Recent evidence suggests that injection drug users who abuse heroin are at increased risk for CNS complications from human immunodeficiency virus (HIV) infection. Opiate drugs may intrinsically alter the pathogenesis of HIV by directly modulating immune function and by directly modifying the CNS response to HIV. Despite this, the mechanisms by which opiates increase the neuropathogenesis of HIV are uncertain. Herein we describe the effect of morphine and the HIV-1 protein toxin Tat 1-72 on astroglial function in cultures derived from ICR mice. Astroglia maintain the blood brain barrier and influence inflammatory signaling in the CNS. Astrocytes can express μ opioid receptors, and are likely targets for abused opiates, which preferentially activate μ-opioid receptors. While Tat alone disrupts astrocyte function, when combined with morphine, Tat causes synergistic increases in [Ca 2+ ] i. . Moreover, astrocyte cultures treated with morphine and Tat showed exaggerated increases in chemokine release including monocyte chemoattractant protein-1 (MCP-1) and regulated on activation, normal T cell expressed and secreted (RANTES), as well as interleukin-6 (IL-6). Morphine-Tat interactions were prevented by the μ-opioid receptor antagonist β-funaltrexamine, or by immunoneutralizing Tat 1-72 or substituting a non-toxic, deletion mutant (Tat Δ31-61 ). Our findings suggest that opiates may increase the vulnerability of the CNS to viral entry (via recruitment of monocytes/macrophages) and ensuing HIV encephalitis by synergistically increasing MCP-1 and RANTES release by astrocytes. The results further suggest ‡ Abbreviations: alpha chemokine receptor (CXCR); beta chemokine ligand (CCL); beta chemokine receptor (CCR); β-funaltrexamine (β-FNA); calcium-induced calcium release (CICR); excitatory amino acid transporter-2 (EAAT2); granulocyte macrophage colony stimulating factor (GM-CSF); granulocyte-colony stimulating factor (G-CSF); human immunodeficiency virus (HIV); human immunodeficiency virus encephalitis (HIVE); inositol trisphosphate (IP 3 ); interferon (IFN); interleukin (IL); intracellular Ca 2+ ([Ca 2+ ] i ); monocyte chemoattractant protein (MCP); nor-binaltorphimine (nor-BNI); phosphatidylinositol 3-kinase (PI3-kinase); phospholipase C-γ (PLCγ); regulated on activation, normal T cell expressed and secreted (RANTES); soluble TNF receptor subunit (sTNFR1); stem cell factor (SCF); thrombopoietin (TPO); transactivator of transcription (Tat); tumor necrosis factor-α (TNF-α); vascular endothelial growth factor (VEGF).
Individuals infected with human immunodeficiency virus-1 who abuse opiates can have a higher incidence of virus-associated neuropathology. Human immunodeficiency virus does not infect neurons, but viral proteins such as transactivator of transcription and glycoprotein 120, originating from infected glia, are neurotoxic. Moreover, functional changes in glial cells that enhance inflammation and reduce trophic support are increasingly implicated in human immunodeficiency virus neuropathology. In previous studies, co-exposure with morphine enhanced transactivator of transcription neurotoxicity towards cultured striatal neurons. Since those cultures contained µ-opioid receptor-expressing astroglia and microglia, and since glia are the principal site of infection in the central nervous system, we hypothesized that morphine synergy might be glially mediated. A 60 hour, repeated measures paradigm and multiple co-culture models were used to investigate the cellular basis for opiate-enhanced human immunodeficiency virus neurotoxicity. Morphine co-exposure significantly enhanced transactivator of transcription-induced neuron death when glia were present. Synergistic effects of morphine on transactivator of transcription neurotoxicity were greatest with neuron-glia contact, but also occurred to a lesser extent with glial conditioned medium. Importantly, synergy was lost if glia, but not neurons, lacked µ-opioid receptors, indicating that opiate interactions with human immunodeficiency virus converge at the level of µ-opioid receptor-expressing glia. Morphine enhanced transactivator of transcription-induced inflammatory effectors released by glia, elevating reactive oxygen species, increasing 3-nitrotyrosine production by microglia, and reducing the ability of glia to buffer glutamate. But neuron survival was reduced even more with glial contact than with exposure to conditioned medium, suggesting that noxious elements associated with cell contact augment the toxicity due to soluble factors. Similar morphine-transactivator of transcription synergy was also observed in studies with the clade C sequence of HIV-1 transactivator of transcription, which did not cause neuron death unless morphine was present. Several paradoxical observations related to opiate effects were noted when µ-opioid receptors were specifically ablated from either glia or neurons. This suggests that µ-opioid receptor loss in isolated cell types can fundamentally distort cell-to-cell signalling, revealing opponent processes that may exist in individual cell types. Our findings show the critical role of glia in orchestrating neurotoxic interactions of morphine and transactivator of transcription, and support the emerging concept that combined exposure to opiates and human immunodeficiency virus drives enhanced pathology within the central nervous system.
Background Human immunodeficiency virus (HIV) associated neurocognitive disorders (HAND), including memory dysfunction, continue to be a major clinical manifestation of HIV type-1 (HIV-1) infection. Viral proteins released by infected glia are thought to be the principal triggers of inflammation and bystander neuronal injury and death, thereby driving key symptomatology of HAND. Methods We used a GFAP-driven, doxycycline (DOX)-inducible HIV-1 Tat (transactivator of transcription) transgenic mouse model and examined structure-function relationships in hippocampal pyramidal CA1 neurons using morphologic (Golgi-silver impregnations, immunohistochemistry, TUNEL detection, synaptic protein markers, electron microscopy), electrophysiological (long-term potentiation (LTP)), and behavioral (Morris water maze, fear-conditioning) approaches. Results Tat induction caused a variety of different inclusions in astrocytes characteristic of lysosomes, autophagic vacuoles, and lamellar bodies, which were typically present within distal cytoplasmic processes. In pyramidal CA1 neurons, Tat induction reduced the number of apical dendritic spines, while disrupting the distribution of synaptic proteins (synaptotagmin 2 and gephyrin) associated with inhibitory transmission, but with minimal dendritic pathology and no evidence of pyramidal neuron death. Electrophysiological assessment of excitatory postsynaptic field potential (fEPSP) at Schaffer collateral/commissural fiber-CA1 synapses showed near total suppression of LTP in mice expressing Tat. The loss in LTP coincided with disruptions in learning and memory. Conclusion Tat expression in the brain results in profound functional changes in synaptic physiology and in behavior that are accompanied by only modest structural changes and minimal pathology. Tat likely contributes to HAND by causing molecular changes that disrupt synaptic organization, with inhibitory presynaptic terminals containing synaptotagmin 2 appearing especially vulnerable.
HIV encephalitis (HIVE) is accompanied by brain inflammation, leukocyte infiltration, and glial activation, and HIV patients who abuse opiates are more likely to develop HIVE. To better understand how opiates could alter HIV-related brain inflammation, the expression of astrocyte (GFAP immunoreactivity) and macrophage/microglial (F4/80 or Mac1 immunoreactivity) markers in the striatum, and the percentage of 3-nitrotyrosine (3-NT) positive macrophages/microglia, was determined following a 2 day exposure to morphine (5 mg/kg/day via time-release, subcutaneous implant) and doxycycline in GFAP-driven, doxycycline-inducible HIV-1 Tat transgenic mice. Data show that both morphine and Tat induction via doxycycline increased astrocyte activation, with significant additive increases achieved with combined morphine and doxycycline exposure. By contrast, combined Tat induction and morphine exposure, but neither manipulation alone, significantly increased the proportion of macrophages/microglia present in the striatum of transgenic mice, although morphine exposure was necessary to elevate 3-NT co-detection in Mac1-positive macrophages/microglia. Finally, Tat induction increased the percentage of neurons expressing active caspase-3, and this was even more significantly elevated by co-administration of morphine. In spite of elevations in caspase-3, neuronal TUNEL reactivity was unchanged in all groups, even after 10 days of Tat induction. Importantly, co-administration of naltrexone completely antagonized the effects of morphine. These findings indicate that morphine rapidly and significantly increases the activation of astrocytes and macrophages/microglia in the brains of inducible Tat transgenic mice, supporting the theory that early inflammatory changes in glia could underlie the development of HIVE in opiate-abusing AIDS patients.
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