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.

Three types of ficolins have been identified in humans: L-ficolin, M-ficolin, and H-ficolin. Similar to mannose-binding lectin, L-ficolin and H-ficolin are the recognition molecules in the lectin complement pathway. Another human ficolin, M-ficolin, is a nonserum ficolin that is expressed in leukocytes and lung; however, little is known about its physiologic roles. In this study, we report the characterization of M-ficolin in terms of its protein localization and lectin activity. M-ficolin was localized in secretory granules in the cytoplasm of neutrophils, monocytes, and type II alveolar epithelial cells in lung. M-ficolin precipitated with mannose-binding lectin-associated serine proteases (MASP)-1 and MASP-2 in a coimmunoprecipitation assay, indicating that M-ficolin forms complexes with MASP-1 and MASP-2. M-ficolin-MASP complexes activated complement on N-acetylglucosamine (GlcNAc)-coated microplates in a C4 deposition assay. M-ficolin bound to several neoglycoproteins bearing GlcNAc, N-acetylgalactosamine, and sialyl-N-acetyllactosamine, suggesting that M-ficolin can recognize the common carbohydrate residues found in microbes. Indeed, M-ficolin bound to Staphylococcus aureus through GlcNAc. These results indicate that M-ficolin, like its family members, functions as a recognition molecule of the lectin complement pathway and plays an important role in innate immunity.

Complement research experienced a renaissance with the discovery of a third activation route, the lectin pathway. We developed a unique model of total lectin pathway deficiency, a mouse strain lacking mannan-binding lectin-associated serine protease-2 (MASP-2), and analyzed the role of MASP-2 in two models of postischemic reperfusion injury (IRI). In a model of transient myocardial IRI, MASP-2-deficient mice had significantly smaller infarct volumes than their wild-type littermates. Mice deficient in the downstream complement component C4 were not protected, suggesting the existence of a previously undescribed lectin pathway-dependent C4-bypass. Lectin pathway-mediated activation of C3 in the absence of C4 was demonstrated in vitro and shown to require MASP-2, C2, and MASP-1/3. MASP-2 deficiency also protects mice from gastrointestinal IRI, as do mAb-based inhibitors of MASP-2. The therapeutic effects of MASP-2 inhibition in this experimental model suggest the utility of anti-MASP-2 antibody therapy in reperfusion injury and other lectin pathway-mediated disorders.

The complement system is an essential component of innate immunity, participating in the pathogenesis of inflammatory diseases and in host defense. In the lectin complement pathway, mannose-binding lectin (MBL) and ficolins act as recognition molecules, and MBL-associated serine protease (MASP) is a key enzyme; MASP-2 is responsible for the lectin pathway activation. The function of other serine proteases (MASP-1 and MASP-3) is still obscure. In this study, we generated a MASP-1– and MASP-3–deficient mouse model (Masp1/3−/−) and found that no activation of the alternative pathway was observed in Masp1/3−/− serum. Mass spectrometric analysis revealed that circulating complement factor D (Df) in Masp1/3−/− mice is a zymogen (pro-Df) with the activation peptide QPRGR at its N terminus. These results suggested that Masp1/3−/− mice failed to convert pro-Df to its active form, whereas it was generally accepted that the activation peptide of pro-Df is removed during its secretion and factor D constitutively exists in an active form in the circulation. Furthermore, recombinant MASP-1 converted pro-Df to the active form in vitro, although the activation mechanism of pro-Df by MASP-1 is still unclear. Thus, it is clear that MASP-1 is an essential protease of both the lectin and alternative complement pathways.

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, ...