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Key Event Title
Activation, Dendritic Cells
|Level of Biological Organization|
Key Event Components
|MHC protein complex assembly||increased|
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP||Point of Contact||Author Status||OECD Status|
|Skin Sensitisation AOP||KeyEvent||Agnes Aggy (send email)||Open for citation & comment||TFHA/WNT Endorsed|
|Covalent binding to proteins leads to Respiratory Sensitisation/Sensitization/Allergy||KeyEvent||Arthur Author (send email)||Under Development: Contributions and Comments Welcome||Under Development|
Key Event Description
Immature epidermal dendritic cells, known as Langerhans cells, and dermal dendritic cells serve as antigen-presenting cells (;;;). In this role, they recognize and internalize the hapten-protein complex formed during covalent binding leading to their activation. Subsequently, the dendritic cell loses its ability to seize new hapten-protein complexes and gains the potential to display the allergen-MHC-complex to naive T-cells; this process is often referred to as dendritic cell maturation. Simultaneously, under the influence of fibroblast- blood endothelial- and lymph endothelial chemokines (e.g. CCL19, CCL21) and epidermal cytokines (e.g. interleukin (IL), IL-1 α, IL-1β, IL-18, tumour necrosis factor alpha (TNF-α)) maturing dendritic cells migrate from the epidermis to the dermis of the skin and then to the proximal lymph nodes, where they can present the hapten-protein complex to T-cells via a major histocompatibility complex (MHC) molecule (;). Dendritic cell activation, upon exposure to hapten-protein complexes also leads to functional changes in the cells. For example, there are changes in chemokine secretion, cytokine secretion and in the expression of chemokine receptors (see). Additionally, during dendritic cell maturation MHC, co-stimulatory and intercellular adhesion molecules (e.g. CD40, CD86, and DC11 and CD54, respectively) are up-regulated (see;;). Signal transduction cascades precede changes in expression of surface proteins markers and chemokine or cytokine secretion. In fact, there is evidence that during the response, hapten-protein complexes can react with cell surface proteins and activate mitogen-activated protein kinase signalling pathway. In particular, the biochemical pathway involving extracellulare signal-regulating kinases- the c-jun N-terminal kinases and the p38 kinases have been shown to be activated upon exposure to protein-binding chemicals. These pathways are of particular importance in keratinocytes and dendritic cell response to protein-hapten complexes. Components of signal transduction pathways are kinases, which phosphorylate and dephosphorylate a variety of substrates in order to elicit a change in the expression or secretion of target molecules. As a result, components of the signal transduction cascade are thought to be biomarkers. Investigations into the possible role of calcium influx as an early event in dendritic cell activation suggest that calcium influx is a second event following reactive oxygen species induction;.
How It Is Measured or Detected
Genomic and proteomic studies also have the potential to reveal biomarkers in dendritic cell-based assays. Custom designed arrays or quantitative polymerase chain reaction (PCR) of selected genes have been used to highlight the reaction of dendritic cells (see). VITOSENS, an assay that uses human CD34+ progenitor-derived dendritic cells (CD34-DC), is based on the differential expression of the cAMP-responsive element modulator (CREM) and monocyte chemotactic protein-1 receptor (CCR2). Genomic signatures have been also developed for the identification of human sensitising chemicals: a biomarker signature, the Genomic Allergen Rapid Detection test (GARD) based on the human myelomonocytic cell line MUTZ-3 and a genomic platform, SENSIS, which consists of measuring the over-expression of 3 sets of genes, that may allow the in vitro assessment of the sensitising potential of a compound.
In Vitro Assays for Cell Surface Markers, Cytokines, and Chemokines
Alterations in intercellular adhesion molecules, cytokines, and chemokines are part of the immunology response which can serve as biomarkers. Since dendritic cell maturation upon exposure to hapten-protein complexes is accompanied by changes in surface marker expression, these surface markers are perceived as promising candidates as primary biomarkers of dendritic cell activation for the development of cell-based in vitro assays. While a variety of surface markers have been described to be up-regulated upon dendritic cell maturation, a review of the literature reveals that CD86 expression, followed by CD54 and CD40, are the most extensively studied intercellular adhesion and co-stimulator molecules to date. The human Cell Line Activation Test (h-CLAT) reported flow cytometry results for CD86 and CD54 expression in THP-1 cells;. An OECD Test Guideline for the h-CLAT is currently under review. The h-CLAT protocol can be found in the EURL ECVAM Database Service on Alternative Methods to animal experimentation (DB-ALM): Protocol No158 for human Cell Line Activation Test (h-CLAT). Other studies with THP-1 cells include that of An et al. (2009). Another assay, the myeloid U937 skin sensitisation test (U-SENS), is based as well on the measurement of CD86 by flow cytometry;;). In addition to that, a variety of cytokines have been studied in relationship to skin sensitizers. IL-8 is a promising chemokine for distinguishing sensitisers from non-sensitisers. Quantification of IL-8 can be performed by Enzyme Linked Immunosorbent Assay, a technique that is far simpler and amenable to high throughput screening than the flow cytometry technique used to measure CD86 expression. The expression of other cytokines linked to skin sensitisers include IL-1 α, IL-1β, IL-18, and TNF-α form the basis for other dendritic cell assays.
While some respiratory sensitizers have been assessed, it is unclear whether this event is distinct between skin and respiratory sensitizers. (dos Santos et al., 2009) The genomic allergen rapid detection (GARD) test is an MUTZ-3-based assay for assessing chemical sensitizers utilizing genomic biomarker prediction signatures to generate prediction calls of unknown chemicals such as skin sensitizers, respiratory sensitizers, or nonsensitizers, including irritants. (Johannsen et al., 2011) Preliminary data on the performance of the GARD for assessing chemical respiratory sensitizers using transcriptional readouts of a genomic biomarker signature indicated 80% accuracy. (Forreryd, et al., 2015)
There are several in vitro assays available to assess DC maturation; the most advanced is the h-CLAT, which determines changes in CD86 and CD54 levels on THP-1 cell.(Ashikaga, et al., 2006, Sakaguchi, et al., 2006) However, only limited data are available substantiating its performance on chemical respiratory sensitizers. (Basketter, et al. 2017) Several assays similar to the h-CLAT have emerged over time and are currently in the process of being validated (e.g., the MUSST measuring CD86 responses by U937 cells), but again no or minimal information is available to assess assay performance in detecting respiratory sensitizers. The MUTZ-3 cell line is also being investigated for the potential to assess the capacity of a chemical to induce LC migration. The discriminating feature of the assay is that irritant-induced migration is CCL5 dependent, while sensitizer-induced migration is CXCL12 dependent. The readout of the test is the ratio between migration toward CXCL12 or to CCL5. Despite its complexity, the assay seems to be relatively well transferable.(Rees et al., 2011)
Overview table: How it is measured or detected
Domain of Applicability
- ↑ Ryan CA, Gerberick GF, Gildea LA, Hulette BC, Bettis CJ, Cumberbatch M, Dearman RJ, Kimber I. 2005. Interactions of contact allergens with dendritic cells: opportunities and challenges for the development of novel approaches to hazard assessment. Toxicol. Sci. 88: 4-11.
- ↑ Ryan CA, Kimber I, Basketter, DA, Pallardy M, Gildea LA, Gerberick GF. 2007. Dendritic cells and skin sensitisation. Biological roles and uses in hazard identification. Toxicol. Appl. Pharmacol. 221: 384-394.
- ↑ 3.0 3.1 3.2 3.3 3.4 3.5 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.
- ↑ 4.0 4.1 4.2 Kimber I, Basketter DA, Gerberick GF, Ryan CA, Dearman, R.J. 2011. Chemical allergy: Translating biology into hazard characterization. Toxicol. Sci. 120(S1): S238-S268.
- ↑ 5.0 5.1 Antonopoulos C, Cumberbatch M, Mee JB, Dearman RJ, Wei XQ, Liew FY, Kimber I, Groves RW. 2008. IL-18 is a key proximal mediator of contact hypersensitivity and allergen induced Langerhans cell migration in murine epidermis. J. Leukoc. Biol. 83: 361-367.
- ↑ Ouwehand K, Santegoets SJAM, Bruynzeel DP, Scheper RJ, de Gruijl TD, Gibbs S. 2008. CXCL12 is essential for migration of activated Langerhans cells for epidermis to dermis. Eur. J. Immunol. 38: 3050-3059.
- ↑ Vandebriel RJ and van Loveren H. 2010. Non-animal sensitisation testing: State-of-the-art. Crit. Rev. Toxicol. 40: 389-404.
- ↑ Trompezinski S, Migdal C, Tailhardat M, Le Varlet B, Courtellemont P, Haftek M and 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.
- ↑ Lambrechts N, Vanheel H, Hooyberghs J, De Boever P, Witters H, Van Den Heuval R, Van Tendeloom V, Nelissen I, Schoeters G. 2010. Gene markers in dendritic cells unravel pieces of the skin sensitisation puzzle. Toxicol. Letters 196: 95-103.
- ↑ Migdal C, Tailhardat M, Courtellemont P, Haftek M, Serres M. 2010. Responsiveness of human monocyte-derived dendritic cells to thimerosal and mercury derivatives. Toxicol. Appl. Pharmacol. 246: 66-73.
- ↑ Aeby P, Ashikaga T, Bessou-Touya S, Schapky A, Geberick F, Kern P, Marrec-Fairley M, Maxwell G, Ovigne JM, Sakaguchi H, Reisinger K, Tailhardat M, Martinozzi-Teisser S, Winkler P. 2010. Identifying and characterizing chemical skin sensitizers without animal testing; Colipa’s research and methods development program. Toxicol. In Vitro 24: 1465-1473.
- ↑ Hooyberghs J, Schoeters E, Lambrechts N, Nelissen I, Witters H, Schoeters G, Van Den Heuvel R. 2008. A cell-based in vitro alternative to identify skin sensitizers by gene expression. Toxicol. Appl. Pharmacol. 231: 103-111.
- ↑ Borrebaeck CA and Wingren C. 2009. Design of high-density antibody microarrays for disease proteomics: key technological issues. J. Proteomics 72: 928-935.
- ↑ Groux H and Sabatier JM. 2010. Polypeptides for the in vitro assessment of the sensitising potential of a test compound. International Application Patent No.: PCT/EP2010/055895.
- ↑ Sakaguchi H, Ashikaga T, Miyazawa M, Kosaka N, Ito Y, Yoneyama K, Sono S, Itagaki H, Toyoda H, Suzuki H. 2009. The relationship between CD86/CD54 expression and THP-1 cell viability in an in vitro skin sensitisation test-human cell line activation test (h-CLAT). Cell Biol. Toxicol. 25: 109-126.
- ↑ Ashikaga T, Sakaguchi H, Sono S, Kosaka N, Ishikawa M, Nukada Y, Miyazawa M, Ito Y, Nishiyama N, Itagaki H. 2010. A comparative evaluation of in vitro skin sensitisation tests: the human cell-line activation test (h-CLAT) versus the local lymph node assay (LLNA). Altern. Lab. Anim. 38:275-84.
- ↑ EURL ECVAM DB-ALM. Protocol No158: Human Cell Line Activation Test (h-CLAT) Available on: http://ecvam-dbalm.jrc.ec.europa.eu/.
- ↑ Ade N, Martinozzi-Teissier S, Pallaardy M, Rousset F. 2006. Activation of U937 cells by contact sensitizers: CD86 expression is independent of apoptosis. J. Immunotoxicol. 3: 189-197.
- ↑ Python F, Goebel C, Aeby P. 2007. Assessment of the U937 cell line for detection of contact allergens. Toxicol. Appl. Pharmacol. 220: 113-124.
- ↑ Ovigne JM, Martinozzi-Teissier S, Verda D, Abdou D, Piroird C, Ade N, Rousset F. 2008. The MUSST for in vitro skin sensitisation prediction: Applicability domains and complementary protocols to adapt to the physico-chemical diversity of chemicals. Toxicology Letters, 180: Supplement 1, 5, S216.
ASHIKAGA T, YOSHIDA Y, HIROTA M, YONEYAMA K, ITAGAKI H, SAKAGUCHI H, MIYAZAWA M, ITO Y, SUZUKI H, TOYODA H. 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. 20(5), 767-73.
BASKETTER, D., POOLE, A., KIMBER, I., 2017. Behaviour of chemical respiratory allergens in novel predictive methods for skin sensitisation, Reg Tox and Pharmacol. 86,101-106,
DOS SANTOS, G. G., REINDERS, J., OUWEHAND, K., RUSTEMEYER, T., SCHEPER, R. J. & GIBBS, S. 2009. Progress on the development of human in vitro dendritic cell based assays for assessment of the sensitizing potential of a compound. Toxicol Appl Pharmacol, 236, 372-82.
FORRERYD A, JOHANSSON H, ALBREKT AS, BORREBAECK CA, LINDSTEDT M. 2015. Prediction of chemical respiratory sensitizers using GARD, a novel in vitro assay based on a genomic biomarker signature. PLoS One.11;10(3):e0118808.
JOHANSSON H, LINDSTEDT M, ALBREKT AS, BORREBAECK CA. 2011. A genomic biomarker signature can predict skin sensitizers using a cell-based in vitro alternative to animal tests. BMC Genomics. 8;12:399.
REES B, SPIEKSTRA SW, CARFI M, OUWEHAND K, WILLIAMS CA, CORSINI E, MCLEOD J.D., GIBBS S. 2011. Inter-laboratory study of the in vitro dendritic cell migration assay for identification of contact allergens. Toxicol In Vitro. 25(8), 2124-34.
SAKAGUCHI H, ASHIKAGA T, MIYAZAWA M, YOSHIDA Y, ITO Y, YONEYAMA K, HIROTA M, ITAGAKI H, TOYODA H, SUZUKI H. 2006. Development of an in vitro skin sensitization test using human cell lines; human Cell Line Activation Test (h-CLAT). II. An inter-laboratory study of the h-CLAT. Toxicol In Vitro. 20(5), 774-84.