Our interest in the role of the macrophage SRAs MARCO and SR-AI/II in regulating lung inflammation after ozone inhalation began with microarray data showing increased pulmonary MARCO mRNA expression in ozone-resistant HeJ mice, but not in ozone-sensitive OuJ mice (Figure ). While this initial hypothesis-generating data came from HeJ and OuJ mice, we subsequently determined that ozone inhalation also increased MARCO expression on AMs from C57BL/6 mice, allowing more detailed analyses using mice deficient in MARCO (available on a C57BL/6 background). We found that absence of MARCO increased lung inflammation after inhalation of ozone and another inhaled oxidant, aerosolized ROFA leachate. These findings prompted further examination of the role of lung macrophage MARCO in uptake and removal of oxidized surfactant lipids such as β-epoxide and PON-GPC. Our analyses showed that MARCO mediated intracellular lipid accumulation after incubation of lung macrophages with β-epoxide in vitro. MARCO acted to protect lungs against inflammation after β-epoxide and PON-GPC instillation in vivo, as shown by increased inflammatory responses in MARCO–/– mice. Taken together, these data suggest what we believe to be a previously unrecognized role for lung macrophage SRAs in lung defense against inhaled oxidants through clearance of otherwise proinflammatory oxidized surfactant lipids from damaged lung lining fluids.
Our data show similar, but not identical, functions for the 2 receptors studied, MARCO and SR-AI/II. We found that SR-AI/II, also contributed to protection of the lungs against acute inflammation, as determined by the number of BAL neutrophils after ROFA and β-epoxide exposure. In contrast to MARCO–/– mice, however, SR-AI/II–/– mice did not show increased inflammation in experiments using 48 hours of exposure to ozone. After such ozone exposure, we observed increased expression of MARCO on AMs from both SR-AI/II+/+ and SR-AI/II–/– mice, suggesting a protective effect of MARCO in both groups. Moreover, it is noteworthy that ozone exposure did not cause increased expression of the gene encoding SR-AI/II in the same microarray analyses that revealed increased MARCO expression in the ozone-resistant HeJ mice (data not shown). One interpretation is that basal levels of SR-AI/II contribute to clearance of proinflammatory oxidized lipids generated by the acute challenges of ROFA aerosols or direct instillation of β-epoxide. Comparison of data from these more acute exposures with those of the 48-hour ozone experiments suggest that the basal and unchanged level of SR-AI/II is insufficient for optimal clearance of oxidized lipids generated during the longer exposure to ozone, a task mediated by the increased MARCO expression we observed in the ozone model. It is also possible that during repair of inflammation, scavenger receptors may be involved in binding and clearing cellular debris, thereby hampering further amplification of the inflammation in the lung. Hence, to the extent that such repair processes are initiated at the 48-hour time point of the ozone exposure, MARCO and SR-AI/II may limit inflammation through mechanisms beyond the proposed binding of oxidized lipids.
It is worth noting that ozone and ROFA generate different lipid oxidation products. Ozone produces specific lipid ozonation products plus nonspecific lipid autoxidation from both ozone-initiated reactions within the epithelial lining fluid and after the onset of the inflammatory response. On the other hand, ROFA likely generates oxidized lipids via transition metal redox cycling as well as inflammation. Production of β-epoxide occurs in both scenarios, while PON-GCP should be relatively specific to ozone reactions. To facilitate comparison of ROFA to ozone exposures, we also analyzed BAL PMNs and isoprostanes using a higher ROFA concentration, which caused levels of PMNs comparable to those seen with ozone. Under these conditions, MARCO–/– mice showed a higher number of BAL PMNs and a trend toward higher 8-isoprostane levels. These data further support the conclusion that MARCO can scavenge a range of oxidatively modified lipids rather than being strictly specific for autoxidation-derived lipids.
When β-epoxide and PON-GCP were administered to MARCO+/+ mice, MARCO appeared to inhibit the inflammatory response to the oxidized lipids in full. When MARCO+/+ mice were exposed to ROFA or ozone, MARCO only partly decreased the inflammatory response, indicating that other oxidized products are capable of inducing inflammation in MARCO+/+ mice. At 100-fold greater doses, both β-epoxide and PON-GCP caused neutrophil influx in MARCO+/+ mice 7 hours after instillation (2.3 × 104 and 17 × 104, respectively). At lower doses, the oxidized surfactant lipids did not seem critical to the inflammatory response in MARCO+/+ mice, probably because MARCO and other scavenger receptors were capable of clearing the lipids sufficiently from the lung surface.
We have previously shown important functions for MARCO and SR-AI/II in innate immune responses against bacteria and environmental particles (9
). The current data indicate that MARCO and SR-AI/II are also involved in clearing oxidized surfactant lipids in the lung. In atherosclerosis, scavenger receptors have long been known to take up oxidized lipids and contribute to foam cell formation (8
). While SR-AI/II is implicated in lipid loading of macrophage-derived foam cells during atherogenesis (18
), the receptor is also expressed on AMs, indicating that it may be engaged in metabolism of modified lipids in the lung. MARCO is upregulated on human foam cells (23
) and on foam cells in atherosclerotic lesions from mice (24
), and a high-fat diet causes increased MARCO mRNA expression in the lungs and livers of C57BL/6 mice (25
), further supporting a role for MARCO in lipid uptake in the lung.
Lipid ozonation products from surfactant are essential for the transmission of toxic signals to the pulmonary epithelium after ozone inhalation (3
). The oxidized surfactant lipids β-epoxide and PON-GPC are important lipid ozonation products implicated in this process. They are created from cholesterol and oleate- and palmitoyl-containing glycerophosphocholines, which constitute about 5% and 20%, respectively, of the lung surfactant (28
). Also, β-epoxide is created from cholesterol in cell membranes. Both lipids are detected in vitro after exposing bovine surfactant to ozone (4
), and β-epoxide and nonanals are detected in vivo in BALs after exposing rodents to ozone (17
). We are not aware of previous studies measuring β-epoxide and PON-GPC using the 48-hour exposure conditions of the present study. However, exposing C57BL/6 mice to 0.5 ppm ozone for 3 hours creates 2.5 ng β-epoxide per ml BAL, and unexposed lungs from C57BL/6 mice have β-epoxide levels of approximately 95 ng per lung (17
). Our data using bolus instillation of 1 μg β-epoxide and PON-GPC provide a proof-of-principle, but future studies would benefit by more detailed measurement of the BAL levels of oxidized lipids during the 48-hour exposure to ozone.
Macrophages have previously been shown to take up β-epoxide (31
), whereas PON-GPC uptake by macrophages is less well described. Lipid ozonation products regulate several proinflammatory cytokines, chemokines, and adhesion molecules (5
). Both β-epoxide and PON-GPC have specifically been associated with cytotoxic activities and expression of IL-8 (5
), an important neutrophil chemoattractant after ozone exposure (3
). In the present study, viability of AMs was relatively unaffected by β-epoxide incubation (16 μg/ml), in contrast to previous studies of cell lines in which this dose has been shown to have cytotoxic effects (4
). One explanation might be that the primary cells used in our assay resist the cytotoxic effect better than cell lines do. Alternatively, differences in our assay compared with previous studies may also account for the better cell survival we observed. Consistent with prior observations of β-epoxide and PON-GPC as potential stimulators of IL-8 release (5
), MIP-2, a rodent homolog of human IL-8, was markedly increased after i.t. instillation of β-epoxide or PON-GPC into MARCO–/–
Both lung epithelial cells and macrophages express CXC chemokine mRNA and protein after ozone exposure in vivo (38
). MIP-2 expression after oxidant exposure has been localized immunohistochemically to both AMs and alveolar epithelial cells (38
). However, these data do not allow quantitation of the relative contribution of epithelial cells versus macrophages to MIP-2 release. One mechanism suggested by our present data is protection of lung epithelial cells from oxidized lipids by AM scavenging. Another possibility is that AM scavenger receptors divert oxidized lipids away from other, more proinflammatory receptors on the same cell and thereby reduce AM-derived MIP-2 and the inflammatory response that follows. In order to begin to address this question, we compared the capacity of AMs from MARCO+/+
mice to respond to oxidants by secretion of MIP-2. We observed similar fold increases in MIP-2 release in AMs exposed in vitro to H2
generated by a glucose oxidase–glucose system (43
) (fold increase at 50 μg/ml glucose oxidase, MARCO+/+
, 1.9 ± 0.6; MARCO–/–
, 1.8 ± 0.4; n
= 3; our unpublished observations). These data suggest that MARCO–/–
AMs retain the ability to respond to oxidants with proinflammatory cytokines, similar to previous observations using LPS (9
). The question of the relative contribution of epithelial versus macrophage cytokine release when AM scavenger receptors are absent could be further analyzed in vitro. However, extrapolation to in vivo biology will remain difficult, as previous coculture experiments show enhanced release of CXC chemokines in response to environmental oxidants when AMs and lung epithelial cells are in contact (44
Oxidants are present in air pollution and tobacco smoke or are released from activated leukocytes during lung inflammation. Increased oxidative stress in terms of increased BAL 8-isoprostane levels has been detected in lungs of smokers, patients with asthma, and patients with COPD (45
). Therefore, we exposed MARCO–/–
mice to the smoke from 4 unfiltered cigarettes and after 6 hours found higher BAL neutrophils in MARCO–/–
than in MARCO+/+
mice (0.51 versus 0.07 × 104
= 8 and n
= 11, respectively; P
= 0.02; our unpublished observations). These data indicate that MARCO protects the lung from acute cigarette smoke exposure. However, because cigarette smoke contains both particulate and gaseous proinflammatory components, whether MARCO protects by scavenging particles, smoke-generated oxidized lipids, or both, remains to be determined in future studies. When the lung lining fluid is under oxidative attack in injuries other than ozone-induced lung disease, significant concentrations of β-epoxide and PON-GPC may be produced and contribute to inflammation. In support of this idea, genetic studies show that the reduced activity of microsomal epoxide hydrolase, an enzyme that metabolizes β-epoxide (46
), is associated with a higher risk of COPD and emphysema (47
). Other members of the epoxide hydrolase family besides microsomal epoxide hydrolase are also expressed in the lung (49
), but their relation to the risk of lung disease is less well described.
In conclusion, our data indicate what we believe to be a previously unrecognized role for MARCO and SR-AI/II in innate defenses against inhaled oxidants, scavenging oxidized surfactant lipids from damaged lung lining fluids. This mechanism may apply to proinflammatory oxidized surfactant lipids other than the β-epoxide and PON-GPC we studied. We also speculate that MARCO and SR-AI/II could be involved in a general mechanism to dampen inflammation caused by the oxidized surfactant lipids generated during many forms of pulmonary inflammation and injury. Similar to the role for scavenger receptors in atherosclerosis (8
), SRAs internalize potentially proinflammatory oxidized lipids without engaging the typical phlogistic response in the lungs.