Morphine and HIV-Tat increase microglial-free radical production and oxidative stress: possible role in cytokine regulation.

Opiate abuse alters the progression of human immunodeficiency virus and may increase the risk of neuroAIDS. As neuroAIDS is associated with altered microglial reactivity, the combined effects of human immunodeficiency virus-Tat and morphine were determined in cultured microglia. Specifically, experiments determined the effects of Tat and morphine on microglial-free radical production and oxidative stress, and on cytokine release. Data show that combined Tat and morphine cause early and synergistic increases in reactive oxygen species, with concomitant increases in protein oxidation. Furthermore, combined Tat and morphine, but not Tat or morphine alone, cause reversible decreases in proteasome activity. The effects of morphine on free radical production and oxidative stress are prevented by pre-treatment with naloxone, illustrating the important role of opioid receptor activation in these phenomena. While Tat is well known to induce cytokine release from cultured microglia, morphine decreases Tat-induced release of the cytokines tumor necrosis factor-alpha and interleukin-6, as well as the chemokine monocyte chemoattractant protein-1 (MCP-1). Finally, experiments using the reversible proteasome inhibitor MG115 show that temporary, non-cytotoxic decreases in proteasome activity increase protein oxidation and decrease tumor necrosis factor-alpha, interleukin-6, and MCP-1 release from microglia. Taken together, these data suggest that oxidative stress and proteasome inhibition may be involved in the immunomodulatory properties of opioid receptor activation in microglia.

Cell-specific actions of HIV-Tat and morphine on opioid receptor expression in glia.

HIV-1 patients who abuse opiate-based drugs, including heroin and morphine, are at a higher risk of developing HIV dementia. The effects of opiates are mediated predominantly through opioid receptors, which are expressed on glial cells. As HIV-1 infection in the CNS is restricted to glial cells, experiments were designed to measure the cell-specific effects of HIV Tat and morphine exposure on opioid receptor expression in both astrocytes and microglia. Specifically, the cell-type-specific pattern of mu opioid receptor (MOR), delta opioid receptor (DOR), and kappa opioid receptor (KOR) localization (surface vs. intracellular) and expression of opioid receptor mRNA were determined after exposure to morphine in the presence and the absence of Tat in primary cultured microglia and astrocytes. Data show that morphine treatment caused significantly decreased cell surface expression of opioid receptors in microglia but not in astrocytes. However, morphine treatment in the presence of Tat significantly increased intracellular expression of opioid receptors and prevented morphine-induced cell surface opioid receptor down-regulation in microglia. These findings document that cell surface opioid receptor expression is divergently regulated by morphine in microglia compared with in astrocytes, and further suggest that HIV-Tat could exacerbate opioid receptor signaling in microglia by increasing receptor expression and/or altering ligand-induced trafficking of opioid receptors.

Gender and estrogen manipulation do not affect traumatic brain injury in mice.

As epidemiological data have suggested that female patients may have improved clinical prognoses following traumatic brain injury (TBI) compared to males, we designed experiments to determine the role of gender and estrogen in TBI-induced brain injury and inflammation in rodents. To this end, male and female C57Bl/6 mice were separated into the following four groups: intact males, intact females with vehicle supplementation, ovariectomized females with vehicle supplementation, and ovariectomized females with estrogen supplementation. All mice were subjected to a controlled cortical impact model of TBI, and cortical injury, hippocampal degeneration, microglial activation, and brain cytokine expression were analyzed after injury. Additionally, the spleens were harvested and cytokine release from cultured splenic cells was measured in response to specific stimuli. Data indicate that TBI-induced cortical and hippocampal injury, as well as injury-related microglial activation were not significantly affected by gender or estrogen manipulation. Conversely, brain levels of MCP-1 and IL-6 were significantly increased in males and intact females following TBI, but not in female mice that had been ovariectomized and supplemented with either estrogen or vehicle. Evaluation of splenic responses showed that the spleen was only moderately affected by TBI, and furthermore that spleens isolated from mice that had been given estrogen supplementation showed significantly higher release of the anti-inflammatory cytokine IL-4, regardless of the presence of absence of TBI. Overall, these data indicate that while estrogen can modulate immune responses, and indeed can predispose splenic responses towards and anti-inflammatory phenotype, these effects do not translate to decreased brain injury or inflammation following TBI in mice.

SOD1 overexpression alters ROS production and reduces neurotoxic inflammatory signaling in microglial cells.

Activation of the oxidative burst is one of the earliest biochemical events in microglial activation, but it is not understood yet how free radicals participate in inflammatory signaling. To determine the role that specific reactive oxygen species play in microglial activation, the levels of SOD1 were manipulated in N9 murine microglia. Stable overexpression of SOD1 caused significant decreases in superoxide and nitric oxide production, with concurrent increases in hydrogen peroxide following LPS. However, LPS-induced activation of NFkappaB, and release of TNFalpha and IL-6 were significantly attenuated in SOD1 overexpressing cells, as was the ability of microglia to induce toxicity in cultured neurons. Conversely, acute inhibition of SOD1 with disulfiram was associated with increased nitric oxide and cytokine release, and increased neurotoxicity. Together, these data suggest that superoxide radicals in microglia play important roles in directing redox-sensitive inflammatory signaling and initiating neurotoxic inflammation.

Immune modulation by estrogens: role in CNS HIV-1 infection.

Experimental and epidemiological data suggest that estrogen can be protective in both brain injury and infection. While estrogens can act directly on neurons to promote neuronal survival, estrogen also has antiinflammatory properties that may contribute to overall neuroprotection. Accordingly, estrogens may have particular relevance in chronic neuroimmune disorders such as HIV dementia. As AIDS is now a leading cause of death among women in their reproductive years, understanding the role that female sex hormones might play in the physiology of HIV-1 infection is especially critical. Indeed, there is accumulating evidence that many manifestations of HIV differ in women. For instance, it is now well established that women present with a lower viral titer at the time of seroconversion, have lower HIV viral loads compared to men at similar stages of disease, and may have altered disease progression during pregnancy. Conversely, while epidemiological studies suggest that women may be more vulnerable to certain late-stage AIDS-related illnesses including HIV dementia, there is accumulating data that strongly suggest an estrogen-deficient state is associated with long-term HIV infection in some women. Evaluated as a whole, existing evidence indicates that estrogen can directly protect neurons from damage, can modulate brain inflammation, and could act to maintain low titers of the HIV-1 virus. Accordingly, it can be hypothesized that maintenance of serum estradiol levels could decrease the incidence of HIV dementia and other AIDS-related neurological syndromes in HIV-1 positive women. In this article, we both summarize current understanding and present new data related to the potential mechanisms whereby estrogen could modulate the mechanics and the consequences of HIV-1 infection in the brain and thereby thwart the development of HIV dementia.

Estrogen and brain inflammation: effects on microglial expression of MHC, costimulatory molecules and cytokines.

To model the effects of estrogen on adaptive immunity in the brain, we examined the effects of 17beta-estradiol on microglial parameters related to antigen presentation and T cell activation. Specifically, the effects of 17beta-estradiol on basal and LPS-induced surface staining of Class I and II MHC, as well as CD40, CD80, CD86, CD152, CD28, CD8, CD11b, Fas, FasL, and also ERalpha and ERbeta, were examined in N9 microglial cells. Additionally, the effects of 17beta-estradiol on basal and LPS-induced release of cytokines (TNF-alpha, IFN-gamma, IL-2, IL-4, and IL-10) were determined. Data indicate that estrogen increases IL-10 while decreasing TNFalpha and IFNgamma release from resting and LPS-stimulated N9 cells. Additionally, LPS-induced surface staining of MHC Class I, CD40, and CD86 was significantly attenuated by estrogen pretreatment. The basal percentage of cells positive for MHC Class I and II, CD40, and CD152, Fas, and FasL was significantly decreased by estrogen exposure. However, CD8, CD86, CD11b, and CD28 were unaffected by estrogen, and CD80 cell surface staining significantly increased following estrogen exposure. Taken together, these data indicate that estrogen can significantly decrease components of adaptive immunity in microglial cells, and highlight the multi-faceted regulatory effects of estrogen on microglial parameters related to antigen presentation and T cell interaction.

Estrogen increases proteasome activity in murine microglial cells.

During inflammation, microglial cells go through phenotypic and functional changes that include the production and release of large amounts of oxygen and nitrogen radicals. As such, activated microglia are subject to heightened oxidative stress. The multicatalytic proteasome clears oxidized and damaged proteins from cells, and has been shown to be an important aspect of the microglial compensatory response to activation. The female sex steroid estrogen is both cytoprotective and anti-inflammatory, and has been shown to affect microglial signaling in particular. To determine if estrogen might affect the proteasome in microglial cells, we examined the effects of 17 beta-estradiol treatment on proteasome activity in N9 microglial cells. Specifically, we measured ATP-dependent and ATP-independent chymotrypsin-like, trypsin-like, and peptidyl glutamyl peptide hydrolase (PGPH)-like activities in response to both 17 beta-estradiol and interferon gamma. Data indicate that estrogen, but not interferon gamma, significantly increases ATP-dependent chymotrypsin-like and PGPH-like activity. Furthermore, this effect was blocked by the p44/42 MAPK inhibitor PD98059. Hence, these data demonstrate that through the MAPK pathway, estrogen can upregulate proteasome activity, suggesting a possible mechanism for estrogen's cytoprotective effects.

Synaptic transport of human immunodeficiency virus-Tat protein causes neurotoxicity and gliosis in rat brain.

Neurodegeneration, synaptic alterations, and gliosis are prominent features of human immunodeficiency virus (HIV) encephalitis, but HIV encephalitis is distinct from other viral encephalitides because neurodegeneration occurs in uninfected neurons at anatomical sites that are often distant from the site of viral replication. The HIV protein Tat is both neurotoxic and proinflammatory; however, its contribution to HIV-related synaptic dysfunction remains unknown. To determine the consequences of continuous Tat production in brain, we genetically engineered rat C6 glioma cells to stably produce Tat and stereotaxically infused these cells into the rat striatum or hippocampus. We discovered that HIV-Tat protein could be transported along anatomical pathways from the dentate gyrus to the CA3/4 region and from the striatum to the substantia nigra, resulting in behavioral abnormalities, neurotoxicity, and reactive gliosis. This demonstrates a unique neuronal transport property of a viral protein and establishes a mechanism for neuroglial dysfunction at sites distant from that of viral replication. Tat may thus be an important participant in brain dysfunction in HIV dementia.

The neuregulin GGF2 attenuates free radical release from activated microglial cells.

The neuregulin glial growth factor 2 (GGF2) is a neural growth factor that is best known for its ability to promote the survival and proliferation of oligodendrocytes and Schwann cells. While it has been shown in recent years that GGF2 is effective in the treatment of autoimmune models of brain injury, it is not known if the beneficial effects of GGF2 are based in part on modulation of brain inflammation. In this report, we document the anti-inflammatory effects of recombinant human GGF2 (rhGGF2) on microglial free radical production in vitro. The presence of the neuregulin receptors ErbB2, 3, and 4 was confirmed in N9 microglial cells by Western blot analysis. Pretreatment of N9 cells with 10-100 ng/ml rhGGF2 24 h before either phorbol 12-myristate 3-acetate (PMA) or interferon gamma (IFNgamma) caused dose-dependent decreases in oxidative burst activity and nitrite release, respectively, with 50 and 100 ng/ml causing significant effects. When cells were co-treated with increasing doses of rhGGF2 and PMA or IFNgamma, only concentrations of 50 ng/ml, but not 10 or 100 ng/ml, were able to decrease oxidative burst activity and nitrite release. Finally, when microglial cell viability following treatment of cells with IFNgamma with or without rhGGF2 was evaluated, it was observed that 50 and 100 ng/ml rhGGF2 conferred significant protection against IFNgamma-induced cell death in microglial cells. Overall, these results indicate that the neuregulin rhGGF2 may have anti-inflammatory and antioxidant properties in the brain, and may also provide trophic support for brain-resident microglial cells.