IMAGING THE SPATIAL-DISTRIBUTION OF MEMBRANE-RECEPTORS DURING NEUTROPHIL PHAGOCYTOSIS

Optical microscopy and image processing have been employed to study th e distribution of several cell surface receptors on living human neutr ophils during opsonin-dependent and opsonin-independent phagocytosis. Receptors were labeled using fluorescein-, rhodamine-, or AMCA-conjuga ted F(ab')(2) fragments of anti-Fc gamma RIIIB (CD16), anti-CR3 (CD11b /CD18), and anti-uPAR (urokinase-type plasminogen activator receptor) antibodies, intact phycoerythrin-labeled interleukin 8, and fluorescei n- or rhodamine-labeled Con A (concanavalin A), Boc-PLPLP rt-butyl-oxy carbonyl-Phe(D)-Leu-Phe(D-Leu-Phe-OH), and N-formyl-Nle-Leu-Phe-Nle-Ty r-Lys. Labeled neutrophils were observed during the phagocytosis of Ig G-opsonized erythrocytes and nonopsonized latex beads, Escherichia col i, and Staphylococcus aureus. To quantitate receptor distribution, cel ls were divided into four quadrants with the first being the point of attachment and the fourth being opposite the point of attachment. Liga ted formyl peptide receptors, and to a lesser extent CR3, accumulated at the sites of target internalization for all forms of phagocytosis e xamined. However, Fc gamma RIIIB, uPAR, IL-8, Con A, and the FPR antag onist FBoc-PLPLP were not polarized on cells during phagocytosis. Thes e data suggest that agonist-labeled formyl peptide receptors may play a broader role in leukocyte function than previously suggested, includ ing possible participation in phagocytosis. (C) 1994 Academic Press, I nc.