Phagocytosis is the primary means of host defense in the animal kingdom. Much progress has been made in understanding the cellular events associated with phagocytosis, yet current knowledge cannot fully explain the basic mechanism of pseudopod formation. Coated pits are specialized structures which are well recognized for their role in receptor-mediated endocytosis. However, their role in both cell locomotion and phagocytosis is only beginning to be appreciated. In the migrating cell, integrins and membrane material are recycled to the leading edge by an endocytic pathway mediated by coated pits. Pseudopod extension during phagocytosis is similar to leading edge formation during cell migration: both are forms of cell protrusion, which suggests that they share a common functional mechanism. Based on the theory that endocytic/exocytic recycling of membrane material allows pseudopod extension in a manner similar to leading edge formation, the hypothesis addressed by this proposal is that coated pits arc necessary components for phagocytosis.
The specific aims are: l) to determine whether increased coated pit formation enhances receptor-mediated phagocytosis; 2) to determine whether decreased coated pit formation diminishes receptor-mediated phagocytosis; 3) to determine whether antibody binding to coated pit proteins prevents receptor-mediated phagocytosis, and 4) to determine whether interruption of the expression of specific coated pit proteins prevents receptor-mediated phagocytosis.
These specific aims will be accomplished by using liposomes as Trojan Horses to directly intervene in the function of living, intact macrophages. Three categories of materials will be packaged into liposomes: I) agents known to affect coated pit formation (Specific Aims #l&2), 2) antibodies directed against coat proteins (Specific Aim #3), and 3) antisense oligonucleotides complementary to the expression messages for coat proteins (Specific Aim #4). Experiments presented in this proposal demonstrate that l) compounds can be entrapped in liposomes, 2) liposomes are phagocytosed by macrophages, 3) ingested liposomal contests achieve cytoplasmic distribution, and 4) liposome-delivered agents can alter phagocytic function. The findings of this project stand to make a significant contribution towards understanding the mechanism of this fundamental cell function.
