Surfactant protein D reduces alveolar macrophage apoptosis in vivo

Surfactant protein D (SP-D) is a molecule of the innate immune system that recognizes the patterns of surface carbohydrate on pathogens and targets them for phagocytosis and killing. SP-D-deficient mice show an increased number of macrophages in the alveolar space, excess surfactant phospholipid, overproduction of reactive oxygen species, and the development of emphysema. We report here that SP-D-deficient mice have a 5- to 10-fold increase in the number of apoptotic and necrotic alveolar macrophages, as defined by annexin V and propidium iodine staining, respectively. Intrapulmonary administration of a truncated 60-kDa fragment of human recombinant SP-D reduces the number of apoptotic and necrotic alveolar macrophages and partially corrects the lipid accumulation in SP-D-deficient mice. The same SP-D fragment binds preferentially to apoptotic and necrotic alveolar macrophages in vitro, suggesting that SP-D contributes to immune homeostasis in the lung by recognizing and promoting removal of necrotic and apoptotic cells.     

Myelin basic protein component C1 in increasing concentrations can elicit fusion, aggregation, and fragmentation of myelin-like membranes

Myelin basic protein (MBP) is considered to have a primary role in the formation and maintenance of the myelin sheath. Many studies using artificial vesicle systems of simple lipid composition, and generally small size, have shown that MBP can elicit vesicle fusion, aggregation, or even fragmentation under different conditions. Here, we have studied the effects of increasing concentrations of bovine MBP charge isomer C1 (MBP/C1) on large unilamellar vesicles (LUVs) composed of phosphatidylcholine and phosphatidylserine (92:8 molar ratio), or with a lipid composition similar to that of the myelin membrane in vivo (Cyt-LUVs). Using absorbance spectrophotometry, fluorescence resonance energy transfer, dynamic light scattering and transmission electron microscopy, we have shown that vesicle aggregation and some vesicle fusion occurred upon addition of MBP/C1, and as the molar protein-lipid ratio increased. Fragmentation of Cyt-LUVs was observed at very high protein concentrations. These results showed that the phenomena of vesicle fusion, aggregation, and fragmentation can all be observed in one in vitro system, but were dependent on lipid composition and on the relative proportions of protein and lipid.     

Activation of P2X(7) receptor by ATP plays an important role in regulating inflammatory responses during acute viral infection

Acute viral infection causes damages to the host due to uncontrolled viral replication but even replication deficient viral vectors can induce systemic inflammatory responses. Indeed, overactive host innate immune responses to viral vectors have led to devastating consequences. Macrophages are important innate immune cells that recognize viruses and induce inflammatory responses at the early stage of infection. However, tissue resident macrophages are not easily activated by the mere presence of virus suggesting that their activation requires additional signals from other cells in the tissue in order to trigger inflammatory responses. Previously, we have shown that the cross-talk between epithelial cells and macrophages generates synergistic inflammatory responses during adenoviral vector infection. Here, we investigated whether ATP is involved in the activation of macrophages to induce inflammatory responses during an acute adenoviral infection. Using a macrophage-epithelial cell co-culture system we demonstrated that ATP signaling through P2X(7) receptor (P2X(7)R) is required for induction of inflammatory mediators. We also showed that ATP-P2X(7)R signaling regulates inflammasome activation as inhibition or deficiency of P2X(7)R as well as caspase-1 significantly reduced IL-1β secretion. Furthermore, we found that intranasal administration of replication deficient adenoviral vectors in mice caused a high mortality in wild-type mice with symptoms of acute respiratory distress syndrome but the mice deficient in P2X(7)R or caspase-1 showed increased survival. In addition, wild-type mice treated with apyrase or inhibitors of P2X(7)R or caspase-1 showed higher rates of survival. The improved survival in the P2X(7)R deficient mice correlated with diminished levels of IL-1β and IL-6 and reduced neutrophil infiltration in the early phase of infection. These results indicate that ATP, released during viral infection, is an important inflammatory regulator that activates the inflammasome pathway and regulates inflammatory responses.     

SP-D counteracts GM-CSF-mediated increase of granuloma formation by alveolar macrophages in lysinuric protein intolerance

Background

Pulmonary alveolar proteinosis (PAP) is a syndrome with multiple etiologies and is often deadly in lysinuric protein intolerance (LPI). At present, PAP is treated by whole lung lavage or with granulocyte/monocyte colony stimulating factor (GM-CSF); however, the effectiveness of GM-CSF in treating LPI associated PAP is uncertain. We hypothesized that GM-CSF and surfactant protein D (SP-D) would enhance the clearance of proteins and dying cells that are typically present in the airways of PAP lungs.

Methods

Cells and cell-free supernatant of therapeutic bronchoalveolar lavage fluid (BALF) of a two-year-old patient with LPI were isolated on multiple occasions. Diagnostic BALF samples from an age-matched patient with bronchitis or adult PAP patients were used as controls. SP-D and total protein content of the supernatants were determined by BCA assays and Western blots, respectively. Cholesterol content was determined by a calorimetic assay or Oil Red O staining of cytospin preparations. The cells and surfactant lipids were also analyzed by transmission electron microscopy. Uptake of Alexa-647 conjugated BSA and DiI-labelled apoptotic Jurkat T-cells by BAL cells were studied separately in the presence or absence of SP-D (1 μg/ml) and/or GM-CSF (10 ng/ml), ex vivo. Specimens were analyzed by light and fluorescence microscopy.

Results

Here we show that large amounts of cholesterol, and large numbers of cholesterol crystals, dying cells, and lipid-laden foamy alveolar macrophages were present in the airways of the LPI patient. Although SP-D is present, its bioavailability is low in the airways. SP-D was partially degraded and entrapped in the unusual surfactant lipid tubules with circular lattice, in vivo. We also show that supplementing SP-D and GM-CSF increases the uptake of protein and dying cells by healthy LPI alveolar macrophages, ex vivo. Serendipitously, we found that these cells spontaneously generated granulomas, ex vivo, and GM-CSF treatment drastically increased the number of granulomas whereas SP-D treatment counteracted the adverse effect of GM-CSF.

Conclusions

We propose that increased GM-CSF and decreased bioavailability of SP-D may promote granuloma formation in LPI, and GM-CSF may not be suitable for treating PAP in LPI. To improve the lung condition of LPI patients with PAP, it would be useful to explore alternative therapies for increasing dead cell clearance while decreasing cholesterol content in the airways.     

Innate immune collectin surfactant protein D simultaneously binds both neutrophil extracellular traps and carbohydrate ligands and promotes bacterial trapping

Neutrophils release DNA-based extracellular traps to capture and kill bacteria. The mechanism(s) and proteins that promote neutrophil extracellular trap (NET)-mediated bacterial trapping are not clearly established. Surfactant protein D (SP-D) is an innate immune collectin present in many mucosal surfaces. We hypothesized that SP-D can bind both the pathogens and NETs to augment NET-mediated bacterial trapping. To test this hypothesis, we used LPS and Pseudomonas aeruginosa pneumonia mouse models and performed in vivo and ex vivo assays. In this study, we show that NETs are produced by the neutrophils recruited to the airways in response to the bacterial ligand. Notably, NETs are detected as short fragments of DNA-protein complexes in the airways as opposed to the long stringlike structures seen in ex vivo cultures. SP-D recognizes both the short NET fragments and the long NET DNA structures. SP-D-NET copurification studies further show that SP-D can simultaneously recognize NETs and carbohydrate ligands in vivo. Similar to the LPS model, soluble DNA-protein complexes and increased amounts of SP-D are detected in the murine model of P. aeruginosa pneumonia. We then tested the effect of SP-D on NET-mediated trapping of P. aeruginosa by means of Western blots, fluorescence microscopy, and scanning electron microscopy. Results of these experiments show that SP-D microagglutinates P. aeruginosa and allows an efficient bacterial trapping by NETs. Collectively, these findings provide a unique biological relevance for SP-D-DNA interactions and places SP-D as an important innate immune protein that promotes bacterial trapping by NETs during neutrophil-mediated host defense.     

Identification and characterization of a novel interaction between pulmonary surfactant protein D and decorin

Surfactant-associated protein D (SP-D) is a collectin that is present in lung surfactant and mucosal surfaces. Although SP-D regulates diverse functions, only a few proteins are known to bind to this collectin. Here we describe the co-purification of decorin, a novel SP-D-binding protein, from amniotic fluid. The human decorin that co-purified with SP-D is a 130-150-kDa proteoglycan, which has a 46-kDa protein core and approximately 90-kDa dermatan sulfate chain. Both native and recombinant decorin can bind to SP-D that is already bound to maltose-agarose matrix, and these SP-D-decorin complexes are dissociated at high salt (0.5-1.0 m NaCl) conditions, releasing the decorin. We further show that SP-D and decorin interact with each other (kd = 4 nm) by two mechanisms. First, the direct binding and competition experiments show that the carbohydrate recognition domain (CRD) of SP-D binds in a calcium dependent-manner to the sulfated N-acetyl galactosamine moiety of the glycosaminoglycan chain. Second, complement component C1q, a complement protein that is known to interact with decorin core protein via its collagen-like region, partially blocks the interaction between decorin and native SP-D. This protein, however, does not block the interaction between decorin and SP-D(n/CRD), a recombinant fragment that lacks the N-terminal and collagen-like regions. Furthermore, the core protein, obtained by chondroitin ABC lyase treatment of decorin, binds SP-D, but not SP-D(n/CRD). These findings suggest that decorin core protein binds the collagen-like region of the SP-D. Concentrations of decorin and SP-D are negatively correlated to each other, in amniotic fluid, implying a functional relevance for SP-D-decorin interaction, in vivo. Collectively, our results show that carbohydrate recognition domains of SP-D interact with the dermatan sulfate moiety of decorin via lectin activity and that the core protein of decorin binds the collagen-like region of SP-D in vitro, and these interactions may be operative in vivo.     

The collagen-like region of surfactant protein A (SP-A) is required for correction of surfactant structural and functional defects in the SP-A null mouse

Pulmonary surfactant isolated from gene-targeted surfactant protein A null mice (SP-A(-/-)) is deficient in the surfactant aggregate tubular myelin and has surface tension-lowering activity that is easily inhibited by serum proteins in vitro. To further elucidate the role of SP-A and its collagen-like region in surfactant function, we used the human SP-C promoter to drive expression of rat SP-A (rSPA) or SP-A containing a deletion of the collagen-like domain (DeltaG8-P80) in the Clara cells and alveolar type II cells of SP-A(-/-) mice. The level of the SP-A in the alveolar wash of the SP-A(-/-,rSP-A) and SP-A(-/-,DeltaG8-P80) mice was 6.1-and 1.3-fold higher, respectively, than in the wild type controls. Tissue levels of saturated phosphatidylcholine were slightly reduced in the SP-A(-/-,rSP-A) mice compared with SP-A(-/-) littermates. Tubular myelin was present in the large surfactant aggregates isolated from the SP-A(-/-,rSP-A) lines but not in the SP-A(-/-,DeltaG8-P80) mice or SP-A(-/-) controls. The equilibrium and minimum surface tensions of surfactant from the SP-A(-/-,rSP-A) mice were similar to SP-A(-/-) controls, but both were markedly elevated in the SP-A(-/-,DeltaG8-P80) mice. There was no defect in the surface tension-lowering activity of surfactant from SP-A(+/+,DeltaG8-P80) mice, indicating that the inhibitory effect of DeltaG8-P80 on surface activity can be overcome by wild type levels of mouse SP-A. The surface activity of surfactant isolated from the SP-A(-/-,rSP-A) but not the SP-A(-/-,DeltaG8-P80) mice was more resistant than SP-A(-/-) littermate control animals to inhibition by serum proteins in vitro. Pressure volume relationships of lungs from the SP-A(-/-), SP-A(-/-,rSP-A), and SP-A(-/-,DeltaG8-P80) lines were very similar. These data indicate that expression of SP-A in the pulmonary epithelium of SP-A(-/-) animals restores tubular myelin formation and resistance of isolated surfactant to protein inhibition by a mechanism that is dependent on the collagen-like region.     

Three-dimensional structure of rat surfactant protein A trimers in association with phospholipid monolayers

Surfactant protein A (SP-A) is a C-type lectin found primarily in the lung and plays a role in innate immunity and the maintenance of surfactant integrity. To determine the three-dimensional (3D) structure of SP-A in association with a lipid ligand, we have used single particle electron crystallography and computational 3D reconstruction in combination with molecular modeling. Recombinant rat SP-A, containing a deletion of the collagen-like domain, was incubated with dipalmitoylphosphatidylcholine:egg phosphatidylcholine (1:1, wt/wt) lipid monolayers in the presence of calcium, negatively stained, and examined by transmission electron microscopy. Images of SP-A-lipid complexes with different angular orientations were used to reconstruct the 3D structure of the protein. These results showed that SP-A subunits readily formed trimers and interacted with lipid monolayers exclusively via the globular domains. A homology-based molecular model of SP-A was generated and fitted into the electron density map of the protein. The plane of the putative lipid-protein interface was relatively flat and perpendicular to the hydrophobic neck region, and the cleft region in the middle of the trimer had no apparent charge clusters. Amino acid residues that are known to affect lipid interactions, Glu(195) and Arg(197), were located at the protein-lipid interface. The molecular model indicated that the hydrophobic neck region of the SP-A did not interact with lipid monolayers but was instead involved in intratrimeric subunit interactions. The glycosylation site of SP-A was located at the side of each subunit, suggesting that the covalently linked carbohydrate moiety probably occupies the spaces between the adjacent globular domains, a location that would not sterically interfere with ligand binding.     

Collectin surfactant protein D binds antibodies and interlinks innate and adaptive immune systems

Innate immune collectins, such as surfactant protein D (SP-D), contain fibrillar collagen-like regions and globular carbohydrate-recognition domains (CRDs). SP-D recognizes carbohydrate arrays present on microbial surfaces via its CRDs, agglutinates microbes and enhances their phagocytosis. In contrast, adaptive immune proteins such as immunoglobulins (Igs) recognize pathogens via binding to specific antigens. Here we show that: SP-D binds various classes of immunoglobins, including IgG, IgM, IgE and secretory IgA, but not serum IgA; the globular domains of SP-D bind both the Fab and Fc domains of IgG; SP-D recognizes IgG via calcium-dependent protein-protein interactions, aggregates IgG-coated beads and enhances their phagocytosis by murine macrophage RAW 264.7 cells. Therefore, we propose that SP-D effectively interlinks innate and adaptive immune systems.     

Mannose-binding lectin recognizes peptidoglycan via the N-acetyl glucosamine moiety, and inhibits ligand-induced proinflammatory effect and promotes chemokine production by macrophages

Peptidoglycan (PGN) is the major cell wall component (90%, w/w) of Gram-positive bacteria and consists of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) disaccharide repeating arrays that are cross-linked by short peptides. We hypothesized that PGN is a ligand for pathogen-associated pattern-recognition proteins. Mannose-binding lectin (MBL) and serum amyloid component P are two carbohydrate-binding innate immune proteins present in the blood. In this study we show that human MBL, but not serum amyloid component P, binds significantly to PGN via its C-type lectin domains, and that the interaction can be more effectively competed by GlcNAc than by MurNAc. Surface plasmon resonance analyses show that native MBL binds immobilized PGN with high avidity. Competition experiments also show that both native MBL and MBL(n/CRD), a 48-kDa recombinant trimeric fragment of MBL containing neck and carbohydrate recognition domains, have higher affinity for GlcNAc than for MurNAc. Protein arrays and ELISA show that PGN increases the secretion of TNF-alpha, IL-8, IL-10, MCP-2, and RANTES from PMA-stimulated human monocytic U937 cells. Interestingly, the presence of MBL together with PGN increases the production of IL-8 and RANTES, but reduces that of TNF-alpha. Our results indicate that Gram-positive bacterial is a biologically relevant ligand for MBL, and that the collectin preferentially binds to the GlcNAc moiety of the PGN via its C-type lectin domains. MBL inhibits PGN-induced production of proinflammatory cytokines while enhancing the production of chemokines by macrophages, which suggests that MBL may down-regulate macrophage-mediated inflammation while enhancing phagocyte recruitment.