Macrophages release plasma membrane-derived particles rich in accessible cholesterol

Abstract

Macrophages are generally assumed to unload surplus cholesterol through direct interactions between ABC transporters on the plasma membrane and HDLs, but they have also been reported to release cholesterol-containing particles. How macrophage-derived particles are formed and released has not been clear. To understand the genesis of macrophage-derived particles, we imaged mouse macrophages by EM and nanoscale secondary ion mass spectrometry (nanoSIMS). By scanning EM, we found that large numbers of 20- to 120-nm particles are released from the fingerlike projections (filopodia) of macrophages. These particles attach to the substrate, forming a "lawn" of particles surrounding macrophages. By nanoSIMS imaging we showed that these particles are enriched in the mobile and metabolically active accessible pool of cholesterol (detectable by ALO-D4, a modified version of a cholesterol-binding cytolysin). The cholesterol content of macrophage-derived particles was increased by loading the cells with cholesterol or by adding LXR and RXR agonists to the cell-culture medium. Incubating macrophages with HDL reduced the cholesterol content of macrophage-derived particles. We propose that release of accessible cholesterol-rich particles from the macrophage plasma membrane could assist in disposing of surplus cholesterol and increase the efficiency of cholesterol movement to HDL.

Cultured macrophages transfer surplus cholesterol into adjacent cells in the absence of serum or high-density lipoproteins

“…After considering these findings, we wanted to make sure that the cholesterol transfer from macrophages to SMCs did not result from nonphysiologic overloading of the macrophage plasma membrane with cholesterol (as a result of loading cells with [ 13 C]cholesterol-MβCD complexes). In subsequent studies, we examined cholesterol transfer from [ 13 C]cholesterol-loaded macrophages in which we had depleted the accessible cholesterol pool (17) from the plasma membrane by incubating the cells for 30 min with 10 mM MβCD. As expected, the MβCD incubation markedly depleted "accessible cholesterol" from the macrophage plasma membrane, as judged by the binding of fluorescently labeled ALO-D4 (21) (Fig.…”

“…Mouse Peritoneal Macrophages. Macrophages were elicited and isolated as described in He et al (17). WT macrophages were elicited by injecting mice intraperitoneally with 1 mL of 3% (wt/vol) Difco Fluid Thioglycollate Medium (Becton, Dickinson & Co.).…”

“…Macrophages were incubated in medium containing 2 mM glutamine, 1 mM sodium pyruvate, and 10% (vol/vol) FBS (HyClone) in DMEM (Thermo Fisher Scientific) overnight before initiating any treatments (e.g., cholesterol loading). [ 13 C]cholesterol was produced as described (24) using a Saccharomyces cerevisiae strain (RH6829) engineered to produce cholesterol rather than ergosterol (17,25,26). The cholesterol contained 94% 13 C, as judged by mass spectrometry.…”

“…The laboratory of Howard Kruth reported that cultured macrophages export cholesterol from the plasma membrane in the form of "cholesterol microdomains" and proposed that the release of these microdomains serves to unload surplus cholesterol (13)(14)(15)(16). More recently, He et al (17) showed that cultured macrophages release 30-nm vesicular particles from the plasma membrane onto the surrounding substrate, and they went on to show that these particles are enriched in "accessible cholesterol" (a pool of cholesterol that is not sequestered by sphingolipids) (18). The accessible pool of cholesterol is mobile and important in regulating the SREBP transcription factors (18,19) in the Significance By ingesting apoptotic cells and other cellular debris, macrophages accumulate cholesterol.…”

Active cholesterol 20 years on

“…It was observed recently that cultured macrophages shed small membrane vesicles enriched in active cholesterol from their filopodia, apparently in a homeostatic response to sterol overload 130 …”