Differential regulation of acid sphingomyelinase in macrophages stimulated with oxidized low-density lipoprotein (LDL) and oxidized LDL immune complexes: role in phagocytosis and cytokine release

Abstract

Oxidized low-density lipoprotein (oxLDL) and oxLDL-containing immune complexes (oxLDL-IC) contribute to the formation of lipid-laden macrophages (foam cells). Fcγ receptors mediate uptake of oxLDL-IC, whereas scavenger receptors internalize oxLDL. We have previously reported that oxLDL-IC, but not free oxLDL, activate macrophages and prolong their survival. Sphingomyelin is a major constituent of cell membranes and lipoprotein particles and acid sphingomyelinase (ASMase) hydrolyses sphingomyelin to generate the bioactive lipid ceramide. ASMase exists in two forms: lysosomal (L-ASMase) and secretory (S-ASMase). In this study we examined whether oxLDL and oxLDL-IC regulate ASMase differently, and whether ASMase mediates monocyte/macrophage activation and cytokine release. The oxLDL-IC, but not oxLDL, induced early and consistent release of catalytically active S-ASMase. The oxLDL-IC also consistently stimulated L-ASMase activity, whereas oxLDL induced a rapid transient increase in L-ASMase activity before it steadily declined below baseline. Prolonged exposure to oxLDL increased L-ASMase activity; however, activity remained significantly lower than that induced by oxLDL-IC. Further studies were aimed at defining the function of the activated ASMase. In response to oxLDL-IC, heat-shock protein 70B' (HSP70B') was up-regulated and localized with redistributed ASMase in the endosomal compartment outside the lysosome. Treatment with oxLDL-IC induced the formation and release of HSP70-containing and IL-1β-containing exosomes via an ASMase-dependent mechanism. Taken together, the results suggest that oxLDL and oxLDL-IC differentially regulate ASMase activity, and the pro-inflammatory responses to oxLDL-IC are mediated by prolonged activation of ASMase. These findings may contribute to increased understanding of mechanisms mediating macrophage involvement in atherosclerosis.

Figure 1

Oxidized low-density lipoprotein-containing immune complexes (oxLDL-IC) but not oxLDL induce acid sphingomyelinase (ASMase) release. After priming with interferon-γ (IFN-γ) overnight, U937 cells were re-suspended in fresh medium containing IFN-γ and 1% fetal bovine serum for 1 hr before treatment with either 3,3′-dioctadecyloxacarbocyanine perchlorate-labelled (DiO-) oxLDL (75 μg/ml) (a), DiO-oxLDL-IC (100 μg/ml) (b), or Dulbecco's PBS-treated for 5 hr (c) then fixed at 30 min or 5 hr post-treatment; ASMase was visualized using anti-ASMase antibody and secondary Alexa-Fluor 594-labelled F(ab)’₂ anti-rabbit antibody. Cells were treated with unlabelled oxLDL-IC, and secondary antibody only as a control (lower panel). Yellow denotes areas of ASMase and oxLDL-IC co-localization. Results are representative of two independent experiments.

Figure 2

Acid sphingomyelinase (ASMase) activity is regulated in a time- and dose-dependent manner. U937 cells were incubated with oxidized low-density lipoprotein (oxLDL; 75 μg/ml), oxLDL-containing immune complexes (oxLDL-IC), or keyhole limpet haemocyanin-containing immune complexes (KLH-IC; 100 μg/ml) and ASMase activity was measured in conditioned media [secretory (S) –ASMase] (a, c) or in cell lysates [lysosomal (L) -ASMase] (b, d). (a) Extracellular S-ASMase activity associated with oxLDL and oxLDL-IC was measured after immunoprecipitation of oxLDL moiety using magnetically labelled Protein G conjugated to anti-oxLDL antibody. (b) A short time–course of L-ASMase activity was measured in cell lysates. ASMase activity was measured in the conditioned media (S-ASMase) (c) or in cell lysates (L-ASMase) (d) from 0 to 18 hr. Different letters denote significant differences among means at each time-point (P < 0·05). S-ASMase (e) and L-ASMase (f) activity was measured after 18 hr-treatment of U937 cells incubated with various doses of oxLDL, oxLDL-IC and KLH-IC. Different letters denote significant difference among means at each concentration (P < 0·05). Data shown are mean ± SE from duplicate samples from two independent experiments (n = 4).

Figure 3

Acid sphingomyelinase (ASMase) co-localizes with the lipid moiety oxidized low-density lipoprotein-containing immune complexes (oxLDL-IC) but not with oxLDL. (a) RAW 264·7 cells were transfected with ASMase-dsRed and treated with either 3,3′-dioctadecyloxacarbocyanine perchlorate-labelled (DiO-) oxLDL (top panel) or DiO-oxLDL-IC (bottom panel) (24 and 32 μg/ml, respectively) for 4 hr before live visualization by confocal microscopy. (b) ASMase is involved with the phagocytosis of DiO-oxLDL-IC. Arrow denotes an area of ASMase-dsRed and DiO-oxLDL-IC co-localization (yellow) in a phagosome.

Figure 4

Exogenous sphingomyelinase enhances the uptake of oxidized low-density lipoprotein-containing immune complexes (oxLDL-IC). U937 cells were incubated with and without 200 mU bacterial sphingomyelinase (bSMase) and with (a) 1,1′-dioctadecyl-3,3,3′,3-tetramethy lindocarbocyanineperchlorate-labelled (DiI-) oxLDL (24 μg/ml, top panels) or DiI-oxLDL-IC (32 μg/ml); or (b) with both 3,3′-dioctadecyloxacarbocyanine perchlorate-labelled (DiO-) oxLDL and DiI-oxLDL-IC simultaneously for 5 hr. Thirty minutes before live visualization by confocal microscopy, the cells were treated with LysoTracker Green (a) or LysoTracker Blue (b). Arrows in (a) denote co-localization (yellow) of DiI-oxLDL (red) with LysoTracker Green. Arrows in (b) top panel denote co-localization (turquoise blue) of DiO-oxLDL (green) with LysoTracker Blue; arrow in lower panel denotes co-localization (yellow) of DiO-oxLDL (green) and DiI-oxLDL-IC (red).

Figure 5

Acid sphingomyelinase (ASMase) co-localizes with heat-shock protein 70B’ (HSP70B’) in the endosomal compartment outside lysosomes. RAW 264·7 cells were co-transfected with both HSP70B’-GFP and ASMase-dsRed before treatment with either unlabelled oxidized low-density lipoprotein (oxLDL; 75 μg/ml) or oxLDL-containing immune complexes (oxLDL-IC; 100 μg/ml) for 5 hr. The cells were fixed in 4% paraformaldehyde and permeabilized using 0·25% saponin and 10% fetal bovine serum in PBS. Lysosomes were probed with anti-LAMP-1 conjugated to Alexa-fluor 647 (blue). Yellow, co-localization of HSP70B’-GFP with ASMase-dsRed; purple, co-localization of ASMase-dsRed with LAMP-1; NS, non-stimulated.

Figure 6

Disruption of lysosomal acid sphingomyelinase (L-ASMase) inhibits internalization of extracellular oxidized low-density lipoprotein-containing immune complexes (oxLDL-IC) aggregated with heat-shock protein 70B’ (HSP70B’) and ASMase. RAW 264.7 cells were transfected with both ASMase-dsRed and HSP70B’-GFP and treated with oxLDL-IC (100 μg/ml) (a) in the presence and absence of desipramine for 4 hr (b). Pretreatment with desipramine (20 μm) was for 2 hr. Cells were visualized live by confocal microscopy. Yellow, co-localization of HSP70B’-GFP with ASMase-dsRed; arrows denote co-localization of HSP70B’-GFP with ASMase-DsRed extracellularly associated with oxLDL-IC aggregates. (c) ASMase activity was measured after 5 hr of incubation with oxLDL (75 μg/ml) or oxLDL-IC (100 μg/ml) in cell lysates (left panel, L-ASMase) or conditioned medium (right panel, S-ASMase). * denotes significant difference between desipramine and no-desipramine treatments (P < 0·05). ** denotes significant difference between oxLDL-IC and each of oxLDL and non-stimulated (NS) treatments. # denotes significant difference between oxLDL-IC and each of the corresponding oxLDL and NS treatments; there was no significant difference in S-ASMase activity in response to desipramine treatment. Data shown are mean ± SE from duplicate samples from two independent experiments (n = 4).

Figure 7

Treatment with oxidized low-density lipoprotein-containing immune complexes (oxLDL-IC) induces the formation of exosomes. U937 cells were either not stimulated (lane 1), or were treated for 5 hr with oxLDL (75 μg/ml) (lane 2), oxLDL-IC (100 μg/ml) (lane 3), or oxLDL-IC (100 μg/ml) in the presence of desipramine (20 μm) (lane 4). Exosomes were isolated from conditioned media, purified using a discontinuous sucrose gradient, and prepared for Western blotting as described in Materials and Methods. Data shown are representative of three independent experiments.

Figure 8

Acid sphingomyelinase (ASMase) activity is required for interleukin-1β (IL-1β) secretion in exsosomes. (a) U937 cells were transfected with ASMase small interfering (si) RNA and treated with oxidized low-density lipoprotein (oxLDL; 75 μg/ml) or oxLDL-immune complexes (oxLDL-IC; 100 μg/ml) and then conditioned media were collected and analysed by ELISA for IL-1β at various time-points. *denotes significant difference between oxLDL-treated cells and oxLDL-IC-treated cells within the control-transfected group at each time-point (P < 0·01). Both oxLDL and oxLDL-IC treatments exhibit significant differences between control and ASMase siRNA-transfected cells (P < 0·01). (b), Monocytes were isolated from ASMase knockout and wild-type mice and treated as mentioned above for U937 cells. *denotes significant difference between oxLDL-treated and oxLDL-IC-treated cells within the wild-type group at each time-point (P < 0·01). #denotes significant difference between ASMase-expressing monocytes and monocytes lacking ASMase expression in oxLDL-treated cells (P < 0·01). Data shown are Mean ± SE from duplicate samples from two independent experiments (n = 4).