Fibronectin-mediated Uptake of Gelatin-coated Latex

The present study demonstrates the ability of plasma fibronectin or cold-insoluble globulin (CIg) to promote the uptake of ' 251-labeled, gelatin-coated latex beads (g-Ltx*) by monolayers of peritoneal macrophages (PM) . The uptake of g-Ltx* by PM was enhanced by CIg in a concentration-dependent fashion and required the presence of heparin (10 U/ml) as an obligatory cofactor for maximal particle uptake . Treatment of PM monolayers with trypsin (1 mg/ml) for 15 min at 37°C after particle uptake removed <15% of the radioactivity incorporated by the monolayers . However, a similar trypsin treatment of the monolayers before the addition of latex particles depressed Clg-dependent uptake by >75% . Pretreatment of PM monolayers with inhibitors of glycolysis effectively reduced the Clg-dependent uptake of latex. Similarly, pretreatment of monolayers with either inhibitors of protein synthesis or agents that disrupt cytoskeletal elements also significantly depressed CIg-dependent particle uptake . Phagocytosis of g-Ltx* by PM in the presence of CIg and heparin was confirmed by electron microscopy . Finally, g-Ltx* could also be effectively opsonized with CIg at 37°C before their addition to the monolayers . These studies suggest that the recognition of g-Ltx* in the presence of CIg required cell surface protein(s) and that subsequent phagocytosis of these particles by PM was energy dependent and required intact intracellular cytoskeleton elements . Thus, PM monolayers provide a suitable system for further studies on the function of Clg in the recognition and phagocytosis of gelatin-coated particles by phagocytic cells. The process ofphagocytosis by vertebrate phagocytes is markedly enhanced by humoral recognition factors called opsonins . Immune opsonins are antibody or complement proteins that interact with foreign antigens and with receptors on the surface of phagocytic cells (26, 36) . There also exist opsonins that enhance the uptake of colloidal material by macrophages of the reticuloendothelial system that are not immune proteins (34). One such nonimmune opsonin has been purified from rat (1, 2, 6, 22) and human serum (4) and was originally designated a-2-macroglobulin on account of its electrophoretic mobility and size (2, 22) . More recent studies have demonstrated that this nonimmune opsonin isolated from human serum is identical to cold-insoluble globulin (CIg) or fibronectin (5), a high molecular weight adhesive glycoprotein, a form of which is also found on the surface of many cell types (10, 38) . Up until now, quantitation ofthe opsonic activity of CIg has depended largely on a liver slice assay utilizing radiolabeled, THE JOURNAL OF CELL BIOLOGY " VOLUME 87 NOVEMBER 1980427-433 © The Rockefeller University Press " 0021-9525/80/11/0427/07 $1 .00 gelatinized lipid emulsion as a test particle (1, 12, 33). The opsonic activity of CIg has also been measured by the agglutination of gelatin-coated colloids (7, 8, 28) . More recently, an electroimmunoassay has been developed for measuring the concentration of CIg in rat or human serum (3) ; however, this method cannot distinguish between biologically inactive and active CIg preparations (14) . Although it has been proposed that CIg promotes the uptake of gelatin-coated particles by liver slices via a phagocytic process, the mechanism of CIgdependent particle uptake by phagocytic cells has not been clearly defined. The present study described an assay utilizing monolayers of elicited peritoneal macrophages (PM) for analyzing the mechanism of CIg-dependent uptake of gelatin-coated latex particles (g-Ltx*) . Evidence will be presented establishing that PM can be employed to quantitate the opsonic activity of CIg present in serum or in purified preparations of CIg . Further427 on Jne 6, 2017 D ow nladed fom Published November 1, 1980