We report the effect of Fab' (anti-60k) to a 60,000 mol wt gelatin binding domain of fibronectin (1981, J. Biol . Chem . 256:5583) on diploid fibroblast (IMR-90) extracellular fibronectin and collagen organization . Anti-60k Fab' did not inhibit IMR-90 attachment or proliferation in fibronectin-depleted medium . Fibroblasts cultured with preimmune Fab' deposited a dense extracellular network of fibronectin and collagen detectable by immunofluorescence, while anti-60k Fab' prevented extracellular collagen and fibronectin fibril deposition . Matrix fibronectin and collagen deposition remained decreased in cultures containing anti-60k Fab' until cells became bilayered or more dense, when fibronectin and collagen began to appear in lower cell layers . Anti-60k Fab' added to confluent cultures 24 h before fixation and staining had no effect on matrix fibronectin or collagen, so anti-60k Fab' did not simply block immunostaining . Confluent cultures grown in anti-60k Fab' and labeled for 24 h with [3H]proline incorporated identical amounts of [3H]proline and [3H]hydroxyproline, but [3H]hydroxyproline deposition in the cell layer was significantly decreased by anti-60k Fab' (P < 0.01) . Extracellular matrix collagen does not appear to form a scaffold for fibronectin deposition, as neither gelatin nor a gelatin-binding fragment of plasma fibronectin inhibited deposition of matrix fibronectin . Our results suggest that interstitial collagens and fibronectin interact to form a fibrillar component of the extracellular matrix, and that fibronectin is required for normal collagen organization and deposition by fibroblasts in vitro. Domain-specific antibodies to fibronectin are powerful tools to study the biological role of fibronectin in extracellular matrix organization and other processes.Fibronectin (FN) is a major synthetic product of diploid fibroblasts (1) organized in discrete extracellular matrix fibrils also containing procollagen types I and 111 (3,5,11,14,42). FN is associated with collagen or collagen precursors in extracellular matrix in vivo in human lung specimens (32), in implanted cellulose sponges (26), and during wound healing (13). The distribution of FN and its multiple binding to other matrix components and cells in vitro (36,39,45 ; and references therein) implicate FN in organization of glycosaminoglycans and collagens in extracellular matrix, and in the interaction of cells with these matrix components . However, direct demonstration of FN-mediated organization of other matrix components is lacking.

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Human pulmonary alveolar macrophages synthesized and secreted several characteristic high molecular weight proteins for at least 7 d in vitro. Immunoprecipitates of medium and cell lysates from metabolically labeled cultures with specific anti-human plasma fibronectin IgG contained one major labeled polypeptide of molecular weight 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, -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, and 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 fibrillarfibronectin . 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 binding sites. Neither plain latex beads nor their cell membrane binding sites stained for fibronectin . These results demonstrate that fibronectin 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. In the lung, alveolar macrophages (PAM) play a key role in the uptake of particulate debris and microorganisms from the terminal airways and alveoli (14). However, unlike blood monocytes and peritoneal macrophages, PAM residing in their

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We observed intense intracellular immunofluorescence of rat lung fibroblasts stained with hybridoma culture supernatant containing monoclonal antibodies to human plasma fibronectin, but no pericellular matrix staining. Immunoprecipitation and absorption experiments revealed that this intracellular staining by hybridoma-conditioned medium was due to binding of fibronectin-antifibronectin immune complexes via the fibronectin to intracellular procollagen . The anomalous staining patterns we encountered were not revealed by the usual controls for immunohistochemical specificity, and also occurred in rat tissue sections . This general phenomena-binding of serum antigens present in hybridoma medium to cellular components-could in principle result in artifactual staining with monoclonal antibodies to other serum components, so investigators using monoclonal antibodies should be aware of this new artifact. Our results also demonstrate that fibronectin binds specifically to native procollagen . Monoclonal antibodies may be useful for studying fibronectin-procollagen and other macromolecular interactions .Monoclonal antibodies (7) are powerful tools for cell biologists, but their use may be accompanied by unusual pitfalls. For example, it is well documented that monoclonals may bind genetically distinct polypeptides (8) . Using hybridoma culture medium containing monoclonal antibody to human fibronectin (FN)' to stain rat lung fibroblasts (RLF), we encountered a previously unreported artifact . Monoclonal antifibronectin-fibronectin complexes bound to intracellular collagenase-sensitive ligands, presumably procollagen, resulting in intense intracellular immunofluorescence. As monoclonals are often used in the form of conditioned medium from serum-containing hybridoma cultures, this general phenomena is important to recognize as a potential artifact of monoclonal antibody staining. Our findings also have implications for fibronectin interactions with procollagen, which are briefly discussed. MATERIALS AND METHODS 1042Immunoreagents and FN: Monoclonal antibodies to human 'Abbreviations used in thispaper: FITC, fluorescein isothiocyanate; FN, fibr6nectin; RLF, rat lung fibroblasts. plasma FN were obtained by fusing spleen cells from immunized BALB/c mice with SP2/O-Ag 14 myeloma cells (19). Positive fusions were cloned in soft agar and antibody-secreting clones detected by enzyme-linked immunosorbent assay (4). Hybridomas were expanded in cell culture using DME plus 10% agammaglobulinemic horse serum, 5% calf serum, and nonessential amino acids ("hybridoma medium") . Ascites fluid containing 2-5 mg/ml of IgG was obtained by intraperitoneal injection of 106 myeloma cells into pristane primed BALB/c mice followed by paracentesis after 1-2 wk. Ascites was clotted at 25°C for 1 h, centrifuged (1,000 g, 10 min), protease inhibitors (I mM phenylmethylsulfonylfluoride, 1 mM N-ethylmaleimide, 10 mM EDTA) and 0.02% NaN3 were added, and the supernate ("ascites") stored at -20°C. In some experiments IgG monoclonals were purifi...