Protection against inhaled oxidants through scavenging of oxidized lipids by macrophage receptors MARCO and SR-AI/II

Abstract

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 5beta,6beta-epoxycholesterol (beta-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 beta-epoxide compared with MARCO-/- AMs, consistent with SRA function in binding oxidized lipids. SR-AI/II-/- mice showed similar enhanced acute lung inflammation after beta-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.

Figure 1. Ozone upregulates MARCO in lungs from ozone-resistant HeJ mice.

HeJ or congenic ozone-sensitive OuJ mice were exposed to 0.3 ppm ozone for up to 48 hours. Microarray analysis (A) and RT-PCR (B) were performed on total RNA isolated from lung samples and showed increased MARCO mRNA expression in HeJ mice (filled symbols) compared with OuJ mice (open symbols). (C) Western blot analysis of lung tissue obtained after 48 hours of ozone exposure also showed increased MARCO protein expression. (D) Ozone upregulates MARCO on the surface of AMs of C57BL/6 mice exposed to 0.3 ppm ozone for 48 hours, as shown by increased fluorescence after flow cytometric analysis. Results shown are representative of 3 independent experiments. MFI, mean fluorescence intensity. *P < 0.05 versus ozone-treated OuJ; ^#P < 0.05 versus air-exposed control.

Figure 2. MARCO decreases inflammation in lungs of mice exposed to ozone and ROFA.

(A–C) BAL samples obtained from MARCO^–/– mice after exposure to 0.3 ppm ozone for 48 hours showed higher levels of neutrophils (A), total protein (B), and 8-isoprostane (C) compared with controls. (D) BAL samples obtained from MARCO^–/– mice 18 hours after exposure to aerosolized leachate from ROFA (100 mg/ml, 1 hour) showed higher levels of neutrophils compared with controls. Number of mice per group is shown for each bar. *P < 0.05 versus treated MARCO^+/+ group; ^#P < 0.05 versus untreated.

Figure 3. MARCO decreases inflammation in lungs of mice exposed to β-epoxide or PON-GPC i.

. BAL samples obtained from MARCO^–/– mice 7 hours after i.t. instillation of 1 μg β-epoxide (Exp) or PON-GPC (PON) showed higher levels of neutrophils (A), MIP-2 (B), and total protein (C) compared with controls. Number of mice per group is shown for each bar. *P < 0.05 versus respective treated MARCO^+/+ group; ^#P < 0.05 versus cholesterol (Chol) control.

Figure 4. SR-AI/II decreases neutrophil accumulation in lungs of mice exposed to ROFA and i.

. β-epoxide, but not ozone. SR-AI/II^–/– mice received aerosolized leachate from ROFA (100 mg/ml for 1 hour; A), 1 μg i.t. β-epoxide (B), or 0.3 ppm ozone (C) for 48 hours. Number of mice per group is shown for each bar. *P < 0.05 versus treated SR-AI/II^+/+ group; ^#P < 0.05 versus untreated control.

Figure 5. Ozone upregulates MARCO on the surface of SR-AI/II^–/– AMs.

SR-AI/II^–/– mice were exposed to 0.3 ppm ozone for 48 hours, after which the AMs were isolated, labeled for MARCO, and analyzed by flow cytometry. Results shown are representative of 2 independent experiments.