0000000000154156
AUTHOR
Uwe Lempertz
Specific stabilization of the 4F7 molecule on dendritic cells by contact allergens.
Our laboratory has recently developed the monoclonal antibody 4F7 which recognizes a molecule on dendritic cells in the dermis of mice that is upregulated after application of contact allergens in vivo. Furthermore, this antibody detects an antigen on dendritic cells in spleen, lymph nodes and colon. In order to study the influence of contact allergens on the surface expression of the 4F7 molecules on dendritic cells, FACScan analysis of splenic dendritic cells was carried out after in vitro application of contact allergens. Freshly isolated splenic dendritic cells were found to be positive for 4F7, 33D1, N418 (CD11c) and MHC class II. After overnight culture the expression of the dendritic…
An approach to predictive testing of contact sensitizers in vitro by monitoring their influence on endocytotic mechanisms.
Endocytotic activation of epidermal Langerhans cells (LC) by immunogenic haptens is an early event during development of allergic contact dermatitis. In this work a fast and objective flow-cytometric assay for predictive in vitro testing of contact sensitizers by monitoring their influence on endocytotic mechanisms in murine LC was developed. Epidermal cell suspensions were labelled with a monoclonal antibody directed to MHC class II molecules and pH-sensitive fluorochrome-coupled second-step reagents. For untreated LC a significant quenching of fluorescence intensity by internalization of the MHC-antibody complexes into acidic compartments was noticed. Similar results were obtained in the …
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
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 …
Contact sensitizers modulate mechanisms of receptor-mediated endocytosis but not fluid-phase endocytosis in murine epidermal Langerhans cells.
In order to define the influence of contact allergens on the fluid-phase endocytosis (FPE) of soluble molecules of murine epidermal Langerhans cells (LC), we studied the internalization of FITC-labeled bovine serum albumin (FITC-BSA), TRITC-labeled dextrane (TRITC-DEX) as well as horseradish peroxidase by LC. A 3-parameter flow-cytometric technique was performed for quantification of internalized FITC-BSA in LC using quantum red-labeled reagents for detection of la-antigen expression by LC and propidium iodide for exclusion of dead cells from analysis. A temperature-dependent rapid accumulation of FITC-BSA was noticed in time-course studies reaching a plateau between 1 and 2 h of in vitro c…