Membrane backtracking at the maximum capacity of nondigestible antigen phagocytosis in macrophages

The zipper model has been dominantly used to describe the driving mechanism of the engulfment process and its specific identification of antigens during phagocytosis in macrophages. However, the abilities and limitations of the zipper model, capturing the process as an irreversible reaction, have not been examined yet under the critical conditions of engulfment capacity. Here, we demonstrated the phagocytic behavior of macrophages after reaching the maximum engulfment capacity by tracking the progression of their membrane extension during engulfment using IgG-coated nondigestible polystyrene beads and glass microneedles. The results showed that, after macrophages reached their maximum engulfment capacity, they induced membrane backtracking (the reverse phenomenon of engulfment) in both polystyrene beads and glass microneedles, regardless of the difference in the shape of these antigens. We evaluated the correlation of engulfment in simultaneous stimulations of two IgG-coated microneedles and found that each microneedle was regurgitated by the macrophage independently of the advancement or backtracking of membranes on the other microneedle. Moreover, assessing the total engulfment capacity determined by the maximum amount the macrophage was capable of engulfing when imposing each antigen geometry showed that the capacity increased as the attached antigen areas increased. These results indicate that the mechanism of engulfment should imply the following: 1) macrophages have a backtracking function to recover their phagocytic activity after reaching maximal engulfment limit, 2) both phagocytosis and backtracking are local phenomena of the macrophage membrane that operates independently, and 3) the maximum engulfment capacity is determined not only by mere local cell membrane capacity but also by the whole-cell volume increase during simultaneous phagocytosis of multiple antigens by the single macrophages. Thus, the phagocytic activity may entail a hidden backtracking function, adding to the conventionally known irreversible zipper-like ligand-receptor binding mechanism during membrane progression to recover the macrophages that are saturated from engulfing targets beyond their capacity.