Purification of a Cell-surface Receptor for Surfactant Protein A

Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 08/1996; 271(27):16375-83. DOI: 10.1074/jbc.271.27.16375
Source: PubMed


In the present report we have characterized the binding of surfactant protein A (SP-A) to bone marrow-derived macrophages, U937 cells, alveolar macrophages, and type II epithelial cells. The binding of SP-A to all cell types is Ca2+-dependent and trypsin-sensitive, but type II cells express distinct Ca2+-independent binding sites. The binding of SP-A to macrophages is independent of known cell surface carbohydrate-specific receptors and of glycoconjugate binding sites on the surface of the cells and is distinct from binding to C1q receptors. Based on ligand blot analysis, both type II cells and macrophages express a 210-kDa SP-A-binding protein. The 210-kDa protein was purified to apparent homogeneity from U937 macrophage membranes using affinity chromatography with noncovalently immobilized surfactant protein A, and was purified from rat lung by differential detergent and salt extraction of isolated rat lung membranes. Polyclonal antibodies against the rat lung SP-A-binding protein inhibit binding of SP-A to both type II cells and macrophages, indicating that the 210-kDa protein is expressed on the cell surface. The polyclonal antibodies also block the SP-A-mediated inhibition of phospholipid secretion by type II cells, indicating that the 210-kDa protein is a functional cell-surface receptor on type II cells. In a separate report we have determined that antibodies to the SP-A receptor block the SP-A-mediated uptake of Mycobacterium bovis, indicating that the macrophage SP-A receptor is involved in SP-A-mediated clearance of pathogens.

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    • "For example, C1q receptor mediates the phagocytosis of SP-A-opsonized Staphylococcus aureus (S. aureus) by monocytes [29]. SP-A-opsonized S. aureus [30] and SP-A-opsonized bacillus Calmette-Guérin (BCG) [31] are ingested by macrophage via SP-R210, which is a specific receptor for SP-A [32]. Pulmonary collectins stimulate phagocytosis of Pseudomonas aeruginosa (P. "
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    ABSTRACT: Pulmonary surfactant is a mixture of lipids and proteins that covers alveolar surfaces and keeps alveoli from collapsing. Four specific proteins have been identified in surfactant. Among them, two C-type lectins, surfactant proteins A and D (SP-A and SP-D), are known to be implicated in host defense and regulation of inflammatory responses of the lung. These host defense lectins are structurally characterized by N-terminal collagen-like domains and lectin domains and are called pulmonary collectins. They prevent dissemination of infectious microbes by their biological activities including agglutination and growth inhibition. They also promote clearance of microbes by enhancing phagocytosis in macrophages. In addition, they interact with the other pattern-recognition molecules, including Toll-like receptors (TLRs) and TLR-associated molecules, CD14 and MD-2, and regulate inflammatory responses. Furthermore, recent studies have demonstrated that these collectins modulate functions of neutrophil-derived innate immune molecules by interacting with them. These findings indicate that pulmonary collectins play critical roles in host defense of the lung.
    BioMed Research International 05/2012; 2012(3):532071. DOI:10.1155/2012/532071 · 2.71 Impact Factor
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    • "More recent studies using mycobacterial components have suggested that mycobacteria might interact with toll-like receptors (TLRs) on the macrophage surface [26,27,57,58]. We have suggested previously that SP-A redirects mycobacteria to interact with the SP-A-specific receptor SPR210 [31,59]. Anti-SPR210 antibodies block SP-A binding, inhibit ingestion of SP-A-BCG complexes, and reduce SP-A-BCG-mediated production of nitric oxide. "
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    ABSTRACT: Surfactant protein A (SP-A) is a C-type lectin involved in surfactant homeostasis as well as host defense in the lung. We have recently demonstrated that SP-A enhances the killing of bacillus Calmette-Guerin (BCG) by rat macrophages through a nitric oxide-dependent pathway. In the current study we have investigated the role of tyrosine kinases and the downstream mitogen-activated protein kinase (MAPK) family, and the transcription factor NFkappaB in mediating the enhanced signaling in response to BCG in the presence of SP-A. Human SP-A was prepared from alveolar proteinosis fluid, and primary macrophages were obtained by maturation of cells from whole rat bone marrow. BCG-SP-A complexes were routinely prepared by incubation of a ratio of 20 microg of SP-A to 5 x 105 BCG for 30 min at 37 degrees C. Cells were incubated with PBS, SP-A, BCG, or SP-A-BCG complexes for the times indicated. BCG killing was assessed using a 3H-uracil incorporation assay. Phosphorylated protein levels, enzyme assays, and secreted mediator assays were conducted using standard immunoblot and biochemical methods as outlined. Involvement of tyrosine kinases was demonstrated by herbimycin A-mediated inhibition of the SP-A-enhanced nitric oxide production and BCG killing. Following infection of macrophages with BCG, the MAPK family members ERK1 and ERK2 were activated as evidence by increased tyrosine phosphorylation and enzymatic activity, and this activation was enhanced when the BCG were opsonized with SP-A. An inhibitor of upstream kinases required for ERK activation inhibited BCG- and SP-A-BCG-enhanced production of nitric oxide by approximately 35%. Macrophages isolated from transgenic mice expressing a NFkappaB-responsive luciferase gene showed increased luciferase activity following infection with BCG, and this activity was enhanced two-fold in the presence of SP-A. Finally, lactacystin, an inhibitor of IkappaB degradation, reduced BCG- and SP-A-BCG-induced nitric oxide production by 60% and 80% respectively. These results demonstrate that BCG and SP-A-BCG ingestion by macrophages is accompanied by activation of signaling pathways involving the MAP kinase pathway and NFkappaB.
    Respiratory research 08/2009; 10(1):60. DOI:10.1186/1465-9921-10-60 · 3.09 Impact Factor
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    • "), which has been initially purified from U937 cells and rat lung as a 210 kDa SP-A binding protein (Chroneos et al., 1996). SP-R210 is expressed on macrophages and alveolar type II cells (Chroneos et al., 1996) and its antibody attenuates the SP-A-stimulated uptake of BCG by macrophages (Weikert et al., 1997). Pulmonary collectins have been shown to bind gp-340 (Holmskov et al., 1997; Tino and Wright, 1999), which is a member of the macrophage scavenger receptor family. "
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    ABSTRACT: Pulmonary collectins, hydrophilic surfactant proteins A and D (SP-A and SP-D), have been implicated in the regulation of pulmonary host defence and inflammation. SP-A and SP-D directly interact with a variety of microorganisms including bacteria and viruses, and attenuate the growth of Gram-negative bacteria, Histoplasma capsulatum and Mycoplasma pneumoniae. The collectins are thought to contribute to bacterial clearance. These lectins augment the phagocytosis of the bacteria by macrophages. SP-A serves as an opsonin and stimulates the uptake of bacteria and bacillus Calmette-Guérin through a C1q receptor- and an SP-R210-mediated processes. The collectin also stimulates FcR- and CR1-mediated phagocytosis by activating the macrophages. In addition, SP-A and SP-D directly interact with macrophages and enhance the phagocytosis of Streptococcus pneumoniae and Mycobacterium by increasing cell surface localization of the phagocytic receptors, scavenger receptor A and mannose receptor. The collectins also modulate pulmonary inflammation. SP-A and SP-D bind to cell surface receptors including Toll-like receptors, SIRPalpha and calreticulin/CD91, and attenuate or enhance inflammation in a microbial ligand-specific manner. In this article we review the immunomodulatory functions of SP-A and SP-D and their possible mechanisms in direct actions on microbes, macrophage phagocytosis and modulation of inflammation.
    Cellular Microbiology 09/2007; 9(8):1871-9. DOI:10.1111/j.1462-5822.2007.00953.x · 4.92 Impact Factor
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