AN IN-VITRO TEST FOR ENDOCYTOTIC ACTIVATION OF MURINE EPIDERMAL LANGERHANS CELLS UNDER THE INFLUENCE OF CONTACT ALLERGENS

Several in vivo and in vitro studies have shown that contact sensitizi ng agents induce enhanced internalization of cell membrane constituent s by epidermal Langerhans cells (LC). However the intracellular distri bution of the internalized material has not yet been clearly defined. For this reason we investigated the uptake of gold-labeled antibodies against MHC class II molecules by cultured murine LC under the influen ce of various contact sensitizing agents, non-sensitizing analogues, a nd irritants. Antigen-antibody complexes were visualized by light micr oscopy using the silver enhancement technique and by pre-embedding ele ctron microscopy. Viability was monitored by staining dead cells with propidium iodide. For light-microscopic evaluation of the intracellula r distribution pattern of gold particles, a stimulation index was defi ned and used for the assessment of endocytotic activation. Untreated a nd solvent treated (control) cells exhibited an accumulation of intern alized gold complexes into large aggregates composed of few intracellu lar vesicles. Cytoplasmic staining was absent and few gold particles w ere detectable in the endocytotic organelles under these conditions. I n contrast to the non-sensitizing compounds DCNB and DNBSO3, which had no effect at all, treatment with subtoxic concentrations of the conta ct sensitizing agents DNFB, DNCB, TNCB, K2Cr2O7, NISO4 and p-phenylene diamine resulted in diffuse intracellular staining which was most pron ounced in the submembraneous region. This was due to the numerous endo cytotic vesicles which were closely associated with the cell membrane. Consequently a significant increase in the stimulation index was note d for these compounds. An irritant such as sodium lauryl sulphate used in subtoxic concentrations did not influence the intracellular distri bution of internalized gold particles whereas toxic amounts of this co mpound induced a diffuse intracellular staining pattern indicative of membrane destruction. This approach represents a practical and reliabl e test for endocytotic activation of murine LC and may be useful for i n vitro tests of the activating and possibly sensitizing properties of new chemical compounds.