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Division of Infectious Diseases and International Medicine, University of Minnesota Medical School
Neuroimmunology Laboratory, Minneapolis Medical Research Foundation, Minneapolis
To investigate the effect of opiates on human immunodeficiency virus type 1 (HIV-1)related neuronal apoptosis, primary human fetal neuronal/glial cell cultures were exposed to gp120IIIB in the absence and the presence of morphine. Although morphine by itself had little effect on neuronal survival, the combination of morphine (10-7 mol/L) and gp120IIIB (1 nmol/L) significantly increased neuronal apoptosis. The mechanism whereby morphine potentiates gp120IIIB-induced neuronal apoptosis appears to involve activation of the p38 mitogen-activated protein kinase intracellular signaling pathway and microglial cells dispersed within the neuronal/glial cell cultures. These results provide additional insight into the molecular basis whereby opiate abuse could promote HIV-1associated dementia.
Substances of abuse have been shown to suppress a number of immune responses [1, 2] and, therefore, have been postulated to serve as cofactors in the progression of HIV-1 infection. That opiates play a role in the development of HIV-1associated dementia (HAD), one of the most devastating complications of AIDS, is supported by a growing body of epidemiological and histopathological evidence [37]. The results of studies of molecular mechanisms whereby substances of abuse and HIV-1 synergistically induce neuronal death also support this role [8]. Neuronal apoptosis, a histopathological hallmark of HAD, appears to be the result of a complex interplay of HIV-1 proteins and neurotoxins released from activated monocytes and microglial cells [911], the resident macrophages of the brain. The viral proteins gp120 and Tat can induce neuronal apoptosis, and, in one study, the opiate morphine was shown to facilitate Tat-induced neuronal death [12]. In the present study, the effect of morphine on gp120IIIB-induced neuronal apoptosis was assessed, and the mechanism underlying its synergistic neurotoxic activity was investigated.
Materials and methods.
The following reagents were purchased from the indicated sources: fetal bovine serum (FBS) (Hyclone); morphine sulfate, Dulbecco's modified Eagle medium (DMEM), penicillin, streptomycin, Hanks' balanced salt solution, trypsin, bovine serum albumin, polyoxyethylenesorbitan monolaurate (Tween 20), PBS, protease inhibitor, paraformaldehyde, and the macrophage activationinhibitor tripeptide Thr-Lys-Pro (TKP; tuftsin fragment 13) (Sigma); naloxone (Sigma-RBI); antineuron nuclei (NeuN; a neuronal marker) and antimicrotubule-associated protein2 (MAP-2; a neuronal marker) antibodies (Chemicon); antiglial fibrillary protein (GFAP; an astrocyte marker) antibody (Sternberger Monoclonals); anti-CD68 (a microglial cell marker) antibody (BD Pharmingen); anti-galactocerebroside (an oligodendrocyte marker) antibody (Polysciences); gp120IIIB (Protein Sciences); SB203580 and SB202474 (Calbiochem); propidium iodide, Hoechst 33342, and Apop Tag in situ apoptosis detection kit (Intergen); antiphosphorylated-p38 mitogen-activated protein kinase (MAPK) antibody (Cell Signaling); and acrylamide/bis solution (Bio-Rad).
Human fetal brain tissue was obtained from women undergoing elective abortion, in accordance with informed-consent guidelines and a protocol approved by the Human Subjects Research Committee at our institution. Neuronal/glial cell cultures were prepared as described elsewhere [13]. In brief, 1618-week-old brain cortical tissues were dissociated and resuspended in DMEM containing 10% heat-inactivated FBS plus penicillin (100 U/mL) and streptomycin (100 g/mL). Dispersed cells were plated onto collagen-coated plates (5 × 105, 106, or 3 × 106 cells/well in 24-, 12-, or 6-well plates, respectively) or chamber slides (4 × 105 cells/well in 4-well chambers). On day 5, cell cultures were treated with uridine (33.6 g/mL) and fluorodeoxyuridine (13.6 g/mL), followed by replacement with DMEM with 10% FBS on day 6 and every 4 days thereafter. On day 12, these brain-cell cultures contained 70%80% neurons (stained by anti-NeuN or antiMAP-2 antibodies), 15%25% astrocytes (stained by anti-GFAP antibody), and 3%7% microglial cells (stained by anti-CD68 antibody).
Apoptosis was assessed by in situ end labeling of fragmented DNA (TUNEL technique), as described elsewhere [13]. After treatment, neuronal/glial cell cultures were fixed in 4% paraformaldehyde, and an Apop Tag in situ apoptotic detection kit was used to label fragmented DNA ends with digoxigenin-11uridine triphosphate by use of terminal deoxynucleotidyl transferase. Fluorescein-labeled anti-digoxigenin antibody was then used to detect the labeled ends in the nuclei. The percentage of apoptotic cells was calculated for 5 fields.
Neuronal apoptosis was also assessed, by use of methods that have been described elsewhere [9, 13]. After treatment, neuronal/glial cell cultures were stained with Hoechst 33342 and propidium iodide, to determine nuclear morphology of apoptotic cells. To stain apoptotic neurons specifically, a double-staining procedure was used. After treatment, paraformaldehyde-fixed neuronal/glial cell cultures were incubated with antiMAP-2 antibody (1 : 200), followed by incubation with fluorescein isothiocyanateconjugated secondary antibody (goat anti-rabbit). Cells were subsequently stained with propidium iodide (1 g/mL) for 10 min in the dark and examined by fluorescence microscopy. The percentage of apoptotic cells was calculated for 5 fields.
Detection of oligonucleosomal cell death by sandwich ELISA (Roche) was used to determine DNA fragmentation subunits generated during apoptosis. In brief, cell lysates from treated and untreated neuronal/glial cell cultures were placed in a streptavidin-coated microtiter plate along with a mixture of antihistone biotinylated antibody and anti-DNA peroxidase-conjugated antibody. Results were expressed as optical densities at 405 nm for equivalent numbers of cells.
To assess activation of p38 MAPK, neuronal/glial cell cultures were lysed, by use of a standard procedure, with 2× SDS sample buffer (125 mmol/L Tris-HCl [pH 6.8], 20% glycerol, 4% SDS [wt/vol], 2% -mercaptoethanol, and 0.001% bromphenol blue) and loaded onto 12% SDS-polyacrylamide gels. After the gel was transblotted to a nitrocellulose membrane, membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with Tween 20 (TTBS) (100 mmol/L Tris-HCl [pH 7.5], 0.9% NaCl, and 0.1% Tween 20) and incubated with antiphosphorylated-p38 MAPK antibody (1 : 1000 dilution in 1% blocking buffer in TTBS). After being washed, membranes were incubated with secondary antibody (alkaline phosphataseconjugated goat antirabbit IgG; 1 : 5000 dilution), followed by chemiluminescent detection (CDP-Star substrates; Applied Biosystems) by use of an image station (Kodak).
Data are expressed as mean ± SE. For comparison of multiple means, analysis of variance was used, followed by Fisher's protected least significant difference test.
Results.
Before assessing the effect of morphine on gp120IIIB-induced neuronal apoptosis, a concentration-response study was performed with gp120IIIB alone. By use of the TUNEL technique, gp120IIIB was found to induce neuronal apoptosis when added to cultures at concentrations 1 nmol/L (figure 1A), an observation corroborated by propidium-iodide staining of apoptotic neurons (data not shown). Thus, a concentration of 1 nmol/L gp120IIIB was used in all subsequent experiments.
Next, the effect of varying concentrations of morphine on gp120IIIB-induced apoptosis was evaluated. Morphine by itself had little effect on neuronal survival, and significantly greater neuronal apoptosis was observed when neuronal/glial cell cultures were exposed to both gp120IIIB and morphine (10-7 or 10-6 mol/L) than when they were exposed to gp120IIIB alone (figure 1B). The synergistic effect of morphine and gp120IIIB on neuronal apoptosis was corroborated by use of an assay that measures histone-associated DNA fragments (figure 1C). The synergistic effect of morphine and gp120IIIB was blocked by pretreatment of the neuronal/glial cell cultures with the opiate antagonist naloxone (figure 1D), indicating that morphine was operating via an opiate receptor.
On the basis of the reported involvement of the p38 MAPK intracellular signaling pathway in gp120IIIB-induced neuronal apoptosis [9], we tested the hypothesis that the synergistic effect of morphine and gp120IIIB is related to enhanced activation of this pathway. In support of this hypothesis, when the p38 MAPK inhibitor SB203580 was added to neuronal/glial cell cultures that had been exposed to morphine plus gp120IIIB, the synergistic effect of morphine was abolished (figure 2A). On the other hand, addition of the inactive compound SB202474 (10 mol/L) had no effect on the synergistic effect of morphine (data not shown). Western-blot analysis of cultures for phosphorylated p38 MAPK showed that this product was present within 15 min of exposure to morphine or gp120IIIB and that, although the activation of this signaling pathway by morphine was relatively short-lived, compared with that by gp120IIIB, cultures exposed to both gp120IIIB and morphine showed a more robust, sustained activation of p38 MAPK, compared with cultures exposed to gp120IIIB alone (figure 2B).
Both neurons and microglial cells [14] express -opioid receptors (MORs), the major binding sites for ligands such as morphine, and both of these brain-cell types also express CXCR4, the chemokine receptor to which gp120IIIB binds. Since the neuronal/glial cell cultures used in the present study contained 3%7% microglial cells, we next investigated whether microglial cells were involved in the synergistic activity of morphine and gp120IIIB, by adding TKP (an inhibitor of macrophage activation) to the cultures. TKP suppressed the synergistic effect of morphine and gp120IIIB-induced neuronal apoptosis by 45.48% ± 1.65% (figure 2C), suggesting that the microglial cells distributed within the neuronal/glial cell cultures used in the present study contributed, in part, to this synergistic interaction.
Discussion.
Previously, other investigators have demonstrated that morphine synergistically increases the neurotoxic activity of HIV-1 Tat protein in murine and human neuronal cell cultures [8, 12]. In the present study, a similar effect was observed when morphine was added along with gp120IIIB to cell cultures and apoptosis of human fetal neurons was assessed. The mechanism underlying the synergistic effect of morphine and gp120IIIB appeared to involve activation of the p38 MAPK intracellular signaling pathway and microglial cells, which were interspersed within the neuronal/glial cell cultures used in the present study. Although the precise mechanism whereby microglial cells contribute to the neurotoxicity of gp120IIIB has not been delineated, their involvement in neuronal apoptosis has been postulated to be of primary importance in HAD [9]. Interestingly, autopsy studies have detected activated microglial cells in the brains of heroin addicts who are not infected with HIV-1, suggesting that opiates may increase the susceptibility of such individuals to development of HAD after HIV-1 infection [7].
Other investigators have demonstrated that morphine induces apoptosis of macrophages via a p38 MAPK signaling pathway [15], and earlier studies in our laboratory have shown that exposure of microglial cells and neurons to morphine for 7 days in culture is associated with apoptosis of both brain-cell types [13]. In the present study, neuronal/glial cells were cultured for only 3 days, and morphine by itself did not induce appreciable neuronal apoptosis during this period. Nonetheless, it may be that microglial cells or neurons are sensitized to gp120IIIB by 3 days of exposure to morphine. Since both neurons and microglial cells express CXCR4 and MOR, it is possible that gp120IIIB and morphine exert their effects on neuronal survival through direct and indirect (i.e., via activation of microglial cells) mechanisms. Taken together with the results of earlier studies that demonstrated that morphine synergistically enhances Tat proteininduced neuronal apoptosis [8, 12], the results of the present study provide additional insight into the molecular basis whereby opiate abuse could promote HAD.
References
1. Eisenstein TK, Hilburger ME. Opioid modulation of immune responses: effects on phagocyte and lymphoid cell populations. J Neuroimmunol 1998; 83:3644. First citation in article
2. Friedman H, Newton C, Klein TW. Microbial infections, immunomodulation, and drugs of abuse. Clin Microbiol Rev 2003; 16:20919. First citation in article
3. Wang F, So Y, Vittinghoff E, et al. Incidence proportion of and risk factors for AIDS patients diagnosed with HIV dementia, central nervous system toxoplasmosis, and cryptococcal meningitis. J Acquir Immune Defic Syndr Hum Retrovirol 1995; 8:7582. First citation in article
4. Davies J, Everall IP, Weich S, McLaughlin J, Scaravilli F, Lantos PL. HIV-associated brain pathology in the United Kingdom: an epidemiological study. AIDS 1997; 11:114550. First citation in article
5. Bouwman FH, Skolasky RL, Hes D, et al. Variable progression of HIV-associated dementia. Neurology 1998; 50:181420. First citation in article
6. Bell JE, Brettle RP, Chiswick A, Simmonds P. HIV encephalitis, proviral load and dementia in drug users and homosexuals with AIDS: effect of neocortical involvement. Brain 1998; 121:204352. First citation in article
7. Tomlinson GS, Simmonds P, Busuttil A, Chiswick A, Bell JE. Upregulation of microglia in drug users with and without pre-symptomatic HIV infection. Neuropathol Appl Neurobiol 1999; 25:36979. First citation in article
8. Nath A, Hauser KF, Wojna V, et al. Molecular basis for interactions of HIV and drugs of abuse. J Acquir Immune Defic Syndr 2002; 31(Suppl 2):S629. First citation in article
9. Kaul M, Garden GA, Lipton SA. Pathways to neuronal injury and apoptosis in HIV-associated dementia. Nature 2001; 410:98894. First citation in article
10. Anderson E, Zink W, Xiong H, Gendelman HE. HIV-1associated dementia: a metabolic encephalopathy perpetrated by virus-infected and immune-competent mononuclear phagocytes. J Acquir Immune Defic Syndr 2002; 31(Suppl 2):S4354. First citation in article
11. Williams KC, Hickey WF. Central nervous system damage, monocytes and macrophages, and neurological disorders in AIDS. Annu Rev Neurosci 2002; 25:53762. First citation in article
12. Gurwell JA, Nath A, Sun Q, et al. Synergistic neurotoxicity of opioids and human immunodeficiency virus1 Tat protein in striatal neurons in vitro. Neuroscience 2001; 102:55563. First citation in article
13. Hu S, Sheng WS, Lokensgard JR, Peterson PK. Morphine induces apoptosis of human microglia and neurons. Neuropharmacology 2002; 42:82936. First citation in article
14. Chao CC, Hu S, Shark KB, Sheng WS, Gekker G, Peterson PK. Activation of mu opioid receptors inhibits microglial cell chemotaxis. J Pharmacol Exp Ther 1997; 281:9981004. First citation in article
15. Singhal PC, Bhaskaran M, Patel J, et al. Role of p38 mitogenactivated protein kinase phosphorylation and Fas-Fas ligand interaction in morphine-induced macrophage apoptosis. J Immunol 2002; 168:402533. First citation in article