Human alveolar macrophage fibronectin: synthesis, secretion, and ultrastructural localization during gelatin-coated latex particle binding

Abstract

Human pulmonary alveolar macrophages synthesized and secreted several characteristic high MW proteins for at least 7 days in vitro. Immunoprecipitates of medium and cell lysates from metabolically labeled cultures with specific anti-human plasma fibronecting IgG contained 1 major labeled polypeptide of MW 440,000 (unreduced) or 220,000 (reduced). An identical polypeptide in conditioned medium from radiolabeled macrophages bound specifically to gelatin-Sepharose, demonstrating that alveolar macrophages synthesized and secreted a molecule immunologically and functionally similar to fibronectin. Fibronectin was the major newly synthesized and secreted polypeptide of freshly harvested alveolar macrophages. Pulse-chase experiments revealed that newly synthesized fibronectin was rapidly secreted into medium, .apprx. 50% appearing by 1 h and 80% by 8 h. Immunoperoxidase staining using antifibronectin F(ab'')2-peroxidase conjugates revealed the majority of immunoreactive fibronectin to be intracellular, localized to endoplasmic reticulum and Golgi apparatus. No extracellular matrix fibronectin was visualized. Cell surface staining was rarely seen, usually appearing only at sites where cells were closely apposed and not at sites of macrophage-substrate attachment. Similar immunostaining of fibroblast cultures revealed cell surface-associated fibrillar fibronectin. Ultrastructural localization of fibronectin during binding and phagocytosis of gelatin-coated and plain latex particles revealed fibronectin only on gelatin-latex beads and at their cell bindng sites. Neither plain latex beads nor their cell membrane binding sites stained for fibronectin. Fibronectin apparently is a major product of human alveolar macrophages, is rapidly secreted, and is localized at cell membrane binding sites for gelatin-coated particles. In view of the known binding properties of fibronectin, it may serve as an endogenous opsonic factor promoting the binding of Staphylococcus, denatured collagen, fibrin, or other macromolecules to macrophages in the lower respiratory tract.