MARCO is the major binding receptor for unopsonized particles and bacteria on human alveolar macrophages

Alveolar macrophages (AMs) avidly bind and ingest inhaled environmental particles and bacteria. To identify the particle binding receptor(s) on human AMs, we used functional screening of anti-human AM hybridomas and isolated a mAb, PLK-1, which inhibits AM binding of unopsonized particles (e.g., TiO2, latex beads; 63 +/- 5 and 67 +/- 4% inhibition, respectively, measured by flow cytometry; n = 11) and unopsonized bacteria ( approximately 84 and 41% inhibition of Escherichia coli and Staphylococcus aureus binding by mAb PLK-1, respectively). The PLK-1 Ag was identified as the human class A scavenger receptor (SR) MARCO (macrophage receptor with collagenous structure) by observing specific immunolabeling of COS cells transfected with human MARCO (but not SR-AI/II) cDNA and by immunoprecipitation by PLK-1 of a protein of appropriate molecular mass (approximately 70 kDa) from both normal human bronchoalveolar lavage cells (>90% AMs) and human MARCO-transfected COS cells. PLK-1 also specifically inhibited particle binding by COS cells, only after transfection with human MARCO cDNA. Immunostaining showed specific labeling of AMs within human lung tissue, bronchoalveolar lavage samples, as well as macrophages in other sites (e.g., lymph node and liver). Using COS transfectants with different truncated forms of MARCO, allowed epitope mapping for the PLK-1 Ab to MARCO domain V between amino acid residues 420 and 431. A panel of Abs to various SRs identified expression on AMs, but failed to inhibit TiO2 or S. aureus binding. The data support a dominant role for MARCO in the human AM defense against inhaled particles and pathogens.     

Defective microarchitecture of the spleen marginal zone and impaired response to a thymus-independent type 2 antigen in mice lacking scavenger receptors MARCO and SR-A

The macrophage scavenger receptor macrophage receptor with a collagenous structure (MARCO) is expressed in mice by the marginal zone macrophages of the spleen and by macrophages of the medullary cords of lymph nodes, as well as the peritoneal macrophages. MARCO is a relative of scavenger receptor A (SR-A), the more widely expressed prototypic member of the scavenger receptor family. In the present study, we found that genetic ablation of MARCO leads to changes in the organization of the splenic marginal zone, and causes a significant reduction in the size of the resident peritoneal macrophage population, possibly due to changes in adhesion and migration capacity. In mice lacking both MARCO and SR-A these effects are even more apparent. During ontogeny, the appearance and organization of the MARCO-expressing cells in the spleen precedes the appearance of other receptors on macrophages in the marginal zone, such as SIGNR1 and Siglec-1. In the absence of MARCO, a clear delay in the organization of the marginal zone was observed. Similar findings were seen when the reappearance of the various subsets from precursors was studied after depleting macrophages from the adult spleen by a liposome treatment. When challenged with a pneumococcal polysaccharide vaccine, a T-independent type 2 Ag for which an intact marginal zone is crucial, the knockout mice exhibited a clearly impaired response. These findings suggest that both MARCO and SR-A, in addition to being important scavenger receptors, could be involved in the positioning and differentiation of macrophages, possibly through interaction with endogenous ligands.     

A phage display screen and binding studies with acetylated low density lipoprotein provide evidence for the importance of the scavenger receptor cysteine-rich (SRCR) domain in the ligand-binding function of MARCO

MARCO is a class A scavenger receptor capable of binding both gram-negative and -positive bacteria. Using the surface plasmon resonance technique, we show here that a recombinant, soluble form of MARCO, sMARCO, binds the major gram-negative and -positive bacterial surface components, lipopolysaccharide and lipoteichoic acid. Yet, the interaction of these two polyanions with sMARCO is of much lower affinity than that of polyinosinic acid, a polyanionic inhibitor of bacterial binding to MARCO. To further elucidate the ligand-binding functions of MARCO, we performed a phage display screen with sMARCO. The screening resulted in the enrichment of only a handful of phage clones. Contrary to expectations, no polyanionic peptides, but only those with a predominantly hydrophobic nature, were enriched. One peptide, VRWGSFAAWL, was displayed on two-thirds of the phages recovered after four rounds of screening. The VRWGSFAAWL phage-sMARCO interaction had significantly slower dissociation kinetics than that between sMARCO and lipopolysaccharide or lipoteichoic acid. Further work with this phage, and the second most enriched phage, displaying the peptide RLNWAWWLSY, demonstrated that both peptides bind to the SRCR domain of MARCO, and that they probably bind to the same site. Data base searches suggested that the VRWGSFAAWL peptide represents complement component C4, but we could not convincingly confirm this suggestion. A study with chimeric scavenger receptors indicated that even minor sequence changes in the MARCO scavenger receptor cysteine-rich (SRCR) domain can have profound effects on the binding of the prototypic scavenger receptor ligand, acetylated low density lipoprotein. As shown by differential binding of glutathione S-transferase-VR-WGSFAAWL, these differences were very likely due to conformational changes. These findings led to experiments that demonstrated a crucial role of the SRCR domain for acetylated low density lipoprotein binding in MARCO. Thus, our results strengthen the notion that the SRCR domain is the major ligand-binding domain in MARCO. Furthermore, they suggest that the domain may contain multiple ligand-binding sites.     

Arginine residues in domain V have a central role for bacteria-binding activity of macrophage scavenger receptor MARCO

MARCO is a bacteria-binding macrophage-specific scavenger receptor that plays a role in innate immune response. MARCO has short intracellular and transmembrane domains, as well as a large extracellular domain composed of a spacer domain, a long collagenous domain, and a C-terminal scavenger receptor cysteine-rich domain (SRCR), domain V. As yet, no specific function has been assigned to the SRCR domain of scavenger receptors. In the present study, we generated several human and mouse MARCO variants with deletions or single amino acid substitutions and localized the primary bacteria-binding region to domain V. Furthermore, analysis of the MARCO variants containing only portions of domain V demonstrated a crucial role for an arginine-rich segment for this function. More precisely, the motif RXR was identified as an essential element for high-affinity bacterial binding. The results indicate that the binding properties of MARCO differ from those of the other class A scavenger receptors, SR-A and SRCL, whose ligand-binding function has been localized to the collagenous domain.     

Expression of macrophage MARCO receptor induces formation of dendritic plasma membrane processes

MARCO is a novel macrophage-specific receptor structurally related to macrophage class A scavenger receptors. It is constitutively expressed in macrophages of the marginal zone of the spleen and in lymph nodes and is up-regulated in other tissues during systemic bacterial infections. In this study, we show that ectopic expression of MARCO in cell lines such as Chinese hamster ovary, HeLa, NIH3T3, and 293 induces dramatic cell shape changes. Typically these changes include formation of large lamellipodia-like structures and of long dendritic processes. The morphological changes are accompanied by disassembly of actin stress fibers and often also by complete loss of focal adhesions. The MARCO-induced changes are dependent on cell adhesion and are inhibited, but not completely abolished, when the cells are plated on fibronectin-coated surfaces. Similarly, a dominant-negative mutant of the Rho family GTPase Rac1 partially inhibited the morphogenic effects of MARCO in Chinese hamster ovary cells, whereas a dominant-negative form of a related protein, Cdc42, did not. Expression studies with a variety of truncated MARCO forms indicated that the proximal segment of the cysteine-rich domain V is important for the morphoregulatory activity. The results indicate that expression of MARCO has a direct effect in generating the phenotype of activated macrophages necessary for the trapping and removal of pathogens and other foreign substances.     

CD163 Binding to Haptoglobin-Hemoglobin Complexes Involves a Dual-point Electrostatic Receptor-Ligand Pairing

Formation of the haptoglobin (Hp)-hemoglobin (Hb) complex in human plasma leads to a high affinity recognition by the endocytic macrophage receptor CD163. A fast segregation of Hp-Hb from CD163 occurs at endosomal conditions (pH <6.5). The ligand binding site of CD163 has previously been shown to involve the scavenger receptor cysteine-rich (SRCR) domain 3. This domain and the adjacent SRCR domain 2 of CD163 contain a consensus motif for a calcium-coordinated acidic amino acid triad cluster as originally identified in the SRCR domain of the scavenger receptor MARCO. Here we show that site-directed mutagenesis in each of these acidic triads of SRCR domains 2 and 3 abrogates the high affinity binding of recombinant CD163 to Hp-Hb. In the ligand, Hp Arg-252 and Lys-262, both present in a previously identified CD163 binding loop of Hp, were revealed as essential residues for the high affinity receptor binding. These findings are in accordance with pairing of the calcium-coordinated acidic clusters in SRCR domains 2 and 3 with the two basic Arg/Lys residues in the Hp loop. Such a two-point electrostatic pairing is mechanistically similar to the pH-sensitive pairings disclosed in crystal structures of ligands in complex with tandem LDL receptor repeats or tandem CUB domains in other endocytic receptors.     

Characterization of a human homologue of the murine peripheral lymph node homing receptor

Lymphocyte trafficking is a fundamental aspect of the immune system that allows B and T lymphocytes with diverse antigen recognition specificities to be exposed to various antigenic stimuli in spatially distinct regions of an organism. A lymphocyte adhesion molecule that is involved with this trafficking phenomenon has been termed the homing receptor. Previous work (Lasky, L., T. Yednock, M. Singer, D. Dowbenko, C. Fennie, H. Rodriguez, T. Nguyen, S. Stachel, and S. Rosen. 1989. Cell. 56:1045-1055) has characterized a cDNA clone encoding a murine homing receptor that is involved in trafficking of lymphocytes to peripheral lymph nodes. This molecule was found to contain a number of protein motifs, the most intriguing of which was a carbohydrate binding domain, or lectin, that is apparently involved in the adhesive interaction between murine lymphocytes and peripheral lymph node endothelium. In this study, we have used the murine cDNA clone to isolate a human homologue of this peripheral lymph node-specific adhesion molecule. The human receptor was found to be highly homologous to the murine receptor in overall sequence, but showed no sequence similarity to another surface protein that may be involved with human lymphocyte homing, the Hermes glycoprotein. The extracellular region of the human receptor contained an NH2 terminally located carbohydrate binding domain followed by an EGF-like domain and a domain containing two repeats of a complement binding motif. Transient cell transfection assays using the human receptor cDNA showed that it encoded a surface glycoprotein that cross reacted with a polyclonal antibody directed against the murine peripheral lymph node homing receptor. Interestingly, the human receptor showed a high degree of sequence homology to another human cell adhesion glycoprotein, the endothelial cell adhesion molecule ELAM.     

Characterization of recombinant soluble macrophage scavenger receptor MARCO

MARCO is a type II transmembrane protein of the class A scavenger receptor family. It has a short N-terminal cytoplasmic domain, a transmembrane domain, and a large extracellular part composed of a 75-residue long spacer domain, a 270-residue collagenous domain, and a 99-residue long scavenger receptor cysteine-rich (SRCR) domain. Previous studies have indicated a role for this receptor in anti-microbial host defense functions. In this work we have produced the extracellular part of MARCO as a recombinant protein, and analyzed its binding properties. The production of this protein, soluble MARCO (sMARCO), has made it possible for the first time to study MARCO and its binding properties in a cell-free system. Using circular dichroism analyses, a protease-sensitive assay, and rotary shadowing electron microscopy, sMARCO was shown to have a triple-helical collagenous structure. Rotary shadowing also demonstrated that the molecules often associate with each other via the globes. sMARCO was found to bind avidly both heat-killed and living bacteria. Lipopolysaccharide, an important component of the outer membrane of Gram-negative bacteria, was shown to be a ligand of MARCO. Studies with different bacterial strains indicated that the O-side chain of lipopolysaccharide is not needed for the bacterial recognition. Finally, the C-terminal SRCR domain was also produced as a recombinant protein, and its bacteria-binding capability was studied. Although the transfection experiments with transmembrane MARCO variants have indicated a crucial role for this domain in bacterial binding, the monomeric domain exhibited low, barely detectable bacteria-binding activity. Thus, it is possible that cooperation between the SRCR domain and the collagenous domain is needed for high-affinity bacterial binding, or that the SRCR domain has to be in a trimeric form to effectively bind to bacteria.     

The scavenger cell pathway for lipoprotein degradation: Specificity of the binding site that mediates the uptake of negatively-charged LDL by macrophages

Macrophages isolated from a variety of organs in several animal species exhibit high affinity binding sites that recognize chemically modified proteins. One of these binding sites recognizes human plasma low density lipoprotein (LDL) in which the positive charges on the epsilon-amino groups of lysine have been removed or neutralized by chemical modification, thus giving the protein an enhanced negative charge. Effective treatments include reaction of LDL with organic acid anhydrides (acetylation or maleylation) and reaction with aldehydes, such as treatment with malondialdehyde. After the negatively-charged LDL binds to the surface receptor sites, it is rapidly internalized by the macrophages by endocytosis and hydrolyzed in lysosomes. The liberated cholesterol is reesterified in the cytoplasm, producing massive cholesteryl ester deposition. The binding site for negatively-charged LDL has been demonstrated so far only on macrophages and other scavenger cells. It is not expressed in cultured fibroblasts, smooth muscle cells, lymphocytes, or adrenal cells. In addition to its affinity for acetylated LDL and malondialdehyde-treated LDL, the macrophage site binds a variety of polyanions. It exhibits a particularly high affinity for certain sulfated polysaccharides (dextran sulfate and fucoidin), certain polynucleotides (polyinosinic acid and polyguanylic acid), polyvinyl sulfate, and maleylated albumin. It is possible that the site that binds negatively-charged LDL may be responsible for the massive accumulation of cholesteryl esters that occurs in vivo in macrophages and other scavenger cells in patients with high levels of circulating plasma LDL.     

A macrophage receptor that recognizes oxidized low density lipoprotein but not acetylated low density lipoprotein

The formation of cholesterol-loaded macrophage foam cells in arterial tissue may occur by the uptake of modified lipoproteins via the scavenger receptor pathway. The macrophage scavenger receptor, also called the acetylated low density lipoprotein (Ac-LDL) receptor, has been reported to recognize Ac-LDL as well as oxidized LDL species such as endothelial cell-modified LDL (EC-LDL). We now report that there is another class of macrophage receptors that recognizes EC-LDL but not Ac-LDL. We performed assays of 0 degrees C binding and 37 degrees C degradation of 125I-Ac-LDL and 125I-EC-LDL by mouse peritoneal macrophages. Competition studies showed that unlabeled Ac-LDL could compete for only 25% of the binding and only 50% of the degradation of 125I-EC-LDL. Unlabeled EC-LDL, however, competed for greater than 90% of 125I-EC-LDL binding and degradation. Unlabeled Ac-LDL was greater than 90% effective against 125I-Ac-LDL; EC-LDL competed for about 80% of 125I-Ac-LDL binding and degradation. Copper-oxidized LDL behaved the same as EC-LDL in all the competition studies. Copper-mediated oxidation of Ac-LDL produced a superior competitor which could now displace 90% of 125I-EC-LDL binding. After 5 h at 37 degrees C in the presence of ligand, macrophages accumulated six times more cell-associated radioactivity from 125I-EC-LDL than from 125I-Ac-LDL, despite approximately equal amounts of degradation to trichloroacetic acid-soluble products, which may imply different intracellular processing of the two lipoproteins. Our results suggest that 1) there is more than one macrophage "scavenger receptor" for modified lipoproteins; and 2) oxidized LDL and Ac-LDL are not identical ligands with respect to macrophage recognition and uptake.     

Identification of uteroglobin-related protein 1 and macrophage scavenger receptor with collagenous structure as a lung-specific ligand-receptor pair

High in normal (HIN)-1 is a secreted protein highly expressed in normal breast epithelium and down-regulated in breast carcinomas. By searching GenBank expressed sequence tag databases, we identified HIN-2, a protein homologous to HIN-1. HIN-2 is identical with a recently identified protein called uteroglobin-related protein 1 (UGRP1). Northern blot analysis demonstrated that UGRP1 is specifically expressed by lung, but not by the other tissues examined. By in situ hybridization experiments, UGRP1 was shown to be expressed by lung Clara-like cells in the bronchial epithelium and to be up-regulated in cystic fibrosis. In a mammalian expression system, secreted recombinant UGRP1 was copurified with apolipoprotein A-I. Using a retroviral vector-mediated expression cloning approach, we identified macrophage scavenger receptor with collagenous structure (MARCO) as a receptor for UGRP1. Northern blot and in situ hybridization experiments indicated that MARCO is expressed by alveolar macrophages in the lung. UGRP1 also bound to bacteria and yeast. LPS, a previously identified MARCO ligand, competed with UGRP1 for binding to MARCO and bacteria. Our findings suggest that UGRP1-MARCO is a ligand-receptor pair that is probably involved in inflammation and pathogen clearance in the lung.     

Crystal structure of the cysteine-rich domain of scavenger receptor MARCO reveals the presence of a basic and an acidic cluster that both contribute to ligand recognition

MARCO is a trimeric class A scavenger receptor of macrophages and dendritic cells that recognizes polyanionic particles and pathogens. The distal, scavenger receptor cysteine-rich (SRCR) domain of the extracellular part of this receptor has been implicated in ligand binding. To provide a structural basis for understanding the ligand-binding mechanisms of MARCO, we have determined the crystal structure of the mouse MARCO SRCR domain. The recombinant SRCR domain purified as monomeric and dimeric forms, and their structures were determined at 1.78 and 1.77 A resolution, respectively. The monomer has a compact globular fold with a twisted five-stranded antiparallel beta-sheet and a long loop covering a single alpha-helix, whereas the dimer is formed via beta-strand swapping of two monomers, thus containing a large eight-stranded beta-sheet. Calculation of the surface electrostatic potential revealed that the beta-sheet region with several arginines forms a basic cluster. Unexpectedly, an acidic cluster was found in the long loop region. In the monomer, the acidic cluster is involved in metal ion binding. Studies with cells expressing various SRCR domain mutants showed that all of the arginines of the basic cluster are involved in ligand binding, suggesting a cooperative binding mechanism. Ligand binding is also dependent on the acidic cluster and Ca2+ ions whose depletion appears to affect ligand binding at least by modulating the electrostatic potential or relative domain orientation. We propose that the SRCR domain dimerization can contribute to the recognition of large ligands by providing a means for the MARCO receptor oligomerization.     

Role of the maleyl-albumin receptor in activation of murine peritoneal macrophages in vitro

It has been previously demonstrated that maley-lated-BSA (maleyl-albumin) induces functional activation in murine peritoneal macrophages. Furthermore, maleyl-albumin has been shown to interact with two distinct sites on human monocytes; one site is the scavenger receptor, a 260-kDa oligomeric protein which recognizes modified forms of low density lipoprotein (LDL), and the second is a lower affinity site which has yet to be structurally characterized. In the present study, we wished to quantitatively assess the number and character of maleyl-albumin-binding sites on murine peritoneal macrophages and to determine which site or sites are involved in signaling the macrophage to undergo extensive functional development. Binding studies. demonstrate at least two distinct receptors for maleyl-albumin on murine peritoneal macrophages. Scatchard analyses of the binding isotherms reveal two sites characterized by dissociation constants (Kd) of 17.6 nM and 4.9 microM and maximal binding of 1.2 x 10(5) and 1 x 10(6) sites/cell, respectively. The contribution of the scavenger receptor, determined by binding analyses of malondialdehyde-LDL, is described by two sites with Kd of 39.4 pM and 9.6 nM, and maximal binding of 2.7 x 10(3) and 1.9 x 10(4) sites/cell, respectively. Maleyl-albumin blocks binding of malondialdehyde-LDL, whereas modified LDL fails to inhibit binding of maleyl-albumin. Maleyl-albumin, at concentrations producing lower affinity binding, stimulates tumor cytolysis, expression of mRNA encoding TNF, and suppression of INF-gamma-induced expression of Ia Ag. Malondialdehyde-LDL fails to elicit these responses. We conclude that macrophage activation produced by maleyl-albumin is mediated by interaction with the low affinity, high capacity binding site for maleyl-albumin rather than the scavenger receptor.     

Oligonucleotide aggregates bind to the macrophage scavenger receptor.

We have prepared a model receptor containing a Lys cluster (320-340) in the collagen-like domain of the bovine macrophage scavenger receptor, and have shown that it has a similar binding specificity to the native scavenger receptor. The native scavenger receptor is reported to bind the quadruplex structure of nucleotides. In this study, we analyzed the model receptor binding of nucleotides with various structures, random, parallel or antiparallel quadruplex and aggregate forms. This was carried out by direct binding assays using labeled oligonucleotides or surface plasmon resonance, and by an inhibition assay using Chinese hamster ovary (CHO) cells expressing the scavenger receptor. The results showed that the nucleotides forming the quadruplex structure did not exhibit any binding. Only the aggregate forms of the nucleotide could bind to the model receptor. They also inhibited the degradation of acetylated low density lipoprotein by CHO cells expressing the native scavenger receptor, whereas nucleotides that did not bind to the model receptor had no effect on cellular acetylated low density lipoprotein degradation. Our results suggest that the quadruplex structure is not essential but may be required for the formation of the nucleotide aggregates, which can interact with the scavenger receptor.     

Species-specific restriction of cell surface expression of mouse MARCO glycoprotein in murine cell lines

The MARCO (macrophage receptor with collagenous structure) glycoprotein belongs to the scavenger receptor type family of pattern-recognition molecules produced by a subset of marginal zone macrophages in the spleen. Stimulation with LPS leads to its appearance on macrophages located at other tissue compartments. In the present work, we report its in vitro expression by various cell lines using transient and stable (lentiviral) gene delivery aimed at investigating the signaling properties of this receptor and its analysis using a novel rat monoclonal antibody against the SRCR-domain of mouse MARCO. When trying to establish stable mouse MARCO-transfectants using lentiviral transduction and other methods, we consistently found that MARCO accumulated intracellularly in various murine host cells. In contrast, such a phenomenon was not observed in non-murine cell lines. Our observations indicate the presence of an unexpected limitation of the in vitro expression of mouse MARCO glycoprotein in murine cell lines. We believe that the failure to express MARCO on the cell surface of the many murine cell lines is likely due to the absence of endoplasmic reticulum molecular chaperones needed for the correct folding and assembly of the trimeric MARCO molecule.     

The macrophage receptor MARCO

MARCO (macrophage receptor with collagenous structure) belongs to the class A scavenger receptor molecules. The structure and function of the molecule is described. Although it is expressed on subsets of macrophages, it can be upregulated on other macrophages after bacterial infection. The strategic position of MARCO-expressing cells in lymphoid organs suggests an important role for this bacteria-binding molecule in removal of pathogens.     

Human platelets exclusively bind oxidized low density lipoprotein showing no specificity for acetylated low density lipoprotein.

The widely studied macrophage scavenger receptor system is known to bind both acetylated low density lipoprotein and oxidized low density lipoprotein. Although only the latter ligand has been shown to occur in vivo, acetylated low density lipoprotein is often used to evaluate the contribution of scavenger receptors to different (patho)physiologic processes, assuming that all existing subtypes of scavenger receptors recognise both lipoproteins. In the present work, we identify human platelets as the first natural cell type to bind oxidized low density lipoprotein without showing specificity for acetylated low density lipoprotein. Consequently, platelets possess exclusive receptor(s) for oxidized low density lipoprotein distinct from the 'classical' scavenger receptor AI/AII. From the data presented in this work, we conclude that the class B scavenger receptor CD36 (GPIV) is responsible for this exclusive oxidized low density lipoprotein binding.