Scavenger Receptors Class A-I/II and CD36 Are the Principal Receptors Responsible for the Uptake of Modified Low Density Lipoprotein Leading to Lipid Loading in Macrophages

Vidya V Kunjathoor,Maria Febbraio,Eugene A Podrez,Kathryn J Moore,Lorna Andersson,Stephanie Koehn,Jeongmi S Rhee,Roy Silverstein,Henry F Hoff,Mason W Freeman

doi:10.1074/jbc.m209649200

Abstract

Modification of low density lipoprotein (LDL) can result in the avid uptake of these lipoproteins via a family of macrophage transmembrane proteins referred to as scavenger receptors (SRs). The genetic inactivation of either of two SR family members, SR-A or CD36, has been shown previously to reduce oxidized LDL uptake in vitro and atherosclerotic lesions in mice. Several other SRs are reported to bind modified LDL, but their contribution to macrophage lipid accumulation is uncertain. We generated mice lacking both SR-A and CD36 to determine their combined impact on macrophage lipid uptake and to assess the contribution of other SRs to this process. We show that SR-A and CD36 account for 75-90% of degradation of LDL modified by acetylation or oxidation. Cholesteryl ester derived from modified lipoproteins fails to accumulate in macrophages taken from the double null mice, as assessed by histochemistry and gas chromatography-mass spectrometry. These results demonstrate that SR-A and CD36 are responsible for the preponderance of modified LDL uptake in macrophages and that other scavenger receptors do not compensate for their absence.

Highlights

Modification of low density lipoprotein (LDL) can result in the avid uptake of these lipoproteins via a family of macrophage transmembrane proteins referred to as scavenger receptors (SRs)
Macrophages from all genotypes produced similar levels of tumor necrosis factor-␣ and IL-6 in response to bacterial LPS (Fig. 1B). These findings indicate that the absence of both SR-A and CD36 does not appear to alter the expression of myeloid differentiation markers, nor does it affect the cytokine responses to the prototypical stimulator of macrophage inflammatory pathways, bacterial endotoxin
Degradation and Binding of Modified LDL—To define the contributions of SR-A and CD36 to modified lipid uptake, we performed binding and degradation studies on macrophages derived from wild type, SR-AϪ/Ϫ, CD36Ϫ/Ϫ, or SR-AϪ/Ϫ/CD36Ϫ/Ϫ mice using LDL modified by acetylation or oxidation, using either copper ions or a myeloperoxidase/hydrogen peroxide/nitrite oxidizing system

Summary

EXPERIMENTAL PROCEDURES

Reagents—Cell culture reagents were from Invitrogen. 125I and 32P were obtained from PerkinElmer Life Sciences. Macrophages were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum overnight, and non-adherent cells were removed by washing. Macrophages prepared in this manner routinely stained positively for CD11b (Ͼ95%) and F4/80 (Ͼ70%) by flow cytometry. Modified LDL Binding, Degradation, and Foam Cell Formation Assays—Binding assays were performed at 4 °C using 10 ␮g/ml modified 125I-LDL (AcLDL or OxLDL) in the presence or absence of a 20-fold excess of unlabeled modified LDL competitor as described previously [9]. Specific degradation was calculated as the total degradation of modified 125I-LDL minus degradation in the presence of unlabelled competitor. Tumor necrosis factor-␣ and IL-6 in cell culture supernatants were assayed by enzyme-linked immunosorbent assay (Pierce) as described previously [17]

RESULTS

DISCUSSION

None OxLDL AcLDL