Alveolar macrophages (AMs) express the class A scavenger receptor macrophage receptor with collagenous structure (MARCO), but its role in vivo in lung defense against bacteria and environmental particles has not been studied. We used MARCO-deficient mice to directly test the in vivo role of AM MARCO in innate defense against pneumococcal infection and environmental particles. In a murine model of pneumococcal pneumonia, MARCO^−/− mice displayed an impaired ability to clear bacteria from the lungs, increased pulmonary inflammation and cytokine release, and diminished survival. In vitro binding of Streptococcus pneumoniae and in vivo uptake of unopsonized particles by MARCO^−/− AMs were dramatically impaired. MARCO^−/− mice treated with the “inert” environmental particle TiO₂ showed enhanced inflammation and chemokine expression, indicating that MARCO-mediated clearance of inert particles by AMs prevents inflammatory responses otherwise initiated by other lung cells. Our findings point to an important role of MARCO in mounting an efficient and appropriately regulated innate immune response against inhaled particles and airborne pathogens.

Alveolar macrophages (AMs) avidly bind and ingest unopsonized environmental particles and bacteria through scavenger-type receptors (SRs). AMs from mice with a genetic deletion of the major macrophage SR (types AI and AII; SR^−/−) showed no decrease in particle binding compared with SR^+/+ mice, suggesting that other SRs are involved. To identify these receptors, we generated a monoclonal antibody (mAb), PAL-1, that inhibits hamster AM binding of unopsonized particles (TiO₂, Fe₂O₃, and latex beads; 66 ± 5, 77 ± 2, and 85 ± 2% inhibition, respectively, measured by flow cytometry). This antibody identifies a protein of ∼70 kD on the AM surface (immunoprecipitation) that is expressed by AMs and other macrophages in situ. A cDNA clone encoding the mAb PAL-1–reactive protein isolated by means of COS cell expression was found to be 84 and 77% homologous to mouse and human scavenger receptor MARCO mRNA, respectively. Transfection of COS cells with MARCO cDNA conferred mAb-inhibitable TiO₂ binding. Hamster MARCO also mediates AM binding of unopsonized bacteria (67 ± 5 and 47 ± 4% inhibition of Escherichia coli and Staphylococcus aureus binding by mAb PAL-1). A polyclonal antibody to human MARCO identified the expected ∼70-kD band on Western blots of lysates of normal bronchoalveolar lavage (BAL) cells (>90% AMs) and showed strong immunolabeling of human AMs in BAL cytocentrifuge preparations and within lung tissue specimens. In normal mouse AMs, the anti-MARCO mAb ED31 also showed immunoreactivity and inhibited binding of unopsonized particles (e.g., TiO₂ ∼40%) and bacteria. The novel function of binding unopsonized environmental dusts and pathogens suggests an important role for MARCO in the lungs' response to inhaled particles.

The class A macrophage scavenger receptor SR-AI/II is implicated as a pattern recognition receptor for innate immunity, but its functional role in lung defense has not been studied. We used mice genetically deficient in SR-AI/II and their wild-type C57BL/6 counterparts to investigate the contribution of this receptor to defense against pneumococcal infection and inhaled particles. SR-AI/II deficiency caused impaired phagocytosis of fluorescent bacteria in vivo, diminished clearance of live bacteria from the lungs, and substantially increased pneumonic inflammation. Survival studies also showed increased mortality in SR-AI/II–deficient mice with pneumococcal lung infection. Similarly, after challenge of the airways with TiO2 particles, SR-AI/II–deficient mice showed increased proinflammatory cytokine levels in lung lavage fluid and a more pronounced neutrophilic inflammation. The data indicate that the lung macrophage class A scavenger receptor SR-AI/II contributes to innate defense against bacteria and inhaled particles.

Alveolar macrophages (AMs) express the class A scavenger receptors (SRAs) macrophage receptor with collagenous structure (MARCO) and scavenger receptor AI/II (SRA-I/II), which recognize oxidized lipids and provide innate defense against inhaled pathogens and particles. Increased MARCO expression in lungs of ozone-resistant mice suggested an additional role protecting against inhaled oxidants. After ozone exposure, MARCO^–/– mice showed greater lung injury than did MARCO^+/+ mice. Ozone is known to generate oxidized, proinflammatory lipids in lung lining fluid, such as 5β,6β-epoxycholesterol (β-epoxide) and 1-palmitoyl-2-(9′-oxo-nonanoyl)-glycerophosphocholine (PON-GPC). Intratracheal instillation of either lipid caused substantial neutrophil influx in MARCO^–/– mice, but had no effect in MARCO^+/+ mice. Normal AMs showed greater uptake in vitro of β-epoxide compared with MARCO^–/– AMs, consistent with SRA function in binding oxidized lipids. SR-AI/II^–/– mice showed similar enhanced acute lung inflammation after β-epoxide or another inhaled oxidant (aerosolized leachate of residual oil fly ash). In contrast, subacute ozone exposure did not enhance inflammation in SR-AI/II^–/– versus SR-AI/II^+/+ mice, reflecting increased AM expression of MARCO. These data identify what we believe to be a novel function for AM SRAs in decreasing pulmonary inflammation after oxidant inhalation by scavenging proinflammatory oxidized lipids from lung lining fluids.

Particulate air pollution is linked to increased pneumonia epidemiologically and diminished lung bacterial clearance experimentally. We investigated the effect of concentrated ambient particles (CAPs, ≤ PM2.5) on the interaction of murine primary alveolar macrophages (AMs) and the murine macrophage cell line, J774 A.1, with Streptococcus pneumoniae. We found that CAPs increased binding of bacteria by both primary AMs and J774 cells (66.7 ± 10.6% and 58.9 ± 4.0%, respectively, n = 4). In contrast to bacterial binding, CAPs decreased internalization in both AMs and J774 (55.4 ± 8.5% and 54.7 ± 5.1%, respectively, n = 4). The rate of killing of internalized bacteria was similar, but CAPs caused a decrease in the absolute number of bacteria killed by macrophages, mainly due to decreased internalization. Additional analyses showed that soluble components of CAPs mediated the enhanced binding and decreased internalization of S. pneumoniae. Chelation of iron in soluble CAPs substantially reversed, while addition of iron as ferric ammonium citrate restored inhibition of phagocytosis of S. pneumoniae in vitro. The results identify phagocytic internalization as a specific target for toxic effects of air pollution particles on AMs.