Fibronectin fragments released from phorbol ester-stimulated pulmonary artery endothelial cell monolayers promote neutrophil chemotaxis.

We have recently shown that monolayer cultures of calf pulmonary artery endothelial (CPAE) cells pretreated with phorbol myristate acetate (PMA) generate a conditioned medium that is chemotactic for human polymorphonuclear leucocytes (PMNL). Fibronectin (Fn) is a multidomain protein found in the plasma and subendothelial extracellular matrix that induces attachment and migration of a variety of cell types. The present study was designed to evaluate the role of Fn or fragments of Fn present in conditioned medium from phorbol ester-stimulated endothelial cells as potential chemotactic factors for human PMNL. A large number of Fn fragments were revealed by Western immunoblotting of serum-free conditioned medium 4 hr after treatment of CPAE monolayers with PMA. Gelatin-Sepharose affinity chromatography of 4-hr conditioned medium demonstrated chemotactic activity for PMNL in both gelatin-binding and non-gelatin-binding fractions. The addition of bovine Fn antiserum to the conditioned medium inhibited PMNL chemotaxis in a dose-dependent manner while having no effect on PMNL chemotaxis generated by zymosan-activated serum. One site on the Fn molecule known to interact with phagocytic cells is the cell-binding domain containing the Arg-Gly-Asp (RGD) sequence. Pretreatment of PMNL with a RGD-containing peptide (1 mM GRGDSPK) for 10 min completely inhibited the expression of chemotactic activity present in conditioned medium and in the gelatin-binding and non-gelatin-binding fractions. PMNL chemotaxis was not stimulated by either intact Fn or the RGD-containing septapeptide tested over a wide concentration range. However, incubation of PMNL with a purified 120,000-MW fragment of Fn containing the cell-binding domain stimulated chemotaxis in a dose-dependent manner. In contrast, a purified 45,000 MW fragment of Fn containing the gelatin-binding domain was not chemotactic for PMNL. When a monoclonal antibody directed against the cell-binding domain of Fn was incubated with conditioned medium, a significant reduction in PMNL chemotaxis was observed. These results demonstrate that phorbol ester-stimulated pulmonary artery endothelial cells release Fn fragments and suggest an important role for Fn fragments containing the cell-binding domain in stimulating the migration of PMNL.