MACROPHAGES FORM CIRCULAR ZONES OF VERY CLOSE APPOSITION TO IGG-COATED SURFACES

When phagocytes spread on surfaces coated with ligands such as IgG, they form a tight seal with the substrate. This seal excludes soluble macromolecules in the medium from the interface between the cell and substrate. In contrast, when cells spread on control surfaces that are not coated with ligands, the underside of the cell remains freely accessible to soluble proteins (Wright and Silverstein: Nature 309:359, 1984). We employed reflection-interference microscopy (RIM) to determine where the seal forms during interaction with ligand (IgG)-coated surfaces. Human monocyte-derived macrophages (MO) were plated at 37°C on dinitrophenylated (DNP)-glass coverslips (control substrate), IgM anti-DNP-DNP-coated glass (control substrate), or on IgG anti-DNP-DNP-coated glass (phagocytosis-promoting substrate). Live or fixed cells were examined by RIM. Spreading on control surfaces at 37°C was complete in 25 minutes, whereas spreading on IgG-coated surfaces was maximal within 15 minutes and resulted in cell-substrate contact area 1.6 x that of control cells. Within 1 h at 37°C, 90% of MO that spread on IgG-coated substrates, but not on control substrates, excluded macromolecules from their underside. A minor population of cells (19%) exhibited a uniform iron gray RIM appearance indicating an even, close approach to the substrate. These cells may represent early stages of frustrated phagocytosis. In contrast to cells on control substrates, 70% of cells on IgG-coated substrates developed continuous peripheral dark rings in RIM indicative of close association with the substrate. Essentially all cells with peripheral dark rings in RIM excluded macromolecules from their underside. Enclosed within this ring was an area of greater separation between the cell membrane and the substrate, as indicated by the lighter grey of this region in RIM and by the accessibility of substrate to anti-substrate antibody when breaks in the dark ring occur. Thus, MO can create a closed compartment between plasma membrane and substrate that excludes proteins in the surrounding medium, thereby protecting substances secreted into this space from potentially inhibitory substances in the medium.