Processing of concanavalin A-receptor complexes by rat osteoclasts in vitro

The osteoclast is a large multinucleate cell that is widely accepted as the primary effector cell responsible for normal bone resorption. In a previous study, we demonstrated that concanavalin A (con A) has a dose-dependent biphasic effect on the bone-resorbing capacity of osteoclasts, using a 45Ca bone-organ culture system; bone resorption was stimulated at low concentrations and inhibited at high concentrations. The mitogenic property of con A in lymphocyte cultures is well documented; therefore con A has been used extensively to study the manner in which lymphocytes and other mononuclear cells process the cell-bound lectin. In this study, we have investigated the processing of con A-receptor complexes by osteoclasts in culture, using con A-FITC to evaluate the redistribution of cell-bound con A via epifluorescence microscopy and using con A-ferritin to determine whether the lectin receptor complexes are internalized. The osteoclasts were obtained from the long bones of newborn rats and allowed to attach to glass coverslips at 37 degrees C. Following attachment, the nonadherent cells were removed by rinsing. The adherent osteoclasts were preincubated in 50 micrograms/ml con A-FITC or con A-ferritin at 4 degrees C for 10 min, washed to remove unbound con A, and incubated at 37 degrees C for 15 or 30 min in the absence of con A. Positive controls were fixed immediately after preincubation at 4 degrees C; negative controls were preincubated in con A-FITC and alpha-methyl mannoside, the haptenic inhibitor of con A binding. The results demonstrate that redistribution and endocytosis of con A-receptor complexes occurs within 30 min. These findings confirm the hypothesis that cell-bound con A can alter the structure and activity of osteoclast membrane components in a manner similar to that observed in mononuclear cell cultures. The internalization of con A may be important in altering osteoclastic activity by mediating intracellular mechanisms involved in the bone-resorbing process.