Recombinant Surfactant Protein D Research Articles

Surfactant protein D in human lung diseases

The lung is continuously exposed to inhaled pollutants, microbes and allergens. Therefore, the pulmonary immune system has to defend against harmful pathogens, while an inappropriate inflammatory response to harmless particles must be avoided. In the bronchoalveolar space this critical balance is maintained by innate immune proteins, termed surfactant proteins. Among these, surfactant protein D (SP-D) plays a central role in the pulmonary host defence and the modulation of allergic responses. Several human lung diseases are characterized by decreased levels of bronchoalveolar SP-D. Thus, recombinant SP-D has been proposed as a therapeutical option for cystic fibrosis, neonatal lung disease and smoking-induced emphysema. Furthermore, SP-D serum levels can be used as disease activity markers for interstitial lung diseases. This review illustrates the emerging role of SP-D translated from in vitro studies to human lung diseases.

Agglutination ofPseudomonas aeruginosa by Surfactant Protein D

Surfactant protein D (SP-D) is part of the innate host defense system, and may bind and agglutinate invading microorganisms to enhance their removal. The ability of bronchoalveolar lavage (BAL) fluid to agglutinate bacteria and the relationship to its SP-D content are of interest and not yet known. A micromethod on slides was used to assess the agglutination of Pseudomonas aeruginosa by recombinant SP-D and native human BAL fluid. The SP-D-induced agglutination was blocked by calcium depletion, alkaline pH, or the presence of maltose. Twenty-three of 30 BAL fluids from outpatients carrying a chronic tracheostoma clearly agglutinated P. aeruginosa, which was completely inhibited by maltose. The extent of the agglutination correlated weakly to the concentration of SP-D in the BAL fluid, but not to that of SP-A. The functional property, i.e., the agglutination of P. aeruginosa by BAL fluid, was characterized and appeared related in part to the concentration of SP-D. Additional factors, such as the multimeric organization of SP-D, are likely to contribute to the agglutination of microorganisms by BAL or other body fluids. The assay presented will allow the systematic evaluation of small-volume samples for SP-D agglutinating ability from subjects with various lung diseases.

A recombinant fragment of human surfactant protein D reduces alveolar macrophage apoptosis and pro-inflammatory cytokines in mice developing pulmonary emphysema.

Rapid removal of apoptotic cells is an important mechanism for immune homeostasis and the resolution of inflammation. Delayed clearance of apoptotic alveolar macrophages may cause activation of healthy bystander macrophages and contribute to high macrophage number and emphysema in surfactant protein D (SP-D) knock-out mice. Using flow cytometry and Annexin V and propidium iodide as markers for apoptosis and necrosis, respectively, SP-D-deficient mice were found to have a 5- to 10-fold increase in the number of apoptotic and necrotic alveolar macrophages in the lungs. SP-D-deficient mice accumulate apoptotic macrophages in the lung, and this accumulation can be reduced by treatment with recombinant SP-D (but not SP-A). The recombinant SP-D binds preferentially to apoptotic cells. The data are consistent with a specific role in vivo for SP-D in promoting apoptotic cell clearance in the lungs to limit macrophage-mediated inflammation and reveal a potential new mechanism for therapeutic targeting in the prevention of emphysema.

Reversibility of Pulmonary Abnormalities by Conditional Replacement of Surfactant Protein D (SP-D) in Vivo

Surfactant protein D (SP-D) gene-targeted mice develop severe pulmonary disease associated with emphysema, pulmonary lipidosis, and foamy macrophage infiltrations. To determine the potential reversibility of these abnormalities, transgenic mice were developed in which SP-D was conditionally replaced in the respiratory epithelium of SP-D(-/-) mice. SP-D was not detected in the absence of doxycycline. Treatment with doxycycline after birth restored pulmonary SP-D concentrations and corrected pulmonary pathology at adulthood. When SP-D was replaced in adult SP-D(-/-) mice, alveolar SP-D was restored within 3 days, pulmonary lipid abnormalities were corrected, but emphysema persisted. In corrected adult SP-D(-/-) mice, loss of SP-D caused focal emphysema and pulmonary inflammation but did not cause phospholipid abnormalities characteristic of SP-D(-/-) mice. Thus, abnormalities in surfactant phospholipid homeostasis and alveolar macrophage abnormalities were readily corrected by restoration of SP-D. However, once established, emphysema was not reversed by SP-D. SP-D-dependent processes regulating surfactant lipid homeostasis were disassociated from those mediating emphysema.

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.

Surfactant protein D inhibition of human macrophage uptake of Mycobacterium tuberculosis is independent of bacterial agglutination.

The innate immune system in the lung is essential for controlling infections due to inhaled pathogens. Mycobacterium tuberculosis (M.tb) encounters components of the innate immune system when inhaled into the lung, but the consequences of these interactions are poorly understood. Surfactant protein D (SP-D) binds to and agglutinates M.tb bacilli, and reduces the uptake of the bacteria by human macrophages. In the current studies, we utilized a recombinant SP-D variant (CDM) that lacks the collagen domain to further characterize the interaction of SP-D with M.tb, and determine the effects of agglutination on bacterial uptake by human monocyte-derived macrophages. These studies demonstrate that the binding of SP-D and CDM to M.tb is saturable and inhibited by carbohydrate competition and Ca(2+) chelation, implicating the carbohydrate recognition domain in the interaction. Fluorescence microscopy reveals that dodecameric SP-D leads to agglutination of the bacilli, whereas the trimeric CDM does not, demonstrating that the multivalent nature of SP-D is essential for agglutination of M.tb. However, preincubation of M.tb with increasing concentrations of SP-D or CDM leads to a concentration-dependent reduction in the uptake of the bacteria by macrophages, indicating that agglutination does not play a direct role in this observation. Finally, the reduced uptake of M.tb by SP-D is associated with reduced growth of M.tb in monocyte-derived macrophages. These studies provide direct evidence that the inhibition of phagocytosis of M.tb effected by SP-D occurs independently of the aggregation process.

Induction of Macrophage Matrix Metalloproteinase Biosynthesis by Surfactant Protein D

Recent studies strongly suggest that surfactant protein D (SP-D) plays important roles in pulmonary host defense and the regulation of immune and inflammatory reactions in the lung. Although SP-D can bind to alveolar macrophages and can elicit their chemotaxis, relatively little is known about the direct cellular consequences of SP-D on the function of these cells. Because matrix metalloproteinases (MMPs) are synthesized in increased amounts in response to various proinflammatory stimuli, we investigated the capacity of SP-D to modulate the production of MMPs by freshly isolated human alveolar macrophages. Unexpectedly we found that recombinant rat SP-D dodecamers selectively induce the biosynthesis of collagenase-1 (MMP-1), stromelysin (MMP-3), and macrophage elastase (MMP-12) without significantly increasing the production of tumor necrosis factor alpha and interleukin-1beta. SP-D did not alter the production of these MMPs by fibroblasts. Phosphatidylinositol, a surfactant-associated ligand that interacts with the carboxyl-terminal neck and carbohydrate recognition domains of SP-D, inhibited the SP-D-dependent increase in MMP biosynthesis. A trimeric, recombinant protein consisting of only the neck and carbohydrate recognition domain did not augment metalloproteinase production, suggesting that the stimulatory effect on MMP production depends on an appropriate spatial presentation of trimeric lectin domains. Although SP-D dodecamers can selectively augment metalloproteinase activity in vitro, this effect may be competitively inhibited by tissue inhibitors of metalloproteinases or surfactant-associated ligands in vivo.

Activity of Pulmonary Surfactant Protein-D (SP-D) in Vivo Is Dependent on Oligomeric Structure

Pulmonary surfactant protein-D (SP-D) is a member of the collectin family of C-type lectins that is synthesized in many tissues including respiratory epithelial cells in the lung. SP-D is assembled predominantly as dodecamers consisting of four homotrimeric subunits each. Association of these subunits is stabilized by interchain disulfide bonds involving two conserved amino-terminal cysteine residues (Cys-15 and Cys-20). Mutant recombinant rat SP-D lacking these residues (RrSP-Dser15/20) is secreted in cell culture as trimeric subunits rather than as dodecamers. In this study, transgenic mice that express this mutant were generated to elucidate the functional importance of SP-D oligomerization in vivo. Expression of RrSP-Dser15/20 failed to correct the pulmonary phospholipid accumulation and emphysema characteristic of SP-D null (mSP-D-/-) mice. Expression of high concentrations of the mutant protein in wild-type mice reduced the abundance of disulfide cross-linked oligomers of endogenous SP-D in the bronchoalveolar lavage fluid and demonstrated a phenotype that partially overlapped with that of the SP-D-/- mice; the animals developed emphysema and foamy macrophages without the associated abnormalities in alveolar phospholipids typical of SP-D-/- mice. Development of foamy macrophages in SP-D-deficient mice is not secondary to the increased abundance of surfactant phospholipids. Disulfide cross-linked SP-D oligomers are required for the regulation of surfactant phospholipid homeostasis and the prevention of emphysema and foamy macrophages in vivo.

Microfibril-associated Protein 4 Is Present in Lung Washings and Binds to the Collagen Region of Lung Surfactant Protein D

We have purified a glycoprotein from bovine lung washings using affinity chromatography on a maltose-affinity column. On SDS-polyacrylamide gel electrophoresis the protein showed a molecular mass of 36 kDa in the reduced state and 66 kDa in the unreduced state. On gel permeation chromatography the apparent molecular mass was 250 kDa. N-terminal sequencing showed homology to the human matrix protein microfibril-associated protein (hMFAP4), and the glycoprotein was designated bovine MFAP4 (bMFAP4). Lung surfactant protein D (SP-D) was also purified from lung washings, and calcium-dependent binding was demonstrated between bMFAP4 and SP-D. hMFAP4 was cloned, and recombinant hMFAP4 showed the same binding pattern to SP-D as bMFAP4. No binding was seen to recombinant SP-D composed of the neck region and carbohydrate recognition domain of SP-D, indicating that the interaction between MFAP4 and SP-D is mediated via the collagen region of SP-D. MFAP4 also showed calcium-dependent binding to mannan, which was partially inhibited by maltose. Our findings indicate that MFAP4 has two binding specificities, one for collagen and one for carbohydrate, and we suggest that MFAP4 may fix the collectins in the extracellular compartment during inflammation.

A recombinant trimeric surfactant protein D carbohydrate recognition domain inhibits respiratory syncytial virus infection in vitro and in vivo.

The pulmonary collectin, lung surfactant protein D (SP-D), plays a role in host defense mediated by the interaction of surface carbohydrates of inhaled pathogens with the lectin domains of SP-D. Respiratory syncytial virus (RSV), the most important viral pathogen of neonates and infants, encodes a highly glycosylated attachment protein, G. Binding studies were performed with G protein from RSV (human, A2 strain) and both native and recombinant human SP-D. The effect of recombinant trimeric SP-D lectin domains (rSP-D) on the interaction between RSV and host cells was determined by two methods: an infectivity study with monolayers of Hep-2C cells and in vivo infections in BALB/c mice. These studies show that full-length and recombinant SP-D bind to RSV G protein in a concentration-dependent manner. Both EDTA and mannan inhibited binding of full-length SP-D. These results indicate that binding occurs via the carbohydrate recognition domain of the SP-D. The recombinant SP-D inhibited RSV infectivity in cell culture in a dose-dependent manner, giving 100% inhibition of replication. Intranasal administration of recombinant SP-D to RSV-infected mice inhibited replication of the virus in the lungs, reducing levels of lung virus by 80%. These results suggest that SP-D plays a major role in clearing RSV from the lungs.

Recombinant SP-D carbohydrate recognition domain is a chemoattractant for human neutrophils.

Human pulmonary surfactant protein D (SP-D) is a collagenous C-type lectin with high binding specificity to alpha-D-glucosyl residues. It is composed of four regions: a short NH2-terminal noncollagen sequence, a collagenous domain, a short linking domain ("neck" region), and a COOH-terminal carbohydrate recognition domain (CRD). Previous studies demonstrated that SP-D is chemotactic for inflammatory cells. To test which domain of SP-D might play a role in this function, a mutant that contains only neck and CRD regions was expressed in Escherichia coli and purified by affinity chromatography on maltosyl-agarose. A 17-kDa recombinant SP-D CRD was identified by two antibodies (antisynthetic SP-D COOH-terminal and neck region peptides) but not by synthetic SP-D NH2-terminal peptide antibody. The recombinant SP-D CRD was confirmed by amino acid sequencing. Gel-filtration analysis found that 84% of CRD was trimeric and the rest was monomeric. Analysis of the chemotactic properties of the trimeric CRD demonstrated that the CRD was chemotactic for neutrophils (polymorphonuclear leukocytes), with peak activity at 10(-10) M equal to the positive control [formyl-Met-Leu-Phe (fMLP) at 10(-8) M]. The chemotactic activity was abolished by 20 mM maltose, which did not suppress the chemotactic response to fMLP. The peak chemotactic activity of the CRD is comparable to the activity of native SP-D, although a higher concentration is required for peak activity (10(-10) vs. 10(-11) M). The chemotactic response to CRD was largely prevented by preincubation of polymorphonuclear leukocytes with SP-D, and the response to SP-D was prevented by preincubation with CRD. These preincubations did not affect chemotaxis to fMLP. These results suggest that trimeric CRD accounts for the chemotactic activity of SP-D.

Structure of a truncated human surfactant protein D is less effective in agglutinating bacteria than the native structure and fails to inhibit haemagglutination by influenza A virus.

Surfactant protein D (SP-D) is a lung-specific protein that is synthesized and secreted by lung epithelial cells and is believed to play an important role in lung host defence. This protein belongs to the C-type lectin family, which is characterized by an N-terminal cysteine-rich domain, a collagen-like domain, a neck domain and a carbohydrate recognition domain (CRD). To elucidate the biological actions of this animal lectin against such pathogens as micro-organisms, the biological activities of a recombinant partial SP-D lacking a collagen-like domain were examined. A recombinant human SP-D, consisting of a short collagen region (two repeats of Gly-Xaa-Yaa amino acid sequences), the neck domain and the CRD, was expressed in Escherichia coli. The recombinant SP-D was purified on a nickel column and then on a maltose-agarose column. This protein can form a trimeric structure owing to the neck domain and exhibits sugar-binding activity and specificity similar to those of native human SP-D. The recombinant SP-D caused dose-dependent and calcium-dependent agglutination of E. coli Y1088. The agglutination titre (the concentration required to achieve a 50% decrease in light transmission by agglutination) of recombinant SP-D was approx. 6-fold that of native SP-D. As for conglutination, the recombinant trimeric conglutinin required 8-16-fold higher concentrations than the native counterpart. In haemagglutination inhibition (HI) of influenza A virus, although native and recombinant conglutinin showed similar levels of HI activity, the recombinant SP-D was unable to inhibit haemagglutination, even at a concentration approx. 120-fold that of the native SP-D. The lectin precipitation and lectin blot assays showed that the truncated SP-D could bind to influenza A virus as well as native SP-D did. These results indicate that the agglutination activity of trimeric collectins can be largely retained, and furthermore that the oligomeric structure with several hands at opposite sites can enhance agglutination activity. The difference in HI activity against influenza A virus between native and recombinant SP-D suggests that SP-D uses a different mechanism from that of conglutinin to inhibit viral haemagglutination.

Interactions of recombinant human pulmonary surfactant protein D and SP-D multimers with influenza A.

To further study the structure and function of surfactant protein D (SP-D), recombinant human SP-D (rhSP-D) was isolated from the culture medium of Chinese hamster ovary (CHO)-K1 cells stably transfected with a full-length hSP-D cDNA. Although a significant fraction of the secreted rhSP-D was recovered as dodecamers similar to recombinant rat SP-D (rrSP-D), a major fraction accumulated as multimers of dodecamers indistinguishable from human proteinosis SP-D. As previously shown for the rat protein, rhSP-D agglutinated specific strains of influenza A virus (IAV), inhibited viral hemagglutinin activity, and protected neutrophils (PMN) from deactivation by IAV. However, the potency of rhSP-D multimers was severalfold greater than for purified dodecamers, comparable to natural, proteinosis hSP-D. Although rhSP-D multimers were also more potent than the serum collectins in mediating viral aggregation and protection of PMN, they were less potent than conglutinin in inhibiting infectivity in vitro. These studies establish that the propensity of hSP-D to form multimers of dodecamers is determined by its primary structure and demonstrate carbohydrate recognition domain valency-dependent interactions of SP-D with IAV.

Site-directed Mutagenesis of Cys-15 and Cys-20 of Pulmonary Surfactant Protein D: EXPRESSION OF A TRIMERIC PROTEIN WITH ALTERED ANTI-VIRAL PROPERTIES

Surfactant protein D (SP-D) molecules are preferentially assembled as dodecamers consisting of trimeric subunits associated at their amino termini. The NH2-terminal sequence of each monomer contains two conserved cysteine residues, which participate in interchain disulfide bonds. In order to study the roles of these residues in SP-D assembly and function, we employed site-directed mutagenesis to substitute serine for cysteine 15 and 20 in recombinant rat SP-D (RrSP-D), and have expressed the mutant (RrSP-Dser15/20) in Chinese hamster ovary (CHO-K1) cells. The mutant, which was efficiently secreted, bound to maltosyl-agarose, but unlike RrSP-D, was assembled exclusively as trimers. The constituent monomers showed a decreased mobility on SDS-polyacrylamide gel electrophoresis resulting from an increase in the size and sialylation of the N-linked oligosaccharide at Asn-70. Although RrSP-Dser15/20 contained a pepsin-resistant triple helical domain, it showed a decreased Tm, and acquired susceptibility to proteolytic degradation. Like RrSP-D, RrSP-Dser15/20 bound to the hemagglutinin of influenza A. However, it showed no viral aggregation and did not enhance the binding of influenza A to neutrophils (PMN), augment PMN respiratory burst, or protect PMNs from deactivation. These studies indicate that amino-terminal disulfides are required to stabilize dodecamers, and support our hypothesis that the oligomerization of trimeric subunits contributes to the anti-microbial properties of SP-D.

Pulmonary surfactant protein D in sera and bronchoalveolar lavage fluids.

Pulmonary surfactant protein D (SP-D) is a hydrophilic glycoprotein with a reduced molecular mass of 43 kDa and a member of the C-type lectin superfamily, along with mannose-binding proteins and surfactant protein A (SP-A). We have recently prepared monoclonal antibodies against human SP-D and developed an enzyme-linked immunosorbent assay (ELISA). In this study, the levels of SP-D in sera and bronchoalveolar lavage (BAL) fluids of patients with lung diseases were determined by ELISA, using human recombinant SP-D as a standard. We demonstrated that the concentrations of SP-D in sera are prominently increased in patients with idiopathic pulmonary fibrosis (IPF), interstitial pneumonia with collagen disease (IPCD), and pulmonary alveolar proteinosis (PAP). Patients with IPF, IPCD, and PAP exhibited levels of serum SP-D 5.1-fold, 7.2-fold, and 7.0-fold, respectively, of those in healthy volunteers; 91.5% of the patients with IPF, 81.3% with IPCD, and 100% with PAP exhibited serum SP-D levels that exceeded the cut-off value (mean + 2 SD of control value). Serum SP-D levels appeared to reflect the disease activity of IPF and IPCD and the disease severity of PAP. High levels of SP-D in BAL fluids were shown in patients with PAP, but not with IPF and IPCD. We conclude that measurement of SP-D in sera can provide an easily identifiable and useful clinical marker for the diagnosis of IPF, IPCD, and PAP, and can predict the disease activity of IPF and IPCD and the disease severity of PAP.

The Role of the Amino-terminal Domain and the Collagenous Region in the Structure and the Function of Rat Surfactant Protein D

Surfactant protein D (SP-D) is a member of the C-type lectin superfamily with four distinct structural domains: an amino terminus involved in forming intermolecular disulfides, a collagen-like domain, a neck region, and a carbohydrate recognition domain. A collagen domain deletion mutant (CDM) of SP-D was created by site-directed mutagenesis. A second variant lacking both the amino-terminal region and the collagen-like domain was generated by collagenase treatment and purification of the collagenase-resistant fragment (CRF). The CDM expressed in CHO-K1 cells formed the covalent trimers, but not the noncovalent dodecamers, typical of native SP-D. The CRF derived from recombinant SP-D formed only monomers. The CDM bound mannose-Sepharose and phosphatidylinositol (PI) as well as SP-D, but the binding to mannosyl bovine serum albumin and glucosylceramide was diminished by approximately 60%. The CRF displayed weak binding to mannose-Sepharose and PI and essentially no binding to mannosyl bovine serum albumin and glucosylceramide. Both SP-D and CDM altered the self-aggregation of PI-containing liposomes. SP-D reduced the density and the light scattering properties of PI aggregates. These results demonstrate that the collagen-like domain is required for dodecamer but not covalent trimer formation of SP-D and plays an important, but not essential, role in the interaction of SP-D with PI and GlcCer. Removal of the amino-terminal domain of SP-D along with the collagen-like domain diminishes PI binding and effectively eliminates GlcCer binding.

Altered Carbohydrate Recognition Specificity Engineered into Surfactant Protein D Reveals Different Binding Mechanisms for Phosphatidylinositol and Glucosylceramide

Pulmonary surfactant protein D (SP-D) is a member of the collection subgroup of the C-type lectin superfamily that binds glycosylated lipids such as phosphatidylinositol (PI) and glucosylceramide (GlcCer). We have previously reported that the carbohydrate recognition domain of SP-D plays an essential role in lipid binding. However, it is unclear how the carbohydrate binding property of SP-D contributes to the lipid binding. To clarify the relationship between the lectin property and the lipid binding activity of rat SP-D, we expressed wild-type recombinant rat SP-D (rSP-D) and a mutant form of the protein with substitutions Glu-321-->Gln and Asn-323-->Asp (SP-DE321Q,N323D) in CHO-K1 cells. The indicated mutations have previously been shown to change the carbohydrate binding specificity of surfactant protein A and mannose-binding protein from mannose > galactose to the converse. rSP-D expressed in mammalian cells was essentially identical to native rat SP-D in its lipid and carbohydrate binding properties. In contrast, SP-DE321Q,N323D was unable to bind GlcCer, but retained binding activity toward PI liposomes and solid-phase PI. The efficiency of SP-DE321Q,N323D binding to PI liposome was approximately 50% of that of rSP-D in the presence of 5 mM Ca2+, but equivalent at 20 mM Ca2+. Carbohydrates competed for SP-D binding to PI such that maltose > galactose for rSP-D, and the order was reversed for SP-DE321Q,N323D. Furthermore, SP-DE321Q,N323D could bind to digalactosyldiacylglycerol more effectively than rSP-D. These results suggest the following. 1) The carbohydrate binding specificity of SP-DE321Q,N323D was changed from a mannose-glucose type to a galactose type; 2) the GlcCer binding property of SP-D is closely related to its sugar binding activity; and 3) the PI binding activity is not completely dependent on its carbohydrate binding specificity.

Interactions of pulmonary surfactant protein D (SP-D) with human blood leukocytes.

Surfactant protein D (SP-D) is believed to contribute to nonimmune host defense within the alveoli and distal airways of the lung. SP-D molecules can bind to specific carbohydrates on the surface of bacterial, fungal, and viral organisms and can also interact with membrane glycoconjugates expressed by alveolar macrophages. Because neutrophils (PMN) and monocytes are recruited into the airspaces in association with many types of infection or lung injury, we examined the interactions of these cells with purified natural and recombinant SP-Ds, using a modified Boyden chamber assay and checkerboard analysis. Natural or recombinant rat SP-D (approximately 10(-9) to 10(-13) M) showed dose-dependent effects on human PMN and monocyte migration with a maximal response at a SP-D concentration of 5 ng/ml (approximately 10(-11) M). The migratory response was comparable to that obtained with the optimum concentration of FMLP (10(-8) M). HL-60 cells, after induction of differentiation with DMSO, responded to SP-D with the same dose-response as neutrophils. The effects of SP-D were abrogated by the simultaneous addition of SP-D to the upper chamber or by the addition of antibodies to the carboxy-terminal lectin domain. Migration toward SP-D was markedly inhibited (< 10% of controls) by 10 mM maltose but was not significantly inhibited by to 50 mM lactose. These studies establish that SP-D can bind to specific sites on neutrophils and monocytes and strongly suggest that these interactions involve the saccharide binding domains of SP-D.

Evidence for a protective role of pulmonary surfactant protein D (SP-D) against influenza A viruses.

We tested the hypothesis that pulmonary surfactant-associated lectins--surfactant proteins A and D (SP-A, and -D)--contribute to initial protective mechanisms against influenza A viruses (IAVs). SP-D potently inhibited hemagglutination activity of several strains of IAV as well as causing viral aggregation. SP-D enhanced neutrophil binding of IAV and neutrophil respiratory burst responses to the virus. Neutrophil dysfunction resulting from IAV exposure was diminished when the virus was pre-incubated with SP-D. Each of these effects was mediated by the calcium-dependent carbohydrate-binding property of SP-D. Native SP-D preparations of both human and rat origin, as well as recombinant rat SP-D, had similar activity. SP-A also inhibited IAV hemagglutination activity. We have previously reported that related mammalian serum lectins (mannose-binding lectin [MBL] and conglutinin) have similar effects. SP-D was at least 10-fold more potent at causing hemagglutination inhibition than were SP-A or MBL. SP-D was shown to contribute to potent anti-IAV activity of human bronchoalveolar lavage fluid. These results suggest that SP-D--alone, and in conjunction with SP-A and phagocytic cells--constitutes an important component of the natural immune response to IAV infection within the respiratory tract.