Imaging the Spatial Distribution of Membrane Receptors during Neutrophil Phagocytosis

Optical microscopy and image processing have been employed to study the distribution of several cell surface receptors on living human neutrophils during opson-independent and opsonin-independent phagocytosis. Receptors were labeled using fluorescein-, rhodamine-, or AMCA-conjugated F(ab′) 2 fragments of anti-FcγRIIIB (CD16), anti-CR3 (CD11b/CD18), and anti-uPAR (urokinase-type plasminogen activator receptor) antibodies, intact phycoerythrin-labeled interleukin 8, and fluorescein- or rhodamine-labeled Con A (concanavalin A), Boc-PLPLP (tert-butyl-oxycarbonyl-Phe( d)-Leu-Phe( d)-Leu-Phe-OH), and N -formyl-Nle-Leu-Phe-Nle-Tyr-Lys. Labeled neutrophils were observed during the phagocytosis of IgG-opsonized erythrocytes and nonopsonized latex beads, Escherichia coli, and Staphylococcus aureus. To quantitate receptor distribution, cells were divided into four quadrants with the first being the point of attachment and the fourth being opposite the point of attachment. Ligated formyl peptide receptors, and to a lesser extent CR3, accumulated at the sites of target internalization for all forms of phagocytosis examined. However, FcγRIIIB, uPAR, IL-8, Con A, and the FPR antagonist FBoc-PLPLP were not polarized on cells during phagocytosis. These data suggest that agonist-labeled formyl peptide receptors may play a broader role in leukocyte function than previously suggested, including possible participation in phagocytosis.