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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'.

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Heat shock proteins (HSPs) have been implicated in the activation and survival of macrophages. This study examined the role of HSP70B, a poorly characterized member of the HSP70 family, in response to oxidatively modified LDL (oxLDL) and immune complexes prepared with human oxLDL and purified human antibodies to oxLDL (oxLDL-IC) in monocytic and macrophage cell lines. Immunoblot analysis of cell lysates and conditioned medium from U937 cells treated with oxLDL alone revealed an increase in intracellular HSP70B protein levels accompanied by a concomitant increase in HSP70B extracellular levels. Fluorescence immunohistochemistry and confocal microscopy, however, demonstrated that oxLDL-IC stimulated the release of HSP70B, which co-localized with cell-associated oxLDL-IC. In HSP70B-green fluorescent protein-transfected mouse RAW 264.7 cells, oxLDL-IC-induced HSP70B co-localized with membrane-associated oxLDL-IC as well as the lipid moiety of internalized oxLDL-IC. Furthermore, the data demonstrated that HSP70B is involved in cell survival, and this effect could be mediated by sphingosine kinase 1 (SK1) activation. An examination of regularly implicated cytokines revealed a significant relationship between HSP70B and the release of the anti-inflammatory cytokine interleukin-10 (IL-10). Small interfering RNA knockdown of HSP70B resulted in a corresponding decrease in SK1 mRNA levels and SK1 phosphorylation as well as increased release of IL-10. In conclusion, these findings suggest that oxLDL-IC induce the synthesis and release of HSP70B, and once stimulated, HSP70B binds to the cellassociated and internalized lipid moiety of oxLDL-IC. The data also implicate HSP70B in key cellular functions, such as regulation of SK1 activity and release of IL-10, which influence macrophage activation and survival.