Phagocytosis of gelatin-latex particles by a murine macrophage line is dependent on fibronectin and heparin.

Abstract

It has been suggested that fibronectin plays a role in clearing particles from the circulation by promoting binding to phagocytes of the reticuloendothelial system . By use of a well-defined system to investigate the possible opsonic role of fibronectin, we have studied the uptake of gelatin-coated latex particles by a murine macrophage cell line (P388D 1 ) .Fibronectin promotes binding of gelatin-coated beads to these cells in both suspension and monolayer cultures . In both cases there is a requirement for heparin as a cofactor . Other glycosaminoglycans (chondroitin sulfates A and C, dermatan sulfate, and keratan sulfate) were inactive, whereas heparan sulfate was somewhat active .Proof that beads were actually endocytosed was obtained by electron microscopy, which showed beads internalized in membrane-bounded vesicles, and by immunofluorescence analyses, using antibodies to fibronectin to stain external beads. Two rapid assays for the opsonic activity of fibronectin were developed based on differential centrifugation of cellassociated beads and on the immunofluorescence procedure . Binding and endocytosis were time-and temperature-dependent and varied with the amount of gelatin on the beads and with the concentrations of fibronectin and heparin added, and could be inhibited by F(ab') 2 antifibronectin . These studies provide a sound basis for a detailed analysis of the interaction of fibronectin with the cell surface and of its involvement in endocytosis.Phagocytosis is the process by which particles (bacteria, damaged cells, fibrin aggregates, etc .) are bound and endocytosed by phagocytic cells . Recognition of these particles by the cells is due to the presence of humoral factors (opsonins) such as immunoglobulins or complement, which coat the particles and promote their phagocytosis (44). Recently, an a-2-globulin from plasma has been shown to facilitate clearance of certain particles from the circulation (31,32,42) . It also promotes the binding of particles to liver slices (12,16,31,40) and the uptake of gelatin-coated particles by rat peritoneal macrophages (13,18) . This a-2-globulin has recently been purified from rat and human sera and found to be immunologically and functionally identical with plasma fibronectin (2, 33), a dimeric glycoprotein with subunits of 230,000 daltons (3) . Fibronectin is known to be involved in adhesion of cells to solid substrata and has specific binding sites for gelatin, glycosaminoglycans (i .e ., heparin), fibrinogen, and fibrin, and can be crosslinked to fibrin or collagen by Factor XIII (reviewed in references 17, 21, 35, 32 47, 49) . Fibronectin has also been shown to bind to bacteria' (27, 36), but has not been shown to promote phagocytosis of bacteria .To define the conditions necessary for endocytosis of fibronectin-coated particles, we have studied an established mouse macrophagelike cell line (P388D,) by using a centrifugation assay, as well as electron and immunofluorescence microscopy. We find that endocytosis of gelatin-conjugated latex beads...

Fibronectin binds to the C1q component of complement.

“…There is evidence that fibronectin plays a role in the clearance of particulate material both in vivo (7,8) and in defined systems in vitro (32)(33)(34). The interaction of fibronectin (whether free or cell bound) with C1q could play a prominent role in clearance of particulate debris coated with C1q, analogous with the clearance mechanisms that exist for removal of material bearing exposed C3b and Fc regions ofimmunoglobulins.…”

Mammalian reticulocytes lose adhesion to fibronectin during maturation to erythrocytes.

“…Loss of fibronectin-adhesive moleules from the cell surface of circulating reticulocytes mi occur by an endocytotic mechanism, since spectrin-free surface invaginations and intracellular vesicles have been observed in reticulocytes within normal rabbit spleen (21). Fibronectin or fibronectinadhesive molecules on the surface of reticulocytes may also serve as an attachment site for splenic macrophages that participate in remodeling of the surface membrane, because macrophages are known to adhere and ingest fibronectincoated particles (22)(23)(24). Interactions of cells with extracellular matrices are essential for many developmental processes (25,26), and it will be important to determine the specificity of interactions among erythroid precursor cells, extracellular matrix proteins, and-7macrophages.…”

Monocyte receptors for fibronectin characterized by a monoclonal antibody that interferes with receptor activity.

“…These receptors are present on most, if not all, human peripheral blood monocytes (5) and on peritoneal macrophages (6,7), alveolar macrophages (8), and macrophage cell lines (9). Receptors can be detected by their ability to mediate the attachment of monocytes to fibronectin-containing surfaces or by the attachment of fibronectin-coated particles to monocytes (1).…”