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 (∼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 (∼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.

The alveolar macrophage (AM)4 is responsible for the binding, ingestion, and, ultimately, clearance of inhaled particles and bacteria (1, 2). If inhaled bacteria are opsonized by Abs and/or complement, AMs can bind and phagocytose them via Fc and complement receptors (3, 4). Despite the absence of specific opsonins for inhaled dusts and inorganic materials, AMs bind and phagocytose such unopsonized environmental particles (and many unopsonized bacteria) with remarkable efficiency (5, 6). The receptors on AMs that mediate binding of unopsonized particles were, until recently, not known. Previous work in our laboratory identified a role for the scavenger receptor (SR) with collagenous structure MARCO (macrophage receptor with collagenous structure) in this process in AMs from hamsters (7) and mice (7, 8). Expression of MARCO on human macrophages is revealed by Ab labeling (7) and by mRNA expression studies (9).

SRs, characterized by their broad ligand specificity, are thought to have evolved as pattern recognition receptors involved in innate immune recognition (10, 11, 12). MARCO (13), SR-AI, SR-AII, SR-AIII, and SR-CL (SR with C-type lectin) form the SR-A subclass in the SR family (10, 11, 12, 14). Although SR-AI/II/III are alternate spliced forms of the same gene, MARCO is a distinct gene product (15, 16, 17, 18). MARCO, like SR-AI/II (SR-AIII is thought to be an intracellular regulatory protein), binds acetylated low-density lipoprotein and bacteria but not yeast (17, 19). We showed that an Ab to hamster MARCO substantially inhibited uptake of unopsonized environmental particles and bacteria (TiO2, Fe2O3, and Escherichia coli; 66 ± 5, 77 ± 2, and 67 ± 5% inhibition, respectively) by AMs in this species (7). Mice with genetically deleted MARCO show significant, but partial, defects in particle binding in vivo (8). However, Ab blockade of MARCO on normal mouse AMs resulted in a much lower (25 ± 4%) inhibition of particle binding (7), suggesting that other receptor(s) also mediate binding and that species may differ in the relative contribution of different receptor(s).

Because human macrophages also express multiple SRs, we sought to determine which receptor(s) are functionally important in human AM interaction with unopsonized particles. We developed a mAb capable of substantially blocking human AM particle binding. Identification of the ligand for this blocking Ab as human MARCO supports a dominant role for MARCO in binding unopsonized inert particles and bacteria.

TiO2 was generously provided by Dr. J. Brain (Harvard School of Public Health, Boston, MA). These particles have been shown to be heterogeneous in size, with a median diameter of 1.3 μm (20). Latex beads (1.0 μM in diameter, sulfated, polystyrene), which show green fluorescence after excitation at 488 nm, were obtained from Interfacial Dynamics. All particles were suspended in balanced salt solution (BSS; 124 mM NaCl, 5.8 mN KCl, 10 mM dextrose, and 20 mM HEPES) as stock solutions and sonicated ∼30 s before use. Anti-CD23 (IgG3), anti-CD44, and a nonspecific mouse IgG3 (Southern Biotechnology Associates) were used as controls. All reagents not otherwise specified were obtained from Sigma-Aldrich. A panel of Abs for known or potential scavenger-type receptors on human macrophages was used and is detailed in Table I.

Table I.

Description of polyclonal (P) and monoclonal (M) Abs tested for inhibition of particle and bacteria binding to AM

AntigenSpecificationsSourceReferences
LOX-1a M; clone no. 23C11 HyCult Biotechnology 26  
 P; goat, no. sc-11650 (anti-C-terminus peptide) Santa Cruz Biotechnology  
SR-AI/SR-AI M; clone no. SRA-C6 Dr. Motohiro Takeya (Kumamoto University, Kumamoto, Japan) 27  
 P; goat no. AB5486 (anti-C-terminus peptide) Chemicon 28  
 P; rabbit anti-peptide Dr. T. Kodama (University of Tokyo, Tokyo, Japan) 24  
SRCL-1a P; rabbit anti-peptide Drs. K. Nakamura, H. Funakoshi, and T. Nakamura, (Osaka University, Osaka, Japan) 29  
SR-PSOXa M; clone no. 22-19-12 Dr. Kimihisa Ichikawa (Sankyo, Tokyo, Japan) 30  
 P; goat, no. AF976 R&D Systems  
CD68 M; clone KP-1 Dako, Cytomation 3132  
SREC P; goat, no.11300 (anti-C-terminus peptide) Santa Cruz Biiotechnology  
Stabilin M; clone MS-1 Dr. Julia Kzhyshkowska (University of Heidelberg, Heidelberg, Germany) 3334  
AntigenSpecificationsSourceReferences
LOX-1a M; clone no. 23C11 HyCult Biotechnology 26  
 P; goat, no. sc-11650 (anti-C-terminus peptide) Santa Cruz Biotechnology  
SR-AI/SR-AI M; clone no. SRA-C6 Dr. Motohiro Takeya (Kumamoto University, Kumamoto, Japan) 27  
 P; goat no. AB5486 (anti-C-terminus peptide) Chemicon 28  
 P; rabbit anti-peptide Dr. T. Kodama (University of Tokyo, Tokyo, Japan) 24  
SRCL-1a P; rabbit anti-peptide Drs. K. Nakamura, H. Funakoshi, and T. Nakamura, (Osaka University, Osaka, Japan) 29  
SR-PSOXa M; clone no. 22-19-12 Dr. Kimihisa Ichikawa (Sankyo, Tokyo, Japan) 30  
 P; goat, no. AF976 R&D Systems  
CD68 M; clone KP-1 Dako, Cytomation 3132  
SREC P; goat, no.11300 (anti-C-terminus peptide) Santa Cruz Biiotechnology  
Stabilin M; clone MS-1 Dr. Julia Kzhyshkowska (University of Heidelberg, Heidelberg, Germany) 3334  
a

LOX-1, Lectin-like oxidized low-density lipoprotein receptor 1; SR-PSOX, SR that binds phosphatidylserine and oxidized lipoprotein; SREC, scavenger receptor expressed by endothelial cells.

Human AMs were collected by bronchoalveolar lavage (BAL) from healthy adults under an institutionally reviewed and approved protocol. AMs obtained by lung lavage were centrifuged at 150 × g and resuspended in BSS+ (124 mM NaCl, 5.8 mM KCl, 10 mM dextrose, 20 mM HEPES, Ca (0.3 mM), and Mg (1 mM)). AMs (2 × 105 in 400 μl of BSS+) were preincubated with mAbs (25 μg/ml mAb) or inhibitors (10 μg/ml) and 2.5 μg/ml cytochalasin D for 5 min on ice in a 1-ml microfuge tube. Following the addition of probe-sonicated particles (25 μg/ml) or beads (10:1 particle:cell ratio) the tubes were rotated at 37°C for 30 min, placed on ice, and analyzed by flow cytometry. Flow cytometry was performed using the Coulter Epics Elite flow cytometer (Beckman Coulter) as described previously (21). AM uptake of particles was measured using the increase in the mean right angle scatter (RAS) caused by these granular materials (21, 22). Latex bead binding is expressed as mean fluorescence intensity (MFI) units. In some cases, AMs were cultured overnight in Costar low binding tissue culture dishes (Corning) before assay. The source of AMs (smoker or nonsmoker donors) and their culture in the laboratory did not significantly alter the particle binding characteristic or the effect of inhibitors on particle binding (data not shown). Therefore, results of particle binding studies shown are pooled data from smoker, nonsmoker, and either fresh or cultured BALs.

Fluorescent-labeled, heat-killed bacteria (E. coli and Staphylococcus aureus) and yeast (Zymosan) were purchased from Molecular Probes. The bacteria binding assay was performed exactly as described above, except that AMs were incubated with either bacteria (5 × 107) or yeast (2 × 105) instead of particles. Binding was measured by detecting AM-associated fluorescence by flow cytometry.

BALB/c mice were immunized by i.p. injection of 2 × 107 human AMs. After 3 wk, mice received another injection of 2 × 107 AMs i.p., and 3 days later spleens of the mice were removed. The splenocytes were fused with a nonsecreting mouse myeloma, P3U1, using PEG 4000 and cultured in DMEM (BioWhittaker) containing hypoxanthine, aminopterin, and thymidine. After 2 wk, supernatants from hybridoma cultures were screened for their ability to inhibit the adhesion of TiO2 to AMs. The clone PLK-1 was isolated and characterized as an IgG3. The Ab was produced and purified on a protein A affinity column by BioExpress Cell Culture Services (BioExpress).

Human AM cell surface proteins were labeled with Sulfo-NHS-LC-Biotin (Pierce), as per the manufacturer’s suggested protocol, and resuspended at a concentration of 4 × 107 cells/ml in a 1% extraction buffer (1% Triton X-100, 50 mM Tris-Hcl, 150 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, and 5 mM iodoacetamide supplemented with 40 μg/ml PMSF, 2 μg/ml aprotinin, and 10 μg/ml phenanthroline as protease inhibitors). The lysates were precleared with pan-mouse-Ig magnetic beads (Dynabeads; Dynal Biotech). Aliquots of lysate were incubated with mAbs PLK-1 or IgG3 bound to pan-mouse-Ig magnetic beads overnight at 4°C. The immunoprecipitates were washed in cold lysis buffer (without protease inhibitors), subjected to SDS-PAGE, electroblotted to membrane filters and probed with avidin-HRP conjugate (Pierce), and developed using a chemiluminence reagent (Supersignal; Pierce).

Full-length and truncation mutants of human MARCO cDNA in pcDNA3 expression vectors were prepared as described previously (23). The human SR-AI cDNA (24) was provided by Dr. T. Kodama (Univeristy of Tokyo, Tokyo, Japan).

COS cells were grown in DMEM (BioWhittaker) with 10% FBS plus 100 IU/ml penicillin and 100 μg/ml streptomycin. For transfection, COS cells were plated at 5 × 105 cells/100-mm tissue culture dish overnight and transfected with 4 μg of cDNA using the LipofectAMINE PLUS reagent (Invitrogen Life Technologies) according to the manufacturer’s instructions. The cells were used after 48 h. COS cell expression of human MARCO was confirmed by immunohistochemical staining with a polyclonal rabbit anti-human MARCO Ab (16). COS cell TiO2 adhesion assays were performed as described for AMs above.

Tissue samples were snap-frozen on OCT (Miles Laboratory). Cryostat sections were fixed in buffered 2% paraformaldehyde for 10 min. Cytocentrifuge preparations of human BAL samples were air-dried before similar fixation. After rinsing, immunostaining was performed by sequential application of primary Ab (mAb PLK-1) 5 μg/ml or IgG3 mAb (5 μg/ml), goat anti-mouse IgG (1:50; Steinberg Monoclonals), mouse peroxidase-anti-peroxidase complex (1:100; Steinberg Monoclonals), followed by labeling with the chromogen diaminobenzidine and H2O2. The slides were washed with water, counterstained with hematoxylin, dehydrated, and mounted for light microscopy.

Data were analyzed using ANOVA (StatView; Abacus Concepts). Significance was accepted when p < 0.05.

To develop a mAb to AM receptor(s) that mediate particle binding, mice were immunized with human AMs, and hybridomas were prepared and screened for mAbs that block human AM binding of TiO2. As shown in Fig. 1, and reported earlier (7, 25), the SR ligand, polyinosinic acid (PI), blocked the majority of AM binding of TiO2 and served as a positive control for these assays. The functional screening led to the identification of a new IgG3 mAb, PLK-1, which inhibits AM binding of TiO2 by 63 ± 5% (n = 11; p < 0.001; Fig. 1, A and C). Chondroitin sulfate (CS; a control polyanion), an isotype-matched control mAb (IgG3) and anti-CD23 mAb, a control Ab that binds to AMs and is also an IgG3, had no effect on AM binding of TiO2. We next examined the effect of mAb PLK-1 on AM binding of another inert particle, fluorescent latex beads. As shown in Fig. 1, B and D, PLK-1, but not other control agents, inhibited AM binding of latex beads by 67 ± 5 (n = 11; p < 0.001). Thus, mAb PLK-1 substantially inhibits human AM binding of unopsonized inert particles.

FIGURE 1.

The mAb PLK-1 specifically blocks binding of inert particles. Human AMs were untreated or pretreated with PI, CS, mAb PLK-1, an isotype-matched control mAb (IgG3), and an IgG3 mAb to an irrelevant AM Ag (CD23), and their binding of TiO2 (A and C) or fluorescent latex beads (B and D) was determined by flow cytometry as described in Materials and Methods. A and C illustrate results of a typical single experiment; B and D show summarized data. TiO2 binding is expressed as increase in flow cytometric RAS, and bead binding is expressed as mean cell-associated fluorescence intensity. The data represent mean (±SEM) of 11 separate experiments. Next to the bars, the percentage inhibition for each agent tested is shown. ∗, Significantly different from TiO2 or beads alone; p < 0.001.

FIGURE 1.

The mAb PLK-1 specifically blocks binding of inert particles. Human AMs were untreated or pretreated with PI, CS, mAb PLK-1, an isotype-matched control mAb (IgG3), and an IgG3 mAb to an irrelevant AM Ag (CD23), and their binding of TiO2 (A and C) or fluorescent latex beads (B and D) was determined by flow cytometry as described in Materials and Methods. A and C illustrate results of a typical single experiment; B and D show summarized data. TiO2 binding is expressed as increase in flow cytometric RAS, and bead binding is expressed as mean cell-associated fluorescence intensity. The data represent mean (±SEM) of 11 separate experiments. Next to the bars, the percentage inhibition for each agent tested is shown. ∗, Significantly different from TiO2 or beads alone; p < 0.001.

Close modal

The SR ligand, PI, consistently caused a slightly greater inhibition than mAb PLK-1 for both particles (79 ± 2; 63 ± 5% inhibition of TiO2 binding by PI and PLK-1, respectively (p ≤ 0.05); 83 ± 3; 67 ± 4% inhibition of latex beads binding by PI and PLK-1, respectively (p ≤ 0.05)). These data suggest a role, albeit minor, for a non-PLK-1 inhibitable SR(s) in this function or alternatively greater affinity of PI than PLK-1 for MARCO.

We used COS cell transfection and surface labeling to identify the receptor-mediating AM binding of particles. A human MARCO cDNA clone transfected into COS cells conferred mAb PLK-1 reactivity (Fig. 2A). mAb PLK-1 did not react with untransfected COS cells or COS cells transfected with a cDNA encoding for human SR-AI. Immunoprecipitation analysis was used to corroborate the surface labeling studies. Human MARCO and human SR-AI-transfected COS cells were surface-labeled with sulfo-NHS-biotin (Pierce), and the cell surface molecules bound by PLK-1 were analyzed by SDS-PAGE (Fig. 2,B). PLK-1 immunoprecipitated bands of 60 kDa and 50 kDa from MARCO, but not untransfected or SR-AI-transfected COS cell lysates (Fig. 2,B). This molecular mass pattern is consistent with previous results regarding human MARCO expressed in COS cells (16). To characterize the AM protein(s) recognized by mAb PLK-1, immunoprecipitation experiments (as described for COS cells above) were conducted on human AMs. A nonspecific background band of ∼50 kDa was observed in both control IgG3 and PLK-1 immunoprecipitates (Fig. 2,B). A major band with an apparent molecular mass of 70 kDa was detected in lysates of normal AMs (Fig. 2,B). This band was absent from cells precipitated with an isotype-matched (IgG3) negative control Ab (Fig. 2 B). This 70-kDa molecular mass size band is consistent with the molecular mass pattern determined by Western blot analysis with a polyclonal anti-human MARCO Ab (7). These data indicate that mAb PLK-1 recognizes the SR MARCO.

FIGURE 2.

Transfection of COS cells with human MARCO cDNA confers mAb PLK-1 reactivity. A, COS cells were untransfected or transfected with a human MARCO cDNA or human SR-AI cDNA; PLK-1-immunostained cells only in MARCO cDNA-transfected samples, whereas anti-SR-AI Ab only reacted with cells after transfection with SR-AI cDNA. Control IgG3 and mouse Ig showed no labeling (not shown). B, Immunoprecipitation of surface proteins recognized by mAb PLK-1. COS (untreated, transfected with cDNAs for human SR-AI or human MARCO) and human AMs were surface labeled with biotin and extracted with Triton X-100, precleared with anti-pan-mouse Ig-coated magnetic beads, and incubated with mAb PLK-1 or a control IgG3 coupled to anti-pan-mouse IgG beads, and the bound proteins were analyzed by SDS-PAGE under reducing conditions. Relative molecular masses in kilodaltons are indicated.

FIGURE 2.

Transfection of COS cells with human MARCO cDNA confers mAb PLK-1 reactivity. A, COS cells were untransfected or transfected with a human MARCO cDNA or human SR-AI cDNA; PLK-1-immunostained cells only in MARCO cDNA-transfected samples, whereas anti-SR-AI Ab only reacted with cells after transfection with SR-AI cDNA. Control IgG3 and mouse Ig showed no labeling (not shown). B, Immunoprecipitation of surface proteins recognized by mAb PLK-1. COS (untreated, transfected with cDNAs for human SR-AI or human MARCO) and human AMs were surface labeled with biotin and extracted with Triton X-100, precleared with anti-pan-mouse Ig-coated magnetic beads, and incubated with mAb PLK-1 or a control IgG3 coupled to anti-pan-mouse IgG beads, and the bound proteins were analyzed by SDS-PAGE under reducing conditions. Relative molecular masses in kilodaltons are indicated.

Close modal

To further test the role of MARCO in particle binding, we transfected COS cells with either human MARCO or human SR-AI cDNA and tested the effect of mAb PLK-1 on TiO2 binding. In line with our previous findings with primary hamster (7) and mouse (8) AMs, TiO2 binding by MARCO-transfected COS cells but not untransfected COS or SR-AI-transfected cells was significantly inhibited by the anti-MARCO mAb PLK-1 (Fig. 3,B). Controls, including untransfected COS cells and COS cells transfected with a plasmid encoding the cDNA for human SR-AI, exhibited binding of TiO2 that was inhibited by PI and heparin, but not mAb PLK-1 (Fig. 3, A and C, and data not shown). The constitutive heparin-sensitive particle binding receptor on COS cells is distinct from MARCO and remains to be identified. Thus, mAb PLK-1 recognizes human MARCO and inhibits MARCO-mediated particle binding.

FIGURE 3.

Transfection of COS cells with human MARCO cDNA confers mAb PLK-1-inhibitable TiO2 binding. COS cells (untransfected (A) or transfected with a human MARCO cDNA (B) or human SR-AI cDNA (C)) were pretreated with mAb PLK-1 and a control mAb, and their binding of TiO2 was determined using a flow cytometric assay. Data are mean percentage inhibition of TiO2 binding by mAb PLK-1 from three separate experiments.

FIGURE 3.

Transfection of COS cells with human MARCO cDNA confers mAb PLK-1-inhibitable TiO2 binding. COS cells (untransfected (A) or transfected with a human MARCO cDNA (B) or human SR-AI cDNA (C)) were pretreated with mAb PLK-1 and a control mAb, and their binding of TiO2 was determined using a flow cytometric assay. Data are mean percentage inhibition of TiO2 binding by mAb PLK-1 from three separate experiments.

Close modal

To map the epitope of the PLK-1 Ab on MARCO receptor, and therefore the ligand binding site, we performed immunohistochemistry on COS cells transfected with either the cDNA encoding the full-length human MARCO or with truncated forms lacking different peptide sequences of the SRCR domain. PLK-1 labels the full-length MARCO, as well as the h442 (MARCO extending 22 residues into the SRCR domain) and h431 (MARCO extending 11 residues into the SRCR domain) mutants, whereas it failed to label the h420 variant (MARCO lacking the SRCR domain) (Fig. 4). This indicates that the epitope is located between residues 420 and 431, consistent with a previous report mapping the ligand binding site on domain V of the MARCO receptor in the human (23) and mouse (16).

FIGURE 4.

PLK-1 binds to the MARCO receptor through the SRCR domain. COS cells were transfected with either the full-length MARCO (A and D) or with the truncated variants h442 (B), h431 (C), and h420 (E and F). Cells were immunostained with PLK-1 Ab (A, B, C, and E), a control IgG3 Ab (D), or a polyclonal anti-cytoplasmic peptide Ab (F). Photomicrographs are shown at ×600 magnification.

FIGURE 4.

PLK-1 binds to the MARCO receptor through the SRCR domain. COS cells were transfected with either the full-length MARCO (A and D) or with the truncated variants h442 (B), h431 (C), and h420 (E and F). Cells were immunostained with PLK-1 Ab (A, B, C, and E), a control IgG3 Ab (D), or a polyclonal anti-cytoplasmic peptide Ab (F). Photomicrographs are shown at ×600 magnification.

Close modal

Immunostaining of cryostat sections of a panel of normal human tissues (lung, lymph node, liver, spleen, kidney) and cytocentrifuge preparations of human BAL cells (n = 10) with PLK-1 showed that it reacted strongly with most or all AMs, macrophages of lymph node sinuses, and Kupffer cells of the liver (Fig. 5,A). Ag expression was also detected on macrophages within intestinal mucosa and to a lesser extent on macrophages in the splenic red pulp (data not shown). Cross-reactions with other cells or tissue components were not observed. Flow cytometric analysis showed that PLK-1 specifically, but weakly, labels AMs (MFI CD44, PLK-1, and IgG3; 503 ± 84, 61 ± 9, and 39 ± 7, respectively; n = 8; p < 0.001) (Fig. 5 B) (see Discussion). Thus, human MARCO is predominantly expressed on AMs and tissue macrophages.

FIGURE 5.

Human AMs express MARCO. A, Immunostaining of normal BAL cytocentrifuge preparations and surgical lung specimens showed strong labeling by mAb PLK-1. PLK-1 also labeled Kupffer cell macrophages in the liver and macrophages in lymph nodes and other tissues (see text). Control IgG3 showed minimal nonspecific background staining (original magnification, ×400). B, Human AMs were labeled with PLK-1 anti-MARCO Ab, IgG3 isotype control, anti-CD23, or anti-CD44, and analyzed by flow cytometry. MFI values shown represent the mean ± SD from eight donors. ∗, p < 0.05 vs control.

FIGURE 5.

Human AMs express MARCO. A, Immunostaining of normal BAL cytocentrifuge preparations and surgical lung specimens showed strong labeling by mAb PLK-1. PLK-1 also labeled Kupffer cell macrophages in the liver and macrophages in lymph nodes and other tissues (see text). Control IgG3 showed minimal nonspecific background staining (original magnification, ×400). B, Human AMs were labeled with PLK-1 anti-MARCO Ab, IgG3 isotype control, anti-CD23, or anti-CD44, and analyzed by flow cytometry. MFI values shown represent the mean ± SD from eight donors. ∗, p < 0.05 vs control.

Close modal

To further investigate the range of ligands for human AM MARCO, we tested the effect of mAb PLK-1 on AM binding of unopsonized microorganisms. As shown in Fig. 6, A and B, mAb PLK-1-inhibited AM binding of fluorescent heat killed E. coli by 84 ± 4% (n = 7) and binding of the Gram-positive bacteria S. aureus by 41 ± 9% (n = 9). PLK-1 inhibition of AM binding of S. aureus was consistently lower than inhibition of E. coli binding, suggesting differences in affinity and/or mechanism in MARCO binding of unopsonized bacteria. PLK-1 had no effect on binding of yeast (Zymosan; Fig. 6 C) by normal AMs. An isotype-matched control Ab did not inhibit bacteria and yeast binding. Thus, MARCO mediates human AM binding of unopsonized bacteria in vitro, as reported previously with hamster (7) and mouse (8) AMs.

FIGURE 6.

Human AM MARCO confers binding of Gram-negative and Gram-positive bacteria. AMs were incubated with either fluorescent E. coli (A), S. aureus (B), or Zymozan (C) in the absence or presence of either PI, CS, PLK-1, IgG3, or anti-CD23. The total amount of AM-associated fluorescence was determined by flow cytometry. Data shown represent the mean of 11 separate experiments. ∗, p < 0.05 vs control.

FIGURE 6.

Human AM MARCO confers binding of Gram-negative and Gram-positive bacteria. AMs were incubated with either fluorescent E. coli (A), S. aureus (B), or Zymozan (C) in the absence or presence of either PI, CS, PLK-1, IgG3, or anti-CD23. The total amount of AM-associated fluorescence was determined by flow cytometry. Data shown represent the mean of 11 separate experiments. ∗, p < 0.05 vs control.

Close modal

Additional, unidentified receptor(s) likely mediate the portion of AM binding of unopsonized particles that is not inhibited by anti-MARCO PLK-1. To evaluate the potential contribution of other SRs to AM binding of unopsonized TiO2 and S. aureus, we tested a panel of Abs raised against various SRs known or likely to be expressed on human AMs including several with known functional blocking activity (see Table I). Irrelevant isotype-matched mAbs or normal polyclonal antisera or IgG were used as controls (Table I). Although some variability was observed, expression of all receptors was identified on human AM samples (Fig. 7,A). However, none of these Abs caused inhibition of binding of either unopsonized TiO2 and S. aureus (Fig. 7, B and C). The data indicate a major role for MARCO in human AM binding of unopsonized particles.

FIGURE 7.

MARCO is the major particle and bacteria binding receptor on human AMs. Abs to different SRs were tested for AM staining by immunohistochemistry (A) and for their blocking ability of TiO2 (B) and S. aureus (C) binding to AMs; ∗, p < 0.05 vs control.

FIGURE 7.

MARCO is the major particle and bacteria binding receptor on human AMs. Abs to different SRs were tested for AM staining by immunohistochemistry (A) and for their blocking ability of TiO2 (B) and S. aureus (C) binding to AMs; ∗, p < 0.05 vs control.

Close modal

This study has identified the class A SR MARCO as a dominant receptor for unopsonized environmental particles and bacteria on human AMs. The data address questions raised by species discordance in the level of inhibition caused by anti-MARCO Abs in vitro, i.e., hamsters (high, ∼70%) vs mice (low, ∼25%) (7). Because human AMs express both class A SRs (SR-AI/II and MARCO) as well as other SRs (Refs.7 , 24 , 35 , 36 ; see also Fig. 6), we sought to determine the relative contribution of these potential candidates by developing blocking mAbs.

By screening anti-human AM hybridomas for the ability to block AM binding of unopsonized particles, we isolated the mAb PLK-1. PLK-1 showed robust and specific inhibition of AM binding of TiO2 and latex beads (Fig. 1). Transfection of COS cells with human MARCO cDNA, but not human SR-AI/II/III cDNA, conferred mAb PLK-1 reactivity (Fig. 2,A). mAb PLK-1 immunoprecipitation from MARCO-transfected COS cell lysate (Fig. 2,B) is similar to immunoprecipitation analysis for COS cell MARCO with a polyclonal anti-human MARCO Ab (16). In addition, transfection with human MARCO confers PLK-1-inhibitable binding of TiO2 by COS cells (Fig. 3 B). These data demonstrate that the receptor recognized by mAb PLK-1 is human MARCO.

The identification of the PLK-1 binding site on domain V (SRCR) allowed us to conclude that this domain is responsible for the binding of TiO2 and bacteria. This observation corroborates previous findings with the murine MARCO receptor (16, 23), and also brings additional proof that, unlike the SR-AI/II and SR-CL, which bind their ligands through the collagenous domain (37, 38), MARCO receptor interacts with its ligands through the SRCR domain.

As reported earlier, COS cells constitutively express an endogenous particle adhesion receptor(s) distinct from MARCO (7). Nevertheless, transfection of COS cells with human MARCO but not control human SR-AI/II cDNA renders a portion of particle binding sensitive to specific inhibition by PLK-1. The constitutive COS cell receptor(s) for particles is sensitive to heparin inhibition (data not shown), whereas the AM receptor-mediated particle binding is not heparin sensitive. Existence of a particle binding receptor on nonphagocytes, such as COS cells, is not unprecedented. The lung epithelial cell line, A549, binds and ingests unopsonized TiO2, and this binding can also be inhibited by PI and heparin (39). Another interesting finding in these studies is that the AMs collected from smokers and nonsmokers bound unopsonized particle and bacteria essentially identically. Smoker AMs had a higher basal flow cytometric RAS, consistent with uptake of smoking-related particles. However, MARCO-mediated binding of TiO2 and latex beads did not significantly differ between smoker and nonsmoker AMs (data not shown). Thus, chronic exposure to inhaled cigarette smoke did not seem to dramatically alter the function of MARCO in particle binding.

The lung is constantly exposed to environmental substances such as microbes, pollutant particles, and allergens. The clearance of inhaled particulate matter is primarily mediated by the AMs through the process of phagocytosis (1, 2). The essential first step in phagocytosis, receptor-mediated target recognition, is either opsonin-dependent or opsonin-independent. Specific receptors on the phagocytes recognize either serum components (opsonins) bound to the particle or directly recognize molecular determinants on the target. The opsonin-dependent phagocytic pathway is the most studied, and several opsonin-receptors have been well characterized, such as FcγR, complement receptor CR3, and collectin receptor, C1q (4, 40, 41, 42, 43). However, the recent studies on opsonin-independent recognition of microorganisms and apoptotic cells have implicated receptors such as the SR SR-AI/II, mannose receptor, vitronectin receptor, asialoglycoprotein receptor, and the β2 integrins (44, 45). In this study, we have identified MARCO as a major receptor on AMs for binding of unopsonized inert particles and certain microorganisms.

Although human MARCO is the major receptor on AMs for unopsonized particles, the SR ligand, PI, caused a slightly higher inhibition of particle binding, suggesting a role for other SR(s) on AMs. We initially considered this receptor to most likely be SR-AI/II, because we have found that an Ab to SR-AI/II partially blocks TiO2 binding by mouse macrophages (our unpublished observation). However, analysis of a panel of Abs to other SRs on human AMs failed to detect inhibition of particle binding, including monoclonal and polyclonal Abs reported to have functional blocking ability against SR-AI/SR-AII (K. Nakamura, personal communication and Ref.28 , respectively). One limitation to these data is that we did not independently confirm the blocking function reported by other investigators, although in some cases we were able to use two or three different Abs with the same (negative) result. Another unanswered question is the structure(s) responsible for the ∼20% of particle binding and the up to 60% of bacteria (S. aureus) that is not blocked by either PI or PLK-1.

There is noteworthy discordance between the rather low expression level of MARCO on human AMs detected by immunofluorescence labeling and flow cytometry and the high capacity for particle binding by these cells via surface structures blocked with the PLK-1 anti-MARCO. We have observed similar findings in mouse AMs and another anti-MARCO mAb, ED31 (data not shown). Immunochemical differences in blocking vs labeling capacities may be a relatively trivial cause for this discordance. Another speculative possibility is that the unopsonized particles studied (dusts, bacteria) are large enough to engage a complex of multiple surface receptors that require MARCO (present in small amounts) to function optimally. It is worth noting that whereas our analysis focused on the cardinal property of SRs, namely the binding of particles and pathogens, the role, if any, of these receptors in the subsequent internalization phase of phagocytosis remains to be characterized.

Our finding that MARCO mediates human AM binding of unopsonized particles and bacteria indicates an important function for MARCO in human lung host defense. Additional investigation is needed to address the potential role of other receptors involved in particle binding and to identify the signaling events (if any) initiated by SRs like MARCO.

The authors have no financial conflict of interest.

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. Section 1734 solely to indicate this fact.

1

This work was supported by National Institutes of Health Grants ES0002 and ES11008.

4

Abbreviations used in this paper: AM, alveolar macrophage; SR, scavenger receptor; MARCO, macrophage receptor with collagenous structure; BSS balanced salt solution; BAL, bronchoalveolar lavage; RAS, right angle scatter; MFI, mean fluorescence intensity; PI, polyinosinic acid; CS, chondroitin sulfate; SRCR, SR cysteine-rich.

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