Surfactant protein A (SP-A)-mediated clearance of Staphylococcus aureus involves binding of SP-A to the staphylococcal adhesin eap and the macrophage receptors SP-A receptor 210 and scavenger receptor class A

Staphylococcus aureus causes life-threatening pneumonia in hospitals and deadly superinfection during viral influenza. The current study investigated the role of surfactant protein A (SP-A) in opsonization and clearance of S. aureus. Previous studies showed that SP-A mediates phagocytosis via the SP-A receptor 210 (SP-R210). Here, we show that SP-R210 mediates binding and control of SP-A-opsonized S. aureus by macrophages. We determined that SP-A binds S. aureus through the extracellular adhesin Eap. Consequently, SP-A enhanced macrophage uptake of Eap-expressing (Eap(+)) but not Eap-deficient (Eap(-)) S. aureus. In a reciprocal fashion, SP-A failed to enhance uptake of Eap(+) S. aureus in peritoneal Raw264.7 macrophages with a dominant negative mutation (SP-R210(DN)) blocking surface expression of SP-R210. Accordingly, WT mice cleared infection with Eap(+) but succumbed to sublethal infection with Eap- S. aureus. However, SP-R210(DN) cells compensated by increasing non-opsonic phagocytosis of Eap(+) S. aureus via the scavenger receptor scavenger receptor class A (SR-A), while non-opsonic uptake of Eap(-) S. aureus was impaired. Macrophages express two isoforms: SP-R210(L) and SP-R210(S). The results show that WT alveolar macrophages are distinguished by expression of SP-R210(L), whereas SR-A(-/-) alveolar macrophages are deficient in SP-R210(L) expressing only SP-R210(S). Accordingly, SR-A(-/-) mice were highly susceptible to both Eap(+) and Eap(-) S. aureus. The lungs of susceptible mice generated abnormal inflammatory responses that were associated with impaired killing and persistence of S. aureus infection in the lung. In conclusion, alveolar macrophage SP-R210(L) mediates recognition and killing of SP-A-opsonized S. aureus in vivo, coordinating inflammatory responses and resolution of S. aureus pneumonia through interaction with SR-A.     

Identification of the surfactant protein A receptor 210 as the unconventional myosin 18A

Mass spectrometric characterization of the surfactant protein A (SP-A) receptor 210 (SP-R210) led to the identification of myosin (Myo) XVIIIA and nonmuscle myosin IIA. Antibodies generated against the unique C-terminal tail of MyoXVIIIA revealed that MyoXVIIIA, MyoIIA, and SP-R210 have overlapping tissue distribution, all being highly expressed in myeloid cells, bone marrow, spleen, lymph nodes, and lung. Western blot analysis of COS-1 cells stably transfected with either MyoXVIIIA or MyoIIA indicated that SP-R210 antibodies recognize MyoXVIIIA. Furthermore, MyoXVIIIA but not MyoIIA localized to the surface of COS-1 cells, and most importantly, expression of MyoXVIIIA in COS-1 cells conferred SP-A binding. Western analysis of recombinant MyoXVIIIA domains expressed in bacteria mapped the epitopes of previously derived SP-R210 antibodies to the neck region of MyoXVIIIA. Antibodies raised against the neck domain of MyoXVIIIA blocked the binding of SP-A to macrophages. Together, these findings indicate that MyoXVIIIA constitutes a novel receptor for SP-A.     

Pulmonary surfactant protein A modulates the cellular response to smooth and rough lipopolysaccharides by interaction with CD14.

Pulmonary surfactant protein A (SP-A) plays an important part in Ab-independent host defense mechanisms of the lung. In this study we investigated how SP-A interacts with distinct serotypes of bacterial LPS and modulates LPS-elicited cellular responses. SP-A bound to rough forms but not to smooth forms of LPS. In the macrophage-like cell line U937, SP-A inhibited mRNA expression and secretion of TNF-alpha induced by smooth LPS, but rough LPS-induced TNF-alpha expression was unaffected by SP-A. When U937 cells and rat alveolar macrophages were preincubated with SP-A, smooth LPS failed to induce TNF-alpha secretion, whereas rough LPS-induced TNF-alpha secretion was modestly increased. To clarify the mechanism by which SP-A modulates LPS-elicited cellular responses, we further examined the interaction of SP-A with CD14, which is known as a major LPS receptor. Western blot analysis revealed that CD14 was one of the SP-A binding proteins isolated from solubilized U937 cells. In addition, SP-A directly bound to recombinant soluble CD14 (rsCD14). When rsCD14 was preincubated with SP-A, the binding of rsCD14 to smooth LPS was significantly reduced but the association of rsCD14 with rough LPS was augmented. These results demonstrate the different actions of SP-A upon distinct serotypes of LPS and indicate that the direct interaction of SP-A with CD14 constitutes a likely mechanism by which SP-A modulates LPS-elicited cellular responses.     

Surfactant protein A inhibits alveolar macrophage cytokine production by CD14-independent pathway

The lung collectin surfactant protein A (SP-A) has both anti-inflammatory and prophagocytic activities. We and others previously showed that SP-A inhibits the macrophage production of tumor necrosis factor (TNF)-alpha stimulated by the gram-negative bacterial component LPS. We propose that SP-A decreases the production of proinflammatory cytokines by alveolar macrophages via a CD14-independent mechanism. SP-A inhibited LPS-simulated TNF-alpha production in rat and mouse macrophages in the presence and absence of serum (72% and 42% inhibition, respectively). In addition, SP-A inhibited LPS-induced mRNA levels for TNF-alpha, IL-1 alpha, and IL-1 beta as well as NF-kappa B DNA binding activity. SP-A also diminished ultrapure LPS-stimulated TNF-alpha produced by wild-type and CD14-null mouse alveolar macrophages by 58% and 88%, respectively. Additionally, SP-A inhibited TNF-alpha stimulated by PMA in both wild-type and TLR4-mutant macrophages. These data suggest that SP-A inhibits inflammatory cytokine production in a CD14-independent manner and also by mechanisms independent of the LPS signaling pathway.     

Pulmonary surfactant protein A augments the phagocytosis of Streptococcus pneumoniae by alveolar macrophages through a casein kinase 2-dependent increase of cell surface localization of scavenger receptor A

Pulmonary surfactant proteins A (SP-A) and D (SP-D), members of the collectin family, play important roles in the innate immune system of the lung. Here, we show that SP-A but not SP-D augmented phagocytosis of Streptococcus pneumoniae by alveolar macrophages, independent of its binding to the bacteria. Analysis of the SP-A/SP-D chimeras, in which progressively longer carboxyl-terminal regions of SP-A were replaced with the corresponding SP-D regions, has revealed that the SP-D region Gly(346)-Phe(355) can be substituted for the SP-A region Leu(219)-Phe(228) without altering the SP-A activity of enhancing the phagocytosis and that the SP-A region Cys(204)-Cys(218) is required for the SP-A-mediated phagocytosis. Acetylated low density lipoprotein significantly reduced the SP-A-stimulated uptake of the bacteria. SP-A failed to enhance the phagocytosis of S. pneumoniae by alveolar macrophages derived from scavenger receptor A (SR-A)-deficient mice, demonstrating that SP-A augments SRA-mediated phagocytosis. Preincubation of macrophages with SP-A at 37 degrees C but not at 4 degrees C stimulated the phagocytosis. The SP-A-mediated enhanced phagocytosis was not inhibited by the presence of cycloheximide. SP-A increased cell surface localization of SR-A that was inhibitable by apigenin, a casein kinase 2 (CK2) inhibitor. SP-A-treated macrophages exhibited significantly greater binding of acetylated low density lipoprotein than nontreated cells. The SP-A-stimulated phagocytosis was also abolished by apigenin. In addition, SP-A stimulated CK2 activity. These results demonstrate that SP-A enhances the phagocytosis of S. pneumoniae by alveolar macrophages through a CK2-dependent increase of cell surface SR-A localization. This study reveals a novel mechanism of bacterial clearance by alveolar macrophages.     

Pulmonary collectins in innate immunity of the lung

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.     

Innate immune responses to bacterial ligands in the peripheral human lung--role of alveolar epithelial TLR expression and signalling

It is widely believed that the alveolar epithelium is unresponsive to LPS, in the absence of serum, due to low expression of TLR4 and CD14. Furthermore, the responsiveness of the epithelium to TLR-2 ligands is also poorly understood. We hypothesised that human alveolar type I (ATI) and type II (ATII) epithelial cells were responsive to TLR2 and TLR4 ligands (MALP-2 and LPS respectively), expressed the necessary TLRs and co-receptors (CD14 and MD2) and released distinct profiles of cytokines via differential activation of MAP kinases. Primary ATII cells and alveolar macrophages and an immortalised ATI cell line (TT1) elicited CD14 and MD2-dependent responses to LPS which did not require the addition of exogenous soluble CD14. TT1 and primary ATII cells expressed CD14 whereas A549 cells did not, as confirmed by flow cytometry. Following LPS and MALP-2 exposure, macrophages and ATII cells released significant amounts of TNFα, IL-8 and MCP-1 whereas TT1 cells only released IL-8 and MCP-1. P38, ERK and JNK were involved in MALP-2 and LPS-induced cytokine release from all three cell types. However, ERK and JNK were significantly more important than p38 in cytokine release from macrophages whereas all three were similarly involved in LPS-induced mediator release from TT1 cells. In ATII cells, JNK was significantly more important than p38 and ERK in LPS-induced MCP-1 release. MALP-2 and LPS exposure stimulated TLR4 protein expression in all three cell types; significantly more so in ATII cells than macrophages and TT1 cells. In conclusion, this is the first study describing the expression of CD14 on, and TLR2 and 4 signalling in, primary human ATII cells and ATI cells; suggesting that differential activation of MAP kinases, cytokine secretion and TLR4 expression by the alveolar epithelium and macrophages is important in orchestrating a co-ordinated response to inhaled pathogens.     

Surfactant protein A enhances apoptotic cell uptake and TGF-beta1 release by inflammatory alveolar macrophages

The phagocytosis of apoptotic inflammatory cells by alveolar macrophages (AMs) is a key component of inflammation resolution within the air space. Surfactant protein A (SP-A) has been shown to stimulate the phagocytosis of apoptotic neutrophils (PMNs) by normal AMs. We hypothesized that SP-A promotes the resolution of alveolar inflammation by enhancing apoptotic PMN phagocytosis and anti-inflammatory cytokine release by inflammatory AMs. Using an LPS lung inflammation model, we determined that SP-A stimulates the phagocytosis of apoptotic PMNs threefold by normal AMs and AMs isolated after LPS injury. Furthermore, SP-A enhances transforming growth factor-beta1 (TGF-beta1) release from both AM populations. Inflammatory AMs release twofold more TGF-beta1 in culture than do normal AMs. SP-A and apoptotic PMNs together stimulate TGF-beta1 release equivalently from normal and inflammatory cultured AMs (330% of unstimulated release by normal AMs). In summary, SP-A enhances apoptotic PMN uptake, stimulates AM TGF-beta1 release, and modulates the amount of TGF-beta1 released when AMs phagocytose apoptotic PMNs. These findings support the hypothesis that SP-A promotes the resolution of alveolar inflammation.     

Interleukin-4 inhibits lipopolysaccharide-induced expression of prostaglandin H synthase-2 in human alveolar macrophages

Several studies have shown that interleukin-4 (IL-4) down-regulates synthesis of prostaglandin E₂ (PGE₂). We evaluated the mechanisms for this suppression in human alveolar macrophages (HAMs). Normal HAMs were obtained from healthy nonsmoking volunteers. The cells either remained unstimulated, or were exposed to 10 μg/ml of lipopolysaccharide (LPS) and/or various amounts of IL-4. LPS alone induced the synthesis of large amounts of PGE₂ and prostaglandin H synthase-2 (PGHS-2) protein. This effect of LPS was suppressed by increasing amounts of IL-4. Expression of LPS-induced PGHS-2 mRNA was also inhibited by IL-4. In addition, IL-4 inhibited expression of CD14, which is a receptor for LPS bound to the LPS-binding protein (LBP). We conclude that IL-4 down-regulates LPS-induced release of PGE₂, by reducing expression of the enzyme, PGHS-2. One potential mechanism for this effect of IL-4 is a reduced expression of CD14, which is the LPS-LBP receptor. © 1995 Wiley-Liss Inc.     

The role of nitric oxide in lung innate immunity: Modulation by surfactant protein-A

Surfactant protein A (SP-A) and alveolar macrophages are essential components of lung innate immunity. Alveolar macrophages phagocytose and kill pathogens by the production of reactive oxygen and nitrogen species. In particular, peroxynitrite, the reaction product of superoxide and nitric oxide, appears to have potent antimicrobial effects. SP-A stimulates alveolar macrophages to phagocytose and kill pathogens and is important in host defense. However, SP-A has diverse effects on both innate and adaptive immunity, and may stimulate or inhibit immune function. SP-A appears to mediate toxic or protective effects depending on the immune status of the lung. In contrast to mouse or rat cells, it has been difficult to demonstrate nitric oxide production by human macrophages. We have recently demonstrated that human macrophages produce nitric oxide and use it to kill Klebsiella pneumoniae. SP-A either stimulates or inhibits this process, depending on the activation state of the macrophage. Given its diverse effects on immune function, SP-A may prove to be an effective therapy for both infectious and inflammatory diseases of the lung.     

Surfactant protein D increases phagocytosis and aggregation of pollen-allergen starch granules

Recent studies have shown that surfactant components, in particular the collectins surfactant protein (SP)-A and -D, modulate the phagocytosis of various pathogens by alveolar macrophages. This interaction might be important not only for the elimination of pathogens but also for the elimination of inhaled allergens and might explain anti-inflammatory effects of SP-A and SP-D in allergic airway inflammation. We investigated the effect of surfactant components on the phagocytosis of allergen-containing pollen starch granules (PSG) by alveolar macrophages. PSG were isolated from Dactylis glomerata or Phleum pratense, two common grass pollen allergens, and incubated with either rat or human alveolar macrophages in the presence of recombinant human SP-A, SP-A purified from patients suffering from alveolar proteinosis, a recombinant fragment of human SP-D, dodecameric recombinant rat SP-D, or the commercially available surfactant preparations Curosurf and Alveofact. Dodecameric rat recombinant SP-D enhanced binding and phagocytosis of the PSG by alveolar macrophages, whereas the recombinant fragment of human SP-D, SP-A, or the surfactant lipid preparations had no effect. In addition, recombinant rat SP-D bound to the surface of the PSG and induced aggregation. Binding, aggregation, and enhancement of phagocytosis by recombinant rat SP-D was completely blocked by EDTA and inhibited by d-maltose and to a lesser extent by d-galactose, indicating the involvement of the carbohydrate recognition domain of SP-D in these functions. The modulation of allergen phagocytosis by SP-D might play an important role in allergen clearance from the lung and thereby modulate the allergic inflammation of asthma.     

Pulmonary surfactant protein A up-regulates activity of the mannose receptor,a pattern recognition receptor expressed on human macrophages

Inhaled particulates and microbes are continually cleared by a complex array of lung innate immune determinants, including alveolar macrophages (AMs). AMs are unique cells with an enhanced capacity for phagocytosis that is due, in part, to increased activity of the macrophage mannose receptor (MR), a pattern recognition receptor for various microorganisms. The local factors that "shape" AM function are not well understood. Surfactant protein A (SP-A), a major component of lung surfactant, participates in the innate immune response and can enhance phagocytosis. Here we show that SP-A selectively enhances MR expression on human monocyte-derived macrophages, a process involving both the attached sugars and collagen-like domain of SP-A. The newly expressed MR is functional. Monocyte-derived macrophages on an SP-A substrate demonstrated enhanced pinocytosis of mannose BSA and phagocytosis of Mycobacterium tuberculosis lipoarabinomannan-coated microspheres. The newly expressed MR likely came from intracellular pools because: 1) up-regulation of the MR by SP-A occurred by 1 h, 2) new protein synthesis was not necessary for MR up-regulation, and 3) pinocytosis of mannose BSA via MR recycling was increased. AMs from SP-A(-/-) mice have reduced MR expression relative to SP-A(+/+). SP-A up-regulation of MR activity provides a mechanism for enhanced phagocytosis of microbes by AMs, thereby enhancing lung host defense against extracellular pathogens or, paradoxically, enhancing the potential for intracellular pathogens to enter their intracellular niche. SP-A contributes to the alternative activation state of the AM in the lung.     

Surfactant Protein-A Modulates LPS-Induced TLR4 Localization and Signaling via β-Arrestin 2

The soluble C-type lectin surfactant protein (SP)-A mediates lung immune responses partially via its direct effects on alveolar macrophages (AM), the main resident leukocytes exposed to antigens. SP-A modulates the AM threshold of lipopolysaccharide (LPS) activity towards an anti-inflammatory phenotype both in vitro and in vivo through various mechanisms. LPS responses are tightly regulated via distinct pathways including subcellular TLR4 localization and thus ligand sensing. The cytosolic scaffold and signaling protein β-arrestin 2 acts as negative regulator of LPS-induced TLR4 activation. Here we show that SP-A neither increases TLR4 abundancy nor co-localizes with TLR4 in primary AM. SP-A significantly reduces the LPS-induced co-localization of TLR4 with the early endosome antigen (EEA) 1 by promoting the co-localization of TLR4 with the post-Golgi compartment marker Vti1b in freshly isolated AM from rats and wild-type (WT) mice, but not in β-arrestin 2^−/− AM. Compared to WT mice pulmonary LPS-induced TNF-α release in β-arrestin 2^−/− mice is accelerated and enhanced and exogenous SP-A fails to inhibit both lung LPS-induced TNF-α release and TLR4/EEA1 positioning. SP-A, but not LPS, enhances β-arrestin 2 protein expression in a time-dependent manner in primary rat AM. The constitutive expression of β-arrestin 2 in AM from SP-A^−/− mice is significantly reduced compared to SP-A^+/+ mice and is rescued by SP-A. Prolonged endosome retention of LPS-induced TLR4 in AM from SP-A^−/− mice is restored by exogenous SP-A, and is antagonized by β-arrestin 2 blocking peptides. LPS induces β-arrestin 2/TLR4 association in primary AM which is further enhanced by SP-A. The data demonstrate that SP-A modulates LPS-induced TLR4 trafficking and signaling in vitro and in vivo engaging β-arrestin 2.     

CD36 Differently Regulates Macrophage Responses to Smooth and Rough Lipopolysaccharide

Lipopolysaccharide (LPS) is the major pathogen-associated molecular pattern of Gram-negative bacterial infections, and includes smooth (S-LPS) and rough (R-LPS) chemotypes. Upon activation by LPS through CD14, TLR4/MD-2 heterodimers sequentially induce two waves of intracellular signaling for macrophage activation: the MyD88-dependent pathway from the plasma membrane and, following internalization, the TRIF-dependent pathway from endosomes. We sought to better define the role of scavenger receptors CD36 and CD204/SR-A as accessory LPS receptors that can contribute to pro-inflammatory and microbicidal activation of macrophages. We have found that CD36 differently regulates activation of mouse macrophages by S-LPS versus R-LPS. The ability of CD36 to substitute for CD14 in loading R-LPS, but not S-LPS onto TLR4/MD-2 allows CD14-independent macrophage responses to R-LPS. Conversely, S-LPS, but not R-LPS effectively stimulates CD14 binding to CD36, which favors S-LPS transfer from CD14 onto TLR4/MD-2 under conditions of low CD14 occupancy with S-LPS in serum-free medium. In contrast, in the presence of serum, CD36 reduces S-LPS binding to TLR4/MD-2 and the subsequent MyD88-dependent signaling, by mediating internalization of S-LPS/CD14 complexes. Additionally, CD36 positively regulates activation of TRIF-dependent signaling by both S-LPS and R-LPS, by promoting TLR4/MD-2 endocytosis. In contrast, we have found that SR-A does not function as a S-LPS receptor. Thus, by co-operating with CD14 in both R- and S-LPS loading onto TLR4/MD-2, CD36 can enhance the sensitivity of tissue-resident macrophages in detecting infections by Gram-negative bacteria. However, in later phases, following influx of serum to the infection site, the CD36-mediated negative regulation of MyD88-dependent branch of S-LPS-induced TLR4 signaling might constitute a mechanism to prevent an excessive inflammatory response, while preserving the adjuvant effect of S-LPS for adaptive immunity.     

Scavenger receptor expressed by endothelial cells I (SREC-I) mediates the uptake of acetylated low density lipoproteins by macrophages stimulated with lipopolysaccharide

Scavenger receptor expressed by endothelial cells I (SREC-I) is a novel endocytic receptor for acetylated low density lipoprotein (LDL). Here we show that SREC-I is expressed in a wide variety of tissues, including macrophages and aortas. Lipopolysaccharide (LPS) robustly stimulated the expression of SREC-I in macrophages. In an initial attempt to clarify the role of SREC-I in the uptake of modified lipoproteins as well as in the development of atherosclerosis, we generated mice with a targeted disruption of the SREC-I gene by homologous recombination in embryonic stem cells. To exclude the overwhelming effect of the type A scavenger receptor (SR-A) on the uptake of Ac-LDL, we further generated mice lacking both SR-A and SREC-I (SR-A(-/-);SREC-I(-/-)) by cross-breeding and compared the uptake and degradation of Ac-LDL in the isolated macrophages. The contribution of SR-A and SREC-I to the overall degradation of Ac-LDL was 85 and 5%, respectively, in a non-stimulated condition. LPS increased the uptake and degradation of Ac-LDL by 1.8-fold. In this condition, the contribution of SR-A and SREC-I to the overall degradation of Ac-LDL was 90 and 6%, respectively. LPS increased the absolute contribution of SR-A and SREC-I by 1.9- and 2.3-fold, respectively. On the other hand, LPS decreased the absolute contribution of other pathways by 31%. Consistently, LPS did not increase the expression of other members of the scavenger receptor family such as CD36. In conclusion, SREC-I serves as a major endocytic receptor for Ac-LDL in LPS-stimulated macrophages lacking SR-A, suggesting that it has a key role in the development of atherosclerosis in concert with SR-A.     

Surfactant proteins A and D bind CD14 by different mechanisms

Surfactant proteins A (SP-A) and D (SP-D) are lung collectins that are constituents of the innate immune system of the lung. Recent evidence (Sano, H., Sohma, H., Muta, T., Nomura, S., Voelker, D. R., and Kuroki, Y. (1999) J. Immunol. 163, 387-395) demonstrates that SP-A modulates lipopolysaccharide (LPS)-induced cellular responses by direct interaction with CD14. In this report we examined the structural elements of the lung collectins involved in CD14 recognition and the consequences for CD14/LPS interaction. Rat SP-A and SP-D bound CD14 in a concentration-dependent manner. Mannose and EDTA inhibited SP-D binding to CD14 but did not decrease SP-A binding. The SP-A binding to CD14 was completely blocked by a monoclonal antibody that binds to the SP-A neck domain but only partially blocked by an antibody that binds to the SP-A lectin domain. SP-A but not SP-D bound to deglycosylated CD14. SP-D decreased CD14 binding to both smooth and rough LPS, whereas SP-A enhanced CD14 binding to rough LPS and inhibited binding to smooth LPS. SP-A also altered the migration profile of LPS on a sucrose density gradient in the presence of CD14. From these results, we conclude that 1) lung collectins bind CD14, 2) the SP-A neck domain and SP-D lectin domain participate in CD14 binding, 3) SP-A recognizes a peptide component and SP-D recognizes a carbohydrate moiety of CD14, and 4) lung collectins alter LPS/CD14 interactions.     

Surfactant protein-D regulates soluble CD14 through matrix metalloproteinase-12

Surfactant protein D (SP-D) and CD14 are important innate immune defense molecules that mediate clearance of pathogens and apoptotic cells from the lung. To test whether CD14 expression and function were influenced by SP-D, the surface expression of CD14 was assessed on alveolar macrophages from SP-D-/- mice. CD14 was reduced on alveolar macrophages from SP-D-/- mice and was associated with reduced uptake of LPS and decreased production of TNF-alpha after LPS stimulation. CD14 is proteolytically cleaved from the cell surface to form a soluble peptide. Soluble CD14 (sCD14) was increased in the bronchoalveolar lavage fluid from SP-D-/- mice. Because matrix metalloproteinase (MMP)-9 and -12 activities were increased in the lungs of SP-D-/- mice, the role of these metalloproteases in the production of sCD14 was assessed. sCD14 was decreased in both MMP(9-/-)/SP-D-/- and MMP12(-/-)/SP-D-/- mice demonstrating MMP-9 and MMP-12 contribute to proteolytic shedding of CD14. The increased sCD14 seen in SP-D-/- mice was dependent upon the activation of MMP-12 via an MMP-9-dependent mechanism. Supporting this observation, MMP-12 caused the release of sCD14 from RAW 264.7 cells in vitro. In conclusion, SP-D influences innate host defense, in part, by regulating sCD14 in a process mediated by MMP-9 and MMP-12.     

Localization of surfactant protein A (SP-A) in alveolar macrophage subpopulations of normal and fibrotic rat lung

The colocalization of surfactant protein A (SP-A) and the alveolar macrophage markers ED1 and RM-1, as well as various lectins of the N-acetyl-galactosamine group [Maclura pomifera lectin (MPA), Dolichos biflorus lectin (DBA), soybean agglutinin (SBA)] and of the mannose group [Canavalia ensiformis lectin (ConA), Galanthus nivalis lectin (GNA)] was studied in normal and fibrotic rat lung tissues. In normal tissue, SP-A was located preferentially in the alveolar macrophage subpopulation lacking specific binding sites for lectins of the N-acetylgalactosamine group (DBA and SBA), although 50% of MPA-binding macrophages contained SP-A. The ED1-positive cells were SP-A-negative, whereas SP-A uptake could be detected among the RM-1 immunoreactive as well as the ConA and GNA binding macrophages. In fibrotic lung tissue, however, a small number of .DBA and SBA binding macrophages contained SP-A and the percentage of GNA and ConA binding alveolar macrophages exhibiting SP-A immunoreactivity was reduced. Additionally, the number of ED1⁺/SP-A⁺ macrophages was found to be increased. Immunoelectron microscopy revealed accumulation of SP-A in the extracellular space. The differing SP-A content in different alveolar macrophage subpopulations suggests a more complex mechanism of uptake and degradation of surfactant proteins in normal and pathological conditions, which cannot simply be explained by the glycoconjugate pattern on the surface of alveolar macrophages.     

Suppression of TLR4-mediated inflammatory response by macrophage class A scavenger receptor (CD204)

The class A scavenger receptor (SR-A, CD204), one of the principal receptors expressed on macrophages, has been found to regulate inflammatory response and attenuate septic endotoxemia. However, the detailed mechanism of this process has not yet been well characterized. To clarify the regulative mechanisms of lipopolysaccharide (LPS)-induced macrophage activation by SR-A, we evaluated the activation of Toll-like receptor 4 (TLR4)-mediated signaling molecules in SR-A-deficient (SR-A^−/−) macrophages. In a septic shock model, the blood levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and interferon (IFN)-β were significantly increased in SR-A^−/− mice compared to wild-type mice, and elevated nuclear factor kappa B (NFκB) activation was detected in SR-A^−/− macrophages. SR-A deletion increased the production of pro-inflammatory cytokines, and the phosphorylation of mitogen-activated protein kinase (MAPK) and NFκB in vitro. SR-A deletion also promoted the nuclear translocation of NFκB and IFN regulatory factor (IRF)-3. In addition, a competitive binding assay with acetylated low-density lipoprotein, an SR-A-specific ligand, and anti-SR-A antibody induced significant activation of TLR4-mediated signaling molecules in wild-type macrophages but not in SR-A^−/− macrophages. These results suggest that SR-A suppresses the macrophage activation by inhibiting the binding of LPS to TLR4 in a competitive manner and it plays a pivotal role in the regulation of the LPS-induced inflammatory response.