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RESEARCH PRODUCT

Contact sensitizers modulate mechanisms of receptor-mediated endocytosis but not fluid-phase endocytosis in murine epidermal Langerhans cells.

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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 culture at 37°C. The quantity of FPE under stimulation with phorbol 12-myristate 13-acetate (PMA), concanavalin A (Con A), sta-phylococcal enterotoxin B (SEB) and contact sensitizers (DNFB, Kathon CG, K2Cr2O7) as well as the irritant SLS was determined. Treatment of LC with PMA and Con A resulted in a significant increase of total FITC-BSA uptake. The contact sensitizers as well as SEB and SLS failed to mediate augmented fluid-phase endocytosis. By use of the pH-insensitive soluble marker, TRITC-DEX and a microscope photometer for evaluation these findings could be confirmed. This excluded any artificial influence of differences in pH values in endocytotic compartments which might have influenced the fluorescence intensity of the pH-sensitive fluoro-chrome FITC. For qualitative analysis of FPE, the intracellular distribution of internalized horseradish peroxidase in LC was studied. An aggregated pattern became apparent in untreated LC and did not change under stimulation with any of the substances used. This was in sharp contrast to a modulation of receptor-mediated endocytosis of antibody-crosslinked MHC class II molecules under the influence of contact sensitizers, and suggested that haplen-mediated endocytotic activation of LC was restricted to this mechanism of inlernalization.