Journal of Leukocyte Biology 50:331-340 (1991) © 1991 Wiley-Liss, Inc. Proteolysis of Gelatin-Bound Fibronectin by Activated Leukocytes: A Role for

Fragmentation of subendothelial matrix-bound fibronectin by proteases released from stimulated leukocytes has been implicated in lung vascular injury. We studied the degradation of fibronectin bound to denatured collagen by inflammatory polymorphonu-clear leukocytes (PMNL). Tissue culture wells coated with denatured collagen (gelatin) were pretreated with 1251 rat plasma fibronectin to allow for fibronectin binding prior to the addition of rat inflammatory PMNL. The release of both intact and fragmented fibronectin from the 125I-Iabelled artificial matrix was quantified following the addition of PMNL stimulated by the phagocytosis of opsonized zymosan as well as leukocyte elastase. Stimulated PMNL released three times more radiolabelled fibronectin from the denatured collagen surface during a 4 h incubation as compared with unstimulated PMNL. This pattern of 125l-fibronectin release could also be elicited by the addition of purified leukocyte elastase alone, in the absence of PMNL. The release of radiolabelled fibronectin by stimulated PMNL was blocked In a dose-dependent manner by the addition of both methoxysuccinyl-alanlne-alanine-valine chloromethyl ketone (AAPVCK), a leukocyte elas-tase specific inhibitor as well as phenylmethylsulfonylfluoride (PMSF), a non-specific se-rine protease inhibitor. Western blot analysis coupled with autoradiography confirmed the presence of fibronectin fragments in the medium after addition of PMNL or leukocyte elastase. The large hiolecular weight fragments (60-200 kD) were not labelled, but the smaller molecular weight fragments (<45 kD), derived from the artifical matrix, were labelled. Thus, fibronectin complexed with denatured collagen is susceptible to prote-olytic degradation by stimulated inflammatory PMNL. Such a process may have a role in the pathogenesis of acute vascular injury following microvascular margination of acti-vated blood leukocytes. Key words: lung vascular injury, polymorphonuclear leukocytes, protease inhibito