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. Author manuscript; available in PMC: 2016 Apr 1.
Published in final edited form as: J Immunol. 2015 Feb 25;194(7):3246–3258. doi: 10.4049/jimmunol.1302647

BUPRENORPHINE DECREASES THE CCL2-MEDIATED CHEMOTACTIC RESPONSE OF MONOCYTES

Loreto Carvallo 1, Lillie Lopez 1, Fa-Yun Che 1, Jihyeon Lim 1, Eliseo Eugenin 2,3, Dionna W Williams 1, Edward Nieves 4, Tina M Calderon 1, Carlos Madrid-Aliste 5, Andras Fiser 5, Louis Weiss 1, Ruth Hogue Angeletti 4, Joan W Berman 1,6
PMCID: PMC4369415  NIHMSID: NIHMS658567  PMID: 25716997

Abstract

Despite successful cART, approximately 60% of HIV infected people exhibit HIV associated neurocognitive disorders (HAND). CCL2 is elevated in the CNS of infected people with HAND and mediates monocyte influx into the CNS, which is critical in neuroAIDS. Many HIV infected opiate abusers have increased neuroinflammation that may augment HAND. Buprenorphine is used to treat opiate addiction. However, there are few studies that examine its impact on HIV neuropathogenesis. We show that buprenorphine reduces the chemotactic phenotype of monocytes. Buprenorphine decreases the formation of membrane projections in response to CCL2. It also decreases CCL2-induced chemotaxis and mediates a delay in reinsertion of the CCL2 receptor, CCR2, into the cell membrane after CCL2-mediated receptor internalization, suggesting a mechanism of action of buprenorphine. Signaling pathways in CCL2-induced migration include increased phosphorylation of p38 MAPK and of the junctional protein JAM-A. We show that buprenorphine decreases these phosphorylations in CCL2-treated monocytes. Using DAMGO, CTAP, and Nor-BNI, we demonstrate that the effect of buprenorphine on CCL2 signaling is opioid receptor mediated. To identify additional potential mechanisms by which buprenorphine inhibits CCL2-induced monocyte migration, we performed proteomic analyses to characterize additional proteins in monocytes whose phosphorylation after CCL2 treatment was inhibited by buprenorphine. Leukosialin and S100A9, were identified and had not been shown previously be involved in monocyte migration. We propose that buprenorphine limits CCL2-mediated monocyte transmigration into the CNS, thereby reducing neuroinflammation characteristic of HAND. Our findings underscore the use of buprenorphine as a therapeutic for neuroinflammation as well as for addiction.

INTRODUCTION

An estimated 34 million people live with HIV worldwide (1). HIV enters the brain early after peripheral infection (2). Despite the success of combined antiretroviral therapy (cART) in reducing viral load, 40–70% of the HIV infected population develop cognitive, behavioral and motor deficits characteristic of HIV-associated neurocognitive disorders (HAND). The prevalence of HAND continues to rise as individuals with HIV live longer (3–6). Infected monocytes transport virus into the CNS, resulting in infection of macrophages and microglia, and to a lesser extent astrocytes. These cells produce cytokines, chemokines and viral proteins, leading to recruitment of additional monocytes into the brain, neuroinflammation, and eventual neuronal damage (7–10). Thus, the transmigration of HIV infected and uninfected monocytes into the CNS plays a key role in the initiation and progression of HAND.

Monocyte transmigration across the blood brain barrier (BBB), mediated in part by the chemokine CCL2, is critical to the neuropathogenesis of HIV (11–13). CCL2 is the most potent monocyte chemoattractant (14, 15) and is highly elevated in the brain tissue and CSF of people with HAND (16). Even with successful antiretroviral therapy, CCL2 levels in the CNS of infected people remain elevated, resulting in ongoing monocyte transmigration into the brain and chronic, low level neuroinflammation (17–19). Cognitive impairment correlates closely with the extent of neuroinflammation and with CCL2 in the CNS (16, 20). CCL2 is released by infected monocytes, macrophages, microglia, and astrocytes, promoting monocyte entry into the CNS (21, 22). Our laboratory previously demonstrated that HIV infected monocyte transmigration across the BBB in response to CCL2 is significantly higher when compared to uninfected monocytes and that CCL2 transiently disrupts BBB integrity (22–24).

Many studies indicate that HIV infected opiate abusers exhibit increased CNS inflammation, neuronal injury and death, and neuropathology as compared with HIV infected non-drug users, all of which contribute to cognitive impairment (25–27). Following acute withdrawal from opiates, people can be maintained on a full opioid agonist, methadone, or a partial opioid agonist, buprenorphine. Buprenorphine is a partial agonist of the mu-opioid receptor and an antagonist of the kappa-opioid receptor. It can be given at higher doses than methadone with fewer adverse effects. Buprenorphine increases the duration of both opioid withdrawal suppression and opioid blockade (28–31). However, the effects of buprenorphine on the mechanisms that mediate neuroinflammation and cognitive impartment during HIV infection have not been examined. Previous studies showed that agonists of specific opioid receptors, including the mu-opioid receptor agonist [D-Ala2-N-Me-Phe4, Gly-ol5]-enkephalin (DAMGO) and the delta-opioid receptor agonist [D-Pen2, D-Pen5]-Enkephalin (DPDPE), suppress the migration of both neutrophils and monocytes in response to multiple factors including chemokines (32–34). Thus, we propose that buprenorphine decreases CCL2 induced monocyte migration by decreasing surface and cytoskeletal protein rearrangements, as well as intracellular signaling pathways that are necessary for these cells to transmigrate into the CNS in response to CCL2. This would result in decreased neuroinflammation and less neuronal damage in HIV infected opioid abusers being treated with buprenorphine, and would therefore be an additional clinical benefit of this therapeutic drug.

MATERIALS AND METHODS

Materials

Buprenorphine, [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO), H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), Norbinaltorphimine (Nor-BNI), acetone, dithiothreitol (DTT) and iodoacetamide were from Sigma (St. Louis, MO) and CCL2 was from R&D Systems (Minneapolis, MN). Sodium dodecyl sulfate (SDS) was from Bio-Rad (Hercules, CA), urea from GE Healthcare (Piscataway, NJ) and sequencing grade modified trypsin from Promega (Madison, WI). TEAB buffer was from Fluka (St. Louis, MO), and detergent removal spin columns, trifluoroacetic acid (TFA) and formic acid were from Pierce (Rockford, IL). Titansphere ™ Phos-TiO Kit was from GL Sciences (Torrance, CA), iTRAQ reagent 4plex kit was from AB Sciex (Foster City, CA), and LC/MS grade acteonitrile and water were from Fisher Scientific (Waltham, MA).

Cell isolation

Leukopaks were obtained from the New York Blood Center in accordance with Albert Einstein College of Medicine guidelines. PBMC were isolated using Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden) density gradient centrifugation. Monocytes were isolated from PBMC by positive selection using the CD14 EasySep selection kit (Stem Cell Technologies, Vancouver, BC, Canada) according to the manufacturer’s protocol.

Immunofluorescence

Human monocytes were treated with 200 ng/ml CCL2 or an equal volume of 0.1% BSA in PBS, the CCL2 diluent, for 15 minutes. Cells were then fixed with 2 % paraformaldehyde in PBS for 1 hour, permeabilizated with 0.01% triton, and incubated in blocking solution containing 0.5 M EDTA, 1% fish gelatin, 1% Ig-free BSA, 1% horse serum and 1% human serum for 30 minutes. After blocking, cells were stained with anti-tubulin antibody (1:10.000, #T9026, Sigma) for 1 hour at room temperature. The cells were washed and then incubated with the secondary antibody, anti-mouse IgG F(ab′)2 fragment-FITC (1:100, #F2883, Sigma) mixed with Texas Red®-X phalloidin ( 15ul/ml, #T7471, Invitrogen, Grand Island, NY) for 2 hours at room temperature. Samples were washed and then mounted in Prolong Gold antifade reagent (P36931, Invitrogen) and examined by confocal microscopy using a Leica SP2 confocal microscope.

Chemotaxis assay

Cell migration was assayed using a 48-well microchemotaxis chamber (Neuro Probe, Gaithersburg, MD). 3×105 monocytes were incubated for 15 minutes at 37°C, in the presence of BSA (CCL2 diluent), buprenorphine (20 nM), CCL2 (200 ng/ml), or buprenorphine plus CCL2. Cells were then washed, resuspended in 50 μl of RPMI 1640 + 2% of FBS, and placed in the upper well of the microchemotaxis chamber, separated from the lower well by a polycarbonate filter with 5 μm pores (Neuro Probe). The chemotactic response of monocytes was assayed after the addition of CCL2 or BSA diluted in RPMI 1640 to the lower well. The chamber was incubated at 37°C with 5% CO2 for 25 minutes. For CCR2 recovery experiments, monocytes were pretreated using the same protocol above, and after 15 minutes of treatment, the cells were washed and incubated at 37°C with 5% CO2 for an additional 30 minutes. Cells were then added to the top well of the microchemotaxis chamber and allowed to migrate as described above. Filters were then removed, fixed and stained using Diff-Quik ™ Stain Set (Siemens Healthcare Diagnostics, Malvern, PA). Migrated cells per well were calculated using densitometry (35) with the computer imaging software, UN-SCAN-IT (Silk Scientific, Orem, UT).

Flow cytometry

FACS analysis was performed on monocytes after 5 and 15 minutes of treatment with BSA (CCL2 diluent), buprenorphine, CCL2, or CCL2 plus buprenorphine. For CCR2 recovery experiments, monocytes were treated using the same protocol described above, and then the cells were washed and incubated in media at 37°C with 5% CO2 for an additional 30 minutes. After treatment, 2×105 cells were immunostained with human CCR2 PE-conjugated antibody (0.15 ug of antibody/assay, clone 48607, R&D Systems) or an isotyped matched negative control PE-conjugated antibody in 1% BSA/PBS in the dark on ice for 30 minutes. Cells were then washed and fixed with 2% paraformaldehyde. Forward and side scatter were used to gate for monocytes. Monocytes were >93% pure, as determined by CD14 positivity, and contained <1% CD19+ B cells, <3% CD3+ T cells, and <1% CD56+ NK cells. At least 10,000 events were acquired with a BD Canto II flow cytometer. Analysis was performed using FlowJo software (v. 9.5.3, TreeStar, Ashland, OR). CCR2 mean fluorescence intensity for each treatment group was calculated after subtracting the fluorescence contribution of the negative control antibody.

Western blot analysis

Monocytes (2×106 cells/ml) were treated with BSA, buprenorphine, CCL2, or buprenorphine plus CCL2, washed with cold PBS and lysed with cell lysis buffer (Cell Signaling, Beverly, Ma) containing protease inhibitor cocktail (Sigma). To demonstrate that buprenorphine was acting specifically through opioid receptors and not by off-target effects, cells were treated with DAMGO (100 nM) (36), DAMGO+CCL2, Nor-BNI (1 μM) (37, 38), or Nor-BNI+CCL2, for 5 and 15 minutes, or pretreated with CTAP (1 μM) (36, 39, 40) for 30 minutes before the addition of CCL2. It is important to note that, due to the number of monocytes necessary for each treatment group, different donors were used for the DAMGO, CTAP, and Nor-BNI experiments. Lysate protein concentrations were quantified using the Bio-Rad protein assay. Lysates were heated to 95–100 °C with 5× loading buffer (300 mM Tris-HCl, pH 6.8–8.0; 10% w/v SDS; 12.5% beta-mercaptoethanol; 50% glycerol; 0.1% bromophenol blue in deionized water) for 5 minutes and 50 μg of lysates were analyzed in each lane of 10% SDS–PAGE gels (Bio-Rad). Proteins were transferred electrophoretically to Protran nitrocellulose (Schleicher and Schuell, Dassel, Germany) and membranes were incubated with primary antibodies to phospho-p38 (1:1000, #4511, Cell Signaling), total p38 (1:1000, #9212, Cell Signaling), phospho-JAM-A (1:500, #sc-17430-R, Santa Cruz Biotechnology), JAM-A (1:500, #sc-53623, Santa Cruz Biotechnology, Dallas, TX) overnight at 4°C with gentle rocking. After washing, membranes were incubated with either anti-rabbit-HRP (1:2500, #7074, Cell Signaling) or anti-mouse-HRP (1:2500, #7076, Cell Signaling) and immunocomplexes were detected by Western Lightning Chemiluminesence Reagent (Perkin Elmer, Waltham, MA). Densitometric analyses were performed with UN-SCAN-IT gel digitizing software (Silk Scientific).

Proteomic assays

Preparation of iTRAQ-labeled membrane protein phosphopeptides

Each experiment was performed with cells isolated from one leukopak. Membrane proteins were enriched from 20–30×106 monocytes for each experimental condition using differential detergent fractionation with a Qproteome Cell Compartment Kit from Qiagen (Valencia, CA) (41). After acetone precipitation, the membrane proteins were solubilized in 1% SDS, 8M urea, 20% acetonitrile (ACN) and 50 mM TEAB, pH 8.5, followed by reduction with DTT and alkylation with iodoacetamide. A solution of 50 mM TEAB buffer, pH 8.5 containing 2 mM CaCl2 and 10% ACN was added to reduce the concentration of SDS to 0.1% before adding trypsin (20 ng/μl) at times 0 and 4 hours of a 12–14 hour incubation at 37°C. SDS was removed by using 2 ml detergent removal spin columns (Pierce, Rockford, IL) before acidifying the sample with 20% TFA to a final pH between 2 to 3 and concentrating on C18 spin columns containing 42 mg of C18 resin. After elution, tryptic peptides were prepared for iTRAQ labeling according to the manufacturer’s instructions (AB Sciex). Aliquots of the labeled samples were analyzed by LC-MS/MS to verify completion of iTRAQ labeling. Samples from all the experimental conditions were pooled after confirming that no unlabeled peptides were identified. Phosphopeptides were enriched from the pooled iTRAQ-labeled sample with a TiO2 column (GL Sciences, Rolling Hills Estates, CA) according to the manufacturer’s instruction and as described elsewhere (42).

LC-MS/MS and Data Analysis

The enriched iTRAQ-labeled phosphopeptides were separated online by 2D-LC-MS/MS using a Waters NanoAcquity nano-UPLC system (Milford, MA) interfaced to an Orbitrap Velos high resolution mass spectrometer (Thermo Scientific, San Jose, CA) as previously described (42). Text files were created from the raw data using Proteome Discoverer 1.2 (ThermoScientific), merged and searched against a Uniprot database of human and mouse protein sequences using the Mascot Protein Search Engine (Matrix Science, Boston, MA). The following parameters for searches were: trypsin 2 missed cleavages; fixed modifications of iTRAQ 4plex (peptide N-terminus and Lys); variable modifications of carbamidomethylation (Cys), phosphorylation (Ser, Thr and Tyr), iTRAQ 4plex (Tyr) and oxidation (Met); monoisotopic masses; peptide mass tolerance of 20 ppm; and product ion mass tolerance of 0.1 Da. A peptide mass error within 7 ppm and product ions within ± 0.05 Da were achieved, in the rare case of LC-MS/MS runs, the accurate observed peptide mass can be determined by an adjustment with the systematic mass error obtained from the same dataset. A false discovery rate (FDR) for peptide identification was assessed by decoy database searching. Proteins were considered identified having at least two bold red peptides unique to that sequence (significance p < 0.05). This method identifies the most logical assignment of a peptide to a specific protein, and prevents duplicate homologous proteins from being reported. The peptide FDR was less than 1%. Scaffold Q+ was used to validate MS/MS based peptide and protein identifications and to quantify isobaric tag peptides and proteins (version 3.6.2; Proteome Software, Portland, OR). FDR was controlled under 0.1%. Intensity based normalization was used for quantitation based on the median ratio and using the individual spectrum’s iTRAQ reporter ion as reference. Only uniquely assigned peptides were quantified. Differential expression was presented as log2 fold change of reference. A change of 0.6 was considered significant. This corresponds to approximately a 1.5 fold difference in expression. Phosphoproteins that were common to all 3 donors were selected for Ingenuity Pathway Analysis (IPA) (Ingenuity Systems, Redwood City, CA). Unphosphorylated peptides from the same peptide mixture were also analyzed to evaluate the stoichiometry of phosphorylation.

RESULTS

Buprenorphine reduces the CCL2 induced migratory phenotype in monocytes

CCL2 is elevated in the CNS of people with HAND and mediates increased monocyte recruitment and transmigration into the brain (16, 20). Monocyte movement is necessary for entry into the CNS and is associated with cytoskeletal changes, including actin and tubulin reorganization, that regulate locomotion and chemotactic migration (43–45). Monocytes express opioid receptors and therefore are able to respond to buprenorphine (46–49). Sublingual doses of 8 mg of buprenorphine once or twice daily are used to treat drug addiction, giving peak plasma concentrations of 10 to 20 nM (50–52). Therefore, we chose 20 nM buprenorphine to examine whether this therapeutic concentration impacts the migratory phenotype induced in monocytes by CCL2.

Human monocytes isolated from PBMC were treated with 200 ng/ml CCL2, an equal volume of 0.1% BSA in PBS (CCL2 diluent), buprenorphine or CCL2 plus buprenorphine for 15 minutes and stained for actin and tubulin. Immunofluorescence was examined by confocal microscopy and the percentage of cells with membrane projections was quantified as an indicator of a CCL2 induced migratory phenotype (43, 44). In control conditions (BSA), the monocytes were spherical and actin was distributed throughout the cell body (Figure 1A). Monocytes treated with CCL2 underwent morphological changes, forming membrane projections or large protrusions from the cell body where actin and tubulin were co-localized (Figure 1A), characteristic of a “polarized” migratory phenotype (43–45). Buprenorphine treatment had no effect on monocytes as evidenced by similar actin and tubulin staining as BSA treated cells. However, in cells treated with buprenorphine and CCL2, membrane projections were almost completely abolished (Figure 1A). Similar results were obtained with monocytes from 4 independent donors (Figure 1B). These data indicate that buprenorphine reduces the formation of CCL2 induced membrane projections in monocytes and this may have an effect on the chemotaxis of these cells in response to CCL2.

Figure 1. Buprenorphine inhibits the migratory phenotype of monocytes induced by CCL2.

Figure 1

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(A) Monocytes were incubated with 0.01% BSA (CCL2 diluent), CCL2 (200 ng/ml), buprenorphine (20 nM), or CCL2 plus buprenorphine for 15 minutes and were analyzed by confocal microscopy after staining with Texas-red phalloidin for actin (red), and anti-tubulin antibody (green). Monocytes treated with BSA had a characteristic round morphology with actin at the membrane and tubulin dispersed throughout the cell. Monocytes treated with CCL2 underwent morphological changes, with a cell projection from the cell body where actin and tubulin were co-localized (arrow). Treatment with buprenorphine only had no effect on the normal cell morphology of monocytes. However, cell projections that were observed with CCL2 treatment were almost completely abolished by co-treatment of cells with CCL2 plus buprenorphine (B) quantification of cells that have cell projections per total number of cells, n=4 independent experiments. Significance was determined using a two-tailed paired t test **p<0.01, ***p<0.005, NS= no significant change.

Buprenorphine reduces CCL2 mediated monocyte chemotaxis

An important function of monocytes is to migrate from the peripheral vasculature to tissue sites of inflammation in response to chemokines, a process termed chemotaxis (53, 54). To determine the effects of buprenorphine on CCL2 induced chemotaxis, CCL2 (200 ng/ml) was added to the lower wells of a microchemotaxis chamber and 3×105 human monocytes from 5 independent donors were added to the upper wells of a chamber. Monocytes were either added directly to the upper wells or were pretreated prior to their addition to the chamber. Monocyte migration across a polycarbonate membrane after 25 minutes at 37°C with 5% CO2 was quantified.

As shown in the first two columns of Figure 2, CCL2 was a strong chemoattractant for untreated monocytes as compared to BSA (diluent control, set to one), in agreement with studies published from our laboratory and others (22, 55, 56). We then tested the response of cells pretreated with CCL2 and buprenorphine for their ability to chemotax to CCL2. Cells were pretreated with CCL2, buprenorphine, or CCL2 plus buprenorphine for 15 minutes at 37°C, washed, and then added to the upper wells of a microchemotaxis chamber. Pretreatment of cells with buprenorphine alone had no effect on monocyte chemotaxis in response to CCL2 (Figure 2, Bup pretreatment). In contrast, pretreating monocytes with CCL2 and buprenorphine significantly reduced the chemotactic response to CCL2 (Figure 2, CCL2+Bup pretreatment). However, pretreatment of monocytes with CCL2 alone also significantly decreased CCL2 induced chemotaxis (Figure 2, CCL2 pretreatment), indicative of receptor desensitization and internalization (57, 58). Thus, additional experiments were necessary to determine whether the reduced chemotaxis exhibited by monocytes pretreated with CCL2 and buprenorphine was due to CCL2 pretreatment or to a buprenorphine mediated effect on CCL2 treated cells.

Figure 2. Buprenorphine reduces monocyte chemotaxis towards CCL2.

Figure 2

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Chemotaxis assays were performed to examine buprenorphine (Bup, 20 nM) mediated changes in monocyte chemotaxis towards CCL2 (200 ng/ml). For migration of monocytes towards wells containing CCL2 or BSA (CCL2 diluent), cells were untreated or pretreated for 15 minutes at 37°C as described in detail in the Materials and Methods section. The pretreated monocytes were then washed and placed in the upper wells of the chamber. The chamber was incubated at 37°C for 25 minutes. Filters were then removed, fixed and stained using Diff-Quik TM Stain Set (Siemens). The total number of migrated cells per well was determined by densitometry using the UN-SCAN-IT computer-assisted imaging system, n=5 independent experiments. Significance was determined using a two-tailed paired t test. *p<0.05, **p<0.01, ***p<0.005.

The binding of CCL2 to its receptor, CCR2, has been shown to induce phosphorylation of CCR2, resulting in rapid receptor internalization (59, 60). Chemotaxis inhibition by buprenorphine may result from a specific effect of buprenorphine on the recycling of CCR2 to the surface after CCL2 induced receptor internalization. Thus, we pretreated cells with CCL2 or CCL2 plus buprenorphine for 15 minutes, washed away the treatment, and incubated cells for an additional 30 minutes prior to addition to the upper wells of a microchemotaxis chamber to allow for recycling of CCR2 to the cell surface. CCL2 was added to the lower wells of the chamber and chemotaxis was quantified. We found that chemotaxis of monocytes pretreated with CCL2 as described (Figure 2, CCL2 pretreatment with Wash and 30 minutes of incubation) was similar to that of untreated monocytes (Figure 2, second column), suggesting pretreatment with CCL2 followed by a 30 minutes incubation allows internalized CCR2 to return to the cell surface and mediate chemotaxis. However, with buprenorphine plus CCL2 pretreatment, even after 30 minutes of additional incubation in media alone, there was a significant decrease in monocyte chemotaxis in response to CCL2 (Figure 2, CCL2+Bup pretreatment with Wash and 30 minutes of incubation). These data indicate that exposure to buprenorphine and CCL2 may decrease subsequent monocyte migration to CCL2 by limiting the recycling of CCR2 to the plasma membrane.

CCL2 reduces the surface expression of CCR2 and buprenorphine delays its recycling to the cell membrane

To determine whether buprenorphine and CCL2 treatment delayed the recycling of CCR2 to the monocyte surface, we performed FACS analyses. Monocytes were treated with BSA, CCL2, buprenorphine, or CCL2 plus buprenorphine and CCR2 surface expression was quantified by FACS analysis. Fold changes in CCR2 mean fluorescent intensity for all treatments were compared to BSA, which was set to one. CCR2 was significantly reduced from the cell surface after treatment with CCL2 for 5 minutes, as shown from one representative donor (Figure 3A). Similar results were obtained from 9 independent donors (Figure 3B), as also reported by others (59–61). CCR2 was also internalized after CCL2 treatment for 15 minutes (data not shown) but maximal internalization occurred after 5 minutes. Monocytes treated with buprenorphine alone for 5 minutes had no change in surface CCR2 expression when compared to BSA (Figure 3A from a representative donor, and Figure 3B from 9 independent donors). Treatment with buprenorphine plus CCL2 decreased CCR2 similarly to treatment with CCL2 alone (Figure 3A from a representative donor, and Figure 3B from 9 independent donors).

Figure 3. Buprenorphine delays the recycling of CCR2, the CCL2 receptor, to the cell membrane after CCL2 induced receptor internalization.

Figure 3

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The surface expression of CCR2 on monocytes was analyzed by flow cytometry after treatment with BSA, CCL2 (200 ng/ml), buprenorphine (20 nM), or CCL2 plus buprenorphine. (A) The change in surface CCR2 was determined by FACS analysis after 5 minutes of treatment. (B) The fold change in the mean fluorescence intensity (MFI) of CCR2 on monocytes from 9 different individuals as compared to BSA (set to one) was calculated after subtracting the contribution of the isotype matched negative control antibody. The fold change in CCR2 after 5 minutes of CCL2 or CCL2 plus buprenorphine treatment was decreased as compared to BSA. (C, D) The change in surface CCR2 was determined after treatments followed by washing and 30 minutes of incubation to allow for receptor recycling. After pretreatment of cells with CCL2 and then washing and incubation for 30 minutes, the recycling of CCR2 resulted in higher CCR2 surface expression when compared to 5 minutes of CCL2 treatment only. In contrast, treatment with CCL2 plus buprenorphine followed by washing and incubation for 30 minutes resulted in reduced recycling of CCR2 to the surface. (E) The fold change in the mean fluorescence intensity (MFI) of CCR2 on monocytes from 5 different individuals as compared to control treatment with BSA (set to one) was calculated after subtracting the contribution of the isotype matched negative control antibody. Data are represented as mean ± standard error of the mean. Significance was determined using a two-tailed paired t test. *p<0.05 **p<0.01.

To evaluate CCR2 recycling to the plasma membrane, cells were pretreated with BSA, CCL2, buprenorphine, or CCL2 plus buprenorphine followed by washing to remove the treatment, and incubation for an additional 30 minutes as performed for the chemotaxis experiments. After pretreatment with CCL2 alone, we found that CCR2 recycled to the membrane after 30 minutes (Figure 3D from a representative donor, and Figure 3E from 5 independent donors) and was significantly increased on the cell surface when compared to monocytes analyzed immediately after 5 minutes of incubation with CCL2 (Figure 3C from a representative donor, and Figure 3E from 5 independent donors). In contrast, after CCL2 plus buprenorphine pretreatment, the amount of CCR2 that recycled to the membrane after 30 minutes of recovery was less when compared to cells pretreated with CCL2 alone (Figure 3D from a representative donor, and Figure 3E from 5 independent donors). These data further demonstrate that one mechanism by which buprenorphine may reduce CCL2 mediated chemotaxis is by delaying CCR2 recycling to the cell surface after internalization induced by CCL2. Thus, exposure of monocytes to buprenorphine and CCL2 may reduce the ability of these cells to subsequently transmigrate across the BBB in response to increased CCL2 in the CNS.

Buprenorphine decreases CCL2 induced p38 MAPK phosphorylation in human monocytes

To determine whether inhibition of CCL2 induced chemotaxis by buprenorphine may be mediated by alterations in CCL2 mediated signaling in monocytes, we examined p38 MAPK phosphorylation in cells treated with buprenorphine and CCL2. p38 has been reported to be important in the migration of many cells, including monocytes (62–65), and its phosphorylation regulates CCL2 induced monocyte chemotaxis (64–66). To determine the effect of buprenorphine on the CCL2 induced phosphorylation of this kinase, monocytes were treated with BSA, CCL2, buprenorphine, or CCL2 plus buprenorphine for 5 and 15 minutes. Cell lysates were prepared and analyzed by western blotting with anti-phospho-p38 antibody. Blots were then stripped and reprobed with anti-p38 antibody. Treatment with buprenorphine alone had no effect on p38 phosphorylation in monocytes from a representative donor (Figure 4A, 5 minutes of treatment). We found that p38 phosphorylation was increased after CCL2 treatment alone and buprenorphine reduced CCL2 induced p38 phosphorylation to near baseline levels. Similar results were obtained with monocytes from 7 independent donors (Figure 4B). These treatments were repeated 23 additional times as positive controls for the studies of opioid receptor specificity illustrated in Figure 6. Thus, this p38 phosphorylation mediated by CCL2 and its downregulation by buprenorphine was observed in a total of 30 independent experiments. Since the time point of CCL2 induced p38 phosphorylation was different in each individual experiment, we used the time point (5 or 15 minutes) of maximal phosphorylation to quantify by densitometry the levels of p38 phosphorylation normalized to the amount of total p38 for each experiment. Fold changes in normalized p38 phosphorylation were compared to BSA, which was set to one. These data demonstrate that buprenorphine reduces CCL2 induced p38 phosphorylation which may contribute to the inhibitory effect of buprenorphine on monocyte chemotaxis in response to CCL2.

Figure 4. Buprenorphine reduces CCL2 induced p38 MAPK phosphorylation.

Figure 4

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Monocytes from 7 different donors were treated with BSA, CCL2 (200 ng/ml), buprenorphine (20 nM) or CCL2 plus buprenorphine. (A) Monocytes were incubated for 5 minutes and protein lysates were analyzed by western blotting using phospho-p38 (anti-p38-P) and total p38 (anti-p38) antibodies. In monocytes from one representative donor, CCL2 induced p38 phosphorylation. Treatment with CCL2+buprenorphine resulted in decreased p38 phosphorylation when compared to CCL2 alone. (B) The time point of p38 phosphorylation was inconsistent due to donor variability so the time point of maximal phosphorylation (5 or 15 minutes) in each experiment was analyzed. At the time point of maximal phosphorylation for each experiment, densitometric analysis of p38 phosphorylation normalized to total p38, indicated a significant increase with CCL2 treatment. Buprenorphine decreased CCL2 induced p38 phosphorylation. N=7 independent experiments. Significance was determined using a two-tailed paired t test *p<0.05, NS: No significance.

Figure 6. Effects of buprenorphine and of opioid receptor–specific agonist and antagonists on CCL2 mediated p38 MAPK phosphorylation.

Figure 6

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(A) Monocytes were treated with BSA, CCL2, buprenorphine, CCL2 plus buprenorphine, DAMGO, and CCL2 plus DAMGO. Monocytes were incubated for 5 and 15 minutes and protein lysates were analyzed by western blotting using phospho-p38 (anti-p38-P) and total p38 (anti-p38) antibodies. As illustrated by the western blot of monocytes from one representative donor, CCL2 induced p38 phosphorylation. Treatment with CCL2+buprenorphine resulted in decreased p38 phosphorylation when compared to CCL2 alone. CCL2+DAMGO also resulted in decreased p38 phosphorylation as compared to CCL2. (B) The time point of p38 phosphorylation was inconsistent due to donor variability inherent in using primary cells. Therefore the time point of maximal phosphorylation (5 or 15 minutes) in each experiment was analyzed. At the time point of maximal phosphorylation for each experiment, densitometric analysis of p38 phosphorylation normalized to total p38, indicated a significant increase with CCL2 treatment. N=7 independent experiments. (C) Monocytes were treated with BSA, CCL2, buprenorphine, CCL2 plus buprenorphine, or pretreated for 30 minutes with the antagonist CTAP prior to the treatments. Monocytes were incubated for 5 and 15 minutes and protein lysates were analyzed by western blotting using phospho-p38 (anti-p38-P) and total p38 (anti-p38) antibodies. As shown in the western blot of monocytes from one representative donor, preincubation with CTAP blocked the inhibitory effect of buprenorphine on CCL2 induced p38 phosphorylation. (D) The time point of maximal phosphorylation (5 or 15 minutes) in each experiment was analyzed and pooled with data from all experiments. N=6 independent experiments. (E–H) Monocytes were treated with BSA, CCL2, buprenorphine, CCL2 plus buprenorphine, Nor-BNI, or CCL2 plus Nor-BNI. Monocytes were incubated for 5 and 15 minutes and protein lysates were analyzed by western blotting using phospho-p38 (anti-p38-P) and total p38 (anti-p38) antibodies. (E) In monocytes from one representative donor, CCL2+Nor-BNI resulted in decreased p38 phosphorylation compared to CCL2 (F) The time point of maximal phosphorylation (5 or 15 minutes) in each experiment was analyzed and pooled with data from all experiments. N=6 independent experiments. (G) CCL2+Nor-BNI did not result in decreased p38 phosphorylation as compared to CCL2. (H) The time point of maximal phosphorylation (5 or 15 minutes) in each experiment was analyzed and pooled with data from all experiments. N=4 independent experiments. Significance was determined using a two-tailed paired t test *p<0.05, NS: No significance.

Buprenorphine reduces CCL2 induced JAM-A phosphorylation in monocytes

Homophilic interactions between adhesion proteins on monocyte and those on the vascular endothelium facilitate the tightly controlled diapedesis of monocytes into tissues. Our laboratory demonstrated that junctional adhesion molecule-A (JAM-A) is important in the transmigration of monocytes across the BBB (23, 24, 67). JAM-A is a single membrane-spanning protein and a member of the immunoglobulin (Ig) superfamily. It is expressed at cell–cell junctions of endothelial cells and on monocytes. JAM-A mediates transendothelial migration of leukocytes by regulating the integrity and permeability of cell junctions (68–72). Phosphorylation is a regulatory mechanism to direct the cellular localization and function of these junctional proteins that enables diapedesis (73–75). To examine the effect of buprenorphine on the CCL2 mediated phosphorylation of JAM-A in monocytes, we used western blotting assays.

Monocytes from 8 independent donors were incubated with BSA, CCL2, buprenorphine, or CCL2 plus buprenorphine for 15 and 30 minutes. Cell lysates were prepared and analyzed by western blotting with anti-phospho-JAM-A (serine 284) antibody. Blots were then stripped and reprobed for total JAM-A. The time point (15 or 30 minutes) of maximal phosphorylation was used to quantify the levels of JAM-A phosphorylation normalized to the amount of total JAM-A for each experiment. Treatment with BSA or buprenorphine alone had no effect on phosphorylation as shown from a representative donor (Figure 5A, 15 minutes of treatment), and in Figure 5B from 8 independent donors. JAM-A was phosphorylated specifically at serine 284 in monocytes with CCL2 treatment. However, treatment of cells with buprenorphine plus CCL2 decreased JAM-A phosphorylation to baseline (Figure 5A from a representative donor, and Figure 5B from 8 independent donors). Thus, buprenorphine inhibits CCL2 induced phosphorylation of the junctional protein JAM-A, which may contribute to an inhibition of monocyte diapedesis into the CNS in response to CCL2.

Figure 5. Buprenorphine reduces CCL2 induced JAM-A phosphorylation in monocytes.

Figure 5

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Monocytes were treated with BSA, CCL2 (200 ng/ml), buprenorphine (20 nM) or CCL2 plus buprenorphine. (A) Monocytes were incubated for 15 minutes and protein lysates were analyzed by western blotting using anti-phospho-JAM-A (JAM-A-P) and anti-JAM-A (JAM-A) antibodies. The western blot from one representative donor shows that CCL2 induced JAM-A phosphorylation, and that buprenorphine decreased CCL2 mediated JAM-A phosphorylation. (B) The time point of JAM-A phosphorylation was inconsistent due to donor variability. Therefore, the time point of maximal phosphorylation (15 or 30 minutes) in each experiment was analyzed. At the time point of maximal phosphorylation for each experiment, densitometric analysis of JAM-A phosphorylation was normalized to total JAM-A. N=8 independent experiments. Significance was determined using a two-tailed paired t test *p<0.05, NS: No significance.

Inhibition of CCL2-mediated p38 MAPK phosphorylation by buprenorphine is specifically through opioid receptors

Buprenorphine is a partial agonist of the mu-opioid receptor and an antagonist of the kappa-opioid receptor. To demonstrate that buprenorphine reduces CCL2 mediated p38 phosphorylation specifically through opioid receptors and not by off target effects, we performed a series of experiments using an agonist and an antagonist of the mu-opioid receptor, DAMGO and CTAP respectively, and an antagonist of the kappa-opioid receptor, Nor-BNI. Primary monocytes were either treated with diluent as control, or with CCL2, buprenorphine, CCL2 plus buprenorphine, DAMGO, or CCL2 plus DAMGO, for 5 and 15 minutes. Cell lysates were prepared and analyzed by western blotting with anti-phospho-p38 antibody. Blots were then stripped and reprobed with anti-p38 antibody. The time point (5 or 15 minutes) of maximal phosphorylation was used to quantify the levels of p38 phosphorylation normalized to the amount of total p38 for each condition. As shown in Figure 4, p38 phosphorylation was increased after CCL2 treatment and buprenorphine reduced CCL2 mediated p38 phosphorylation to baseline levels (Figure 6A from a representative donor, and Figure 6B from 7 independent donors). DAMGO inhibited CCL2 mediated phosphorylation of p38 similarly as buprenorphine, that is it reduced CCL2-mediated phosphorylation (Figure 6A from a representative donor, and Figure 6B from 7 independent donors). When monocytes from different donors were pretreated with CTAP for 30 minutes and then exposed to control, CCL2, buprenorphine or CCL2 plus buprenorphine treatments for 5 or 15 minutes, we found that CTAP blocked the inhibitory effect of buprenorphine on CCL2 induced p38 phosphorylation (Figure 6C from a representative donor, and Figure 6D from 6 independent donors). Thus, our data demonstrated that the effect of buprenorphine on CCL2 signaling is mu-opioid receptor mediated.

To determine whether buprenorphine is acting through the kappa-opioid receptor as well, we treated the monocytes with diluent as control, CCL2, buprenorphine, CCL2 plus buprenorphine, Nor-BNI, or CCL2 plus Nor-BNI, for 5 and 15 minutes. We used monocytes from different donors than were used for the DAMGO or CTAP experiments due to the large number of cells required for each treatment group. Interestingly, as shown in Figures 6E-H, the effect of Nor-BNI on CCL2-induced p38 phosphorylation was highly donor dependent. In 60% (6 out of 10 independent donors) of the donors, Nor-BNI reduced the p38 phosphorylation induced by CCL2, as did CCL2+buprenorphine (Figure 6E from a representative donor, and Figure 6F from 6 independent donors). In contrast, for 40% of the donors (4 out of 10 independent donors), Nor-BNI had no effect on the increase of p38 phosphorylation after CCL2 treatment (Figure 6G from a representative donor, and Figure 6H from 4 independent donors). These results suggest that the effect of buprenorphine on CCL2 signaling is mediated by kappa-opioid receptor in some donors but not for others, and that kappa-opioid receptor expression may be variable on these primary cells.

Proteomic identification of membrane phosphopeptides on monocytes that are regulated by CCL2 and buprenorphine

A large-scale quantitative proteomics analysis of phosphorylated membrane peptides on monocytes was used to identify additional CCL2 induced phosphorylated proteins, with a focus on proteins whose phosphorylation was inhibited by buprenorphine. This proteomic analysis was performed to identify additional mechanisms by which exposure to buprenorphine and CCL2 may inhibit subsequent monocyte transmigration across the BBB in response to CCL2 in the CNS. Monocytes were treated with BSA, CCL2, buprenorphine, or CCL2 plus buprenorphine for 15 minutes. Membranes were isolated and proteins were concentrated by acetone precipitation followed by trypsin digestion. The membrane peptides from the four different treatment groups were labeled with one form of the stable isotope label iTRAQ, 114,115, 116 or 117 reagents. After pooling the peptides from all the treatment groups, the iTRAQ-labeled phosphopeptides were enriched 3 times using a TiO2 column and analyzed by LC-MS/MS. This experiment was repeated with monocytes from three different donors. From these different donors, we identified 210, 361 and 388 proteins respectively, that were phosphorylated in response to any of the four treatments. The Venn Diagram in Figure 7A shows the number of phosphorylated proteins that were expressed by monocytes from each donor, with the number of proteins that were unique to each donor, as well as the number of proteins that were common to two or all three donors. We focused our subsequent studies on the 149 proteins that were phosphorylated in monocytes from all three donors (Figure 7A, gray highlight). Ingenuity Pathway Analysis (IPA) of these 149 proteins was used to identify molecular networks and biological functions in categories related to monocyte chemotaxis and the results were as follows: 29 proteins involved in cellular movement, 20 in immune cell trafficking, and 5 in cell-mediated immune responses (Figure 7B). These proteins were then examined for differences in phosphorylation in monocytes treated with BSA, CCL2, buprenorphine, or CCL2 plus buprenorphine. We identified 25 proteins whose phosphorylation was increased by CCL2 when compared to BSA (Figure 7C). Two phosphoproteins of interest, S100-A9 and leukosialin/CD43, exhibited a buprenorphine mediated decrease in CCL2 induced phosphorylation as defined in the methods section (Figure 7C, gray highlight).

Figure 7. Identification of monocyte membrane proteins altered by CCL2 plus buprenorphine treatment using proteomics.

Figure 7

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Monocytes from 3 different donors were treated with BSA, CCL2 (200 ng/ml), buprenorphine (20 nM) or CCL2 plus buprenorphine. Membrane proteins were isolated, concentrated by acetone precipitation, followed by trypsin digestion and analyzed by LC-MS/MS. Membrane proteins phosphorylated in monocytes treated with any of the four treatments were identified by proteomics. (A) Venn Diagram illustrates the 149 phospo-proteins induced by any of the four treatments that were common to all three donors (gray highlight). (B) Ingenuity Pathway Analysis (Ingenuity Systems) was performed on these 149 proteins. (C) List of the 25 phosphoproteins involved in cellular movement, immune cell trafficking and cell-mediated immune response. The list includes log2 fold change differences in phosphorylation in monocytes treated with BSA, CCL2, buprenorphine or CCL2 plus buprenorphine, peptide sequence, and the phosphorylated residue in red. The proteins highlighted, S100-A9 and leukosialin, are discussed in the text. STD, standard deviation.

S100-A9, or MRP14, is a calcium-binding protein highly expressed in monocytes, and is involved in cytoskeletal-membrane interactions and regulation of cytoskeletal reorganization (76–79). Leukosialin, or CD43, is a sialoglycoprotein found on the cell membrane of all leukocytes and has been implicated in the regulation of CD4+ T cell trafficking and T cell migration and activation (80–82). CD43 is expressed in microglia, with higher expression in reactive cells as compared to resting cells (83). The proteomic results of this study suggest that phosphorylation of S100-A9/MRP14 and leukosialin/CD43 may regulate cytoskeletal reorganization involved in cellular chemotaxis in response to CCL2 and that buprenorphine reduces this phosphorylation, therefore limiting CCL2 induced monocyte chemotaxis. We are currently examining the role of these phosphoproteins in monocyte diapedesis across the BBB.

DISCUSSION

In HIV infection, the monocyte chemoattractant protein, CCL2 is elevated in the brain tissue and CSF of people with HAND (16–19). CCL2 mediates the transmigration of uninfected and HIV infected monocytes across the BBB into the CNS, thereby playing a key role in neuroinflammation and HAND (84–86). Despite the effectiveness of antiretroviral therapy in the treatment of HIV infection, the prevalence of cognitive, behavioral and motor abnormalities has increased as infected individuals live longer, and greater than 40–70% of the HIV infected population exhibit HAND (3–5).

Injection drug use is responsible for 16% of all new HIV infections in the United States (87), and several studies showed that drug abuse increased the severity of cognitive dysfunction in HIV infected people (88–90). Opiate drug abusers with HIV often have increased neuroinflammation and neuronal damage that may result in accelerated progression of HAND (25–27). As HIV infected people who abuse drugs live longer, these comorbidities remain a critical public health issue.

Buprenorphine and methadone are the main therapeutics prescribed for people dependent on opiates. Buprenorphine has a different mechanism of action than methadone, indicating that buprenorphine may be a better therapeutic for opioid addiction, particularly in HIV infected people. Buprenorphine is a partial agonist of the mu-opioid receptor and antagonist of the kappa-opioid receptor, while methadone is an agonist of the mu-opioid receptor. In some studies, buprenorphine therapy improved decision-making, and resulted in fewer deficits in working memory and verbal list learning than did methadone treatment. These improvements may be due to its distinct pharmacological action as a partial kappa-opioid antagonist (29–31). In addition, buprenorphine has high affinity and slower dissociation from opioid receptors than does methadone. Buprenorphine also has longer physiologic effects and provides more effective relief from withdrawal symptoms during detoxification from opioids than methadone (50, 91–94). Importantly for the HIV infected population, buprenorphine also appears to have fewer interactions with antiretroviral drugs than does methadone (94).

We examined the effects of buprenorphine on several aspects of CCL2-mediated chemotaxis. CCL2 increased the formation of monocyte cell membrane projections characteristic of a migratory phenotype, and, additionally, increased the phosphorylation of p38 MAPK and JAM-A. Buprenorphine reduced all of these effects. We also showed that buprenorphine acted through opioid receptors. Thus, exposure to buprenorphine and CCL2 may limit subsequent monocyte diapedesis across the BBB in response to CCL2 in the CNS, therefore reducing neuroinflammation.

We demonstrated that CCL2 reduced CCR2 from the monocyte cell surface. Buprenorphine plus CCL2 treatment also reduced CCR2 from the surface. However, the receptor recycled to the membrane within a defined time frame in cells treated with CCL2 alone when the chemokine was removed. In contrast, CCR2 recycling to the cell membrane was delayed in monocytes treated with CCL2 plus buprenorphine within the 30 minutes time frame of our experiments. These findings suggest that one mechanism by which buprenorphine may inhibit CCL2 induced monocyte chemotaxis is by delaying the recycling of CCR2 to the cell membrane after CCL2 induced internalization, thus inhibiting the chemotactic response.

p38 signaling has been described to have a key role in the chemotaxis and migration of many cells (62–65). p38 is activated in monocytes by numerous extracellular mediators of inflammation, including cytokines, chemokines, and bacterial lipopolysaccharide (95, 96). p38 phosphorylation is important in the migration of corneal epithelial cells during wound healing and of renal proximal tubular cells after mechanical injury (62, 63). p38 also regulates CCL2 induced monocyte chemotaxis (64–66). Of importance to this study, we showed that buprenorphine inhibited CCL2 induced p38 phosphorylation in monocytes. We propose that this inhibition of p38 phosphorylation may limit the ability of monocytes to transmigrate across the BBB in response to elevated levels of CCL2 in the CNS of HIV infected opioid abusers on buprenorphine therapy.

JAM-A is a transmembrane tight junction protein that is phosphorylated specifically on serine 284 by PKC upon platelet activation. This phosphorylation regulates the localization of JAM-A in these cells (75). Overexpression of a JAM-A cytoplasmic domain deletion mutant lacking several phosphorylation sites as well as the use of a PKC inhibitor inhibited JAM-A mediated endothelial cell migration (97). Our finding in monocytes indicates that buprenorphine may reduce neuroinflammation by limiting the phosphorylation of JAM-A induced by CCL2, thereby inhibiting monocyte diapedesis across the BBB.

We suggest a mechanism by which buprenorphine may decrease the chemotactic response of human primary monocytes in the presence of CCL2. We showed that the effects of buprenorphine are opioid receptor depended and not off target. To demonstrate this, we used DAMGO and CTAP, an agonist and an antagonist of the mu-opioid receptor respectively, and Nor-BNI, an antagonist of the kappa-opioid receptor. Our results indicated that buprenorphine mediates the decrease in the phosphorylation of p38 induced by CCL2 through the mu-opioid receptor. The role of kappa-opioid receptor is less clear. In 60% of our experiments, buprenorphine mediated its effects through kappa-opioid receptor, but for primary monocytes from 40% of the donors, this receptor did not appear to play a role. One possible explication is the variability inherent in primary human cells, which may lead to different responsiveness to buprenorphine and/or different kinetics of this response. Preliminary data from our laboratory indicate significant variation in opioid receptor expression among donors, which also could contribute to the differences in sensitivity to buprenorphine. We could not address whether mu-opioid and kappa-opioid receptors are mediating the effect of buprenorphine in the same donor due to the large number of cells required to perform the different treatments for each experiment, and the limited number of monocytes obtained from one leukopak. Studies of the role of kappa-opioid receptor in the reduction by buprenorphine of CCL2 mediated effects as well as of the role of both receptors in the same cells are ongoing in our laboratory.

Using proteomics, we identified multiple phosphorylated proteins in our treated monocytes that were grouped by IPA analysis according to their functions. Among these, we focused on two proteins involved in cellular movement and immune cell trafficking that have differential phosphorylation when cells are treated with CCL2 or CCL2 plus buprenorphine. One of these proteins, leukosialin/CD43, is a sialoglycoprotein found on the cell membrane of all leukocytes. It is a transmembrane protein with a highly 0-glycosylated extracellular domain (98, 99). The CD43 cytoplasmic tail has many serines and threonines and phosphorylation at these sites has been implicated in the regulation of CD4 T cell trafficking, and T cell migration and activation (80–82). There are no published reports addressing the role of phosphorylated leukosialin/CD43 in monocyte diapedesis.

The second protein, S100-A9 or MRP14, is a calcium-binding protein of the S100 protein family and is highly expressed in neutrophils and monocytes. S100 proteins are involved in various cellular processes including cell cycle progression and modulation of cytoskeletal-membrane interactions (76–79). S100-A9 is phosphorylated in the cytoplasmic tail specifically on Thr113 by p38 MAPK, translocates to the plasma membrane and increases actin binding to regulate cytoskeletal reorganization (100–102). We found that CCL2 increased phosphorylation of leukosialin and of S100-A9 (Figure 7C). For leukosialin we found two different residues involved (Thr326 and Ser355) and for S100-A9, we showed phosphorylation at Thr113 (Figure 7C). We propose that with CCL2 treatment, leukosialin and p38 are phosphorylated, and p38 leads to phosphorylation of S100-A9, resulting in an increase in the movement of monocytes. Exposure of monocytes to CCL2 plus buprenorphine decreases all of these phosphorylation events and consequently decreases subsequent CCL2 induced cellular movement.

The proteomic data in Figure 7B and 7C were obtained from the 149 phosphoproteins common to all three donors. TiO2 enrichment identified one phosphopeptide of JAM-A (AA#281-290, KVIYSQPpSAR) from 2 donors and is the same site identified by western blot. While the trend for phosphorylation at this site was the same as that observed by western blot, the changes did not reach the threshold of significance usually required for mass spectrometry (see Materials and Methods). There were no JAM-A peptides detected in the flow-through fraction. The lack of sequence coverage suggests that these proteins are low in abundance. In the case of S100-A9, for example, the sequence coverage is 60, 63 and 78% for the three donors, suggesting that it is present in a relatively high concentration. While mass spectrometry is not inherently quantitative without the use of stable isotope labels, it is recognized that the number of peptides identified in a protein sequence is somewhat representative of protein abundance. Unphosphorylated peptides of JAM-A were not identified while one phosphopeptide may simply be related to this issue. Phosphopeptides underwent an extra enrichment step decreasing the total number of complexity of peptides undergoing LC-MS/MS as compared to the unphosphorylated flow-through fraction.

In summary, our study demonstrates that CCL2 induces a chemotatic phenotype in monocytes characterized by the formation of cell surface projections and phosphorylation of p38 MAPK and the tight junction protein JAM-A. Buprenorphine treatment decreases CCL2-induced monocyte migration, membrane projections, and p38, and JAM-A phosphorylation, and is dependent on the mu-opioid receptor and, in approximately half of the donors, is kappa-opioid receptor dependent. We also demonstrate that buprenorphine reduces the recycling of CCR2 to the membrane after CCL2 mediated internalization. All of these buprenorphine mediated effects are possible mechanisms for the inhibition of CCL2 induced monocyte chemotaxis.

Despite the success of cART in increasing the survival of HIV infected people, HAND remains a critical public health issue, in particular among the drug abusing HIV-infected population. Our data suggest a positive impact of buprenorphine in the CNS of HIV infected opiate drug abusers. We propose that buprenorphine limits CCL2 mediated transmigration of uninfected and HIV infected monocytes into the CNS, thereby reducing neuroinflammation and HAND. Thus, buprenorphine is not only an effective treatment for this population with regard to managing withdrawal and reducing addiction, it may have significant therapeutic benefits in reducing HAND.

Acknowledgments

This work was supported by NIH grants R01MH075679 (JWB), R01DA025567 (JWB, TMC), R01MH090958 (JWB, LC), R21MH102113-01A1 (JWB, LC), P20DA026149 (JWB, RHA), 1S10RR029398 (RHA), MH080663 (Pilot funds, Mount Sinai Institute for NeuroAIDS Disparities DWW), UNCF/Merck Graduate Science Dissertation Fellowship (DWW), Einstein Proteomics Center for Study of the Neurological Consequences of HIV and Substance Abuse and CFAR.

We thank Jacqueline Coley for her assistance with the cells.

Footnotes

DISCLOSURES

The authors have no financial conflicts of interest.

References

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