Differential Trafficking of Oxidized LDL and Oxidized LDL Immune Complexes in Macrophages: Impact on Oxidative Stress

Oxidized low-density lipoproteins (oxLDL) and oxLDL-containing immune complexes (oxLDL-IC) contribute to formation of lipid-laden macrophages (foam cells). It has been shown that oxLDL-IC are considerably more efficient than oxLDL in induction of foam cell formation, inflammatory cytokines secretion, and cell survival promotion. Whereas oxLDL is taken up by several scavenger receptors, oxLDL-IC are predominantly internalized through the FCgamma receptor I (FCgamma RI). This study examined differences in intracellular trafficking of lipid and apolipoprotein moieties of oxLDL and oxLDL-IC and the impact on oxidative stress.Fluorescently labeled lipid and protein moieties of oxLDL co-localized within endosomal and lysosomal compartments in U937 human monocytic cells. In contrast, the lipid moiety of oxLDL-IC was detected in the endosomal compartment, whereas its apolipoprotein moiety advanced to the lysosomal compartment. Cells treated with oxLDL-IC prior to oxLDL demonstrated co-localization of internalized lipid moieties from both oxLDL and oxLDL-IC in the endosomal compartment. This sequential treatment likely inhibited oxLDL lipid moieties from trafficking to the lysosomal compartment. In RAW 264.7 macrophages, oxLDL-IC but not oxLDL induced GFP-tagged heat shock protein 70 (HSP70) and HSP70B', which co-localized with the lipid moiety of oxLDL-IC in the endosomal compartment. This suggests that HSP70 family members might prevent the degradation of the internalized lipid moiety of oxLDL-IC by delaying its advancement to the lysosome. The data also showed that mitochondrial membrane potential was decreased and generation of reactive oxygen and nitrogen species was increased in U937 cell treated with oxLDL compared to oxLDL-IC.Findings suggest that lipid and apolipoprotein moieties of oxLDL-IC traffic to separate cellular compartments, and that HSP70/70B' might sequester the lipid moiety of oxLDL-IC in the endosomal compartment. This mechanism could ultimately influence macrophage function and survival. Furthermore, oxLDL-IC might regulate the intracellular trafficking of free oxLDL possibly through the induction of HSP70/70B'

Development of Capture Assays for Different Modifications of Human Low-Density Lipoprotein

Antibodies to malondialdehyde (MDA)-modified low-density lipoprotein (LDL), copper-oxidized LDL (oxLDL), N(ɛ)(carboxymethyl) lysine (CML)-modified LDL, and advanced glycosylation end product (AGE)-modified LDL were obtained by immunization of rabbits with in vitro-modified human LDL preparations. After absorption of apolipoprotein B (ApoB) antibodies, we obtained antibodies specific for each modified lipoprotein with unique patterns of reactivity. MDA-LDL antibodies reacted strongly with MDA-LDL and also with oxLDL. CML-LDL antibodies reacted strongly with CML-LDL and also AGE-LDL. oxLDL antibodies reacted with oxLDL but not with MDA-LDL, and AGE-LDL antibodies reacted with AGE-LDL but not with CML-LDL. Capture assays were set with each antiserum, and we tested their ability to capture ApoB-containing lipoproteins isolated from precipitated immune complexes (IC) and from the supernatants remaining after IC precipitation (free lipoproteins). All antibodies captured lipoproteins contained in IC more effectively than free lipoproteins. Analysis of lipoproteins in IC by gas chromatography-mass spectrometry showed that they contained MDA-LDL and CML-LDL in significantly higher concentrations than free lipoproteins. A significant correlation (r = 0.706, P < 0.019) was obtained between the MDA concentrations determined by chemical analysis and by the capture assay of lipoproteins present in IC. In conclusion, we have developed capture assays for different LDL modifications in human ApoB/E lipoprotein-rich fractions isolated from precipitated IC. This approach obviates the interference of IC in previously reported modified LDL assays and allows determination of the degree of modification of LDL with greater accuracy

Characterization of oxLDL labeling and uptake by U937 cells.

<p>(<b>A</b>) Migration of fluorescently labeled oxLDL analyzed by agarose gel electrophoresis: lane 1: native LDL, lane 2: oxLDL, lane 3: DiI-oxLDL, lane 4: DiO-oxLDL, lane 5: Alexa 546-oxLDL, lane 6: LPDS. (<b>B</b>) Uptake of fluorescently labeled oxLDL. U937 cells were treated with labeled oxLDL: DiO-oxLDL, DiI-oxLDL, or Alexa 546-oxLDL (24 µg/ml) for 5 h then fixed with 4% formaldehyde and visualized in sealed capillaries using confocal microscopy. (<b>C</b>) FACS analysis showing dose-dependent uptake of labeled oxLDL 90 min post treatment in U937 cells. Each data point represents the mean ± range of duplicate determinations (1×10⁴ cells/determination), and data presented are representative of four independent experiments.</p

Localization of labeled lipoprotein moieties of oxLDL and oxLDL-IC in endosomal vesicles.

<p>U937 cells were treated with endosomal marker (Alexa 488-transferrin, green) and with either DiI-labeled lipid moiety (red) (<b>A</b>), or Alexa 546-labeled protein moiety (red) (<b>B</b>) for 90 min and 5 h. Cells were treated with Alexa 488-transferrin (5 µg/ml) and with either labeled oxLDL (24 µg/ml) or labeled oxLDL-IC (32 µg/ml), fixed with 4% formaldehyde, suspended in sealed capillaries and visualized using Zeiss LSM 510 laser scanning confocal microscope. Arrows point at co-localization of lipid and apolipoprotein moities of oxLDL and oxLDL-IC with Alexa 488-transferrin.</p

Lipid moieties of oxLDL and oxLDL-IC co-localize when administered sequentially but not simultaneously.

<p>U937 cells were incubated with DiI-oxLDL-IC (red) and DiO-oxLDL (green) (<b>A</b>) sequentially and (<b>B</b>) in parallel in U937 cells. They were incubated for a total of 5 h. Sequential experiment involved 2 h incubation of oxLDL-IC (32 µg/ml), prior to addition of oxLDL (24 µg/ml) for 3 h. Lyso Tacker Blue DND-22, (50 nM) was used and applied for the last 30 min of incubation. Cells were fixed with 4% formaldehyde, suspended in sealed capillaries and visualized using Zeiss LSM 510 Laser scanning confocal microscope. Arrows point at co-localization of oxLDL in the lysosomal compartment in the upper panel, and the co-localization of oxLDL and oxLDL-IC in the lower panel.</p

Lipid moiety of oxLDL-IC but not oxLDL co-localizes with induced HSP70/70B' in endosomal vesicles.

<p>RAW 264.7 cells were transfected with HSP70-GFP (<b>A</b>) or HSP70B'-GFP (<b>B</b>), then treated with DiI-oxLDL (24 µg/ml), DiI-oxLDL-IC (32 µg/ml), or DPBS vehicle in serum-free DMEM for 3 h. Alexa 633-transferrin (10 µg/ml) was then added for an additional 2 h. Cells were then fixed with 4% formaldehyde, washed with DPBS, and visualized using confocal microscopy.</p

Localization of labeled lipoprotein moieties of oxLDL and oxLDL-IC in lysosomal compartment.

<p>U937 cells were treated with either DiI-labeled lipid moiety (red) (<b>A</b>), or Alexa 546-labeled protein moiety (red) (<b>B</b>) for 90 min and 5 h, with lysosomal marker (Lyso Tracker Green DND-26, 50 nM) applied for the last 30 min of incubation. Cells were treated with labeled oxLDL and oxLDL-IC at 18 µg/ml and 24 µg/ml, respectively. Live cells were washed with DPBS then suspended in sealed capillaries and visualized using confocal microscopy. Arrows point at co-localization of lipid and apolipoprotein moieties of oxLDL and oxLDL-IC with lysosomal compartment.</p

Differential effect of oxLDL and oxLDL-IC on mitochondrial membrane potential and ROS/RNS production.

<p>U937 cells were grown in phenol red-free IMDM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 50 µg/ml streptomycin, and IFN-γ (200 ng/ml) for 18 h then treated with oxLDL (90 µg/ml), oxLDL-IC, KLH-IC (120 µg/ml) in IMDM up to 5 h. (<b>A</b>) Detection of H₂O₂: Cells were treated with CM-H2DCFDA (5 µM), and Mito Tracker (100 nM) for 30 and 15 min, respectively, prior to conclusion of incubation time with the treatments (5 h). Cells were fixed, suspended in sealed capillaries and visualized using Zeiss LSM 510 Laser scanning confocal microscope. (<b>B</b>) Detection of NO: IFN-γ-treated cells were incubated with L-arginine (100 µM), and DAF-FM diacetate (10 µM) for 1 h, then washed with IMDM twice. Cells were then treated with oxLDL, oxLDL-IC, and KLH-IC as indicated above. Mito Tracker (100 nM) were added 15 min prior to conclusion of incubation time with the treatments (90 min). Live cells were washed, suspended in sealed capillaries and visualized using Zeiss LSM 510 Laser scanning confocal microscope.</p