Endogenous fibronectin of blood polymorphonuclear leukocytes: stimulus-induced secretion and proteolysis by cell surface-bound elastase.
In an accompanying study, we described the presence of intact fibronectin, a large adhesive molecule, in the specific granules of blood PMNs. Secretion of fibronectin by blood PMNs is poorly understood, and the fate of this fibronectin is practically unknown. In the present study we demonstrate that nanomolar concentrations of phorbol ester or the chemoattractants fMLP, PAF, and LTB4 induce fibronectin secretion from blood PMNs. Phorbol ester induced secretion of approximately 85% of the total fibronectin content, as well as expression of small amounts on the cell surface of the activated PMNs. Secreted fibronectin was proteolytically cleaved and, after 20 min, four major fragments of 150, 120, 90, and 80 kDa containing a midchain epitope were identified by Western blot analysis. Kinetic studies indicated that fibronectin was rapidly secreted as an intact molecule and that proteolysis started within minutes and proceeded for at least 1 h. If cells were removed after 5 min TPA treatment, no further proteolysis of the secreted fibronectin was observed, indicating participation of cell-bound proteinases. From a cocktail of proteinase inhibitors, PMSF was the most active in suppressing fibronectin proteolysis. Studies with specific peptidyl inhibitors of human leukocyte elastase and cathepsin G, major serine proteinases of PMNs, demonstrated some inhibition with the cathepsin G inhibitor, while the human leukocyte elastase inhibitor almost completely abolished fibronectin proteolysis. A monoclonal antibody to the elastase had a similar effect. The results indicate that intact fibronectin is a secretory product of blood PMNs and that this endogenous adhesive molecule is within minutes extracellularly processed by cell surface-bound elastase.
Salcedo
R
R
Microbiology and Tumorbiology Center, Karolinska Institute, Stockholm, Sweden.
Wasserman
K
K
Patarroyo
M
M
D000911:Antibodies, Monoclonal; D015815:Cell Adhesion Molecules / Q000276:immunology / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology*; D002630:Chemotactic Factors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D005353:Fibronectins / Q000276:immunology / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D019272:Leukocyte Elastase / Q000378:metabolism*; D007975:Leukotriene B4 / Q000494:pharmacology; D018375:Neutrophil Activation; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D010972:Platelet Activating Factor / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D015815:Cell Adhesion Molecules; D002630:Chemotactic Factors; D000939:Epitopes; D005353:Fibronectins; D010972:Platelet Activating Factor; D007975:Leukotriene B4; D019272:Leukocyte Elastase; D013755:Tetradecanoylphorbol Acetate