Key Event Title
|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|
|Skin Sensitisation AOP||KeyEvent|
|Respiratory Sensitisation/Allergy induced by covalent binding to proteins||KeyEvent|
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.
Overview table: How it is measured or detected
|h-CLAT||draft TG under discussion at OECD||||X|
|EURL ECVAM Recommendation|||
|Ashiga et al., 2015|||
|Genomic Allergen Rapid Detection test (GARD)||Johansson et al., 2013||||X|
|VitroSens||Hooyberghs et al., 2008||||X|
Domain of Applicability
The main in vitro assays currently used and based on dendritic cells activation use human dendritic-cell-like cell lines (e.g. THP-1, U-937, MTZ-3). In addition to that some assays were performed on murine models.
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