Methamphetamine Enhances HIV Infection of Macrophages
2008; Elsevier BV; Volume: 172; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2008.070971
ISSN1525-2191
AutoresHao Liang, Xu Wang, Hui Chen, Song Li, Li Ye, Shi-Hong Wang, Yanjian Wang, Lin Zhou, Wen‐Zhe Ho,
Tópico(s)HIV/AIDS Research and Interventions
ResumoEpidemiological studies have demonstrated that the use of methamphetamine (meth), a sympathomimetic stimulant, is particularly common among patients infected with HIV. However, there is a lack of direct evidence that meth promotes HIV infection of target cells. This study examined whether meth is able to enhance HIV infection of macrophages, the primary target site for the virus. Meth treatment resulted in a significant and dose-dependent increase of HIV reverse transcriptase activity in human blood monocyte-derived macrophages. Dopamine D1 receptor antagonists (SCH23390 and SKF83566) blocked this meth-mediated increase in the HIV infectivity of macrophages. Investigation of the underlying mechanisms of meth action showed that meth up-regulated the expression of the HIV entry co-receptor CCR5 on macrophages. Additionally, meth inhibited the expression of endogenous interferon-α and signal transducer and activator of transcription-1 in macrophages. These findings provide direct in vitro evidence to support the possibility that meth may function as a cofactor in the immunopathogenesis of HIV infection and may lead to the future development of innate immunity-based intervention for meth users with HIV infection. Epidemiological studies have demonstrated that the use of methamphetamine (meth), a sympathomimetic stimulant, is particularly common among patients infected with HIV. However, there is a lack of direct evidence that meth promotes HIV infection of target cells. This study examined whether meth is able to enhance HIV infection of macrophages, the primary target site for the virus. Meth treatment resulted in a significant and dose-dependent increase of HIV reverse transcriptase activity in human blood monocyte-derived macrophages. Dopamine D1 receptor antagonists (SCH23390 and SKF83566) blocked this meth-mediated increase in the HIV infectivity of macrophages. Investigation of the underlying mechanisms of meth action showed that meth up-regulated the expression of the HIV entry co-receptor CCR5 on macrophages. Additionally, meth inhibited the expression of endogenous interferon-α and signal transducer and activator of transcription-1 in macrophages. These findings provide direct in vitro evidence to support the possibility that meth may function as a cofactor in the immunopathogenesis of HIV infection and may lead to the future development of innate immunity-based intervention for meth users with HIV infection. Methamphetamine (meth) and related amphetamine compounds are among the most commonly used illicit drugs, with more than 35 million users worldwide. In the United States, approximately 1.5 million individuals regularly use/abuse meth.1Colfax G Shoptaw S The methamphetamine epidemic: implications for HIV prevention and treatment.Curr HIV/AIDS Rep. 2005; 2: 194-199Crossref PubMed Scopus (161) Google Scholar, 2Rawson RA Anglin MD Ling W Will the methamphetamine problem go away?.J Addict Dis. 2002; 21: 5-19Crossref PubMed Scopus (154) Google Scholar An estimated 11 million Americans at the age of 12 and older reported trying meth at least once during their lifetime. Meth use and HIV type 1 infection frequently coexist because of the association of meth use with engagement of high-risk behaviors.3Semple SJ Patterson TL Grant I Motivations associated with methamphetamine use among HIV+ men who have sex with men.J Subst Abuse Treat. 2002; 22: 149-156Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 4Copeland AL Sorensen JL Differences between methamphetamine users and cocaine users in treatment.Drug Alcohol Depend. 2001; 62: 91-95Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 5Crofts N Hopper JL Milner R Breschkin AM Bowden DS Locarnini SA Blood-borne virus infections among Australian injecting drug users: implications for spread of HIV.Eur J Epidemiol. 1994; 10: 687-694Crossref PubMed Scopus (50) Google Scholar, 6Harris NV Thiede H McGough JP Gordon D Risk factors for HIV infection among injection drug users: results of blinded surveys in drug treatment centers, King County, Washington 1988–1991.J Acquir Immune Defic Syndr. 1993; 6: 1275-1282PubMed Google Scholar The risk for HIV infection attributable to meth use continues to increase.7HIV & drugs: meth use develops stronger link to HIV risk.AIDS Policy Law. 2005; 20: 5Google Scholar, 8Boddiger D Metamphetamine use linked to rising HIV transmission.Lancet. 2005; 365: 1217-1218Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 9Volkow ND Wang GJ Fowler JS Telang F Jayne M Wong C Stimulant-induced enhanced sexual desire as a potential contributing factor in HIV transmission.Am J Psychiatry. 2007; 164: 157-160Crossref PubMed Scopus (79) Google Scholar Several studies have shown that there is a high prevalence of HIV infection among meth users10Frosch D Shoptaw S Huber A Rawson RA Ling W Sexual HIV risk among gay and bisexual male methamphetamine abusers.J Subst Abuse Treat. 1996; 13: 483-486Abstract Full Text PDF PubMed Scopus (171) Google Scholar, 11Halkitis PN Fischgrund BN Parsons JT Explanations for methamphetamine use among gay and bisexual men in New York City.Subst Use Misuse. 2005; 40: 1331-1345Crossref PubMed Scopus (112) Google Scholar, 12Urbina A Jones K Crystal methamphetamine, its analogues, and HIV infection: medical and psychiatric aspects of a new epidemic.Clin Infect Dis. 2004; 38: 890-894Crossref PubMed Scopus (149) Google Scholar and that among men who sell sex to men, those who use meth have a higher HIV risk than nonusers.13Shoptaw S Reback CJ Freese TE Patient characteristics, HIV serostatus, and risk behaviors among gay and bisexual males seeking treatment for methamphetamine abuse and dependence in Los Angeles.J Addict Dis. 2002; 21: 91-105Crossref PubMed Scopus (83) Google Scholar Active meth users displayed higher levels of HIV loads than nonusers,14Ellis RJ Childers ME Cherner M Lazzaretto D Letendre S Grant I Increased human immunodeficiency virus loads in active methamphetamine users are explained by reduced effectiveness of antiretroviral therapy.J Infect Dis. 2003; 188: 1820-1826Crossref PubMed Scopus (177) Google Scholar which may be attributable to increased viral replication, as was shown in an animal study.15Gavrilin MA Mathes LE Podell M Methamphetamine enhances cell-associated feline immunodeficiency virus replication in astrocytes.J Neurovirol. 2002; 8: 240-249Crossref PubMed Scopus (69) Google Scholar However, the direct effects of meth on HIV infection and HIV disease progression are still poorly understood.16Kopnisky KL Bao J Lin YW Neurobiology of HIV, psychiatric and substance abuse comorbidity research: workshop report.Brain Behav Immun. 2007; 21: 428-441Crossref PubMed Scopus (27) Google Scholar In particular, the deleterious effect of meth on the host's immune response and its role in the immunopathogenesis of HIV infection remain to be elucidated. Therefore, study of the interactions between meth and HIV has become a greater research priority.17Berman JW Berman MJ Masliah E Chang L Cox BW Fox H Gonzalez RG Hanson GR Hauser KF Ho WZ Hong JS Major EO Maragos W Masliah E McArthur JC Miller DB Nath A O'Callaghan JP Persidsky Y Power C Rogers TJ Royal III, W NeuroAIDS, drug abuse, and inflammation: building collaborative research activities.J Neuroimmune Pharmacol. 2006; 1: 351-399Crossref PubMed Scopus (21) Google Scholar The microenvironment in which the interactions between HIV and target cells take place has a crucial role in modulating HIV infectivity. Besides CD4+ T lymphocytes, cells from the mononuclear phagocyte system are the primary targets for HIV infection. Monocytes and macrophages as the primary sites of HIV replication are among the first cells infected by HIV and later function as reservoirs for the virus.18Embretson J Zupancic M Beneke J Till M Wolinsky S Ribas JL Burke A Haase AT Analysis of human immunodeficiency virus-infected tissues by amplification and in situ hybridization reveals latent and permissive infections at single-cell resolution.Proc Natl Acad Sci USA. 1993; 90: 357-361Crossref PubMed Scopus (166) Google Scholar, 19Embretson J Zupancic M Ribas JL Burke A Racz P Tenner-Racz K Haase AT Massive covert infection of helper T lymphocytes and macrophages by HIV during the incubation period of AIDS.Nature. 1993; 362: 359-362Crossref PubMed Scopus (1242) Google Scholar Although abuse of drug such as opioids have been implicated in modulation of functions of monocytes/macrophages20Guo CJ Li Y Tian S Wang X Douglas SD Ho WZ Morphine enhances HIV infection of human blood mononuclear phagocytes through modulation of beta-chemokines and CCR5 receptor.J Investig Med. 2002; 50: 435-442Crossref PubMed Google Scholar and microglia,21Hu S Chao CC Hegg CC Thayer S Peterson PK Morphine inhibits human microglial cell production of, and migration towards RANTES.J Psychopharmacol. 2000; 14: 238-243Crossref PubMed Scopus (43) Google Scholar there is limited information about the impact of meth on functions of monocytes/macrophages. Meth inhibited polyinosinic:polycytidylic acid-induced antiviral activity in murine peritoneal macrophages.22In SW Son EW Rhee DK Pyo S Modulation of murine macrophage function by methamphetamine.J Toxicol Environ Health A. 2004; 67: 1923-1937Crossref PubMed Scopus (23) Google Scholar Meth also modulated the patterns of gene expression in monocyte-derived immature and mature dendritic cell.23Mahajan SD Hu Z Reynolds JL Aalinkeel R Schwartz SA Nair MP Methamphetamine modulates gene expression patterns in monocyte derived mature dendritic cells: implications for HIV-1 pathogenesis.Mol Diagn Ther. 2006; 10: 257-269Crossref PubMed Scopus (44) Google Scholar, 24Reynolds JL Mahajan SD Sykes DE Schwartz SA Nair MP Proteomic analyses of methamphetamine (METH)-induced differential protein expression by immature dendritic cells (IDC).Biochim Biophys Acta. 2007; 1774: 433-442Crossref PubMed Scopus (40) Google Scholar Although these findings suggest that meth is immunosuppressive, there is a lack of direct evidence at cellular and molecular levels to demonstrate that meth has the ability to enhance HIV infection of macrophages, the primary target for the virus. In the present study, we investigated the impact of meth on HIV infection of human blood monocyte-derived macrophages and explored the mechanisms underlying the meth action on HIV infection. Peripheral blood samples from healthy adult donors were provided by the University of Pennsylvania Center for AIDS Research, which has Institutional Review Board review and approval for the sample collection. These blood samples were screened for all normal viral blood-borne pathogens and certified to be pathogen free. Monocytes were purified according to a previously described technique.25Hassan NF Campbell DE Douglas SD Purification of human monocytes on gelatin-coated surfaces.J Immunol Methods. 1986; 95: 273-276Crossref PubMed Scopus (91) Google Scholar In brief, heparinized blood was separated by centrifugation over lymphocyte separation medium (Organon Teknika, Durham, NC) at 400 to 500 × g for 45 minutes. The mononuclear cell layer was collected and incubated with Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) in a 2% gelatin-coated flask for 45 minutes at 37°C, followed by removal of the nonadherent cells with Dulbecco's modified Eagle's medium. Adherent monocytes were detached with 10 mmol/L EDTA. After the initial purification, greater than 97% of the cells were monocytes, as determined by nonspecific esterase staining and flow cytometry analysis using monoclonal antibody against CD14, the marker specific for monocytes and macrophages. Isolated monocytes were plated in 24- or 48-well culture plates at a density of 5 or 2.5 × 105 cells/well in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Whereas monocytes refer to freshly isolated (within 24 hours) monocytes, macrophages refer to 7-day-cultured monocytes in vitro. Monocyte and macrophage viability was monitored by trypan blue exclusion and maintenance of cell adherence. Methamphetamine and the dopamine D1 receptor (D1R) antagonists (SCH23390 and SKF83566) were purchased from Sigma-Aldrich Co. (St. Louis, MO). Fluorescein isothiocyanate-conjugated antibodies against CD14, CD4, and CCR5 and the control IgGs (IgG1, IgG2a, and IgG2b) were obtained from PharMingen (San Diego, CA). Enzyme-linked immunosorbent assay kit for interferon-α (IFN-α) protein was purchased from PBL Biomedical Laboratories (Piscataway, NJ). Rabit polyclonal antibodies against actin and signal transducer and activator of transcription1 (STAT1) were obtained from Sigma-Aldrich. Rabbit polyclonal antibody against Dopamine D1 receptor was purchased from Calbiochem (La Jolla, CA). Fluorescein isothiocyanate-conjugated goat anti-rabbit IgG antibody was purchased from Southern Biotechnology Associates, Inc. (Birmingham, AL). Seven-day-cultured macrophages (2.5 × 105 cells/well) were treated with different concentrations (1, 10, 100, and 250 μmol/L) of meth for 3, 6, and 24 hours. These concentrations of meth are comparable with the levels found in the blood, urine, or tissue samples of meth-using subjects.23Mahajan SD Hu Z Reynolds JL Aalinkeel R Schwartz SA Nair MP Methamphetamine modulates gene expression patterns in monocyte derived mature dendritic cells: implications for HIV-1 pathogenesis.Mol Diagn Ther. 2006; 10: 257-269Crossref PubMed Scopus (44) Google Scholar, 26Gjerde H Hasvold I Pettersen G Christophersen AS Determination of amphetamine and methamphetamine in blood by derivatization with perfluorooctanoyl chloride and gas chromatography/mass spectrometry.J Anal Toxicol. 1993; 17: 65-68Crossref PubMed Scopus (67) Google Scholar, 27Melega WP Cho AK Harvey D Lacan G Methamphetamine blood concentrations in human abusers: application to pharmacokinetic modeling.Synapse. 2007; 61: 216-220Crossref PubMed Scopus (109) Google Scholar, 28Schepers RJ Oyler JM Joseph Jr, RE Cone EJ Moolchan ET Huestis MA Methamphetamine and amphetamine pharmacokinetics in oral fluid and plasma after controlled oral methamphetamine administration to human volunteers.Clin Chem. 2003; 49: 121-132Crossref PubMed Scopus (185) Google Scholar, 29Takayasu T Ohshima T Nishigami J Kondo T Nagano T Screening and determination of methamphetamine and amphetamine in the blood, urine and stomach contents in emergency medical care and autopsy cases.J Clin Forensic Med. 1995; 2: 25-33Abstract Full Text PDF PubMed Scopus (26) Google Scholar To investigate whether D1 receptor antagonists block meth-induced up-regulation of HIV infection, 10 μmol/L D1 receptor antagonist (SCH23390 or SKF83566) was added to the macrophages cultures 1 hour before meth treatment for 24 hours. The cell cultures were re-fed with fresh media containing meth and/or SCH23390 or SKF83566 every 4 days. There were no cytotoxic effects of meth, SCH23390, and SKF83566 treatment on macrophages as demonstrated by trypan blue dye staining (data not shown). The macrophage-tropic R5 strain (Bal) was obtained from the AIDS Research and Reference Reagent Program (NIH, Bethesda, MD). Macrophages were infected with equal amounts of cell-free HIV Bal (p24 20 ng/106 cells) for 2 hours at 37°C after 24 hours of treatment with or without meth. The cells were then washed three times with Dulbecco's modified Eagle's medium to remove unabsorbed virus, and fresh media containing meth and/or SCH23390 or SKF83566 were added to the cell cultures. The final wash was tested for HIV reverse transcriptase (RT) activity and shown to be free of residual inocula. Untreated cells served as a control. Culture supernatants were collected for HIV RT activity assay at days 4, 8, and 12 after infection. HIV RT activity was determined based on the technique of Guo et al20Guo CJ Li Y Tian S Wang X Douglas SD Ho WZ Morphine enhances HIV infection of human blood mononuclear phagocytes through modulation of beta-chemokines and CCR5 receptor.J Investig Med. 2002; 50: 435-442Crossref PubMed Google Scholar and Ho et al30Ho WZ Lioy J Song L Cutilli JR Polin RA Douglas SD Infection of cord blood monocyte-derived macrophages with human immunodeficiency virus type 1.J Virol. 1992; 66: 573-579PubMed Google Scholar with modifications. In brief, 10 μl of culture supernatants from macrophages infected with or without HIV was added to a cocktail containing poly(A), oligo(dT) (Amersham Biosciences, Inc., Piscataway, NJ), MgCl2, and [32P]dTTP (Amersham Biosciences, Inc.) and incubated for 20 hours at 37°C. Then, 30 μl of the cocktail was spotted onto DE81 paper, dried, and washed five times with 2× saline-sodium citrate buffer and once with 95% ethanol. The filter paper was then air-dried. Radioactivity was counted in a liquid scintillation counter (PerkinElmer Life Sciences, Boston, MA). To determine whether meth affects the expression of CD14, CD4, and CCR5 receptors on macrophages, the cells were incubated with or without 100 μmol/L meth for 24 hours and then removed from the culture plate and resuspended in 100 μl of PBS. After incubation with 20 μl of fluorescein isothiocyanate-conjugated antibodies against CD14, CD4, and CCR5 for 45 minutes at 4°C, the cells were washed twice with PBS and fixed with 1% paraformaldehyde in PBS. Fluorescein isothiocyanate-conjugated control IgG was used as a control antibody. Fluorescence-positive cells were analyzed on an EPICS-elite flow cytometer (Beckman Coulter, Inc., Hialeah, FL). Total RNA was extracted from macrophages using Tri-Reagent (Molecular Research Center, Cincinnati, OH) as previously described.31Li Y Zhang T Douglas SD Lai JP Xiao WD Pleasure DE Ho WZ Morphine enhances hepatitis C virus (HCV) replicon expression.Am J Pathol. 2003; 163: 1167-1175Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar Total cellular RNA (1 μg) was subjected to reverse transcription using the reverse transcription system from Promega (Madison, WI). The real time RT-PCR for the quantification of D1R, IFN-α, and glyceraldehyde-3-phosphate dehydrogenase mRNA was performed with the iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA) as previously described.32Zhang T Lin RT Li Y Douglas SD Maxcey C Ho C Lai JP Wang YJ Wan Q Ho WZ Hepatitis C virus inhibits intracellular interferon alpha expression in human hepatic cell lines.Hepatology. 2005; 42: 819-827Crossref PubMed Scopus (56) Google Scholar The amplified products were visualized and analyzed using the software MyiQ provided with the thermocycler (iCycler iQ real time PCR detection system; Bio-Rad Laboratories). The levels of glyceraldehyde-3-phosphate dehydrogenase mRNA were used as an endogenous reference to normalize the quantities of targets mRNA. The special oligonucleotide primers used in this study were listed as follows: D1R, 5′-AAACCCACAAGCCCCTCTGA-3′ (sense) and 5′-GATGAATTAGCCCACCCAAAC-3′ (antisense)33Ostadali MR Ahangari G Eslami MB Razavi A Zarrindast MR Ahmadkhaniha HR Boulhari J The detection of dopamine gene receptors (DRD1-DRD5) expression on human peripheral blood lymphocytes by real time PCR.Iran J Allergy Asthma Immunol. 2004; 3: 169-174PubMed Google Scholar; IFN-α, 5′-TTTCTCCTGCCTGAAGGACAG-3′ (sense) and 5′-GCTCATGATTTCTGCTCTGACA-3′ (antisense); and glyceraldehyde-3-phosphate dehydrogenase, 5′-GGTGGTCTCCTCTGACTTCAACA-3′ (sense) and 5′-GTTGCTGTAGCCAAATTCGTTGT-3′ (anti-sense). The oligonucleotide primers were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). The macrophages were cultured on glass coverslips at a density of 0.25 × 106/well in 24-well plates. The macrophages were washed with 1 × cold PBS (with Ca2+ and Mg2+) twice. Cells were fixed at 4°C in 4% paraformaldehyde-4% sucrose in PBS for 20 minutes and then permeated in cold methanol (100%) for additional 10 minutes followed by 0.2% Triton X-100 for additional 10 minutes. Cells were blocked in Block Solution (Pierce, Rockford, IL) for 1 hour at room temperature. To examine expression of D1R, rabbit polyclonal antibody (1:500) against D1R was used as the primary antibody. After washing five times with 1× PBS, the cells were incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG antibody (green, 1:100) for 1 hour. After washing five times with 1× PBS, the cells were mounted on glass coverslips in mounting media (Biomeda, Foster City, CA) and viewed with a fluorescence microscopy (Zeiss, Jena, Germany). Hoechst 33342 was used for nuclei morphology. Total cell lysates of macrophages were prepared using a radioimmune precipitation assay buffer (Promega) with 1% protease inhibitor cocktail (Sigma-Aldrich). Protein concentrations were determined by the protein assay kit (Bio-Rad Laboratories). Western blot assay was carried out as previously described.31Li Y Zhang T Douglas SD Lai JP Xiao WD Pleasure DE Ho WZ Morphine enhances hepatitis C virus (HCV) replicon expression.Am J Pathol. 2003; 163: 1167-1175Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar STAT1 and actin proteins were detected using rabbit anti-STAT1 (1:1000; Sigma-Aldrich) and anti-actin (1:3000; Sigma-Aldrich) polyclonal antibodies, respectively. Peroxidase-conjugated goat anti-rabbit antibody (1:10,000; Jackson ImmunoResearch Laboratories, West Grove, PA) was used as the second antibody. The bound antibodies were recognized by using SuperSignal West Pico Chemiluminescent Substrate Kit (Pierce) according to the manufacturer's instruction. Prestained molecular markers (Bio-Rad Laboratories) were used to determine molecular weight of immunoreactive bands. Total cell lysates from the cultured macrophages were prepared using a radioimmune precipitation assay buffer (Promega). Enzyme-linked immunosorbent assay for IFN-α was performed according to the protocol provided by the manufacturer (PBL Biomedical Laboratories). Student's t-test was used to evaluate the significance of difference between groups, and multiple comparisons were performed by regression analysis and one-way analysis of variance. P values of less than 0.05 were considered significant. All data are presented as mean ± SD. Statistical analyses were performed with SPSS 11.5 for Windows. Statistical significance was defined as P < 0.05. We first determined the effect of meth on HIV infection of macrophages. As shown in Figure 1, meth treatment resulted in increase of HIV RT activity. This meth-mediated increase of HIV RT activities is statistically significant. In addition, the increase of HIV RT activity was dose- and time-dependent (Figure 1, A and B). The highest enhancement of HIV by meth was observed with a dose of 250 μmol/L (Figure 1A) at day 8 after infection (Figure 1B). D1R has been implicated in the pathological effect of meth on the target cells. Thus, it is of importance to determine whether macrophages express D1R that is involved in the meth action on HIV replication. We performed a conventional RT-PCR assay using the primary pair specific for D1R detection. Ethidium bromide staining of RT-PCR-amplified products from macrophages showed a visible 471-bp band that is identical to that from human neuronal cells (NT2-N) (Figure 2A). We also observed that there is expression of D1R in macrophages at protein level (Figure 2B). We then examined whether the enhancing effect of meth on the HIV Bal infection was mediated through the D1R. The D1R antagonists (SCH23390 and SKF83566) completely abrogated the enhancing effect of meth on HIV RT activity (Figure 3), whereas the antagonists alone had little effect on HIV infection of macrophages (Figure 3).Figure 3Effect of the D1R antagonists on meth-mediated up-regulation of HIV Bal infection. Seven-day-cultured macrophages were incubated with or without the D1R antagonist, 10 μmol/L SCH23390, or 10 μmol/L SKF83566 for 1 hour before treatment with or without 100 μmol/L meth for 24 hours and then infected with HIV Bal strain for 2 hours in the presence or absence of meth and/or SCH23390 or SKF83566. Culture supernatants were collected at day 8 after infection for HIV RT assay. Data are expressed as HIV RT activity in meth-treated cells (percentage of control) to those in untreated cells, which is defined as 100%. The results represent the mean ± SD of three independent experiments. Statistical analysis was performed using one-way analysis of variance, and significance is shown with *P < 0.05 and **P < 0.01 (METH + SCH or METH + SKF versus METH).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because CCR5 receptor is a primary co-receptor for HIV M-tropic strain entry into macrophages,34Deng H Liu R Ellmeier W Choe S Unutmaz D Burkhart M Di Marzio P Marmon S Sutton RE Hill CM Davis CB Peiper SC Schall TJ Littman DR Landau NR Identification of a major co-receptor for primary isolates of HIV-1.Nature. 1996; 381: 661-666Crossref PubMed Scopus (3186) Google Scholar we examined whether meth has the ability to modulate the expression of CCR5 receptor. Meth treatment (100 μmol/L) significantly up-regulated CCR5 expression on macrophages as determined by flow cytometry (Figure 4). This meth action on CCR5 expression, however, was not mediated by D1R, because the D1R antagonist (SCH23390 and SKF 83566) did not block the meth effect (data not shown). To determine the specificity of the effect of meth on CCR5 expression, we also examined whether macrophage marker (CD14) and CD4 receptor are affected by meth. Meth, when added to macrophage cultures, had little effect on the expression of CD14 and CD4 receptors (Figure 4). To look for potential mediators involved in meth-mediated enhancement of HIV replication, we examined the impact of meth on IFN-α expression by macrophages. IFN-α is a potent antiviral cytokine that impedes HIV infection of macrophages.35Samuel CE Antiviral actions of interferons.Clin Microbiol Rev. 2001; 14: 778-809Crossref PubMed Scopus (2088) Google Scholar, 36Baca-Regen L Heinzinger N Stevenson M Gendelman HE Alpha interferon-induced antiretroviral activities: restriction of viral nucleic acid synthesis and progeny virion production in human immunodeficiency virus type 1-infected monocytes.J Virol. 1994; 68: 7559-7565PubMed Google Scholar, 37Meylan PR Guatelli JC Munis JR Richman DD Kornbluth RS Mechanisms for the inhibition of HIV replication by interferons-alpha, -beta, and -gamma in primary human macrophages.Virology. 1993; 193: 138-148Crossref PubMed Scopus (124) Google Scholar We hypothesized that meth inhibits endogenous IFN-α expression in macrophages, which could be a mechanism involved in meth-mediated up-regulation of HIV. Meth treatment of macrophages resulted in a significant decrease of endogenous IFN-α at both mRNA and protein levels (Figure 5, A and B, respectively). This meth-mediated down-regulation of IFN-α in macrophage was completely blocked by the D1R antagonists (SCH23390 and SKF83566) (Figure 6). To further determine whether meth, through suppressing IFN signaling pathway, enhances HIV replication, we investigated the effect of meth on the expression of STAT1, the major component of the IFN signaling cascade.38Darnell Jr, JE STATs and gene regulation.Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3330) Google Scholar, 39Darnell Jr, JE Kerr IM Stark GR Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins.Science. 1994; 264: 1415-1421Crossref PubMed Scopus (4912) Google Scholar Macrophages treated with meth expressed lower levels of STAT1 proteins than untreated macrophages (Figure 7).Figure 6Effect of the D1R antagonists on meth-mediated down-regulation of IFN-α. Seven-day-cultured macrophages were incubated with or without the D1R antagonist, 10 μmol/L SCH23390, or 10 μmol/L SKF83566 for 1 hour before treatment with or without 100 μmol/L meth for 3 hours. Cellular RNA was subjected to the real-time RT-PCR for IFN-α mRNA. Data are expressed as IFN-α mRNA levels in meth-treated cells (percentage of control) to those in untreated cells, which are defined as 100%. The results represent the mean ± SD of three independent experiments. Statistical analysis was performed using one-way analysis of variance, and significance is shown with *P < 0.05 and **P < 0.01 (METH + SCH or METH + SKF versus METH).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Effect of meth on STAT1 expression. Seven-day-cultured macrophages were incubated with or without 100 μmol/L meth for 24 hours. Equal amount of proteins extracted from the cells was subjected to the Western blot assay using rabbit antibodies against STAT1 and actin. The numbers in the right panel are the signal intensities expressed as densitometry scanning units (DSU) of protein bands of Western blot shown in the left panel. One representative experiment is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Epidemiological studies have demonstrated that meth use is particularly common among HIV-infected patients.10Frosch D Shoptaw S Huber A Rawson RA Ling W Sexual HIV risk among gay and bisexual male methamphetamine abusers.J Subst Abuse Treat. 1996; 13: 483-486Abstract Full Text PDF PubMed Scopus (171) Google Scholar, 40Garofalo R Mustanski BS McKirnan DJ Herrick A Donenberg GR Methamphetamine and young men who have sex with men: understanding patterns and correlates of use and the association with HIV-related sexual risk.Arch Pediatr Adolesc Med. 200
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