Heat shock protein 70B' (HSP70B') expression and release in response to human oxidized low density lipoprotein immune complexes in macrophages

Abstract

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 cell-associated 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.

FIGURE 1.

Immunoblot analysis of HSP70 and HSP70B′ expression in U937 cells in response to oxLDL and oxLDL-IC. A and B, total cell lysates; C, conditioned media; D, membrane/cytoplasmic fractions. A–C, cells (1.5 × 10⁶ cells/ml) were treated with oxLDL, oxLDL-IC, KLH-IC (150 μg/ml), or PBS vehicle for 2, 6, 12, and 24 h. Equal protein (20 μg) was loaded in each lane and probed for cellular HSP70 and HSP70B′ (A); densitometry of HSP70 and HSP70B′ in A were normalized to actin and presented in B. Total protein secreted into the conditioned media was captured using StrataClean resin and subjected to immunoblot analysis (C). Results in A–C are representative of three independent experiments. D, cells (1.5 × 10⁶ cells/ml) were treated with oxLDL, oxLDL-IC, KLH-IC (150 μg/ml), or PBS vehicle for 6 h and then lysed using the NE-PER^TM nuclear and cytoplasmic extraction reagent kit to obtain membrane and cytoplasmic fractions. Equal protein (30 μg) was loaded in each lane and probed for HSP70B′. Results in D are representative of two independent experiments. m, membrane fractions; c, cytoplasmic fractions.

FIGURE 2.

oxLDL-IC induce up-regulation and release of HSP70B′ in U937 cells. Cells were incubated with DiI-oxLDL-IC (30 μg/ml) (A) or DiI-oxLDL (10 μg/ml) (B) for 5 h and then fixed and permeabilized using the Cytofix/Cytoperm^TM kit. Cells were probed with an Alexa Fluor® 488-labeled monoclonal antibody against HSP70B′ and visualized by confocal microscopy. C, quantification of mean green channel intensity (n = 10; difference between means is significant at p < 0.05). Results are representative of two independent experiments. The arrows point at co-localization of HSP70B′ with membrane-associated and extracellular insoluble oxLDL-IC. Error bars, S.D.

FIGURE 3.

oxLDL-IC induce co-localization of transfected HSP70B′-GFP with DiI-labeled lipid moiety. A and B, RAW 264.7 cells were transfected with GFP-HSP70B′ and then incubated with DiI-oxLDL-IC (30 μg/ml) (A), or DiI-oxLDL (10 μg/ml) (B) for 4 h, fixed with 3% paraformaldehyde, and visualized by confocal microscopy. Live Cell Imaging panel, HSP70B′ co-localization with DiI-labeled lipid moiety of DiI-oxLDL-IC (see supplemental Video 1). C, quantification of mean green channel intensity (n = 10; difference between means is significant at p < 0.05). Results are representative of three independent experiments. The arrows point at co-localization of HSP70B′-GFP with the lipid moiety of oxLDL-IC. Error bars, S.D.

FIGURE 4.

oxLDL-IC induce up-regulation of both HSP70-GFP and HSP70B′-GFP in RAW 264.7 cells. Cells were transfected with HSP70-GFP or HSP70B′-GFP and then treated with oxLDL, oxLDL-IC (150 μg/ml), or PBS vehicle for 6 h, fixed with 4% formaldehyde, and visualized by confocal microscopy. Results are representative scoped fields in three independent experiments.

FIGURE 5.

HSP70B′ regulates cell proliferation and survival. A, transfection with HSP70B′-GFP induced decreased cell proliferation compared with HSP70-GFP in RAW 264.7 cells. Cells were transfected with HSP70B′-GFP or HSP70-GFP, maintained in complete media, and then counted at 6, 12, 24, 48, 72, and 96 h post-transfection using a Nexcelom Auto T4 Cellometer^TM. Plotted values are means ± S.D. (error bars) of triplicate values. Data were analyzed by one-way analysis of variance; different letters denote significant differences among means at each time point (p < 0.05). Results are representative of three independent experiments. B, knockdown of HSP70B′ inhibited oxLDL-induced cell death in U937 cells. Upper panel, cells were transfected with scrambled siRNA (sc) or HSP70B′ siRNA and then treated with oxLDL or oxLDL-IC (150 μg/ml) for 72 h; cell viability was assessed using a CyQUANT DNA assay. Plotted values are means ± S.D. of triplicate values. Differences between means within treatment were evaluated by Student's t test (p < 0.05). *, significant difference between scrambled siRNA-transfected and HSP70B′ siRNA-transfected cells treated with oxLDL-IC but not oxLDL. Results are representative of three independent experiments. Lower panel, cells transfected with scrambled or HSP70B′ siRNA then induced with oxLDL (150 μg/ml) for 2, 6, and 12 h; an equal amount of protein (20 μg) was separated on SDS-PAGE, probed for HSP70B′, and then re-probed for HSP70.

FIGURE 6.

HSP70B′ knockdown results in decreased SK1 mRNA and protein expression in U937 cells. Cells were transfected with scrambled siRNA (sc) or HSP70B′ siRNA, primed with IFN-γ for 18 h, and then incubated in serum-free medium for 2 h prior to treatment with oxLDL, oxLDL-IC (150 μg/ml), or PBS vehicle for 6 h. A, Q-PCR analysis of HSP70B′ mRNA levels; B, Q-PCR analysis of SK1 mRNA levels. Quantification of RNA was performed using the cycle threshold of HSP70B′ and SK1 cDNA relative to that of GAPDH. Data are expressed as means ± S.D. (error bars) of triplicate values. Data were analyzed by two-way analysis of variance with transfection and treatment as variables. Data are representative of two experiments. C, cells transfected with control or HSP70B′ siRNA and then induced with oxLDL (150 μg/ml) for 2 and 6 h; an equal amount of protein (20 μg) was separated on SDS-PAGE, probed for phosphorylated SK1, and then reprobed for HSP70B′. *, significantly different from all other groups (p < 0.05). **, significantly different from scrambled siRNA-transfected oxLDL-IC-treated cells (p < 0.05).

FIGURE 7.

HSP70B′ knockdown results in increased IL-10 secretion in U937 cells. Cells were transfected with HSP70B′ siRNA, primed with IFN-γ, and treated with oxLDL, oxLDL-IC, or vehicle as described in the legend to Fig. 6. Conditioned medium was collected after 12 h, and IL-10 was quantified using the Bio-Plex human cytokine kit (Bio-Rad). Plotted values are means ± S.D. (error bars) of triplicate values. Differences between means were evaluated by Student's t test (p < 0.05). Results are representative of three independent experiments. *, significantly different from control transfected cells within each treatment; #, significantly different from PBS and oxLDL-IC in control transfected cells; †, significantly different from PBS and oxLDL-IC in HSP70B′ siRNA-transfected cells.