|Home | About | Journals | Submit | Contact Us | Français|
HIV-associated increase in monocyte adhesion and trafficking is exacerbated by cocaine abuse. The underlying mechanisms involve cocaine-mediated up-regulation of adhesion molecules with subsequent disruption of the blood brain barrier (BBB). Recently, a novel activated leukocyte cell adhesion molecule (ALCAM) has been implicated in leukocyte transmigration across the endothelium. We now show that up-regulation of ALCAM in the brain endothelium seen in HIV+/cocaine drug abusers paralleled increased CD68 immunostaining compared with HIV+/no cocaine or uninfected controls, suggesting the important role of ALCAM in promoting leukocyte infiltration across the BBB. Furthermore, ALCAM expression was increased in cocaine-treated mice with concomitant increase in monocyte adhesion and transmigration in vivo, which was ameliorated by pre-treating with the neutralizing antibody to ALCAM, lending further support to the role of ALCAM. This new concept was further confirmed by in vitro experiments. Cocaine mediated induction of ALCAM in human brain microvascular endothelial cells through the translocation of sigma receptor to the plasma membrane, followed by phosphorylation of platelet-derived growth factor (PDGF)-β receptor. Downstream activation of mitogen-activated protein kinases, Akt, and NF- κB pathways resulted in induced expression of ALCAM. Functional implication of up-regulated ALCAM was confirmed using cell adhesion and transmigration assays. Neutralizing antibody to ALCAM ameliorated this effect. Taken together, these findings implicate cocaine-mediated induction of ALCAM as a mediator of increased monocyte adhesion/transmigration into the CNS.
While the advent of anti-retroviral therapy has decreased the incidence of HIV-associated neurocognitive disorders (HAND), its prevalence is actually on a rise. Drug abuse has been implicated as a contributing risk factor for HIV-1 infection. Intriguingly, cocaine has been shown to facilitate transmigration of inflammatory leukocytes into the brain (Fiala et al., 1998; Fiala et al., 2005). Viral entry into the CNS is mediated, in part, by the transmigration of HIV-infected monocytes into the brain. Leukocyte transmigration is a dynamic, multistep process involving initial “rolling” of cells on the vessel endothelium in response to inflammatory mediators and, subsequent adhesion and diapedesis across the systemic vasculature (Carlos and Harlan, 1994). Interaction of endothelial adhesion molecules with their cognate ligands on monocytes is critical for this process.
Up-regulation of adhesion molecules such as ICAM-1/VCAM-1 is pivotal for development of inflammatory responses. Recently, discovery of a novel activated leukocyte cell adhesion molecule (ALCAM/CD166) has been shown to play important roles in the migration of THP-1 monocytes (Masedunskas et al., 2006) and regulatory T cells (Nummer et al., 2007). ALCAM also participates in the migration of leukocytes from the periphery into the CNS in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) (Cayrol et al., 2008). Interactions between ALCAM expressed on endothelial cells and leukocytes may thus be critical for leukocyte transmigration across the endothelium (Swart, 2002; Masedunskas et al., 2006).
Aberrant expression of adhesion molecules has been implicated as an early step in the development of neurologic disease associated with HIV-infection and cocaine abuse. Cocaine abuse is known to exacerbate HIV-associated neuroinflammation through multiple mechanisms involving up-regulation of adhesion molecules (Gan et al., 1999) and potentiation of neurotoxicity (Bagasra and Pomerantz, 1993; Koutsilieri et al., 1997). Although cocaine-mediated increase in ICAM-1 and VCAM-1 has been demonstrated in BMVEC (Gan et al., 1999), its role in regulating ACLAM remains elusive.
The present study was aimed at exploring the molecular mechanisms by which cocaine mediates the induction of ALCAM in human brain microvascular endothelial cells (HBMECs). Understanding the regulation of ALCAM expression by cocaine may provide insights into the development of therapeutic targets aimed at blocking neuroinflammation.
Sigma receptor antagonist BD1047 and IκB kinase-2 inhibitor SC514 were purchased from Sigma Chemicals (St. Louis, MO). STI-571, an inhibitor of PDGF receptor-tyrosine kinase, was obtained from Novartis, Basel, Switzerland. The specific MEK1/2 inhibitor U0126, JNK inhibitor SP600125, p38 inhibitor SB 203580, Src kinase inhibitor-PP2 and its inactive ortholog PP3, PI3-kinase inhibitor LY294002 were purchased from Calbiochem (San Diego, CA). The concentrations of these inhibitors were based the concentration-curve study and our previous reports (Yao et al., 2010). Adenovirus vectors expressing either the WT or DN forms of Akt were abtained from Dr. K Walsh (Tufts University School of Medicine). Recombinant adenovirus vectors co-expressing either GFP and full length RelA (RelAFL), or mutant were provided by Dr. S Maggirwar (University of Rochester Medical Center). HBMECs were pre-treated with pharmacological inhibitors (BD1047: 20μM; STI571: 1μM; PP2: 10μM; PP3: 1 μM; U0126: 20μM; SP600125: 20μM; SB203580: 20μM; LY294002: 20 μM; SC514: 5μM) for 1 h followed by cocaine exposure.
C57BL/6N male mice and the nude mice were purchased from (Charles River Laboratories). All of the animals were housed under conditions of constant temperature and humidity on a 12-h light/ dark cycle, with lights on at 0700 h. Food and water were available ad libitum. For systemic inflammation, mice were injected with LPS (E.coli O111:B4) intraperitoneally (i.p) at 10mg/kg. All animal procedures were performed according to the protocols approved by the Institutional Animal Care and Use Committee of the University of Nebraska Medical Center.
Primary HBMECs obtained from Dr. Monique Stins (Johns Hopkins University) were cultured in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum, 10% Nu-Serum, 2 mM glutamine, 1 mM pyruvate, penicillin (100 units/ml), streptomycin (100 μg/ml), essential amino acids, and vitamins. Purified HBMECs were positive for endothelial makers DiI-AcLDL (left panel), ZO-1 (middle panel) and β-catenin (right panel) and were found to be >99% pure after exclusion of staining for non-endothelial cell type markers (GFAP, smooth muscle actin, cytokeratin and macrophage antigens). Early passages P3-P7 were used in this study.
Lipid raft were isolated from confluent HBMEC treated with cocaine according to the previous study (Cayrol et al., 2008). Briefly, lysates were mixed with 1 ml of 85% (wt/vol) sucrose, were overlaid with 35% sucrose and 5% sucrose (5 ml each) and were centrifuged for 24 h at 39,000 rpm (Beckman SW 4 rotor) and 4°C. Twelve 1-ml fractions were collected, from the top to bottom. The concentration of cholesterol and protein in each fraction was analyzed with a cholesterol assay kit (Molecular Probes) and a BCA protein assay kit (Pierce).
The quantitative polymerase chain reaction (qPCR) primers for human ALCAM were obtained from SABiosciences (Cat.# PPH00622A-200). Total RNA was extracted with Trizol reagent (Invitrogen) according to the manufacturer's instructions and our previous report (Yao et al., 2010).
HBMECs treated with cocaine were collected in cold PBS and EDTA (5 mM) followed by incubation with anti-ALCAM (3A6, 1:100), anti-ICAM-1 (HA58, 1:1000) and anti-VCAM-1(51-10C9, 1:100). LSR II (BD Biosciences) was used for fluorescence acquisition and data were analyzed with FACSDiva software (BD Biosciences)
HBMECs were transfected with siRNA for σ-1R and PDGF-βR that were obtained from Dharmacon (Boulder,CO) and used according to the manufacturer's instructions. The knockdown efficiency of siRNAs was determined after 2 days of transfection by Western blotting.
The intracellular domain of human PDGF-βR (557-end) with an N-terminal glutathione-S-transferase (GST) tag (GST-PDGF-βR) was expressed and purified via a baculovirus/Sf9 insect cell expression system. To express σ-1R proteins, HEK293T cells were transiently transfected (48 h) with a mammalian TrueORF expression plasmid (5 μg) encoding full-length human σ-1R cDNA (Origene) followed by lysis in modified RIPA buffer and centrifugation. The quantity of σ-1R was determined by immunoblots using a rabbit anti-σ-1R antibody that stably expressed high levels of full length σ-1R. Binding reactions (buffer: 200 mM NaCl, 0.2% TritonX-100, 0.1 mg/ml BSA, and 50 mM Tris, pH7.5) were initiated by adding GST or GST-PDGF-βR (2 μg) and σ-1R (2 μg) proteins and were maintained at 4°C (2-3 h). GST-fusion proteins were precipitated using 100 μl of 10% glutathione Sepharose and the precipitate washed thrice with binding buffer. Bound proteins were eluted with 2X SDS loading buffer, resolved by SDS-PAGE, and immunoblotted with an anti-σ-1R antibody.
CHO cells were transfected for 6h with Lipofectamine-2000 to express PDGFβR-GFP and σ-1R-RFP. Transfected cells were seeded on poly-D-lysine-coated round cover slips. One day following transfection, cells were cultured in serum-free medium for 4 h, and subjected to FRET analysis. Briefly, three images were acquired with the PerkinElmer UltraView confocal system: a donor image (excite with a green laser, emit with a GFP filter), an acceptor image (excite with a red laser, emit with a RFP filter), and a FRET image (excite with a green laser, emit with a RFP filter). Bleed-through constants and cross-talk of each dye were calculated with the Velocity software (PerkinElmer) based on fluorescence intensities obtained from samples expressing only PDGFβR-EGFP or σ-1R-RFP. The “corrected” FRET was calculated from the net FRET according to those constants, and further normalized to the intensity of the donor fluorophore (i.e., normalized FRET). The normalized FRET image was presented with a rainbow color scale.
PDGF antibody array was performed according to the manufacturer's instructions (Full Moon Biosystems). Briefly, confluent cultures of HBMECs treated with cocaine (10 μM) or untreated were used for isolation of lysates. Total proteins in the lysates were biotinylated and incubated with the antibody-coated slides, followed by washes and incubation with Cy3-Streptavidin. The slides were scanned on Axon GenePix Array Scanner (Molecular Devices) to detect bound biotinylated proteins. The fold change was calculated using the ratio of phosphorylated PDGF-βR in the presence versus absence of cocaine. The antibody array analysis was repeated twice independently.
ChIP assay was performed according to the manufacturer's instructions (Millipore). Purified DNA was subjected to PCR to identify the promoter region containing NF-κB binding site “GGA GGG TCC G”. The sequence of the primers used to identify the ALCAM promoter bound by transcription factor NF-κB was as follows: sense: 5′-GAACGGACCAAGACGGACTT-3′, anti-sense:5′-GCCCGGTACCAACAGAAA-3′.
Monocytes were obtained from HIV-1/HIV-2/Hepatitis B seronegative donor leukopacks, and separated by countercurrent centrifugal elutriation as previously described (Gendelman et al., 1988; Chaudhuri et al., 2008b). Freshly elutriated monocytes were cultured in DMEM containing 10% heat-inactivated human serum, 2 mmol/l L-glutamine (Invitrogen), 100 mg/ml gentamicin, and 10 mg/ml ciprofloxacin (Sigma) and were exposed to HIV-1ADA at a multiplicity of infection (MOI) of 0.01 and used for co-culture experiments at day 5 post-infection.
For monocyte adhesion as previously reported (Ramirez et al., 2008; Ramirez et al., 2010), HBMECs were seeded on 96-well plates at a density of 2.5×104cells/well and following confluence were treated with different concentrations of cocaine for 24 h. HIV-infected or non-infected monocytes at 5×106cells/ml were labeled with 10μM Cell tracker green (Molecular Probes) for 10 min. Monocytes and HBMECs were then incubated for 15 min and rinsed three times with PBS to eliminate the non-adherent monocytes. The fluorescence intensity of adherent monocytes was measured using a Synergy Mx fluorescence plate reader (BioTek Instruments).
For monoctye transmigration, cells were washed with PBS and fluorescently labeled with 10μM Cell tracker green (Molecular Probes, Eugene, OR) for 10 min at room temperature. Labeled cells (2×105 cells) were added to the upper compartments of trans-well inserts and allowed to transmigrate at 37°C in a humid atmosphere of 5%CO2 for 24 hrs. Transmigrated monocytes were counted by two individuals ‘blinded’ to the identity of the treatment group according to previously published reports (Eugenin et al., 2006; Kanmogne et al., 2007; Cayrol et al., 2008; Chaudhuri et al., 2008a; Ramirez et al., 2010).
HBMECs were transduced with adenoviral constructs containing either the wild type (WT) or dominant negative (DN) forms of Akt. In addition, HBMEC cells were also transduced with recombinant adenovirus vectors co-expressing both GFP and full length RelA (RelAFL), or GFP and a transcriptionally inert RelA mutant (RelA1-300) used at a multiplicity of infection of 50 as previously described (Kaul et al., 2007).
Treated cells were lysed using the Mammalian Cell Lysis kit (Sigma, St. Louis, MO) and the NE-PER Nuclear and Cytoplasmic Extraction kit (Pierce,). Equal amounts of the corresponding proteins were electrophoresed in a sodium dodecyl sulfate-polyacrylamide gel (12%) in reducing conditions followed by transfer to PVDF membranes. The blots were blocked with 5% non-fat dry milk in phosphate buffered saline. The Western blots were then probed with antibodies recognizing the phosphorylated forms of PDGF-βR, c-Src, ERK1/2, JNK, p38, Akt, NF-κB p65 (1:200; Cell Signaling), ALCAM (1:1000; sc-25624, Santa Cruz Biotechnology), GM1 (1:1000; gift from Dr. Prat) and β-actin (1:4000; Sigma). The secondary antibodies were alkaline phosphatase conjugated to goat anti mouse/rabbit IgG (1:5000). Signals were detected by chemiluminescence (Yao et al., 2009c).
The procedure for immunoprecipitation was performed as described previously (Yao et al., 2009b). HBMECs with different treatments were lysed in RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.1% SDS, 1.0% NP-40 and 0.5% sodium deoxycholate) containing proteinase and phosphatase inhibitors. For each sample, 200 μg of protein was used for co-immunoprecipitation. Sample protein was incubated with 2 μg diluted anti-4G10 (Millipore) overnight at 4°C followed by incubation with 20 μl of protein A-Sepharose for 3 h at 4°C. The mixture was then centrifuged (at 6000 g for 30 s) and the cell pellets were rinsed twice with RIPA, followed by boiling in 2X Western blot loading buffer for 4 min. After spinning (at 6000 g for 30 s) the supernatants were subjected to Western blot analysis as described above.
Male C57 BL/6 mice (Charles River Laboratory) 4-5 wk of age were used as BMM donors. Briefly, the femur was removed, bone marrow cells were dissociated into single-cell suspensions, and were cultured for 10 days supplemented with 1000 U/ml M-CSF (Wyeth). Cultured BMMs proved to be 98% CD11b+ by flow cytometric analysis using a FACSC flow cytometer (BD Biosciences). For tracking experiments, cells were labeled with the membrane dye PKH26 according to the manufacturer's instructions (Sigma).
Assay of monocyte transmigration into the brain was performed in C57BL/6 mice. Animals were divided into 4 groups (n=6): (a) Saline, (b) Cocaine, (c) Cocaine plus anti-ALCAM and (d) Cocaine plus isotope control antibody. Cocaine was injected at a dose of 20 mg/kg i.p for 7 days. For the ALCAM neutralizing antibody study mice were injected with cocaine for 7 days as described above; additionally on days 3, 5 and 7 of cocaine injection, co-administration of either anti-ALCAM or isotope control antibody (250 μg each i.p; monoclonal MAB 656 or mouse IgG1, MAB 002, R&D Systems, Minneapolis, MN) was also performed. Antibody concentrations and treatment regimens were based on previously published report (Cayrol et al., 2008). On the 8th day post-cocaine injection, animals were injected with PKH 26-labeled BMM at a concentration of 107 cells/100 μl through tail vein. Twenty four hrs following cell infusion, the animals were sacrificed and subjected to transcardial perfusion with saline to remove labeled monocytes from tissue blood vessels. Brain tissues were then removed and frozen at -80°C until cryosection. In all the six animals per group perivascular and parenchyma were counted in the entire area of the three coronal brain sections: 1.94, 1.34 and 0.14 mm to bregma (Franklin and Paxinons, 1997) according to the previously described report (Schilling et al., 2009).
Brain microvessels were isolated as described previously (Huang et al., 2010). Under anesthesia, animals were perfused with saline; the brains were removed and immediately immersed in ice-cold isolation buffer A [103 mM NaCl, 4.7mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4,1.2 mM MgSO4, and 15 mM HEPES; pH 7.4 with Complete Protease Inhibitor (Roche)]. The choroid plexus, meninges, cerebellum, and brain stem, were removed followed by homogenization of the brains in 5 ml isolation buffer B [Buffer A with 25 mM NaHCO3, 10 mM glucose, 1 mM Na pyruvate, and dextran (molecular weight 64kD; 10 g/l); pH 7.4] with Complete Protease Inhibitor. 26% dextran (was then added to the homogenates, followed by centrifugation at 5,800×g for 20 min. Cell pellets were re-suspended in isolation buffer B and filtered through a 70 μm mesh filter (Becton Dickinson). Filtered homogenates were repelleted by centrifugation and smeared on slides.
For immunofluorescence staining, brain microvessels smeared on slides were fixed for 10 min at 95°C, followed by incubation with 3% formaldehyde in PBS for 10 min at 25°C. The slides were washed five times with PBS, permeabilized with 0.1% Triton X-100 for 30 min, rewashed five times in PBS, and blocked in 1% BSA in PBS for 30 min at 25°C. Samples were then incubated with rabbit ALCAM (polyclonal rabbit, 1:100 dilution; sc-25624 Santa Cruz Biotechnology) and Cav-1 (monoclonal mouse, 1:100 dilution; sc-70516 Santa Cruz Biotechnology) antibodies (1:100) overnight at 4°C. The slides were washed with PBS and incubated with Alexa Fluor 594-conjugated anti-rabbit or AlexaFluor 488-conjugated anti-mouse IgG for 1 h at RT. After a final washing with PBS, the slides were mounted with ProLong Gold Antifade reagent to visualize the nuclei as previously reported (Huang et al., 2010). The immunofluorescent images were captured using confocal microscopy.
Formalin-fixed, paraffin-embedded sections (5 μm) of basal ganglia from HIV-, HIV+, and HIV+/drug abusers (cocaine included) were obtained from the National NeuroAIDS Tissue Consortium and stained with antibodies specific for ALCAM (polyclonal goat, 1:40 dilutions: AF1172; R & D Systems) , Cav-1 (monoclonal mouse , 1:100 dilution; sc-70516 Santa Cruz Biotechnology), or CD-68 (1:100; Dako). Sections were washed thrice in PBS followed by incubation in secondary AlexaFluor 488 Donkey anti-Goat antibody (1:200) and AlexaFluor 594 Goat anti-mouse antibody (1:200) or AlexaFluor 594 Goat anti-rabbit antibody (1:200). For quantification of ALCAM and Cav-1 signal, Z-stack images were acquired on a Carl Zeiss confocal laser scanning microscope and were analyzed with LSM 510 software (Release Version 4.0 SP1). Signal intensities were collected in duplicate and microscopy data were acquired by two investigators ‘blinded’ to the intensity of the disease group.
Statistical analysis was performed using one-way analysis of variance with a post hoc Student t-test. Results were judged statistically significant if p < 0.05 by analysis of variance.
Since the degree of neurologic deficit in HIV-infected individuals is strongly correlated with the number of activated macrophages and microglia within the basal ganglia, we verified the CD68-positive staining in postmortem brain tissues from HIV-, HIV+/no cocaine and HIV+/cocaine drug abusers. Due to the inherent difficulty of finding tissues from uni-drug abusers, we resorted to samples from poly-drug abusers that included a history of cocaine abuse. In order to establish clinical relevance of ALCAM expression in the context of HIV-1 infection with drug (cocaine) abusers, we examined ALCAM expression and double-labeling with CD68. As shown Fig.1A, the increased CD68 immunostaining seen in HIV+/cocaine drug abusers paralleled with up-regulation of ALCAM in the brain endothelium compared with HIV+/no cocaine or uninfected controls. This new finding suggested that ALCAM plays a role in promoting leukocyte infiltration across the BBB.
Further validation of up-regulated expression of ALCAM, sections from basal ganglia region of the brain were stained for both ALCAM and caveolin-1 (Cav-1, endothelial cell membrane marker). ALCAM expression, although weak in microvessels from both HIV- and HIV+/no cocaine individuals, was significantly enhanced in vessels from HIV+ /cocaine drug abusers. Merged images of ALCAM and Cav-1 staining confirmed co-localization of ALCAM on the surface of endothelial cells (Supplementary Fig.1).
Cav-1 and ALCAM signal intensities in CNS blood vessels from HIV-(Fig. 1B-upper panel), HIV+/no cocaine (Fig. 1B-middle panel) and HIV+/cocaine drug abusers (Fig. 1B-lower panel) were quantified by recording x-y planar images (0.1 μm in thickness) on two distinct channels, followed by reconstruction of 3-μm z-stack images. ALCAM staining was significantly stronger in HIV+/cocaine drug abusers compared with the HIV-or the HIV+/no cocaine group (Fig. 1C). Intriguingly, there was no significant change in the expression of other adhesion molecules such as ICAM-1 or VCAM-1 in the brains tissues in response to cocaine as shown in Supplementary Fig.2 & 3, thus underscoring the specificity of ALCAM induction by cocaine.
To further validate cocaine-mediated induction of ALCAM in vivo, we examined the expression level of ALCAM in brain capillaries isolated from cocaine versus saline-treated mice. Following administration of cocaine, there was an increase in the expression level of ALCAM in isolated microvessels from cocaine-exposed mice compared with controls (Fig. 2A).
Since ALCAM expression was increased in cocaine-treated mice, to validate the role of ALCAM in monocyte transmigration in vivo, mice were treated with cocaine, followed by tail vein injection of labeled mouse BMM and their detection in the brain. Of particular note, the distribution of labeled monocytes was primarily within the perivascular cuffs (See arrowheads in Fig.2B), with some localization in the parenchyma (See arrows in Fig.2B). Quantification of the brain sections revealed increased transmigration in the CNS of cocaine-treated mice compared with controls. Specificity of ALCAM action was further demonstrated in mice pre-treated with the neutralizing antibody to ALCAM. Monocyte transmigration in these mice was ameliorated lending further support to the role of ALCAM (Fig.2C).
To better understand how cocaine regulates ALCAM expression, we examined the effects of cocaine on ALCAM induction in HBMECs. As an initial screening study to identify the effective concentration of cocaine on ALCAM expression, HBMECs were treated with varying concentrations of cocaine (1,10 and 100μM) with a maximal response at 10μM (Supplementary Fig. 4 A). This concentration of cocaine was therefore chosen for all our further studies. The next step was to examine the time course of ALCAM induction. Cocaine-mediated induction of ALCAM was observed as early as 6 hours following treatment and was significantly up-regulated even at 24 hours post-treatment in HBMECs (shown in supplementary Fig. 4 B & C). Furthermore, using flow cytometry it was demonstrated that under normal conditions HBMECs expressed almost equal levels of ALCAM and ICAM-1, and lower levels of VCAM-1 (Fig.3A). Intriguingly, activation of cells with cocaine elicited robust up-regulation of ALCAM, but a weaker induction of ICAM-1 (Fig.3A).
The next step was to examine the distribution of ALCAM on these cells. As evidenced by confocal microscopy (Fig.3B), following cocaine exposure, ALCAM immunoreactivity concentrated around the plasma membrane. Since ALCAM is present in cholesterol-enriched membrane microdomains (Cayrol et al., 2008), as expected, ALCAM was primarily recruited to cholesterol-enriched membrane microdomains (fractions 4-5), positive for ganglioside marker of lipid raft, GM1 (Fig.3C). Following cocaine treatment, ALCAM expression in lipid raft fractions of HBMECs was increased significantly compared to controls (Fig.3D).
σ-1R belonging to the non-opioid receptor family bind to diverse classes of psychotropic drugs including cocaine (Hayashi and Su, 2003). As shown by RT-PCR (Fig.4A) and Western blotting (Fig.4C), HBMECs expressed σ-1R mRNA and protein. Cocaine-mediated induction of ALCAM was significantly attenuated by pre-treatment of cells with the σ-1R antagonist BD1047 (Fig.4B). Transfection of cells with σ-1R siRNA also resulted in significant knock-down of expression of σ-1R (Fig.4C) with significant abrogation of cocaine-mediated induction of ALCAM expression (Fig.4D).
Engagement of σ-1R can regulate both the activation and signaling of tyrosine kinase receptors, including PDGF-βR, which in turn, regulate the expression of cell adhesion molecules (Lin et al., 2007). We thus rationalized that cocaine/σ-1R dyad could result in activation of PDGF-βR, and subsequently, induction of ALCAM. Lysates from HBMECs treated with cocaine induced time-dependent phosphorylation of PDGF-βR at Tyr 751 (Fig.5 A), that was abrogated by both STI571 (Fig.5 B) and BD1047 (Fig.5 C). Pretreatment with STI571 (Fig.5 D) and transfection with PDGF-βR siRNA (Fig.5 E-F) attenuated ALCAM expression.
These findings were further validated using human phospho-receptor tyrosine kinase (RTK) antibody arrays specific for PDGF-βR (Supplementary Table 1 online). Cocaine specifically induced greater than 2-fold increase in phosphorylation of PDGF-βR at Tyr 751. On the other hand, phosphorylation of PDGF-βR (Tyr 1021 &741) and PDGF-αR (Tyr 849) were not affected by cocaine. Phosphorylation at Tyr 771 but not Tyr 1009 was also observed with cocaine treatment (Supplementary Fig.5).
To explore protein-protein interaction of the two receptors, GST-fusion protein containing PDGF-βR was synthesized and used as GST immobilized baits in the pull-down assay. σ-1R was readily precipitated by GST-PDGF-βR (Fig.6A).
Protein-protein interaction of σ-1R and PDGF-βR was further confirmed by FRET analysis. As shown in Fig.6B (left panels), CHO cells transfected with GFP and the σ-1R-RFP failed to demonstrate any FRET signal. In contrast, cells co-transfected with both PDGF-βR-GFP and σ-1R-RFP demonstrated enhanced protein-protein interaction as shown in Fig.6B (right panels).
To further assess this interaction, series of co-immunoprecipitations using lysates from cocaine-treated HBMECs were performed. σ-1R immunoreactive band was consistently seen in the protein precipitated by anti-PDGF-βR antibody (Fig.6C-upper panel) and reciprocally, PDGF-βR band was displayed in the σ-1R precipitates (Fig.6C-lower panel). These data demonstrate an evident interaction between σ-1R and PDGF-βR in response to cocaine. Further validation of this interaction was performed using confocal microscopy. As shown in Fig.6D, there was a diffuse pattern of σ-1R expression in the cytoplasm of untreated HBMECs. Treatment with cocaine resulted in clustering and polarization of σ-1R with the cell membrane. A 1-μm z-stack reconstruction demonstrated co-localization of both σ-1R and PDGF-βR in the presence of cocaine (see merged image and Fig.6E).
Activation of c-Src has been documented as an upstream event of PDGF-βR activation (Lin et al., 2007). We thus examined its involvement in cocaine-mediated activation of PDGF-βR and ALCAM induction. Treatment with cocaine resulted in increased c-Src phosphorylation, which was inhibited by Src tyrosine kinase inhibitor PP2, but not by its inactive ortholog PP3 (Fig.7 A-B). Intriguingly, cocaine-stimulated PDGF-βR phosphorylation was also blocked by PP2, but not by PP3 (Fig.7C). In contrast, cocaine-induced activation of Src was not blocked by STI-571(Fig.7D), thereby suggesting the role of c-Src activation in transactivation of PDGF-βR. Pre-treatment with PP2 significantly blocked cocaine-induced expression of ALCAM, thus confirming the role of c-Src in this process (Fig.7E).
MAPK kinase and PI3K-Akt pathways play critical roles in both cocaine (Yao et al., 2009a) and PDGF-βR signaling (Lin et al., 2005; Yao et al., 2009a). We next examined the involvement of these pathways in cocaine-mediated induction of ALCAM. Treatment with cocaine resulted in time-dependent increase in phosphorylation of MAPK (Fig.8A) and Akt pathways (Fig.8C). Pretreatment of cells with either MEK1/2 (U0126), JNK (SP600125), p38 (SB203580), or PI3K (LY294002) inhibitor resulted in amelioration of cocaine-mediated induction of ALCAM, thus underpinning their roles in the process (Fig.8 B and D). Further validation of the Akt pathway in this process was confirmed by transfecting cells with either the WT or DN Akt, followed by treatment with cocaine. Cocaine-mediated induction of ALCAM was attenuated by the DN-Akt, but not the WT-Akt construct (Fig.8E).
Having determined that cocaine mediated translocation of the σ-1R to the cell membrane and, that subsequent activation of the c-Src, PDGF-βR, and MAPKs pathways were critical processes involved in the induction of ALCAM, we next sought to link σ-1R & PDGF-βR activation with the signal transduction pathways. HBMECs pretreated with either the σ-1R or PDGF-βR (antagonists and siRNAs) followed by treatment with cocaine, were assessed for activation of signaling pathways. Both σ-1R and PDGF-βR antagonists inhibited cocaine-mediated activation of MAPKs (ERK1/2, JNK and p38) and Akt (Fig. 9 A). In addition to the pharmacological approach, silencing using σ-1R (Fig. 9 B) and PDGF-βR siRNAs (Fig. 9 C) was also able to inhibit cocaine-mediated activation of these pathways. Similarly, we also found abrogation of cocaine-mediated activation of both MAPK and Akt pathways in cells pretreated with Src kinase inhibitor (Fig. 9 D).
Members of the NF-κB family are considered to play essential roles in both cocaine and PDGF-βR-mediated signaling (Huang et al., 2003; Lin et al., 2005). Treatment of HBMECs with cocaine resulted in translocation of NF-κB p65 into the nucleus (Fig.10 A). Pretreatment of cells with either IκB kinase-2 inhibitor-SC514 (Fig.10 B) or mutant NF-κB adenovirus (Fig.10 C) abrogated cocaine-induced ALCAM expression, thereby underscoring the role of NF-κB p65 in cocaine-mediated induction of ALCAM.
Next step was to examine whether there existed a link that could tie activation of σ-1R and PDGF-βR with NF-κB translocation. Both σ-1R (Fig.10 D) and PDGF-βR siRNAs (Fig.10 E) were able to inhibit cocaine-mediated activation of NF-κB. To further confirm the involvement of NF-κB binding of NF-κB to ALCAM promoter was explored using ChIP assays. These experiments revealed increased binding of NF-κB to the ALCAM promoter in HBMECs treated with cocaine (Fig.10 F-G).
To identify the functional relevance of up-regulated ALCAM, we examined the ability of cocaine-induced ALCAM to induce monocyte adhesion. Treatment of HBMECs with cocaine resulted in increase in both HIV-infected and uninfected monocyte adhesion (Fig.11 A-B). To confirm the role of ALCAM in this process, monocyte adhesion was evaluated in the presence of blocking antibodies. ALCAM antibody significantly restricted monocyte adhesion compared with the control (Fig.11C), suggesting thereby that ALCAM was critical for monocyte adhesion.
Roles of σ-1R and PDGF-βR in this process were confirmed by pre-treating cells with either the σ-1R or PDGF-βR antagonist, both of which resulted in inhibition of monocyte adhesion (Fig.11D). Similarly, a link between cocaine-mediated adhesion of monocytes and activation of c-Src with the signaling pathways was also established as evidenced by the fact that Src kinase inhibitor abolished cocaine-mediated monocyte adhesion (Fig.11D). Inhibitors specific for MAPK, PI3K & IkB kinase also blocked cocaine-induced monocyte adhesion (Fig.11E), further supporting the notion that in HBMECs sequential activation of σ-1R/Src/PDGF-βR/MAPK/PI3K pathways results in up-regulation of ALCAM and monocyte adhesion.
Next, we examined the ability of cocaine to induce monocyte transmigration in an in vitro model of human BBB. Exposure of HBMECS to cocaine resulted in increased monocyte transmigration (Fig.11F). Pre-treatment of HBMECs with anti-ALCAM antibody significantly restricted monocyte migration compared with the isotype control, thereby underpinning the role of ALCAM in transmigration of monocytes.
To understand whether cocaine-mediated induction of ALCAM was dependent on systemic inflammation, two mouse models: a) systemic inflammation model of LPS injection and, b) T cell deficiency model using nude mice, were exposed to cocaine and examined for induction of ALCAM in the isolated brain microvessels. As shown in Fig.12A, administration of LPS resulted in induction of ALCAM expression compared with the control group without LPS exposure. This effect was potentiated in the presence of cocaine. Intriguingly, in T cell deficient mice, cocaine failed to induce ALCAM expression. These findings were also validated by confocal microscopy in sections of microvessels stained with ALCAM and caveolin-1 from the three groups of mice exposed to cocaine as shown in Fig.12B.
Although antiviral therapies have had a profound impact on controlling systemic HIV-1 load leading to increased longevity in AIDS patients, the inability of some of these drugs to cross the BBB results in a slow and smoldering infection/cell activation in the CNS. As these patients continue to live longer, the brain becomes a sanctuary for virus-induced toxicity leading to increased prevalence of HAND. Adding fuel to this problem is the increased use of illicit drugs that is rapidly becoming a burgeoning problem in HIV+ individuals (Goodkin et al., 1998). Intriguingly, abuse of the psychostimulant cocaine in these patients has been linked to increased HIV seroprevalence and disease progression (Goodkin et al., 1998). In cell culture studies, cocaine has been shown to disrupt BBB, thereby enhancing increased extravasation of infected/activated cells across the endothelium (Gan et al., 1999). Cocaine has also been shown to impair BBB by modulating transcriptional regulation of key cellular functional genes (Fiala et al., 1998). Although cocaine has been shown to induce ICAM-1 and VCAM-1 expression, very few studies have actually explored direct effects of cocaine on the expression of ALCAM.
In the present study we report cocaine-mediated transcriptional and translational induction ALCAM in HBMECs. Our findings complement earlier studies on the induction of ICAM-1 and VCAM-1 expression by cocaine (Gan et al., 1999). Triggering interaction of ALCAM-ALCAM or ALCAM-CD6 initiates monocyte transmigration through endothelial junctions both in the periphery (Masedunskas et al., 2006) and the CNS (Cayrol et al., 2008). Using human brain sections from HIV+/cocaine drug abusers, we found that prominent up-regulation of ALCAM in brain endothelium. ALCAM expression paralleled with macrophage recruitment. The study reported here provides first evidence of ALCAM up-regulation in brain vasculature of HIV+ drug abusers. Next, the functional significance of cocaine-mediated induction of ALCAM was examined in both the in vitro and in vivo models of monocyte adhesion and transmigration. Cocaine treatment of HBMECs resulted in increased monocyte adhesion and transmigration across an in vitro BBB model. Furthermore, both these effects were inhibited by treating cells with the neutralizing antibody specific for ALCAM. These findings were also validated in vivo wherein following cocaine injections there was increased expression of ALCAM resulting in increased transmigration of labeled monocytes into the brains. Furthermore, we also demonstrated that cocaine-mediated induction of ALCAM was dependent on systemic inflammation, as this effect of cocaine was not observed in the T-cell deficient mouse model (Fig.12). In concordance with cell culture studies, cocaine-mediated transmigration of monocytes was also abrogated in mice pretreated with the ALCAM neutralizing antibody. These findings are in agreement with a recent study investigating the role of ALCAM in monocyte infiltration in MS and in EAE (Cayrol et al., 2008). It is likely that ALCAM expressed in response to cocaine binds either to the ALCAM or CD6 expressed on the blood-derived monocytes. Both the ALCAM ligands, CD6 and ALCAM are expressed on monocytes isolated from the peripheral blood of healthy donors (Cayrol et al., 2008). In our study cocaine treatment exerted no effect on the expression of ALCAM or CD6 in human monocytes (data not shown).
To delve deeper into the molecular mechanisms of cocaine-mediated induction of ALCAM, role of σ-1R was assessed. Cocaine-mediated induction of ALCAM involved σ-1R activation since pretreatment of cells with σ-1R antagonist, abrogated induction of ALCAM. σ-1R is known to translocate and remodel the plasma membrane following activation (Hayashi and Su, 2003, 2005; Monnet, 2005). Consistent with these findings, we demonstrated translocation of σ-1R from the cytoplasm to the plasma membrane following cocaine treatment. Intriguingly, activation of σ-1R with cocaine resulted in specific phosphorylation of PDGF-βR. We demonstrated direct binding of σ-1R with PDGF-βR specifically at Tyr 751. Plasma membranes thus serve as “platform or hubs” for coalescing the interaction of key signaling molecules triggered by cocaine. Furthermore the interaction of σ-1R with the PDGF-βR is in concordance with earlier published reports suggesting σ-1R-mediated regulation of signaling of the tropic factors such as EGF receptor (Takebayashi et al., 2004).
The role of c-Src in cocaine-mediated induction of ALCAM expression reported here is consistent with previous reports implicating its involvement in TNF-α/IL-1β-mediated induction of ICAM-1 expression (Lin et al., 2005; Lin et al., 2007). Another key point of our findings was that cocaine-mediated phosphorylation of PDGF-βR was dependent on c-Src activation since inhibition of Src kinase significantly blocked cocaine-induced PDGF-βR phosphorylation.
Intracellular signaling events such as MAPK kinases are known to be triggered by cocaine and during inflammation in the CNS (Yao et al., 2009a). Using both the pharmacological and genetic approaches we examined the activation of MAPK kinase and PI3K/Akt pathways in cocaine-mediated induction of ALCAM. These findings are consistent with the previous reports implicating the role of these signaling pathways in the induction of ICAM-1(Lin et al., 2005; Xia et al., 2009). Cocaine-mediated induction of signaling was thus regulated by sequential upstream activation of σ-1R, c-Src and PDGF-βR.
Dissection of the downstream mediators of signaling pathways indicated phosphorylation and translocation of NF-κB. The requirement of NF-κB signaling in cell adhesion expression has been confirmed in previous studies (Ramudo et al., 2009; Sutcliffe et al., 2009). Additionally, MAPKs are also known to transduce their signals via activation and translocation of NF-κB in various cells systems (Meucci et al., 2000). Consistent with these findings, we also demonstrated the role of MAPKs and p65/RelA nuclear translocation in cocaine-mediated induction of ALCAM expression.
In summary, our findings have chalked out a detailed molecular pathway of cocaine-mediated induction of ALCAM, involving activation and translocation of σ-1R, activation of c-Src, and subsequently of PDGF-βR. It is likely that σ-1R translocation brings together signaling molecules such as c-Src and PDGF-βR in close proximity for interaction/activation. Subsequently, the activation of MAPKs and PI3K/Akt pathways leads to translocation NF-κB into the nucleus, ultimately resulting in increased ALCAM. This in turn, can lead to adhesion and recruitment of increased numbers of inflammatory cells onto the vessel endothelium. These findings have implications for HIV-1-infected cocaine abusers that have increased risk of stroke and CNS-associated neuroinflammation. Neutralizing ALCAM antibody can thus be considered as an adjunct therapeutic strategy for the treatment of cocaine addicts that are HIV-1 infected.
This work was supported by grants MH-068212, DA020392, DA023397, DA024442 (SB) and DA030285 (HY) from the National Institutes of Health and by grant M01-RR-00071 from the Clinical Research Center of the Mount Sinai School of Medicine (SM).
Conflict of interest disclosure: The authors declare no competing financial interests.