ABSTRACT
Surfactant protein D (SP-D) plays important roles in innate immunity including the defense against bacteria, fungi, and respiratory viruses. Because SP-D specifically interacts with neutrophils that infiltrate the lung in response to acute inflammation and infection, we examined the hypothesis that the neutrophil-derived serine proteinases (NSPs): neutrophil elastase, proteinase-3, and cathepsin G degrade SP-D. All three human NSPs specifically cleaved recombinant rat and natural human SP-D dodecamers in a time- and dose-dependent manner, which was reciprocally dependent on calcium concentration. The NSPs generated similar, relatively stable, disulfide cross-linked immunoreactive fragments of approximately 35 kDa (reduced), and sequencing of a major catheptic fragment definitively localized the major sites of cleavage to a highly conserved subregion of the carbohydrate recognition domain. Cleavage markedly reduced the ability of SP-D to promote bacterial aggregation and to bind to yeast mannan in vitro. Incubation of SP-D with isolated murine neutrophils led to the generation of similar fragments, and cleavage was inhibited with synthetic and natural serine proteinase inhibitors. In addition, neutrophils genetically deficient in neutrophil elastase and/or cathepsin G were impaired in their ability to degrade SP-D. Using a mouse model of acute bacterial pneumonia, we observed the accumulation of SP-D at sites of neutrophil infiltration coinciding with the appearance of approximately 35-kDa SP-D fragments in bronchoalveolar lavage fluids. Together, our data suggest that neutrophil-derived serine proteinases cleave SP-D at sites of inflammation with potential deleterious effects on its biological functions.
Subject(s)
Lectins/genetics , Neutrophils/enzymology , Pulmonary Surfactant-Associated Protein D/antagonists & inhibitors , Serine Endopeptidases/metabolism , Amino Acid Sequence , Animals , Calcium/chemistry , Calcium/metabolism , Cathepsins/deficiency , Cathepsins/genetics , Cathepsins/metabolism , Conserved Sequence , Humans , Klebsiella Infections/metabolism , Klebsiella pneumoniae/metabolism , Klebsiella pneumoniae/pathogenicity , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Sequence Data , Protein Structure, Tertiary , Pulmonary Alveolar Proteinosis/metabolism , Pulmonary Alveolar Proteinosis/microbiology , Pulmonary Surfactant-Associated Protein D/chemistry , Pulmonary Surfactant-Associated Protein D/genetics , Pulmonary Surfactant-Associated Protein D/metabolism , Rats , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Serine Endopeptidases/physiology , Serine Proteinase Inhibitors/pharmacology , TemperatureABSTRACT
Surfactant protein (SP)-D gene targeted (SP-D-/-) and wild-type mice were infected with respiratory syncytial virus (RSV) by intratracheal instillation. Decreased clearance of RSV was observed in SP-D-/- mice. Deficiency of SP-D was associated with increased inflammation and inflammatory cell recruitment in the lung after infection. In vitro, SP-D bound RSV-infected Vero cells. Binding was inhibited with ethylenediamine tetraacetic acid and maltose, suggesting that the carbohydrate recognition domain of SP-D recognizes RSV glycoproteins in a calcium-dependent manner. SP-D bound specifically to the RSV proteins G and F. Phagocytosis of RSV by alveolar macrophages was reduced in the absence of SP-D in vivo, and SP-D enhanced phagocytosis of RSV by alveolar macrophages and neutrophils but not peritoneal macrophages in vitro. Oxygen radical production by alveolar macrophages from SP-D+/+ and SP-D-/- mice was decreased after RSV infection, and SP-D ameliorated the inhibitory effects of RSV on oxygen radical production by macrophages and neutrophils in vitro. Because the airway is the usual portal of entry for RSV and other respiratory pathogens, the local production of SP-D is likely to play a role in innate defense responses to inhaled viruses.
Subject(s)
Phagocytosis/physiology , Pulmonary Surfactant-Associated Protein D/pharmacology , Respiratory Syncytial Viruses/physiology , Animals , Cell Line , Female , Macrophages, Alveolar/virology , Male , Mice , Rats , Recombinant Proteins/pharmacologyABSTRACT
Surfactant Protein D (SP-D) plays important roles in antimicrobial host defense, inflammatory and immune regulation, and pulmonary surfactant homeostasis. The best-characterized endogenous ligand is phosphatidylinositol; however, this lipid interaction at least in part involves the carbohydrate moiety. In this study we observed that SP-D binds specifically to saturated, unsaturated, and hydroxylated fatty acids (FA). Binding of biotinylated-SP-D to FAs or biotinylated FA to SP-D was dose-dependent, saturable, and specifically competed by the corresponding unlabeled probe. Specific binding to FA chains was also demonstrated by solution phase competition for FA binding to acrylodan-labeled FA binding protein (ADIFAB), and by overlay of thin layer chromatograms with SP-D. Maximal binding to FA was dependent on calcium, and binding was localized to the neck and carbohydrate recognition domains (CRD) using recombinant trimeric neck+CRDs. Saccharide ligands showed complex, dose-dependent effects on FA binding, and FAs showed dose- and physical state-dependent effects on the binding of SP-D to mannan. In addition, CD spectroscopy suggested alterations in SP-D structure associated with binding to monomeric FA. Together, the findings indicate specific binding of FA to one or more sites in the CRD. We speculate that the binding of SP-D to the fatty acyl chains of surfactant lipids, microbial ligands, or other complex lipids contributes to the diverse biological functions of SP-D in vivo.
