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Objective-To compare the global effects of oxidized LDL (oxLDL) and oxLDL-containing immune complexes (oxLDL-IC) on gene expression in human monocytic cells and to identify differentially expressed genes involved with inflammation and survival.Methods and Results-U937 cells were treated with oxLDL-IC, oxLDL, Keyhole limpet hemocyanin immune complexes (KLH-IC), or vehicle for 4 h. Transcriptome profiling was performed using DNA microarrays. oxLDL-IC uniquely affected the expression of genes involved with pro-survival (RAD54B, RUFY3, SNRPB2, and ZBTB24). oxLDL-IC also regulated many genes in a manner similar to KLH-IC. Functional categorization of these genes revealed that 39% are involved with stress responses, including the unfolded protein response which impacts cell survival, 19% with regulation of transcription, 10% with endocytosis and intracellular transport of protein and lipid, and 16% with inflammatory responses including regulation of I-κB/NF-κB cascade and cytokine activity. One gene in particular, HSP70 6, greatly up-regulated by ox-LDL-IC, was found to be required for the process by which oxLDL-IC augments IL1-β secretion. The study also revealed genes uniquely up-regulated by oxLDL including genes involved with growth inhibition (OKL38, NEK3, and FTH1), oxidoreductase activity (SPXN1 and HMOX1), and transport of amino acids and fatty acids (SLC7A11 and ADFP).Conclusions-These findings highlight early transcriptional responses elicited by oxLDL-IC that may underlie its cytoprotective and pro-inflammatory effects. Cross-linking of Fcγ receptors appears to be the trigger for most of the transcriptional responses to oxLDL-IC. The findings further strengthen the hypothesis that oxLDL and oxLDL-IC elicit disparate inflammatory responses and play distinct roles in the process of atherosclerosis.

Summary 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.

BackgroundOxidized 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 FCγ receptor I (FCγ RI). This study examined differences in intracellular trafficking of lipid and apolipoprotein moieties of oxLDL and oxLDL-IC and the impact on oxidative stress.Methodology/FindingsFluorescently 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.Conclusions/SignificanceFindings 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'.

Macrophages play a central role in innate immune responses, in disposal of cholesterol, and in tissue homeostasis and remodeling. To perform these vital functions macrophages display high endosomal/lysosomal activities. Recent studies have highlighted that acid sphingomyelinase (ASMase), which generates ceramide from sphingomyelin, is involved in modulation of membrane structures and signal transduction in addition to its metabolic role in the lysosome. In this review, we bring together studies on ASMase, its different forms and locations that are necessary for the macrophage to accomplish its diverse functions. We also address the importance of ASMase to several disease processes that are mediated by activated macrophages.