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.

U50488 inhibits HIV-1 expression in acutely infected monocyte-derived macrophages.

Opioids may play an immunomodulatory role in the pathogenesis of human immunodeficiency virus-1 (HIV-1) infection. Recently, synthetic kappa-opioid receptor (KOR) ligands have been found to have anti-human immunodeficiency virus type 1 activity in acutely infected brain macrophages. In the present study, we investigated whether the selective KOR ligand U50488 would exert such an anti-HIV-1 effect in acutely infected blood monocyte-derived macrophages (MDM). Treatment of acutely infected MDM with U50488 induced a concentration-dependent inhibition of HIV-1 expression. The dose--response relationship of U50488 was U-shaped with a peak effect observed at 10(-13) M, which was evident at both 7 and 14 days post-infection. The KOR antagonist nor-binaltorphimine blocked the anti-HIV-1 effect of U50488 by 73%, indicating involvement of a KOR-mediated mechanism. Also, expression of KOR mRNA and binding activity with a fluorescence-labeled KOR ligand supported the existence of KOR on MDM. Antibodies to the beta-chemokine, RANTES (regulated on activation normal T-cell expressed and secreted), but not to various other cytokines, blocked U50488 inhibition by 56% suggesting that the anti-HIV-1 effect of U50488 involved, in part, the production of RANTES by MDM. Taken together, these in vitro findings support the anti-HIV-1 property of U50488, and suggest that KOR ligands may have therapeutic potential for treating patients with acquired immunodeficiency syndrome.

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.

Kappa-opioid potentiation of tumor necrosis factor-alpha-induced anti-HIV-1 activity in acutely infected human brain cell cultures.

Opioids have been postulated to play an immunomodulatory role in the pathogenesis of HIV-1. Synthetic kappa-opioid receptor (KOR) ligands have been found to inhibit HIV-1 expression in acutely infected microglial cell cultures. We recently found that interleukin(IL)-1beta and tumor necrosis factor(TNF)-alpha have antiviral effects in acutely infected mixed glial/neuronal cell cultures. In the present study, we investigated whether selective KOR ligands would exert antiviral effects in acutely infected brain cell cultures. While the KOR ligand trans-3,4-dichloro-N-methyl-N[2-(1-pyrolidinyl)cyclohexyl]benze neaceamide methanesulfonate (U50,488) alone had little anti-HIV-1 activity, this opioid potentiated in a concentration-dependent manner the antiviral activity of TNF-alpha, but not of IL-1beta. The potentiating effect of U50,488 was detected after a 6-hr pretreatment and peaked at 24 hr. The KOR antagonist nor-binaltorphimine completely blocked the potentiating effect of U50,488, suggesting the involvement of a KOR-mediated mechanism. Antibodies to TNF-alpha completely blocked the potentiating effect of U50,488, suggesting a critical role for TNF-alpha. Antibodies to IL-1beta blocked the potentiating effect of U50,488, suggesting that IL-1beta was released following U50,488 treatment, which might contribute to the potentiating effect of U50,488. These in vitro findings support the notion that synthetic kappa-opioids could be considered as potential adjunctive therapeutic agents in HIV-1-related brain disease.

Effect of cytokines on anticryptococcal activity of human microglial cells.

The effect of selected cytokines on the antifungal activity of human microglia was studied with encapsulated and acapsular strains of Cryptococcus neoformans. None of the cytokines tested increased the fungistatic activity of microglia, suggesting that killing of cryptococci within the central nervous system is dependent on other host defense mechanisms.

Renal cell cytokine production stimulates HIV-1 expression in chronically HIV-1-infected monocytes.

Renal infiltration of human immunodeficiency virus type 1 (HIV-1)-infected monocytes might play an important role in the development of HIV-associated nephropathy (HIVAN). In the present study, we investigated the effects of cytokines produced by cultured human mesangial cells (HMC) and proximal tubular epithelial cells (PTEC) on HIV-1 expression in chronically HIV-1-infected promonocytes (U1 cells). Human mesangial cells constitutively secreted interleukin-6 (IL-6) but not tumor necrosis factor-alpha (TNF-alpha) into the culture medium, whereas PTEC constitutively secreted both IL-6 and TNF-alpha. Coculture of U1 cells with HMC or PTEC for 72 hours markedly stimulated HIV-1 expression, with the p24 antigen concentration in the coculture supernatants ranging from approximately 200 to 1850 pg/ml. The presence of anti-IL-6 antibody in the coculture medium nearly completely blocked HIV-1 expression in the HMC/U1 cell cocultures (P < 0.05). Anti-IL-6 antibody and anti-TNF-alpha antibody blocked HIV-1 expression in the PTEC/U1 cell cocultures by 40% and 53%, respectively (P < 0.05). Moreover, the combination of anti-IL-6 and anti-TNF-alpha antibodies additively reduced coculture HIV-1 expression by 87% (P < 0.05). We conclude that renal cell production of IL-6 and TNF-alpha might provide a potent stimulus for HIV-1 expression in HIV-1-infected monocytes that infiltrate the kidney, and that this may play an important role in the pathogenesis of HIVAN.

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.

Multinucleated giant cell formation of swine microglia induced by Mycobacterium bovis.

Multinucleated giant cells (MGC) have been long recognized as a histopathologic feature of tuberculosis, yet little is known about the underlying mechanism of tubercle bacillus-induced formation of these fused macrophages. The main purpose of this study was to characterize cellular mechanisms involved in MGC formation of swine microglia, the resident macrophages of the brain, in cultures containing nonopsonized Mycobacterium bovis. Within 2 h of incubation, MGC were readily detected in these cultures by light and transmission electron microscopy. MGC formation was blocked by anti-CD14 and anti-CD18 antibodies and by thalidomide, a potent inhibitor of tumor necrosis factor-alpha (TNF-alpha) production my microglia. Also, TNF-alpha alone induced MGC formation. These findings suggest that two microglial cell receptors, CD14 and a beta2 integrin, and the cytokine TNF-alpha participate in M. bovis-induced swine microglial MGC formation.

Cytokine and free radical production by porcine microglia.

Swine have been used increasingly as an animal model for a variety of immunologic purposes. Because the functional activities of porcine microglia, the resident macrophages of the brain, have not been elucidated, highly enriched porcine microglial cell cultures were developed in the present study to assess cytokine and free radical production by these cells compared to microglia of human and murine origin. Porcine microglial cells were found to behave similarly to both human and murine cells in releasing tumor necrosis factor-alpha and interleukin-1 and in generating superoxide anion. In contrast to murine cells, porcine microglial cells, like human cells, failed to generate NO in response to cytokine stimulation. These findings suggest that swine will serve as an excellent model for investigations of central nervous system diseases in which microglia are involved in host defense or neuronal injury.

Upregulation of HIV-1 expression in cocultures of chronically infected promonocytes and human brain cells by dynorphin.

Using cocultures of human fetal brain cells and a chronically human immunodeficiency virus-1 (HIV-1)-infected promonocytic line U1, we investigated the effect of dynorphin, an endogenous opioid peptide found in the CNS, on upregulation of HIV-1 expression. Dynorphin and the synthetic kappa receptor agonist U50,488 promoted HIV-1 expression with a bell-shaped concentration-response relationship in which maximal effects were observed at 10(-13) and 10(-11) M, respectively. Pretreatment for 30 min with the kappa receptor antagonist nor-binaltorphimine completely blocked the stimulatory effect of dynorphin and U50,488. The involvement of cytokines on HIV-1 expression was tested. Dynorphin-induced upregulation of HIV-1 in the cocultures was largely blocked by antibodies to tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 but not by antibodies to IL-10. Also, dynorphin stimulated TNF-alpha and IL-6 in the brain cell cultures at both mRNA and protein levels, suggesting the involvement of these cytokines in opioid-induced HIV-1 expression. These findings suggest that endogenous opioid peptides such as dynorphin may have an immunomodulatory function in the CNS and could act as a cofactor in the neuropathogenesis of HIV-1.

CT-2576, an inhibitor of phospholipid signaling, suppresses constitutive and induced expression of human immunodeficiency virus.

Viruses such as human immunodeficiency virus (HIV) require cellular activation for expression. Cellular activation in lymphoid cells is associated with augmented accumulation of certain phosphatidic acid (PA) species derived from the hydrolysis of glycan phosphatidylinositol (GPI). This suggests that activation of a phospholipid pathway may play a role in initiation of viral replication. To test this hypothesis, we examined the effect of tat gene expression on the production of cellular PA species, as the Tat protein is essential for HIV expression and has been implicated in activating the expression of multiple host cellular genes. Expression of tat increased the expression of PA. We then tested whether synthetic inhibitors of PA metabolism would inhibit activation of the HIV long terminal repeat by Tat and tumor necrosis factor alpha (TNF-alpha). CT-2576 suppressed both PA generation induced by Tat and HIV long terminal repeat-directed gene expression in response to Tat or TNF-alpha at a posttranscriptional step. CT-2576 also inhibited constitutive as well as TNF-alpha- and interleukin 6-induced expression of HIV p24 antigen in chronically infected U1 cells and in peripheral blood lymphocytes acutely infected with a clinical isolate of HIV. Pharmacological inhibition of synthesis of selected PA species may therefore provide a therapeutic approach to suppression of HIV replication.

Endogenous opioid peptides suppress cytokine-mediated upregulation of HIV-1 expression in the chronically infected promonocyte clone U1.

Opioid peptides appear to have an immunomodulatory activity. Using the chronically HIV-1-infected promonocyte clone U1, we investigated the effect of endogenous and synthetic opioid agonists on cytokine-induced HIV-1 expression. None of the endogenous or synthetic opioid agonists had an effect on constitutive HIV-1 expression. Opioid agonists such as methionine-enkephalin, dynorphin, and the kappa receptor agonist, U50,488, dose-dependently suppressed (> 40%) interleukin (IL)-6-induced upregulation of HIV-1 expression. Interestingly, opioid receptor antagonists (mu, delta, and kappa types) also inhibited (> 60%) IL-6-induced upregulation of HIV-1 expression. All opioid agonists and antagonists tested only modestly suppressed (< 20%) tumor necrosis factor-alpha-induced upregulation of HIV-1 expression in U1 cell cultures. These data suggest that certain opioid peptides alter an IL-6-induced signal transduction pathway which triggers HIV-1 expression in the chronically infected promonocyte U1.

Anti-human immunodeficiency virus type 1 activities of U-90152 and U-75875 in human brain cell cultures.

Antiviral activities of the reverse transcriptase inhibitors U-90152 and 3'-azido-2',3'-dideoxythymidine and the protease inhibitor U-75875 were compared in two culture models of human immunodeficiency virus type 1 brain infection. In a model involving acutely infected microglial cells, U-90152 was the most active, whereas in a model using chronically infected promonocytes, U-75875 was the most active.

Priming effect of morphine on the production of tumor necrosis factor-alpha by microglia: implications in respiratory burst activity and human immunodeficiency virus-1 expression.

Opiates alter a variety of functional activities of the somatic immune system; within the central nervous system, however, their effects on immune responses are unknown. In the present study, we investigated the effect of morphine on the release of tumor necrosis factor (TNF)-alpha from murine neonatal microglia. Microglial cell cultures did not release TNF-alpha when incubated with morphine alone; however, an enhanced (P < .01) release of TNF-alpha was observed when cultures were first primed with morphine for 24 h and then stimulated with lipopolysaccharide. A bell-shaped dose-response curve was observed for the priming effect of morphine; maximal enhancement of TNF-alpha release (310 +/- 15% of control) was detected at a concentration of 10(-10) M morphine. Pretreatment of microglia for 30 min with opioid receptor antagonists (i.e. naloxone and beta-funaltrexamine) completely blocked the priming effect of morphine. In addition, morphine treatment amplified (P < .01) the priming effect of lipopolysaccharide on phorbol myristate acetate-triggered superoxide anion production by microglial cell cultures, and this effect was abrogated (P < .01) by anti-TNF-alpha antibody. Furthermore, culture supernatants derived from microglial cell cultures that had been treated with morphine before stimulation with lipopolysaccharide had an increased capacity to upregulate human immunodeficiency virus-1 expression in the latently infected promonocytic clone U1. This effect was also blocked by anti-TNF-alpha antibody. These findings suggest that morphine primes microglia for enhanced production of TNF-alpha which could alter several functional activities of these cells within the brain.

Morphine amplifies HIV-1 expression in chronically infected promonocytes cocultured with human brain cells.

Previous studies have shown that morphine promotes the replication of human immunodeficiency virus (HIV)-1 in peripheral blood mononuclear cell cocultures. In the present study, we tested the hypothesis that morphine would amplify HIV-1 expression in the chronically infected promonocytic clone U1 when cocultured with lipopolysaccharide-stimulated human fetal brain cells. Marked upregulation of HIV-1 expression was observed in these cocultures (quantified by measurement of HIV-1 p24 antigen levels in supernatants), and treatment of brain cells with morphine resulted in a bell-shaped dose-dependent enhancement of viral expression. The mechanism of morphine's amplifying effect appears to be opioid receptor-mediated and to involve enhanced production of tumor necrosis factor-alpha by microglial cells.

Human microglial cell defense against Toxoplasma gondii. The role of cytokines.

Microglia may play an important role in host defense of the central nervous system against Toxoplasma gondii, and cytokines produced by these glial cells may participate in their antitoxoplasma activity. In our study, the antitoxoplasma activity of human fetal microglia was investigated. The RH strain of T. gondii multiplied readily in these glial cells. IFN-gamma/LPS-treated microglia limited (p < 0.01) T. gondii growth by reducing entry of this parasite rather than intracellular multiplication. More than 90% of the antitoxoplasma activity of activated microglia was blocked (p < 0.01) by neutralizing antibodies to TNF-alpha or IL-6 (but not to IL-1 or TGF-beta), suggesting that these proinflammatory cytokines play a role in the inhibitory process. Consistent with this hypothesis, treatment of microglia with TNF-alpha or IL-6 (in the presence or absence of IFN-gamma) inhibited (p < 0.01), in a dose-dependent manner, T. gondii growth. Inasmuch as NGMA did not affect cytokine-mediated antitoxoplasma activity of microglia, nitric oxide appears not to be involved in this host defense function of human fetal microglia. Results of our study suggest that the host defense activity of human microglia against T. gondii is dependent primarily on the activating properties of IFN-gamma, TNF-alpha, and IL-6.

Intracellular survival and multiplication of Toxoplasma gondii in astrocytes.

Primary neonatal murine astrocyte cultures were used to investigate the role of these glial cells in host defense of the central nervous system (CNS) against Toxoplasma gondii. For comparison, neonatal murine microglial cells were also studied. Microscopic analyses revealed that uptake of T. gondii into astrocytes was parasite-driven and was followed by uniform intracellular survival and multiplication of tachyzoites. Treatment of astrocytes with interferon (IFN)-gamma and lipopolysaccharide (LPS) had no apparent effect on the survival or growth of T. gondii. Microglia, on the other hand, had both an intrinsic phagocytosis-associated antitoxoplasma activity and a nitric oxide-dependent inhibitory activity that was up-regulated by IFN-gamma and LPS. The results of this study suggest that in contrast to microglial cells, astrocytes may provide a safe harbor within the CNS for T. gondii.

Activated microglia inhibit multiplication of Toxoplasma gondii via a nitric oxide mechanism.

The role of microglia in host defense against Toxoplasma gondii is unknown. In the present study, we investigated the multiplication of T. gondii tachyzoites in murine microglial cell cultures. T. gondii multiplied readily in these cells; multiplication was prevented when microglia were activated with interferon-gamma plus lipopolysaccharide, a treatment that also upregulates nitric oxide (NO) synthase activity. Simultaneous treatment of microglial cell cultures with activation signals and the NO synthase inhibitor NG-monomethyl-L-arginine (NGMA) prevented the antitoxoplasmic activity. Transmission electron microscopic analysis demonstrated degenerative tachyzoites in activated microglia but not in control or NGMA groups. These findings support the view that the host defense function of activated microglia against T. gondii involves generation of the free radical NO.

Effects of cytokines on multiplication of Toxoplasma gondii in microglial cells.

Microglia may play a role in host defense against Toxoplasma gondii in the central nervous system. In our study, the antitoxoplasma activity of murine microglia was investigated. T. gondii multiplied readily in cultured microglial cells, and activation of these cells with IFN-gamma plus LPS suppressed (p < 0.01) intracellular growth of this parasite. This antitoxoplasma defense of activated microglia was significantly blocked by neutralizing antibodies against TNF-alpha and transforming growth factor-beta, suggesting that these cytokines play a role in the inhibitory process. Consistent with this hypothesis, activation of microglia with IFN-gamma plus TNF-alpha or transforming growth factor-beta inhibited, in a dose-dependent manner, T. gondii multiplication. Treatment with pentoxifylline or dexamethasone, drugs that suppress production of TNF-alpha, prevented (p < 0.01) the microglia from exerting an anti-T. gondii effect. These findings suggest that the host defense function of microglia against T. gondii is dependent primarily on the activating properties of IFN-gamma and TNF-alpha, and that agents that interfere with cytokine-mediated activation may promote the pathogenesis of this infection.