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.     

Carbon dioxide enhances nitration of surfactant protein A by activated alveolar macrophages

We assessed whether reactive oxygen-nitrogen intermediates generated by alveolar macrophages (AMs) oxidized and nitrated human surfactant protein (SP) A. SP-A was exposed to lipopolysaccharide (100 ng/ml)-activated AMs in 15 mM HEPES (pH 7.4) for 30 min in the presence and absence of 1.2 mM CO(2). In the presence of CO(2), lipopolysaccharide-stimulated AMs had significantly higher nitric oxide synthase (NOS) activity (as quantified by the conversion of L-[U-(14)C]arginine to L-[U-(14)C]citrulline) and secreted threefold higher levels of nitrate plus nitrite in the medium [28 +/- 3 vs. 6 +/- 1 (SE) nmol. 6.5 h(-1). 10(6) AMs(-1)]. Western blotting studies of immunoprecipitated SP-A indicated that CO(2) enhanced SP-A nitration by AMs and decreased carbonyl formation. CO(2) (0-1.2 mM) also augmented peroxynitrite (0.5 mM)-induced SP-A nitration in a dose-dependent fashion. Peroxynitrite decreased the ability of SP-A to aggregate lipids, and this inhibition was augmented by 1.2 mM CO(2). Mass spectrometry analysis of chymotryptic fragments of peroxynitrite-exposed SP-A showed nitration of two tyrosines (Tyr(164) and Tyr(166)) in the absence of CO(2) and three tyrosines (Tyr(164), Tyr(166), and Tyr(161)) in the presence of 1.2 mM CO(2). These findings indicate that physiological levels of peroxynitrite, produced by activated AMs, nitrate SP-A and that CO(2) increased nitration, at least partially, by enhancing enzymatic nitric oxide production.     

Innate immune and non-immune mediators of erythrocyte clearance.

Erythrocyte clearance is reviewed in the context of what is known in 2003 on clearance of apoptotic cells in vitro and in vivo. Thus, emphasis is put on the role of the innate immune system comprised of naturally occurring autoantibodies (NAbs) and complement. Oxidative damage, cellular senescence and diffusion-controlled exoplasmic cross-linking appear to generate oligomers of band 3 (anion transport protein) that are a prerequisite for anti-band 3 NAb binding to human red blood cells (RBC). Similar processes seem to be responsible for premature RBC clearance in hemoglobinopathies and membrane protein deficiencies. The review discusses why NAb binding alone is insufficient and how bound NAbs may enhance complement deposition. Clearance of RBC is not only the result of cell-bound opsonins, but is enhanced by the loss of RBC membrane constituents, such as CD47 and sialic acids. As long as these constituents are present on RBC in normal numbers and topologic arrangement, they bind to their respective receptors on macrophages, elicit a negative signal that appears to prevent the macrophage from engulfing bound RBC. Exposure of phosphatidylserine is not a primary signal for RBC removal and where exposed it initiates binding of CRP or of beta-2-glycoprotein I and NAbs.     

Lung surfactant protein A (SP-A) enhances serum-independent phagocytosis of bacteria by alveolar macrophages.

Surfactant protein A (SP-A) is the main protein component of lung surfactant. We studied the involvement of SP-A in body defense, i.e., effect of SP-A on the phagocytosis of bacteria by alveolar macrophages. We show here that SP-A enhances the phagocytosis of some non-opsonized bacteria: Escherichia coli growing logarithmically (E. coli/log), Pseudomonas aeruginosa/log as well as from stationary phase (P. aeruginosa/stat) and Staphylococcus aureus/log. Furthermore, not only serum-independent phagocytosis was effected by SP-A but also phagocytosis of serum-opsonized S. aureus/stat. No effect of SP-A on phagocytosis was observed with E. coli/stat neither on serum-independent nor on serum-dependent phagocytosis and on phagocytosis of non-opsonized S. aureus/stat. Thus, effect of SP-A on phagocytosis is dependent on bacterial species and on the growth phase of the microorganisms, and this effect is concentration dependent. We studied two different human recombinant SP-As and SP-A isolated from lung lavage material from proteinosis patients. These SP-A molecules contain different isomeric chains, and they differ in complexity of their structure. Qualitatively, we found the same effect with all three substances. Quantitatively, the proteinosis SP-A that forms the most complex structure was the most effective. Taken together, we demonstrated a stimulating effect of SP-A on serum-independent as well as on serum-dependent phagocytosis of bacteria by alveolar macrophages, both depending on species and growth phase of the bacteria.     

Effect of antibody excess on the size, stoichiometry, and DNAse resistance of DNA anti-DNA immune complexes

The binding of antibodies to DNA was examined under conditions of increasing antibody excess. DNA anti-DNA immune complexes (IC) formed at increasing antibody to DNA ratios were digested with excess DNAse I, and the DNAse-resistant (protected) IC were analyzed. With increasing antibody excess, the size of the IC that were resistant to DNAse digestion increased, and the size of the protected DNA within the IC also increased. This suggested that IgG molecules could bind in close proximity along the DNA molecule, preventing access of DNAse to the DNA between adjacent IgG. To further define the binding of adjacent IgG, DNAse digested IC containing one or two IgG were isolated, and the DNA contained within these IC was analyzed on DNA sequencing gels. Binding of a single IgG to DNA resulted in the protection of a DNA fragment 35 to 45 base pairs (bp) long, corresponding to the distance between binding sites of a single IgG molecule. Binding of two IgG to DNA protected a DNA fragment 50 to 60 bp long, 1 1/2 times the size of the fragment protected by one IgG. These data suggest that in conditions of Ab excess, IgG molecules can interdigitate along the DNA molecule, resulting in small, stable, DNAse-resistant IC of high antibody density.     

Acrolein in cigarette smoke attenuates the innate immune responses mediated by surfactant protein D

BackgroundSurfactant proteins (SP) A and D belong to collectin family proteins, which play important roles in innate immune response in the lung. We previously demonstrated that cigarette smoke (CS) increases the acrolein modification of SP-A, thereby impairing the innate immune abilities of this protein. In this study, we focused on the effects of CS and its component, acrolein, on the innate immunity role of another collectin, SP-D.MethodsTo determine whether aldehyde directly affects SP-D, we examined the lungs of mice exposed to CS for 1 week and detected aldehyde-modified SP-D using an aldehyde reactive probe. The structural changes in CS extract (CSE) or acrolein-exposed recombinant human (h)SP-D were determined by western blot, liquid chromatography-electrospray ionization tandem mass spectrometry, and blue native-polyacrylamide gel electrophoresis analyses. Innate immune functions of SP-D were determined by bacteria growth and macrophage phagocytosis.ResultsAldehyde-modified SP-D as well as SP-A was detected in the lungs of mice exposed to CS for 1 week. Exposure of hSP-D to CSE or acrolein induced an increased higher-molecular -weight of hSP-D and acrolein induced modification of five lysine residues in hSP-D. These modifications led to disruption of the multimer structure of SP-D and attenuated its ability to inhibit bacterial growth and activate macrophage phagocytosis.ConclusionCS induced acrolein modification in SP-D, which in turn induced structural and functional defects in SP-D.General SignificanceThese results suggest that CS-induced structural and functional defects in SP-D contribute to the dysfunction of innate immune responses in the lung following CS exposure.     

Degradation of pulmonary surfactant protein D by Pseudomonas aeruginosa elastase abrogates innate immune function

The alveolar epithelium is lined by surfactant, a lipoprotein complex that both reduces surface tension and mediates several innate immune functions including bacterial aggregation, alteration of alveolar macrophage function, and regulation of bacterial clearance. Surfactant protein-D (SP-D) participates in several of these immune functions, and specifically it enhances the clearance of the pulmonary pathogen Pseudomonas aeruginosa, a common cause of morbidity and mortality in cystic fibrosis (CF) patients. P. aeruginosa secretes a variety of virulence factors including elastase, a zinc-metalloprotease, which degrades both SP-A and SP-D. Here we show that SP-D is cleaved by elastase to produce a stable 35-kDa fragment in a time-, temperature-, and dose-dependent manner. Degradation is inhibited by divalent metal cations, a metal chelator, and the elastase inhibitor, phosphoramidon. Sequencing the SP-D degradation products localized the major cleavage sites to the C-terminal lectin domain. The SP-D fragment fails to bind or aggregate bacteria that are aggregated by intact SP-D. SP-D fragment is observed when normal rat bronchoalveolar lavage (BAL) is treated with Pseudomonas aeruginosa elastase, and SP-D fragments are present in the BAL of CF lung allograft patients. These data show that degradation of SP-D occurs in the BAL environment and that degradation eliminates many normal immune functions of SP-D.     

Enhanced clearance of surfactant protein D during LPS-induced acute inflammation in rat lung

Pulmonary surfactant participates in the regulation of alveolar compliance and lung host defense. Surfactant homeostasis is regulated through a combination of synthesis, secretion, clearance, recycling, and degradation of surfactant components. The extracellular pool size of surfactant protein (SP) D fluctuates significantly during acute inflammation. We hypothesized that changes in SP-D levels are due, in part, to altered clearance of SP-D. Clearance pathways in rats were assessed with fluorescently labeled SP-D that was instilled into control lungs or lungs that had been treated with lipopolysaccharide (LPS) 16 h earlier. SP-D clearance from lavage into lung tissue was time dependent from 5 min to 1 h and 1.7-fold greater in LPS-treated lungs than in control lungs. Analysis of cells isolated by enzymatic digestion of lung tissue revealed differences in the SP-D-positive cell population between groups. LPS-treated lungs had 28.1-fold more SP-D-positive tissue-associated neutrophils and 193.6-fold greater SP-D association with those neutrophils compared with control lungs. These data suggest that clearance of SP-D into lung tissue is increased during inflammation and that tissue-associated neutrophils significantly contribute to this process.     

Pulmonary surfactant protein A enhances endolysosomal trafficking in alveolar macrophages through regulation of Rab7

Surfactant protein A (SP-A), the most abundant pulmonary soluble collectin, modulates innate and adaptive immunity of the lung, partially via its direct effects on alveolar macrophages (AM), the most predominant intra-alveolar cells under physiological conditions. Enhanced phagocytosis and endocytosis are key functional consequences of AM/SP-A interaction, suggesting a SP-A-mediated modulation of small Rab (Ras related in brain) GTPases that are pivotal membrane organizers in both processes. In this article, we show that SP-A specifically and transiently enhances the protein expression of endogenous Rab7 and Rab7b, but not Rab5 and Rab11, in primary AM from rats and mice. SP-A-enhanced GTPases are functionally active as determined by increased interaction of Rab7 with its downstream effector Rab7 interacting lysosomal protein (RILP) and enhanced maturation of cathepsin-D, a function of Rab7b. In AM and RAW264.7 macrophages, the SP-A-enhanced lysosomal delivery of GFP-Escherichia coli is abolished by the inhibition of Rab7 and Rab7 small interfering RNA transfection, respectively. The constitutive expression of Rab7 in AM from SP-A(-/-) mice is significantly reduced compared with SP-A(+/+) mice and is restored by SP-A. Rab7 blocking peptides antagonize SP-A-rescued lysosomal delivery of GFP-E. coli in AM from SP-A(-/-) mice. Activation of Rab7, but not Rab7b, by SP-A depends on the PI3K/Akt/protein kinase Cζ (PKCζ) signal transduction pathway in AM and RAW264.7 macrophages. SP-A induces a Rab7/PKCζ interaction in these cells, and the disruption of PKCζ by small interfering RNA knockdown abolishes the effect of SP-A on Rab7. The data demonstrate a novel role for SP-A in modulating endolysosomal trafficking via Rab7 in primary AM and define biochemical pathways involved.     

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.     

High-resolution structural insights into ligand binding and immune cell recognition by human lung surfactant protein D

Lung surfactant protein D (SP-D) can directly interact with carbohydrate residues on pulmonary pathogens and allergens, stimulate immune cells, and manipulate cytokine and chemokine profiles during the immune response in the lungs. Therapeutic administration of rfhSP-D, a recombinant homotrimeric fragment of human SP-D comprising the alpha-helical coiled-coil neck plus three CRDs, protects mice against lung allergy and infection caused by the fungal pathogen Aspergillus fumigatus. The high resolution crystal structures of maltose-bound rfhSP-D to 1.4A, and of rfhSP-D to 1.6A, define the fine detail of the mode and nature of carbohydrate recognition and provide insights into how a small fragment of human SP-D can bind to allergens/antigens or whole pathogens, and at the same time recruit and engage effector cells and molecules of humoral immunity. A previously unreported calcium ion, located on the trimeric axis in a pore at the bottom of the funnel formed by the three CRDs and close to the neck-CRD interface, is coordinated by a triad of glutamate residues which are, to some extent, neutralised by their interactions with a triad of exposed lysine residues in the funnel. The spatial relationship between the neck and the CRDs is maintained internally by these lysine residues, and externally by a glutamine, which forms a pair of hydrogen-bonds within an external cleft at each neck-CRD interface. Structural links between the central pore and the cleft suggest a possible effector mechanism for immune cell surface receptor binding in the presence of bound, extended natural lipopolysaccharide and phospholipid ligands. The structural requirements for such an effector mechanism, involving both the trimeric framework for multivalent ligand binding and recognition sites formed from more than one subunit, are present in both native hSP-D and rfhSP-D, providing a possible explanation for the significant biological activity of rfhSP-D.     

Surfactant protein D enhances clearance of influenza A virus from the lung in vivo.

Mice lacking surfactant protein surfactant protein D (SP-D(-/-)) and wild-type mice (SP-D(+/+)) were infected with influenza A virus (IAV) by intranasal instillation. IAV infection increased the endogenous SP-D concentration in wild-type mice. SP-D-deficient mice showed decreased viral clearance of the Phil/82 strain of IAV and increased production of inflammatory cytokines in response to viral challenge. However, the less glycosylated strain of IAV, Mem/71, which is relatively resistant to SP-D in vitro, was cleared efficiently from the lungs of SP-D(-/-) mice. Viral clearance of the Phil/82 strain of IAV and the cytokine response were both normalized by the coadministration of recombinant SP-D. Since the airway is the usual portal of entry for influenza A virus and other respiratory pathogens, SP-D is likely to play an important role in innate defense responses to IAV.     

Pathways for clearance of surfactant protein A from the lung

Uptake and degradation of (125)I-surfactant protein A (SP-A) over a 1-h period was studied in alveolar cells in culture and in isolated perfused lungs to elucidate the mechanism for clearance of the protein from the alveolar space. Specific inhibitors of clathrin- and actin-dependent endocytosis were utilized. In type II cells, uptake of SP-A, compared with controls, was decreased by 60% on incubation with clathrin inhibitors (amantadine and phenylarsine oxide) or with the actin inhibitor cytochalasin D. All agents reduced SP-A metabolism by alveolar macrophages. Untreated rat isolated perfused lungs internalized 36% of instilled SP-A, and 56% of the incorporated SP-A was degraded. Inhibitors of clathrin and actin significantly reduced SP-A uptake by approximately 54%, whereas cytochalasin D inhibited SP-A degradation. Coincubation of agents did not produce an additive effect on uptake of SP-A by cultured pneumocytes or isolated perfused lungs, indicating that all agents affected the same pathway. Thus SP-A clears the lung via a clathrin-mediated pathway that requires the polymerization of actin.     

Ligand specificity of human surfactant protein D: expression of a mutant trimeric collectin that shows enhanced interactions with influenza A virus

Surfactant protein D is a pattern recognition molecule that plays diverse roles in immune regulation and anti-microbial host defense. Its interactions with known ligands are calcium-dependent and involve binding to the trimeric, C-type carbohydrate recognition domain. Surfactant protein D preferentially binds to glucose and related sugars. However, CL-43, a bovine serum lectin, which evolved through duplication of the surfactant protein D gene in ruminants, prefers mannose and mannose-rich polysaccharides. Surfactant protein D is characterized by two relatively conserved motifs at the binding face, along the edges of the shallow carbohydrate-binding groove. For CL-43, sequence alignments demonstrate a basic insertion, Arg-Ala-Lys (RAK), immediately N-terminal to the first motif. We hypothesized that this insertion contributes to the differences in saccharide selectivity and host defense function and compared the activities of recombinant trimeric neck + carbohydrate recognition domains of human surfactant protein D (NCRD) with CL-43 (RCL-43-NCRD) and selected NCRD mutants. Insertion of the CL-43 RAK sequence or a control Ala-Ala-Ala sequence (AAA) into the corresponding position in NCRD increased the efficiency of binding to mannan and changed the inhibitory potencies of competing saccharides to more closely resemble those of CL-43. In addition, RAK resembled CL-43 in its greater capacity to inhibit the infectivity of influenza A virus and to increase uptake of influenza by neutrophils.     

Clearance and organ localization of small DNA anti-DNA immune complexes in mice

DNA anti-DNA immune complexes (IC) play a major role in the pathogenesis of SLE. We studied the clearance and organ localization of small DNA anti-DNA IC formed at different Ag/antibody ratios in normal mice. IC formed at Ag excess, containing areas of "exposed" DNA not covered by IgG, showed rapid Ag-mediated clearance from the circulation by the liver. DNAse digestion of these IC in vitro yielded small IC devoid of exposed DNA that were cleared more slowly from the circulation. IC formed at antibody excess were cleared by an Ag-independent mechanism at rates proportional to the number of IgG in the IC. None of the IC studied bound significantly to complement receptors on circulating cells in vivo or in vitro. For all IC, after initial rapid clearance, 10 to 20% of the injected material persisted in the circulation. Analysis of these IC showed that they were processed in vivo to yield complexes similar to those generated by in vitro DNAse digestion. We conclude that IC containing exposed DNA are removed rapidly from the circulation by Ag-mediated clearance. However, in vivo processing of IC occurs to yield smaller IC that are cleared slowly. We propose that these IC containing small DNA may persist in the circulation and accumulate in tissues, thereby playing an important role in the pathogenesis of tissue injury in SLE.     

Mutagenesis of surfactant protein D informed by evolution and x-ray crystallography enhances defenses against influenza A virus in vivo

The recognition of influenza A virus (IAV) by surfactant protein D (SP-D) is mediated by interactions between the SP-D carbohydrate recognition domains (CRD) and glycans displayed on envelope glycoproteins. Although native human SP-D shows potent antiviral and aggregating activity, trimeric recombinant neck+CRDs (NCRDs) show little or no capacity to influence IAV infection. A mutant trimeric NCRD, D325A/R343V, showed marked hemagglutination inhibition and viral neutralization, with viral aggregation and aggregation-dependent viral uptake by neutrophils. D325A/R343V exhibited glucose-sensitive binding to Phil82 hemagglutinin trimer (HA) by surface plasmon resonance. By contrast, there was very low binding to the HA trimer from another virus (PR8) that lacks glycans on the HA head. Mass spectrometry demonstrated the presence of high mannose glycans on the Phil82 HA at positions known to contribute to IAV binding. Molecular modeling predicted an enhanced capacity for bridging interactions between HA glycans and D325A/R343V. Finally, the trimeric D325A/R343V NCRD decreased morbidity and increased viral clearance in a murine model of IAV infection using a reassortant A/WSN/33 virus with a more heavily glycosylated HA. The combined data support a model in which altered binding by a truncated mutant SP-D to IAV HA glycans facilitates viral aggregation, leading to significant viral neutralization in vitro and in vivo. These studies demonstrate the potential utility of homology modeling and protein structure analysis for engineering effective collectin antivirals as in vivo therapeutics.