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 sensitizing agents induce enhanced internalization of cell membrane constituents by epidermal Langerhans cells (LC). However the intracellular distribution 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 influence of various contact sensitizing agents, non-sensitizing analogues, and irritants. Antigen-antibody complexes were visualized by light microscopy using the silver enhancement technique and by pre-embedding electron microscopy. Viability was monitored by staining dead cells with propidium iodide. For light-microscopic evaluation of the intracellular distribution pattern of gold particles, a stimulation index was defined and used for the assessment of endocytotic activation. Untreated and solvent treated (control) cells exhibited an accumulation of internalized gold complexes into large aggregates composed of few intracellular vesicles. Cytoplasmic staining was absent and few gold particles were detectable in the endocytotic organelles under these conditions. In contrast to the non-sensitizing compounds DCNB and DNBSO3, which had no effect at all, treatment with subtoxic concentrations of the contact sensitizing agents DNFB, DNCB, TNCB, K2Cr2O7, NISO4 and p-phenylenediamine resulted in diffuse intracellular staining which was most pronounced in the submembraneous region. This was due to the numerous endocytotic vesicles which were closely associated with the cell membrane. Consequently a significant increase in the stimulation index was noted for these compounds. An irritant such as sodium lauryl sulphate used in subtoxic concentrations did not influence the intracellular distribution of internalized gold particles whereas toxic amounts of this compound induced a diffuse intracellular staining pattern indicative of membrane destruction. This approach represents a practical and reliable test for endocytotic activation of murine LC and may be useful for in vitro tests of the activating and possibly sensitizing properties of new chemical compounds.