6533b7d7fe1ef96bd1267bb3

RESEARCH PRODUCT

Flow-cytometric screening for the modulation of receptor-mediated endocytosis in human dendritic cells: implications for the development of an in vitro technique for predictive testing of contact sensitizers

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The aim of this study was to explore the usefulness of human blood dendritic cells (DC) in the development of an in vitro model for predictive testing of contact sensitizers. A method was established to monitor the influence of chemicals on the intracellular targeting of antibody-crosslinked MHC class II molecules after their uptake by human DC. Using a three-colour flow-cytometric technique, freshly prepared DC were distinguished from other MHC class II-bearing cell types such as B-cells and monocytes in unseparated mononuclear cell suspensions of healthy volunteers. The assay is based on the pH-sensitivity of internalized fluorescein-coupled MHC class II specific antibodies. Quenching of fluorescence intensity due to internalization into acidic intracellular compartments was observed with untreated DC whereas internalization into less acidic structures following stimulation with strong contact sensitizers ensured that the fluorescence intensity was conserved. The usefulness of this approach for predictive testing of the preservatives MI/MCI, imidazolidinyl urea, methyl-4-hydroxy-benzoate and 2-phenoxyethanol in comparison to the strong allergen DNFB and the irritants sodium lauryl sulphate and dithranol was explored. Whereas low concentrations of MI/MCI resembled the strong allergen DNFB, high concentrations of imidazolidinyl urea were required for a moderate response. Methyl-4-hydroxy-benzoate and 2-phenoxyethanol as well as the irritants SLS and dithranol failed to induce a significant effect in this assay. The non-responsiveness to the latter compounds reflected their minor or absent capacity to induce contact hypersensitivity in humans, whereas DNFB, MI/MCI and imidazolidinyl urea are well established contact sensitizers. These data suggest that the capacity of a chemical to modulate endocytotic mechanisms in dendritic cells in vitro seems to reflect the probability of that substance acting as a hapten in vivo.