Heat Shock Protein 70B′ (HSP70B′) Expression and Release in Response to Human Oxidized Low Density Lipoprotein Immune Complexes in Macrophages*

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.

Heat shock proteins (HSPs) 2 belong to a group of more than 20 highly conserved stress proteins that are routinely employed by cells as cytoprotective agents against a variety of stress stimuli, including heat shock, oxidative and mechanical stress, and inflammation (1,2). Evidence exists that HSP expression is higher at sites of atherosclerotic lesions than it is in normal tissue (3,4). Elevated HSP70 levels are associated with atherosclerotic plaques, particularly in areas with abundant activated macrophages, and seem to coincide around sites of necrosis and lipid accumulation (5,6).
Of particular interest in the development of atherogenesis is the role of oxidatively modified LDL (oxLDL). It is established that oxLDL particles are taken up by activated macrophages, resulting in lipid accumulation (7). oxLDL also triggers an immune response, initiating the production of predominantly proinflammatory IgG antibodies, which then form circulating complexes with oxLDL (oxLDL-IC) (8,9). These immune complexes activate macrophages through the Fc␥RI receptor, resulting in the release of proinflammatory cytokines (interleukin-1␤ (IL-1␤) and tumor necrosis factor-␣ (TNF-␣)) and the associated acceleration of foam cell formation (10 -13). Whereas free oxLDL have been shown to be cytotoxic to monocytic cells (14 -16), oxLDL complexed to IgG was found to promote survival (17)(18)(19).
Studies have demonstrated that circulating HSP60 is linked to cardiovascular disease (20). However, the role HSP70 family members play in the development of atherosclerotic plaques is still unclear. Increasing evidence suggests that HSPs may serve as cytokines themselves. Asea et al. (21) determined that activated macrophages secrete HSP70, which then binds to CD14 on the outer membrane, triggering the production and subsequent release of proinflammatory cytokines. This finding supports earlier experiments that induced cytokine production with the addition of exogenous HSP70 (22,23).
Elevated oxLDL, a hallmark of increased risk of atherosclerosis, has been implicated as the initial factor in the HSP70linked proinflammatory pathway of activated macrophages. Svensson et al. (24) demonstrated that high levels of oxLDL directly up-regulate and initiate release of HSP70 in macrophages, resulting in a corresponding increase in cytokine (IL-1␤ and IL-12) production. Whether exposure of human macrophages to elevated levels of oxLDL-IC elicits HSP70 regulation has not been previously examined.
We have recently shown that one member of the HSP70 family, HSP70BЈ (also known as HSP70 protein 6; gene HSPA6), displayed a considerable increase in expression in response to oxLDL-IC but not oxLDL alone (25). Furthermore, evidence was provided that IL-1␤ secretion is HSP70BЈ-dependent, suggesting a novel, oxLDL-IC-dependent proinflammatory mechanism involving the little known HSP70BЈ species (25). The HSP70BЈ gene is unique to the human genome, probably arising after the divergence of rodents and humans (26), and although HSP70BЈ and HSP70 are over 80% homologous, differences are present in sequences coding for substrate-binding and activation sites, evidence of a unique cellular function (27). Until recently, sequence data and immunological reagents for HSP70BЈ were unavailable, and due to the sequence homology, standard antibodies designed for the common HSP70A/B members most likely included the detection of HSP70BЈ. HSP70, while functionally inducible, is fairly ubiquitous at basal conditions in many different cell types, whereas HSP70BЈ is strictly inducible (27)(28)(29). Our recent findings have led to the hypothesis that HSP70BЈ expression could be induced by Fc␥-RI activation in human macrophages, resulting in increased proinflammatory cytokine release, prolonged foam cell survival, and thus the associated increased risk of atherosclerotic plaque development (18,25). Herein, we show that HSP70BЈ was induced and released in response to oxLDL-IC and that HSP70BЈ could bind to the cell-associated and internalized lipid moiety of oxLDL-IC. The presented data also implicated HSP70BЈ in cellular functions that underlie the ability of oxLDL-IC to promote prolonged activation of foam cells, such as regulation of sphingosine kinase 1 (SK1) activity and release of IL-10.

EXPERIMENTAL PROCEDURES
Cells-The human monocytic cell line U937 was obtained from the American Type Culture Collection (Manassas, VA) (ATCC CRL-1593.2). This line is a promonocytic lymphoma cell line, which originates from resident macrophages (30). Cells were maintained in Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 50 g/ml streptomycin at 37°C and 5% CO 2 . Unless otherwise indicated, cells were seeded at 1.5 ϫ 10 6 cells/ml and incubated in serum-free medium in the presence of 200 ng/ml interferon-␥ (IFN-␥) (EMD, Bioscience, San Diego, CA) for 18 h prior to the addition of experimental treatments. RAW 264.7 cells are a macrophage-like, Abelson leukemia virustransformed cell line derived from BALB/c mice. RAW 264.7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin, and strep-tomycin at 37°C and 5% CO 2 . For routine maintenance, cells were grown to 80% confluence and subcultured every 2 days.
Lipoprotein Isolation and Oxidation-LDL (d ϭ 1.019 -1.063 g/ml) was isolated from plasma of donors who were free from clinically apparent disease and oxidatively modified using Cu 2ϩ as described previously (25,31,32). The degree of LDL oxidation was monitored continuously by fluorescence emission at 234 nm using a fluorescence spectrophotometer (SLM-AMINCO Series 2; Spectronic Instruments, Rochester, NY) and stopped when the fluorescence reached a value of Ն1.1 fluorescence units (31,32). LDL modification was verified by particle migration on the Paragon electrophoresis system (Beckman Coulter, Fullerton, CA).
Preparation of Immune Complexes-oxLDL-IC were prepared with human oxLDL and purified human anti-oxLDL antibodies as described previously (13,25,33). Keyhole limpet hemocyanin immune complexes (KLH-IC) were used as a control immune complex because keyhole limpet hemocyanin has a molecular weight comparable with LDL and because it can engage Fc␥ receptors similar to oxLDL-IC but does not contain lipoproteins. Human KLH-IC was prepared as described previously (13). Immune complexes were suspended in phosphatebuffered saline (PBS), and the concentrations of total protein were determined using the BCA protein assay (Pierce).
Detection of HSP70 and HSP70BЈ in Cell Lysates and Conditioned Media-Cells were treated with oxLDL, oxLDL-IC, and KLH-IC (150 g/ml) for 2, 6, 12, and 24 h. Cells were pelleted and lysed using Extraction Reagent, a Tris-based buffer (Assay Designs, Ann Arbor, MI). Protein concentrations in the extracts were determined by the BCA protein assay. Equal amounts (10 g) of protein were electrophoresed in 4 -12% NuPAGE BisTris precast polyacrylamide gels (Invitrogen) under reducing conditions, transferred to nitrocellulose membranes, blocked in 5% nonfat milk in TBST (Tris-buffered saline, 0.1% Tween 20), and probed with mouse monoclonal antibodies against human HSP70 (BD Biosciences) and HSP70BЈ (Assay Designs). Membranes were then exposed to horseradish peroxidase-conjugated anti-mouse-IgG and visualized using Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences).
Protein in the media was captured using StrataClean TM resin (hydroxylated silica particles) (Stratagene) according to the manufacturer's instructions and then electrophoresed using 4 -12% precast NuPAGE gels under non-reducing conditions. HSP70 and HSP70BЈ were immunoblotted using monoclonal mouse anti-human antibodies as mentioned above.
Detection of HSPs in Detergent-insoluble Cell Membrane Fractions-To examine whether HSP70BЈ is translocated/associated with the cell membrane in response to oxLDL-IC, the NE-PER TM Nuclear and Cytoplasmic Extraction Reagents kit (Pierce) was used. The kit was used according to the manufacturer's instructions with modification to accommodate the insolubility of oxLDL-IC bound to membranes. Briefly, treated cells (8 ϫ 10 6 ) were pelleted by centrifugation at 400 ϫ g for 3 min. After isolating the cytoplasmic extract fraction using the designated detergent-containing reagent, the insoluble pellet including the immune complex was resuspended in 100 l of ice-cold nuclear extraction reagent, vortexed for 15 s every 10 min, for a total of 40 min, and then centrifuged at 16,000 ϫ g for 10 min. The supernatant was isolated immediately and mixed with 5 l of StrataClean TM resin. Captured proteins were analyzed by immunoblot as mentioned above.
Fluorescent Labeling of oxLDL and oxLDL-IC-Fluorescent labeling of oxLDL was performed as described previously (34) with modification. Briefly, oxLDL (1.0 ml, 1.0 mg of protein) was mixed with lipoprotein-deficient serum (1.0 ml) and then filter (0.22 m)-sterilized. A 50-l aliquot of DiI:1,1Ј-dioctadecyl-3,3,3Ј,3-tetramethylindocarbocyanine perchlorate (DiI) (Invitrogen), 3.0 mg/ml in dimethyl sulfoxide (Sigma), was added to the oxLDL/lipoprotein-deficient serum mixture. The mixture was gently mixed and incubated at 37°C for 8 h. To isolate the labeled oxLDL, the density of the solution containing the fluorescent labeled LDL was raised to 1.225 g/ml with solid KBr, and the solution was loaded into a polymer ultracentrifuge tube (13-ml tube, Beckman). Tubes were then filled with a saline solution whose density was adjusted to 1.21 g/ml with solid KBr. The labeled LDL was then floated out of the mixture by ultracentrifugation with a Beckman SW41 Ti rotor at 41,000 rpm, 36 h, 4°C. The labeled oxLDL floating at the top of the tube was aspirated, the density of this solution was raised to 1.225 g/ml, and the solution was again centrifuged at 41,000 rpm, 36 h, 4°C. The top layer was aspirated, dialyzed against NaCl-EDTA solution (150 mM NaCl/300 M EDTA, pH 8.6), and then filter-sterilized and stored at 4°C. The concentration of protein in the labeled oxLDL was determined using the BCA protein assay. Labeled oxLDL was used to prepare oxLDL-IC as described above.
Fluorescent Labeling of Anti-HSP70BЈ Antibody with Alexa Fluor 488-Mouse monoclonal anti-human HSP70BЈ antibody was labeled using the Alexa Fluor 488 monoclonal antibody labeling kit (Invitrogen) according to the manufacturer's protocol.
Detection of HSP70BЈ by Immunocytochemistry-Fluorescently labeled oxLDL (10 g/ml) and oxLDL-IC (30 g/ml) were incubated with cells for 5 h. Cells were then pelleted by centrifugation at 400 ϫ g for 3 min and then fixed and permeabilized using the Cytofix/Cytoperm TM kit (Pharmingen, San Diego, CA) according to the manufacturer's instructions. Cells were probed with Alexa Fluor 488-labeled monoclonal antihuman HSP70BЈ and were mounted using the VECTASH-IELD HardSet TM with 4Ј,6-diamidino-2-phenylindole antifade mounting medium (Vector Laboratories, Burlingame, CA) and visualized using confocal microscopy (Zeiss LSM 510 laserscanning confocal microscope, Carl Zeiss MicroImaging, Inc. (Thornwood, NY)). Fluorescent images were taken from representative fields of two independent experiments using the same gain and exposure settings, and the amount of green fluorescence was quantified using Adobe Photoshop CS2 to reveal the mean green channel intensity of individual cells as previously described (35).
Knockdown of HSP70 and HSP70BЈ-U937 cells were transfected with non-targeting or HSP70BЈ ON-TARGETplus SMARTpool siRNAs (Dharmacon RNA Technologies, Chicago, IL) (D-001206-13 and L-019455-00) using the Nucleofector TM device (Amaxa Inc., Gaithersburg, MD) according to the manufacturer's instructions. Knockdown was verified using quantitative PCR (Q-PCR) analysis. 48 h post-transfection, cells were primed with IFN-␥ and incubated in serum-free medium as described above or as indicated otherwise. Cells were then treated with oxLDL and oxLDL-IC (150 g/ml) for 6, 12, 24, 48, and 72 h. To detect HSP70, HSP70BЈ, and SK1 in cell lysates, equal amounts of protein (20 g) were separated on SDS-PAGE, transferred to nitrocellulose membranes, and probed with antibodies against HSP70 and HSP70BЈ as described above. SK1 was probed with a polyclonal antibody against phosphorylated SK1 (a gift from Dr. Stuart Pitson, Centre for Cancer Biology, Adelaide, Australia).
Expression of GFP-tagged human HSP70 and HSP70BЈ-HSP70 and HSP70BЈ cDNAs were purchased from OriGene (Rockville, MD) and used to generate a 1700-bp PCR amplicon using high fidelity Taq polymerase (Roche Applied Science) and custom designed primers (Table 1), with KpnI and ApaI restriction enzyme sites. The PCR products were cloned into a shuttle vector using the TOPO TA cloning kit (Invitrogen) and transformed in OneShot Top10 competent cells (Invitrogen). The product was sequenced in both directions to verify the clone. The cloned HSP sequences were then cloned into the pEGFP-N1 expression vector (Clontech, Mountain View, CA), coding for an enhanced green fluorescent protein. Ligation of HSP70 or HSP70BЈ sequences was confirmed by DNA sequencing. Transfection with LipoD293 TM DNA in vitro transfection reagent (SignaGen Laboratories) was performed according to the manufacturer's instructions. Briefly, 1 g of plasmid DNA and 3 l of LipoD293 TM reagent were each diluted into 50 l of serum-free Dulbecco's modified Eagle's medium. The mixtures were then combined and incubated for 15 min at room temperature. RAW 264.7 cells (80 -90% confluent) were incubated with the combined mixture under normal growing conditions for 18 h. Transfection reagent-containing medium was then replaced with complete medium and incubated for an additional 24 h prior to treatment. The amount of green fluorescence was quantified as described above. For live imaging, cells were maintained in serum-free treatment-containing medium in glass bottom 96-well plates (MatTek Corp., Ashland, MA) at 37°C and 5% CO 2 for the duration of the experiment.
Cell Toxicity-Numbers of non-viable cells were determined using trypan blue. Cell survival was also assessed using the CyQUANT cell proliferation assay kit from Molecular Probes, Inc. (Eugene, OR), a highly sensitive, fluorescence-based assay for determining the number of cultured cells (36). Briefly, cells were rinsed with PBS and lysed, and the DNA was stained using the CyQUANT fluorescent dye. Fluorescence was measured using a GENios multidetection plate fluorescence reader (MTX Lab Systems, Vienna, VA). Cell numbers were extrapolated using a standard curve generated from fluorescence readings of known counts of U937 cells. Alternatively, RAW 264.7 cells were trypsinized, neutralized, and counted using the Nexcelom Auto T4 Cellometer TM (Nexcelom Biosciences, Lawrence, MA).
Real-time Q-PCR-PCR primers were designed using the Beacon Designer 5 software (Premier Biosoft International, Palo Alto, CA). The forward and reverse primer sequences for HSP70BЈ (CCCTAAGGCTTTCCTCTTGC and CAT-GAAGCCGAGCAGTACAA) and for SK1 (CTGGCAGCT-TCCTTGAACCAT and TGTGCAGAGACAGCAGGTTCA) were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). IFN-␥-treated U937 cells were exposed to oxLDL-IC, oxLDL (150 g/ml), or PBS vehicle for 8 h. The RNAeasy mini kit was used to isolate mRNA (Qiagen), and complementary DNA (cDNA) was synthesized using iScript TM cDNA synthesis kit (Bio-Rad). Q-PCR was performed using the iCycler TM real-time detection system (Bio-Rad) with a two-step method using iQ TM SYBR Green Supermix (Bio-Rad). Amplification of glyceraldehyde-3-phosphate dehydrogenase was performed to standardize the amount of sample cDNA. Quantification was performed using the cycle threshold of receptor cDNA relative to that of glyceraldehyde-3-phosphate dehydrogenase cDNA in the same sample.
Cytokine Analysis-Conditioned medium was harvested and processed in duplicate with a custom Bio-Rad Bio-Plex human cytokine reagent kit for IL-1␤, IL-6, IL-10, IL-12 (p70), and TNF-␣ according to the manufacturer's instructions (Bio-Rad). Briefly, conditioned media containing 0.5% BSA were incubated with anti-cytokine-conjugated beads, followed by incubation with biotinylated detection antibody. The reaction mixture was detected with streptavidin-phycoerythrin and analyzed using a BioPlex 200 machine (Bio-Rad). Unknown cytokine concentrations were calculated by BioPlex Manager Software using standard curves derived from a recombinant cytokine standard.
Statistics-Significant differences between two groups were evaluated by Student's t test and between more than two groups by one-way or two-way analysis of variance followed by Tukey's post hoc test for mean separation (p Ͻ 0.05). All data are expressed as mean Ϯ S.D.

RESULTS
Differential Induction of HSP70 and HSP70BЈ by oxLDL and oxLDL-IC-To determine the expression levels of HSP70 and HSP70BЈ and the kinetics of this expression in response to oxLDL and oxLDL-IC in U937 monocytic cells, immunoblot analysis of HSP70 and HSP70BЈ in cells and conditioned media was performed. Fig. 1, A and B, demonstrates that in total cell lysate, HSP70 levels increased in cells treated with oxLDL, whereas base-line levels remained unchanged in cells treated with oxLDL-IC, KLH-IC, or the PBS vehicle. Increases in HSP70 in response to oxLDL occurred at 6 h and continued through 24 h post-treatment. HSP70BЈ expression levels, however, were undetectable in response to PBS or to either of the immune complex treatments but were significantly induced in response to oxLDL at 6 -24 h post-treatment (Fig. 1, A and B).
To determine whether HSP70 and HSP70BЈ were secreted, conditioned media were collected and probed for HSP70 and HSP70BЈ using immunoblot analysis. Fig. 1C shows that HSP70 was present in media collected from cells induced by oxLDL with a considerable increase at 24 h post-treatment. Interestingly, HSP70 was detected in media collected from cells exposed to the PBS vehicle but not from cells exposed to either of the immune complex treatments. In contrast, HSP70BЈ was only detected in the conditioned media of oxLDL-treated cells.
To examine whether HSP70BЈ was translocated to the cell membrane and therefore not detected in the whole cell lysate of cells induced with oxLDL-IC or KLH-IC, the cytoplasmic and membrane fractions were isolated and probed for HSP70BЈ. HSP70BЈ was detected at higher levels in the membrane fraction of oxLDL-treated cells than in oxLDL-IC-or KLH-ICtreated cells (Fig. 1D), consistent with data from whole cell lysates shown in Fig. 1A. These data suggested that immune complex-stimulated HSP70BЈ message was not translated or that the protein immediately degraded or complexed to undetermined molecule(s).
HSP70BЈ Is Up-regulated and Released in Response to oxLDL-IC-To investigate whether there was an association between HSP70BЈ and oxLDL-IC, we used fluorescently labeled oxLDL (DiI-oxLDL) and Alexa Fluor 488-labeled antibody specific against HSP70BЈ. Fig. 2 shows that HSP70BЈ was expressed at higher levels in response to oxLDL-IC compared with oxLDL alone. Furthermore, HSP70BЈ co-localized with extracellular membrane-associated oxLDL-IC. Although both oxLDL and oxLDL-IC induced the release of HSP70BЈ, as shown in Figs. 1C and 2A, respectively, the membrane-associated HSP70BЈ induced by oxLDL-IC was modestly detected using immunoblot analysis (Fig. 1D). This could be explained by poor extractability of membrane-associated HSP70BЈ, which might have become part of lipid rafts and/or the detergent-resistant membrane fraction.
Transfected HSP70BЈ-GFP Colocalizes with DiI-labeled Lipid Moiety of oxLDL-IC-To verify HSP70BЈ co-localization with insoluble oxLDL-IC and to avoid membrane permeabilization required for antibody-based visualization, we cloned HSP70BЈ into the pEGFP-N1 expression vector (HSP70BЈ-GFP) and transfected it in RAW 264.7 cells, an adherent mouse leukemic monocyte macrophage cell line. Fig. 3 shows that HSP70BЈ-GFP was expressed at higher levels in response to oxLDL-IC compared with oxLDL alone. This is consistent with data shown in Fig. 2. Interestingly, stimulation with oxLDL-IC resulted in co-localization of HSP70BЈ-GFP with the lipid moiety of oxLDL-IC both in extracellular and intracellular compartments. Fig. 3A shows also live cell imaging of HSP70BЈ-GFP co-localizing with intracellular DiI-oxLDL-IC between 3 and 4 h post-treatment (see supplemental Video 1).
HSP70-GFP and HSP70BЈ-GFP Are Up-regulated in Response to oxLDL-IC-HSP70 was also cloned into pEGFP-N1 expression vector (HSP70-GFP) to compare the response of HSP70BЈ-GFP and HSP70-GFP to oxLDL and oxLDL-IC. Fig. 4 shows that oxLDL-IC but not free oxLDL induced up-regulation of both HSP70-GFP and HSP70BЈ-GFP. KLH-IC induced also up-regulation of both HSP70-GFP and HSP70BЈ-GFP similar to oxLDL-IC (data not shown).
HSP70BЈ Regulates Cell Proliferation and Survival-In an effort to investigate the physiological relevance of HSP70BЈ expression, we examined the effect of overexpression of the HSP70BЈ in RAW 264.7 cells over 5 days post-transfection with HSP70BЈ-GFP (Fig. 5A). Interestingly, transfection with HSP70BЈ induced decreased cell proliferation compared with cells transfected with the empty GFP control vector. Although both HSP70BЈ-GFP-and HSP70-GFP-transfected cells showed decreased cell proliferation compared with control cells, HSP70BЈ-GFP-expressing cells exhibited significantly less proliferation than HSP70-GFP-expressing cells (Fig. 5A), suggesting a similar but possibly selective role for HSP70BЈ in macrophage cell growth.

FIGURE 2. oxLDL-IC induce up-regulation and release of HSP70B in U937 cells. Cells were incubated with
DiI-oxLDL-IC (30 g/ml) (A) or DiI-oxLDL (10 g/ml) (B) for 5 h and then fixed and permeabilized using the Cytofix/Cytoperm TM kit. Cells were probed with an Alexa Fluorா 488-labeled monoclonal antibody against HSP70BЈ and visualized by confocal microscopy. C, quantification of mean green channel intensity (n ϭ 10; difference between means is significant at p Ͻ 0.05). Results are representative of two independent experiments. The arrows point at co-localization of HSP70BЈ with membrane-associated and extracellular insoluble oxLDL-IC. Error bars, S.D. MAY 21, 2010 • VOLUME 285 • NUMBER 21 in U937 cells. Fig. 5B shows no significant difference in cell survival in response to oxLDL-IC between cells transfected with HSP70BЈ siRNA and scrambled siRNA. However, U937 cells showed decreased mortality following knockdown of HSP70BЈ in cells treated with oxLDL. This finding suggests that oxLDL-induced HSP70BЈ may serve as a proapoptotic mediator.

HSP70B Induction by Oxidized LDL Immune Complexes
HSP70BЈ Knockdown Results in Decreased SK1 mRNA and Protein Expression-We have previously shown that a prosurvival mechanism associated with oxLDL-IC signaling in U937 cells is mediated by activation of SK1 and that oxLDL-IC, but not oxLDL alone, induce an immediate membrane translocation and release of SK1 (18). To determine a regulatory relationship between HSP70BЈ, cell survival, and SK1, we examined the effect of HSP70BЈ knockdown on SK1 mRNA expression in the presence of oxLDL and oxLDL-IC (Fig. 6). A significant increase in HSP70BЈ mRNA expression was induced by oxLDL-IC at 6 h compared with other treatments in cells transfected with control siRNA (Fig.  6A). Interestingly, siRNA knockdown of HSP70BЈ inhibited SK1 mRNA expression in response to oxLDL-IC but not oxLDL alone (Fig. 6B), suggesting a regulatory link between HSP70BЈ and SK1. Using an antibody raised against phosphorylated SK1, Fig. 6C shows that activated SK1 protein was also decreased in response to knockdown of HSP70BЈ.
HSP70BЈ Knockdown Results in Increased IL-10 Secretion-We also examined the effect of HSP70BЈ on cell activation and cytokine release in U937 cells induced by oxLDL and oxLDL-IC. Using a multiplex human cytokine detection assay, the secretion of both pro-and anti-inflammatory cytokines known to be involved with macrophage activation was examined. Among the cytokines tested, the anti-inflammatory IL-10 displayed significant differences between control and HSP70BЈ siRNA-transfected cells (Fig. 7). In cells treated with oxLDL, whether HSP70BЈ was knocked down or not, IL10 levels were significantly decreased compared with both oxLDL-IC and vehicle treatments, with no difference between oxLDL-IC and vehicle treatments. Intriguingly, knockdown of HSP70BЈ resulted in an increase in IL10 levels whether cells were treated with oxLDL, oxLDL-IC, or PBS vehicle (Fig. 7).

DISCUSSION
In this study, we examined the role of HSP70BЈ, a poorly characterized member of the heat shock protein 70 family, in foam cell activation and survival induced by oxLDL-IC. HSP70BЈ appears to play a unique and complex role in macrophage activation associated with oxLDL-IC signaling. The results showed for the first time that human monocytic cells respond to oxLDL-IC by stimulating and secreting HSP70BЈ. The results also showed that HSP70BЈ appears to become associated with the intracellular as well as membraneassociated oxLDL-IC lipid moiety. Furthermore, the data demonstrated that HSP70BЈ is involved in cell survival, and this effect could be mediated by SK1 activation. The regulation of the antiinflammatory cytokine IL-10 by HSP70BЈ provides further evidence of the involvement of the little known member of the HSP70 family, HSP70BЈ, in foam cell functionality. Investigating the significance of HSPs, not only as classical chaperones and stress proteins, but as complex signaling molecules is of increasing importance in understanding the mechanisms involved in the activity of foam cells, a hallmark of atherosclerotic plaques.
In a recent study, we identified 83 genes as being similarly regulated by oxLDL-IC in the human leukemic monocyte lymphoma U937 cell line (25). Among the up-regulated genes, HSP70BЈ showed the highest increase in expression. Despite the substantial increase of HSP70BЈ at the gene level in response to oxLDL-IC, the fate of the protein could not be determined using immunoblot analysis (Fig. 1). The lack of HSP70BЈ protein suggested aborted translation, improper folding, and/or complexing to an undetermined molecule(s) concealing the antibody binding sites. Due to cross-linking of the Fc␥ receptors with immune complexes (oxLDL-IC and KLH-IC), we believe that the HSP70BЈ protein and possibly other stimulated proteins, such as SK1 (18,37), become part of lipid rafts and/or the detergent-resistant membrane fraction. To extract the cell membrane-associated HSP70BЈ, which is dem- onstrated clearly in the confocal microscopy images, we used several different detergents and extraction buffers (38) (data not shown). These did not improve the extractability of HSP70BЈ, from cells induced by immune complexes beyond what is shown in Fig. 1D. Interestingly, we were able to detect more HSP70BЈ protein in response to KLH-IC than oxLDL-IC (Fig. 1D), which reflects appropriately the higher gene expression of HSP70BЈ in response to KLH-IC compared with oxLDL-IC (25). Apparently, the protein expression of HSP70BЈ in response to oxLDL alone is mainly post-translational because it reached a plateau at 6 h post-treatment.
HSP70 has been shown to form complexes with a number of intracellular proteins (39), and more recently HSPs have been shown to bind to intracellular lipids (29,40,41). Our data suggest that oxLDL-IC on the surface of the cells become associated with secreted HSP70BЈ (Figs. 2 and 3). This association may inhibit the presumed autocrine effect of HSP70BЈ on cell signaling.
The differential trafficking of oxLDL and oxLDL-IC probably plays a role in the action of HSP70BЈ. Differences in trafficking could be due to differences in receptor binding, uptake, and delivery to lysosomes and/or to lysosomal and post-lysosomal processing. Several studies have previously shown that the lipid and protein moieties are metabolized in lysosomes within hours after internalization of oxLDL (42)(43)(44)(45). Live cell imaging shown in Fig.  3A demonstrated internalization of oxLDL-IC and co-localization with HSP70BЈ-GFP at 4 h post-treatment. This novel finding suggests that induced HSP70BЈ could bind to cytoplasmic oxLDL-IC but not to oxLDL in the lysosomal compartment.
There have been several reports implicating HSPs in survival of immune cells (46 -48), mostly suggesting a protective func-  Differences between means within treatment were evaluated by Student's t test (p Ͻ 0.05). *, significant difference between scrambled siRNA-transfected and HSP70BЈ siRNA-transfected cells treated with oxLDL-IC but not oxLDL. Results are representative of three independent experiments. Lower panel, cells transfected with scrambled or HSP70BЈ siRNA then induced with oxLDL (150 g/ml) for 2, 6, and 12 h; an equal amount of protein (20 g) was separated on SDS-PAGE, probed for HSP70BЈ, and then re-probed for HSP70. tion against inflammation and other types of stressors. Our data suggest that inducible HSP70BЈ may have an inhibitory effect on macrophage cell growth and survival. For example, cells overexpressing HSP70BЈ showed significantly decreased cell proliferation and knockdown of HSP70BЈ-attenuated oxLDLinduced cell death compared with controls.
A mechanism regularly implicated in mediating prosurvival and inflammatory responses to macrophage cell survival is regulation of SK1, an enzyme that is responsible for the generation of the signaling molecule sphingosine 1-phosphate (49). We have recently shown that SK1 mRNA levels increased in response to oxLDL-IC in U937 cells (25), and we also showed that oxLDL-IC prompt the release of SK1 into the medium, suggesting the generation of sphingosine 1-phosphate extracellularly (18). It has recently been reported that overexpression of HSP70 in RAW 264.7 macrophages resulted in both increased SK1 protein and mRNA levels, contributing to the partial reversal of cell death caused by the combination of LPS and SK1 inhibitor (50). In this study, we show that knockdown of HSP70BЈ reduced the expression of SK1 by half in response to oxLDL-IC, suggesting that HSP70BЈ is an upstream mediator of SK1. Furthermore, phosphorylated SK1 is significantly reduced in U937 cells transfected with HSP70BЈ siRNA compared with control cells treated with oxLDL. These data are the first to suggest an interaction between HSP70BЈ and SK1 and could significantly influence further research to define SK1 effects mediating macrophage activation and survival.
The anti-inflammatory cytokine, IL-10, has been shown to be cytoprotective in macrophages (51) and induced by the addition of exogenous HSP70 in synovial cells (52,53), suggesting that HSPs play an active role in suppressing inflammation by the regulation of IL-10. Here we provide evidence that secretion of IL-10 may be regulated by HSP70BЈ.
Although HSPs are classically viewed as intracellular proteins, we provided evidence that HSP70BЈ is induced and secreted in response to cell activation by oxLDL-IC. This is in agreement with previous reports demonstrating that HSPs may be released by viable cells under inflammatory stress (3,22). Recently, it has been shown that HSP70 can be secreted from cells via exosomes (54,55). Moreover, it has been shown that in stressed cells, HSPs may be inserted into the plasma membrane before release into the extracellular environment in membrane-associated structures (56). The molecular mechanisms that regulate the mobilization of secretory vesicles and secretion of mediators from inflammatory cells, including macrophages, are still obscure (57, 58). FIGURE 6. HSP70B knockdown results in decreased SK1 mRNA and protein expression in U937 cells. Cells were transfected with scrambled siRNA (sc) or HSP70BЈ siRNA, primed with IFN-␥ for 18 h, and then incubated in serum-free medium for 2 h prior to treatment with oxLDL, oxLDL-IC (150 g/ml), or PBS vehicle for 6 h. A, Q-PCR analysis of HSP70BЈ mRNA levels; B, Q-PCR analysis of SK1 mRNA levels. Quantification of RNA was performed using the cycle threshold of HSP70BЈ and SK1 cDNA relative to that of GAPDH. Data are expressed as means Ϯ 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 HSP70BЈ siRNA and then induced with oxLDL (150 g/ml) for 2 and 6 h; an equal amount of protein (20 g) was separated on SDS-PAGE, probed for phosphorylated SK1, and then reprobed for HSP70BЈ. *, significantly different from all other groups (p Ͻ 0.05). **, significantly different from scrambled siRNA-transfected oxLDL-IC-treated cells (p Ͻ 0.05). In conclusion, our current data suggest that HSP70BЈ is induced and released by macrophages stimulated by oxLDL-IC and that once released into the extracellular space, HSP70BЈ immediately associates with oxLDL-IC. We also provided evidence that a variety of cellular responses, including regulation of SK1 and release of the anti-inflammatory prosurvival cytokine IL-10, could be mediated by HSP70BЈ. Our findings contribute to the understanding of foam cell survival and activation and may advance efforts to reveal additional therapeutic targets that could mediate the stabilization of vulnerable atherosclerotic plaques.