The lung collectin surfactant protein A (SP-A) has been implicated in the regulation of pulmonary host defense and inflammation. Zymosan induces proinflammatory cytokines in immune cells. Toll-like receptor (TLR)2 has been shown to be involved in zymosan-induced signaling. We first investigated the interaction of TLR2 with zymosan. Zymosan cosedimented the soluble form of rTLR2 possessing the putative extracellular domain (sTLR2). sTLR2 directly bound to zymosan with an apparent binding constant of 48 nM. We next examined whether SP-A modulated zymosan-induced cellular responses. SP-A significantly attenuated zymosan-induced TNF-α secretion in RAW264.7 cells and alveolar macrophages in a concentration-dependent manner. Although zymosan failed to cosediment SP-A, SP-A significantly reduced zymosan-elicited NF-κB activation in TLR2-transfected human embryonic kidney 293 cells. Because we have shown that SP-A binds to sTLR2, we also examined whether SP-A affected the binding of sTLR2 to zymosan. SP-A significantly attenuated the direct binding of sTLR2 to zymosan in a concentration-dependent fashion. From these results, we conclude that 1) TLR2 directly binds zymosan, 2) SP-A can alter zymosan-TLR2 interaction, and 3) SP-A down-regulates TLR2-mediated signaling and TNF-α secretion stimulated by zymosan. This study supports an important role of SP-A in controlling pulmonary inflammation caused by microbial pathogens.

Surfactant proteins A (SP-APulmonary surfactant is a complex mixture of lipids and proteins that functions to keep alveoli from collapsing at the end of expiration (1). Surfactant proteins A (SP-A) 1 and D (SP-D) are glycoprotein constituents of lung surfactant (2). SP-A and SP-D belong to the collectin subgroup of the C-type lectin superfamily along with mannose-binding proteins A and C, conglutinin, and CL43 (3). These proteins possess similar characteristic structures consisting of a short intersubunit disulfide forming the NH 2 terminus region, a collagen-like domain, a coiled-coil motif neck domain, and a carbohydrate recognition domain (CRD) (2). The CRD region of SP-A is essential for dipalmitoylphosphatidylcholine and galactosylceramide binding, liposome aggregation, the inhibitory effect on lipid secretion, and the augmentation of lipid uptake by alveolar type II cells (4 -11). Likewise, the CRD region of SP-D functions in the recognition of the ligands phosphatidylinositol and glucosylceramide (12,13). In addition to their interaction with lipids and alveolar type II cells, lung collectins interact with macrophages (14 -16) and enhance phagocytosis of a wide spectrum of microorganism (17-25). Lung collectins are now thought to be important components of the innate immune system of the lung (26 -28).Studies with transgenic mice provide strong support for a role of pulmonary collectins in host defense properties. Mice homozygous for null alleles of SP-A exhibit increased susceptibility to group B streptococcal and Pseudomonas aeruginosa infections (29 -31). These mice clear the bacteria from the lungs at a slower rate than wild-type mice. Phagocytosis of P. aeruginosa by alveolar macrophages in SP-A Ϫ/Ϫ mice is also significantly decreased (30). Coadministration of SP-A with bacteria into the airway of SP-A Ϫ/Ϫ mice enhance phagocytosis of the bacteria by alveolar macrophages. Secretion of proinflammatory cytokines into the alveolar space is significantly elevated in SP-A Ϫ/Ϫ mice compared with SP-A ϩ/ϩ mice after intratracheal challenge with P. aeruginosa. These studies suggest that SP-A modulates innate immune responses by several different mechanisms. Although these in vivo studies explicitly indicate that SP-A plays a crucial role in host defense of the lung, the molecular basis of SP-A-mediated modification of inflammatory responses remains to be elucidated.Lipopolysaccharide (LPS), derived from Gram-negative bacteria, is a potent stimulator of inflammation (32). Smooth LPS is composed of O-antigen, core oligosaccharides, and lipid A, while rough LPS lacks O-antigen and a part of the core oligosaccharides (33). The cellular responses to physiological amounts of LPS depend on membrane CD14 that is phosphatidylinositol-anchored to the plasma membrane of myeloid cells (34). A soluble form of CD14 which exists in serum also facilitates the responsiveness of the cells to LPS (35,36). The principal role of CD14 is to bind LPS, but how CD14 acts in transmitting LPS signal remains to be resolved. Recently, ...

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Pulmonary surfactant protein A (SP-A

Toll-like receptor 2 (TLR2) has been recognized to mediate cell signaling in response to peptidoglycan (PGN), a major cell wall component of Gram-positive bacteria. The mechanism by which TLR2 recognizes PGN is unknown. It is not even clear whether TLR2 directly binds to PGN. In this study, we generated a soluble form of recombinant TLR2 (sTLR2) possessing only its putative extracellular domain by using the baculovirus expression system to examine the direct interaction between sTLR2 and PGN. sTLR2 bound avidly to insoluble PGN (iPGN) from Staphylococcus aureus coated onto microtiter wells in a concentration-dependent manner. In contrast, sTLR2 exhibited a very weak binding to lipopolysaccharide. iPGN cosedimented sTLR2 after the mixture of iPGN and sTLR2 had been incubated and centrifuged. sTLR2 partially attenuated the iPGN-induced NF-B activation in TLR2-transfected HEK 293 cells and the iPGN-induced IL-8 secretion in U937 cells. One of anti-human TLR2 monoclonal antibodies, which blocked iPGN-induced NF-B activation in TLR2-transfected cells, inhibited the binding of sTLR2 to iPGN. In addition, we found that sCD14 interacted with sTLR2 and increased the binding of sTLR2 to iPGN. From these results, we conclude that the extracellular TLR2 domain directly binds to PGN.

Long-term prognosis of Henoch-Schönlein purpura is dependent on the severity of renal involvement. In those patients who have the risk factors of renal involvement, especially significant proteinuria, close attention should be paid to a urinalysis for at least 3 months from the onset of the disease.

The purpose of the current study was to examine the binding of pulmonary surfactant protein A (SP-A) to TLR4 and MD-2, which are critical signaling receptors for lipopolysaccharides ( In innate immune systems, toll-like receptors (TLRs) 2 are implicated in recognition and signaling of pathogen-associated molecular patterns (1). Stimulation of different TLRs induces distinct patterns of gene expression, which leads to the activation of innate immunity and instructs the development of antigen-specific acquired immunity (2). Among the TLR family, TLR4 plays a critical role in recognition and signaling of bacterial lipopolysaccharide (LPS) (3). TLR4 requires accessory protein MD-2 for an efficient response to LPS (4). We have recently demonstrated the direct interaction between MD-2 and extracellular TLR4 domain (5, 6). MD-2 binds LPS (7), but LPS has been demonstrated to be cross-linked with TLR4 and MD-2 only when coexpressed with CD14 (8), suggesting that LPS is in close proximity to the receptor complex.The lung is constantly challenged by inhaled pathogens, pollutants, and particles that are present in the environment. Pulmonary surfactant, a mixture of lipids and proteins that serves to reduce the surface tension of the alveoli, is involved in the innate immune system of the lung. Recent studies demonstrate that the most abundant component of surfactant protein, surfactant protein A (SP-A), plays important roles in pathogen clearance and inflammatory responses (9 -12). SP-A belongs to the collectin subgroup of the C-type lectin superfamily along with surfactant protein D (SP-D) and mannose-binding lectin. The primary structure of SP-A subunits are composed of a short amino-terminal segment, a collagen-like sequence characterized by Gly-X-Y repeats with an interruption near the midpoint of the domain, a neck domain, and a carbohydrate recognition domain (CRD) (13). Trimeric association occurs by the folding of collagenous domains into triple helices (14) and coiled-coil bundling of ␣-helices in the neck (15). Fully assembled SP-A is a bouquet-like octadecamer consisting of six trimeric subunits that are stabilized by the amino-terminal sequences and disulfide bonds (16).Recent studies from this and other laboratories have demonstrated that SP-A modulates inflammation by interacting with cell surface receptors including CD14 (17), TLR2 (18, 19), signal-inhibitory regulatory protein ␣, and calreticulin/CD91 (20). Although it has been suggested that SP-A activates cellular responses dependent on TLR4 (21), the interactions of SP-A * This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture, Japan and by Akiyama Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.

Pulmonary surfactant protein D (SP-D), a member of the collectin group of innate immune proteins, plays important roles in lipopolysaccharide (LPS) recognition. We have previously shown that surfactant protein A (SP-A), a homologous collectin, interacts with Toll-like receptor (TLR) 2, resulting in alteration of TLR2-mediated signaling. In this study, we found that natural and recombinant SP-Ds exhibited specific binding to the extracellular domains of soluble forms of recombinant TLR2 (sTLR2) and TLR4 (sTLR4). Binding was concentration- and Ca2+-dependent, and SP-D bound to N-glycosidase F-treated sTLRs on ligand blots. Anti-SP-D monoclonal antibody 7A10 blocked binding of SP-D to sTLR2 and sTLR4, but there was no inhibitory effect of monoclonal 7C6. Epitope mapping with recombinant proteins consisting of the carbohydrate recognition domain (CRD) and the neck domain plus CRD (NCRD) localized binding sites for 7A10 and 7C6 to sequential epitopes associated with the CRD and the neck domain, respectively. Interactions with 7A10 but not 7C6 were blocked by prior binding of the NCRD to sTLRs. Although antibody 7A10 significantly inhibited the binding of SP-D to its major surfactant-associated ligand, phosphatidylinositol (PI), and Escherichia coli Rc LPS, 7C6 enhanced binding to both molecules. An SP-D(E321Q, N323D) mutant with altered carbohydrate specificity exhibited attenuated PI binding but showed an increased level of binding to sTLRs. Thus, human SP-D binds the extracellular domains of TLR2 and TLR4 through its CRD by a mechanism different from its binding to PI and LPS.