They report a 79%, concordance, 79% sensitivity and 81% specificity[17]. An in vitro assay based on IL-18 induction in human keratinocytes (cell line NCTC 2544) can also distinguish between sensitizers and irritants ([18];[19]). Other studies have described chemokines (e.g. CCL2, CCL4) and receptor (e.g. CCR7) (see [11]).

The Keap1/Nrf2/ARE/EpRE cell signalling assay is also the mechanistic basis for the work on skin sensitisation chemicals at CeeTox Inc.[9]. Briefly, this work includes quantitative realtime polymerase chain reaction measurements of the relative abundance of mRNA for eleven selected genes. While most of the data is proprietary, the reported results are highly promising. Interestingly, both Emter et al.[8] and McKim et al.[9] 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.


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 DC activation. Using human keratinocytes (HaCaT cells), McKim et al.[9] evaluated selected genes associated with three cell signalling pathways (Keap1/Nrf 2/ARE/EpRE, ARNT/AhR/XRE, and Nrf1/MTF/MRE) which are known to be activated by sensitizing agents. Briefly, the relative abundance of eleven genes whose expression is controlled by one of these pathways, was measured. The Nrf2/ARE/EpRE-controlled genes are: 1) NQO1, 2) AKR1C2, 3) thioredoxin (TXN), 4) interleukin 8 (IL8), 5) aldehyde dehydrogenase 3A (ALDH3A), 6) heme-oxygenase 1 (HMOX1), 7) musculoaponeurotic fibrosarcoma (MafF), and 8) GCLC. The XRE-controlled gene is 9) CYP1A1. The MRE-controlled genes are 10) metallothionein 1 (MT1), and 11) metallothionein 2 (MT2). Gene expression was reported on a scale of 4+, 3+, 2+, + and NC). For DNCB McKim et al, report: NQO1 expression = 4+; AKR1C2 = 2+; TXN = NC; IL8 = +; CYP1A1 = +; ALDH3A = NC; HMOX = NC; MafF = +; GCLC = NC; MT1A = NC, and MT2A = 2+. Microarray analysis of DCNB-treated HaCaT cells by Vanderbriel et al. (2010) show similar results.

The KeratinoSens assay[8] examines dose-responses (routinely twelve concentrations in triplicate) for significant induction of gene activity in an in vitro assay with a luciferase reporter gene under control of a single copy of the ARE-element of the human AKR1C2 gene stably inserted into HaCaT keratinocyte cell line. Using a standard operating procedure[8], experimental data was generated and the average maximal induction of gene activity (Imax) and the average concentration inducing gene activity >50% above control values (EC1.5) were determined. The latter calculations were performed with linear extrapolation from the values above and below the induction threshold (as for the EC3 value determination in the LLNA. Intra- and inter-laboratory testing of DNCB with the KeratioSens assay[16] report repeatable and reproducible results (Table 2).
Table 2. Intra- and inter-laboratory testing of DNCB with the KeratinoSens assay.


Imax (fold induction)

EC 1.5 (μM)

A (historical)


















Cultures of human keratinocyte cell line NCTC 2544 were exposed to DNCB and cell-associated IL-18 evaluated 24 hours later[18]. Intracellular IL-18 content was assessed by specific sandwich ELISA, with results expressed in pg/mg of total intracellular protein. DNCB induces a significant dose-response increase in IL-18 production; however, production is modest as compared to other sensitizers that were tested.

Overview table: How it is measured or detected

Method(s) Reference URL Regulatory


Validated Non


ARE-Nrf2 Luciferase Test Method (Keratinosens) TG442D [1] X X
DB-ALM [2]
RhE-IL-18 Gibbs et al., 2013 [3] X

Domain of Applicability




  1. Weltzien H, Corsini E, Gibbs S, Lindstedt M, Borrebaeck C, Budde P, Schulz-Knappe P, Thierse H-J, Martin S, Roggen E. 2009. Safe cosmetics without animal testing? Contributions of the EU Project Sens-it-iv. J. für Verbraucherschutz und Lebensmittelsicherheit. 4: 41-48.
  2. Martinon, F., Mayor, A. and Tschopp, J. 2009. The inflammasomes: guardians of the body. Ann. Rev. Immunol. 27: 229-265.
  3. 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.
  4. 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.
  5. 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.
  6. 6.0 6.1 6.2 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
  7. 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.
  8. 8.0 8.1 8.2 8.3 8.4 8.5 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.
  9. 9.0 9.1 9.2 9.3 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.
  10. Trompezinski S, Migdal C, Tailhardat M, Le Varlet B, Courtellemont P, Haftek M, Serres M. 2008. Charaterization of early events involved in human dendritic cell maturation induced by sensitizers: cross talk between MAPK signalling pathways. Toxicol. Appl. Pharmacol. 230: 397- 406.
  11. 11.0 11.1 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.
  12. Miyazawa, M. Ito, Y., Kosaka, N., Nukada, Y., Sakaguchi, H., Suzuki, H., and Nishiyama, N. 2008a. Role of MAPK signalling pathway in the activation of dendritic type cell line, THP-1, induced by DNBC and NiSO4. Toxicol. Sci. 33: 51-59.
  13. Miyazawa, M., Ito, Y., Kosaka, N., Nukada, Y., Sakaguchi, H., Suzuki, H., and Nishiyama, N. 2008b. Role of TNF-alpha and extracellular ATP in THP-1, cell activation following allergen exposure. Toxicol. Sci. 33: 51-59.
  14. Dinkova-Kostova AT, Holtzclaw WD, Kensler TW. 2005. The role of Keap1 in cellular protective responses. Chem. Res. Toxicol. 18: 1779-1791.
  15. Natsch A. 2010. The Nrf2-Keap1-ARE toxicity pathway as a cellular sensor for skin sensitizers-functional relevance and a hypothesis on innate reactions to skin sensitizers. Toxicol. Sci. 113: 284-292.
  16. 16.0 16.1 Natsch A, Bauch C, Foertsch L, Gerberick F, Norman K, Hilberer A, Inglis H, Landsiedel R, Onken S, Reuter H, Schepky A, Emter R. 2011. The intra- and inter-laboratory reproducibility and predictivity of the KeratinoSens assay to predict skin sensitizers in vitro: results of a ring-study in five laboratories. Toxicol. In Vitro 25: 733-44.
  17. 17.0 17.1 Natsch A, Emter R, Ellis G. 2009. Filling the concept with data: Integrating data from different in vitro and in silico assays on skin sensitizers to explore the battery approach for animal-free skin sensitisation testing. Toxicol. Sci. 107: 106-121.
  18. 18.0 18.1 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.
  19. 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.