Induction of interleukin-1 and tumor necrosis factor alpha in brain cultures by human immunodeficiency virus type 1.

Cytokines such as interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) are produced by leukocytes and play a role in immune responses. They also function in normal brain physiology as well as in pathological conditions within the central nervous system, where they are produced by brain macrophages (microglia) and brain astrocytes. In this study, we document the ability of human immunodeficiency virus type 1 (HIV-1) to induce TNF alpha and IL-1 in primary rat brain cultures. While productive infection did not occur in these cells, it was not required for cytokine induction. Using monocyte/macrophage-tropic (JRFL) and T-cell-tropic (IIIB) strains of HIV-1, we were able to induce cytokines in both microglia and astrocytes. In addition to whole virus, recombinant envelope proteins also induced these cytokines. The induction of IL-1 and TNF alpha could be blocked by a panel of antibodies recognizing epitopes in the gp120 and gp41 areas of the envelope. Soluble recombinant CD4 did not block TNF alpha and IL-1 production. If TNF alpha and IL-1 can be induced in brain tissue by HIV-1, they may contribute to some of the neurologic disorders associated with AIDS.     

Differential regulation and function of Fas expression on glial cells

Fas/Apo-1 is a member of the TNF receptor superfamily that signals apoptotic cell death in susceptible target cells. Fas or Fas ligand (FasL)-deficient mice are relatively resistant to the induction of experimental allergic encephalomyelitis, implying the involvement of Fas/FasL in this disease process. We have examined the regulation and function of Fas expression in glial cells (astrocytes and microglia). Fas is constitutively expressed by primary murine microglia at a low level and significantly up-regulated by TNF-alpha or IFN-gamma stimulation. Primary astrocytes express high constitutive levels of Fas, which are not further affected by cytokine treatment. In microglia, Fas expression is regulated at the level of mRNA expression; TNF-alpha and IFN-gamma induced Fas mRNA by approximately 20-fold. STAT-1alpha and NF-kappaB activation are involved in IFN-gamma- or TNF-alpha-mediated Fas up-regulation in microglia, respectively. The cytokine TGF-beta inhibits basal expression of Fas as well as cytokine-mediated Fas expression by microglia. Upon incubation of microglial cells with FasL-expressing cells, approximately 20% of cells underwent Fas-mediated cell death, which increased to approximately 60% when cells were pretreated with either TNF-alpha or IFN-gamma. TGF-beta treatment inhibited Fas-mediated cell death of TNF-alpha- or IFN-gamma-stimulated microglial cells. In contrast, astrocytes are resistant to Fas-mediated cell death, however, ligation of Fas induces expression of the chemokines macrophage inflammatory protein-1beta (MIP-1beta), MIP-1alpha, and MIP-2. These data demonstrate that Fas transmits different signals in the two glial cell populations: a cytotoxic signal in microglia and an inflammatory signal in the astrocyte.     

HIV-1, macrophages, glial cells, and cytokines in AIDS nervous system disease

Hallmarks of central nervous system (CNS) disease in AIDS patients are headaches, fever, subtle cognitive changes, abnormal reflexes, and ataxia. Dementia and severe sensory and motor dysfunction characterize more severe disease. Autoimmune-like peripheral neuropathies, cerebrovascular disease, and brain tumors are also observed. Histological changes include inflammation, astrocytosis, microglial nodule formation, and diffuse de- or dysmyelination. Focal demyelination can also be seen. It is clear that AIDS-associated neurological diseases are correlated with greater levels of HIV-1 antigen or genome in tissues. In AIDS dementia, macrophages and microglial cells of the CNS are the predominant cell types infected and producing HIV-1. However, manifestations of the disease make it unlikely that direct infection by HIV-1 is responsible. It seems more likely that the effects are mediated through secretion of viral proteins or viral induction of cytokines that bind to glial cells and neurons. HIV-1 induction of such cytokines as interleukin 1 (IL 1) and tumor necrosis factor-alpha (TNF alpha) may lead to an autocrine feedback loop involving further productive virus replication and induction of other cytokines such as interleukin 6 (IL 6) and granulocyte-macrophage colony-stimulating factor (GMCSF). Interleukin 1 and TNF alpha in combination with IL 6 and GMCSF could account for many clinical and histopathological findings in AIDS nervous system diseases. As HIV-1 infected patients produce elevated levels of IL 1, TNF alpha, and IL 6, it will be important to make a formal connection between the presence of these factors in the CNS, which are all products of activated macrophages, astroglia, and microglia, their in vivo induction directly by virus or indirectly by virus-induced intermediates, and the clinical and pathological conditions seen in the nervous system in this disease.     

HIV-1 Tat induces monocyte chemoattractant protein-1-mediated monocyte transmigration across a model of the human blood-brain barrier and up-regulates CCR5 expression on human monocytes.

AIDS dementia is characterized by neuronal loss in association with synaptic damage. A central predictor for clinical onset of these symptoms is the infiltration of monocytes and macrophages into CNS parenchyma. Chronic HIV-1 infection of monocytes also allows these cells to serve as reservoirs for persistent viral infection. Using a coculture of endothelial cells and astrocytes that models several aspects of the human blood-brain barrier, we examined the mechanism whereby the HIV-derived factor Tat may facilitate monocyte transmigration. We demonstrate that treatment of cocultures on the astrocyte side with HIV-1 Tat induced significant monocyte chemoattractant protein (MCP)-1 protein. Astrocytes, but not endothelial cells, were the source of this MCP-1 expression. Supernatants from Tat-treated cocultures induced significant monocyte transmigration, which was detected by 2.5 h after the addition of PBMC. Pretreatment of the supernatants from Tat-stimulated cocultures with an Ab to MCP-1 completely blocked monocyte transmigration. Flow cytometric analysis of Tat-stimulated PBMC demonstrated that Tat up-regulated expression of the chemokine receptor, CCR5, on monocytes in a time-dependent manner. Taken together, our data indicate that HIV-1 Tat may facilitate the recruitment of monocytes into the CNS by inducing MCP-1 expression in astrocytes. These recruited monocytes may contribute to the pathogenesis of HIV-1-associated AIDS encephalitis and dementia.     

Tumor necrosis factor alpha and interleukin-1 alpha inhibit through different pathways interferon-gamma-induced antigen presentation, processing and MHC class II surface expression on astrocytes, but not on microglia

Astrocytes and microglia, two glial cell populations of the CNS, have been described to be involved in many immune processes. We used defined combinations of cytokines, interferon gamma (IFN-gamma)/interleukin-1 alpha (IL-1 alpha) and IFN-gamma/tumor necrosis factor alpha (TNF alpha), to simulate different in vitro immune environments observed in disease or inflammation. In these conditions, we analyzed and compared the regulating effects of these cytokines on cell surface and total expression of MHC II and on the capacity of murine astrocytes and microglia to present peptide and native antigens to specific primed T cells. Neither IL-1 alpha nor TNF alpha affected the IFN-gamma-induced antigen presentation capacity of microglia. Astrocytes, however, were severely impaired in their capacity to present native antigens and, to a minor extent, a peptide antigen. Total expression of MHC II was not affected by these cytokines in microglia, whereas in astrocytes it was reduced by IL-1 alpha and increased by TNF alpha. Both cytokines downregulated MHC II expression at the surface of astrocytes, but not of microglia. This shows that TNF alpha affects the of IFN-gamma-immunocompetent astrocytes to process and present antigen, probably either by altering membrane traffic of MHC II and of antigen and/or enzymatic activities associated with these mechanisms, while IL-1 alpha does so by downregulating MHC II expression. Altogether, our results illustrate how differently astrocytes and microglia react toward a defined, similar immune environment. One type of cell, the astrocytes, downregulate their T-cell stimulation and MHC II trafficking, and probably also their antigen processing, functions while the other, the microglia, maintain their antigen presentation potential.     

Evolving paradigms in the pathogenesis of HIV-1-associated dementia

HIV-1-associated dementia (HIV-D) remains a significant consequence of HIV-1 infection and AIDS. Since the clinical introduction of highly active antiretroviral therapy (HAART), the incidence of HIV-D has decreased, yet the prevalence has increased as patients are living longer under treatment. Additionally, a less severe form of HIV-D, minor cognitive motor disorder, has become an increasing issue. Two different models have been proposed for virus entry in the central nervous system (CNS) in HIV-D. In the 'Trojan horse' model, the virus enters the CNS early carried by macrophages and infects resident glia; later in the course of infection, virus replication is activated and additional monocyte/macrophages are recruited into the CNS via cytokine/chemokine networks and endothelial-cell-leukocyte interactions at the blood-brain barrier. In the 'late invasion' model, an inherently invasive activated monocyte subset is expanded from bone marrow as a result of immune dysregulation in the periphery in the setting of AIDS. In this review we discuss these two separate, although not mutually exclusive, means for virus entry and persistence in the CNS. Additionally, we explore mechanisms for neuronal injury and apoptosis, including the role of virus, viral and host proteins, oxidative stress and products of infected or uninfected activated microglia and astrocytes. Potential therapeutic strategies are also briefly discussed.     

HIV-1-infected macrophages induce astrogliosis by SDF-1alpha and matrix metalloproteinases

Brain macrophages/microglia and astrocytes are known to be involved in the pathogenesis of HIV-1-associated dementia (HAD). To clarify their interaction and contribution to the pathogenesis, HIV-1-infected or uninfected macrophages were used as a model of brain macrophages/microglia, and their effects on human astrocytes in vitro were examined. The culture supernatants of HIV-1-infected or uninfected macrophages induced significant astrocyte proliferation, which was annihilated with a neutralizing antibody to stromal cell-derived factor (SDF)-1alpha or a matrix metalloproteinase (MMP) inhibitor. In these astrocytes, CXCR4, MMP, and tissue inhibitors of matrix metalloproteinase mRNA expression and SDF-1alpha production were significantly up-regulated. The supernatants of infected macrophages were always more effective than those of uninfected cells. Moreover, the enhanced production of SDF-1alpha was suppressed by the MMP inhibitor. These results indicate that the activated and HIV-1-infected macrophages can indirectly induce astrocyte proliferation through up-regulating SDF-1alpha and MMP production, which implies a mechanism of astrogliosis in HAD.     

Motility and ramification of human fetal microglia in culture: an investigation using time-lapse video microscopy and image analysis

Microglia are mononuclear phagocytes of the central nervous system and are considered to derive from circulating bone marrow progenitors that colonize the developing human nervous system in the second trimester. They first appear as ameboid forms and progressively differentiate to process-bearing "ramified" forms with maturation. Signals driving this transformation are known to be partly derived from astrocytes. In this investigation we have used cocultures of astrocytes and microglia to demonstrate the relationship between motility and morphology of microglia associated with signals derived from astrocytes. Analysis of progressive cultures using time-lapse video microscopy clearly demonstrates the dynamic nature of microglia. We observe that ameboid microglial cells progressively ramify when cocultured with astrocytes, mirroring the "differentiation" of microglia in situ during development. We further demonstrate that individual cells undergo morphological transformations from "ramified" to "bipolar" to "tripolar" and "ameboid" states in accordance with local environmental cues associated with astrocytes in subconfluent cultures. Remarkably, cells are still capable of migration at velocities of 20-35 microm/h in a fully ramified state overlying confluent astrocytes, as determined by image analysis of motility. This is in keeping with the capacity of microglia for a rapid response to inflammatory cues in the CNS. We also demonstrate selective expression of the chemokines MIP-1alpha and MCP-1 by confluent human fetal astrocytes in cocultures and propose a role for these chemotactic cytokines as regulators of microglial motility and differentiation. The interchangeable morphological continuum of microglia supports the view that these cells represent a single heterogeneous population of resident mononuclear phagocytes capable of marked plasticity.     

Manganese modulates pro-inflammatory gene expression in activated glia

Redox-active metals are of paramount importance for biological functions. Their impact and cellular activities participate in the physiological and pathophysiological processes of the central nervous system (CNS), including inflammatory responses. Manganese is an essential trace element and it is required for normal biological activities and ubiquitous enzymatic reactions. However, excessive chronic exposure to manganese results in neurobehavioral deficits. Recent evidence suggests that manganese neurotoxicity involves activation of microglia or astrocytes, representative CNS immune cells. In this study, we assessed the molecular basis of the effects of manganese on the modulation of pro-inflammatory cytokines and nitric oxide (NO) production in primary rat cortical glial cells. Cultured glial cells consisted of 85% of astrocytes and 15% of microglia. Within the assayed concentrations, manganese was unable to induce tumor necrosis factor alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS) expression, whereas it potentiated iNOS and TNF-alpha gene expression by lipopolysaccharide/interferon-gamma-activated glial cells. The enhancement was accompanied by elevation of free manganese, generation of oxidative stress, activation of mitogen-activated protein kinases, and increased NF-kappaB and AP-1 binding activities. The potentiated degradation of inhibitory molecule IkappaB-alpha was one of underlying mechanisms for the increased activation of NF-kappaB by manganese. However, manganese decreased iNOS enzymatic activity possibly through the depletion of cofactor since exogenous tetrahydrobiopterin reversed manganese's action. These data indicate that manganese could modulate glial inflammation through variable strategies.     

Neurovirulent simian immunodeficiency virus infection induces neuronal, endothelial, and glial apoptosis.

BACKGROUND: Studies of human immunodeficiency virus type 1 (HIV-1) associated dementia have shown neuronal loss in discrete areas. The presence and mechanism of neuronal death, however, has remained quite elusive. One mechanism of cell death, apoptosis, has been clearly demonstrated outside the central nervous system (CNS) in HIV-1 infection but has not been firmly established within the CNS. Therefore, we set out to ascertain whether neuronal cell loss in simian immunodeficiency virus (SIV) encephalitis, an animal model of HIV-1-associated dementia, is a result of apoptosis. MATERIALS AND METHODS: With the aid of an in situ technique for identifying the 3'-OH ends of newly fragmented DNA characteristic of apoptosis, in conjunction with specific detected morphological criteria via light microscopy, we have examined encephalitic and nonencephalitic brains of macaques infected with a neurovirulent, neuroendotheliotropic strain of SIV to see if virus is spatially associated with apoptosis of neurons and non-neuronal cell types. RESULTS: We demonstrate the presence of DNA damage, indicative of apoptosis, in neurons, endothelial cells, and glial cells of the CNS of SIV-infected macaques. Furthermore, we observe an association between the localization of cells with significant DNA fragmentation and perivascular inflammatory cell infiltrates containing SIV-infected macrophages and multinucleated giant cells. Quantitative analysis reveals significantly more cells with DNA fragmentation in the CNS of macaques infected with neurovirulent, neuroendotheliotropic SIV strains as compared with strictly lymphocyte-tropic SIV strains and SIV negative controls. CONCLUSIONS: Our findings of apoptosis in SIV-infected CNS may potentially lead to a better understanding of the AIDS dementia complex, ultimately providing a basis for better treatments.     

Macrophage-inflammatory protein-1alpha receptor expression on normal and chronic myeloid leukemia CD34+ cells

We have assessed expression of MIP-1alpha binding sites on the surface of CD34+ cells from normal bone marrow (NBM) and chronic myeloid leukemia (CML) peripheral blood. This study has highlighted a small subpopulation of CD34+ (15.7 +/- 6.2% in NBM and 9 +/- 4% in CML), which has specific macrophage-inflammatory protein-1alpha (MIP-1alpha) cell surface binding sites. Further phenotypic characterization of the receptor-bearing cells has shown that they do not express the Thy-1 Ag, suggesting that they are committed progenitor cells rather than CD34+ Thy+ stem cells. However, more than 80% of methanol-fixed CD34+ cells do bind MIP-1alpha, suggesting that these cells may possess a pool of internal receptors, although we were unable to induce cell surface expression by cytokine stimulation. The percentage of these CD34+, MIP-1alpha-R+ cells present in the CD34 compartment of NBM is significantly higher than in CML, implicating lack of binding sites as part of the mechanism for the loss of response to this chemokine seen in CML. Specific Ab to the MIP-1alpha receptor implicated in HIV infection, CCR5, revealed that very few CD34+ cells expressed these receptors and that expression was confined to the CD34+ Thy- progenitor population. Data presented in this work suggest that active binding sites for the stem cell growth inhibitor MIP-1alpha are not constitutively expressed on the surface of most resting primitive multipotent cells, and that these cells are not potential targets for HIV-1 infection through CCR5.