Vacuolar acidification and bafilomycin-sensitive proton translocating ATPase in human epidermal Langerhans cells

Langerhans cells (LC) are the principal antigen-presenting cells (APC) of squamous epithelia. We have previously shown that freshly isolated LC (fLC) are able to deliver endocytosed membrane MHC class II molecules into acidic environments, and that this capacity is lost when LC are placed in culture (cLC). Inasmuch as processing of antigens requires their passage through acidic compartments, we undertook the present study to examine the ability of fLC and cLC to take up acridine orange, and to identify proton-translocating ATPases in these cells. Using flow cytometry and fluorescence microscopy, acridine orange was observed to accumulate in acidic compartments in both fLC and cLC. Using a radioactive ATPase assay, crude membrane preparations from both fLC and cLC were shown to possess three types of ion-translocating ATPase, based on sensitivity to the following inhibitors: ouabain (Na+, K+ ATPase), oligomycin (mitochondrial F1F0 ATPase), and bafilomycin (vacuolar-type proton pump ATPase); the last type is responsible for acidification in vacuolar compartments. cLC displayed markedly less (less than 50%) total ATPase activity compared to fLC; however, the relative proportions of specific ATPases were similar in fLC and cLC. Combined use of the three inhibitors resulted in abrogation of only 25-40% of the total ATPase activity. Finally, treatment of LC with bafilomycin inhibited both acridine orange uptake and acidification of internalized HLA-DR molecules. These results confirm the ability of both fLC and cLC to acidify vacuolar compartments, thereby suggesting that lack of acidification of endocytosed membrane class II molecules in cultured cells is due to alternative routing to non-acidic organelles.