The Role of Plasma Membrane Receptors and the Kinetics of Macrophage Activation by Lymphokines Encapsulated in Liposomes1

The kinetics of activation of tumoricidal functions in mouse macrophages incubated with macrophage-activating factors (MAP) released by mitogen-stimulated lymphocytes (free MAP) and MAP encapsulated within liposomes (liposome-MAF) have been compared. Development of tumoricidal activity requires incubation of macrophages with free or liposome-encapsulated MAP for a minimum of 4 hr. Macrophages incubated with MAP for 4 hr were not cytotoxic when tumor target cells were added immediately after removal of MAP, but they were highly cyto toxic when allowed to complete a "lag" phase before being exposed to tumor cells. The duration of the lag phase varied with different activation protocols. The level of cytotoxic activity induced by liposome-encapsulated MAP was consistently higher than that obtained with free MAP. Studies using inhibi tors of endocytosis demonstrated that internalization of the liposome carrier is required for activation by liposome-MAF and that activation does not result from MAP leaking from liposomes and binding to MAP receptors on either the plasma membrane or the membrane of endocytic vesicles. Comparison of the efficiency of macrophage activation by MAP encapsu lated in liposomes of differing internal volume revealed that large multilamellar and large unioligolamellar liposomes were more efficient in activating peritoneal exÃodate macrophages than were small unilamellar liposomes. Measurement of the volume of liposome contents internalized by macrophages from these three types of liposomes revealed that maximum cytotoxicity required internalization of a given volume of MAFcontaining lymphocyte supernatants, after which no further increase in cytotoxicity occurred.