Previously, we reported that heat shock protein (HSP)65 impairs the effects of high-density lipoprotein on macrophages. We also showed that immune response activation adversely affects reverse cholesterol transport (RCT). In this study, we investigated the effects of the Src family kinase lymphocyte-specific protein tyrosine kinase (Lck) and elucidated the mechanism underlying HSP65-regulated cholesterol efflux in T cells. We evaluated cell proliferation, Lck expression, and inflammatory cytokine production in Jurkat cells and CD4+ T cells. HSP65-mediated inhibition of RCT was assessed by evaluating ABCA1, ABCG1, SR-BI, PPAR-γ, and liver X receptor-α expression. A dose-dependent relationship was found between the levels of these proteins and the suppression of cholesterol efflux. Stimulation of Lck-silenced T cells with ionomycin resulted in a decrease in intracellular calcium levels. Treatment of Jurkat cells with PP2, an inhibitor of Src family kinase, inhibited calcium-induced, but not PMA-induced, ERK phosphorylation. NF-κB activation in response to PMA was minimally inhibited in cells stimulated with PP2. HSP65 failed to trigger downstream ERK or JNK phosphorylation or to activate NF-κB or protein kinase C-γ in Lck-silenced cells. Additionally, elevation of intracellular calcium was also impaired. However, HSP65 significantly enhanced cholesterol efflux and decreased cellular cholesterol content by inducing the expression of cholesterol transport proteins in Lck-silenced cells. The treatment of Jurkat cells with PP2 also inhibited cell proliferation and promoted RCT. In conclusion, Lck is a key molecule in the TCR signaling cascade that inhibits cholesterol efflux and upregulates intracellular cholesterol ester content in T cells. Our results demonstrate that the immune response plays a previously unrecognized role in RCT.

Atherosclerosis (AS) is now viewed as an immune-inflammatory syndrome (13).Vulnerable atherosclerotic plaques are occupied by tissue-injurious CD4+ T cells, which destabilize tissue integrity through multiple damage pathways (4).T cells secrete signature cytokines that magnify and sustain inflammatory responses in the intima (58).When engaged by Ags, TCR is thought to initiate protein tyrosine kinase–dependent signaling events (9). A central molecule in this process is the Src family kinase lymphocyte-specific protein tyrosine kinase (Lck), which phosphorylates downstream signaling proteins, resulting in changes in gene expression that are critical for T cell activation and the development of AS (10, 11). There has been evidence to suggest that heat shock protein (HSP)65, a major autoantigen, contributes to the initiation and development of autoimmunity and AS (12, 13). T cells specifically responding to HSP65 have been found in atherosclerotic lesions (14). HSP65 can also specifically bind to TCR to initiate immune responses, resulting in the production of proinflammatory cytokines (15). Many studies focusing on atherosclerotic plaques have used the Jurkat cell line to simulate cellular immune responses during the progression of AS (16, 17).

Cholesterol efflux capacity, a new biomarker that has been used to characterize a key step in reverse cholesterol transport (RCT), is inversely associated with incidence of cardiovascular events (18, 19). Recent studies investigating the underlying mechanism of this association have suggested that lipid homeostasis and immunity may be intrinsically coupled. Activation of inflammation is critically sensitive to RCT; conversely, activation of immune-inflammatory responses modifies RCT (2022). TCR signaling is essential to cholesterol efflux and the upregulation of ATP-binding cassette transporters in T cells (23, 24). Liver X receptor (LXR) signaling is a metabolic checkpoint that modulates cell proliferation and immunity (25). Collectively, the above evidence suggests that immune signaling has a potential role in cholesterol homeostasis.

Our previous studies have indicated that HSP65 decreases the expression of cholesterol transport proteins in macrophages. These changes in expression hinder the process of RCT and accelerate the progression of AS (26). However, the inhibitory effects of HSP65 on RCT in T cells are unknown. In the present study, we report a novel mechanism through which Lck may inhibit cholesterol efflux in T cells by negatively regulating cholesterol transport proteins. Our data indicate that HSP65 suppresses cholesterol efflux and increases cellular cholesterol content through an Lck-mediated pathway in T cells. Targeting cholesterol metabolism in immune cells might therefore be a novel therapeutic approach toward preventing atherogenesis.

[3H]cholesterol was purchased from PerkinElmer (Shanghai, China). ApoA-I and PP2 were acquired from Calbiochem (Darmstadt, Germany). CCK-8 was obtained from Dojindo (Kumamoto, Japan). Ficoll was obtained from Amersham Biosciences (Uppsala, Sweden). PMA and ionomycin were purchased from Beyotime (Shanghai, China). Anti-human CD4 PE (clone, OKT4) and functional-grade, purified anti-CD3 and anti-CD28 (clones HIT3a and CD28.2, respectively) were obtained from eBioscience. A human CD4+ T cell isolation kit was obtained from Miltenyi Biotec (Bergisch Gladbach, Germany). An ELISA kit was purchased from Cusabio Biotech. Western blotting reagents, including mAbs against β-actin (bs-0061R), ABCA1 (ab151585), ABCG1 (ab52617), SR-B1 (ab106572), PPAR-γ (ab19481), LXR-α (ab106464), protein kinase C (PKC)-γ (ab71558), and Lck (ab3885), were acquired from Abcam; mAbs against phosphorylated and nonphosphorylated ERK (Thr202/Tyr204) (no. 9101), phosphorylated and nonphosphorylated JNK (Thy183/Thy185) (no. 4671), and NF-κB p65 (no. 8242) were acquired from Cell Signaling Technology. Ca2+ Fluo-3 AM was obtained from Sigma-Aldrich (St. Louis, MO). A Fluo-8 no wash calcium assay kit was acquired from AAT Bioquest. RT-PCR primers were obtained from Sango (Shanghai, China). RNA TRIzol was obtained from Life Technologies. A PrimeScript reverse transcription reagent kit and quantitative PCR mix were acquired from Toyobo (Osaka, Japan).

Recombinant HSP65 was obtained from StressMarq Biosciences (Victoria, BC, Canada). We measured the endotoxicity of the recombinant HSP65 protein using a commercial kit (test kit for the detection of Gram-negative bacteria LPS with photometric assay; Zhanjiang A&C Biological). The sensitivity was <0.05 endotoxin unit/ml. Based on two experiments that resulted in a coefficient of variation < 15%, the level of LPS in HSP65 was <0.05 endotoxin unit/ml.

Jurkat cells (human acute T lymphocyte leukemia cell lines TCHU123 and CCTCC) were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin (all from Life Technologies) at 37°C in 5% CO2. Jurkat cells, a continuously growing T lymphoma cell line, are derived from humans and show many similarities to primary lymphocytes. These cells are commonly used to investigate the properties of the major cell types present in atherosclerotic plaques.

EDTA-anticoagulated blood was obtained from young adult volunteers aged between 20 and 50 y from Southern Medical University. Informed consent was obtained from each donor, and the study was approved by the Institutional Review Committee at Nanfang Hospital. PBMCs were separated on Ficoll by density gradient centrifugation, washed in PBS, and resuspended in complete RPMI 1640 supplemented with 10% FBS. CD4+ T cells were purified by negative selection. After an initial step of monocyte adherence (1 h at 37°C), nonadherent cells were purified using a CD4 negative selection kit according to the manufacturer’s instructions. Purity was confirmed at >95% by flow cytometry. The cell density was estimated by counting the cells in a suspension aliquot using a hemocytometer under a microscope. Experiments were initiated by seeding the plates with 5 × 105 cells and incubated with 1 μg/ml functional-grade, purified anti-CD3 and anti-CD28 under the above-mentioned normal growth conditions for 24 h.

Small interfering RNA interference targeting the human Lck gene was screened by cotransfection with a human Lck cDNA plasmid into HEK293T cells using Lipofectamine 2000. The optimal sequence of small interfering RNA against human Lck (no. 1, 5′-CCATTAACTACGGGACATT-3′, no. 2, 5′-CGCCAGAAGCCATTAACTA-3′) was then cloned into a pGCL-GFP plasmid, which encodes an HIV-derived lentiviral vector containing a multiple cloning site for insertion of short hairpin RNA (shRNA) constructs to be driven by an upstream U6 promoter and a downstream CMV promoter–GFP fluorescent protein cassette flanked by loxP sites. Lentivirus preparations were produced by Shanghai Genechem (Shanghai, China). The resulting lentiviral vector containing human Lck shRNA was named GV115 lentivirus. An empty vector was used as negative control (5′-TTCTCCGAACGTGTCACGT-3′). Jurkat cells were infected with GV115 lentivirus or negative control virus for 72 h, and the transfection efficiency was analyzed using flow cytometry (LSRFortessa; BD Biosciences). The cells were then lysed for RT-PCR and Western blotting to determine the effects of RNA interference on Lck expression. The following Lck primer sequences were used: forward, 5′-GCCCACCTTTGACTACCTG-3′, reverse, 5′-GGCACAAGAACTCCATCTCC-3′.

Jurkat cells were seeded into a 96-well plate at a concentration of 5000 cells per well. After 24 h of treatment with different concentrations of HSP65, cell proliferation was evaluated after the addition of 10 μl of CCK-8 solution to each well. Following this, the plate was incubated at 37°C for 2 h. OD value was measured at a wavelength of 450 nm using a multimode microplate reader (SpectraMax M5; Molecular Devices).

Production of IL-10 and IFN-γ in cell supernatants was determined using a human ELISA development kit according to the manufacturer’s instructions.

Cells were incubated for 24 h in RPMI 1640 medium containing 0.2% BSA, 30 μg/ml oxidized low-density lipoprotein (ox-LDL), and 1 μCi/ml [1α,2α-3H]cholesterol. After washing with PBS, the cells were incubated in RPMI 1640 medium with 0.2% BSA containing various concentrations of HSP65 (0–1 μg/ml) for another 24 h. The cells were then incubated in serum-free medium (without BSA) with or without 10 μg/ml apolipoprotein A-I (apoA-I) for 6 h. Subsequently, the incubation medium was collected and the cells were washed with PBS and then lysed with 0.1 M NaOH. Finally, the radioactivity of the medium and the cell lysate was measured using liquid scintillation spectrometry. Cholesterol efflux rate was presented as the 3H-cholesterol radioactivity of the medium normalized to the total 3H-cholesterol radioactivity.

Cells were incubated for 24 h in RPMI 1640 medium containing 30 μg/ml ox-LDL. The cells were then submitted to fixation with 4% paraformaldehyde and subsequent staining with 0.5% Oil Red O. Hematoxylin was used as a counterstain, and the cells were photographed at ×200 magnification using a microscope fitted with a digital camera. Three regions of each image were chosen for analysis. The density of the red color in each photo represents the content of lipid droplets within the cells.

Cell-free cholesterol and total cholesterol were analyzed using HPLC. Briefly, cells were placed in a 0.9% NaCl solution on ice and homogenized by sonication for 10 s. Following this, an equal volume of freshly prepared cold (−20°C) potassium hydroxide in ethanol (150 g/l) was added to the supernatant of each cell lysate, and the mixture was vortexed until a clear solution was obtained. Then, an equal volume of solvent (hexane/isopropanol of 3:2, v/v) was added, and the mixture was vortexed for 5 min and then centrifuged at 800 × g for 5 min. Organic phases were collected and dried in a SpeedVac. Each organic phase was then dissolved in 100 μl of isopropanol/acetonitrile (20:80, v/v) and placed in an ultrasonic water bath at room temperature for 5 min. Finally, the samples were subjected to HPLC analysis (Agilent 1100; Agilent Technologies). HPLC was performed using a Hypersil C18 column with isopropanol/acetonitrile (20:80, v/v) as the eluent at a flow rate of 1 ml/min. Cholesterol ester content was determined by subtracting the cell-free cholesterol values from the total cholesterol values.

Total RNA was isolated from cells, and mRNA levels for specific genes were measured using quantitative real-time RT-PCR. β-Actin mRNA was used as an internal control. The following primer sequences were used: ABCA1, forward, 5′-ATGGCACTGAGGAAGATGCT-3′, reverse, 5′-CAGATAATGCGGGAAAGAGG-3′; ABCG1, forward, 5′-TGCCAGGAAACAGGAAGATT-3′, reverse, 5′-GAGACACCCACAAACCCAAC-3′; SR-BI, forward, 5′-TCGGAGAGCGACTACATCG-3′, reverse, 5′-GTGGTGAATGCCAAGGTCA-3′; PPAR-γ, forward, 5′-CAGAAATGCCTTGCAGTGG-3′, reverse, 5′-CTGGATTCAGCTGGTCGATA-3′; LXR-α, forward, 5′-AGTTTGCCTTGCTCATTGCT-3′, reverse, 5′-CATCCGTGGGAACATCAGTC-3′; β-actin, forward, 5′-TGTTACAGGAAGTCCCTTGCCATC-3′, reverse, 5′-CTGTGTGGACTTGGGAGAGGAC-3′.

Cells were harvested, washed with PBS, lysed for 30 min in an ice bath, and centrifuged (15 min at 14,000 × g and 4°C). Half of the resultant supernatant was applied to an 8% gel for NaDodSO4-PAGE and the other half was applied to a 10% gel. Following electrophoresis, the gels were transferred to polyvinyl fluoride membranes. The membranes were blocked with 5% milk and then probed overnight with protein-specific Abs. Finally, the membranes were incubated with an HRP-conjugated secondary Ab (Protein Tech Group, Chicago, IL) and detected using an ECL Western blotting detection kit (Sigma-Aldrich). Protein band intensity was measured using ImageJ software (National Institutes of Health).

Intracellular Ca2+ was measured by incubating Fluo-3 AM (4 μM, final) with cells for 30 min at 37°C in incubation medium. After this, the cells were washed with PBS three times. Fluorescence measurements of intracellular Ca2+ concentration in the cells were performed by an FV10i-W confocal laser scanning microscopy (Olympus, Tokyo, Japan) with an excitation wavelength of 488 nm and an emission wavelength of 520 nm. The resultant images were quantitatively analyzed using Olympus FV500 Vision software to assess changes in fluorescence intensities within designated regions of interest.

Ca2+ flux was measured by adding an equal volume of Fluo-8 dye-loading solution to the cell plate, after which the plate was incubated at 37°C for 30 min and then at room temperature for 30 min. Calcium levels were determined by flow cytometry in a FACScan flow cytometer (BD Biosciences) using excitation/emission wavelengths of 490/525 nm.

All data are presented as the means ± SD. Statistical analysis was conducted using a Student t test or one-way ANOVA coupled with a Bonferroni–Dunn post hoc test. All analyses were performed using Statistical Product and Service Solutions for Windows (version 13.0; Statistical Product and Service Solutions, Chicago, IL). A p value <0.05 was considered statistically significant.

HSP65 possesses cytokine-like activity in its ability to evoke proinflammatory immune responses. Thus, identifying the specific receptors used by HSP65 and the associated signaling pathways in T cells have been subjects of interest in the research community (27, 28). To examine whether HSP65 modulates cellular immune response, we examined cellular proliferation, Lck protein expression, and the secretion of inflammatory cytokine IFN-γ and anti-inflammatory cytokine IL-10. When HSP65 (in the range of 0–1 μg/ml) was incubated with Jurkat cells, increased levels of Ag-induced proliferative response were observed (Fig. 1A). Furthermore, HSP65 dose-dependently increased Lck expression in Jurkat cells and purified CD4+ T cells (Fig. 1B, 1C). Additionally, HSP65-treated Jurkat cells (1 μg/ml) exhibited lower IL-10 and higher IFN-γ levels than did the control group, indicating the successful induction of proinflammatory status by HSP65 (Fig. 1C, 1D). These results indicate that HSP65 can stimulate T cells by increasing cell proliferation, activating Lck protein expression, and inducing inflammatory cytokine secretion.

FIGURE 1.

HSP65 induces Lck expression in CD4+ T cells and affects proliferation and cytokine production in Jurkat cells. Jurkat cells were stimulated with different concentrations of HSP65 (0, 0.25, 0.5, 0.75, and 1 μg/ml) for 24 h. Effects of HSP65 on cell proliferation were detected by CCK-8 (A), and Lck protein expression was measured by Western blotting (B). (C) Purified CD4+ T cells from healthy donors were stimulated with anti-CD3/CD28 (1 μg/ml) for 24 h, and Lck protein expression was assessed. Production of IFN-γ (D) and IL-10 (E) in Jurkat cell supernatant was measured by ELISA. Data were pooled from four independent experiments and are presented as the mean ± SD. Data that are not significantly different (p > 0.05) are indicated with the same letter.

FIGURE 1.

HSP65 induces Lck expression in CD4+ T cells and affects proliferation and cytokine production in Jurkat cells. Jurkat cells were stimulated with different concentrations of HSP65 (0, 0.25, 0.5, 0.75, and 1 μg/ml) for 24 h. Effects of HSP65 on cell proliferation were detected by CCK-8 (A), and Lck protein expression was measured by Western blotting (B). (C) Purified CD4+ T cells from healthy donors were stimulated with anti-CD3/CD28 (1 μg/ml) for 24 h, and Lck protein expression was assessed. Production of IFN-γ (D) and IL-10 (E) in Jurkat cell supernatant was measured by ELISA. Data were pooled from four independent experiments and are presented as the mean ± SD. Data that are not significantly different (p > 0.05) are indicated with the same letter.

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In a previous report, we demonstrated that HSP65 can inhibit cholesterol efflux to apoA-I in macrophages (26). To examine the inhibitory effect of HSP65 on RCT in T cells, apoA-I–stimulated cholesterol efflux was assessed. Compared with control cells, 1 μg/ml HSP65 significantly reduced cholesterol efflux to apoA-I (49.7% reduction) (Fig. 2A). A similar trend of decreased cholesterol transport protein expression was also observed. Furthermore, compared with the control group, treatment with 1 μg/ml HSP65 reduced the protein expression levels of ABCA1, ABCG1, SR-BI, PPAR-γ, and LXR-α (Fig. 2B), and the mRNA levels for the corresponding genes were respectively decreased to 54, 71, 68, 62.1, and 49.2% of the control (Fig. 2C). We further found that 1 μg/ml HSP65 significantly inhibited cholesterol efflux in CD4+ T cells (20.1% reduction compared with the control group), whereas there was a lower level of cholesterol efflux (24.1% reduction compared with the control group) upon anti-CD3/CD28 stimulation (Fig. 2D). Furthermore, we observed that the expression levels of the membrane transporters ABCA1, ABCG1, and SR-BI following both 1 μg/ml HSP65 and anti-CD3/CD28 stimulation were decreased in CD4+ T cells compared with the control group (Fig. 2E). This evidence suggests that HSP65 inhibits apoA-I–mediated cholesterol efflux as well as the expression of cholesterol transport proteins in T cells.

FIGURE 2.

HSP65 inhibits cholesterol efflux and downregulates cholesterol transport protein expression in Jurkat cells and CD4+ T cells. (A) 3H-cholesterol efflux in Jurkat cells was detected in each group. The cells were treated with 30 μg/ml ox-LDL and 1 μCi/ml [3H]cholesterol for 24 h, followed with HSP65 for an additional 24 h. The cells were then incubated with or without 10 μg/ml apoA-I for 6 h. 3H-cholesterol radioactivity was measured. Data were pooled from four independent experiments. (B) Protein expression of ABCA1, ABCG1, SR-BI, PPAR-γ, and LXR-α in Jurkat cells was detected by Western blotting. (C) Cholesterol transporter mRNA expression in Jurkat cells was detected by RT-PCR. (D) CD4+ T cells were stimulated with anti-CD3/CD28 and different concentration of HSP65. 3H-cholesterol radioactivity was detected by liquid scintillation spectrometry as described above. Each symbol represents an individual donor. The results were from two independent experiments. (E) The membrane proteins ABCA1, ABCG1, and SR-BI were detected when CD4+ T cells were treated with the TCR stimuli anti-CD3/CD28 and HSP65. Values that are not significantly different (p > 0.05) are indicated with the same letter.

FIGURE 2.

HSP65 inhibits cholesterol efflux and downregulates cholesterol transport protein expression in Jurkat cells and CD4+ T cells. (A) 3H-cholesterol efflux in Jurkat cells was detected in each group. The cells were treated with 30 μg/ml ox-LDL and 1 μCi/ml [3H]cholesterol for 24 h, followed with HSP65 for an additional 24 h. The cells were then incubated with or without 10 μg/ml apoA-I for 6 h. 3H-cholesterol radioactivity was measured. Data were pooled from four independent experiments. (B) Protein expression of ABCA1, ABCG1, SR-BI, PPAR-γ, and LXR-α in Jurkat cells was detected by Western blotting. (C) Cholesterol transporter mRNA expression in Jurkat cells was detected by RT-PCR. (D) CD4+ T cells were stimulated with anti-CD3/CD28 and different concentration of HSP65. 3H-cholesterol radioactivity was detected by liquid scintillation spectrometry as described above. Each symbol represents an individual donor. The results were from two independent experiments. (E) The membrane proteins ABCA1, ABCG1, and SR-BI were detected when CD4+ T cells were treated with the TCR stimuli anti-CD3/CD28 and HSP65. Values that are not significantly different (p > 0.05) are indicated with the same letter.

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Fluctuating intracellular calcium ion concentrations is an immediate consequence of TCR engagement (29). To evaluate the effect of Lck on TCR signal strength intensity, we transfected cells with two shRNA sequences (LV1 and LV2) targeted to interfere with Lck expression (Fig. 3A, 3B). As shown by confocal microscopy, both LV1 and LV2 inhibited the intracellular accumulation of free calcium ions (Fig. 3C). To determine whether Lck was required for calcium flux, we incubated Lck-shRNA–transfected cells with a calcium ionophore. Ionomycin strongly promotes intracellular calcium flux in Jurkat cells, and this effect was decreased in the Lck-silenced cells (Fig. 3D). In addition to repressing Lck gene expression, Lck-shRNA inhibited the mobilization of calcium downstream of TCR. Western blotting analysis demonstrated that silencing the Lck gene led to a lack of PKC-γ and MAPK pathway activation, including a lack of ERK1/2, JNK, and NF-κB phosphorylation (Fig. 3E). These results indicated that Lck plays a key role in initiating TCR-mediated signaling.

FIGURE 3.

Lck-shRNA decreased intracellular calcium concentration and downregulated the expression of TCR downstream signaling proteins in Jurkat cells. Jurkat cells were transfected with either a negative control shRNA (NC) or targeted Lck-shRNA (LV1 or LV2) for 72 h. (A) Cell lysates were probed with an anti-Lck mAb and detected by Western blotting. (B) Lck mRNA expression was detected by RT-PCR. Data were pooled from three independent experiments. (C) Changes in intracellular calcium concentrations were detected using laser confocal scanning microscopy after incubation with Fluo-3 AM. The bright dots in the Jurkat cells and NC cells indicate a high concentration of calcium, and the dim color in the LV1 and LV2 groups indicates a low concentration of calcium. Intracellular calcium intensity is shown as the mean ± SD from at least four equivalent-sized regions of interest in five independent experiments. (D) Calcium flux was detected by flow cytometry following stimulation with 5 μM ionomycin for 30 min in Lck-silenced cells after incubation with Fluo-8 dye-loading solution for the indicated time. (E) The cytoplasmic and nuclear proteins were extracted. The phosphorylation of ERK1/2 and JNK, the cytoplasmic expression of PKC-γ and the nuclear expression of NF-κB were determined by Western blotting. Data that are not significantly different (p > 0.05) are indicated with the same letter.

FIGURE 3.

Lck-shRNA decreased intracellular calcium concentration and downregulated the expression of TCR downstream signaling proteins in Jurkat cells. Jurkat cells were transfected with either a negative control shRNA (NC) or targeted Lck-shRNA (LV1 or LV2) for 72 h. (A) Cell lysates were probed with an anti-Lck mAb and detected by Western blotting. (B) Lck mRNA expression was detected by RT-PCR. Data were pooled from three independent experiments. (C) Changes in intracellular calcium concentrations were detected using laser confocal scanning microscopy after incubation with Fluo-3 AM. The bright dots in the Jurkat cells and NC cells indicate a high concentration of calcium, and the dim color in the LV1 and LV2 groups indicates a low concentration of calcium. Intracellular calcium intensity is shown as the mean ± SD from at least four equivalent-sized regions of interest in five independent experiments. (D) Calcium flux was detected by flow cytometry following stimulation with 5 μM ionomycin for 30 min in Lck-silenced cells after incubation with Fluo-8 dye-loading solution for the indicated time. (E) The cytoplasmic and nuclear proteins were extracted. The phosphorylation of ERK1/2 and JNK, the cytoplasmic expression of PKC-γ and the nuclear expression of NF-κB were determined by Western blotting. Data that are not significantly different (p > 0.05) are indicated with the same letter.

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To confirm that the tyrosine kinase Lck is the key protein in the TCR signaling cascade that affects calcium and PMA-induced ERK and NF-κB activation, we treated Jurkat cells with the Src family kinase inhibitor PP2. PP2 strongly inhibited Lck expression in a dose-dependent manner (Fig. 4A). Cells treated with PP2 showed a marked reduction in the level of ERK1/2 phosphorylation in response to ionomycin (Fig. 4B). Furthermore, when cells were treated with PP2 and then stimulated with PMA, ERK1/2 phosphorylation and NF-κB activation were at similar levels to those observed in the untreated PMA cells (Fig. 4C). These data support our conclusion that increased intracellular calcium is required for Lck to induce ERK phosphorylation.

FIGURE 4.

The Src family kinase inhibitor PP2 blocks calcium-induced ERK1/2 phosphorylation but not PMA-induced NF-κB activation in Jurkat cells. (A) Jurkat cells were treated with 5 or 10 μM PP2 for 30 min. Lck protein expression was then analyzed by Western blotting with an anti-Lck mAb. (B) Cells were incubated with 10 μM PP2 for 30 min prior to being stimulated with DMSO (1:100), ionomycin (5 μM), or PMA (1 μM) for 20 min. The phosphorylation of ERK1/2 was detected. (C) Cells were incubated with 10 μM PP2 for 30 min prior to stimulation with PMA (1 μM) for 20 min. Nuclear proteins were extracted. The activation of NF-κB in the nucleus was determined by Western blotting.

FIGURE 4.

The Src family kinase inhibitor PP2 blocks calcium-induced ERK1/2 phosphorylation but not PMA-induced NF-κB activation in Jurkat cells. (A) Jurkat cells were treated with 5 or 10 μM PP2 for 30 min. Lck protein expression was then analyzed by Western blotting with an anti-Lck mAb. (B) Cells were incubated with 10 μM PP2 for 30 min prior to being stimulated with DMSO (1:100), ionomycin (5 μM), or PMA (1 μM) for 20 min. The phosphorylation of ERK1/2 was detected. (C) Cells were incubated with 10 μM PP2 for 30 min prior to stimulation with PMA (1 μM) for 20 min. Nuclear proteins were extracted. The activation of NF-κB in the nucleus was determined by Western blotting.

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Subsequently, we examined the capacity of Lck to inhibit RCT. As shown in Fig. 5A, both Lck-shRNA sequences tested increased cholesterol efflux. Increasing evidence suggests that cellular immune-inflammatory responses produce conflicting effects on RCT (2022). ERK1/2, JNK, and NF-κB have been shown to affect ABCA1 and ABCG1 mRNA and protein stability in lymphocytes (3032). In contrast to the above-described changes in TCR signaling pathway proteins, Lck-shRNA effectively increased the levels of ABCA1, ABCG1, SR-BI, PPAR-γ, and LXR-α proteins (Fig. 5B). These data demonstrate that inhibition of Lck leads to activation of cholesterol transport proteins. Because cellular cholesterol content results from cholesterol transport, we monitored this parameter as an indirect measure of RCT. Representative images of intracellular lipid droplets stained using Oil Red O are shown in Fig. 5C. Nontransfected cells and cells transfected with a negative control sequence were stained dark red and showed a greater residual of lipid droplets, whereas experimentally transfected cells produced a faded color. Lipid droplets were counted using ImageJ Plus, and the results demonstrated that Lck-shRNA effectively decreased intracellular lipid content. Intracellular cholesterol ester concentration was also analyzed using HPLC, and the results confirmed that cholesterol ester content was also decreased in Lck-silenced cells (Fig. 5D). Similar to Lck-shRNA transfection, PP2 treatment upregulated the expression of ABCA1, ABCG1, and SR-BI (Fig. 5E). Collectively, these results suggest that Lck inhibits T cell RCT.

FIGURE 5.

Lck inhibits RCT and downregulates cholesterol transport proteins in Jurkat cells. (A) 3H-cholesterol radioactivity was detected by liquid scintillation spectrometry in cells transfected with negative control (NC) or Lck-shRNA. Cholesterol efflux increased in the Lck-shRNA groups. (B) Expression of cholesterol transport proteins was detected by Western blotting. (C) The cells were incubated with ox-LDL for 24 h and with 1 μg/ml HSP65 for another 24 h. After fixation in 4% paraformaldehyde, the cells were stained with Oil Red O to detect lipid content. Cellular nuclei were stained with hematoxylin. Original magnification, ×200. The arrows indicate lipid droplets. The red portion in each cell was quantified using ImageJ software. At each time point, the mean value and SD were obtained. All of the data were merged into a bar graph. The bar graph represents the data from three independent experiments. (D) The ratio of cholesterol ester (CE) to total cholesterol (TC) was calculated. Cellular free cholesterol and TC were measured using HPLC. (E) The membrane proteins ABCA1, ABCG1, and SR-BI were detected by Western blotting after Jurkat cells were treated with 10 μM PP2 for 30 min. Data pooled from at least three to four independent experiments are presented as the mean ± SD. Data that are not significantly different (p > 0.05) are indicated with the same letter.

FIGURE 5.

Lck inhibits RCT and downregulates cholesterol transport proteins in Jurkat cells. (A) 3H-cholesterol radioactivity was detected by liquid scintillation spectrometry in cells transfected with negative control (NC) or Lck-shRNA. Cholesterol efflux increased in the Lck-shRNA groups. (B) Expression of cholesterol transport proteins was detected by Western blotting. (C) The cells were incubated with ox-LDL for 24 h and with 1 μg/ml HSP65 for another 24 h. After fixation in 4% paraformaldehyde, the cells were stained with Oil Red O to detect lipid content. Cellular nuclei were stained with hematoxylin. Original magnification, ×200. The arrows indicate lipid droplets. The red portion in each cell was quantified using ImageJ software. At each time point, the mean value and SD were obtained. All of the data were merged into a bar graph. The bar graph represents the data from three independent experiments. (D) The ratio of cholesterol ester (CE) to total cholesterol (TC) was calculated. Cellular free cholesterol and TC were measured using HPLC. (E) The membrane proteins ABCA1, ABCG1, and SR-BI were detected by Western blotting after Jurkat cells were treated with 10 μM PP2 for 30 min. Data pooled from at least three to four independent experiments are presented as the mean ± SD. Data that are not significantly different (p > 0.05) are indicated with the same letter.

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To explore the mechanism underlying the observed HSP65-mediated decrease in cholesterol efflux in T cells, we detected intracellular calcium levels, ERK1/2 and JNK phosphorylation, and PKC-γ and NF-κB protein expression in Lck-silenced T cells following treatment with 1 μg/ml HSP65. Treatment of Jurkat cells with HSP65 increased intracellular free calcium levels, whereas diminished intracellular calcium levels were observed following the application of LV1 or LV2 (Fig. 6A). When 10 μM PP2 was incubated with Jurkat cells, a decrease in the level of HSP65-induced proliferative response was observed (Fig. 6B). Furthermore, we assessed whether the application of Lck-shRNA impaired the activation of various downstream proteins. Both LV1 and LV2 inhibited HSP65-induced ERK and JNK activation; PKC-γ and NF-κB expression exhibited the same trend (Fig. 3E). Collectively, these results suggest that loss of Lck greatly impairs HSP65-mediated TCR signaling pathway.

FIGURE 6.

Intracellular calcium concentration and cell proliferation are dependent on Lck activity after stimulation with HSP65 in Jurkat cells. (A) Jurkat cells were transfected with negative control (NC) or Lck-shRNA prior to being stimulated with HSP65 (1 μg/ml; 24 h). Changes in intracellular calcium concentration were evaluated by laser confocal scanning microscopy after incubation with Fluo-3 AM. (B) The effects of PP2 on cell proliferation were detected by CCK-8 with or without HSP65 treatment. The results from four independent experiments are presented as the mean ± SD. Data that are not significantly different (p > 0.05) are indicated with the same letter.

FIGURE 6.

Intracellular calcium concentration and cell proliferation are dependent on Lck activity after stimulation with HSP65 in Jurkat cells. (A) Jurkat cells were transfected with negative control (NC) or Lck-shRNA prior to being stimulated with HSP65 (1 μg/ml; 24 h). Changes in intracellular calcium concentration were evaluated by laser confocal scanning microscopy after incubation with Fluo-3 AM. (B) The effects of PP2 on cell proliferation were detected by CCK-8 with or without HSP65 treatment. The results from four independent experiments are presented as the mean ± SD. Data that are not significantly different (p > 0.05) are indicated with the same letter.

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T cell activation has previously been shown to regulate RCT (23, 24). In the present study, we showed that HSP65 inhibited cholesterol efflux and that Lck inhibition enhanced RCT. We therefore investigated whether HSP65 inhibited RCT through an Lck-mediated pathway. Cholesterol efflux in the LV1 and LV2 groups following treatment with HSP65 was 17.9 and 17.8%, respectively, whereas in the negative control plus 1 μg/ml HSP65 group it was 10.8% (Fig. 7A). When 10 μM PP2 was incubated with Jurkat cells, an increase in the level of HSP65-mediated cholesterol efflux was also observed (Fig. 7B). These results suggest that silencing Lck could weaken the inhibitory effect of HSP65 on cholesterol efflux in Jurkat cells. Contrary to the above-described decreases in the expression of proteins involved in TCR-mediated signaling, HSP65 treatment increased ABCA1, ABCG1, SR-BI, PPAR-γ, and LXR-α protein expression following the application of Lck-shRNA (Fig. 5B).

FIGURE 7.

Lck inhibits HSP65-mediated reverse cholesterol transport in Jurkat cells. (A) Cellular cholesterol efflux to apoA-I was detected in Jurkat cells transfected with negative control (NC) or Lck-shRNA prior to stimulation with HSP65 (1 μg/ml; 24 h). (B) Cells were incubated with 10 μM PP2 for 30 min prior to stimulation with HSP65 (1 μg/ml) for 24 h. 3H-cholesterol efflux was detected. (C) Cells were stained with Oil Red O. Representative images of intracellular lipid droplets are shown. The arrows indicate lipid droplets. Original magnification, ×200. (D) The ratio of cholesterol ester (CE) to total cholesterol (TC) was calculated. Data pooled from at least three to four independent experiments are presented as the mean ± SD. Data that are not significantly different (p > 0.05) are indicated with the same letter.

FIGURE 7.

Lck inhibits HSP65-mediated reverse cholesterol transport in Jurkat cells. (A) Cellular cholesterol efflux to apoA-I was detected in Jurkat cells transfected with negative control (NC) or Lck-shRNA prior to stimulation with HSP65 (1 μg/ml; 24 h). (B) Cells were incubated with 10 μM PP2 for 30 min prior to stimulation with HSP65 (1 μg/ml) for 24 h. 3H-cholesterol efflux was detected. (C) Cells were stained with Oil Red O. Representative images of intracellular lipid droplets are shown. The arrows indicate lipid droplets. Original magnification, ×200. (D) The ratio of cholesterol ester (CE) to total cholesterol (TC) was calculated. Data pooled from at least three to four independent experiments are presented as the mean ± SD. Data that are not significantly different (p > 0.05) are indicated with the same letter.

Close modal

The role of Lck in HSP65-mediated changes in intracellular cholesterol content was also examined. Representative images of stained intracellular lipid droplets are shown in Fig. 7C. Nontransfected cells appeared dark red and contained more lipid droplets than did transfected cells. In the transfected cells, the cell color faded and became more pink following stimulation with HSP65. Therefore, HSP65 treatment decreased the number of intracellular lipid droplets in the Lck-silenced group. Consistent with the above results, cholesterol ester content decreased in Lck-silenced cells relative to nontransfected cells following stimulation with HSP65 (Fig. 7D), as detected by HPLC. These findings indicate that Lck inhibits HSP65-mediated RCT by promoting TCR signaling (Fig. 8).

FIGURE 8.

Proposed Lck-mediated signaling pathway might be involved in cellular immune response and RCT. Following HSP65-mediated TCR engagement, Lck is activated and triggers a signaling cascade that leads to PLCγ activation, which generates DAG and IP3. These messengers provoke Ca2+ flux from the endoplasmic reticulum and phosphorylation of MAPK family members, which in turn promotes nuclear NF-κB activation. Following that, inhibition of the nuclear cholesterol transporters LXR-α and PPAR-γ could downregulate the membrane transporters SR-BI, ABCG1, and ABCA1, which blocks intracellular cholesterol efflux in T cells. The arrows indicate the direction of signaling.

FIGURE 8.

Proposed Lck-mediated signaling pathway might be involved in cellular immune response and RCT. Following HSP65-mediated TCR engagement, Lck is activated and triggers a signaling cascade that leads to PLCγ activation, which generates DAG and IP3. These messengers provoke Ca2+ flux from the endoplasmic reticulum and phosphorylation of MAPK family members, which in turn promotes nuclear NF-κB activation. Following that, inhibition of the nuclear cholesterol transporters LXR-α and PPAR-γ could downregulate the membrane transporters SR-BI, ABCG1, and ABCA1, which blocks intracellular cholesterol efflux in T cells. The arrows indicate the direction of signaling.

Close modal

Atherogenesis begins with the recruitment of inflammatory cells to the intima. Although in fewer numbers than mononuclear phagocytes, T cells also enter the intima and send decisive regulatory signals. Although HSP65 has been shown to inhibit both RCT and the anti-inflammatory capacity of high-density lipoprotein (HDL) in macrophages, it remains unknown whether RCT suppression in T cells is involved in this inhibition or its associated mechanism. Exploring RCT in T cells might not only uncover the mechanism underlying immune response to AS but also may verify whether additional mechanisms, such as lipid metabolism disorder, are relevant. In the present study, we assessed the role of Lck in the process of RCT and demonstrated that HSP65 might inhibit RCT via an Lck-mediated pathway (Fig. 8). To the best of our knowledge, this is the first report that HSP65, an important inflammatory substance, impairs RCT in T cells. Although the immunoreaction to HSP65 is considered a critical promoter of AS, the effects caused by intracellular cholesterol transportation resulting from an immune-inflammatory response cannot be ruled out. Our findings also suggest that blocking Lck may provide a novel therapeutic approach to promote RCT.

In a previous study, we demonstrated that the proatherosclerotic effects of HSP65 occur via its promotion of HDL dysfunction (26). However, it is not clear whether HSP65 has a similar inhibitory effect on RCT in T cells. Our results illustrate that HSP65 inhibits T cell RCT through its effects on cholesterol transport proteins, leading to an inhibition of cholesterol efflux. These results suggest that HSP65 is an autoantigen that induces HDL dysfunction in immune cells.

Accumulating evidence suggests that Lck is involved in modulating T cell responses (33). It has been shown that the Src family protein kinase Lck plays a crucial role in activation of the MAPK pathway and the phospholipase C (PLC)γ–diacylglycerol (DAG)/inositol 1,4,5-triphosphate (IP3) pathway following TCR stimulation (34). Lck phosphorylates the ITAM in TCR/CD3 ζ-chains. Following this, ITAM activates the PLC signaling cascade. PLC then cleaves the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) into the second messengers DAG and IP3. This cleavage leads to intracellular calcium release and PKC activation (10). MAPK pathways that are further downstream are also affected. Our analysis of these downstream signaling pathways demonstrated that cells either transfected with Lck-shRNA or treated with PP2 were incapable of activating the PIP2 pathway following TCR stimulation. Both PKC-γ expression and intracellular calcium concentration were dependent on Lck activation. Furthermore, as with the PIP2 pathway, TCR stimulation with HSP65 failed to induce ERK1/2 and JNK phosphorylation and NF-κB activation in Lck-silenced cells. Thus, Lck activity is required for calcium-induced ERK1/2 phosphorylation. These results indicate that silencing Lck interrupts TCR-mediated signaling and thereby attenuates the immune-inflammatory response.

It is possible that enhancing T cell RCT could prevent the progression of AS or even induce AS regression. Among several potential pathological mechanisms, the cellular immune response is the most conceptually attractive because it causes both inflammatory injury and HDL dysfunction. During the acute phase response, RCT is reduced at multiple levels, including in relationship to cholesterol transporter expression, HDL quality, and HDL cholesterol uptake and excretion by the liver (20). LPS downregulates ABCA1 and ABCG1 in macrophages, thus impairing cholesterol efflux (21, 22). TLR induces cholesterol transporter downregulation at least in part through the inhibitory action of IFN regulatory factor-3 at LXR-binding sites in the ABCA1 promoter (35). Many proinflammatory cytokines, such as C reactive protein, have been shown to downregulate ABCA1 in mononuclear cells (22). The MAPK pathway has also been reported to impact RCT. Recently, it was observed that blocking ERK1/2 activity upregulated ABCA1 and ABCG1 protein levels in human THP1 macrophages. These results indicate that the Ras/MAPK pathway has opposite roles in the regulation of PPAR- and LXR-dependent ABCA1 and ABCG1 transporter activity in macrophages (30). Recently, we also reported that JNK inhibition enhanced apoA-I–mediated cholesterol efflux in Raw264.7 cells. Moreover, ABCA1 and SR-BI expression was enhanced following treatment with a JNK inhibitor (31). Additionally, NF-κB has also been shown to suppress ABCA1 and ABCG1 expression by regulating T cell activation, which in turn induces the production of oxysterol-metabolizing enzyme SREBP-2 and miR-33a (32).

Given the potent effects of TCR signaling on RCT, it is essential to determine whether Lck inhibition induces the activation of RCT. In the present study, we used several different assays to evaluate the relationship between Lck and RCT. We found that application of Lck-shRNA and PP2 could increase cholesterol efflux, whereas treatment with HSP65 repressed this effect to a certain degree (Fig. 7). To further delineate the relationship between Lck and RCT, the expression levels of five transport proteins were monitored. In Lck-silenced cells, PPAR-γ, LXR-α, SR-BI, ABCG1, and ABCA1 expression increased. Previous reports have demonstrated that ABCA1 and ABCG1, the most prominent transporters used to eliminate intracellular cholesterol, are targets of LXR-α and that LXR-α is a target of PPAR-γ (36). Furthermore, recent data from a knockout mouse model support the conclusion that LXR signaling acts as a metabolic checkpoint to modulate cell proliferation and immunity. Unlike other cholesterol transport proteins, LXR can be inhibited via the transcription factor IFN regulatory factor-3 or the actions of the SREBP pathway after TLR activation (25). Our studies in cells transfected with Lck-shRNA showed that TCR-mediated signaling pathways could alter PPAR-γ expression. Thus, our studies indicate that PPAR-γ, an upstream mediator of LXR-α, may play a key role in regulating the crosstalk between inflammatory and RCT pathways.

We hypothesized that Lck may inhibit lipid droplet efflux in lymphocytes and accelerate the formation of atherosclerotic plaques. Therefore, we analyzed intracellular cholesterol content. Contrary to our negative control cells, intracellular cholesterol content was remarkably reduced in Lck-silenced cells. Indeed, HSP65 treatment resulted in decreased mobilization of cholesterol. Our results demonstrated that silencing Lck abrogated the effects of HSP65 on TCR-mediated signaling and increased cholesterol efflux; as a result, intracellular cholesterol content was reduced. This process may represent a cellular immune mechanism that accelerates atherogenesis.

In summary, this work outlines a previously unknown role for Lck signaling in regulating HSP65-mediated RCT. The observed changes in transport protein expression levels suggest that cholesterol efflux may be controlled by regulating Lck activity. Furthermore, HSP65 enhanced Lck-mediated signaling, which inhibited RCT in T cells. Because the key findings of our study were derived from T cell culture line and primary CD4+ T cells, future in vivo studies using mouse AS models are needed to validate the role of Lck and its downstream signaling in modulating HSP65-mediated RCT.

We thank the members of the Cardiology Laboratory and Medical Research Center in Nanfang Hospital for critical comments and technical support.

This work was supported by National Natural Science Foundation of China 81370380, Natural Science Foundation of Guangdong Province of China S2013010014739, and by Science and Technology Foundation of Guangdong Province of China 2012B091100155 and 2011B031800065.

Abbreviations used in this article:

apoA-I

apolipoprotein A-I

AS

atherosclerosis

DAG

diacylglycerol

HDL

high-density lipoprotein

HSP

heat shock protein

IP3

inositol 1,4,5-triphosphate

Lck

lymphocyte-specific protein tyrosine kinase

LXR

liver X receptor

ox-LDL

oxidized low-density lipoprotein

PIP2

phosphatidylinositol 4,5-bisphosphate

PKC

protein kinase C

PLC

phospholipase C

RCT

reverse cholesterol transport

shRNA

short hairpin RNA.

1
Hansson
G. K.
,
Libby
P.
.
2006
.
The immune response in atherosclerosis: a double-edged sword.
Nat. Rev. Immunol.
6
:
508
519
.
2
Hansson
G. K.
,
Berne
G. P.
.
2004
.
Atherosclerosis and the immune system.
Acta Paediatr. Suppl.
93
:
63
69
.
3
Hansson
G. K.
2005
.
Inflammation, atherosclerosis, and coronary artery disease.
N. Engl. J. Med.
352
:
1685
1695
.
4
Boyle
J. J.
2005
.
Macrophage activation in atherosclerosis: pathogenesis and pharmacology of plaque rupture.
Curr. Vasc. Pharmacol.
3
:
63
68
.
5
Libby
P.
,
Ridker
P. M.
,
Hansson
G. K.
.
2011
.
Progress and challenges in translating the biology of atherosclerosis.
Nature
473
:
317
325
.
6
Sato
K.
,
Niessner
A.
,
Kopecky
S. L.
,
Frye
R. L.
,
Goronzy
J. J.
,
Weyand
C. M.
.
2006
.
TRAIL-expressing T cells induce apoptosis of vascular smooth muscle cells in the atherosclerotic plaque.
J. Exp. Med.
203
:
239
250
.
7
Pryshchep
S.
,
Sato
K.
,
Goronzy
J. J.
,
Weyand
C. M.
.
2006
.
T cell recognition and killing of vascular smooth muscle cells in acute coronary syndrome.
Circ. Res.
98
:
1168
1176
.
8
Nakajima
T.
,
Schulte
S.
,
Warrington
K. J.
,
Kopecky
S. L.
,
Frye
R. L.
,
Goronzy
J. J.
,
Weyand
C. M.
.
2002
.
T-cell-mediated lysis of endothelial cells in acute coronary syndromes.
Circulation
105
:
570
575
.
9
Dumont
C.
,
Blanchard
N.
,
Di Bartolo
V.
,
Lezot
N.
,
Dufour
E.
,
Jauliac
S.
,
Hivroz
C.
.
2002
.
TCR/CD3 down-modulation and ζ degradation are regulated by ZAP-70.
J. Immunol.
169
:
1705
1712
.
10
Palacios
E. H.
,
Weiss
A.
.
2004
.
Function of the Src-family kinases, Lck and Fyn, in T-cell development and activation.
Oncogene
23
:
7990
8000
.
11
Smith-Garvin
J. E.
,
Koretzky
G. A.
,
Jordan
M. S.
.
2009
.
T cell activation.
Annu. Rev. Immunol.
27
:
591
619
.
12
Parada
C. A.
,
Portaro
F.
,
Marengo
E. B.
,
Klitzke
C. F.
,
Vicente
E. J.
,
Faria
M.
,
Sant’Anna
O. A.
,
Fernandes
B. L.
.
2011
.
Autolytic Mycobacterium leprae Hsp65 fragments may act as biological markers for autoimmune diseases.
Microb. Pathog.
51
:
268
276
.
13
Kilic, A., and K. Mandal. 2012. Heat shock proteins: pathogenic role in atherosclerosis and potential therapeutic implications. Autoimmune Dis. 2012: 502813. doi:10.1155/2012/502813.
14
Hansson
G. K.
2001
.
Immune mechanisms in atherosclerosis.
Arterioscler. Thromb. Vasc. Biol.
21
:
1876
1890
.
15
Shimizu
J.
,
Izumi
T.
,
Suzuki
N.
.
2012
.
Aberrant activation of heat shock protein 60/65 reactive T cells in patients with Behcet’s disease.
Autoimmune Dis.
2012
:
105205
.
16
Ouimet
T.
,
Lancelot
E.
,
Hyafil
F.
,
Rienzo
M.
,
Deux
F.
,
Lemaître
M.
,
Duquesnoy
S.
,
Garot
J.
,
Roques
B. P.
,
Michel
J. B.
, et al
.
2012
.
Molecular and cellular targets of the MRI contrast agent P947 for atherosclerosis imaging.
Mol. Pharm.
9
:
850
861
.
17
Mazière
C.
,
Trécherel
E.
,
Ausseil
J.
,
Louandre
C.
,
Mazière
J. C.
.
2011
.
Oxidized low density lipoprotein induces cyclin A synthesis. Involvement of ERK, JNK and NFkappaB.
Atherosclerosis
218
:
308
313
.
18
Rohatgi
A.
,
Khera
A.
,
Berry
J. D.
,
Givens
E. G.
,
Ayers
C. R.
,
Wedin
K. E.
,
Neeland
I. J.
,
Yuhanna
I. S.
,
Rader
D. R.
,
de Lemos
J. A.
,
Shaul
P. W.
.
2014
.
HDL cholesterol efflux capacity and incident cardiovascular events.
N. Engl. J. Med.
371
:
2383
2393
.
19
Khera
A. V.
,
Cuchel
M.
,
de la Llera-Moya
M.
,
Rodrigues
A.
,
Burke
M. F.
,
Jafri
K.
,
French
B. C.
,
Phillips
J. A.
,
Mucksavage
M. L.
,
Wilensky
R. L.
, et al
.
2011
.
Cholesterol efflux capacity, high-density lipoprotein function, and atherosclerosis.
N. Engl. J. Med.
364
:
127
135
.
20
Khovidhunkit
W.
,
Kim
M. S.
,
Memon
R. A.
,
Shigenaga
J. K.
,
Moser
A. H.
,
Feingold
K. R.
,
Grunfeld
C.
.
2004
.
Effects of infection and inflammation on lipid and lipoprotein metabolism: mechanisms and consequences to the host.
J. Lipid Res.
45
:
1169
1196
.
21
Khovidhunkit
W.
,
Moser
A. H.
,
Shigenaga
J. K.
,
Grunfeld
C.
,
Feingold
K. R.
.
2003
.
Endotoxin down-regulates ABCG5 and ABCG8 in mouse liver and ABCA1 and ABCG1 in J774 murine macrophages: differential role of LXR.
J. Lipid Res.
44
:
1728
1736
.
22
Wang
X.
,
Liao
D.
,
Bharadwaj
U.
,
Li
M.
,
Yao
Q.
,
Chen
C.
.
2008
.
C-reactive protein inhibits cholesterol efflux from human macrophage-derived foam cells.
Arterioscler. Thromb. Vasc. Biol.
28
:
519
526
.
23
Lo
J. C.
,
Wang
Y.
,
Tumanov
A. V.
,
Bamji
M.
,
Yao
Z.
,
Reardon
C. A.
,
Getz
G. S.
,
Fu
Y. X.
.
2007
.
Lymphotoxin beta receptor-dependent control of lipid homeostasis.
Science
316
:
285
288
.
24
Jiang
H.
,
Badralmaa
Y.
,
Yang
J.
,
Lempicki
R.
,
Hazen
A.
,
Natarajan
V.
.
2012
.
Retinoic acid and liver X receptor agonist synergistically inhibit HIV infection in CD4+ T cells by up-regulating ABCA1-mediated cholesterol efflux.
Lipids Health Dis.
11
:
69
80
.
25
Bensinger
S. J.
,
Bradley
M. N.
,
Joseph
S. B.
,
Zelcer
N.
,
Janssen
E. M.
,
Hausner
M. A.
,
Shih
R.
,
Parks
J. S.
,
Edwards
P. A.
,
Jamieson
B. D.
,
Tontonoz
P.
.
2008
.
LXR signaling couples sterol metabolism to proliferation in the acquired immune response.
Cell
134
:
97
111
.
26
Sun
H.
,
Shen
J.
,
Liu
T.
,
Tan
Y.
,
Tian
D.
,
Luo
T.
,
Lai
W.
,
Dai
M.
,
Guo
Z.
.
2014
.
Heat shock protein 65 promotes atherosclerosis through impairing the properties of high density lipoprotein.
Atherosclerosis
237
:
853
861
.
27
Srivastava
P.
2002
.
Roles of heat-shock proteins in innate and adaptive immunity.
Nat. Rev. Immunol.
2
:
185
194
.
28
Kol
A.
,
Lichtman
A. H.
,
Finberg
R. W.
,
Libby
P.
,
Kurt-Jones
E. A.
.
2000
.
Cutting edge: heat shock protein (HSP) 60 activates the innate immune response: CD14 is an essential receptor for HSP60 activation of mononuclear cells.
J. Immunol.
164
:
13
17
.
29
Acuto
O.
,
Cantrell
D.
.
2000
.
T cell activation and the cytoskeleton.
Annu. Rev. Immunol.
18
:
165
184
.
30
Zhou
X.
,
Yin
Z.
,
Guo
X.
,
Hajjar
D. P.
,
Han
J.
.
2010
.
Inhibition of ERK1/2 and activation of liver X receptor synergistically induce macrophage ABCA1 expression and cholesterol efflux.
J. Biol. Chem.
285
:
6316
6326
.
31
Liu
T.
,
Li
C.
,
Sun
H.
,
Luo
T.
,
Tan
Y.
,
Tian
D.
,
Guo
Z.
.
2014
.
Curcumin inhibits monocyte chemoattractant protein-1 expression and enhances cholesterol efflux by suppressing the c-Jun N-terminal kinase pathway in macrophage.
Inflamm. Res.
63
:
841
850
.
32
Zhao
G. J.
,
Tang
S. L.
,
Lv
Y. C.
,
Ouyang
X. P.
,
He
P. P.
,
Yao
F.
,
Tang
Y. Y.
,
Zhang
M.
,
Tang
Y. L.
,
Tang
D. P.
, et al
.
2014
.
NF-κB suppresses the expression of ATP-binding cassette transporter A1/G1 by regulating SREBP-2 and miR-33a in mice.
[Published erratum appears in 2014 Int. J. Cardiol. 176: e76.]
Int. J. Cardiol.
171
:
e93
e95
.
33
Picard
C.
,
Dogniaux
S.
,
Chemin
K.
,
Maciorowski
Z.
,
Lim
A.
,
Mazerolles
F.
,
Rieux-Laucat
F.
,
Stolzenberg
M. C.
,
Debre
M.
,
Magny
J. P.
, et al
.
2009
.
Hypomorphic mutation of ZAP70 in human results in a late onset immunodeficiency and no autoimmunity.
Eur. J. Immunol.
39
:
1966
1976
.
34
Merino
E.
,
Ávila-Flores
A.
,
Shirai
Y.
,
Moraga
I.
,
Saito
N.
,
Mérida
I.
.
2008
.
Lck-dependent tyrosine phosphorylation of diacylglycerol kinase α regulates its membrane association in T cells.
J. Immunol.
180
:
5805
5815
.
35
Castrillo
A.
,
Joseph
S. B.
,
Vaidya
S. A.
,
Haberland
M.
,
Fogelman
A. M.
,
Cheng
G.
,
Tontonoz
P.
.
2003
.
Crosstalk between LXR and toll-like receptor signaling mediates bacterial and viral antagonism of cholesterol metabolism.
Mol. Cell
12
:
805
816
.
36
Yvan-Charvet
L.
,
Wang
N.
,
Tall
A. R.
.
2010
.
Role of HDL, ABCA1, and ABCG1 transporters in cholesterol efflux and immune responses.
Arterioscler. Thromb. Vasc. Biol.
30
:
139
143
.

The authors have no financial conflicts of interest.