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Infiltrating activated monocytes are important mediators of damaging inflammation during influenza A virus (IAV) infection. We show that soluble respiratory proteins [collectins, surfactant proteins D (SP-D) and mannose binding lectin (MBL), H-ficolin and LL-37] inhibit replication of seasonal IAV in human monocytes. The collectins and H-ficolin also increased viral uptake by the cells, while LL-37 did not. H-ficolin was able to inhibit replication of the 2009 pandemic H1N1 strain (Cal09) in monocytes, but SP-D and LL-37 had significantly fewer inhibitory effects on this strain than on seasonal IAV. All of these proteins reduced IAV-induced TNF-α production, even in instances when viral replication was not reduced. We used modified recombinant versions of SP-D, MBL and ficolin to elucidate mechanisms through which these proteins alter monocyte interactions with IAV. We demonstrate the importance of the multimeric structure, and of binding properties of the lectin domain, in mediating antiviral and opsonic activity of the proteins. Hence, soluble inhibitors present in airway lining fluid may aid clearance of IAV by promoting monocyte uptake of the virus, while reducing viral replication and virus-induced TNF-α responses in these cells. However, SP-D and LL-37 have reduced ability to inhibit replication of pandemic IAV in monocytes.

Host defense roles for the lung collectins, surfactant protein A (SP-A) and surfactant protein D (SP-D), were first suspected in the 1980s when molecular characterization revealed their sequence homology to the acute phase reactant of serum, mannose-binding lectin. Surfactant protein A and SP-D have since been shown to play diverse and important roles in innate immunity and pulmonary homeostasis. Their location in surfactant ideally positions them to interact with air-space pathogens. Despite extensive structural similarity, the two proteins show many functional differences and considerable divergence in their interactions with microbial surface components, surfactant lipids, and other ligands. Recent crystallographic studies have provided many new insights relating to these observed differences. Although both proteins can participate in calcium-dependent interactions with sugars and other polyols, they display significant differences in the spatial orientation, charge, and hydrophobicity of their binding surfaces. Surfactant protein D appears particularly adapted to interactions with complex carbohydrates and anionic phospholipids, such as phosphatidylinositol. By contrast, SP-A shows features consistent with its preference for lipid ligands, including lipid A and the major surfactant lipid, dipalmitoylphosphatidylcholine. Current research suggests that structural biology approaches will help to elucidate the molecular basis of pulmonary collectin-ligand recognition and facilitate development of new therapeutics based upon SP-A and SP-D.

Surfactant protein D (SP-D) plays important roles in innate defense against respiratory viruses [including influenza A viruses (IAVs)]. Truncated trimers composed of its neck and carbohydrate recognition domains (NCRDs) bind various ligands; however, they have minimal inhibitory activity for IAV. We have sought to find ways to increase the antiviral activity of collectin NCRDs. Cross-linking of the SP-D NCRD with nonblocking monoclonal antibodies (mAbs) markedly potentiates antiviral activity. In the present report, we demonstrate that F(ab')2 [but not F(ab')1] fragments of a cross-linking mAb have similar effects. Hence, cross-linking activity, but not the Fc domain of the mAb, is needed for increased antiviral activity. In contrast, the Fc domain of the mAb was important for increasing viral uptake or respiratory burst responses of human neutrophils. Our NCRD constructs contain an S protein binding site. Herein, we show that a multivalent S protein complex caused cross-linking and also increased the antiviral activity of NCRDs. NCRDs of conglutinin and CL43 had greater intrinsic antiviral activity than those of SP-D or mannose-binding lectin. Based on motifs found in these serum collectins, we have constructed mutant versions of the human SP-D NCRD that have increased antiviral activity. These mutant NCRDs also had potentiated activity after cross-linking with F(ab')2 fragments or S protein complexes. Hence, the antiviral activity of NCRDs can be increased by 2 distinct, complementary strategies, namely cross-linking of NCRDs through various means and mutagenesis of CRD residues to increase viral binding. These findings may be relevant for antiviral therapy., ((c) 2009 S. Karger AG, Basel.)

Collectins are collagenous lectins present in blood, respiratory lining fluid, and other mucosal secretions that play important roles in innate defense against infection. The collectin, surfactant protein D (SP-D), limits infection by viruses and bacteria in the respiratory tract, eye, and female genital tract. Multimeric SP-D has strong antiviral activity and is a potent viral and bacterial agglutinin and opsonin; however, trimers composed of the neck and carbohydrate recognition domain (hSP-D-NCRD) of SP-D lack these activities. We now show that, in contrast, a trimeric neck and CRD construct of bovine serum collectin CL-46 induces aggregation of influenza A virus (IAV) and potently increases IAV uptake by neutrophils. CL-46-NCRD showed calcium-dependent and sugar-sensitive binding to both neutrophils and IAV. Replacement of specific residues of the CRD of human SP-D with those found in bovine serum collectins conferred opsonizing activity. The most effective substitution involved replacement of arginine 343 with valine (hSP-D-NCRD/R343V). hSP-D-NCRD/R343V greatly increased viral uptake by neutrophils and monocytes and also potentiated neutrophil respiratory burst responses. These effects were further increased by cross-linking of hSP-D-NCRD/R343V trimers with MAbs directed against areas of the hSP-D-NCRD not involved in viral binding. Unlike the wild-type human SP-D hSP-D-NCRD, hSP-D-NCRD/R343V also induced viral aggregation. These results indicate that collectins can act as opsonins for IAV even in the absence of the collagen domain or higher order multimerization. This may involve increased affinity of individual CRDs for glycoconjugates displayed on host cells or the viral envelope.

Collectins play important roles in innate defence against viral, fungal and bacterial pathogens. CL-43, a bovine serum collectin, which appears to have evolutionarily evolved from surfactant protein D (SP-D), shows unique structural and functional properties. In the present study, we describe the initial characterization of a recombinant CL-43 expressed in mammalian cells. Like natural CL-43, the recombinant is secreted as trimeric forms that show a preference for mannose and N-acetyl mannosamine. The natural and recombinant proteins have significantly higher haemagglutination-inhibiting activity against influenza A virus (IAV) than recombinant trimeric forms of SP-D. In contrast with the more highly multimerized forms of SP-D, namely conglutinin or mannose-binding lectin, CL-43 did not induce viral or bacterial aggregation and did not enhance IAV-induced neutrophil H(2)O(2) generation. Like SP-D, CL-43 also strongly enhanced neutrophil uptake of IAV. However, the mechanism of this enhanced internalization is different from that of SP-D in that it did not require viral aggregation. These studies establish that the trimeric structure of CL-43 is specified by its primary sequence and indicate that this naturally occurring trimeric collectin has unique antiviral activities. These findings could facilitate the development of recombinant collectins with novel antimicrobial properties.

Surfactant protein D (SP-D) and serum conglutinin are closely related members of the collectin family of host defense lectins. Although normally synthesized at different anatomic sites, both proteins participate in the innate immune response to microbial challenge. To discern the roles of specific domains in the function of SP-D in vivo, a fusion protein (SP-D/Cong(neck+CRD)) consisting of the NH(2)-terminal and collagenous domains of rat SP-D (rSP-D) and the neck and carbohydrate recognition domains (CRDs) of bovine conglutinin (Cong) was expressed in the respiratory epithelium of SP-D gene-targeted (SP-D(-/-)) mice. While SP-D/Cong(neck+CRD) fusion protein did not affect lung morphology and surfactant phospholipid levels in the lungs of wild type mice, the chimeric protein substantially corrected the increased lung phospholipids in SP-D(-/-) mice. The SP-D/Cong(neck+CRD) fusion protein also completely corrected defects in influenza A clearance and inhibited the exaggerated inflammatory response that occurs following viral infection. However, the chimeric protein did not ameliorate the ongoing lung inflammation, enhanced metalloproteinase expression, and alveolar destruction that characterize this model of SP-D deficiency. By contrast, a single arm mutant (RrSP-D(Ser15,20)) partially restored antiviral activity but otherwise failed to rescue the deficient phenotype. Our findings directly implicate the CRDs of both SP-D and conglutinin in host defense in vivo. Our findings also strongly suggest that the molecular mechanisms underlying impaired pulmonary host defense and abnormal lipid metabolism are distinct from those that promote ongoing inflammation and the development of emphysema.

Abstract Surfactant protein-D (SP-D) participates in the innate response to inhaled microorganisms and organic antigens, and contributes to immune and inflammatory regulation within the lung. SP-D is synthesized and secreted by alveolar and bronchiolar epithelial cells, but is also expressed by epithelial cells lining various exocrine ducts and the mucosa of the gastrointestinal and genitourinary tracts. SP-D, a collagenous calcium-dependent lectin (or collectin), binds to surface glycoconjugates expressed by a wide variety of microorganisms, and to oligosaccharides associated with the surface of various complex organic antigens. SP-D also specifically interacts with glycoconjugates and other molecules expressed on the surface of macrophages, neutrophils, and lymphocytes. In addition, SP-D binds to specific surfactant-associated lipids and can influence the organization of lipid mixtures containing phosphatidylinositol in vitro. Consistent with these diverse in vitro activities is the observation that SP-D-deficient transgenic mice show abnormal accumulations of surfactant lipids, and respond abnormally to challenge with respiratory viruses and bacterial lipopolysaccharides. The phenotype of macrophages isolated from the lungs of SP-D-deficient mice is altered, and there is circumstantial evidence that abnormal oxidant metabolism and/or increased metalloproteinase expression contributes to the development of emphysema. The expression of SP-D is increased in response to many forms of lung injury, and deficient accumulation of appropriately oligomerized SP-D might contribute to the pathogenesis of a variety of human lung diseases.

Surfactant protein D (SP-D) plays important roles in host defense against a variety of pathogens including influenza A virus (IAV). Ligand binding by SP-D is mediated by the trimeric neck and carbohydrate recognition domain (NCRD). We used monoclonal antibodies (mAbs) against human SP-D and a panel of mutant collectin NCRD constructs to identify functionally and structurally important epitopes. The ability of SP-D to bind to IAV and mannan involved partially overlapping binding sites that are distinct from those involved in binding to the glycoprotein-340 (gp-340) scavenger receptor protein. A speciesspecific motif (D324,D325,R343), which has been implicated in the specific binding of several ligands, contributes to recognition by mAbs that block antiviral or mannan binding activity. D325, in particular, is involved in the epitopes of these blocking mAbs. Conversely, the interspecies substitution of arginine for Lys343 in the rat NCRD (rK343R) conferred binding to two of the mAbs. The single site substitution of alanine for R349 or E347 resulted in highly selective alterations in mAb binding and caused decreased antiviral activity. Mutations at Glu333 (E333A), Trp340 (W340F), and Phe335 (F335A), which abrogated antiviral activity, were associated with decreased binding to multiple blocking mAbs, consistent with critical structural roles. More conservative substitutions at 335, which showed a significant increase in neutralization activity, caused selective loss of binding to one mAb. The analysis reveals, for the first time, an extended binding site for IAV; calcium-dependent antiviral activity involves residues flanking the primary carbohydrate binding site as well as more remote residues displayed on the carbohydrate recognition domain surface. [ABSTRACT FROM AUTHOR]

Host defense roles for the lung collectins, surfactant protein A (SP-A) and surfactant protein D (SP-D), were first suspected in the 1980s when molecular characterization revealed their sequence homology to the acute phase reactant of serum, mannose-binding lectin. Surfactant protein A and SP-D have since been shown to play diverse and important roles in innate immunity and pulmonary homeostasis. Their location in surfactant ideally positions them to interact with air-space pathogens. Despite extensive structural similarity, the two proteins show many functional differences and considerable divergence in their interactions with microbial surface components, surfactant lipids, and other ligands. Recent crystallographic studies have provided many new insights relating to these observed differences. Although both proteins can participate in calcium-dependent interactions with sugars and other polyols, they display significant differences in the spatial orientation, charge, and hydrophobicity of their binding surfaces. Surfactant protein D appears particularly adapted to interactions with complex carbohydrates and anionic phospholipids, such as phosphatidylinositol. By contrast, SP-A shows features consistent with its preference for lipid ligands, including lipid A and the major surfactant lipid, dipalmitoylphosphatidylcholine. Current research suggests that structural biology approaches will help to elucidate the molecular basis of pulmonary collectin-ligand recognition and facilitate development of new therapeutics based upon SP-A and SP-D. [ABSTRACT FROM AUTHOR]

Mast cells play a key role in allergy and asthma. They reside at the host-environment interface and are among the first cells to make contact with inhaled microorganisms and particulate antigens. Pulmonary surfactant proteins A and D (SP-A and SP-D) function in lung host defense by enhancing microbe phagocytosis and mediating other immune cell functions, but little is known about their effects on mast cells. We hypothesized that SP-A and/or SP-D modulate IgE-dependent mast cell functions. Pollen starch granules (PSG) extracted from Dactylis glomerata and coated with trinitrophenol (TNIP) were used as a model of an inhaled organic particulate allergen. Our data revealed that SP-D inhibited by 50% the release of β-hexosaminidase by peritoneal mast cells sensitized with IgE antiTNP and stimulated with TNP-PSG. In contrast, SP-A had no effect. Furthermore, SP-D aggregated PSG in a dose-dependent manner, and this aggregation was mediated by SP-D's carbohydrate recognition domain. A single arm SP-D mutant (RrSP-Dser15,20) neither aggregated PSG nor inhibited degranulation, suggesting that multimerization of SP-D is required for maximal PSG aggregation and inhibition of PSG-induced mast cell degranulation. This study is the first to demonstrate that SP-D modulates IgE-mediated mast cell functions, which are important in asthma and allergic inflammation. [ABSTRACT FROM AUTHOR]