Integrative Experimental and Theoretical Approach Exposes Fundamental Mechanisms of J774 Macrophage Phagocytosis

Macrophage cell lines like murine J774 cells are ideal model systems to study phagocytosis. Our unique methodology combines single-cell/single-target experiments with advanced computer simulations to elucidate the fundamental mechanisms of J774 macrophage phagocytosis. As a first step, the baseline mechanical properties of the murine macrophages were established. Micropipette-aspiration experiments were used to characterize the cortical tension and cytoplasmic viscosity of the J774 cells. Next, we used a dual-micropipette manipulation system to quantify the time courses of a number of key parameters during Fcγ-target phagocytosis. A passive cell is selected and picked up with a micropipette by partial aspiration. Another micropipette is used to bring an opsonized target bead (5-30 um diameter) into soft contact with the cell, which usually results in immediate adhesion. The target is released and images of the ensuing phagocytosis are recorded directly to a computer hard disk. Their analysis provides the time course of cell morphology, bead position, and cortical tension. Intriguingly, the macrophages maintained a constant cortical tension when engulfing targets that required a surface area expansion of up to ∼250%, indicating an extremely large membrane reservoir. The tension rose when the cells increased their surface area by an amazing ∼550%. These and other experimental observations were compared side-by-side with finite-element models of macrophage phagocytosis. This comparison allows us to assess the viability of different mechanistic models used for phagocytosis. Optimal models include a repulsion between the cytoskeleton and the free membrane (which drives protrusion), and an attraction between cytoskeleton and surface membrane newly adherent to the target (which results in thin pseudopods). Confocal inspection of the engulfment of 20 um beads by GFP-actin transfected macrophages revealed excellent agreement between the cytoskeletal density predicted by the optimal model and the observed actin distribution.