点击显示 收起
【摘要】
Opioid withdrawal is a crucial and recurring event during the course of opioid abuse that has a negative impact on the immune system. In this study, we investigated whether abrupt withdrawal (AW) or precipitated withdrawal (PW) potentiates human immunodeficiency virus (HIV) infection of human T lymphocytes. AW and PW enhanced HIV infection of peripheral blood lymphocytes and T-cell lines (Jurkat and CEMX174). In addition, both AW and PW induced HIV replication in a latently HIV-infected human T-cell line (J1.1). The enhancing effect of AW and PW was associated with the induction of neuropeptide substance P in both peripheral blood lymphocytes and the T-cell lines. The substance P receptor antagonist, CP-96,345, not only blocked AW- or PW-induced endogenous substance P expression but also abrogated AW- or PW-induced HIV replication in T cells. These findings provide a cellular mechanism that supports the notion that opioids have a co-factor role in promoting HIV infection of the immune cells.
--------------------------------------------------------------------------------
Because as many as 96% of opiate abusers use injection as the primary route of administration, opiate abuse contributes significantly to human immunodeficiency virus (HIV) transmission. Recent data from the Center for Disease Control (CDC) indicated that more than 27% of total AIDS cases in the United States were associated with injection drug use.1 Although it is known that injection drug use of heroin contributes significantly to HIV transmission, there is limited information available regarding whether opiate use such as heroin increases HIV replication and promotes HIV disease progression in HIV-infected opiate abusers or enhances susceptibility to HIV infection in HIV-seronegative individuals exposed to opiates such as heroin. Clinical and epidemiological evidence from early, pre-AIDS2,3 studies supports the concept that drug abuse, such as opiates, is a co-factor in the pathogenesis of HIV disease. Several lines of evidence have also shown that opioids are involved in HIV infection of different cell systems.4-7 Opioids alter cytokine production and cell trafficking, enhancing susceptibility of the immune cells to HIV infection.4,5 Morphine enhances HIV replication in chronically infected promonocytes co-cultured with human brain cells.7 Although general immunosuppression contributes to HIV infection and replication, the cellular mechanism(s) by which opiates effectively enhance HIV infection of immune cells remain to be determined. There are several potential mechanisms by which opiates could effectively enhance HIV infection. One is direct action through lymphoid opioid receptor on viral replication. Another would be indirect action through the modulation of other HIV-enhancing factors.
There is substantial evidence supporting the existence of a complex, bi-directional link between the central nervous system and the immune system in HIV infection. Both opioids and neuropeptide substance P (SP) play important roles as modulators of neuroimmunoregulation and are involved in modulation of the immune system and HIV infection.8 SP and its receptor have been implicated in opioid dependence and withdrawal.9-15 Opioid withdrawal is a crucial and recurring event during the course of opioid abuse. The phenomenon of opioid tolerance is well established; once tolerance develops, termination of the drug by withdrawal (drug cessation) or precipitated withdrawal (PW) (administration of an opioid antagonist with or without drug cessation) can lead to an abstinence syndrome indicating a state of physical dependence,16 which is among the defining characteristics of opioid addiction. Although opiate addicts may not suffer immune dysfunction when getting the drug on a regular basis,17 opioid withdrawal has a negative impact on the immune system,16,18-24 thus having a co-factor role in promoting viral infections. Drug withdrawal adversely affects the immune function of macrophages, perhaps making the addict more susceptible to infections.17 We recently demonstrated that morphine withdrawal enhances hepatitis C virus replicon expression.25 Donahoe and colleagues26 have reported that precipitated morphine withdrawal increases viral load in SIV-infected monkeys. Our previous studies have shown that morphine treatment enhances HIV R5 strain infection of macrophages and microglia.27,28 In the present study, we examined the effect of morphine withdrawal on HIV infection of human T lymphocytes. We also investigated the role of SP in morphine withdrawal-mediated enhancement of HIV infection of T lymphocytes.
【关键词】 morphine withdrawal manifests enhancing immunodeficiency infection lymphocytes induction substance
Materials and Methods
Cell Isolation and Cell Lines
Peripheral blood was obtained from six healthy adult donors without any known history of drug abuse. Informed consent was obtained, and the Institutional Research Board of our institution approved this study. Heparinized blood samples were identified as HIV antibody-negative by anonymous testing with the enzyme-linked immunosorbent assay method (Coulter Immunology, Hialeah, FL). Peripheral blood lymphocytes (PBLs) were prepared as previously reported29 and maintained in a culture of RPMI 1640 medium containing 10% fetal calf serum and 1 µg/ml of phytohemagglutinin-P (PHA-P) for 72 hours. Cells were then transferred to a T-25 flask (5 x 106 cells/flask) and treated with interleukin-2 (50 ng/ml). CEMX174, 1G5 cell lines, and HIV-infected J1.1 cell line were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institutes of Health, Bethesda, MD. 1G5 is a Jurkat T-cell line that harbors two copies of a stably transfected plasmid containing the luciferase reporter gene downstream of the HIV long terminal repeat (LTR).30 J1.1 is a subclone of the Jurkat E6.1 T-cell line, which is latently infected with HIV.31 Because of the low expression level of µ-opioid receptor in human immune cells, we used the human lymphocyte cell lines J-MOR5.1 and CEMX174. The human T lymphoblastoid cell line (Jurkat) transfected with the plasmid containing the µ-opioid receptor gene (J-MOR5.1) was kindly provided by Dr. Thomas J. Rogers (Temple University, Philadelphia, PA).32,33 CENX174 cells express high levels of human µ-opioid receptor at both the mRNA and protein level.34 CEMX174, 1G5, and J1.1 cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum, 2 mmol/L glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. The cell viability was assessed using trypan blue dye exclusion. In all cases, limulus amebocyte lysate assay demonstrated that the media and reagents were endotoxin-free.
Reagents
Morphine sulfate was obtained from Elkins-Sinn, Inc. (Cherry Hill, NJ). SP and naloxone was obtained from Sigma (St. Louis, MO). Neurokinin-1 receptor (NK-1R) antagonist (CP-96,345) and its inactive enantiomer (CP-96,344)35 were generously provided by Pfizer Central Research (Groton, CT). CP-96,345 and CP-96,344 were dissolved in fast-performance liquid chromatography-grade water at the concentration of 10C3 mol/L, filtered through a 0.22-µm filter (Millipore, Billerica, MA), and stored at C70??C.
HIV Strains
Based on the different use of the major HIV co-receptors (CCR5 and CXCR4), HIV isolates used in this study are referred to as R5, X4, or R5X4 strains.36 The T-cell-tropic X4 strain (NL4-3)37 and dual-tropic R5X4 strain (89.6) were obtained from the Center for AIDS Research at the University of Pennsylvania, School of Medicine.
Morphine Abrupt Withdrawal (AW) and Precipitated Withdrawal (PW)
PHA-stimulated (72 hours) PBLs, J-MOR5.1, and CEMX174 cells were incubated with or without morphine (10C8 mol/L) for 4 days and then subjected to AW or PW (blocking opioid receptors by naloxone after morphine cessation) for 24 hours. At the time of AW or PW, the cells were incubated with or without CP-96,345 or CP-96,344 (inactive enantiomer of CP-96,345). For AW, we removed morphine from the cell cultures by washing the cells three times with plain RPMI. For PW, the cells were treated with naloxone (10C8 mol/L) immediately after AW. J1.1 cells and 1G5 cells were incubated with or without morphine (10C8 mol/L) in T-25 flasks for 4 days and then plated in triplicate at a density of 105 cells/well in 48-well culture plates, subjecting them to AW or PW as described above. The cells were stimulated with tumor necrosis factor (TNF)- (2 ng/ml) 2 hours after withdrawal. For J1.1 cells, HIV-1 reverse transcriptase (RT) activity was measured in culture supernatants collected at 48 hours after withdrawal. For 1G5 cells, luciferase activity assay was performed in cell extracts at 24 hours after withdrawal.
HIV Infection of T Cells
The cells (PBLs, J-MOR5.1, and CEMX174) that underwent AW or PW as described above were infected with equal amounts (HIV p24, 20 ng/106 cells) of cell-free HIV strains (NL4-3 or 89.6) for 24 hours. The cells were then washed three times with plain RPMI to remove unabsorbed HIV 24 hours after infection. Supernatants were collected from HIV-infected cell cultures for HIV RT activity assay at day 9 after infection. Selection of the day 9 time point is based on our earlier study38 showing that HIV RT activity in the infected PBL cultures is not detectable until day 6 or day 9 (dependent on donors). The time line for morphine treatment, withdrawal, and infection is illustrated in Figure 1 .
Figure 1. Experimental design to study the effect of morphine withdrawal on HIV infection of human T lymphocytes. PHA-stimulated (72 hours) PBLs, J-MOR5.1, and CEMX174 cells were incubated with or without morphine (10C8 mol/L) for 4 days and then subjected to AW or PW for 24 hours. At the time of AW or PW, the cells were incubated with or without CP-96,345 or CP-96,344 (inactive enantiomer of CP-96,345). For AW, we removed morphine from the cell cultures by washing the cells three times with plain RPMI. For PW, the cells were treated with naloxone (10C8 mol/L) immediately after AW. The cells undergoing AW or PW as described above were infected with equal amounts (HIV p24, 20 ng/106 cells) of cell-free HIV strains (NL4-3 or 89.6). The cells were then washed three times with plain RPMI to remove unabsorbed HIV 24 hours after infection. Supernatants were collected from HIV-infected cell cultures for HIV RT activity assay at day 9 after infection (day 14 after morphine treatment).
RT and Luciferase Assays
HIV RT activity was determined based on a modified technique of Willey and colleagues39 In brief, 10 µl of collected culture supernatants were added to a cocktail containing poly(A), oligo(dT) (Pharmacia Inc., Piscataway, NJ), MgCl2, and dTTP (Amersham Corp., Arlington Heights, IL) 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 2x saline-sodium citrate buffer and once with 95% ethanol. The filter paper was then air-dried. Radioactivity was counted in a liquid scintillation counter (Packard Instrument Inc., Palo Alto, CA). The HIV LTR-driven luciferase activity in 1G5 cells was determined using a luciferase assay kit (Promega Biotec, Madison, WI) according to the manufacturer??s instruction.
RNA Extraction and Reverse Transcription
Total cellular RNA was extracted from PBLs, J-MOR5.1, and CEMX174 using Tri-reagent (Molecular Research Center, Cincinnati, OH). In brief, the total cellular RNA was extracted by a single step, guanidium thiocyanate-phenol-chloroform extraction. After centrifugation at 13,000 x g for 15 minutes at 4??C, the RNA-containing aqueous phase was collected and precipitated in isopropanol. The RNA precipitates were then washed once in 75% ethanol and resuspended in 30 µl of RNase-free water. Total RNA (1 µg) was subjected to reverse transcription using the reverse transcription system (Promega, Madison, WI) with specific primers (anti-sense) for 1 hour at 42??C. The reaction was terminated by incubating the reaction mixture at 99??C for 5 minutes and then kept at 4??C. The resulting cDNA was ready to serve as a template for polymerase chain reaction (PCR) amplification.
Real-Time PCR for SP mRNA
We have developed a highly sensitive and specific real-time RT-PCR assay to quantitatively measure SP mRNA.40 The PCR primers and probe (molecular beacon, MB) used for SP mRNA qualification were designed with Primer Express software (PE Applied Biosystems, Foster City, CA) and were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA).
Statistical Analysis
Where appropriate, data were expressed as mean ?? SD. For comparison of the mean of the two groups (AW- or PW-treated versus untreated control cells), statistical significance was assessed by analysis of variance with the appropriate post hoc test. Calculations were performed with the use of Stata Statistical Software (StataCorp., College Station, TX). Statistical significance was defined as P < 0.05.
Results
AW or PW Enhances Acute HIV Infection of T Cells
We first investigated whether AW and PW enhanced HIV infection of PBLs and the T-cell lines (J-MOR5.1, CEMX174). In comparison to control cells, the increased HIV RT activity was observed in both PBLs and the T-cell lines (J-MOR5.1, CEMX174) undergoing AW (Figure 2) . Although there are differential effects of AW or PW on HIV infection of PBLs from different donors, PBLs from five of six donors showed enhanced susceptibility to HIV infection in response to AW or PW (Figure 2A) . Naloxone alone had no effect on HIV replication in these cells (data not shown). In addition, although we did not observe morphine withdrawal- and/or HIV infection-induced cytotoxicity in J-MOR5.1 and CEMX174 cell cultures, we observed that there was HIV-induced PBL death (presumably CD4+ T-cell death) in the cultures. However, there were no differences in the HIV-caused cell death between the PBL cultures undergoing morphine withdrawal and control cultures (data not shown).
Figure 2. Effect of AW or PW on HIV infection of T cells. A: PHA-stimulated (72 hours) PBLs were incubated with or without morphine (10C8 mol/L) for 4 days, followed by AW or PW for 24 hours. For PW, naloxone (10C8 mol/L) was added to the cell cultures immediately after morphine was removed. The cells were then infected with HIV (NL4-3). B: T-cell lines (J-MOR5.1 and CEMX174) were treated and infected with HIV (89.6) in the same way as for PBLs. HIV RT activity was measured in culture supernatants collected at day 9 after infection. For PBLs, data shown are the mean ?? SD of triplicate determinations, representative of six independent experiments using PBLs from six different donors. For T-cell lines, data shown are the mean ?? SD of triplicate determinations, representative of three independent experiments (**P < 0.01, *P < 0.05, AW or PW versus control).
AW or PW Induces HIV Replication in Latently Infected T Cells and Activates HIV LTR
We then examined whether AW or PW activates HIV replication in J1.1 cells, a latently infected T-cell line.31 A significant increase in HIV RT activity in J1.1 cells was observed at 48 hours after AW or PW (Figure 3A) . To investigate the mechanism(s) responsible for AW- or PW-mediated up-regulation of HIV replication, we examined whether AW or PW has a stimulatory effect on HIV promoter LTR. HIV-LTR promoter activity was increased in 1G5 cells undergoing AW or PW (Figure 3B) . Naloxone alone had no effect on HIV activation in both J1.1 cells and 1G5 cells (data not shown).
Figure 3. Effect of AW or PW on latent HIV replication and HIV LTR activation. A: J1.1 cells were incubated with or without morphine (10C8 mol/L) for 96 hours and then subjected to AW or PW. The cells were stimulated with TNF- (2 ng/ml) 2 hours after withdrawal. HIV RT activity was measured in culture supernatants 48 hours after withdrawal. B: 1G5 cells were incubated with or without morphine (10C8 mol/L) for 96 hours and then subjected to AW or PW. The cells were stimulated with TNF- (2 ng/ml) 2 hours after withdrawal. The luciferase activity assay was measured in the cell extracts 48 hours after withdrawal. Data shown are the mean ?? SD of triplicate determinations, representative of three independent experiments (**P < 0.01, *P < 0.05, AW or PW versus control).
The Role of the SP and NK-1R in AW- or PW-Mediated Action
Using PBLs isolated from six different donors, we demonstrated that AW or PW induced SP mRNA expression in PBLs by as much as 20-fold, although there is variability in SP mRNA expression in PBLs from different donors (Figure 4A) . A significant increase of SP mRNA expression in both J-MOR5.1 and CEMX174 cells was also observed at 24 hours after AW or PW (Figure 4B) . The role of SP in enhancing HIV infection of T cells was confirmed in the experiments showing that exogenous SP, in a dose-dependent manner, significantly enhanced HIV infection of PBLs and CEMX174 cells (Figure 5) , and the NK-1R antagonist (CP-96,345) inhibited exogenous SP-mediated enhancement of HIV infection of PBLs and CEMX174 cells (Figure 5) .
Figure 4. Effect of AW and PW on SP mRNA expression in PBLs, J-MOR5.1, and CEMX174 cells. PBLs (A) and J-MOR5.1, CEMX174 (B) were treated with or without morphine (10C8 mol/L) for 4 days and followed by withdrawal for 24 hours. Cells were then subjected for RNA extraction and real-time PCR. For PBLs, data shown are the mean ?? SD of triplicate determinations, representative of six independent experiments using PBLs from six different donors. For J-MOR5.1 and CEMX174 cells, data shown are the mean ?? SD of triplicate determinations, representative of three independent experiments (**P < 0.01, *P < 0.05, AW or PW versus control).
Figure 5. Effect of SP and/or CP-96,345 on HIV infection of PBLs and CEMX174 cells. PHA-stimulated (72 hours) PBLs and CEMX174 were treated with SP and/or CP-96,345 for 3 hours and then infected with HIV strains (NL-43 for PBLs and 89.6 for CEMX714). HIV RT activity was determined at day 9 after infection. Data shown are the mean ?? SD of triplicate determinations, representative of four independent experiments with PBLs from four different donors or CEMX174 cells (CP: CP-96,345; **P < 0.01, *P < 0.05, SP versus control).
CP-96,345 Blocks AW- and PW-Induced SP Expression and HIV Replication in PBLs
The biological interaction of morphine with the SP-NK-1R pathway in T cells was further examined in experiments showing that CP-96,345 abrogated the effects of AW or PW on SP expression in PBLs (Figure 6A) . To further determine the role of endogenous SP in AW- or PW-mediated HIV enhancement, we examined whether CP-96,345 antagonizes the effect of AW or PW. CP-96,345 inhibited AW- or PW-induced HIV replication in PBLs (Figure 6B) whereas its inactive enantiomer CP-96,344 had no effect.
Figure 6. Effects of NK-1R antagonist CP-96,345 on AW- or PW-mediated enhancement of SP expression (A) and HIV replication (B) in PBLs. PBLs (PHA-stimulated) were incubated with or without morphine (10C8 mol/L) for 6 days and followed by AW or PW for 24 hours. At the time of withdrawal, the cells were incubated with or without CP-96,345 or CP-96,344. Cells were then subjected to RNA extraction and real-time PCR. For HIV infection, PBLs (24 hours after AW or PW) were infected with HIV (NL-43). HIV RT activity was measured in culture supernatants collected on day 9 after infection. Data shown in both A and B are the mean ?? SD of triplicate determinations, representative of four independent experiments using four different donors (**P < 0.01, *P < 0.05, AW or PW versus control).
Discussion
Our earlier studies showed that morphine, the principal metabolite of heroin, enhanced HIV infection of macrophages and microglia.27,28,41 Others4-7 also showed that morphine has a co-factor role in facilitating HIV infection of the immune cells. There have been no studies, however, to examine the impact of complete morphine removal (AW) or PW (addition of naloxone to the cell cultures immediately after removal of morphine) on HIV infection of T lymphocytes. The common outcomes of repetitive use of opiate are tolerance, physical dependence, and abstinence syndrome.42,43 Naloxone-precipitated opioid withdrawal is also a validated model for testing medications to treat opiate withdrawal.44 In this study, we pretreated the T cells with morphine for 4 days to induce a tolerant/dependent state, followed by AW or PW. Although this model system does not reflect the in vivo situation in which opiate withdrawal occurs repetitively during the course of opiate abuse, this in vitro system should provide direct evidence and initial evidence about the effect of AW on the T cells in the context of HIV infection. Using this cell system, we examined the effect of AW or PW on HIV infection, a common infection among injecting opioid users. We demonstrated that both AW and PW significantly enhanced HIV infection of human T lymphocytes. In addition, AW and PW activated HIV replication in latently infected T cells. Although the mechanism(s) underlying the morphine action on HIV remain to be determined, we showed that the enhancing effect of AW or PW on HIV was associated with the induction of SP mRNA, an important proinflammatory neuropeptide that has an important role in modulating the immune system.
The neuropeptide SP, the most extensively studied member of the tachykinin family, is a modulator of neuroimmunoregulation. Both in vitro and in vivo studies have clearly indicated that SP is involved in the physiological changes accompanying opioid withdrawal.9,10,13 SP levels were altered during opiate dependence after withdrawal. Morley and colleagues10 found that SP levels in the brain were elevated during long-term morphine treatment and attenuated after an injection of naloxone. Blockade of the SP-preferring receptor (neurokinin 1 receptor, NK-1R) induced a decrease in the expression of naloxone-precipitated morphine withdrawal syndrome in rats.13 Murtra and colleagues14 reported a loss of the rewarding properties of morphine in mice with a genetic disruption of the SP receptor NK-1R. Our observation that CP-96,345 suppressed AW- or PW-induced SP expression in the T cells (Figure 6) is in agreement with the in vivo observation that CP-96,345 inhibited SP production and morphine withdrawal response in guinea pigs.15 Our early study also showed that CP-96,345 down-regulated SP mRNA expression in human mononuclear phagocytes.45
Our earlier study showed that there is SP and NK-1R expression in human immune cells,46,47 and morphine induced SP expression in immune cells, including T cells.48 We also reported that SP enhances HIV expression in macrophages49 and latently infected T lymphocytes.50 Thus, we hypothesized that AW or PW induces SP expression in T cells, through which it enhances HIV replication. The role of SP in AW- or PW-mediated HIV infection of T cells was evidenced by the following observations: 1) AW and PW induced endogenous SP expression in the T cells, 2) exogenous SP enhanced HIV infection of T cells, and 3) the SP receptor antagonist (CP-96,345) not only abolished AW- or PW-induced endogenous SP expression but also antagonized AW- and PW-mediated enhancement of HIV infection. The effect of CP-96,345 is specific because its inactive enantiomer (CP-96,344) had little effect on AW- or PW-induced SP and HIV expression in T cells (Figure 6) . The role of SP in the morphine action is also supported by our earlier findings that human T lymphocytes express SP and SP receptors47 and that SP enhanced HIV infection of macrophages49 and activated HIV replication in chronically infected immune cells.50 The interaction between SP and HIV in the immune cells is bi-directional because HIV infection and activation also induced SP expression in human immune cells.8,51 SP released from HIV-infected immune cells, in return, may enhance HIV infection by directly facilitating virus replication and/or by indirectly affecting HIV proliferation through induction of inflammatory cytokines, such as, interleukin-1, interleukin-6, and TNF-, which are HIV-enhancing cytokines.52,53 SP modulates expression of these cytokines through activation of nuclear factor (NF)-B,54 which is supported by our earlier study showing that SP activates NF-B promoter in human T cells.55 In addition, treatment with SP enhanced HIV-LTR activation in the immune cells.56 These collective findings strongly indicate that AW- or PW-induced endogenous SP in T cells is likely to be one of the mechanisms responsible for the morphine withdrawal action on HIV infection. Although SP may play a major role in morphine withdrawal-mediated HIV replication, other factors such as norepinephrine are also involved in the withdrawal action in vivo. For example, a recent study showed that norepinephrine is widely implicated in opiate withdrawal.57 Interestingly, norepinephrine accelerates HIV replication in quiescently infected peripheral blood mononuclear cells that were subsequently activated with antibodies to CD3 and CD28.58 These collective data suggest that morphine withdrawal-mediated HIV enhancement is complex and is involved in multiple factors in the microenvironment.
Taken together, we and others have demonstrated that there are biological as well as pathological interactions between opiates and SP in both the central nervous system and immune systems. The interaction of opioids and SP in human T lymphocytes is likely to have a role in the immunopathogenesis of HIV disease among opiate abusers. Because SP is an important modulator of immune regulation and a possible co-factor in HIV infection of immune cells, morphine withdrawal-induced SP is likely to have a critical role in morphine withdrawal-mediated enhancement of HIV infection of the human immune system. Future investigations are necessary to understand molecular mechanism(s) involved in both the in vitro and in vivo interactions between opioids and SP in the human immune system in the context of HIV infection.
Acknowledgements
We thank Stephen Jasionowski for editorial assistance.
【参考文献】
Brzuskiewicz J, Carr L, Costa S: Estimated numbers of diagnoses of HIV/AIDS, by year of diagnosis and selected characteristics of persons, 1999C2002 2002, vol 14.:pp 10-15 HIV/AIDS Surveillance Report CDC
Pillai R, Nair BS, Watson RR: AIDS, drugs of abuse and the immune system: a complex immunotoxicological network. Arch Toxicol 1991, 65:609-617
Pakesch G, Loimer N, Grunberger J, Pfersmann D, Linzmayer L, Mayerhofer S: Neuropsychological findings and psychiatric symptoms in HIV-1 infected and noninfected drug users. Psychiatry Res 1992, 41:163-177
Peterson PK, Molitor TW, Chao CC: The opioid-cytokine connection. J Neuroimmunol 1998, 83:63-69
McCarthy L, Wetzel M, Sliker JK, Eisenstein TK, Rogers TJ: Opioids, opioid receptors, and the immune response. Drug Alcohol Depend 2001, 62:111-123
Stefano GB, Scharrer B, Smith EM, Hughes TK, Magazine HI, Bilfinger TV, Hartman AR, Fricchione GL, Liu Y, Makman MH: Opioid and opiate immunoregulatory processes. Crit Rev Immunol 1996, 16:109-144
Peterson PK, Gekker G, Hu S, Anderson WR, Kravitz F, Portoghese PS, Balfour HH, Jr, Chao CC: Morphine amplifies HIV-1 expression in chronically infected promonocytes cocultured with human brain cells. J Neuroimmunol 1994, 50:167-175
Ho WZ, Douglas SD: Substance P and neurokinin-1 receptor modulation of HIV. J Neuroimmunol 2004, 157:48-55
Tiong GK, Pierce TL, Olley JE: Sub-chronic exposure to opiates in the rat: effects on brain levels of substance P and calcitonin gene-related peptide during dependence and withdrawal. J Neurosci Res 1992, 32:569-575
Morley JE, Yamada T, Walsh JH, Lamers CB, Wong H, Shulkes A, Damassa DA, Gordon J, Carlson HE, Hershman JM: Morphine addiction and withdrawal alters brain peptide concentrations. Life Sci 1980, 26:2239-2244
Bergstrom L, Sakurada T, Terenius L: Substance P levels in various regions of the rat central nervous system after acute and chronic morphine treatment. Life Sci 1984, 35:2375-2382
McCarson KE, Krause JE: The formalin-induced expression of tachykinin peptide and neurokinin receptor messenger RNAs in rat sensory ganglia and spinal cord is modulated by opiate preadministration. Neuroscience 1995, 64:729-739
Maldonado R, Girdlestone D, Roques BP: RP 67580, a selective antagonist of neurokinin-1 receptors, modifies some of the naloxone-precipitated morphine withdrawal signs in rats. Neurosci Lett 1993, 156:135-140
Murtra P, Sheasby AM, Hunt SP, De Felipe C: Rewarding effects of opiates are absent in mice lacking the receptor for substance P. Nature 2000, 405:180-183
Chahl LA, Johnston PA: Effect of the nonpeptide NK-1 receptor antagonist CP-96,345 on the morphine withdrawal response of guinea-pigs. Regul Pept 1993, 46:373-375
Rahim RT, Adler MW, Meissler JJ, Jr, Cowan A, Rogers TJ, Geller EB, Eisenstein TK: Abrupt or precipitated withdrawal from morphine induces immunosuppression. J Neuroimmunol 2002, 127:88-95
Tomei EZ, Renaud FL: Effect of morphine on Fc-mediated phagocytosis by murine macrophages in vitro. J Neuroimmunol 1997, 74:111-116
Govitrapong P, Suttitum T, Kotchabhakdi N, Uneklabh T: Alterations of immune functions in heroin addicts and heroin withdrawal subjects. J Pharmacol Exp Ther 1998, 286:883-889
West JP, Dykstra LA, Lysle DT: Immunomodulatory effects of morphine withdrawal in the rat are time dependent and reversible by clonidine. Psychopharmacology (Berl) 1999, 146:320-327
Dougherty PM, Pellis NR, Dafny N: The brain and the immune system: an intact immune system is essential for the manifestation of withdrawal in opiate addicted rats. Neuroscience 1990, 36:285-289
Dafny N, Pellis NR: Evidence that opiate addiction is in part an immune response. Destruction of the immune system by irradiation-altered opiate withdrawal. Neuropharmacology 1986, 25:815-818
Feng P, Wilson QM, Meissler JJ, Jr, Adler MW, Eisenstein TK: Increased sensitivity to Salmonella enterica serovar Typhimurium infection in mice undergoing withdrawal from morphine is associated with suppression of interleukin-12. Infect Immun 2005, 73:7953-7959
Feng P, Meissler JJ, Jr, Adler MW, Eisenstein TK: Morphine withdrawal sensitizes mice to lipopolysaccharide: elevated TNF-alpha and nitric oxide with decreased IL-12. J Neuroimmunol 2005, 164:57-65
Kelschenbach J, Barke RA, Roy S: Morphine withdrawal contributes to Th cell differentiation by biasing cells toward the Th2 lineage. J Immunol 2005, 175:2655-2665
Wang CQ, Li Y, Douglas SD, Wang X, Metzger DS, Zhang T, Ho WZ: Morphine withdrawal enhances hepatitis C virus replicon expression. Am J Pathol 2005, 167:1333-1340
Donahoe RM: Neuroimmunomodulation by opiates: relationship to HIV infection and AIDS. Adv Neuroimmunol 1993, 3:31-46
Li Y, Wang X, Tian S, Guo CJ, Douglas SD, Ho WZ: Methadone enhances human immunodeficiency virus infection of human immune cells. J Infect Dis 2002, 185:118-122
Guo 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 Invest Med 2002, 50:435-442
Hassan NF, Campbell DE, Douglas SD: Purification of human monocytes on gelatin-coated surfaces. J Immunol Methods 1986, 95:273-276
Aguilar-Cordova E, Chinen J, Donehower L, Lewis DE, Belmont JW: A sensitive reporter cell line for HIV-1 tat activity, HIV-1 inhibitors, and T cell activation effects. AIDS Res Hum Retroviruses 1994, 10:295-301
Perez VL, Rowe T, Justement JS, Butera ST, June CH, Folks TM: An HIV-1-infected T cell clone defective in IL-2 production and Ca2+ mobilization after CD3 stimulation. J Immunol 1991, 147:3145-3148
Rogers TJ, Steele AD, Howard OM, Oppenheim JJ: Bidirectional heterologous desensitization of opioid and chemokine receptors. Ann NY Acad Sci 2000, 917:19-28
Szabo I, Wetzel MA, Zhang N, Steele AD, Kaminsky DE, Chen C, Liu-Chen LY, Bednar F, Henderson EE, Howard OM, Oppenheim JJ, Rogers TJ: Selective inactivation of CCR5 and decreased infectivity of R5 HIV-1 strains mediated by opioid-induced heterologous desensitization. J Leukoc Biol 2003, 74:1074-1082
Suzuki S, Miyagi T, Chuang TK, Chuang LF, Doi RH, Chuang RY: Morphine upregulates mu opioid receptors of human and monkey lymphocytes. Biochem Biophys Res Commun 2000, 279:621-628
Snider RM, Constantine JW, Lowe JA, III, Longo KP, Lebel WS, Woody HA, Drozda SE, Desai MC, Vinick FJ, Spencer RW: A potent nonpeptide antagonist of the substance P (NK1) receptor. Science 1991, 251:435-437
Berger EA, Doms RW, Fenyo EM, Korber BT, Littman DR, Moore JP, Sattentau QJ, Schuitemaker H, Sodroski J, Weiss RA: A new classification for HIV-1. Nature 1998, 391:240
Adachi A, Gendelman HE, Koenig S, Folks T, Willey R, Rabson A, Martin MA: Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol 1986, 59:284-291
Wang X, Douglas SD, Peng JS, Metzger DS, O??Brien CP, Zhang T, Ho WZ: Naltrexone inhibits alcohol-mediated enhancement of HIV infection of T lymphocytes. J Leukoc Biol 2006, 79:1166-1172
Willey RL, Smith DH, Lasky LA, Theodore TS, Earl PL, Moss B, Capon DJ, Martin MA: In vitro mutagenesis identifies a region within the envelope gene of the human immunodeficiency virus that is critical for infectivity. J Virol 1988, 62:139-147
Lai JP, Douglas SD, Shaheen F, Pleasure DD, Ho WZ: Quantification of substance P mRNA in human immune cells by real-time reverse transcriptase PCR assay. Clin Diagn Lab Immunol 2002, 9:138-143
Li Y, Merrill JD, Mooney K, Song L, Wang X, Guo CJ, Savani RC, Metzger DS, Douglas SD, Ho WZ: Morphine enhances HIV infection of neonatal macrophages. Pediatr Res 2003, 54:282-288
Jeffe JH, Martuin WR: Opioid analgesics and antagonists. Gilman AG Rall TW Nies AS Taylor P Goodman LS eds. The Pharmacological Basis of Therapeutics. 1990:pp 485-521 Macmillan Publishing Co., Inc., New York
Jeffe JH: Drug addiction and drug use. Gilman AG Rall TW Nies AS Taylor P eds. The Pharmacological Basis of Therapeutics. 1990:pp 522-573 Macmillan Publishing Co., Inc., New York
Rosen MI, McMahon TJ, Hameedi FA, Pearsall HR, Woods SW, Kreek MJ, Kosten TR: Effect of clonidine pretreatment on naloxone-precipitated opiate withdrawal. J Pharmacol Exp Ther 1996, 276:1128-1135
Lai JP, Ho WZ, Yang JH, Wang X, Song L, Douglas SD: A non-peptide substance P antagonist down-regulates SP mRNA expression in human mononuclear phagocytes. J Neuroimmunol 2002, 128:101-108
Ho WZ, Lai JP, Zhu XH, Uvaydova M, Douglas SD: Human monocytes and macrophages express substance P and neurokinin-1 receptor. J Immunol 1997, 159:5654-5660
Lai JP, Douglas SD, Ho WZ: Human lymphocytes express substance P and its receptor. J Neuroimmunol 1998, 86:80-86
Li Y, Tian S, Douglas SD, Ho WZ: Morphine Up-regulates expression of substance P and its receptor in human blood mononuclear phagocytes and lymphocytes. Cell Immunol 2000, 205:120-127
Ho WZ, Cnaan A, Li YH, Zhao H, Lee HR, Song L, Douglas SD: Substance P modulates human immunodeficiency virus replication in human peripheral blood monocyte-derived macrophages. AIDS Res Hum Retroviruses 1996, 12:195-198
Li Y, Douglas SD, Song L, Sun S, Ho WZ: Substance P enhances HIV-1 replication in latently infected human immune cells. J Neuroimmunol 2001, 121:67-75
Ho WZ, Lai JP, Li Y, Douglas SD: HIV enhances substance P expression in human immune cells. FASEB J 2002, 16:616-618
Folks TM, Justement J, Kinter A, Dinarello CA, Fauci AS: Cytokine-induced expression of HIV-1 in a chronically infected promonocyte cell line. Science 1987, 238:800-802
Folks TM, Justement J, Kinter A, Schnittman S, Orenstein J, Poli G, Fauci AS: Characterization of a promonocyte clone chronically infected with HIV and inducible by 13-phorbol-12-myristate acetate. J Immunol 1988, 140:1117-1122
Marriott I, Mason MJ, Elhofy A, Bost KL: Substance P activates NF-kappaB independent of elevations in intracellular calcium in murine macrophages and dendritic cells. J Neuroimmunol 2000, 102:163-171
Guo C-J, Lai J-P, Luo H-M, Douglas SD, Ho W-Z: Substance P up-regulates macrophage inflammatory protein-1 beta expression in human T lymphocytes. J Neuroimmunol 2002, 131:160-167
Lai JP, Ho WZ, Zhan GX, Yi Y, Collman RG, Douglas SD: Substance P antagonist (CP-96,345) inhibits HIV-1 replication in human mononuclear phagocytes. Proc Natl Acad Sci USA 2001, 98:3970-3975
Olson VG, Heusner CL, Bland RJ, During MJ, Weinshenker D, Palmiter RD: Role of noradrenergic signaling by the nucleus tractus solitarius in mediating opiate reward. Science 2006, 311:1017-1020
Cole SW, Korin YD, Fahey JL, Zack JA: Norepinephrine accelerates HIV replication via protein kinase A-dependent effects on cytokine production. J Immunol 1998, 161:610-616
作者单位：From the Department of Pediatrics,* Division of Allergy and Immunology, Joseph Stokes Jr. Research Institute at the Children??s Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania; the Virology Laboratory, Wuhan Center for Disease Prevention and Contro