Cannabinoids and morphine differentially affect HIV-1 expression in CD4(+) lymphocyte and microglial cell cultures.

The influence of substances of abuse on the progression of HIV-1 infection is controversial, and pharmacologic factors have been postulated as a potential explanation for conflicting data arising from epidemiological studies and animal models. In the present study, cell culture models of HIV-1 infection were used to test this hypothesis. The synthetic cannabinoid WIN 55,212-2 was found to potently inhibit HIV-1 expression in a concentration- and time-dependent manner in CD4(+) lymphocyte and microglial cell cultures. In sharp contrast, morphine either inhibited or stimulated viral expression, depending upon the time of drug exposure, and marked differences were observed between CD4(+) and microglial cells. Also, WIN 55,212-2 inhibited the stimulatory effect of morphine in HIV-1 infected CD4(+) cells. These in vitro findings support the notion that pharmacologic factors need to be considered in epidemiological studies and animal models that pertain to HIV-1 infection.

Diazepam inhibits HIV-1 Tat-induced migration of human microglia.

During HIV-1 encephalitis, the chemotaxis-inducing activity of Tat may enhance the viral life cycle through recruitment of additional susceptible microglial cells to foci of infection. Benzodiazepines (BDZs) readily penetrate the blood-brain barrier and are known to possess anti-inflammatory properties. Pretreatment of human microglial cells with peripheral (Ro5-4864) and mixed (diazepam), but not central (clonazepam), benzodiazepine receptor ligands was found to potently suppress HIV-1 Tat-induced chemotaxis. Application of Tat to microglial cells evokes an increase in intracellular calcium concentration ([Ca(2+)]i) that rapidly desensitizes the cells. Diazepam's inhibitory effect was associated with its ability to block Tat-induced [Ca(2+)]i mobilization. These data support the notion that through their effects on microglia, peripheral BDZ receptor ligands could alter the neuropathogenesis of HIV-1.

Naltrexone potentiates anti-HIV-1 activity of antiretroviral drugs in CD4+ lymphocyte cultures.

CD4(+) T lymphocytes are the primary cell target for human immunodeficiency virus-1 (HIV-1), and these cells are known to express opioid receptors. Due to the need for new treatment approaches to HIV-1 infection, we sought to determine whether the non-selective opioid receptor antagonist naltrexone would affect HIV-1 expression in CD4(+) lymphocyte cultures and whether naltrexone would alter the antiviral properties of zidovudine (AZT) or indinavir. Activated CD4(+) lymphocytes were infected with a monocytotropic or T-cell tropic HIV-1 isolate, and p24 antigen levels were measured in supernatants of drug-treated or untreated (control) cultures. While naltrexone alone did not affect HIV-1 expression, at a concentration of 10(-12)-10(-10) M naltrexone increased the antiviral activity of AZT and indinavir 2-3-fold. Similar findings with a kappa-opioid receptor (KOR) selective antagonist supported the possible involvement of KOR in naltrexone's potentiation of the antiretroviral drugs. The results of this in vitro study suggest that treatment of alcohol or opiate dependent HIV-1-infected patients with naltrexone is unlikely to interfere with the activity of antiretroviral drugs. Also, based upon naltrexone's safety profile and its synergistic activity in vitro, these findings suggest clinical trials should be considered of naltrexone as an adjunctive therapy of HIV-1 infection.

Cytomegalovirus induces cytokine and chemokine production differentially in microglia and astrocytes: antiviral implications.

Glial cells function as sensors for infection within the brain and produce cytokines to limit viral replication and spread. We examined both cytokine (TNF-alpha, IL-1beta, and IL-6) and chemokine (MCP-1, MIP-1alpha, RANTES, and IL-8) production by primary human glial cells in response to cytomegalovirus (CMV). Although CMV-infected astrocytes did not produce antiviral cytokines, they generated significant quantities of the chemokines MCP-1 and IL-8 in response to viral infection. On the other hand, supernatants from CMV-stimulated purified microglial cell cultures showed a marked increase in the production of TNF-alpha and IL-6, as well as chemokines. Supernatants from CMV-infected astrocyte cultures induced the migration of microglia towards chemotactic signals generated from infected astrocytes. Antibodies to MCP-1, but not to MIP-1alpha, RANTES, or IL-8, inhibited this migratory activity. These findings suggest that infected astrocytes may use MCP-1 to recruit antiviral cytokine-producing microglial cells to foci of infection. To test this hypothesis, cocultures of astrocytes and microglial cells were infected with CMV. Viral gene expression in these cocultures was 60% lower than in CMV infected purified astrocyte cultures lacking microglia. These results support the hypothesis that microglia play an important antiviral role in defense of the brain against CMV. The host defense function of microglial cells may be directed in part by chemokines, such as MCP-1, produced by infected astrocytes.

Robust expression of TNF-alpha, IL-1beta, RANTES, and IP-10 by human microglial cells during nonproductive infection with herpes simplex virus.

Cytokine (TNF-alpha/beta, IL-1beta, IL-6, IL-18, IL-10, and IFN-alpha/beta/gamma) and chemokine (IL-8, IP-10, MCP-1, MIP-1alpha/beta, and RANTES) production during herpes simplex virus (HSV) 1 infection of human brain cells was examined. Primary astrocytes as well as neurons were found to support HSV replication, but neither of these fully permissive cell types produced cytokines or chemokines in response to HSV. In contrast, microglia did not support extensive viral replication; however, ICP4 was detected by immunochemical staining, demonstrating these cells were infected. Late viral protein (nucleocapsid antigen) was detected in <10% of infected microglial cells. Microglia responded to nonpermissive viral infection by producing considerable amounts of TNF-alpha, IL-1beta, IP-10, and RANTES, together with smaller amounts of IL-6, IL-8, and MIP-1alpha as detected by RPA and ELISA. Surprisingly, no interferons (alpha, beta, or gamma) were detected in response to viral infection. Pretreatment of fully permissive astrocytes with TNF-alpha prior to infection with HSV was found to dramatically inhibit replication, resulting in a 14-fold reduction of viral titer. In contrast, pretreatment of astrocytes with IL-1beta had little effect on viral replication. When added to neuronal cultures, exogenous TNF-alpha or IL-1beta did not suppress subsequent HSV replication. Exogenously added IP-10 inhibited HSV replication in neurons (with a 32-fold reduction in viral titer), however, similar IP-10 treatment did not affect viral replication in astrocytes. These results suggest that IP-10 possesses direct antiviral activity in neurons and support a role for microglia in both antiviral defense of the brain as well as amplification of immune responses during neuroinflammation.

Kappa-opioid receptor agonist suppression of HIV-1 expression in CD4+ lymphocytes.

Synthetic kappa-opioid receptor (KOR) agonists have been shown to suppress HIV-1 expression in acutely infected macrophages. In the present study, we examined the effects of the KOR ligand trans-3,4-dichloro-N-methyl-N[2-(1-pyrolidinyl)cyclohexyl]benzeneaceamide methanesulfonate (U50,488) on HIV-1 expression in CD4+ lymphocytes, the main target cell of this virus. When U50,488 was added to activated CD4+ lymphocytes, HIV-1 expression was inhibited in a concentration- and time-dependent manner with maximal suppression (approximately 60%) at 10(-7) M U50,488. The KOR selective antagonist nor-binaltorphimine (nor-BNI) had no effect by itself on viral expression but blocked the antiviral property of U50,488, suggesting that U50,488 was acting via a KOR-related mechanism. Support for the involvement of KOR was provided by the findings that 34% of activated CD4+ lymphocytes were positive for KOR, using an immunofluorescence technique, and that seven additional synthetic KOR ligands also inhibited HIV-1 expression. The results of this study broaden understanding of the antiviral properties of KOR ligands to include cells outside of the nervous system and suggest a potential role for these agents in the treatment of HIV-1 infection.

Effect of thalidomide on chemokine production by human microglia.

Thalidomide, a psychoactive drug that readily crosses the blood-brain barrier, has been shown to possess immunomodulatory attributes, including the inhibition of cytokine production by monocytes and microglia. In this study, we investigated the effect of thalidomide on chemokine production by human microglial cells. Microglial cells were stimulated with lipopolysaccharide, a key cell-wall component of gram-negative bacteria responsible for meningitis, and production of chemokines (regulated upon activation normally T cell expressed and secreted [RANTES], monocyte chemoattractant protein [MCP]-1, macrophage inflammatory protein [MIP]-1beta, and interleukin [IL]-8) was examined by ELISA. Thalidomide treatment was found to cause potent and selective inhibition of IL-8 production in a dose-responsive manner. This inhibition was associated with decreased intracellular IL-8 staining as well as reduced transcription of IL-8 mRNA. In addition, thalidomide treatment of lipopolysaccharide-stimulated microglia inhibited the activation of protein NF-kappaB, a transcription factor known to be important for IL-8 production. These results suggest thalidomide could have a therapeutic role in acute bacterial meningitis through inhibition of IL-8-mediated neutrophil chemotaxis.

CD4(+) lymphocyte-mediated suppression of cytomegalovirus expression in human astrocytes.

Cytomegalovirus-stimulated CD4(+) lymphocytes from seropositive but not seronegative donors suppressed viral gene expression in primary human astrocytes. This suppressive activity was mediated through soluble factors. These findings suggest that CD4(+) lymphocytes play a role in defense of the brain against cytomegalovirus.

U50,488 protection against HIV-1-related neurotoxicity: involvement of quinolinic acid suppression.

The pathogenesis of human immunodeficiency virus type 1 (HIV-1) encephalopathy has been associated with multiple factors including the neurotoxin quinolinate (an endogenous N-methyl-D-aspartate [NMDA] receptor ligand) and viral proteins. The kappa opioid receptor (KOR) agonist U50,488 recently has been shown to inhibit HIV-1 p24 antigen production in acutely infected microglial cell cultures. Using primary human brain cell cultures in the present study, we found that U50,488 also suppressed in a dose-dependent manner the neurotoxicity mediated by supernatants derived from HIV-1-infected microglia. This neuroprotective effect of U50,488 was blocked by the KOR selective antagonist nor-binaltorphimine. The neurotoxic activity of the supernatants from HIV-1-infected microglia was blocked by the NMDA receptor antagonists 2-amino-5-phosphonovalerate and MK-801. HIV-1 infection of microglial cell cultures induced the release of quinolinate, and U50,488 dose-dependently suppressed quinolinate release by infected microglial cell cultures with a corresponding inhibition of HIV-1 p24 antigen levels. These findings suggest that the kappa opioid ligand U50,488 may have therapeutic potential in HIV-1 encephalopathy by attenuating microglial cell production of the neurotoxin quinolinate and viral proteins.

Decreased cytomegalovirus expression following proinflammatory cytokine treatment of primary human astrocytes.

Understanding the influence of immune effector mechanisms on CMV infection of the CNS may facilitate the development of immunotherapies for viral encephalitis. Using cultures of highly purified, fully permissive primary human astrocytes, proinflammatory cytokines, but not antiinflammatory cytokines or beta-chemokines, were found to inhibit CMV expression, DNA synthesis, and replication. Treatment with certain proinflammatory cytokines 24 h before CMV infection markedly suppressed viral expression in astrocytes. TNF-alpha, IL-1beta, and IFN-gamma all inhibited CMV expression (70 +/- 4.2%, 65 +/- 3.4%, and 82 +/- 3.6% inhibition of viral expression, respectively, n = 5). In contrast, no viral suppression was observed following IL-6 treatment. Suppressive activity was dependent on the addition of cytokines before CMV infection. Cytokine pretreatment did not affect CMV entry into primary astrocytes, and the observed cytokine-induced suppressive activity was not affected by the NO synthase inhibitor NG-monomethyl- -arginine (NGMA). Instead, the suppressive effect appeared to be mediated through a mechanism involving inhibition of CMV major immediate early promoter activity. These results support the hypothesis that proinflammatory cytokines possess anti-CMV activity in brain cells and may lead to new interventions for CMV encephalitis based upon immunotherapy.

Human cytomegalovirus replication and modulation of apoptosis in astrocytes.

OBJECTIVES: To characterize replication patterns and cytopathic effects during human cytomegalovirus (HCMV) infection of brain cells. DESIGN: Primary human mixed glial/neuronal cells, as well as purified microglial, astroglial, and enriched neuronal cell cultures, were infected with HCMV strains AD169 and RC256 to determine the ability of the different brain cell types to support viral replication. RESULTS: Mixed glial/neuronal cell cultures were fully permissive for viral replication. Based on previous studies, we hypothesized that human microglial cells would preferentially support productive HCMV replication. However, HCMV did not replicate or display genomic expression in microglial cells. In contrast, primary astrocytes were fully permissive and displayed HCMV-induced cytopathic effects resulting in cell death. In highly enriched neuronal cultures, productive infection and viral expression occurred only in scattered astrocytes. Early in the infection, apoptotic plasma membrane changes were induced in astrocytes. However, nuclear fragmentation was not apparent until later during the course of infection. CONCLUSIONS: These results suggest that HCMV possesses astrocytotropic properties that confer preferential expression and cytopathic replication in astrocytes over microglia or neuronal cells. Apoptotic cell death, which is a result of HCMV infection, appears to be delayed until peak viral replication has occurred.

Benzodiazepines, glia, and HIV-1 neuropathogenesis.

Although the precise mechanisms whereby HIV-1 infection induces neurodegeneration have yet to be determined, a great deal of evidence has incriminated glial cells and the production of proinflammatory mediators in this pathologic process. For this reason, ideal therapeutic agents for the treatment of AIDS dementia would attenuate HIV-1 neuropathogenesis through both direct inhibition of viral expression and suppression of brain cell-produced immune mediators. Benzodiazepines (BDZs), such as Valium, are extensively prescribed drugs for anxiety disorders, which readily cross the blood-brain barrier and have demonstrated immunomodulatory properties. BDZs bind to primary human microglial cells, the principal site of HIV-1 replication in the brain, and inhibit lipopolysaccharide (LPS) induced tumour necrosis factor (TNF-alpha) production by these cells in a concentration-dependent manner. Treatment of HIV-1-infected primary human microglial, as well as mixed glial/neuronal, cell cultures with BDZs inhibits the expression of HIV-1 p24 antigen. BDZ-induced inhibition of HIV-1 expression in chronically infected promonocytic (U1) cells has been found to be associated with decreased activation of the nuclear transcription factor kappa B (NF-kappa B). Because HIV-1 expression is critically dependent on the cellular transcription machinery, inhibition of the activation of transcription factors, which participate in both HIV-1 expression and the production of neurotoxic immune mediators, by BDZ analogs may provide new therapeutic options for AIDS dementia.

Diazepam-mediated inhibition of human immunodeficiency virus type 1 expression in human brain cells.

Treatment of acutely infected human brain cell and enriched microglial cell cultures with diazepam inhibited human immunodeficiency virus type 1 (HIV-1) p24 antigen expression. Similarly, diazepam suppressed HIV-1 expression in chronically infected promonocytic (U1) cells and acutely infected monocyte-derived macrophages, and this antiviral activity was associated with decreased activation of nuclear factor kappa B.

IL-1-induced iNOS expression in human astrocytes via NF-kappa B.

Nitric oxide (NO) plays an important role in host defense as well as cell injury within the CNS. In contrast to rodent species, human astrocytes are the major glial source of NO. Although interleukin (IL)-1 stimulates astrocyte inducible NO synthase (iNOS) expression, the mechanism is poorly defined. In the present study using primary human fetal astrocyte cultures, we found that IL-1 beta stimulated activation of nuclear factor kappa B (NF-kappa B) within 2 h, iNOS mRNA expression at 8 h, and maximal NO production by 5 days post-treatment. This IL-1-induced activation of astrocyte iNOS was suppressed by pyrrolidine dithiocarbamate, an inhibitor of NF-kappa B activation, suggesting involvement of a NF-kappa B mechanism.

The latency-associated promoter of herpes simplex virus type 1 requires a region downstream of the transcription start site for long-term expression during latency.

The latency-associated transcript (LAT) promoter of herpes simplex virus type 1 (HSV-1) is unique among the many promoters on the viral genome in that it remains active during the latent state. We have previously shown that a DNA fragment comprising the LAT promoter element through the cap site, when moved from the LAT locus to the glycoprotein C gene, is capable of only short-term expression. These and other data suggested that an HSV DNA element from the repeat region, not included in the LAT promoter itself, might be needed to preserve long-term expression. Based on a number of recombinant viruses, we narrowed our search for this putative element to a region 3' of the LAT transcription start site. In the present study, we have shown that a 1.1-kb DNA fragment containing the putative long-term expression element (LTE) is able to restore latent-phase gene expression to the LAT promoter. The element appeared to function best when it was placed in its natural location, which is 3' of the LAT promoter; however, partial function was obtained when the LTE was inserted upstream of the LAT promoter in the reverse direction. These data indicate that the LAT promoter region is more complex than originally anticipated and that in addition to requiring both core promoter and neuronal transcription factor binding sites, the promoter requires a specific region of DNA to prevent its shutoff during a latent infection.

Proinflammatory cytokines inhibit HIV-1(SF162) expression in acutely infected human brain cell cultures.

An understanding of how viral replication in glial cells responds to proinflammatory cytokines is important in delineating HIV-1 neuropathogenesis. Because no information is available in the literature regarding the regulatory effects of exogenous cytokines on acute HIV-1 replication in human brain cells, we studied the impact of cytokine treatment on viral p24 Ag expression. Based upon reports using mononuclear phagocytes derived from somatic sources, we hypothesized that TNF-alpha, IL-1 beta, and IL-6 would up-regulate the expression of HIV-1(SF162) (a monocytotropic strain) in purified microglial cells and in mixed brain cell cultures. This hypothesis was not supported. In fact, a contrary, unexpected result was obtained; whereas in purified microglial cultures TNF-alpha displayed a mild stimulatory effect on HIV-1 expression (15% increase in p24 Ag production compared with control cultures), surprisingly, IL-1 beta and IL-6 were highly suppressive (91 and 83% inhibition of HIV expression, respectively). In contrast to the findings in microglial cell cultures, TNF-alpha profoundly suppressed (84%) HIV-1 expression in mixed brain cell cultures, as did IL-1 beta (82%), and IL-6 was moderately suppressive (55% inhibition). In an attempt to identify factors responsible for the differential effects of TNF-alpha in the two brain cell infection models, it was found that compared with microglial cell cultures, TNF-alpha treatment of mixed brain cell cultures released significantly greater amounts of RANTES (regulated upon activation, normal T cell expressed and secreted) and macrophage inflammatory protein-1 alpha, beta-chemokines that have been suggested to have anti-HIV-1 effects. Thus, these data suggest that proinflammatory cytokines possess anti-HIV-1 activity in the central nervous system.

Long-term promoter activity during herpes simplex virus latency.

The ability to direct foreign gene expression from the herpes simplex virus type 1 (HSV-1) genome during an acute or latent infection is a subject of increasing importance in the utilization of HSV vectors for gene therapy. Little is known about the types of transcription factors present in neurons or about whether different neuronal populations within a ganglion vary in their complement of these factors. With respect to HSV-1 latency, it is not known how or why the latency-associated transcript (LAT) promoter is able to function continually during latency while all other viral promoters are inactive. To further studies of these two phenomena, we constructed seven recombinant viruses with various promoter constructs driving expression of the lacZ reporter gene. Each construct was inserted into HSV-1 at the glycoprotein C locus, and recombinant viruses were evaluated for the ability to express beta-galactosidase during acute and latent viral infections in murine dorsal root ganglia. During acute infection of murine dorsal root ganglia, the activities of the promoters varied over a wide range. Constructs containing the murine metallothionein promoter (MT1), the phosphoglycerate kinase promoter, the Moloney murine leukemia virus long terminal repeat (LTR), or the region upstream of and including the HSV LAT core promoter (LAT) were active during the acute but not the latent phase of infection. The addition of transcription factor binding sites present in the upstream LAT region to the MT1 and LTR promoters (LAT-MT1 and LAT-LTR, respectively) significantly increased acute-phase expression. Despite these high initial rates of transcription, of all the promoter constructs only LAT-LTR was able to remain transcriptionally active after the establishment of a latent state. Thus, the Moloney murine leukemia virus LTR provides a DNA element which functions to prevent promoter inactivation during latency. An analogous HSV long-term-expression element is evidently not present in the upstream LAT promoter, indicating that the HSV long-term-expression function is provided by a region outside of that which gives high-level neuronal expression during the acute phase of infection.