The effects of opioids on HIV reactivation in latently-infected T-lymphoblasts

In order to investigate the effects of the opioids heroine and morphine on reactivation of proviral HIV in latently-infected T cells, we incubated ACH-2 cells with different concentrations of morphine-sulfate and heroine. After incubation for 24 h we measured intracellular HIV-p24 protein expression by flow cytometry. We found a dose-dependent reactivation of HIV for morphine (Figure 1A) and heroine (Figure 1B). The EC₅₀ concentrations for the two substances were 2.82 mM and 1.96 mM, respectively (Figure 1). Figures 1C and D display representative dot-plots analyzing p24-expression (y-axis) of untreated (Figure 1C) or 4 mM heroin-treated (D) cells. The background expression visible in Figure 1C is typical for ACH-2 cells and originates from intrinsic stimulation of the cells. We also tested the effects of heroin on HIV replication at very low concentrations, similar to the plasma concentrations found in IDUs and found no activating effects (Figure 1D).

Opioid receptor signaling has been described on lymphocytes[1] and opioid receptors that bind the opioid antagonist naloxone have been identified on T cells (reviewed in[17]). We therefore investigated whether the observed HIV-activating effect of opioids was mediated by opioid-specific receptor stimulation. For that we triggered HIV reactivation with heroine in the absence or presence of different concentrations of naloxone. As depicted in Figure 1F, naloxone did not interfere with heroine-mediated HIV reactivation, indicating that opioid receptor-independent mechanisms account for the observed effects.

Heroin- and morphine-mediated activation of HIV replication was completely prevented by the antioxidants N-acetylcystein (NAC) or reduced glutathione (GSH) (Figure 2), indicating that oxidative stress might be involved in opioid-mediated reactivation of HIV. The relatively high opioid concentrations needed to stimulate HIV replication were also associated with induction of necrotic cell death. A3.01 T cells, the uninfected parental cell line of ACH-2 cells, showed a significant amount of necrosis induction following treatment with 2.5 mM heroin as assessed by staining with the cell dye 7-AAD (Figure 3A). This necrosis induction could again be completely prevented by the antioxidant N-acetylcystein (Figure 3A). We used for this experiment the uninfected parental cell line because the 7-AAD dye requires nonfixated cells, which in case of the HIV-infected ACH-2 cells we could not subject to our flow cytometer for safety reasons. In order to demonstrate that ACH-2 cells react in a similar way to opioids as A3.01 cells, we analyzed cell death in ACH-2 cells by a characteristic shift of the cell population in the forward-scatter/sideward-scatter dot plot analysis, which correlates with 7-AAD-staining. This shift is depicted in Figure 3C from representative analyses for different heroin concentrations. The corresponding p24-expression profiles are depicted in Figure 3D. As shown in Figure 3B, activation of HIV in ACH-2 cell completely paralleled with opioid-triggered necrosis. We reported earlier that necrotic signaling triggers NF-κB and HIV-reactivation in ACH-2 T lymphoblasts[18]. These data indicate that the observed opioid-mediated activation of HIV replication was secondary to the opioid-triggered necrosis observed at high concentrations.

A3.01 (human CD4 + -T-lymphoblasts, NIAID) and ACH-2 (latently HIV-infected A3.01, Folks 1989, NIAID) were cultured in standard tissue flasks using RPMI-medium (Invitrogen, Karlsruhe, Germany) supplemented with 10% FCS (Linaris, Bettingen, Germany) at 37°C and 5% CO2 atmosphere. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll/Paque density-separation centrifugation from human whole blood donated by healthy volunteers. For the different experiments 10⁵ cells per well were cultured in flat-bottom 96 well plates in a total volume of 200 μl for 24 h in the absence or presence of the indicated substances.

Morphine and Heroine were obtained from the University of Wurzburg’s Department of Forensic Medicine. The Use and Discard of those substances were documented in accordance with the German Narcotics Act. Naloxone, Glutathion and N-Acetylcysteine were purchased through Sigma, St.Lewis, MO.

Reactivation of HIV in ACH-2 cells was quantified by the expression of viral p24-antigen detected by flow cytometry (Becton-Dickinson, Heidelberg, Germany). Cells were fixated for 20 min with 4% formalin in PBS and later permeabilized with 0.5% saponin in PBS/5%BSA. The following antibodies were used: 183-H12-5C-anti-HIV-p24-mouse-antibody and goat-anti-mouse antibodies conjugated with FITC or PE (BD Biosciences). Cell death in uninfected A3.01 cells was quantified by 7-AAD staining according to the instructions of the manufacturer (BD Biosciences). Besides staining with 7-AAD, necrotic cells also displayed a characteristic shift in a forward-scatter (FSC)/sideward scatter (SSC) dot plot. This shift was used to quantify necrotic cells also for HIV-infected ACH-2 cells, for which a 7-AAD staining was not possible for safety reasons (7-AAD staining requires nonfixated cells which in case of ACH-2 cells would contaminate the flow cytometer with HIV).