The role of MMP-9 and serine proteases in the cleavage of surfactant protein D by stimulated PMNs and in sputum from CF patients (P3131)

Abstract

Abstract Although the ability of serine proteases to cleave surfactant protein D (SP-D) has been well characterized, it has recently been reported by our group that matrix metalloprotease 9 (MMP-9) is able to cleave SP-D as well. However, our initial study examined MMP-9-mediated cleavage of SP-D in vitro using purified proteins. In order to expand upon this study in a more physiological context, we collected supernatants from primary polymorphonuclear leukocytes stimulated with LPS and fMLP and incubated them with purified SP-D in the presence of a general serine protease inhibitor (PMSF) and/or specific inhibitors against MMP-9 and neutrophil elastase (NE). All inhibitors were able to decrease the amount of detectable cleavage of SP-D, and none of them alone were able to completely inhibit cleavage. We were only unable to detect cleavage when a combination of PMSF and the MMP-9 inhibitor were used, suggesting that MMP-9 is the only non-serine protease released from PMNs with the ability to cleave SP-D. In addition, we examined the cleavage of endogenous SP-D in sputum samples from patients with cystic fibrosis (CF), and found that the MMP-9 inhibitor also significantly attenuated SP-D degradation seen when CF sputum was incubated at 37°C over time. These experiments suggest that MMP-9 is able to cleave SP-D in vivo, and that MMP-9 degrades SP-D in the airways of CF patients.