Overexpression of Uncoupling Protein 2 in THP1 Monocytes Inhibits ?2 Integrin-Mediated Firm Adhesion and Transendothelial Migration
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《动脉硬化血栓血管生物学》
From the Department of Internal Medicine (J-W.R., K.H.H., J.H.M., J.K., J.Y.P., K.-U.L., M.K.H., S.W.P., Y.H.K., K.H.H), Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea; and the School of Biological Sciences (J.-W.R., J.-B.K.), Seoul National University, Seoul, Republic of Korea.
ABSTRACT
Objective— Uncoupling protein 2 (UCP2) belongs to the mitochondrial anion carrier family and regulates production of reactive oxygen species in macrophages. Previous studies have shown that selective genetic disruption of UCP2 in bone marrow cells results in excess accumulation of monocytes/macrophages in the vascular wall of hypercholesterolemic low-density lipoprotein receptor-deficient (LDLR–/–) mice. Here we investigated whether UCP2 regulates expression of genes involved in monocyte recruitment.
Methods and Results— UCP2 overexpression in THP1 monocytes, which induced a 10-fold increase in mitochondrial UCP2 protein levels, reduced steady-state level of intracellular reactive oxygen species (ROS) and H2O2-induced ROS production. THP1 monocytes with UCP2 overexpression showed lower intracellular calcium levels and less H2O2-triggered intracellular calcium mobilization, and less protein and mRNA levels of ?2 integrins, most notably CD11b. UCP2 overexpression reduced ?2 integrin-mediated firm adhesion of monocytes to either tumor necrosis factor- (TNF-)–stimulated human aortic endothelial cell (HAEC) monolayers or to plates coated with intercellular adhesion molecule-1, not vascular cell adhesion molecule-1. UCP2 overexpression also inhibited cell spreading and actin polymerization in monocytes treated with TNF- and monocyte chemoattractant protein-1 (MCP-1), and reduced MCP-1–induced transmigration of monocytes through HAEC monolayers.
Conclusions— Mitochondrial UCP2 in circulating monocytes may prevent excessive accumulation of monocytes/macrophages in the arterial wall, thereby reducing atherosclerotic plaque formation.
Key Words: UCP2 ? monocytes ? integrins ? adhesion ? atherosclerosis
Introduction
Atherosclerosis exhibits features of chronic inflammatory diseases.1 Macrophages that are derived from recruited monocytes accumulate in the atheroma from the earliest stage of atherogenesis and play pivotal roles in development of atherosclerosis. The process of monocyte recruitment is complex. E- and P-selectins are involved in the initial reversible adherence of monocytes to the endothelial cell layer.2 The following irreversible firm adhesion is mediated by monocyte integrins that recognize vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) on endothelial cells.3 Monocytes express both ?1 (CD49d/CD29) and ?2 integrins (CD11a/CD18, CD11b/CD18, and CD11c/CD18).3 Firm adhesion of monocytes requires activation of integrins, which can be triggered by agonist-induced activation of G protein–coupled chemokine receptors.4 Monocytes express CC chemokine receptor 2 (CCR2), which binds monocyte chemoattractant protein-1 (MCP-1), leading to ? integrin-mediated firm adhesion and subsequent transmigration of adhered monocytes through the vascular endothelium.5
Uncoupling protein 2 (UCP2) belongs to the mitochondrial anion carrier family, and is ubiquitously expressed in various tissues and by monocytes and macrophages. UCP2 is located on the mitochondrial inner membrane. Uncoupling process by mitochondrial UCP2 dissipates the proton electrochemical gradient that builds up across the mitochondrial membrane and results in reduced ATP synthesis, greater heat generation, and less intracellular reactive oxygen species (ROS) production.6,7 Transplantation of UCP2–/– bone marrow cells to low-density lipoprotein receptor-deficient (LDLR –/–) mice accelerated the process of atherosclerosis under hypercholesterolemic conditions, which may result from higher mitochondrial ROS production by UCP2–/– macrophages. Interestingly, arterial walls isolated from hypercholesterolemic LDLR–/– mice with UCP2–/– bone marrow cells had increased macrophage accumulation in atherosclerotic plaques.8
These findings are consistent with the possibility that UCP2 regulates expression of genes involved in monocyte recruitment. To explore this possibility, we examined the effect of UCP2 overexpression on ? integrins and CCR2 expression and the related cellular functions in monocytes.
Methods
Cell Culture and Transfection
THP1 monocytes (American Type Culture Collection) and human aortic endothelial cells (HAECs) obtained from Cell Applications Inc. (San Diego, CA) were cultured and maintained as described.5 Complete sequence of human UCP2 was amplified, inserted into the expression vector pcDNA3.1(Invitrogen), and confirmed by DNA sequencing. L--phosphatidylcholine (ePC), L--phosphatidylserine (bPS), 1,2-Dioleoyl-sn-Glycero-3-phosphoethanolamine (DOPE), and sphingomyelin (eSph) and cholesterol (Chol) (Avanti Polar Lipids, Alabaster, AL) in chloroform, were mixed in an approximate weight ratio of ePC:DOPE:eSph:Chol:bPS=1.6:3:1.47:3:1.3 (10.37 mg/mL), filtered (0.2 μm pore size), dried in a N2-filled rotary evaporator to make a thin film, and mixed with 200 μg UCP2-pcDNA3.1 (1 μg/μL stock). Hemagglutinating virus of Japan (HVJ) was prepared as described.9 Briefly, HVJ was irradiated by ultraviolet light (198mJ/cm2) to inactivate the RNA genome, and 1 mL HVJ suspension (20,000 hemagglutinating units) was mixed with the liposome-UCP2-pcDNA3.1 complex. The resultant liposome-UCP2-pcDNA3.1-HVJ complex was separated from free HVJ by centrifugation on a discontinuous 30% sucrose gradient (25,000 rpm at 4°C for 2 hours), and then collected and suspended in 2 mL balanced salt solution. Liposome-UCP2-pcDNA3.1-HVJ complex (250 μL) was added dropwise to 5 million THP1 monocytes in 20 mL RPMI medium 1640 containing 2% heat-inactivated FBS. After 24 hours, monocytes were harvested and used for further analysis. Flow cytometry analysis of monocytes stained with propidium iodide showed that the liposome complex was not cytotoxic.
Immunoblot Analysis of Human UCP2 Protein
THP1 monocytes in 2 mL ice-cold buffer (10 mmol/L NaCl; 1.5 mmol/L MgCl2; 10 mmol/L Tris·HCl, pH 7.5; 1 mmol/L phenylmethylsulfonyl fluoride (PMSF); 2 μmol/L leupeptin; 1x Aprotinin) were homogenized in a Dounce homogenizer. Then 0.5 mL 2.5x buffer (525 mmol/L mannitol; 175 mmol/L sucrose; 12.5 mmol/L Tris·HCl, pH 7.5; 2.5 mmol/L EDTA, pH 7.5; 1 mmol/L PMSF; 2 μmol/L leupeptin; 1x Aprotinin) was added and centrifuged at 1300g for 5 minutes. Supernatant containing whole-cell protein extract was saved. To isolate mitochondrial and cytosolic protein, the decanted supernatant was further centrifuged twice at 17,000g for 15 minutes, and supernatant containing cytosolic protein was saved. The pellet containing mitochondrial protein was lysed using a Pro-prep protein extraction solution (iNtRON Biotech, Seoul, Korea), agitated on ice for 30 minutes, centrifuged at 12,000g for 30 minutes, and supernatant was saved.
Ten μg protein was separated by electrophoresis (60V for 2 hours) on a 15% polyacrylamide reducing gel and electroblotted onto an Immobilon-P polyvinylidene difluoride transfer membrane (Millipore Corporation). The membrane was blocked for 60 minutes with Tris-buffered saline containing 0.01% Tween-20 and 10% skim milk, and UCP2 protein was visualized using rabbit anti-human UCP2 IgG (1:600, Alpha Diagnostic International, San Antonio, TX), horseradish peroxidase–conjugated goat anti-rabbit IgG (1:15,000, Santa Cruz Biotechnology, Santa Cruz, CA), and a chemiluminescent detection kit (ECL, Amersham). UCP2 protein levels were quantified by scanning photodensitometry using a MULTI-IMAGE analysis system and Quantitation Software (Bio-Rad Laboratories).
Analysis of CCR2 and Integrin mRNA Expression
Expression of CCR2, ? integrins (CD11a, CD11b, CD11c, and CD49d), and adhesion molecules (VCAM-1 and ICAM-1) in THP1 monocytes were estimated by semi-quantitative polymerase chain reaction (PCR) and real-time PCR. The following specific primers were used in PCRs: CCR2, sense 5'-ATGCTGTCCACATCTC-GTTCTCG-3', antisense 5'-TTATAAACCAGCCGAGACTT-CCTGC-3', 1083-bp product; CD11a, sense 5'-CCAAAGACA-TCATCCGCTAC-3', antisense 5'-CACGGTGTAACCCA-AATAGC-3', 398-bp product; CD11b, sense 5'-CTCTGCT-TCCTGTTTGGATC-3', antisense 5'- GGCAGCATAACCC-AAGTAAG-3', 807-bp product; CD11c, sense 5'-CAAGGGTT-TACATACACGGC-3', antisense 5'-CATGTTGATGAAGGT-AGGGC-3', 513-bp product; CD49d, sense 5'-TTTCGGAGCC-AGCATACTAC-3', antisense 5'-AAGACAGCAGAATCA-GACCG-3', 602-bp product; VCAM-1, sense 5'-GAAGATG-GTCGTGATCCTTG-3', antisense 5'-ACTTGACTGTGATC-GGCTTC-3', 401-bp product; and ICAM-1, sense 5'-AGC-TGTTTGAGAACACCTCG-3', antisense 5'-TTTAGACACTTGAGCTCGGG-3', 706-bp product.
As an internal standard, GAPDH was amplified and analyzed under identical conditions using specific primers (sense 5'-GACCCCTTCATTGACCTC-3', antisense 5-GCTAAGCAGTTG-GTGGTG-3', 360-bp product). PCRs involved 25 to 33 cycles, and each cycle was at 94°C for 30 seconds, 60°C for 1 minute, and 72°C for 1 minute. PCR products were electrophoresed on ethidium bromide–containing agarose gels, and band intensity was measured by densitometric scanning. The linearity of amplification was established using serial dilutions of template DNA.
To estimate the relative amount of transcript, real time PCR with SYBR Green I was performed using the LightCycler rapid thermal cycler system (Roche Diagnostics), following the manufacturer’s instructions. The PCRs were done for 40 cycles and each cycle was set as follows: 95°C for 5 seconds, 60°C for 5 seconds, and 72°C for 5 seconds. Detection of the fluorescent product was carried out at end point of the 72°C extension period, and the Ct value (the cycle number at which emitted fluorescence exceeded an automatically determined threshold) was recorded. Ct values for specific genes were corrected by the Ct value for the GAPDH housekeeping gene and expressed as Ct. Data represent mean ± S.D, values of Ct.
Confocal Microscopy
THP1 monocytes were incubated with 50 ng/mL MitoTracker Red CM-H2 XROS (Molecular Probes) to visualize mitochondria. Those monocytes were plated on coverslips by centrifugation, fixed with 3.7% paraformaldehyde in PBS for 10 minutes at 37°C, permeabilized with 0.2% Triton X-100 in PBS for 5 minutes, and blocked with PBS containing 0.8 μg/mL IgG Fc portion (Jackson ImmunoResearch Laboratory), 5% non-fat milk, and 0.2% Triton X-100. In order to detect UCP2 protein, monocytes were serially incubated with rabbit anti-human UCP2 IgG and FITC-conjugated F(ab)2 fragments of goat anti-rabbit IgG (Jackson ImmunoResearch Laboratory, West Grove, PA).
The polymerization of actin filaments in THP1 monocytes, induced by pretreatment with tumor necrosis factor- (TNF-; 1 or 10 ng/mL) or MCP-1 (1 or 10 nM) for 30 minutes, was determined using rhodamine-conjugated phalloidin staining (Molecular Probes), following the manufacturer’s protocol. Coverslips were mounted on glass microscope slides with DAKO fluorescent mounting medium (DAKO Corp.). Images were captured by confocal microscopy using a TCS-SP2 system (Leica Microsystems).
Flow Cytometry
CCR2 or ? integrins surface expressions were detected using flow cytometry as described.5 The relative surface expression was estimated by subtracting the mean fluorescence intensity (MFI) of cells labeled with the nonspecific antibody from that of cells labeled with the antibodies detecting ? integrins (CD11a, CD11b, CD11c, and CD49d) and CCR2. To estimate the intracellular production of ROS, monocytes were labeled with 5 μmol/L H2DCFDA (Molecular Probes) for 30 minutes at room temperature as described,10 stimulated with 1 or 10 μmol/L H2O2 at 37°C for an hour, and cell-associated fluorescence was measured in FL1 window. To estimate mobilization of intracellular calcium, THP1 monocytes were labeled for 30 minutes at 37°C with 3 μmol/L Ca2+-Indicator Fluo-3 AM (Molecular Probes) in calcium-free Hanks’ balanced salt solution, stimulated with 10 μmol/L H2O2 or 1 mmol/L ATP, and the change of cell-associated fluorescence (emission wavelength=530 nm) was detected in FL1 window for 3 minutes by continuous monitoring. All studies consisted of at least three independent experiments. Flow cytometry was performed on a FACScan instrument and data were analyzed using CELLQUEST software (BD Biosciences).
Adhesion Assay
HAEC monolayers in 24-well plates were activated with 25 ng/mL TNF- for 6 hours to induce VCAM-1 and ICAM-1 expression, after which an adhesion assay was performed. In separate experiments, 24-well plates were coated for 2 hours with 10 μg/mL human recombinant VCAM-1 or ICAM-1. THP1 monocytes were suspended in phenol red-free RPMI 1640 medium containing 0.1% BSA (Buffer A) and stimulated with 10 nM MCP-1 for 30 minutes to activate integrins. In a subset of experiments, monocytes were preincubated with 50 μmol/L dibutyl-cAMP (Bt2cAMP) for 30 minutes prior to stimulation with MCP-1 to inhibit chemokine-mediated integrin activation. THP1 monocytes(105 cells in 200 μL buffer A) were added to the HAEC monolayer and incubated for 30 minutes at 37°C. Nonadherent monocytes were removed by gentle washing (2x) with PBS and the number of bound monocytes was counted using high-power microscopy.
Transmigration Assay
The transmigration assay was performed in a Transwell system (BD Biosciences) as described.5 Transwells (polycarbonate membranes with 8 μm pore sizes) in 24-well plates were used, and HAEC monolayers on Transwell membranes were activated with 25 ng/mL TNF- for 6 hours to induce adhesion molecule expression. THP1 monocytes (5x105 cells in 200 μL buffer A) were added to the upper compartment and allowed to equilibrate for 15 minutes, after which MCP-1 (1 or 10 nM) was added to the lower compartment, and cells were incubated for 2 hours at 37°C under 5% CO2. Cells that had transmigrated to the lower compartment were harvested in 1 mL PBS 0.1% BSA and labeled with phycoerythrin (PE)-conjugated anti-human CD14 mouse IgG. Fluorescein isothiocyanate (FITC)-conjugated 106 standard beads (PharMingen, La Jolla, CA) were added to the 1 mL cell suspension and the number of monocytes was counted until 10,000 beads were gated and counted by flow cytometry. All experiments were repeated in at least three independent studies and data represent the calculated number of transmigrated monocytes.
Results
Mitochondrial UCP2 Overexpression Reduces Intracellular ROS Production
The delivery of the pcDNA3.1 vector encoding human UCP2 into THP1 monocytes profoundly enhanced UCP2 expression. Immunoblot analysis showed that the amount of UCP2 protein in UCP2-transfected monocytes was 10-times higher than that in mock-transfected monocytes. UCP2 protein was mostly detected in mitochondrial protein, not in cytosolic protein (Figure I, available online at http://atvb.ahajournals.org). Confocal microscope images of UCP2-transfected monocytes showed newly synthesized UCP2 protein was localized in mitochondria (Figure II, available online at http://atvb.ahajournals.org). UCP2 overexpression reduced both steady-state intracellular ROS levels and production of ROS in response to H2O2, indicating overexpressed UCP2 was functional (Figure 1A). UCP2 overexpression also reduced TNF-- and MCP-1–induced intracellular ROS generation (data not shown).
Figure 1. UCP2 overexpression in transfected THP1 monocytes. THP1 monocytes were transfected with the empty pcDNA3.1 vector (CONT) or the UCP2-encoding pcDNA3.1 vector (UCP2). A, UCP2- (UCP2) or mock- (CONT) transfected THP1 monocytes were labeled with H2DCFDA and stimulated with H2O2 for 30 minutes. Cell-associated fluorescence was measured by flow cytometry. *P<0.05, **P<0.01, determined by Mann–Whitney U test. B, UCP2- (UCP2) or mock- (CONT) transfected THP1 monocytes were labeled with fluo-3 AM. The labeled THP1 monocytes were stimulated with 10 μmol/L H2O2 or 1 mmol/L ATP, and the change of cell-associated fluorescence was monitored by flow cytometry.
UCP2 Overexpression Reduces Intracellular Calcium Mobilization
Cytosolic calcium level, as determined by labeling THP1 monocytes with fluo-3 AM, was measured to be lower in UCP2-transfected THP1 monocytes under steady state. Stimulation of the labeled THP1 monocytes with 10 μmol/L H2O2 and 1 mmol/L ATP triggered elevation of cytosolic calcium(i) in calcium-free media. UCP2 overexpression selectively reduced the magnitude of i triggered by H2O2, not ATP (Figure 1B).
UCP2 Overexpression Reduces Monocyte ?2 Integrin Expression
THP1 monocytes express ? integrins at levels comparable to human circulating monocytes.5 To estimate surface protein expression, UCP2- (UCP2) or mock- (CONT) transfected monocytes were labeled with PE-conjugated mouse IgG specific for CD11a, CD11b, CD11c, CD49d, and CCR2, and surface expression was analyzed using flow cytometry. The results showed that UCP2 overexpression in THP1 monocytes reduced cell surface expression of ?2 integrins, not ?1 integrins (Figures III and IV, available online at http://atvb.ahajournals.org). Of the ?2 integrins, expression of CD11b was the most reduced by UCP2 overexpression (Figure 2A). Real time PCR analysis also indicated reduced expression of CD11b and CD11c mRNA in UCP2 overexpressing monocytes (Table 1). UCP2 overexpression also decreased CD11a, CD49d, and CCR2 transcript levels, but had no effect on cell surface levels of these proteins. Transcript levels of NADPH oxidase, catalase, and superoxide dismutase were not affected by UCP2 overexpression (data not shown), indicating UCP2 overexpression does not induce general downregulation of monocyte gene expression.
Figure 2. The effect of UCP2 overexpression on surface expression of ? integrins and CCR2 in THP1 monocytes. A, UCP2- (UCP2; white bars) or mock- (CONT; black bars) transfected monocytes were incubated with 1 to 10 μmol/L H2O2 or 1 mmol/L ATP for 1 hour, and CD11b surface expression was measured using flow cytometry. *P<0.05, determined by Mann–Whitney U test. B, The effect of UCP2 overexpression on mRNA levels of ? integrins, adhesion molecules, and CCR2 in THP1 monocytes. UCP2- (U) or mock- (C) transfected monocytes were treated with 1 or 10 μmol/L H2O2 for an hour, incubated for 6 hours, and steady-state mRNA levels of ? integrins, ICAM-1, VCAM-1, and CCR2 were determined by RT-PCR.
Effect of UCP2 Overexpression on CCR2 and ? Integrin mRNA Expression in THP1 Monocytes
Oxidative stress induction by an hour incubation with 1 to 10 μmol/L H2O2 or 1 mmol/L ATP enhanced CD11b expression on cell surface. UCP2 overexpression only inhibited upregulation of CD11b surface expression by H2O2, not by ATP (Figure 2A). In mock-transfected THP1 monocytes, H2O2 increased mRNA levels of ?2 integrins CD11a, CD11b, and CD11c, and adhesion molecules ICAM-1 and VCAM-1 in 6 hours. In THP1 monocytes with UCP2 overexpression, this stimulatory effect of H2O2 on expression levels of ?2 integrins and adhesion molecules was reduced (Figure 2B).
UCP2 Overexpression in THP1 Monocytes Reduces ?2 Integrin-Mediated Firm Adhesion
Chemokines such as MCP-1 immediately activate integrins on the cell surface, triggering integrin-mediated firm adhesion and subsequent transmigration of monocytes.5 We confirmed that MCP-1–stimulated THP1 monocytes showed integrin-mediated firm adhesion to TNF-–stimulated HAEC monolayers, and monocyte UCP2 overexpression mostly inhibited this firm adhesion (Figure 3A). Since TNF-–stimulated HAEC monolayers express both VCAM-1 and ICAM-1, the specific ligands for ?1 and ?2 integrins, respectively, additional adhesion assays were performed using plates coated with human recombinant ICAM-1 or VCAM-1. To estimate background adhesion, control adhesion assays were performed using THP1 monocytes pretreated with Bt2cAMP, a permeable analogue of cAMP that blocks integrin-dependent firm adhesion triggered by MCP-1.5 UCP2-transfected THP1 monocytes did not show MCP-1–induced firm adhesion on plates coated with ICAM-1, while adhesion on plates coated with VCAM-1 was intact (Figure 3B). Oxidative stress induction by an hour incubation with 10 μmol/L H2O2 increased adhesion of MCP-1–stimulated THP1 monocytes on ICAM-1–coated plates, and UCP2 overexpression inhibited this stimulatory effect of H2O2 (Figure V, available online at http://atvb.ahajournals.org).
Figure 3. UCP2 overexpression in THP1 monocytes decreases MCP-1-dependent adhesion. A, UCP2- or mock-transfected monocytes were stimulated with 10 nM MCP-1 for 30 minutes (MCP-1 (+)) and then added to HAEC monolayers. HAECs were pretreated with 25 ng/mL TNF- for 6 hours (TNF-(+)) to induce adhesion molecule expression. Micrographs show monocytes adhered to HAEC monolayers. MCP-1–dependent adhesion of monocytes to HAEC monolayers is shown as histograms. B, MCP-1–dependent adhesion of UCP2- (UCP2) or mock- (CONT) transfected THP1 monocytes to plates coated with ICAM-1 or VCAM-1 and adhesion assay was performed as described in Methods. In control experiments, monocytes were pretreated with 50 μmol/L dibutyryl-cAMP (Bt2cAMP) to determine background integrin-independent nonspecific adhesion. NS indicates not significant; *P<0.05, **P<0.01, determined by Mann–Whitney U test.
Cell Spreading and Actin Polymerization in THP1 Monocytes is Inhibited by UCP2 Overexpression
Integrin-mediated firm adhesion initiates cell shape changes and spreading of monocytes, events that must occur for subsequent cellular locomotion and transmigration. We investigated the effect of UCP2 overexpression on TNF-- and MCP-1–stimulated actin polymerization and cell spreading in THP1 monocytes. In control experiments, monocytes were pretreated with cytochalasin D (cyt.D) to inhibit actin polymerization. Confocal microscope image analysis showed that THP1 monocytes exposed to TNF- and MCP-1 underwent morphological changes resulting in multiple pseudopods with abundant actin filaments, and that this process was inhibited by cyt.D. In contrast, less TNF-- and MCP-1–stimulated actin polymerization and cell spreading had occurred in monocytes overexpressing UCP2 (Figure 4).
Figure 4. TNF-- and MCP-1–induced cell spreading and actin polymerization in THP1 monocytes. UCP2- (UCP2) or mock- (CONT) transfected THP1 monocytes were stimulated with TNF- or MCP-1 for 30 minutes, fixed, and labeled with rhodamine-conjugated phalloidin to visualize actin filaments. As a negative control, mock-transfected THP1 monocytes were pretreated with cytochalasin D, an inhibitor of actin polymerization (CONT + Cyt.D). Labeled monocytes were mounted on coverslips and images were obtained using confocal microscopy.
UCP2 Overexpression in THP1 Monocytes Reduces Transendothelial Migration in Response to MCP-1
We previously reported that chemokine-mediated activation of ? integrins was essential for monocyte adhesion and subsequent transmigration through endothelial monolayers.5 The migration of THP1 monocytes across the endothelial layer in response to MCP-1 was found to be dependent on firm adhesions mediated by ?2 integrins.5 Therefore, the effect of UCP2 overexpression on THP1 monocyte transendothelial migration was examined by transmigration assay. In mock-transfected THP1 monocytes, MCP-1 triggered transendothelial migration. UCP2 overexpression in THP1 monocytes reduced transendothelial migration in response to MCP-1 (Figure 5). In both cases, spontaneous migration of THP1 monocytes in the absence of MCP-1 was minimal (data not shown).
Figure 5. The effect of monocyte UCP2 overexpression on transmigration of THP1 monocytes through HAEC monolayers. HAEC monolayers were grown in Transwell plates and pretreated with 25 ng/mL TNF- for 6 hours to induce adhesion molecule expression. Transendothelial migration of UCP2- (UCP2) or mock- (CONT) transfected monocytes in response to 1 to 10 nM MCP-1 was performed as described in Methods. *P<0.05, **P<0.01, determined by Mann–Whitney U test.
Discussion
Intracellular ROS generation is controlled by numerous modulators such as the NADPH oxidase system as well as mitochondrial UCP2. As confirmed by immunoblot assay in the present study, substantially low expression of endogenous UCP2 in monocytes indicates a relatively minor role of UCP2 in the regulation of ROS production under steady state. On the other hand, we found that oxidative stress induction with H2O2 stimulation rapidly induced UCP2 expression in monocytes, suggesting a cytoprotective role of UCP2 from deleterious oxidative insults. Overexpression of mitochondrial UCP2 reportedly protected cardiomyocytes from mitochondrial apoptosis under oxidative stress condition.11 The present study demonstrates that UCP2 overexpression reduces intracellular oxidative stress in monocytes. Transient overexpression of UCP2 in monocytes, which increased mitochondrial UCP2 up to 10 fold, reduced both steady-state level of intracellular ROS and the production of ROS in response to H2O2, MCP-1, and TNF-. Previous studies with macrophages support our observations that UCP2 overexpression markedly reduced ROS production,10 while UCP2–/– mouse macrophages generated excess ROS in response to Toxoplasma gondii infection.12
The present study proves that the reduction of intracellular oxidative stress by UCP2 overexpression decreases CD11b expression and inhibits the positive regulatory effects of H2O2 on ?2 integrins, including CD11b, and adhesion molecules in monocytes. The expression of ?2 integrins and adhesion molecules in inflammatory cells is reportedly regulated by cellular red-ox state. H2O2, a biologically occurring oxidant molecule, increased gene expression of CD11b and CD18 in neutrophils and ICAM-1 in endothelial cells13 and stimulated CD11b/CD18-dependent cell adhesion in the U937 monocyte cell line.14 Dietary supplementation with antioxidants such as vitamin E was found to decrease monocyte CD11b levels.15 Previous studies demonstrated that the elevation of intracellular cytosolic calcium (i) was directly triggered by both intra- and extracellular ROS,16 and i mediated upregulation of CD11b in monocytes.5 This is the first study that shows UCP2 overexpression reduced steady-state cytosolic calcium level and the magnitude of H2O2-mediated i in the absence of extracellular calcium. On the other hand, i and subsequent CD11b upregulation triggered by a nonoxidative stimulus, ie, ATP,17 was not affected by UCP2 overexpression in this study. Such a highly selective inhibition of ROS-triggered i in UCP2-overexpressing monocytes suggests that less ROS production induced by UCP2 overexpression results in less i and eventually lower CD11b expression.
The current study clearly indicates that the change in monocyte CD11b expression is not only a marker for the cellular red-ox state, but also directly regulates the process of monocyte recruitment. UCP2 overexpression, which reduced CD11b surface expression by 75% to 80%, inhibited ?2 integrin-dependent firm adhesion to endothelial monolayers or plates coated with ICAM-1 under steady state and oxidative stress condition. We previously reported that CD11b-dependent firm adhesion was required for transendothelial migration of monocytes in response to MCP-1.5 Although UCP2 overexpression did not affect surface expression of CCR2, the receptor for MCP-1, the resulted CD11b reduction profoundly inhibited MCP-1–mediated transendothelial migration in this study. The positive correlation between CD11b expression in circulating monocytes and the degree of monocyte infiltration into the proatherogenic vascular wall has been documented.5,18,19 In hypercholesterolemic LDLR–/– and apoE–/– mice, CD11b downregulation led to a reduction in lipid accumulation and monocyte infiltration in arterial walls.18 The increased expression of monocyte CD11b under hypercholesterolemic conditions enhanced MCP-1–mediated chemotaxis and transendothelial migration of monocytes in vitro,5 induced excess monocyte adhesion to vascular endothelium, and increased formation of neointima and atherosclerotic plaques in vivo.19 In addition to CD11b reduction, this study demonstrates that THP1 monocytes overexpressing UCP2 display less cell spreading and actin polymerization in response to TNF- and MCP-1. It had been reported that the generation of intracellular ROS was required for actin polymerization in response to TNF-20 and MCP-1.21 Taken together, UCP2 overexpression may potently inhibit the process of monocyte recruitment, especially under oxidative stress conditions.
In summary, the present study shows convincing evidence that intracellular red-ox state determines functional activities of monocytes. The induction of mitochondrial UCP2 overexpression in monocytes led to a reduction of mitochondrial ROS, which decreased the magnitude of i, and eventually resulted in less ?2 integrin-mediated activities, cellular spreading, and actin polymerization. This observation suggests that UCP2 in vivo may function to decrease transendothelial migration of circulating monocytes, reduce the burden of monocytes/macrophages in atheroma, and ultimately decrease plaque mass under atherogenic conditions. Such an outcome would indicate UCP2 overexpression in monocytes/macrophages has an antiatherogenic effect. Future studies are necessary to confirm the effects of UCP2 overexpression on the process of monocyte recruitment and the process of atherosclerosis in vivo.
Acknowledgments
This work was supported by Korean Ministry of Science Grant FPR02A624110 and Asan Institute for Life and Sciences Grants 2002-282, 2002-075, and 2003-288. J.-W.R., J.Y.P., and K.-U.L. were supported by NRL Grant M1-0104-00-0103 from the Korean Ministry of Science.
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ABSTRACT
Objective— Uncoupling protein 2 (UCP2) belongs to the mitochondrial anion carrier family and regulates production of reactive oxygen species in macrophages. Previous studies have shown that selective genetic disruption of UCP2 in bone marrow cells results in excess accumulation of monocytes/macrophages in the vascular wall of hypercholesterolemic low-density lipoprotein receptor-deficient (LDLR–/–) mice. Here we investigated whether UCP2 regulates expression of genes involved in monocyte recruitment.
Methods and Results— UCP2 overexpression in THP1 monocytes, which induced a 10-fold increase in mitochondrial UCP2 protein levels, reduced steady-state level of intracellular reactive oxygen species (ROS) and H2O2-induced ROS production. THP1 monocytes with UCP2 overexpression showed lower intracellular calcium levels and less H2O2-triggered intracellular calcium mobilization, and less protein and mRNA levels of ?2 integrins, most notably CD11b. UCP2 overexpression reduced ?2 integrin-mediated firm adhesion of monocytes to either tumor necrosis factor- (TNF-)–stimulated human aortic endothelial cell (HAEC) monolayers or to plates coated with intercellular adhesion molecule-1, not vascular cell adhesion molecule-1. UCP2 overexpression also inhibited cell spreading and actin polymerization in monocytes treated with TNF- and monocyte chemoattractant protein-1 (MCP-1), and reduced MCP-1–induced transmigration of monocytes through HAEC monolayers.
Conclusions— Mitochondrial UCP2 in circulating monocytes may prevent excessive accumulation of monocytes/macrophages in the arterial wall, thereby reducing atherosclerotic plaque formation.
Key Words: UCP2 ? monocytes ? integrins ? adhesion ? atherosclerosis
Introduction
Atherosclerosis exhibits features of chronic inflammatory diseases.1 Macrophages that are derived from recruited monocytes accumulate in the atheroma from the earliest stage of atherogenesis and play pivotal roles in development of atherosclerosis. The process of monocyte recruitment is complex. E- and P-selectins are involved in the initial reversible adherence of monocytes to the endothelial cell layer.2 The following irreversible firm adhesion is mediated by monocyte integrins that recognize vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) on endothelial cells.3 Monocytes express both ?1 (CD49d/CD29) and ?2 integrins (CD11a/CD18, CD11b/CD18, and CD11c/CD18).3 Firm adhesion of monocytes requires activation of integrins, which can be triggered by agonist-induced activation of G protein–coupled chemokine receptors.4 Monocytes express CC chemokine receptor 2 (CCR2), which binds monocyte chemoattractant protein-1 (MCP-1), leading to ? integrin-mediated firm adhesion and subsequent transmigration of adhered monocytes through the vascular endothelium.5
Uncoupling protein 2 (UCP2) belongs to the mitochondrial anion carrier family, and is ubiquitously expressed in various tissues and by monocytes and macrophages. UCP2 is located on the mitochondrial inner membrane. Uncoupling process by mitochondrial UCP2 dissipates the proton electrochemical gradient that builds up across the mitochondrial membrane and results in reduced ATP synthesis, greater heat generation, and less intracellular reactive oxygen species (ROS) production.6,7 Transplantation of UCP2–/– bone marrow cells to low-density lipoprotein receptor-deficient (LDLR –/–) mice accelerated the process of atherosclerosis under hypercholesterolemic conditions, which may result from higher mitochondrial ROS production by UCP2–/– macrophages. Interestingly, arterial walls isolated from hypercholesterolemic LDLR–/– mice with UCP2–/– bone marrow cells had increased macrophage accumulation in atherosclerotic plaques.8
These findings are consistent with the possibility that UCP2 regulates expression of genes involved in monocyte recruitment. To explore this possibility, we examined the effect of UCP2 overexpression on ? integrins and CCR2 expression and the related cellular functions in monocytes.
Methods
Cell Culture and Transfection
THP1 monocytes (American Type Culture Collection) and human aortic endothelial cells (HAECs) obtained from Cell Applications Inc. (San Diego, CA) were cultured and maintained as described.5 Complete sequence of human UCP2 was amplified, inserted into the expression vector pcDNA3.1(Invitrogen), and confirmed by DNA sequencing. L--phosphatidylcholine (ePC), L--phosphatidylserine (bPS), 1,2-Dioleoyl-sn-Glycero-3-phosphoethanolamine (DOPE), and sphingomyelin (eSph) and cholesterol (Chol) (Avanti Polar Lipids, Alabaster, AL) in chloroform, were mixed in an approximate weight ratio of ePC:DOPE:eSph:Chol:bPS=1.6:3:1.47:3:1.3 (10.37 mg/mL), filtered (0.2 μm pore size), dried in a N2-filled rotary evaporator to make a thin film, and mixed with 200 μg UCP2-pcDNA3.1 (1 μg/μL stock). Hemagglutinating virus of Japan (HVJ) was prepared as described.9 Briefly, HVJ was irradiated by ultraviolet light (198mJ/cm2) to inactivate the RNA genome, and 1 mL HVJ suspension (20,000 hemagglutinating units) was mixed with the liposome-UCP2-pcDNA3.1 complex. The resultant liposome-UCP2-pcDNA3.1-HVJ complex was separated from free HVJ by centrifugation on a discontinuous 30% sucrose gradient (25,000 rpm at 4°C for 2 hours), and then collected and suspended in 2 mL balanced salt solution. Liposome-UCP2-pcDNA3.1-HVJ complex (250 μL) was added dropwise to 5 million THP1 monocytes in 20 mL RPMI medium 1640 containing 2% heat-inactivated FBS. After 24 hours, monocytes were harvested and used for further analysis. Flow cytometry analysis of monocytes stained with propidium iodide showed that the liposome complex was not cytotoxic.
Immunoblot Analysis of Human UCP2 Protein
THP1 monocytes in 2 mL ice-cold buffer (10 mmol/L NaCl; 1.5 mmol/L MgCl2; 10 mmol/L Tris·HCl, pH 7.5; 1 mmol/L phenylmethylsulfonyl fluoride (PMSF); 2 μmol/L leupeptin; 1x Aprotinin) were homogenized in a Dounce homogenizer. Then 0.5 mL 2.5x buffer (525 mmol/L mannitol; 175 mmol/L sucrose; 12.5 mmol/L Tris·HCl, pH 7.5; 2.5 mmol/L EDTA, pH 7.5; 1 mmol/L PMSF; 2 μmol/L leupeptin; 1x Aprotinin) was added and centrifuged at 1300g for 5 minutes. Supernatant containing whole-cell protein extract was saved. To isolate mitochondrial and cytosolic protein, the decanted supernatant was further centrifuged twice at 17,000g for 15 minutes, and supernatant containing cytosolic protein was saved. The pellet containing mitochondrial protein was lysed using a Pro-prep protein extraction solution (iNtRON Biotech, Seoul, Korea), agitated on ice for 30 minutes, centrifuged at 12,000g for 30 minutes, and supernatant was saved.
Ten μg protein was separated by electrophoresis (60V for 2 hours) on a 15% polyacrylamide reducing gel and electroblotted onto an Immobilon-P polyvinylidene difluoride transfer membrane (Millipore Corporation). The membrane was blocked for 60 minutes with Tris-buffered saline containing 0.01% Tween-20 and 10% skim milk, and UCP2 protein was visualized using rabbit anti-human UCP2 IgG (1:600, Alpha Diagnostic International, San Antonio, TX), horseradish peroxidase–conjugated goat anti-rabbit IgG (1:15,000, Santa Cruz Biotechnology, Santa Cruz, CA), and a chemiluminescent detection kit (ECL, Amersham). UCP2 protein levels were quantified by scanning photodensitometry using a MULTI-IMAGE analysis system and Quantitation Software (Bio-Rad Laboratories).
Analysis of CCR2 and Integrin mRNA Expression
Expression of CCR2, ? integrins (CD11a, CD11b, CD11c, and CD49d), and adhesion molecules (VCAM-1 and ICAM-1) in THP1 monocytes were estimated by semi-quantitative polymerase chain reaction (PCR) and real-time PCR. The following specific primers were used in PCRs: CCR2, sense 5'-ATGCTGTCCACATCTC-GTTCTCG-3', antisense 5'-TTATAAACCAGCCGAGACTT-CCTGC-3', 1083-bp product; CD11a, sense 5'-CCAAAGACA-TCATCCGCTAC-3', antisense 5'-CACGGTGTAACCCA-AATAGC-3', 398-bp product; CD11b, sense 5'-CTCTGCT-TCCTGTTTGGATC-3', antisense 5'- GGCAGCATAACCC-AAGTAAG-3', 807-bp product; CD11c, sense 5'-CAAGGGTT-TACATACACGGC-3', antisense 5'-CATGTTGATGAAGGT-AGGGC-3', 513-bp product; CD49d, sense 5'-TTTCGGAGCC-AGCATACTAC-3', antisense 5'-AAGACAGCAGAATCA-GACCG-3', 602-bp product; VCAM-1, sense 5'-GAAGATG-GTCGTGATCCTTG-3', antisense 5'-ACTTGACTGTGATC-GGCTTC-3', 401-bp product; and ICAM-1, sense 5'-AGC-TGTTTGAGAACACCTCG-3', antisense 5'-TTTAGACACTTGAGCTCGGG-3', 706-bp product.
As an internal standard, GAPDH was amplified and analyzed under identical conditions using specific primers (sense 5'-GACCCCTTCATTGACCTC-3', antisense 5-GCTAAGCAGTTG-GTGGTG-3', 360-bp product). PCRs involved 25 to 33 cycles, and each cycle was at 94°C for 30 seconds, 60°C for 1 minute, and 72°C for 1 minute. PCR products were electrophoresed on ethidium bromide–containing agarose gels, and band intensity was measured by densitometric scanning. The linearity of amplification was established using serial dilutions of template DNA.
To estimate the relative amount of transcript, real time PCR with SYBR Green I was performed using the LightCycler rapid thermal cycler system (Roche Diagnostics), following the manufacturer’s instructions. The PCRs were done for 40 cycles and each cycle was set as follows: 95°C for 5 seconds, 60°C for 5 seconds, and 72°C for 5 seconds. Detection of the fluorescent product was carried out at end point of the 72°C extension period, and the Ct value (the cycle number at which emitted fluorescence exceeded an automatically determined threshold) was recorded. Ct values for specific genes were corrected by the Ct value for the GAPDH housekeeping gene and expressed as Ct. Data represent mean ± S.D, values of Ct.
Confocal Microscopy
THP1 monocytes were incubated with 50 ng/mL MitoTracker Red CM-H2 XROS (Molecular Probes) to visualize mitochondria. Those monocytes were plated on coverslips by centrifugation, fixed with 3.7% paraformaldehyde in PBS for 10 minutes at 37°C, permeabilized with 0.2% Triton X-100 in PBS for 5 minutes, and blocked with PBS containing 0.8 μg/mL IgG Fc portion (Jackson ImmunoResearch Laboratory), 5% non-fat milk, and 0.2% Triton X-100. In order to detect UCP2 protein, monocytes were serially incubated with rabbit anti-human UCP2 IgG and FITC-conjugated F(ab)2 fragments of goat anti-rabbit IgG (Jackson ImmunoResearch Laboratory, West Grove, PA).
The polymerization of actin filaments in THP1 monocytes, induced by pretreatment with tumor necrosis factor- (TNF-; 1 or 10 ng/mL) or MCP-1 (1 or 10 nM) for 30 minutes, was determined using rhodamine-conjugated phalloidin staining (Molecular Probes), following the manufacturer’s protocol. Coverslips were mounted on glass microscope slides with DAKO fluorescent mounting medium (DAKO Corp.). Images were captured by confocal microscopy using a TCS-SP2 system (Leica Microsystems).
Flow Cytometry
CCR2 or ? integrins surface expressions were detected using flow cytometry as described.5 The relative surface expression was estimated by subtracting the mean fluorescence intensity (MFI) of cells labeled with the nonspecific antibody from that of cells labeled with the antibodies detecting ? integrins (CD11a, CD11b, CD11c, and CD49d) and CCR2. To estimate the intracellular production of ROS, monocytes were labeled with 5 μmol/L H2DCFDA (Molecular Probes) for 30 minutes at room temperature as described,10 stimulated with 1 or 10 μmol/L H2O2 at 37°C for an hour, and cell-associated fluorescence was measured in FL1 window. To estimate mobilization of intracellular calcium, THP1 monocytes were labeled for 30 minutes at 37°C with 3 μmol/L Ca2+-Indicator Fluo-3 AM (Molecular Probes) in calcium-free Hanks’ balanced salt solution, stimulated with 10 μmol/L H2O2 or 1 mmol/L ATP, and the change of cell-associated fluorescence (emission wavelength=530 nm) was detected in FL1 window for 3 minutes by continuous monitoring. All studies consisted of at least three independent experiments. Flow cytometry was performed on a FACScan instrument and data were analyzed using CELLQUEST software (BD Biosciences).
Adhesion Assay
HAEC monolayers in 24-well plates were activated with 25 ng/mL TNF- for 6 hours to induce VCAM-1 and ICAM-1 expression, after which an adhesion assay was performed. In separate experiments, 24-well plates were coated for 2 hours with 10 μg/mL human recombinant VCAM-1 or ICAM-1. THP1 monocytes were suspended in phenol red-free RPMI 1640 medium containing 0.1% BSA (Buffer A) and stimulated with 10 nM MCP-1 for 30 minutes to activate integrins. In a subset of experiments, monocytes were preincubated with 50 μmol/L dibutyl-cAMP (Bt2cAMP) for 30 minutes prior to stimulation with MCP-1 to inhibit chemokine-mediated integrin activation. THP1 monocytes(105 cells in 200 μL buffer A) were added to the HAEC monolayer and incubated for 30 minutes at 37°C. Nonadherent monocytes were removed by gentle washing (2x) with PBS and the number of bound monocytes was counted using high-power microscopy.
Transmigration Assay
The transmigration assay was performed in a Transwell system (BD Biosciences) as described.5 Transwells (polycarbonate membranes with 8 μm pore sizes) in 24-well plates were used, and HAEC monolayers on Transwell membranes were activated with 25 ng/mL TNF- for 6 hours to induce adhesion molecule expression. THP1 monocytes (5x105 cells in 200 μL buffer A) were added to the upper compartment and allowed to equilibrate for 15 minutes, after which MCP-1 (1 or 10 nM) was added to the lower compartment, and cells were incubated for 2 hours at 37°C under 5% CO2. Cells that had transmigrated to the lower compartment were harvested in 1 mL PBS 0.1% BSA and labeled with phycoerythrin (PE)-conjugated anti-human CD14 mouse IgG. Fluorescein isothiocyanate (FITC)-conjugated 106 standard beads (PharMingen, La Jolla, CA) were added to the 1 mL cell suspension and the number of monocytes was counted until 10,000 beads were gated and counted by flow cytometry. All experiments were repeated in at least three independent studies and data represent the calculated number of transmigrated monocytes.
Results
Mitochondrial UCP2 Overexpression Reduces Intracellular ROS Production
The delivery of the pcDNA3.1 vector encoding human UCP2 into THP1 monocytes profoundly enhanced UCP2 expression. Immunoblot analysis showed that the amount of UCP2 protein in UCP2-transfected monocytes was 10-times higher than that in mock-transfected monocytes. UCP2 protein was mostly detected in mitochondrial protein, not in cytosolic protein (Figure I, available online at http://atvb.ahajournals.org). Confocal microscope images of UCP2-transfected monocytes showed newly synthesized UCP2 protein was localized in mitochondria (Figure II, available online at http://atvb.ahajournals.org). UCP2 overexpression reduced both steady-state intracellular ROS levels and production of ROS in response to H2O2, indicating overexpressed UCP2 was functional (Figure 1A). UCP2 overexpression also reduced TNF-- and MCP-1–induced intracellular ROS generation (data not shown).
Figure 1. UCP2 overexpression in transfected THP1 monocytes. THP1 monocytes were transfected with the empty pcDNA3.1 vector (CONT) or the UCP2-encoding pcDNA3.1 vector (UCP2). A, UCP2- (UCP2) or mock- (CONT) transfected THP1 monocytes were labeled with H2DCFDA and stimulated with H2O2 for 30 minutes. Cell-associated fluorescence was measured by flow cytometry. *P<0.05, **P<0.01, determined by Mann–Whitney U test. B, UCP2- (UCP2) or mock- (CONT) transfected THP1 monocytes were labeled with fluo-3 AM. The labeled THP1 monocytes were stimulated with 10 μmol/L H2O2 or 1 mmol/L ATP, and the change of cell-associated fluorescence was monitored by flow cytometry.
UCP2 Overexpression Reduces Intracellular Calcium Mobilization
Cytosolic calcium level, as determined by labeling THP1 monocytes with fluo-3 AM, was measured to be lower in UCP2-transfected THP1 monocytes under steady state. Stimulation of the labeled THP1 monocytes with 10 μmol/L H2O2 and 1 mmol/L ATP triggered elevation of cytosolic calcium(i) in calcium-free media. UCP2 overexpression selectively reduced the magnitude of i triggered by H2O2, not ATP (Figure 1B).
UCP2 Overexpression Reduces Monocyte ?2 Integrin Expression
THP1 monocytes express ? integrins at levels comparable to human circulating monocytes.5 To estimate surface protein expression, UCP2- (UCP2) or mock- (CONT) transfected monocytes were labeled with PE-conjugated mouse IgG specific for CD11a, CD11b, CD11c, CD49d, and CCR2, and surface expression was analyzed using flow cytometry. The results showed that UCP2 overexpression in THP1 monocytes reduced cell surface expression of ?2 integrins, not ?1 integrins (Figures III and IV, available online at http://atvb.ahajournals.org). Of the ?2 integrins, expression of CD11b was the most reduced by UCP2 overexpression (Figure 2A). Real time PCR analysis also indicated reduced expression of CD11b and CD11c mRNA in UCP2 overexpressing monocytes (Table 1). UCP2 overexpression also decreased CD11a, CD49d, and CCR2 transcript levels, but had no effect on cell surface levels of these proteins. Transcript levels of NADPH oxidase, catalase, and superoxide dismutase were not affected by UCP2 overexpression (data not shown), indicating UCP2 overexpression does not induce general downregulation of monocyte gene expression.
Figure 2. The effect of UCP2 overexpression on surface expression of ? integrins and CCR2 in THP1 monocytes. A, UCP2- (UCP2; white bars) or mock- (CONT; black bars) transfected monocytes were incubated with 1 to 10 μmol/L H2O2 or 1 mmol/L ATP for 1 hour, and CD11b surface expression was measured using flow cytometry. *P<0.05, determined by Mann–Whitney U test. B, The effect of UCP2 overexpression on mRNA levels of ? integrins, adhesion molecules, and CCR2 in THP1 monocytes. UCP2- (U) or mock- (C) transfected monocytes were treated with 1 or 10 μmol/L H2O2 for an hour, incubated for 6 hours, and steady-state mRNA levels of ? integrins, ICAM-1, VCAM-1, and CCR2 were determined by RT-PCR.
Effect of UCP2 Overexpression on CCR2 and ? Integrin mRNA Expression in THP1 Monocytes
Oxidative stress induction by an hour incubation with 1 to 10 μmol/L H2O2 or 1 mmol/L ATP enhanced CD11b expression on cell surface. UCP2 overexpression only inhibited upregulation of CD11b surface expression by H2O2, not by ATP (Figure 2A). In mock-transfected THP1 monocytes, H2O2 increased mRNA levels of ?2 integrins CD11a, CD11b, and CD11c, and adhesion molecules ICAM-1 and VCAM-1 in 6 hours. In THP1 monocytes with UCP2 overexpression, this stimulatory effect of H2O2 on expression levels of ?2 integrins and adhesion molecules was reduced (Figure 2B).
UCP2 Overexpression in THP1 Monocytes Reduces ?2 Integrin-Mediated Firm Adhesion
Chemokines such as MCP-1 immediately activate integrins on the cell surface, triggering integrin-mediated firm adhesion and subsequent transmigration of monocytes.5 We confirmed that MCP-1–stimulated THP1 monocytes showed integrin-mediated firm adhesion to TNF-–stimulated HAEC monolayers, and monocyte UCP2 overexpression mostly inhibited this firm adhesion (Figure 3A). Since TNF-–stimulated HAEC monolayers express both VCAM-1 and ICAM-1, the specific ligands for ?1 and ?2 integrins, respectively, additional adhesion assays were performed using plates coated with human recombinant ICAM-1 or VCAM-1. To estimate background adhesion, control adhesion assays were performed using THP1 monocytes pretreated with Bt2cAMP, a permeable analogue of cAMP that blocks integrin-dependent firm adhesion triggered by MCP-1.5 UCP2-transfected THP1 monocytes did not show MCP-1–induced firm adhesion on plates coated with ICAM-1, while adhesion on plates coated with VCAM-1 was intact (Figure 3B). Oxidative stress induction by an hour incubation with 10 μmol/L H2O2 increased adhesion of MCP-1–stimulated THP1 monocytes on ICAM-1–coated plates, and UCP2 overexpression inhibited this stimulatory effect of H2O2 (Figure V, available online at http://atvb.ahajournals.org).
Figure 3. UCP2 overexpression in THP1 monocytes decreases MCP-1-dependent adhesion. A, UCP2- or mock-transfected monocytes were stimulated with 10 nM MCP-1 for 30 minutes (MCP-1 (+)) and then added to HAEC monolayers. HAECs were pretreated with 25 ng/mL TNF- for 6 hours (TNF-(+)) to induce adhesion molecule expression. Micrographs show monocytes adhered to HAEC monolayers. MCP-1–dependent adhesion of monocytes to HAEC monolayers is shown as histograms. B, MCP-1–dependent adhesion of UCP2- (UCP2) or mock- (CONT) transfected THP1 monocytes to plates coated with ICAM-1 or VCAM-1 and adhesion assay was performed as described in Methods. In control experiments, monocytes were pretreated with 50 μmol/L dibutyryl-cAMP (Bt2cAMP) to determine background integrin-independent nonspecific adhesion. NS indicates not significant; *P<0.05, **P<0.01, determined by Mann–Whitney U test.
Cell Spreading and Actin Polymerization in THP1 Monocytes is Inhibited by UCP2 Overexpression
Integrin-mediated firm adhesion initiates cell shape changes and spreading of monocytes, events that must occur for subsequent cellular locomotion and transmigration. We investigated the effect of UCP2 overexpression on TNF-- and MCP-1–stimulated actin polymerization and cell spreading in THP1 monocytes. In control experiments, monocytes were pretreated with cytochalasin D (cyt.D) to inhibit actin polymerization. Confocal microscope image analysis showed that THP1 monocytes exposed to TNF- and MCP-1 underwent morphological changes resulting in multiple pseudopods with abundant actin filaments, and that this process was inhibited by cyt.D. In contrast, less TNF-- and MCP-1–stimulated actin polymerization and cell spreading had occurred in monocytes overexpressing UCP2 (Figure 4).
Figure 4. TNF-- and MCP-1–induced cell spreading and actin polymerization in THP1 monocytes. UCP2- (UCP2) or mock- (CONT) transfected THP1 monocytes were stimulated with TNF- or MCP-1 for 30 minutes, fixed, and labeled with rhodamine-conjugated phalloidin to visualize actin filaments. As a negative control, mock-transfected THP1 monocytes were pretreated with cytochalasin D, an inhibitor of actin polymerization (CONT + Cyt.D). Labeled monocytes were mounted on coverslips and images were obtained using confocal microscopy.
UCP2 Overexpression in THP1 Monocytes Reduces Transendothelial Migration in Response to MCP-1
We previously reported that chemokine-mediated activation of ? integrins was essential for monocyte adhesion and subsequent transmigration through endothelial monolayers.5 The migration of THP1 monocytes across the endothelial layer in response to MCP-1 was found to be dependent on firm adhesions mediated by ?2 integrins.5 Therefore, the effect of UCP2 overexpression on THP1 monocyte transendothelial migration was examined by transmigration assay. In mock-transfected THP1 monocytes, MCP-1 triggered transendothelial migration. UCP2 overexpression in THP1 monocytes reduced transendothelial migration in response to MCP-1 (Figure 5). In both cases, spontaneous migration of THP1 monocytes in the absence of MCP-1 was minimal (data not shown).
Figure 5. The effect of monocyte UCP2 overexpression on transmigration of THP1 monocytes through HAEC monolayers. HAEC monolayers were grown in Transwell plates and pretreated with 25 ng/mL TNF- for 6 hours to induce adhesion molecule expression. Transendothelial migration of UCP2- (UCP2) or mock- (CONT) transfected monocytes in response to 1 to 10 nM MCP-1 was performed as described in Methods. *P<0.05, **P<0.01, determined by Mann–Whitney U test.
Discussion
Intracellular ROS generation is controlled by numerous modulators such as the NADPH oxidase system as well as mitochondrial UCP2. As confirmed by immunoblot assay in the present study, substantially low expression of endogenous UCP2 in monocytes indicates a relatively minor role of UCP2 in the regulation of ROS production under steady state. On the other hand, we found that oxidative stress induction with H2O2 stimulation rapidly induced UCP2 expression in monocytes, suggesting a cytoprotective role of UCP2 from deleterious oxidative insults. Overexpression of mitochondrial UCP2 reportedly protected cardiomyocytes from mitochondrial apoptosis under oxidative stress condition.11 The present study demonstrates that UCP2 overexpression reduces intracellular oxidative stress in monocytes. Transient overexpression of UCP2 in monocytes, which increased mitochondrial UCP2 up to 10 fold, reduced both steady-state level of intracellular ROS and the production of ROS in response to H2O2, MCP-1, and TNF-. Previous studies with macrophages support our observations that UCP2 overexpression markedly reduced ROS production,10 while UCP2–/– mouse macrophages generated excess ROS in response to Toxoplasma gondii infection.12
The present study proves that the reduction of intracellular oxidative stress by UCP2 overexpression decreases CD11b expression and inhibits the positive regulatory effects of H2O2 on ?2 integrins, including CD11b, and adhesion molecules in monocytes. The expression of ?2 integrins and adhesion molecules in inflammatory cells is reportedly regulated by cellular red-ox state. H2O2, a biologically occurring oxidant molecule, increased gene expression of CD11b and CD18 in neutrophils and ICAM-1 in endothelial cells13 and stimulated CD11b/CD18-dependent cell adhesion in the U937 monocyte cell line.14 Dietary supplementation with antioxidants such as vitamin E was found to decrease monocyte CD11b levels.15 Previous studies demonstrated that the elevation of intracellular cytosolic calcium (i) was directly triggered by both intra- and extracellular ROS,16 and i mediated upregulation of CD11b in monocytes.5 This is the first study that shows UCP2 overexpression reduced steady-state cytosolic calcium level and the magnitude of H2O2-mediated i in the absence of extracellular calcium. On the other hand, i and subsequent CD11b upregulation triggered by a nonoxidative stimulus, ie, ATP,17 was not affected by UCP2 overexpression in this study. Such a highly selective inhibition of ROS-triggered i in UCP2-overexpressing monocytes suggests that less ROS production induced by UCP2 overexpression results in less i and eventually lower CD11b expression.
The current study clearly indicates that the change in monocyte CD11b expression is not only a marker for the cellular red-ox state, but also directly regulates the process of monocyte recruitment. UCP2 overexpression, which reduced CD11b surface expression by 75% to 80%, inhibited ?2 integrin-dependent firm adhesion to endothelial monolayers or plates coated with ICAM-1 under steady state and oxidative stress condition. We previously reported that CD11b-dependent firm adhesion was required for transendothelial migration of monocytes in response to MCP-1.5 Although UCP2 overexpression did not affect surface expression of CCR2, the receptor for MCP-1, the resulted CD11b reduction profoundly inhibited MCP-1–mediated transendothelial migration in this study. The positive correlation between CD11b expression in circulating monocytes and the degree of monocyte infiltration into the proatherogenic vascular wall has been documented.5,18,19 In hypercholesterolemic LDLR–/– and apoE–/– mice, CD11b downregulation led to a reduction in lipid accumulation and monocyte infiltration in arterial walls.18 The increased expression of monocyte CD11b under hypercholesterolemic conditions enhanced MCP-1–mediated chemotaxis and transendothelial migration of monocytes in vitro,5 induced excess monocyte adhesion to vascular endothelium, and increased formation of neointima and atherosclerotic plaques in vivo.19 In addition to CD11b reduction, this study demonstrates that THP1 monocytes overexpressing UCP2 display less cell spreading and actin polymerization in response to TNF- and MCP-1. It had been reported that the generation of intracellular ROS was required for actin polymerization in response to TNF-20 and MCP-1.21 Taken together, UCP2 overexpression may potently inhibit the process of monocyte recruitment, especially under oxidative stress conditions.
In summary, the present study shows convincing evidence that intracellular red-ox state determines functional activities of monocytes. The induction of mitochondrial UCP2 overexpression in monocytes led to a reduction of mitochondrial ROS, which decreased the magnitude of i, and eventually resulted in less ?2 integrin-mediated activities, cellular spreading, and actin polymerization. This observation suggests that UCP2 in vivo may function to decrease transendothelial migration of circulating monocytes, reduce the burden of monocytes/macrophages in atheroma, and ultimately decrease plaque mass under atherogenic conditions. Such an outcome would indicate UCP2 overexpression in monocytes/macrophages has an antiatherogenic effect. Future studies are necessary to confirm the effects of UCP2 overexpression on the process of monocyte recruitment and the process of atherosclerosis in vivo.
Acknowledgments
This work was supported by Korean Ministry of Science Grant FPR02A624110 and Asan Institute for Life and Sciences Grants 2002-282, 2002-075, and 2003-288. J.-W.R., J.Y.P., and K.-U.L. were supported by NRL Grant M1-0104-00-0103 from the Korean Ministry of Science.
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