EXTRACELLULAR PROTEOLYSIS OF FIBRONECTIN BY NEUTROPHILS - CHARACTERIZATION AND THE EFFECTS OF RECOMBINANT CYTOKINES

We have used I-125-labeled fibronectin (FN) as an extracellular substrate for neutrophils (PMN) in order to investigate the mechanism responsible for FN solubilization by PMN and the effects of recombinant cytokines on this process. Pure active alpha-1-antitrypsin (alpha-1AT), when added to PMN before or during, but not after, adherence to FN, inhibited solubilization of the substrate in a dose-dependent manner, but alpha-1AT that had been inactivated by proteolysis or oxidation and alpha-1AT Pittsburgh (alpha-1AT 358Met-Arg) had no significant effect. The solubilization of FN was also inhibited by the PMN elastase inhibitor N-methoxysuccinyl-alanyl-alanyl-prolyl-valine-chloromethylketone but not by the chymotrypsin and cathepsin G inhibitor N-Cbz-glycyl-glycyl-phenylalanine-chloromethylketone, nor by catalase or superoxide dismutase. The products of solubilization of FN by PMN, analyzed by sodium dodecyl sulphate polyacrylamide electrophoresis, were similar to those produced by pure PMN elastase but not cathepsin G. These results suggest that FN solubilization by PMN is caused largely by the pericellular activity of PMN elastase. The solubilization of FN by PMN was increased significantly by adding tumor necrosis factor-alpha, interleukin-1-alpha, or interferon-gamma to the adherent cells but without a significant general release of elastase into the culture supernatants. Granulocyte/macrophage colony-stimulating factor (GM-CSF) had no significant effect. None of the cytokines had any effect when preincubated with the cells in suspension, and none increased FN solubilization by PMN incubated with the optimal (10(-6) mol/liter) or suboptimal dose (10(-8) mol/liter) of the peptide formylmethionylleucylphenylalanine. Each of the cytokines, including GM-CSF, increased PMN adherence, suggesting that their stimulating effect on FN solubilization was not mediated solely through their ability to increase adherence of the cells to the substrate.