ABSTRACT
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.
Subject(s)
Astrocytes/virology , Cytomegalovirus Infections/immunology , Cytomegalovirus/immunology , Encephalitis, Viral/immunology , Microglia/virology , Astrocytes/cytology , Astrocytes/immunology , Brain/cytology , Brain/virology , Cells, Cultured , Chemokine CCL3 , Chemokine CCL4 , Chemokine CCL5/genetics , Chemokine CCL5/immunology , Chemotaxis/immunology , Coculture Techniques , Cytomegalovirus/growth & development , Encephalitis, Viral/virology , Fetus/cytology , Gene Expression Regulation, Viral/immunology , Humans , Interleukin-1/genetics , Interleukin-1/immunology , Interleukin-6/genetics , Interleukin-6/immunology , Interleukin-8/genetics , Interleukin-8/immunology , Macrophage Inflammatory Proteins/genetics , Macrophage Inflammatory Proteins/immunology , Microglia/cytology , Microglia/immunology , RNA, Messenger/analysis , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunology , Virus ReplicationABSTRACT
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.
Subject(s)
Cytokines/immunology , Encephalitis, Herpes Simplex/immunology , Herpesvirus 1, Human/growth & development , Microglia/virology , Astrocytes/cytology , Astrocytes/virology , Brain/cytology , Brain/virology , Butadienes/pharmacology , Cell Death/immunology , Cells, Cultured , Chemokine CCL5/biosynthesis , Chemokine CCL5/immunology , Chemokine CXCL10 , Chemokines, CXC/biosynthesis , Chemokines, CXC/immunology , Cytokines/biosynthesis , Enzyme Inhibitors/pharmacology , Fetus/cytology , Humans , Imidazoles/pharmacology , Interleukin-1/biosynthesis , Interleukin-1/immunology , Microglia/cytology , Microglia/metabolism , Neurons/cytology , Neurons/virology , Nitriles/pharmacology , Pyridines/pharmacology , Signal Transduction/drug effects , Signal Transduction/immunology , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/immunology , Virus ReplicationABSTRACT
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.
Subject(s)
Astrocytes/virology , CD4-Positive T-Lymphocytes/immunology , Cytomegalovirus/physiology , Astrocytes/immunology , CD4-Positive T-Lymphocytes/virology , Humans , Virus Replication/immunologyABSTRACT
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.
Subject(s)
Astrocytes/immunology , Astrocytes/virology , Cytokines/pharmacology , Cytomegalovirus/immunology , Inflammation Mediators/pharmacology , Viral Proteins , Antiviral Agents/pharmacology , Astrocytes/metabolism , Cells, Cultured , Cytomegalovirus/genetics , DNA, Viral/antagonists & inhibitors , DNA, Viral/biosynthesis , Dose-Response Relationship, Immunologic , Gene Expression Regulation, Viral/immunology , Genes, Immediate-Early/immunology , Humans , Immediate-Early Proteins/antagonists & inhibitors , Immediate-Early Proteins/biosynthesis , Immediate-Early Proteins/genetics , Nitric Oxide/physiology , Promoter Regions, Genetic/immunology , RNA, Messenger/antagonists & inhibitors , RNA, Messenger/biosynthesis , Time Factors , Virus Replication/immunologyABSTRACT
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.